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studies in
Natural Products Chemistry
Volume 28
Bioactive Natural Products (Part I)
studies in Natural Products Chemistry
edited by Atta-ur-Rahman
Vol. 1 Stereoselective Synthesis (Part A)

Vol. 2 Structure Elucidation (Part A)
Vol. 3 Stereoselective Synthesis (Part B)
Vol. 4 Stereoselective Synthesis (Part C)
Vol. 5 Structure Elucidaton (Part B)
Vol. 6 Stereoselective Synthesis (Part D)
Vol. 7 Structure and Chemistry (Part A)
Vol. 8 Stereoselective Synthesis (Part E)
Vol. 9 Structure and Chemistry (Part B)
Vol. 10 Stereoselective Synthesis (Part F)
Vol. 11 Stereoselective Synthesis (Part G)
Vol. 12 Stereoselective Synthesis (Part H)
Vol. 13 Bioactive Natural Products (Part A)
Vol. 14 Stereoselective Synthesis (Part I)
Vol. 15 Structure and Chemistry (Part C)
Vol. 16 Stereoselective Synthesis (Part J)
Vol. 17 Structure and Chemistry (Part D)
Vol. 18 Stereoselective Synthesis (Part K)
Vol. 19 Structure and Chemistry (Part E)
Vol. 20 Structure and Chemistry (Part F)
Vol. 21 Bioactive Natural Products (Part B)
Vol. 22 Bioactive Natural Products (Part C)
Vol. 23 Bioactive Natural Products (Part D)
Vol. 24 Bioactive Natural Products (Part E)
Vol. 25 Bioactive Natural Products (Part F)
Vol. 26 Bioactive Natural Products (Part G)
Vol. 27 Bioactive Natural Products (Part H)
Vol. 28 Bioactive Natural Products (Part I )
studies in
natural Products Chemistry
Volume 28
Bioactive natural Products (FM I)
Edited by
Atta-ur-Rahman
H.E.J. Research Institute of Chemistry,
University of Karachi, Karachi 75270, Pakistm
2003
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FOREWORD
"Studies in Natural Products Chemistry" has become the world's leading series of
volumes in the field of isolation, structure elucidation, biological activity and synthesis of
natural products, with comprehensive reviews written by leading experts. Volume 1 of the
series was published in March 1988 and during the last 15 years 27 volumes have been
published. From Volume 21 onwards, the series has been devoted to bioactive natural
products. Natural products present a huge source of organic substances with differing
structural features. The very large number of terrestrial and marine natural products
found in differing environmental conditions with correspondingly different biosynthetic
patterns gives medicinal chemists access to millions of substances with differing
bioactivity profiles. This represents a genuine treasure chest for discovering new
medicinal agents against a variety of diseases. The series on bioactive natural products
should therefore be of considerable interest not only to natural product chemists but also
to medicinal chemists, pharmacologists, and synthetic organic chemists working in
academia and industry. I hope that the present volume will be received with the same
excitement and enthusiasm as the previous volumes of his encyclopaedic series.
I would like to express my thanks to Mr. Shakeel Ahmad for his assistance in the
preparation of the index. I am also grateful to Mr. Waseem Ahmad for typing and to Mr.
Mahmood Alam for secretarial assistance.
Atta-ur-Rahman
Ph.D. (Cantab.), Sc.D. (Cantab.)
Chairman, Higher Education Commission
Government of Pakistan
January, 2003
This Page Intentionally Left Blank
PREFACE
In various segments of the scientific community, natural products research comes in

and out of favor In a cyclic manner as a function of time. Irrespective of this rhythmic
pattern, the true and absolute value of natural products research is immutable, in the
context of both applied and basic investigations. Clearly, therapeutic modalities
throughout the world rely heavily on natural product drugs and formulations. Similarly,
although acknowledgements may be less overt or absent, many aspects of basic
research programs are intimately related to natural products. In essence, natural
products play an integral and ongoing role in promoting numerous aspects of scientific
advancement.
Obviously, for progress to be realized on a widespread basis, general dissemination of

contemporary information is necessary. Incredibly, we now see the 28th volume in the
series Studies in Natural Product Ctiemistry edited by Professor Atta-ur-Rahman. The
significance of this indelible effort cannot be overestimated. More specifically, in the
same impeccable manner as the former volumes, we are again presented with cutting-
edge contributions of great importance. The first paper presents over 100 compounds
obtained from Broussonetia spp., and discusses biological activities. This is followed by
similar contributions dealing with the genus Licania and Ginkgo biloba. Additional
papers describe in detail a number of interesting and important natural compounds or
structural classes: retinoids, tetramic acid metabolites, isoprenylated flavonoids, plant
polyphenols, crocin, marcfortine and paraherquamide, acarlcides, podolactones,
triterpene glycosides and sulfur-containing marine compounds. An additional paper
focuses on the antitumor activities of lipids, and a final contribution deals with natural
product amelioration of cancer chemotherapy-Induced adverse reactions.
These astute summaries are provided by well-respected authors from seven different
countries. Assembly of the volume is a notable achievement; the work as a whole
nicely illustrates the types of critical discoveries that emanate from the interface of
chemistry and biology. Volume 28 can stand as a proud member of this great family of
useful reference books.
John M. Pezzuto
Professor and Dean
Schools of Pharmacy, Nursing
and Health Sciences
Purdue University
CONTENTS
Foreword v
Preface vii
Contributors xi
Bioactive compounds from the genus Broussonetia

DONGHO LEE and A. DOUGLAS KINGHORN 3
Chemical and biological studies on Licania genus

ALESSANDRA BRACA, ANNA RITA BILIA, JEANNETTE MENDEZ,
COSIMO PIZZA, IVANO MORELLI and NUNZIATINA DE TOMMASI 35
Recent progress in retinoid chemistry

ALAIN R. VALLA, DOMINQUE L. CARTIER and ROGER LABIA 69
Bioactive tetramic acid metabolites

EMILIO L. GHISALBERTI 109
Chemistry and biological activities oi Ginkgo biloba

K. SASAKI, K. WADA and M. HAGA 165
Chemistry and biological activities of isoprenylated flavonoids from

medicinal plants (moraceous plants and Glycyrrhiza species)
TARO NOMURA, TOSfflO FUKAI and YOSfflO HANO 199
Plant polyphenols: Structure, occurrence and bioactivity

PIERGIORGIO PIETTA, MARKUS MINOGGIO and LORENZO BRAMATI 257
Promising pharmacological actions of crocin in Crocus sativus on the central

nervous system
SHINJI SOEDA, TAKASHIOCHIAI, HIROSHI SfflMENO, fflROSHI SAITO
KAZUO ABE, MINORU SUGIURA, HIROYUKI TANAKA,
FUTOSHI TAURA, SATOSHI MORIMOTO and YUKIHIRO SHOYAMA 313
Synthesis and modification of marcfortine and paraherquamide class

of anthelmintics
BYUNG H.LEE 331
Acaricides of natural origin, personal experiences and review of

literature (1990-2001)
GUIDOFLAMINI 381
Podolactones: A group of biologically active norditerpenoids

ALEJANDRO F. BARRERO, JOSE F. QUILEZ DEL MORAL
and M. MAR HERRADOR 453
Antitumoral activity of lipids A studies in animal models and cancer patients
DANIELE REISSER, NOLWENN GAUTHIER, ALENA PANCE and
JEAN-FRANCOIS JEANNIN 517
Prevention of cancer chemotherapy drug-induced adverse reaction, antitumor

and antimetastatic activities by natural products
YOSHIYUKIKIMURA 559
Biologically active triterpene glycosidesfromsea cucumbers (holothuroidea,

echinodermata)
HUGO D. CHLUDIL, ANA P. MURRAY, ALICIA M. SELDES and
MARTA S. MAIER 587
Sulfur-containing natural products from marine invertebrates

MICHELE R. PRINSEP 617
Subject Index 753

CONTRIBUTORS
Kazuo Abe Graduate School of Pharmaceutical Sciences, The

University of Tokyo, Tokyo 113-0033, Japan
Alejandro F. Barrero Department of Organic Chemistry, Institute of

Biotechnology, University of Granada, Avda.,
Fuentenueva, 18071, Granada, Spain
Anna Rita Bilia Dipartimento di Scienze Farmaceutiche, Universita di

Firenze, Via Gino Capponi 9, 55100 Firenze, Italy
Alessandra Braca Dipartimento di Chimica Bioorganica e Biofarmacia,

Universita di Pisa, Via Bonanno 33, 56126, Pisa, Italy
Lorenzo Bramati ITB-CNR, Via F. Ui Cervi, 93 - 20090 (MI), Italy
Dominique L. Cartier FRE 2125 CMIS, 6 rue de TUniversite 29000 Quinq)er,

France
Hugo D. Chludil Departamento de Quimica Organica, Facultad de Ciencias

Exactas y Naturales, Universidad de Buenos Aires,
Pabellon 2, Ciudad Universitaria, (1428) Buenos Aires,
Argentina
Guido Flamini Dipartimento di Chimica Bioorganica e Biofarmacia, Via

Bonanno 33, 56126 Pisa, Italy
Toshio Fukai School of Pharmaceutical Sciences, Toho University, 2-2-

1 Miyama, Fimabashi, Chiba 274-8510, Japan
Nolwenn Gauthier Cancer Immunotherapy Research Laboratory, Ecole

Pratique des Hautes Etudes, INSERM U517, Faculty of
Medicine, 7 Bd Jeanne d'Arc, 21079 Dijon, France
Emilio L. Ghisalberti Department of Chemistry, University of Western

Australia, Nedlands, 6009 W.A., Australia
M. Haga Department of Hygienic Chemistry, Faculty of

Pharmaceutical Sciences, Health Sciences, University of
Hokkaido, Ishikari-Tobetsu, Hokkaido 061-0293, Japan
Yoshio Hano School of Pharmaceutical Sciences, Toho University, 2-2-

1 Miyama, Funabashi, Chiba 274-8510, Japan
M. Mar Herrador Department of Organic Chemistry, Institute of
Jean-Francois Jeannin Cancer Immunotherapy Research Laboratory, Ecole

Yoshiyuki Kimura Second Department of Medical Biochemistry, School of

Medicine, Ehime University, Shigenobu-cho, Onsen-gun,
Ehime 791-0295, Japan
A. Douglas Kinghom Program for Collaborative Research in the Pharmaceutical

Sciences and Department of Medicinal Chemistry and
Pharmacognosy, College of Pharmacy, University of
Illinois at Chicago, Chicago, Illinois 60612, USA
Roger Labia FRE 2125 CNRS, 6 rue de I'Universite 29000 Quimper,

France
Byung H. Lee Preclinical Development, Pharmacia Animal Health, 7000

Portage Road, Kalamazoo, MI 49001, USA
Dongho Lee Chemistry and Life Sciences, Research Triangle Institute,

P.O. Box 12194, Research Triangle Park, North Carolina
27709, USA
Departamento de Quimica Organica, Facultad de Ciencias

Marta S. Maier Exactas y Naturales, Universidad de Buenos Aires,
Argentina
Grupo de Productos Naturales, Centro de Quimica

Jeannette Mendez
Organica, Escuela de Quimica, Facultad de Ciencias,
Universidad Central de Venezuela, Apartado de Correos
47102, Caracas 1020-A, Venezuela
Markus Minoggio ITB-CNR, Via F. Hi Cervi, 93 - 20090 (MI), Italy
Jose F. Quilez Del Moral Department of Organic Chemistry, Institute of

Ivano Morelli Dipartimento di Chimica Bioorganica e Biofarmacia,

Universita di Pisa, Via Bonanno 33, 56126, Pisa, Italy
Satoshi Morimoto Graduate School of Pharmaceutical Sciences, Kyushu

University, Fukuoka 812-8582, Japan
Ana P. Murray Departamento de Quimica Organica, Facultad de Ciencias
Argentina
Taro Nomura School of Pharmaceutical Sciences, Toho University, 2-2-

1 Miyama, Funabashi, Chiba 274-8510, Japan
Takashi Ochiai Faculty of Pharmaceutical Sciences, Fukuoka University,

Fukuoka 814-0180, Japan
Alena Pance Cancer Immunotherapy Research Laboratory, Ecole

Piergiorgio Pietta ITB-CNR, Via F. Ui Cervi, 93 - 20090 (MI), Italy
Cosimo Pizza Dipartimento di Scienze Farmaceutiche, Universita di

Salemo, Via Ponte Don Melillo, 84084 Fisciano, Salemo,
Italy
Michele R. Prinsep Department of Chemistry, University of Waikato, Private

Bag 3105, Hamilton, New Zealand
Daniele Reisser Cancer Immunotherapy Research Laboratory, Ecole

Hiroshi Saito Graduate School of Pharmaceutical Sciences, The

K. Sasaki Department of Hygienic Chemistry, Faculty of

Alicia M. Seldes Departamento de Quimica Organica, Facultad de Ciencias

Argentina
Hiroshi Shimeno Faculty of Pharmaceutical Sciences, Fukuoka University,

Yukihiro Shoyama Graduate School of Pharmaceutical Sciences, Kyushu

Shinji Soeda Faculty of Pharmaceutical Sciences, Fukuoka University,

Minoru Sugiura Graduate School of Pharmaceutical Sciences, The
Hiroyuki Tanaka Graduate School of Pharmaceutical Sciences, Kyushu

Futoshi Taura Graduate School of Pharmaceutical Sciences, Kyushu

Nunziatina De Tommasi Dipartimento di Scienze Farmaceutiche, Universita di

Salerno, Via Ponte Don Melillo, 84084 Fisciano, Salerno,
Italy
Alain R. Valla VA R&D Pepiniere d'entreprises, 140 Bd de Creac'h

Gween 29561 Quimper, France
K. Wada Department of Hygienic Chemistry, Faculty of

Bioactive Natural Products
AUa-ur>Rahinan (Ed.) Studies in Natural Products Chemistry, Vol. 28
© 2003 Elsevier Science B.V. All rights reserved.
BIOACTIVE COMPOUNDS FROM THE GENUS

BROUSSONETIA
DONGHO LEE^ and A. DOUGLAS KINGHORN*
Program for Collaborative Research in the Pharmaceutical Sciences

and Department ofMedicinal Chemistry and Pharmacognosy, College of
Pharmacy, University ofIllinois at Chicago, Chicago, Illinois 60612,
USA,
ABSTRACT: The genus Broussonetia of the Moraceae (mulberry family) is of both

ethnomedical and industrial interest. Of the approximately 30 species in this genus, only
three have been subjected to previous phytochemical investigation, namely, B. kazinoki,
B. papyriferay and B, zeylanica. From over 100 compounds isolated from these species,
the major secondary metabolites reported thus far are alkaloids of the pyrrolidine type
and several types of flavonoids. Some of these compounds have exhibited various
biological activities, such antioxidative, aromatase inhibitory, cytotoxic, glycosidase
inhibitory, and platelet aggregation inhibitory effects. The biologically active
constituents of the species in the genus Broussonetia are discussed in detail.
INTRODUCTION
The genus Broussonetia L*Her. ex Vent, of the Moraceae (mulberry

family) is represented by lactiferous trees or shrubs. Broussonetia
comprises about 30 species and is distributed throughout various regions
of the wôrld including Africa, East Asia, and North America [1,2]. Thus
far, only three species of the genus Broussonetia have been studied for
their secondary metabolites, namely, B, kazinoki, B. papyrifera, and B.
zeylanica.
Broussonetia kazinoki Siebold & Zucc. is a deciduous tree
growing to 4.5 m that flowers in August. It occurs in mainland China,
Japan, and Korea [1]. The plant requires well-drained soil but can grow in
Address correspondence to this author at Program for Collaborative Research in the

Pharmaceutical Sciences and Department of Medicinal Chemistry and Pharmacognosy
(M/C 781), College of Pharmacy, University of Illinois at Chicago, 833 South Wood
Street, Chicago, Illinois 60612, U.S.A. E-mail: kinghom@uic.edu.
^Current address: Chemistry and Life Sciences, Research Triangle Institute, P.O. Box
12194, Research Triangle Park, North Carolina 27709, U.S.A.
nutritionally poor soil [3]. Preparations made from B. kazinoki have been
used as a tonic to increase vision and sexual potency, and to treat boils,
eczema, infant colic, and leukorrhea [4,5]. Various extracts of 5. kazinoki
have exhibited antifungal, antiinflammatory, antioxidant, and
antispasmodic activities [6-10].
Broussonetia papyrifera (L.) L'Her. ex Vent, is a deciduous tree
growing up to 15 m that is commonly called the paper mulberry. It is
native to East Asia, then later introduced and naturalized in the United
States. It flowers from August to September, and the seeds ripen from
September to November [1,11]. The plant prefers light and well-drained
soil and' is easily cultivated in a warm sunny position in any soil of
reasonable quality [3]. Fibers from the bark are used in making paper,
cloth, and rope. These fibers can be produced by beating strips of bark on
a flat surface with a wooden mallet [12]. 5. papyrifera has been used for
cancer, dyspepsia, and pregnancy [13]. In mainland China, the fruits of 5.
papyrifera have been employed for impotency and ophthalmic disorders
[4,14], Also, the leaf juice of 5. papyrifera is diaphoretic and laxative and
the stembark is hemostatic [4]. Antifungal and antioxidant activities of the
extracts ofB. papyrifera were reported [6,7,9].
Broussonetia zeylanica (Thwait.) Comer is endemic to Sri Lanka
and its tough bark-fibers were used to make string [15].
Several types of bioactive compounds have been reported from the
genus Broussonetia including glycosidase inhibitory alkaloids and
aromatase inhibitory or cytotoxic flavonoids. This chapter reviews the
biologically active constituents from the genus Broussonetia reported by
the end of 2001.
BIOACTIVE COMPOUNDS FROM BROUSSONETIA KAZINOKI
The bioactive secondary metabolites reported from Broussonetia kazinoki

can be classified into major two groups, alkaloids and flavonoids (Table
1), Fig. (1). The Kusano group at Osaka University of Pharmaceutical
Sciences in Japan reported over 20 pyrrolidine alkaloids, broussonetines
A-H, K-M, 0-T, V-X, and Mi, and broussonetinines A and B, four
pyrrolidinyl piperidine alkaloids, broussonetines I, J, Ji, and J2, two
pyrroline alkaloids, broussonetines U and Ui, and one pyrrolizidine
alkaloid, broussonetine N, from hot water extracts of 5. kazinoki [16-24].
As shown in Table 1, some of these alkaloids exhibited strong
Table 1. Bioactive Compounds from Broussonetia kazinoki
Compound type/name Activity Reference
ALKALOIDS
Pyrrolidines
Broussonetine C (1) Inhibition of glycosidases* [16]
Broussonetine D (2) Inhibition of glycosidases* [16]
Broussonetine E (3) Inhibition of glycosidases" [17]
Broussonetine F (4) Inhibition of glycosidases* [17]
Broussonetine G (5) Inhibition of glycosidases* [18]
Broussonetine H (6) Inhibition of glycosidases* [18]
Broussonetine K (7) Inhibition of glycosidases* [20]
Broussonetine L (8) Inhibition of glycosidases* [20]
Broussonetine M (9) Inhibition of glycosidases* [21]
Broussonetine 0 (10) Inhibition of glycosidases* [21]
Broussonetine P (11) Inhibition of glycosidases* [21]
Broussonetine Q (12) Inhibition of glycosidases* [21]
Broussonetinine A (13) Inhibition of glycosidases* I [17]
Broussonetinine B (14) Inhibition of glycosidases* [17]
Pyrrolizidine
Broussonetine N (15) Inhibition of glycosidases* [22]
FLAVONOIDS
Diphenylpropanes
KazinolD(16) Cytotoxicity against human tumor cell lines'* [25]
Ka2inolK(17) Cytotoxicity against human tumor cell lines** [25]

Table 1. Bioactive Compounds from Broussonetia kazinoki (continued)
Compound type/name Activity Reference
Flavans
7,4'-Dihydroxyflavan (18) Cytotoxicity against human tumor cell lines'* [25]
Antioxidant activity*" [26]

KazinolA(19)
Inhibition of tyrosinase** [26]
Antioxidant activity*^ [26]
KazinolE(20)
Inhibition of tyrosinase' [26]
KazinolQ(21) Cytotoxicity against human tumor cell lines'* [25]
KazinolR(22) Cytotoxicity against human tumor cell lines'* [25]
Flavonols
Broussonol A (23) Cytotoxicity against human tumor cell lines'* [27]
Broussonol B (24) Cytotoxicity against human tumor cell lines'* [27]
Broussonol C (25) Cytotoxicity against human tumor cell lines'* [27]
Broussonol D (26) Cytotoxicity against human tumor cell lines'* [27] 1

"Glycosidase inhibitory activity expressed as ICso value (jiM); 1: p-Gal = 0.036, p-Man = 0.32; 2: P-Gal =
0.029, P-Man = 0.34; 3: a-Glc = 3.3, P-Glc == 0.055, P-Gal = 0.002, p-Man = 0.023; 4: a-Glc = 1.5, p-Glc =
0.01, P-Gal = 0.004, P-Man = 0.028; 5: P-Glc = 0.024, P-Gal = 0.003, P-Man = 0.76; 6: P-Glc = 0.036, p-Gal =
0.002, P-Man = 0.32; 7: P-Glc = 0.026, P-Gal = 0.005, P-Man = 0.3; 8: P-Glc = 0.017, P-Gal = 0.004, p-Man =
0.2; 9: P-Gal = 8.1; 10: P-Glc = 1.4, P-Gal = 0.17, P-Man = 8.2; 11: P-Glc = 2.4, P-Gal = 0.2, P-Man = 7.6; 12:
P-Glc = 1.4, P-Gal = 0.6, P-Man = 20.0; 13: P-Gal = 0.016, a-Man = 0.3; 14: P-Gal = 0.01, a-Man = 0.29; 15:
P-Glc = 6.7, p-Gal = 2.9, P-Man = 3.3 (P-Gal = p-Galactosidase; a-Glc = a-Glucosidase; P-Glc = p-
Glucosidase; a-Man = a-Mannosidase; P-Man = P-Mannosidase).
'*Cytotoxicity expressed as ED50 value (îg/mL); 16: PLC/PRF/5 = 3.3, 212 = 7.0, HT3 = 3.6; 17: HT3 = 8.6:
18: HT3 = 11.6, SiHa = 8.9, CaSki = 17.4; 21: PLC/PRF/5 = 3.5, T24 = 2.3, 212 = 3.8, HT3 = 4.3, SiHa - 4.7;
22: HT3 = 9.3, SiHa = 9.3, CaSki = 8.2; 23: A546 = 8.7, HCT-8 = 9.1; 24: A546 = 5.52, HCT-8 = 8.8; 25:
A546 = 7.8, HCT-8 = 9.6; 26: KB = 4.5 (key to cell lines; 212 = inducible Ha-ras oncogene transformed
NIH/3T3; A549 = human lung carcinoma; CaSki = human cervical carcinoma; HCT-8 = human ileocecal
carcinoma; HT3 = human cervical carcinoma; KB = human epidermoid carcinoma; PLC/PRF/5 = human
hepatoma; SiHa = human cervical carcinoma; T24 = human hepatoma).
'Ântioxidant activity shown by l,l-diphenyl-2-picryl-hydrazyl (DPPH) radical scavenging activity (IC50 pM);
19:41.4,20:33.4.
''Activity not specified.
nCso 241.3 nM.
H
CH2OR2
H(? OH
1 R,=H,R2 = H
3 R, = OH, R2 = H
7 R, = 0H,R2 = Glc
10 R, = H,R2 = H,A-3',4'
H
HOHjCî^^V-*'^ CH2OR2
H(f OH 2 R, = H,R2 = H
4 R, = 0H,R2 = H
8 R, = 0H,R2 = Glc
11 Ri=H,R2 = H,A-3',4'
HOH2C*^^\.-*'^
Ha OH
OH
H Ô
H0H2CM^^'\.»^'^
O
H(f OH
CH2OH
Fig. (1). Continued
H
CH2OR
HO OR
12 R = Glc
13 R==H
CH2OH
HOF^C^^'^V.^^^'
HC) OH
14
CH2OH
HOH2C I?
OH
16
17 A^3,4 18
HO.
TCO'
19
Fig. (1). Continued
OH
OH O
24
Fig. (1). Structures of bioactive constituents of Broussonetia kazinoki.
glycosidase inhibitory activity with IC50 values ranging from 0.002 to 8.2
[xM. Selective inhibition of glycosidase enzymes has a number of
potential therapeutic uses, including the treatment of cancer, diabetes, and
HIV-AIDS [28-32]. Also, the prenylated flavonoid derivatives, kazinols
D (16) and K (17) (diphenylpropanes), 7,4'-dihydroxyflavan (18),
10
kazinols Q (21), and R (22) (flavans), and broussonols A-D (23-26)

(flavonols), were isolated as moderate to weak cytotoxic principles
against several human cancer cell lines with ED50 values ranging from 2.3
to 17.4 îg/mL [25,27]. Two flavans, kazinols A (19) and E (20), were
reported as antioxidative principles using the l,l-diphenyl-2-picryl-
hydrazyl (DPPH) radical scavenging assay (IC50 41.4 and 33.4 |iM,
respectively) [26]. These compounds (19 and 20) also exhibited inhibitory
activity against tyrosinase, which is a key enzyme in melanin biosynthesis
and plays a role in the conversion of tyrosine to DOPA and DOFA to
dopaquinone [26,33]. An antioxidative effect and the suppression of
melanin biosynthesis are useful for cosmetic products in relation to
hyperpigmentation [34].
Broussonetine C (1), a monocyclic polyhydroxy pyrrolidine
alkaloid, showed a yellow spot on TLC when sprayed with ninhydrin
reagent and heated (ninhydrin reaction), and its molecular formula was
determined by a positive high-resolution mass spectrometry (C18H36NO5,
[M + H]^, m/z 346.2579). The IR spectrum displayed a hydroxy band at
3370 cm'^ and a carbonyl band at 1706 c m \ The ^H- and '^^C-NMR
signals were assigned using the ^H-^H correlated spectroscopy (^H-^H
COSY), heteronuclear single quantum coherence (HSQC), and
distortionless enhancement by polarization transfer (DEFT) pulse
sequences. The position of the carbonyl carbon and the linkage of the
pyrrolidine ring and the aliphatic side chain were determined using the
heteronuclear multiple bond coherence (HMBC) NMR technique [HMBC
correlations were observed for the carbonyl carbon signal (5c 210.8) with
the proton signals at 6H 2.71 (H-11') and 6H 2.12 (H-120, and for the C-5
carbon signal (5c 62.9) of the pyrrolidine ring with the proton signals at
5H 4.44 (H-4) and 5H 2.04 (H-l'), respectively] [16].
The relative stereochemistry of the pyrrolidine ring of
broussonetine C (1) was determined from its coupling constants (vicinal
coupling, ^2,3 = •/3,4 = •/4,5 = 6.4 Hz) and nuclear Overhauser enhancement
effects (H-2/H-4 and H-3/H-5). The absolute stereostructure was
disclosed as (2i?,3/?,4i?,5/?) using the benzoate chirality method [35]. A
diacetylacetoamide was prepared from broussonetine C (1) by treatment
with acetic anhydride in pyridine at room temperature, and then a
dibenzoate (la) was obtained by benzoylation of the diacetylacetoamide.
The circular dichroism (CD) curve of l a displayed a negative Cotton
11
effect (A8237 -15.9) and a positive effect (AS223 +16.4), which indicated a
negative chirality as shown in Fig. (2) [16].
C0CH3
BzO OBz
la
Ae(nin): +16.4(223)
-15.9(237)
Fig. (2). Determination of the absolute stereostnicture of broussonetine C (1) by the benzoate
chirality method.
Broussonetine L (8) showed similar physical and spectroscopic

properties to those of broussonetine C (1) [16], except for proton signals
of a p-glucose (anomeric proton, 8H4.78, 1H, doublet, J- 7.8 Hz) moiety
in the H-NMR spectrum. Hydrolysis of broussonetine L (8) with 1 N
HCl provided broussonetine F (4) [17] and D-glucose ([a]D +40.6°).
Therefore, the structure of broussonetine L (8) was determined to be 13'-
O-p-D-glucopyranosylbroussonetine F due to the glycosylation shift of C-
13' (6c 69.3) of broussonetine L (8) (broussonetine F, 4, 6c-i3' 61.6) and
HMBC long-range correlations observed between H-13' (6H 3.69 and
4.09) and an anomeric carbon (5c 104.4), and between an anomeric
proton and C-13'[20].
The absolute stereochemistry of broussonetine L (8) was
determined by the combination of the benzoate chirality method and the
Mosher's method [35-37]. A carbamate (8a) was prepared from
broussonetine F (4) by reaction with phenyl chloroformate in
tetrahydrofuran-H20 (7:3), and a diacetate (8b) was prepared from 8a
with acetic anhydride in pyridine. Finally, a dibenzoate (8c) was obtained
by benzoylation of 8b. The CD curve of 8c showed a negative Cotton
effect (AE237 -30.9) and a positive effect (Ae223 +15.9) to confirm a
counter-clockwise chirality between two benzoyl groups. Fig. (3) [20].
12
OH
IN. CH2OGIC
nd OH
OH
H
HOHjC*^'^.*^'^ CH2OH
Hcf OH
PhOCOCl
NaHCOj
O—CO QH
CH2OH
CH2OAC
HO OH
PhCOCl
pyridine
O—CO OAc
CH2OAC
Bz(f OBz 8c
OBz
BzQ A8(nm): +15.9(223)
-30.9 (237)
Fig. (3). Determination of the absolute stereostructure of the pyrrolidine ring of broussonetine L
(8) by the benzoate chirality method.
13
The absolute configuration of C-l' of 8 was then investigated by

the Mosher's method. The di- (R)- and (S)-2-methoxy-2-phenyl-2-
(trifluoromethyl)-acetic acid (MTPA) esters (8dR and 8d5) and tri- (R)-
and (iS)-MTPA esters (SeR and SeS) prepared from 8a, were analyzed by
' H - ' H C O S Y N M R (500 MHz) and A6 values (SS-SR) were measured.
These values established the R configuration of C-l' of 8 by comparison
of the di-MTPA esters (8d/? and 8d5) and the tri-MTPA esters (SeR and
8fty),Fig.(4)[20].
O—CO OR4
CH2OR1
R-,(f OR,
8dif, 8d5: Ri = R2 = MTPA, R3 - R4 = H

8ei?, ScJ: R, = R2 = R4 = MTPA. R3 = H
A^(^S-^R)
1" 2 3 4 5 r r
8d +0.030 +0.100 -0.039 -0.019 0.000 +0.020 0.000 1
1 8e -0.154 -0.332 -0.161 -0.037 -0.060 +0.013 +0.050
Fig. (4). Determination of the absolute configuration of C-l' of broussonetine L (8) by the
Mosher's method.
Also, the absolute stereochemistry of the pyrrolizidine ring and C-

r of broussonetine N (15) was established by the Mosher's method. The
tri- (Ry and (5)-MTPA esters (15a/? and 15a5) and penta- (Ry and (5)-
MTPA esters (15bif and IShS) were prepared from 15 and A6 values (65-
8R) were measured. Accordingly, the R configuration of C-l of the
pyrrolizidine ring from 15a and the R configuration of C-l' from 15b
were determined, respectively, Fig. (5) [22].
A biosynthetic study of the 18-carbon chain skeleton of
broussonetines was reported [38]. To verify the biosynthetic route of
these alkaloids, the plant was grown on an aseptic medium and the
enriched ^^C of the isolated alkaloids was analyzed by NMR after feeding
with [l-^^C]glucose. The labeling pattem of broussonetine J (27) obtained
14
MTPAO !?-^^^_,,,
-Z. H +0.013
-0.006 "L r +0.001
C-0.029 c V l
MTPAOH2C H CHoOMTPA
+0.061
OH
MTPAO ''-^\,ri^
-0.075'^ - -^-^'^
CH2OMTPA
OMTPA
15b
Fig. (5). Determination of the absolute configuration of broussonetine N (15) by the Mosher's
method.
from the feeding experiment indicated that C-4 through C-18 were
formed via palmitoyl CoA through the acetate-malonate pathway,
whereas C-1 through C-3 were derived via serine from 3-phosphoglyceric
acid. Therefore, the 18-carbon chain of broussonetine J (27) was assumed
to be formed initially by condensation of serine and palmitoyl CoA [38],
As shown in Fig. (6), the absolute stereochemistry of the pyrrolidine rings
of the broussonetines is related to o-serine and that of broussonetine U
(28) is related to L-serine.
Out of a series of over 30 alkaloids obtained from B, kazinoki,
some of them showed potent glycosidase inhibitory activity as shovm in
Table 1. Interestingly, only broussonetines E and F (3 and 4), which have
a hydroxyl group on C-T, demonstrated potent inhibitory activity against
a-glucosidase [17]. However, broussonetines G and H (5 and 6), which
also have a hydroxyl group on C-l', did not inhibit a-glucosidase [18].
These results suggested that the inhibition of a-glucosidase might be
attributed to the hydroxyl groups on both C-T and C-13' and the keto
groups of C-9' or C-10' [17,18]. However, additional studies seem to be
required to verify this suggestion [24].
15
CHjOH
-O.
COOH
OH OH
OH OH
OH
D-[l-^^C]glucose
O
II ^ HO
.COOH
H O - ^ '
COOH
SCoA NH2 NH2

D-serine L-serine
CoAS
OH
HO
HO OH
Fig. (6). Biosynthesis of broussonetines J and U (27 and 28).

16
BIOACTIVE COMPOUNDS FROM BROUSSONETIA

PAPYRIFERA
The major types of bioactive constituents reported from Broussonetia

papyrifera are the prenylated flavonoids, which include compoxmds of the
diphenylpropane, chalcone, flavan, flavanone, flavone, flavonol, and
aurone classes (Table 2), Fig. (7). An early study on B, papyrifera
resulted in the isolation of two diphenylpropanes, broussonins A (29) and
B (30), and a coumarin, marmesin (52), with antifungal activity [39].
Also, a diprenylated diphenylpropane derivative, kazinol F (31) [40], was
reported as an antioxidant and tyrosinase inhibitory constituent [34].
Table 2. Bioactive Compounds from Broussonetia papyrifera
Compound type/name Activity Reference(s)
FLAVONOroS
Diphenylpropanes
Antifungal activity* [39] 1

Broussonin A (29)
Inhibition of aromatase** [41]
Broussonin B (30) Antifungal activity* [39] 1

Antioxidant activity (scavenging free radicals)^ [34]
Kazinol F (31)
Inhibition of tyrosinase** [34] 1
Chalcones
Antioxidant activity (inhibition of lipid peroxidation)^ [42] 1
Inhibition of cyclooxygenase* [43]
Broussochalcone A (32) Inhibition of nitric oxide production'^ [42]
Inhibition of respiratory burst in neutrophils* [44]
Platelet aggregation inhibitory activity** [431
1 3'-[y-Hydroxymethyl-(£)-y-
methylallyl]-2,4,2',4'.
tetrahydroxychalcone 1 r - 0 -
coumarate (33)
Isogemichalcone C (34) Inhibition of aromatase*' [41]
1 2,4,2',4'-Tetrahydroxy-3'-
1 prenylchalcone (35)
Flavans
Antioxidant activity (inhibition of lipid peroxidation)*^ 1 [43] 1

Broussoflavan A (36)
1 Platelet aggregation inhibitory activity** 1 [45]
17
Table 2. Bioactive Compounds from Broussonetia papyrifera (continued)
Compound type/name Activity RefereDce(s)
Antioxidant activity' 1 [26]

KazinolA(19) Inhibition of tyrosinase' [26]
Platelet aggregation inhibitory activity** [43]
KazinolB(37)
Inhibition of cyclooxygenase* [43] 1
Platelet aggregation inhibitory activity** [43] 1
Flavanones
(25)-Abyssinone II (38) Inhibition of aromatase** [41]
(2.S)-2',4'-Dihydroxy-2"-(l-
hydroxy-1-methylethyl)- Inhibition of aromatase*' [41]
dihydrofuro[2,3-Alflavanone (39)
(2iS)-Euchrenone a? (40) Inhibition of aromatase*' [41]
(2.S)-Naringenin (41) Inhibition of aromatase** [41]
(25)-5,7,2',4'-
1 Tetrahydroxyflavanone (42)
Flavone
1 5,7,2',4'-Tetrahydroxy-3-
1 geranylflavone (43)
Flavonols
Broussoflavonol £ (44) Platelet aggregation inhibitory activity*" [43]

Antioxidant activity (inhibition of lipid peroxidation)' [45]
Antiproliferative activity* [45]
Broussoflavonol F (45) Inhibition of aromatase** [41]
Inhibition of cyclooxygenase* [43]
Platelet aggregation inhibitory activity*" [43]
Broussoflavonol G (46)
Antioxidant activity (inhibition of lipid peroxidation)*^ [45] 1
Antiproliferative activity* [45]
Isolicoflavonol (47) Inhibition of aromatase*' [41]
Aurone
'
Antioxidant activity (inhibition of lipid peroxidation)' [45]
Broussoaurone A (48) Inhibition of cyclooxygenase* [43]
Platelet aggregation inhibitory activity*" L. _ [43I_.
MISCELLANEOUS
AlbanoIA(49) Inhibition of aromatase** [41]
Selective cytotoxic activity against melanoma cell

Betulinic acid (50) [46]
lines'"
18
Table 2. Bioactive Compounds from Broussonetia papyrifera (continued)
Compound type/name Activity Reference(s)
Demethylmoracin I (51) Inhibition of aromatase** [41]
Marmesin(52) Antifungal activity* [39]
MoracinN(53) Inhibition of aromatase** [41]
Ursolic acid (54) Inhibition of HIV-1 protease dimerization'' [47]

"Antifungal activity (presented as a range) expressed as the minimum concentration (mM) required for
complete inhibition of fungal growth including Fusarium roseum, F. lateritium, F. solani, Diaporthe nomuraU
Stigmina mori, Sclerotinia sclerotiorum, Bipolaris leersiae, and Rosellinia necatrix\ 29: 0.2-0.9, 30: 0.05-0.9,
52: 0.9-4.0.
''Aromatase inhibitory activity determined as IC50 value (îM); 29: 30.0, 33: 0.5, 34: 7.1, 35: 4.6, 38: 0.4, 39:
0.1,40: 3.4,41: 17.0,42: 2.2,43: 24.0,45: 9.7,47: 0.1,49: 7.5,51: 31.1,53: 31.1.
Ântioxidant activity expressed as IC50 value (pM); 31: 6.7 (jig/mL); 32:0.63,36: 2.1,45:2.7,46:1.0,48: 1.2.
''The tyrosinase inhibitory activity of 31 was IC50 0.39 |ig/mL.
*Cyclooxygenase inhibitory effect determined as IC50 value (ng/mL); 32: 19.4,37: 155.3,45: 17.5,48: 22.7.
Înhibitory effect (IC50) of 32 on nitric oxide production was 11.3 îM.
^Compound 32 inhibited O2 consumption in formylmethionyl-leucyl-phenylalanine- and phoibol 12-myristate
13-acetate-stimulated rat neutrophils with IC50 values of 70.3 and 63.9 ^M, respectively.
•Ântiplatelet activity induced by arachidonic acid was expressed by IC50 value (^M); 19: 11.4, 32: 6.8, 36:
86.7,37: 32.6,44: 39.9,45:16.9,48: 15.4.
'Activity found as a constituent of Broussonetia kazinofd.
Ântiproliferation activity shown by the inhibition of ['H]thymidine incorporation into DNA in the proliferation
of rat vascular smooth muscle cells. The effect was expressed as % of control; 45: 0-7.8,46: 0-0.4.
''Activity found as a constituent of a plant other than a Broussonetia species.
Broussochalcone A (32) [48], a prenylated chalcone, is one of the

most completely studied constituents of B. papyrifera biologically.
Broussochalcone A (32) inhibited platelet aggregation induced by
arachidonic acid with an IC50 value of 6.8 |aM as well as induction by
adrenaline in human platelet-rich plasma. The antiplatelet effect of 32 was
partially due to an inhibitory effect on cyclooxygenase activity and by
reducing thromboxane fomiation [43]. Also, broussochalcone A (32)
inhibited O2 consiraiption in fomiylmethionyl-leucyl-phenylalanine- and
phorbol 12-myristate 13-acetate-stimulated rat neutrophils with IC50
values of 70.3 and 63.9 jiM, respectively. This inhibitory effect of 32 on
respiratory burst in neutrophils was not mediated by the reduction of
phospholipase C activity, but was mediated by the suppression of protein
kinase C activity through interference with the catalytic region and by the
19
RiO
29Ri = CH3,R2 = H
30Rj = H,R2 = CH3 31
33R = H
34R = OCH3
OH
36
20
Fig. (7). Continued
OH
OH
37
OH
OH
41R = H
42R = OH
21
Fig. (7). Continued
HO,
48 49
22
Fig. (7). Continued
COOH
50 51
HO-
o ^ ^ ^ -o- - o
52
CCX)H
54
Fig. (7). Structures of bioactive constituents of Broussonetia papyrifera.
attenuation of O2*" generation from the NADPH oxidase complex, which

might inhibit the generation of toxic oxygen radicals and terminate the
tissue damage [43]. Furthermore, broussochalcone A (32) showed
antioxidant activity in iron-induced lipid peroxidation in a rat brain
23
homogenate model with an IC50 value of 0.63 |iM as well as in the DPPH
system, and exhibited an inhibitory effect on nitric oxide (NO) production
with an IC50 value of 11.3 jaM. This potent inhibitory effect on NO
production was mediated by suppression of nuclear factor (NF)-KB
activation, phosphorylation and degradation of iKBa (an inhibitory
protein of NF-KB), and inducible NO synthesis expression, which have
been associated with autoimmune or inflammatory diseases [42].
In an effort to investigate antioxidant constituents with
antiproliferative effects in rat vascular smooth muscle cells (VSMC),
broussoflavan A (36) [49], broussoflavonols F (45) [50] and G (46) [51],
and broussoaurone A (48) [49] were found to inhibit the Fe^^-induced
thiobarbituric acid-reactive substance formation in rat brain homogenate.
Furthermore, broussoflavonols F (45) and G (46) inhibited fetal calf
serum-, 5-hydroxytryptamine-, or ADP-induced [^H]thymidine
incorporation into rat VSMC [45]. Antioxidant activities and inliibitory
effects on proliferation of rat VSMC with potent antiplatelet activities of
45 and 46 may be useful for vascular diseases and atherosclerosis [43,45].
The concept of cancer chemoprevention is becoming well-
established and refers to the pharmacological intervention to arrest or
reverse the process of carcinogenesis, and thus prevent cancer [52,53]. It
has become evident that various phytochemical components of the diet
are able to prevent cancer formation in full-term carcinogenesis inhibition
studies in animal models [54]. As part of a U.S. National Cancer Institute-
funded program project conducted at the University of Illinois at Chicago
[55-57], an ethyl acetate extract of the whole plants ofB, papyrifera was
found to significantly inhibit aromatase activity in an in vitro assay
[58,59] (74% inhibition at 80 |ig/mL) [41]. This was only one of a
handful of extracts found to significantly inhibit aromatase activity with
the bioassay protocol used, out of over 1,000 extracts screened [60]. This
target was chosen for investigation, because aromatase catalyzes the final,
rate-limiting step in estrogen biosynthesis [61], and is regarded as a target
relevant to the treatment or prevention of breast and prostate cancers [62].
Several synthetic aromatase-inhibitory drugs have been developed,
including aminoglutethimide, substrate androstenedione derivatives,
imidazoles, and triazoles [63-65].
From the active extract of B, papyrifera were isolated several
aromatase inhibitors with IC50 values in the range 0.1-31.1 ^M, inclusive
of broussonin A (29) [66], 3'-[Y-hydroxymethyl-(£)-y-methylallyl]-
24
2,4,2',4'-tetrahydroxychalcone 11 '-O-coumarate (33) [41],

isogemichalcone C (34) [41], 2,4,2',4'-tetrahydroxy-3'-prenylchalcone
(35) [67], (25)-abyssinone II (38) [68], (25)-2',4'.dihycIroxy-2"-(l-
hydroxy-1 -methylethyl)-dihydrofuro[2,3-A]flavanone (39) [41], (25)-
euchrenone a7 (40) [69], (25)-naringenin (41) [70], (25)-5,7,2',4'-
tetrahydroxyflavanone (42) [71], 5,7,2',4'-tetrahydroxy-3-geranylflavone
(43) [41], broussoflavonol F (45) [50], isolicoflavonol (47) [72], albanol
A (49) [73], demethylmoracin I (51) [41], moracin N (53) [74]. Of these
aromatase inhibitors, five of the compounds were new (33, 34, 39, 43,
51), and details of structure elucidation of 33, 34, and 43 are presented as
examples in the following two paragraphs.
The isolates 3'-[y-hydroxymethyl-(£^-Y-methylallyl]-2,4,2',4'.
tetrahydroxychalcone 11'-O-coumarate (33) and 3'-[Y-hydroxymethyl-
(£)-y-methylallyl]-2,4,2',4'-tetrahydroxychalcone 11 '-O-ferulate
(isogemichalcone C, 34) were obtained as orange powders and were
shown by positive HRFABMS to possess molecular formulas of C29H26O8
(m/z [M + Na]^ 525.1884) and C30H28O9 (m/z [M + N a ] \ 555.1577),
respectively. The ^H- and ^^C-NMR spectra of 33 and 34 exhibited
characteristic chalcone signals, and signals for a coumarate group for 33
at 6H 7.54 (2H, 7 = 8.6 Hz, H-2" and H-6"), 6H 6.87 (2H, J = 8.5 Hz, H-
3" and H.5''), 5H 7.59 (IH, 7 = 16.0 Hz, H-T'), and 5H 6.35 (IH, J = 16.0
Hz, H-8") and signals for a ferulate group for 34 at 6H 7.34 (IH, 7 = 1.6
Hz, H-2"), 6H 6.85 (IH, / = 8.1 Hz, H.5"), 6H 7.12 (IH, 7 = 1.7 and 8.2
Hz, H-6"), 5H 7.57 (IH, J = 16.0 Hz, H.7"), 5H 6.40 (IH, J= 15.9 Hz, H-
8"), and 6H 3.91 (3H, singlet, OCH3). Based on these observations, the
structures of 33 and 34 were concluded to be prenylated chalcones with a
coumarate and a ferulate unit attached, respectively, which were
confirmed by 2D-NMR techniques. Fig. (8). In case of isogemichalcone C
(34), it was concluded to be a regioisomer of gemichalcone C by
comparing its spectra with those of the latter compound [75]. This was
confirmed using a NOESY NMR experiment. Thus, the NOE correlations
between H-7' and H-10', and H-8' and H-IT clearly indicated E
stereochemistry of the prenyl group. Moreover, the chemical shift
differences at positions C-10' and C-1T of the E and Z isomers supported
the stereochemistry proposed. Fig. (8) [41,75,76].
5,7,2',4'-Tetrahydroxy-3-geranylflavone (43) exhibited a
molecular ion [M]"^ at m/z 422.1719 by HREIMS, consistent with an
25
6c
Carbon
33 34 Gemichalcone C [75]
10' 14.2 14.2 64.2 '

ir 70.2 70.2 22.8
Fig. (8). Selected HMBC (->) and NOE (<^) correlations of isogemichalcone C (34),
and comparison of *^C NMR data of 3'-[y-hydroxymethyl-(£)-y-methylallyl]-2,4,2',4'-
tetrahydroxychalcone 1 T-O-coumarate (33), 34, and gemichalcone C.
elemental formula of C25H26O6. In its ^H-NMR spectrum, characteristic

proton signals for a geranyl unit [5H 3.12 (2H, •/= 6.9 Hz, H-l"), 5H 5.14
(IH, multiplet, H-2"), 5H 1.89 (2H, multiplet, H-4"), 6H 1.43 (3H, singlet,
H-5"), 5H 2.00 (2H, multiplet, H>6"), 6H 5.04 (IH, multiplet, H-?''), 5H
1.61 (3H, singlet, H.9"), and 5H 1.55 (3H, singlet, H-IO")], a set of meta-
coupled proton signals [5H 6.25 (IH, broad singlet, H-6) and 6H 6.33 (IH,
broad singlet, H-8)], and proton signals of an ABX system [6H 6.57 (IH,
broad singlet, H-3'), 6H 6.51 (IH, 7 = 8.3 Hz, H-5'), and 5H 7.19 (IH, J =
8.3 Hz, H-6')] were observed. These data suggested that 43 has a flavone
skeleton with four hydroxyl groups and one geranyl substituent, and these
inferences were confirmed using the APT, COSY, and HMQC NMR
techniques. The positions of the substituents were deduced as occurring at
C-5, C-7, C-2', and C-4' (four hydroxyls) and C-3 (geranyl) using the
HMBC NMR technique, Fig. (9). Additionally, NOE correlations
26
between H-6' and H-l", and H-2" and H-4" confirmed the position of
attachment and the E stereochemistry of the geranyl group [41].
Fig. (9). Selected HMBC (-^) and NOE (<^) correlations of 5,7,2',4'-tetrahydroxy-3-
geranylflavone (43).
Out of a series of 42 compounds isolated and characterized in our

investigation on B. papyrifera [41], comprising benzofurans, coumarins,
and various types of flavonoids (biphenylpropanes, chalcones, flavans,
flavanones, and flavones), only certain representatives of the latter class
of compounds showed potent aromatase inhibition activity (Table 2).
Flavanone 39 {(25)-2',4'-dihydroxy-2"-( 1 -hydroxy-1 -methylethyl)-
dihydrofuro[2,3-/z]flavanone, IC50 0.1 \xM) [41] and flavone 47
(isolicoflavonol, IC50 0.1 \\M) [72] were the most potent flavonoids
isolated, exhibiting potencies that were approximately 60-fold greater
than aminoglutethimide, the positive control used for this assay. The
functionalized chalcone 33 {3'-[Y-hydroxymethyl-(jE)-y-methylallyl]-
2,4,2',4'-tetrahydroxychalcone 1 T-O-coumarate, IC50 0.5 |aM} [41] and
the flavanone 38 [(25)-abyssinone II, IC50 0.4 |iM] [68] were both
approximately 10 times more active than aminoglutethimide.
Interestingly, the various benzofurans {demethylmoracin I (51) [41],
moracins D [41], I [77], M [77], and N (53) [74]}, biphenylpropanes
{broussonins A (29) [39], B (30) [39], E [66], and F [66], l-(2,4-
dihydroxyphenyl)-3-(4-hydroxyphenyl)propane [41], l-(2,4-dihydroxy-3-
prenylphenyl)-3-(4-hydroxyphenyl)propane [41], and 1 -(4-hydroxy-2-
methoxyphenyl)-3-(4-hydroxy-3-prenylphenyl)propane [41]},
27
flavanonols {(2i?,3i?)-lespedezaflavanone C [78], (2i?,3i2)-katiiranin [79],

and (2/?,3/?)-5,7,2',4'-tetrahydroxyflavanonol [80]}, and flavans {(25)-
7,4'-dihydroxy-3'-prenylflavan [41], (25)-2',4'-dihydroxy-7-methoxy-8-
prenylflavan [81], and (25)-7,4'-dihydroxyflavan [66]} tested, which are
quite closely related structurally to the active compounds, did not show
potent anti-aromatase activity. It was noted that a carbonyl group in
compounds of the chalcone, flavone, andflavanoneclasses is required for
the exhibition of potent aromatase inhibition activity. However, the
presence of a C-5 hydroxyl group among the flavanones decreased
activity significantly {(25)-naringenin (41) [70] and (25)-5,7,2',4'-
tetrahydroxyflavanone (42) [71]} and flavones or flavanones with a
prenyl or geranyl unit at C-6 {bavachin [82], gancaonin P [83], 5,7,3',4'-
tetrahydroxy-6-geranylflavonol [41], and 5,7,3',4'-tetrahydroxy-3-
methoxy-6-geranylflavone [41]} were not active. Presumably, such a
bulky substituent at C-6 prevents these compounds from interacting with
the enzyme [41].
In our study, the inhibition of aromatase was achieved at
physiologically relevant concentrations (100-1000 nM) of dietary
flavonoids. Initially, some of the compounds isolated from B, papyrifera
were tested for binding to the estrogen receptor - a or -p [84].
Interestingly, none of the aromatase-active compounds showed significant
binding to the either of these receptors. Also, the effectiveness of some of
the flavonoids against the inhibition of quinone reductase, a phase II
enzyme involved in detoxification mechanisms, was evaluated [85]. No
significant inhibition of the enzyme was observed by any of the agents
tested. (25)-2',4'-Dihydroxy.2"-( 1 -hydroxy-1 -methylethyl)-
dihydroftiro[2,3-/i]flavanone (39) also was effective in inhibiting (50%)
the formation of alveolar lesions in a mouse mammary organ culture
model when tested at 100 ng/ml [86]. Moreover, the fiiiits of B,
papyrifera have been consumed by individuals in the People's Republic
of China, albeit for the treatment of various medical disorders, rather than
as an edible plant [14,87], Accordingly, these potent aromatase inhibitor
compounds show significant potential to be developed as cancer
chemopreventive agents [41,88].
28
COMPOUNDS FROM BROUSSONETIA ZEYLANICA
Only three constituents have been reported from Broussonetia zeylanica,

all by a group at the University of Peradeniya in Sri Lanka [89-91], A
major alkaloid, 8-hydroxyquinoline-4-carbaldehyde (55), was identified
as an antimicrobial agent active against Staphyllococus aureus and
Candida albicans (the levels of activity were not specified) [89] and then
two minor compounds, 3,4'-dihydroxy-2,3'-bipyridine (56) and 3,4-bis(8-
hydroxyquinolin-4-yl)-y-butyrolactone (broussonetine, 57), were
reported. Fig. (10) [90,91], However, the structure of 3,4'-dihydroxy-2,3'-
bipyridine (56) was revised to 8-hydroxyquinoline-4-carbaldehyde oxime
(58) by synthesis [92,93]. Also, it was noted that an artefactual origin of
this oxime (58) could not be ruled out due to the presence of the
corresponding aldehyde (55) [93].
CHO
OH N
56
58
Fig. (10). Structures of constituents of Broussonetia zeylanica.

29
CONCLUSIONS
Two species of the genus Broussonetia, B. kazinoki and B. papyrifera,

have been of considerable interest for their ethnomedical uses for the
treatment of various human diseases. There is a substantial literature on
the utilization of 5. papyrifera, in particular, in the paper industry in East
Asia. Extracts of Broussonetia species have exhibited various biological
activities including antifungal, antiinflammatory, antioxidant, and
antispasmodic activities.
Broussonetines, a class of pyrrolidine alkaloids from B. kazinoki,
have shown potent but generally non-specific glycosidase inhibitory
activity. The structures of these alkaloids were elucidated by analyzing
NMR data including ^H-^H COSY, ^H-^^C HSQC (HMQC), and ^H-^^C
HMBC 2D-NMR techniques and the absolute stereochemistry was
determined by a combination of the benzoate chirality and Mosher's
methods. Also, prenylated flavonoids were reported with antioxidant,
cytotoxic, and tyrosinase inhibitory activities from this species. From B.
papyrifera, variousflavonoidswere isolated as active principles. Of these,
broussochalcone A (32) and broussoflavonol F (45) showed broad
activities in several types of assay system, and (25)-2',4'-dihydroxy-2"-
(1 -hydroxy-1 -methylethyl)-dihydrofuro[2,3-/z]flavanone (39) and
isolicoflavonol (47) displayed the most potent aromatase inhibitory
activity (IC50 100 nM) of any natural products reported to date.
Furthermore, compound 39 was effective in inhibiting the formation of
alveolar lesions in a mouse mammary organ culture model. Therefore, the
secondary metabolites from Broussonetia seem worthy of more extensive
biological and chemical investigation, particularly from the point of view
of their role as potential cancer chemopreventive agents.
ACKNOWLEDGEMENTS
The laboratory work on potential cancer chemopreventive agents carried

out at the University of Illinois at Chicago was supported by program
project POl CA48112 (Principal Investigator, John M. Pezzuto, Ph.D.),
funded by the National Cancer Institute, NIH, Bethesda, Maryland,
U.S.A.
30
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol, 28
© 2003 Elsevier Science B.V. All rights reserved. 35
CHEMICAL AND BIOLOGICAL STUDIES ONLICANIA

GENUS
ALESSANDRA B R A C A \ ANNA RITA BILIA^ JEANNETTE MENDEZ^

COSIMO PIZZA^ IVANO MORELLl\ NUNZIATINA DE TOMMASf
^ Dipartimento di Chimica Bioorganica e Biofarmacia, Universitd di Pisa,

Via Bonanno 33, 56126, Pisa, Italy
^ Dipartimento di Scienze Farmaceutiche, Universitd di Firenze, Via Gino
Capponi 9, 55100 Firenze, Italy
^ Grupo de Productos Naturales, Centra de Quimica Organica, Escuela
de Quimica, Facultad de Ciencias, Universidad Central de Venezuela,
Apartado de Correos 47102, Caracas 1020'A, Venezuela
"^ Dipartimento di Scienze Farmaceutiche, Universitd di Salerno, Via
Ponte Don Melillo, 84084 Fisciano, Salerno, Italy
ABSTRACT: This paper reports the ph)ôchemical and biological studies carried out on
several species of the Licania genus (Chrysobalanaceae). This genus includes many
species mainly distributed in neotropical South American countries such as Venezuela,
Brasil, and Mexico, most of them not yet investigated from the chemical and biological
point of view. The name Licania derives from the anagram of the indigenous
Venezuelan name "Calignia".
Phytochemical studies of these species led to the isolation and structural
characterization, by NMR and MS analysis, of many secondary metabolites, mainly
flavonoids, and expecially flavonol glycosides, clerodane diterpenes, and triterpenes
having the lupane, ursane, and oleanane skeletons. Particularly flavonoids and their
glycosides have a chemotaxonomic interest in the genus and in general in the
Chrysobalanaceae family.
Furthermore, several biological studies were performed on some crude extracts of these
plants. They were found to be cytotoxic to cultured human hepatoma (HepG2), colon
carcinoma (Caco-2), melanoma B16, and CA-9KB cells. In addition a moUuscicidal
activity against Biomphalaria glabrata was also demonstrated. Pure triterpenes showed
antibacterial activity on Gram positive and yeast and a moderate cytotoxic action against
KB assay, while diterpenes showed antifungal properties. Flavonoids were also tested
for their antioxidative action as radical scavengers.
INTRODUCTION
The Licania genus, until the last decade, vâs almost completely
unexplored and little, if anything, was knov^n about the chemistry of its
family: the Chrysobalanaceae. This family includes 17 genus and 420
36
species of which Chrysobalanus, Acioa, Licania, Parinari,

Hexellodendrum, Hirtella, and Couepia are the neotropical ones growing
in Venezuela. In the past the Chrysobalanaceae family was separated
from the Rosaceae due to a difference in the sexual organ morphology:
the flowers in the Chrysobalanaceae have linked and partially sterile
stamen, while the gynaecium has lateral styles and two ovules [1].
However, a chemotaxonomic study on 31 species of Parinari genus
showed a similar flavonoid pattern between the two families [2]. This
study evidenced also that the Neotropical and Asiatic taxa of Parinari
genus, a complex of closely related species, were chemically very similar
to each other and lack myricetin; African species were split in two groups
based on the presence or the absence of myricetin glycosides. Since
myricetin was considered a primitive flavonoid character, it suggested
that the African species producing this metabolite represented a primitive
nucleus from which a non-myricetin group may have evolved giving rise,
by subsequent eastward and westward expansion, to two myricetin-
lacking phytogeographic lines. These chemical/phytogeographic
correlations could be extended also to Licania genus which is mainly
neotropical.
The Licania genus (the name Licania derives from the anagram of the
indigenous Venezuelan name "Calignia") comprises 150 species, mainly
trees or shrubs, distributed in neotropical South American countries such
as Venezuela, Brazil, Guyana, and Mexico. In Venezuela these plants
grow in the "bosque nublado", a rainy zone between 1500 and 3000 m in
altitude [3]. The aims of our work were to investigate the selected plants:
(1) to isolate as many secondary metabolites as possible for better
phytochemical knowledge of the genus with particular interest to their
flavonoidic fractions, whose occurrence in Licania could be useftil for
the taxonomy of the Chrysobalanaceae;
(2) to evaluate biological effects of both extracts and isolated compounds
on the basis of their structural relationship with other active
molecules.
GENERAL STRATEGIES
The phytochemical study consisted of fractionating plant extracts leading

to the isolation of various "novel" and known derivatives. The choice of
appropriate separation methods was crucial for this type of research: the
extracts may be very complex, thus separation of one single component
37
can be difficult. Multistep chromatographic separations were the norm

for the isolation of pure compounds. In our chemical and biological
investigations the isolation and purification steps were mainly performed
by extraction of the plant material with solvents of varied polarity (i.e.
with petroleum ether, chloroform, chloroform-methanol, methanol,
water) followed by crude separation by silica gel column for hydrophobic
extracts and by gel filtration on dextran gel (Sephadex LH-20 and/or LH-
60) columns and droplet counter current chromatography (DCCC) for
polar extracts. Less polar glycosides were usually isolated from the
chloroform-methanol 9:1 and methanol extracts. Highly polar water-
soluble glycosides present in the aqueous extracts were separated by
purification through an Amberlite XAD-2 column of the lyophilized
aqueous extract followed by gel filtration on Sephadex LH-20. Bioassay-
guided fractionation was employed in both cases. Prior to final separation
by HPLC on RP-18 columns, an optimization of separation parameters
was performed whereby mobile phase, stationary phase, and flow rate
were adjusted by using TLC and other suitable techniques.
The structures of the compounds were elucidated by a combination of
NMR techniques (^H-, ^^C-, and ^^C-DEPT NMR) and chemical
transformation, enzymatic degradation, and as well as mass spectrometry,
which gives information on the saccharide sequence. A more recent
approach consists of an extensive use of high-resolution 2D NMR
techniques, such as homonuclear and heteronuclear correlated
spectroscopy (DQF-COSY, HOHAHA, HSQC, HMBC) and NOE
spectroscopy (NOESY, ROESY), which now play the most important
role in the structural elucidation of intact glycosides. These techniques
are very sensitive and non destructive and allow easy recovery of the
intact compounds for subsequent biological testing.
Detailed biological or pharmacological screenings were performed
also in cooperation with scientists in different laboratories.
PHYTOCHEMICAL STUDIES OF PLANTS BELONGING TO

LICANIA GENUS
Our standard procedure consisted of first defatting the air-dried plant

material with «-hexane followed by extraction with solvents of increasing
polarity starting from chloroform, chloroform-methanol 9:1, methanol,
and water in a Soxhlet apparatus or at room temperature. Only in a few
38
cases, after defatting with «-hexane, the plant material was extracted
directly with methanol, and the crude methanol residue was then
partitioned between solvents with different polarity. Thus, non-polar
compounds, such as triterpenes were found in the less polar extract, while
flavonoids in the more polar fractions. The chloroform extracts were
usually subjected to fractionation over silica gel columns with CHCI3-
MeOH gradients, followed by reversed phase HPLC, Lobar on C-18 and
RP-18 columns with MeOH-H20 mixtures as the eluents. Fractionation of
the CHCb-MeOH 9:1 and MeOH extracts was conducted by gel filtration
over Sephadex LH-20 using MeOH as eluent sometimes preceded by
partition of the extracts between A2-butanol and H2O. The Sephadex LH-
20 fractions were finally purified by RP-HPLC, Lobar RP-8 and/or RP-
18, and flash column chromatography.
The structures of the isolated compounds were elucidated by a
combination of NMR techniques, chemical transformation, and EI-MS or
FAB-MS spectroscopy in positive or negative ion mode. In addition, high
resolution NMR techniques, which are very sensitive and non-destructive
were used.
In the course of our phytochemical work we studied seven Licania
species all belonging to Venezuelan flora, collected in Puerto Ayacucho,
Estado Amazonas and in the Parque Nacional Henry Pittieri, Maracay,
Estado Aragua. A number of new and known secondary metabolites,
mainly flavonoids, especially flavonol glycosides, sterols, and triterpenes
having the lupane, ursane, and oleanane skeleton were isolated and
characterized. The last part of this chapter deals also with the isolation of
clerodane diterpenes from the methanol extract of L, intrapetiolaris by
Oberlies et al, 2001 [4]. All the Licania species investigated up to now
are reported in Table 1.
Table 1. Licania species investigated
Species Source References

Licania pittieri Prance Venezuela rsi
Licania carii Cardozo Venezuela [6,7]
Licania pyrifolia Grisebach Venezuela [8,91
Licania densiflora Kleinhoonte Venezuela no. Ill
Licania heteromorpha var. heteromorpha Bentham Venezuela ri5,161
Licania licaniaeflora (Sagot) Blake Venezuela ri7,181
Licania apetaia var. apetala (E. Mey) Fritsch Venezuela [181
Licania intrapetiolaris Spruce (ex. Hook) Ecuador [4]
Table 2 and Table 3 list all triterpenes and flavonoids obtained from
the genus, respectively, to date. Flavonoids and their glycosides were
39
used as chemotaxonomic markers in the Chrysobalanaceae family and it

was noted that in the genus Licania glycosylation at C-3 of kaempferol,
quercetin, and myricetin and at C-4' only of the myricetin nucleus was
found to be the most common trend. Glucose, galactose, arabinose,
rhamnose, and rutinose are the most common sugars found as glycones of
the flavonoids glycosides. The presence of the aglycon myricetin in all
our investigated species except L. pittieri suggests that
chemical/phytogeographic correlations between and within genera of
Chrysobalanaceae family are probably more complex than those observed
by Coradin et al, 1985 [2]. On the other hand, our results support a strong
relationship between the families of Chrysobalanaceae and Rosaceae; thus
the presence of the flavonoids and triterpenoids in the species investigated
could justify the previous classification that included the
Chrysobalanaceae family into the Rosaceae, and the uselessness of their
separation, at least from a phytochemical point of view.
40
Table 2. Distribution of trierpenes in the genus Licania
Species A 1 B 1 C I E 1 F
Lupanes Betulinic acid + 1 + 1 +

Alphitolic acid 1 + 1
Z^-O'trans-p-coMvasnoyX 1 +
alphitolic acid
3p-0-c/5-/7-coumaroyl 1 +
alphitolic acid
1 la-hydroxybetulinic acid | + 1
6p-hydroxybetulinic acid | + 1
2a-hydroxybetulinic acid +
(3',4'-dihydroxy)-benzoyl ester
2a, 27-dihydroxybetulinic acid +
(3',4'-dihydroxy)-benzoyl ester
Oleananes Oleanolic acid , + +
Oleanolic acid +
3-0-a-L-arabinoside
Maslinic acid + + 4-
3 p-0-rran5-/7-coumaroyl +
maslinic acid
3P-0-cz.s-/7-coumaroyl +
maslinic acid
Arjunic acid +
28-p-D-glucosyl ester
Olean-12-ene-2a,3p-diol +
Ursanes Ursolic acid + + +
2a-hydroxy ursolic acid + +
Ursolic acid +
3 -0-a-L-arabinoside
2a,3a-dihydroxyurs +
-12-ene-28-oic
3 p-0-/ran5-/7-coumaroyl +
-2a-hydroxy ursolic acid
3 P-0-cw-/7-coumaroyl +
-2a-hydroxy ursolic acid
Euscaphic acid +
Euscaphic acid +
28-P-D-glucosyl ester
Tormentic acid +
Tormentic acid + +
28-P-D-glucosyl ester
Pomolic acid +
Legend: A=L. pittieri; B=L. carii; C=L pyrifolia; E=L. heteromorpha var. heteromorpha; ¥=L. licaniaeflora
41
Table 3. Distribution of flavonoids in the genus Licania
Species A B C D E F G
Flavonols Kaempferol (Kae) +
Kae 3-rha + +
Kae 3-ara +
Kae 3-rut +
Kae 3-(2"-xyl)rha + +
Kae 3-(6"-/?-coum)glc +
Quercetin (Que) + + +
Que 3-gIc + + +
Que 3-gal + + + + +
Que 3-ara + + + +
Que 3-rha + + + + +
Que 3'0Me-3-glc -)-
Que 3-rut + + +
Que 3-(2"-xyl)rha + +
Myricetin (Myr) + +
Myr 3-glc + +
Myr 3-gal + + + +
Myr 3-ara +
Myr 3-rha + + +
Myr 3-xyl +
Myr 3'0Me-3-glc +
Myr 3'0Me-3-gal +
Myr 4'0Me-3-glc +
Myr 4'0Me-3-gal +
Myr4'OMe-3-rha + +
Myr 3'0Me-3-rut +
Myr 3',4'-cliOMe +
-3-glc
Myr 3',5'-diOMe +
-3-glc
Myr3',5'-diOMe +
-3-rha
Myr 3-rut +
Myr 3-(2"-xyI)rha + +
Myr3-(2"-rha)rha +
Myr 4'-rha +
Myr 3,4'-dirha +
Myr 7-OMe-3,4'-dirha +
Dihydro Taxifolin 3-rha + +
flavonol
Dihydromyr 3-rha +
Catechins (+)-Catechin + +
(-)-£/7/catechin +
Flavones 8-OMe apigenin +
8-OH luteolin +
Flavanones 8-OH eriodictyol +
8-OH naringenin + +
4'-OMe-8-OH naringenin 1 +
Legend: A=I. pittieri; B=L. carii; C=I. pyrifolia; D=Ldensiflora; E=I. heteromorpha var. heteromorpha;
F=I. licaniaeflora\ G=JL. apetala var. apetala; rha=rhamnopyranoside; ara=arabinopyranoside;
rut=rutinopyranoside; xyl=xylopyranosyl or xylopyranoside; coum=coumaroyl; glc=glucopyranoside; gal=
galactopyranoside
42
Licania pittieri Prance
Licania pittieri Prance is a tree up to 15 meters high growing in the cloud

forests at 1800 m in altitude. From the methanol extract of the leaves,
collected in the Parque Nacional Henry Pittieri, oleanolic acid (1), ursolic
acid (2), catechin (3), epicatechin (4), quercetin (5), and four glycosidic
derivatives, quercetin 3-0-(3-D-galactopyranoside (6), quercetin 3-0-(3-D-
glucopyranoside (7), quercetin 3-0-a-L-rhamnopyranoside (8), and
quercetin 3-0-a-L-arabinopyranoside (9) were identified directly by
comparing their spectroscopic data with those reported in the literature
and/or authentic samples [see Fig. (1)] [5].
Licania carii Cardozo
Licania carii Cardozo is a tree up to 18-20 meters high, growing in the

cloud forests at 1800 m in altitude and is a new neotropical species
discovered and collected in the Parque Nacional Henry Pittieri, Venezuela
[6]. The chloroform-methanol residue afforded a series of triterpenes:
ursolic acid (2), 2a-hydroxyursolic acid (10), betuHnic acid (11), maslinic
acid (12), together with p-sitosterol 3-(9-|3-D-glucopyranoside (13) and
several flavonol glycosides: myricetin 3-0-(2"-0-P-D-xylopyranosyl)-a"
L-rhamnopyranoside (14), quercetin 3-0-(2"-0-p-D-xylopyranosyl)-a-L-
rhamnopyranoside (15), quercetin 3-O-P-D-galactopyranoside (6),
quercetin 3-0-p-D-glucopyranoside (7), myricetin 3 - O - p - D -
glucopyranoside (16), myricetin 3-(9-P-D-galactopyranoside (17),
quercetin 3-0-rutinoside (18), myricetin 3'-methyl-3-(9-rutinoside (19),
and myricetin 3-0-rutinoside (20) that were identified by comparison of
their spectroscopic data with those reported in the literature and/or
authentic samples [see Fig. (1) and (2)] [7].
Licania pyrifolia Grisebach
Licania pyrifolia Grisebach is a small tree widely cultivated in the

Amazonian regions of Venezuela for their edible fruits called "merecure".
The chloroform extract yielded a-amyrin, p-sitosterol, lupeol, betulin.
43
Fig. (1). Compounds 1-9 and 12 from L pittierU L carii, L, pyrifolia, L.

licaniaeflora^ and L apetala
COOH
3 OHcq
R-R"-H R'«CHj 4 OH ax
2 R-CHj R'«R''-H
12 R«H R'«CHj R"«OH
5 R=H
6 R"gal
7 R-gIc
8 R»rha
9 R = ara
Fig. (2). Triterpenes and flavonoids from L. cariUL, pyrifolia.L,

heteromorpha, L licaniajlora, and L apetala
COOPT
10 R=OH R'^R^-R-'-H
25 R-R''»R"'»H R'=ara
R'O
27 R«R-=0H R'=H R-'-gIc
30 R«R-=OH R'R'^^H
59 R«R'=R"^'=H R - - 0 H
COOR
26 R-glc R'«OH
2S R-R'-H
29 R=ll R'=OH
44
14 R«H R*-Tha(l-2)xyl
16 R-H R'-glc 15 R=rha(l-2)xyl
17 R«H R'«g«l 18 R=glc(I-*6)rha
19 R=CHj R'«glc(1-6)rha
20 R-H R'-g'c(l-6)rha
uvaol, oleanolic acid (1), ursolic acid (2), and betulinic acid (11). The
CHCl3-MeOH extract was fractionated by column chromatography on
Sephadex LH-20 with MeOH. The portion containing the bulk triterpenes
was chromatographed over Si gel with CHCla/MeOH mixtures followed
by low-pressure CC on Lichroprep RP-18 with MeOH/HiO mixtures to
yield four new compounds: lla-hydroxybetulinic acid (21), 6(3-
hydroxybetulinic acid (22), 2,3-dihydroxylup-12-en-28-oic acid 3-(3\4*-
45
dihydroxybenzoyl ester) (23), and 2,3,27-trihydroxylup-12-en-28-oic acid

3-(3\4*-dihydroxybenzoyl ester) (24) [see Fig. (3)]. The following
constituents of known structures were also obtained: p-sitosterol 3-(9-(3-
D-glucopyranoside (13), ursolic acid 3-(9-a-L-arabinopyranoside (25),
euscaphic acid 28-p-D-glucopyranosyl ester (26), tormentic acid 28-|3-D-
glucopyranosyl ester (27), 2a-hydroxyursolic acid (10), 2a,3a-
dihydroxyurs-12-ene-28-oic acid (28), euscaphic acid (29), tormentic acid
(30), and maslinic acid (12). All compounds were identified by
comparison of their spectroscopic data with those reported in the literature
and/or authentic samples [8].
The new derivatives 21-22 were identified by means of their
spectroscopic data. The EIMS spectra of both compounds 21 and 2 2
showed a molecular peak [M]^ at m/z 472 corresponding to the formula
C30H48O4 (confirmed by ^^C-NMR and DEPT analysis). These data
indicated a triterpenoid skeleton with one carboxyl and two hydroxyl
moieties and one double bond. The ^^C-NMR spectra of both compounds
21 and 22 revealed 30 carbon signals which were sorted by ^^C-DEPT
NMR into six methyls, nine methylenes, five methines, five quartemary
carbons, two alcoholic methines, one carboxylic acid, and two olefinic
carbons (one =CH2 and one quartemary). The 20,29-functionality of a
lupene skeleton was inferred for both compounds from the resonances of
the sp^ carbons at C-29 at 109 ppm and C-20 at 150 ppm. A detailed
analysis of the ^H-NMR of 21 confirmed the characteristic features for a
betulinic acid parent structure bearing one -OH group at C-11 (one
carbinolic proton at 8 3.85, ddd, J = 10.5, 10.5, and 5.0 Hz). The 7 values
(two diaxial couplings and one axial/equatorial spin-spin coupling) were
in accordance with an a-hydroxyl moiety. The substitution at C-11 was
revealed by a shift of the carbon signal at 20.9 (C-11) of betulinic acid to
69.8 (d) and by the downfield shifts of signals of C-1 (2.9 ppm), C-9 (2.0
ppm), and C-12 (1.6 ppm) due to 8 and y steric effects of the a
configuration of this hydroxyl moiety. This conclusion was confirmed by
the downfield shift in the ^H-NMR spectrum of the methyl protons linked
at C-10 (Me-25) due to the same steric effects. The 6|3-OH substitution of
compound 22 was deduced in an analogous manner.
46
Fig. (3). Lupane derivatives 21-24fromL pyrifolia
COOH
21 R=OH R'=H
22 R»H R'«OH
COOH
23 R-H
24 R«OH
The EIMS of compounds 23 and 24 showed molecular peaks [M]^ at

m/z 608 and 624 corresponding to the formulas C37H52O7 and C37H52O8,
respectively. The ^^C-NMR spectrum of both compounds revealed 37
carbon signals. Those of 23 were sorted by ^^C-DEPT NMR into seven
methyls, eight methylenes, five methines, five quatemaries, two alcoholic
methines, one carboxylic acid, one -COOR, and two olefinic carbons (one
=CH and one quatemary). Furthermore, a nonsymmetrically trisubstituted
aromatic ring was also present. The signals of 24 were similar to those of
23 except that one -CH3 was replaced by a -CH2OH. These data indicated
the presence in both 23 and 24 of a triterpenoid skeleton with two
secondary hydroxyls (plus one primary OH in 24) and one carboxyl
moiety, one double bond, and a 3,4-hydroxybenzoic unit. A 12,13-double
bond and the fact that both derivatives had a lup-12-ene skeleton was
indicated by resonances of the sp^ carbons C-12 (methine) at 126.3 ppm
47
and C-13 (quaternary carbon) at 138.0 ppm and by the analysis of the
methine and methylene resonances. A detailed analysis of the ^H-NMR
spectrum of 23 confirmed the characteristic features for a lup-12-en-28-
oic acid derivative bearing an a-OH at C-2 and a |3-0H at C-3. The
carbinohc region revealed a doublet of doublets at 6 3.82 (IH, J= 4.5,
9.6, and 10.8 Hz) and a doublet at 8 4.63 (IH, / = 9.6 Hz), whose
chemical shifts and J couplings were typical for a 2a,3(3-dihydroxyl
substitution pattern. Furthermore, the ^H-NMR spectrum revealed the
presence of a 3,4-dihydroxybenzoic unit by the presence of signals at 8
6.90 (2H, m, H-5' and H-6') and 8 7.02 (IH, br s, H-20. Moreover, the
linkage of this moiety to C-3 of the triterpene was derived from the
downfield shift (1.6 ppm) of C-3 when compared with other derivatives
with the same substitution pattem. Compound 24 had resonances similar
to those apparent in the ^H-NMR spectrum of 23 for a 2a,3^-
dihydroxylup-12-en-28-oic acid derivative. In addition, two signals for an
oxymethylene group replaced the signal due to a rer/-methyl of the
aliphatic region (Me-27). Further support for this additional substitution
was obtained from NOESY experiments (Table 4). Thus, NOEs were
observed between H-5 and H-9, H-23 and H-6a; between H-9 and H-23,
H-3, H-27a, and H-27b; between H-27b and H-20; as well as between H-
18 and H-19. These results showed that the primary hydroxyl and
isopropyl groups both had a-dispositions, whereas the OHfimctionat C-3
was p-oriented.
Table 4. Interactions observed in the NOESY NMR spectrum of compound 24
Correlated signal
proton ^6H Proton 5H
5 0.72 9 1.56
5 0.72 23 1.04
5 0.72 6a 1.52
9 1.56 6a 1.52
9 1.56 23 1.04
9 1.56 27a 3.18
9 1.56 27P 3.52
18 1.38 19 1.40
27b 3.52 20 0.88
Successively from the chloroform-methanol and methanol extracts also

some flavonoids were obtained: kaempferol 3-0-a-L-rhamnopyranoside
(31), quercetin 3-0-a-L-rhamnopyranoside (8), myricetin 3-0-a-L-
rhamnopyranoside (32), myricetin 3-0-(2"-0-p-D-xylopyranosyl)-a-L-
48
rhamnopyranoside (14), quercetin 3-0-(2"-(9-p-D-xylopyranosyl)-a-L-

rhamnopyranoside (15), kaempferol 3-0-(2"-(9-p-D-xylopyranosyl)-a-L-
rhamnopyranoside (33), kaempferol 3-0-a-L-arabinopyranoside (34),
quercetin 3-(9-a-L-arabinopyranoside (9), myricetin (35), quercetin (5),
kaempferol (36), 8-hydroxy-naringenin (37), 8-hydroxy-eriodictyol (38),
and 8-hydroxy luteolin (39) [see Fig. (1), (2), and (4)]. Compound 38 was
a new natural product: the unusual 5,7,8-trihydroxy substitution of ring A
was determined by the presence of the singlet due to H-6 in the ^H NMR
spectrum and by the evaluation of the resonances of C-2—C-10 of y-
pyronic ring A. The upfield shift of C-8 and the downfield shifts of C-7,
C-5, and C-9 with respect to the same signals of eriodictyol led to the
complete structural elucidation of compound 38 [9].
^^^» (4). Flavonoids from L pyrifolia and L licaniaeflora
31 R-rha
33 R = rha(l-2)xyl
34 R = ara 32 R - r h a
36 R » H 35 R » H
63 R-ara
OH 0
37 R - H
38 R - O H
49
Licania densiflora Kleinhoonte
Licania densiflora Kleinhoonte, synonym Licania kanukuensis Standley,

is a tree 30 meters high, growing in forests and hills of Venezuela, Brasil,
and Guyanes. Especially in Venezuela it grows in the Bolivar and Delta
Amacuro regions; its common names are "Guanay", "Hierrito", and
"Merecurillo" [3]. The chloroform-methanol and methanol extracts of the
plant's leaves afforded eight flavonoids [compounds 40-47; see Fig. (5)]
from which three were new natural products: naringenin 8-hydroxy-4'-
methyl ether (40), myricetin 3-0-(2"-0-a-L-rhamnopyranosyl)-a-L-
rhamnopyranoside (41), 3',4'-dimethylmyricetin-3-0-P-D-
glucopyranoside (42), myricetin 3'-methylether-3-0-(3-D-glucopyranoside
(43), myricetin 3'-methylether-3-0-P-D-galactopyranoside (44), myricetin
4'-methylether-3-0-a-L-rhamnopyranoside (4 5 ), myricetin 3\5'-
dimethylether-3-0-(3-D-glucopyranoside (46), and myricetin 3',5'-
dimethylether-3-O-a-L-rhamnoside (47) [10, 11]. All compounds were
characterised by spectroscopic methods including mono- and bi-
dimensional NMR techniques.
Fig. (5). Compounds 40-47fromL densiflora
.OCH,
41
50
42
43 R = glc R'^CHj R-'R^'H
44 R«gal R'«CHj R-'-R'-^H
45 R-rha R'-R^^H R"«CHj
46 R«glc R'=R'"=CH3 R*»H
47 R-rhi R'-R^-CHj R--H
In particular compound 40 was purified by Sephadex LH-20 column

from the chloroform-methanol extract, while compounds 41-42 were
obtained similarly by using Sephadex LH-20 column and Lobar
Lichroprep RP 8 chromatography.
The structures and molecular formulas of these new derivatives were
determined bv positive ion FAB MS and UV spectra, ID and 2 D - ' H , ' ^ C ,
and C DEPT NMR data. Furthermore, the absolute configurations of the
sugar moieties were confirmed referring to the optical rotation after
hydrolysis of the glycosides. Compound 40 had molecular formula
C16H14O6. Mass spectrometry, ^H, and ^^C NMR analysis indicated its
flavanoidic structures and particularly it was evidenced from the ^H-NMR
spectrum for the presence of the ABX spin system: H-2 (X part of the
system) appeared as a dd at 8 5.49 (/BX= 3.4 Hz and /AX= 13.0 Hz), while
H2-3, (AB part), were represented by two dd, one at 8 2.89 and one at 6
2.72 (7AB= 17.2 Hz). The presence of one singlet at 6 5.83, attributed to
H-6, determined the unusual 5,7,8-trihydroxy substitution of ring A.
Furthermore, the ^H NMR data also revealed one signal at 8 3.75 (3H, s)
ascribable to one methoxyl group that resonated at 56.1 ppm in the ^^C
NMR spectrum. The exact position of the methoxyl group was established
by typical methoxylation shift observed in the C NMR spectrum with
respect to 8-hydroxy naringenin: downfield shift of C-4' (ca. 5 ppm) and
upfield shifts of C-3* (ca. 1.3 ppm), C-5' (ca. 1.3 ppm), and C-T (ca. 0.5
ppm) suggested the presence of the methoxyl group at C-4'. This was
confirmed by bidimensional NOESY experiment which showed a
correlation between the signal at 8 3.75 with the one at 8 6.95 (H-3* and
H-5').
51
Mass spectrometry, ^^C, and ^^C DEPT NMR analysis indicated the
flavonoidic nature also for compound 41, and in particular 15 carbon
atoms ascribable to the aglycon and 12 to the sugar moieties. In the H
NMR spectrum the chemical shifts and the proton coupling constants
indicated a 5,7-dihydroxylated pattern for ring A (two meta-couplQd
doublets at 6 6.21 and 6.42, J= 2.0 Hz) and a 3',4\5*-trihydroxylation for
ring B (a two-proton singlet at 86.95), permitting recognition of the
aglycon as myricetin. Furthermore two anomeric protons were easily
identified in the ^H NMR spectrum. Mass spectra evidenced the presence
of two flanked deoxyhexose moieties due to the presence of a molecular
peak at 611 m/z and two prominent peaks were evidenced at 465 m/z
[(M-f-H)-146]"^ due to the loss of a deoxyhexose unit and at 319 m/z
[(M+H)-(146+146)]^ due to the loss of another deoxyhexose unit;
actually they were identified by ^^C NMR data as two rhamnopyranosyl
moieties with a-configuration at the anomeric carbon [12]. The position
of the disaccaridic moiety at C-3 was determined by typical glycosylation
shift observed in the ^^C NMR spectrum with respect to the aglycon
myricetin: upfield shifts of C-2 and C-4 (about 5.0 and 3.7 ppm,
respectively), and a downfield shift of C-3 (about 3.5 ppm). Actually
myricetin 3-dirhamnoside was previously isolated from Azara
microphylla leaves [13], but the authors did not specify sugar
interlinkage. Therefore, from high digital resolution ^H NMR spectral
data and from ^^C NMR, we determined without a doubt this linkage
which was confirmed by bidimensional COSY and HETCOR
experiments. After hydrolysis L-rhamnose was also identified by TLC
and optical rotation value by comparing their data with those of an
authentic sample. ^^C NMR of compounds 40 and 41 are reported on
Table 5.
52
Table 5."CNMR< lata (200 MHz) for compounds 4( and 41.

Carbon DEPT 40 (DMSO-c/6) DEPT 41 (CD3OD)
2 CH 78.0 C 157.4
3 CH2 42.0 C 136.0
41 C J 196.4 C 178.2
5 1 c 156.8 C 163.9
6 CH 95.3 CH 99.8
7 C 160.8 C 165.9
8 C 128.2 i CH 94.7
9 C 154.3 C 158.5
10 C 103.4 C 104.5
r C 130.4 C 121.8
2' CH 128.4 CH 109.5
3' CH 114.6 C 146.9
4' C 162.4 C 137.3
5' CH 114.6 C 146.9
6' CH 128.4 CH 109.5
OCH3 CH3 56.1 - -
rha 1" - - CH 103.8
2" - - CH 79.1
3" - - CH 71.8
4" - - CH 72.0
5" - - CH 70.3
6" - - CH3 17.8
rha 1'" - - CH 102.4
-^111
- - 1 CH 72.2
3'" - - CH 73.6
4"» - - 1 CH 73.9
5"' - - CH 70.8
6'" - - CH3 17.8
Compound 42 had molecular foraiula C23H24O13. In the positive ion

FAB MS spectrum, a molecular peak was observed at 509 m/z and two
prominent peaks were evidenced at 479 m/z [(M+H)-30]^ due to the loss
of two methyl groups and at 347 miz [(M+H)-162]'^ due to the loss of a
hexose unit. Comparing spectral data of compound 42 with those of 41,
close similarities were observed between the signal values of the aglycon
of both compounds, while sugar moiety provided the point of difference.
Except for myricetin signals, ^H NMR spectrum revealed the presence of
a one-proton doublet at 6 5.27, J= 7.5 Hz, representative of one anomeric
proton, and two singlets at 6 3.93 (3H), and 3.95 (3H), ascribable to two
methoxyl groups. The sugar was identified as P-D-glucopyranoside from
absolute values of the coupling constants in the ^H-NMR spectrum and
the evaluation of TLC and optical rotation of the sugar moiety after
hydrolysis of the glycoside by comparison with an authentic sample.
Relative positions of p-D-glucopyranoside and of the methoxyl groups
were determined by UV spectra registered with AICI3, AICI3 + HCl, and
53
NaOMe establishing respectively the absence of two ortho OH residues

and substituted 3, 3', and 4' positions.
L. densiflora produced methoxylated flavonoid glycosides that are
quite unusual in the plant kingdom and often play an important role of
protection from attacks by herbivores or pathogens, so that their
biosynthesis could be related to local environmental factors [14].
Licania heteromorpha van heteromorpha Bentham
Licania heteromorpha var. heteromorpha Bentham is a tree up to 30 m

high native to the Amazonian forest. Phytochemical study of its aerial
parts yielded both triterpenes and flavonoids; triterpenes were obtained
from the chloroform extract by silica gel column followed by RP-HPLC
and were characterised as: betulinic acid (11), alphitolic acid (48), 3|3-6>-
trans'P'COumaiVoyX alphitolic acid (49), 3p-0-c/5-/7-coumaroyl alphitolic
acid (50), 3p-C>-/ra«5-/7-coumaroyl maslinic acid (51), 3P-0-c/5-/7-
coumaroyl maslinic acid (52), 3(3-0-fraw5-/?-coumaroyl-2a-hydroxy-urs-
12-en-28-oic acid (53), 3(3-0-d5-p-coumaroyl-2a-hydroxy-urs-12-en-28-
oic acid (54) [see Fig. (2) and (6)] [15]. Compounds 11 and 48-54 were
identified comparing their ^H and ^^C NMR data with those previously
described. Triterpenoids 48-54 were found for the first time in Licania,
while betulinic acid had been isolated previously from L carii [9]. On
the other hand, flavonoids were isolated from the chloroform-methanol
and methanol residues by Sephadex LH-20 and HPLC; they were
identified as myricetin 3-(9-p-D-galactopyranoside (17), myricetin 3-0-
a-L-rhamnopyranoside (32), myricetin 4'-methylether-3-(9-(3-D-
glucopyranoside (55), myricetin 4'-methylether-3-0-a-L-
rhamnopyranoside (45), myricetin 3,4*-di-(9-a-L-rhamnopyranoside (56),
myricetin 7-methylether 3,4*-di-(9-a-L-rhamnopyranoside (57), and
myricetin 4'-methylether-3-0-p-D-galactopyranoside (58) [see Fig. (2),
(4), and (6)]. The last three myricetin derivatives were new natural
compounds [16]. Known compounds were identified by comparison of
their ^H and ^^C NMR spectra with those reported in the literature [15].
Compounds 56-58 were purified by Sephadex LH-20 column and RP-
HPLC from the methanol extract. After spraying the TLC with Naturstoff
reagent, they gave red spots typical of 3*, A\ 5'-trihydroxyflavonols
showing that they all have the same aglycon. The structure and molecular
54
Fig. (6). Compounds 48-58 from L heteromorpha
COOH COOH
R'O'
51 R«H m^trans-p-cQWMtxoyX R^-CHj

48 R«H 52 R»H R'«cfe-/'-coumaroyl R^^CHj
49 R«franj-p-coumaroyl 53 R*CHj R'e(7-an5-p-couinaroyi R''"H
50 R«c»-p-coumaroyt 54 R-CHj R'"ar-p-coumaroyi R-^H
.OR-
R'O,
55 R-gIc R'-H R--CH,

56 R=R"«rha R'-H
57 R=R"=Tha R'^CHj
58 R=gal R'=H R"=CHj
formulae of new compounds 56-58 were determined by negative ion ESI-

MS spectra, ID and 2D-^H, ^^C and ^^C DEPT NMR data.
Compound 56 showed in its ^^C, and ^^C DEPT NMR analysis 15
carbon atoms ascribable to the aglycon and 12 to the sugar moieties. In
the ^H NMR spectrum the chemical shifts and the coupling constants of
protons indicated a 5,7-dihydroxylated pattern for ring A (two meta-
coupled doublet at 8 6.22 and 6.36, 7= 1.8 Hz) and a 3\4\5*-
trihydroxylation for ring B (two-proton singlet at 6 6.94), permitting
recognition of the aglycon as myricetin. Two anomeric protons were
easily identified in the ^H NMR spectrum. They resonated at 8 5.31 {J=
1.5 Hz) and 5.57 (y= 1.5 Hz), and correlated respectively with 103.9 and
103.1 ppm in HSQC spectra. Chemical shifts, multipHcity of the signal.
55
absolute values of the coupling constant and the magnitude in the ^H

NMR spectrum as well as ^C NMR data indicated the presence of two
rhamnopyranosyl moieties with a-configuration at the anomeric carbon.
The exact position of rhamnose units was determined by typical
glycosilation shifts observed in the ^^C NMR spectrum with respect to
the aglycon myricetin: upfield shifts of C- 2 (ca. 5.0 ppm ), and C-4 (ca.
3.7 ppm), and a downfield shift of C-3 (ca. 3.5 ppm) suggested the
presence of a rhamnose unit at C-3; in the same way an upfield shift of
C-4' (ca. 2.0 ppm) and downfield shifts of C-3' and C-5' (ca. 0.4 ppm)
implied the position of the other a-L-rhamnose at C-4' [6]. This was
confirmed by HMBC experiment correlations [8 5.31 with 136.8 ppm (C-
3) and 8 5.57 with 151.9 ppm (C-4')].
Compound 57 had molecular formula C28H32O16. Its ^H NMR and ^^C
spectra compared with those of 56 each revealed one more signal
respectively at 6 3.76, (3H, s) and 51.9 ppm assigned to one methoxyl
group. Its position is established from HMBC correlation data. The
methoxylation shift observed at C-6 and C-8 supported the position of the
methoxyl group at C-7.
When 56 is used as reference compound in the spectral analysis of
compound 58, close similarities are observed between spectral data of the
aglycon of both compounds, while sugar moiety provided the point of
difference. Except for aglycon signals, ^H NMR spectrum revealed the
presence of a one-proton doublet at 8 5.24, / = 7.5 Hz representative of
one anomeric proton of a hexose unit, and one singlet at 8 3.92 (3H)
again ascribable to one methoxyl group. Selected ID-TOCSY obtained
irradiating anomeric proton signal (8 5.24) yielded the subspectrum of
sugar residue with high digital resolution. The result of ID-TOCSY
compared with those of ^^C NMR experiment allowed the identification
of sugar as galactopyranoside; its (J-configuration at anomeric position is
derived combining ^H NMR and ^^C NMR data. The relative position of
p-D-galactopyranose and of the methoxyl group was again established
from HMBC experiment correlations. ^^C NMR data of compounds 56-
58 are reported in Table 6.
56
Table 6/^CNMR data (600 MHz, CD3OD) for compounds 56-58.

Carbon I DEPT 56 I 57 I 58
2 C 158^6 1583 157.8
3 c 136.8 136.5 136.7
4 c 179.5 179.3 179.1
5 c 162.8 162.8 162.8
6 CH 100.5 99.5 100.8
7 C 167.7 165.6 165.6
s 1 CH 95.1 94.4 95.5
9 C 158.6 158.2 158.0
10 C 105.6 105.56 104.4
1' C 127.3 126.8 126.9
T CH 109.9 109.6 110.1
y C 136.3 136.0 138.0
4' C 152.3 151.9 151.4
5' C 136.3 136.0 138.0
6' CH 109.9 109.6 110.1
RhaatC-3 1" CH 103.9 103.9 105.5
2" CH 73.8 73.4 73.2
3" CH 72.2 71.8 75.1
4" CH 72.1 71.7 70.1
5" CH 71.9 71.0 77.3
6" CH3 18.0 17.6
CH2 62.0
Rha at C-4' -
1'" CH 103.1 102.7
•îii
CH 73.3 72.9
3.,, CH 72.1 71.7 1 -
4'" CH 72.0 71.5 -
5'" CH 71.3 70.6
6"' CH3 17.8 17.4 1 -_
OCH3 CH3 51.9 60.8
Similarly to L. densiflora, L, heteromorpha is unusual for its

biosynthesis of methoxylated flavonoids glycosides (with the methoxyl
groups at C-4' of ring B and C-7 of ring A). The production of such
flavonoids could be a response to local environmental factors. In
comparison to the other Licania species, L. heteromorpha predominantly
yields flavonol glycosides instead of flavones or flavanones.
Licania licaniaeflora (Sagot) Blake
Licania licaniaeflora (Sagot) Blake is a tree native to the Amazonian

forest where the leaves were collected. The plant's leaves were defatted
with «-hexane and then extracted at room temperature with methanol to
yield a residue which was subsequently partitioned between AcOEt, n-
BuOH, and H2O. From the AcOEt fraction, nine triterpenes were isolated
57
and identified as: oleanolic acid (1), ursolic acid (2), betulinic acid (11),
maslinic acid (12), tormentic acid 28-P-D-glucosyl ester (27), pomolic
acid (59), oleanolic acid 3-O-a-L-arabinopyranoside (60), arjunic acid
28-(3-D-glucosyl ester commonly known as arjunetin (61), and olean-12-
ene-2a,3|3-diol (62) [see Fig. (1), (2), and (7)] [17]. Their structural
identification was achieved by ^H and ^^C NMR experiments. The n-
butanol fraction submitted to Sephadex LH-20 and RP-HPLC afforded
10 flavonoids, both flavonol and dihydroflavonol glycosides that were
characterised on the basis of spectral data (UV, MS, ^H and ^^C NMR)
and by comparison with literature data or authentic samples: quercetin 3-
0-p-D-galactopyranoside (6), quercetin 3-0-a-L-rhamnopyranoside (8),
myricetin 3-0-(3-D-galactopyranoside (17), myricetin 3-0-a-L-
arabinopyranoside (63), kaempferol 3-0-a-L-rhamnopyranoside (31),
kaempferol 3-(9-(2"-p-D-xylopyranosyl)-a-L-rhamnopyranoside (33),
quercetin 3-(9-a-L-arabinopyranoside (9), 8-hydroxy naringenin (37),
taxifolin 3-0-a-L-rhamnopyranoside (64), and dihydromyricetin 3-0-a-
L-rhamnopyranoside (65) [(see Fig. (1), (2), (4), and (7)] [18]. The
presence of dihydroflavonol glycosides such as taxifolin 3-0-a-L-
rhamnoside and dihydromyricetin 3-O-a-L-rhamnoside in the genus is
reported now for the first time and thus may have some chemotaxonomic
implications.
Fig. (7). Compounds 60-62 and 64-65 from L licaniaeflora and L.

apetala
60 R«ara R'-R-'-H R-«C00H

61 R=H R'^R-'OH R"«COO-glc 64 R»H
62 R-R-'H R'=OH R-=CH3 65 R«OH
58
Licania apetala var. apetala (E. May) Fritsch
Licania apetala var. apetala (E. May) Fritsch is a tree native to the
Amazonian forest. Its leaves were extracted with the same method used
for L. licaniaeflora. From the w-butanol extract of the leaves seven
flavonoids were obtained: quercetin 3-0-(3-D-galactopyranoside (6),
quercetin 3-O-a-L-rhamnopyranoside (8) quercetin 3-0-rutinoside (18),
quercetin 3-0-a-L-arabinopyranoside (9), taxifolin 3-O-a-L-
rhamnopyranoside (64), myricetin 4*-0-a-L-rhamnopyranoside (66), and
kaempferol 3-0-rutinoside (67), [see Fig. (1), (2), (7), and (8)] [18].
Fig. (8). Compounds 66-67 from L apetala
Orha
'Oglc(1-^)rh«
66
Licania intrapetiolaris Spruce ex Hook
Licania intrapetiolaris Spruce ex Hook, a large tree that may grow up to

30 m in height and is widely distributed in tropical South America,
including Columbia and Peru, was recently studied by Oberlies et al.,
2001 [4]. The plant root, collected in a tropical rain forest of Ecuador,
was subjected to a bioactivity-directed fractionation procedure through
KB cytotoxicity assay. Using a series of chromatographic techniques,
including flash silica gel and RP-HPLC, three bioactive compounds
intrapetacin A, intrapetacin B, and cucurbitacin B were isolated.
Intrapetacin A and B are new structures in a class of similar compounds
that have been termed variously as either the clerodane diterpenoids, the
kolovane diterpenoids, or the kovalene diterpenoids, while cucurbitacin
B is a triterpenoid [see Fig. (9)] [4]. The structures of the new
compounds were deduced from ID- and 2D-NMR experiments.
59
including relative stereochemical assignments based on NOESY

correlations and COSY coupling constants. All these compounds have
been unreported in the genus Licania until now.
Fig. (9). Intrapetacin A and B and cucurbitacin B from L intrapetiolaris
I \'*°"J
intrapetacin A R^CHj
intrapetacin B R"Ac
OAc
cucurbitacin B
BIOLOGICAL STUDIES OF PLANTS BELONGING TO LICANIA

GENUS
MoUuscicidal activity
Schistosomiasis is a parasitic disease on the increase, affecting millions

of people in Africa, South America, and the Far East. A general strategy
for schistosomiasis control is to remove a link in the vital cycle, i.e. the
60
snail. The use of molluscicides provides a way to control the

multiplication and spreading of snails. In this context, we performed in
our laboratories a permanent programme for random screening of plants
of South American flora, especially from Venezuela, with attributed
moUuscicidal properties. Leaves of six species of Licania genus, L.
pittieri^ L. carii^ L, pyrifolia, L, densiflora, L. heteromorpha var.
heteromorpha, andZ. licaniaeflora, were investigated for a total of 15
extracts. Previously only L, tomentosa had been investigated for this
activity [21]. A preliminary screening, by testing a standard
concentration in water (1.000 ppm/24 hours), was carried out to
differentiate extracts without any moUuscicidal activity from those with
strong, some or weak toxicity. As a first step in our studies we
distinguished the extracts with high or weak toxicity from those without
any moUuscicidal response. Then the LCioo values (the concentration in
water that kills 100% of a target snail population) of the moUuscicide
were evaluated, and finally, for the active extracts, the toxicity species
was tested on two fishes to consider the risks to nontarget organisms.
MoUuscicidal tests were carried out with snails of Biomphalaria
glabrata Say reared in aquaria with a continuous circulation of water
through a filter system, with a temperature of about 24°C. Aquatic snails
employed for the tests were young-mature and relatively uniform in age
and size (average diameter of shell 8 mm). The snails were placed in
dechlorinated tap water at 26-28° C under laboratory lighting conditions
with normal diurnal light changes. Exposure of 24 hours to potential
molluscicides was performed and death was established by examination
(discoloration, heart rate, activity of muscle) and/or crushing. Acute
toxicity on B, glabrata was evaluated by rapid screening procedure: the
extracts were dissolved or suspended in water at a concentration of 1.000
ppm. Two containers at this concentration with two snails each were
used, with the volume per snail not less than 40 ml. As control, one
container with two snails each was included. In the event of both snails'
death, a series of dilutions (e.g. 400, 200, 100, 50 ppm), were tested. For
these assays, two containers at each concentration with 10 snails each
were used, with the volume per snail not less than 40 ml. As control, one
or two containers with 10 snails each were included as well. In order, to
confirm the mortality of the snails, they were placed in a beaker
containing distilled water alone; after 24 hours their condition was re-
checked. The minimal lethal concentration required to kiU both snails
after 24 hours was thus established and tested for the potential toxicity
61
on two fish species with a great adaptabihty: Poecilia reticolata XIPHO

Viemef and Carassius auratus L. Fishes were reared in aquaria under the
same conditions previously described for snails. The piscicidal test of the
active extracts was carried out in duplicate also, with a reference, using
an analogue method described for the rapid screening procedure of the
moUuscicidal activity; in this case the volume per fish was not less than
1000 ml. The tested extract was considered piscicidal if the mortality of
both fishes was recorded after 24 hours.
All methanol extract and one-less polar extracts of the species
examined killed snails at 1.000 ppm within 24 h (Table 7).
Table 7. MoUuscicidal activity of Licania genus plant extracts.*
Species Activity (ppm)

Extract
400 200 100 50 25

L. carii MeOH ++ -H- ++ ++ + 1
L, pittieri MeOH ++ ++ ++ ++ +
L, pyrifolia CHCl3/Me0H ++ ++ +
MeOH ++ ++ ++ ++ +
L, densiflora MeOH ++ -
L. heteromorpha MeOH ++ ++ -
L. licaniaeflora MeOH -
* Activity at a maximun concentration of 1000 ppm after a time interval of 24 hours.
Legend: ++ means LCioo; + for LC50; and - for LCo
On the contrary watery residues were completely inactive at the same

concentration. The active fractions were susequently diluted to the
minimum toxic concentration to determine the lethal concentration for
100% of exposed snails. As suggested from the literature, the data were
judged positively if a plant extract killed snails at concentrations of 100
ppm or less [21]. Among the extracts investigated, four extracts showed
LCioo at 100 ppm and three of them had the same toxicity in snails at 50
ppm. Therefore, these active samples were tested on two species of
fishes, Poecilia reticularis and Carassius auratus, starting from the
minimum effective toxic dose on snails and continuing with increasing
concentrations, to find the LCioo/24 h concentration (Table 8). All
extracts showed a lack of toxicity in fishes (toxicities were generally
comparable in the two fish species examined) even when tested at 10.000
ppm, a value 2.000 times greater than the LCioo/24 h for snails.
62
Table 8. Toxicity (LCioo/24 h) on fishes of Licania leaf extracts
Species (MeOH Extract) Toxic dose (mg/1)

Poecilia reticolata XIPHO Carassius auratus L.
ViERNEF
L, carii > 10,000 >10,000
L. pittieri > 10,000 > 10,000
L, pyrifolia > 10,000 >10,000
Three methanol extracts from the six Licania plants showed activity at
50 ppm within 24 hours on snails, a potency comparable with those
reported in the literature for other tested plants of this family; one killed
snails at 200 ppm, another one at 400 ppm, and the last one is active only
at 1.000 ppm. Furthermore, extracts from L, carii, L. pittieri, and L.
pyrifolia had an high selectivity for snails without toxicity in fishes. All
the tested extracts contained mostly compounds belonging to the class of
flavonoids and triterpenes, derivatives. Studies on the moUuscicidal
activity of triterpenes with oleanane, ursane, and lupane nuclei isolated
from Z. pyrifolia and I . licaniaeflora showed no mortality on snails and
catechins did not exhibit any moUuscicidal activity up to 400 ppm. On
the contrary flavonols glycosides of myricetin were lethal at 100 ppm;
their high content in L carii, L. pittieri, and L. pyrifolia is assumed to be
responsible for the strong moUuscicidal activity of these plants. These
constituents, although characteristic of Z. densiflora andZ. heteromorpha
were not in such high concentration to exhibit moUuscicidal activity at
100 ppm [22].
Antimicrobial activity
Pure compounds 11 and 48-52 isolated from the aerial parts of Z.

heteromorpha var. heteromorpha showed antimicrobial activity, with a
different spectrum of action, on Gram-positive bacteria and yeasts (Table
9).
Table 9. Antimicrobial activity of compounds 11 and 49-53: Minimum Inhibitory Concentration,
Minimum Bactericidal Concentration is reported in parentheses (M-g/ml)
Triterpenoids S. aureus S. capitis S. agalacticae C albicans C krusei

11 - ~ - >- -
48 100(100) ~ - 50(50) ~
49 — 12.5 (12.5) 25 (50) 12.5(12.5) 100(>100)
50 ~ ~ 100(100) ~ ~
51 25 (>100) - 100(>100) 25 (25) -
52 - 100(>100) 25 (50) 25 (25) 25 (100)
— no effect
11 betulinic acid, 48 alphitolic acid, 49 3^-0-trans-p-couTmiroy\ alphitolic acid, 50 3P-0-cw-/7-coumaroyl
alphitolic acid, 51 3P-0-fra/is-p-coumaroyl maslinic acid, 52 3p-0-cw-p-coumaroyl maslinic acid.
63
The minimal inhibitory concentration (MIC) was determined on 96-

well culture plates by a microdilution method using MuUer-Hinton Broth.
Eleven two-fold dilutions of test substances were carried out starting
from the concentration of 100 jxg/ml (2.5 % of ethanol). The wells were
inoculated with a microorganism suspensions at a density of 10^ cells/ml.
The plates were incubated for 24 hr (or 48 hr for the yeast) at 37 °C. The
cultures that did not present growth were used to inoculate plates of solid
medium in order to determine the minimal bactericidal concentration
(MBC). Proper blanks were assayed simultaneously. The broadest
activity was observed for compounds 49 and 52 which inhibited the
growth of S. capitis, S. agalacticae, C. albicans, and C krusei; in
particular 49 showed a MIC of 12.5 jxg/ml on S. capitis and C albicans,
AlphitoUc acid (48) inhibited S. aureus (100 jxg/ml) and C. albicans (50
(xg/ml); 50 was active at the maximum concentration tested (100 |xg/ml)
only against S, agalacticae. Compound 51 inhibited S. aureus, S.
agalacticae, and C albicans with different activity. The antimicrobial
activity of the triterpenoids tested was generally bactericidal. Betulinic
acid (11) was inactive against all microorganisms tested. None of these
triterpenoids inhibited the Gram-negative bacteria Klebsiella
pneumoniae, Escherichia coli, Pseudomonas aeruginosa. The
stereoisomery of the compounds appeared to affect their activity
particularly in the case of Sp-O-^ran^-p-coumaroyl alphitolic acid (49)
and 3(3-0-c/5-/?-coumaroyl alphitolic acid (50); the latter inhibits only
one bacterium at the maximal concentration tested, while the former
inhibited almost all tested microrganisms at low concentration.
Antioxidant activity
Pure flavonoids 6, 8, 9, 31, 33, 37, and 64, both glycosides and aglycons,
isolated from the leaves of Z. licaniaeflora, were tested for their
antioxidant activity by DPPH assay [23]. Oxidation is well known to be a
major cause of material degradation. More recently, oxygen-reactive
species, in particular free radicals, have been recognized as involved in
several diseases, including cancer and atherosclerosis. Ageing may also
be the result of the deleterious free-radical reactions which occur
throughout cells and tissues. In this context, natural antioxidants are
receiving increasing attention; particularly, flavonoids have been
64
reported to be efficient antioxidants by scavenging oxygen radicals,

having interesting anti-cancer, hypolidaemic, anti-ageing, and anti-
inflammatory activities.
The use of the DPPH radical as TLC spray reagent proposed for the
first time in 1994 [24] for screening antioxidants in marine bacteria,
appears to be also well suited for the detection of antioxidants in crude
plant extracts or pure compounds isolated from plant material. The DPPH
assay measured hydrogen atom (or one electron) donating activity and
hence provided an evaluation of antioxidant activity due to free radical
scavenging. DPPH (2,2-diphenyl-l-picrylhydrazyl radical), a purplcr
coloured stable free radical, is reduced into the yellow coloured
diphenylpicryl hydrazine. The test was first performed with a rapid TLC
screening method using a 0.2% DPPH in MeOH. 30 min after spraying
active compounds appear as yellow spots against purple background.
Subsequently, spectrophotometric assay was carried out by the following
method: 30 \xl of a methanolic solution containing the pure compound
were added to 3 ml of a 0.004% MeOH solution of DPPH. Absorbance at
517 nm was determined after 30 min, and the percentage of activity was
calculated. Quercetin was used as reference compound.
The results of our experiments demonstrated that flavonoids possess a
potent antioxidant activity which is consistent with other reports [25].
DPPH on TLC revealed all tested compounds as yellow spots against
purple background, but 8-hydroxy-naringenin (37) and kaempferol 3-(9-
a-L-rhamnopyranoside (31) gave pale yellow spots when compared with
the others. All compounds were also tested against DPPH in a
spectrophotometric assay. As shown in Fig. (10), the activity of quercetin
3-0-(3-D-galactopyranoside (6), quercetin 3-O-a-L-arabinopyranoside
(9), and quercetin-3-O-a-L-rhamnopyranoside (8) remained slightly
lower than that of the reference aglycon, indicating that glycosylation at
3-0 position with different sugar moieties reduced the activity of the
quercetin. Taxifolin 3-0-a-L-rhamnopyranoside (64), the
dihydroflavonol derivative of quercetin 3-(9-a-L-rhamnopyranoside (8),
showed stronger radical-scavenger activity compared with this one.
Interestingly, kaempferol 3-0-(2''-13-D-xylopyranosyl)-a-L-
rhamnopyranoside (33) had stronger activity than its biosynthetic
precursor kaempferol 3-(9-a-L-rhamnopyranoside (31), but both were
less efficient compared with quercetin derivatives: this is due to the
presence in kaempferol derivatives of only one hydroxy group in the ring
B of the flavonol. Finally 8-hydroxy-naringenin (37) exhibited similar
65
activity to kaempferol 3-(9-a-L-rhamnopyranoside (31) but with a

different behaviour according to the concentration: stronger activity was
shown up to 50 |uiM, while lower activity between 50 and 80 îM.
Fig (10). Scavenging activity of flavonoidsfromL licaniaeflora leaves on DPPH radical.
100
1 »• » » »
90 1 1 — : r ^ ^ ^
80
^ 70
60
1 III
50 ^ '"^"'^ # Qiiefcetin
40
—A—33
30
B 64
20
• 9
10 VM[ ^^X^^""^
——37 J
0
10 20 30 40 50 60 70 80
Concentration i&M
Cytotoxic activity
The first screening on cytotoxic activity regarding plants of Licania

genus was accomplished by Suffness et al. in 1988 on the ethanolic
extract of Z. heteromorpha [19]. The plant showed cytotoxic activity in
vitro in colon carcinoma 38 and B16 melanoma models.
Subsequently, also the L. michauxii root methanol extract was found
to be cytotoxic to cultured human hepatoma (HepG2) and colon
carcinoma (Caco-2) cells, inducing necrotic death. There was no report
on the isolation and characterisation of the active compounds present in
this extract [20].
Finally, the triterpenoid cucurbitacin B, isolated from L
intrapetiolaris, showed activity in the KB (human oral epidermoid
carcinoma) assay with EC50 value of 0.008 tig/ml. The clerodane
diterpenes intrapetacin A and intrapetacin B displayed moderate activity
vs KB as well [4].
66
Antifungal activity
Intrapetacin A and intrapetacin B isolated from L. intrapetiolaris, when

examined in antifungal assay with Aspergillus niger spores, induced a
good zone of inhibition of fungal growth if compared with amphotericin
B activity [4].
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[I] Chaffaud, M; Emberger L. Traite' de Botanique Systematique, Vol.11, pp.

1338-40, Masson et C. Editeurs, Paris, 1960.
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[3] Toledo, C.L.; Kubitzki, K.; Prance G.H. Flora de Venezuela, Vol.1 V, pp. 326-
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[13] Sagareishvili, T.G.; Alaniya, M.D.; Kemertelidze, E.P.; Khim. Prir. Soedin,
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67
[22] Bilia, A.R.; Braca, A.; Mendez, J.; Morelli, I.; Life Sciences, 2000, 66, PL 53-
59.
[23] Braca, A.; Sortino, C ; Politi, M.; Morelli, I.; Mendez, J.; J. EthnopharmacoL,
2002, 7P, 379-381.
[24] Takao, T.; Kitatani, F.; Wataanabe, N.; Yagi, A.; Sakata, K.; Biosci.
Biotechnol. Biochem., 1994, 51, 1780-1783.
[25] Hanasaki, Y.; Ogawar, S.; Fukui, S.; Free Rad. Biol Med., 1994,16, 845-850.
Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry^ Vol. 28
RECENT PROGRESS IN RETINOID CHEMISTRY
ALAIN R. VALLA
VA R&D Pepiniere d'entrephses, 140 Bd de Creac 'h Gween 29561

Quimper France
DOMINIQUE L. CARTIER and ROGER LABIA
FRE 2125 CNRS, 6 rue de IVniversite 29000 Quimper France
ABSTRACT: Natural retinoids, especially retinol, are present in all living organisms.
They are known as fundamental mediators for many biological processes, e.g. vision,
cellular growth, differentiation of epithelial tissue, reproduction, etc. Although retinol is
an omnipotent compound, natural retinoids like all E, 9Z and dehydroretinoic acids are
clearly more potent outside the retina and trigger gene expression via binding to nuclear
retinoid receptors. Retinaldehyde occupies an intermediate position in this respect, with
the ability to convert to both retinol and retinoic acid (RA). Retinol has an exceptional
position among the retinoids, both in terms of production and its field of applications. In
1995, the sales of retinol reached US $ 500 million. Considering the interplay between
the stereochemistry of retinoids and their biological activities, any synthetic approach to
these compounds must meet the requirement of stereochemical control, in order to
obtain the desired isomer in a highly stereoselective manner. Despite the recent
achievements in the preparation of conjugated E/Z-dienes and trienes using a variety of
synthetic procedures, there is still a need for versatile and efficient approaches to higher
unsaturated E/Z-polyolefins with the appropriated configuration. This review covers the
major synthetic literature on natural retinoids from 1990 to the present.
INTRODUCTION
Retinol (vitamin A) is found in foods of mammalian origin in the form of

retinyl ester, or in fruits and vegetables as carotenoids wîth provitamin A
activity, especially P-carotene (provitamin A). In enterocytes, retinol
binds to cellular retinol-binding protein type II (CRBPII), w^hich directs
the esterification by the enzyme lecithin: retinol acyltransferase (LRAT).
The p-carotene may undergo central or eccentric cleavage with the
formation of retinoids, or may be released unchanged into the blood-
flow^.
70
Retinyl esters and the P-carotene are incorporated into chylomicrons

and taken up mainly by hepatocytes. In the liver retinol may be stored in
stellate cells as retinyl esters, oxidized to retinoic acid or liberated into
cells bound to retinol-binding proteins (RBP). All E retinoic acid and its
9Z isomer have an affinity for nuclear receptors. They activate the
transcription and bind as dimers to specific nucleotide sequences, present
in promoters of target genes.
Retinoids also modulate gene expression acting and the post-
transcriptional level. The P-carotene has also important implications from
the clinical point of view. All E retinoic acid is used for patients with
acute promyelocytic leukemia [1].
They have been found to be active in the regulation and differentiation
of many cell types, including epithelial. Therefore, many natural
retinoids have been tested therapeutically against skin diseases, including
some forms of malignancies.
The best effects were achieved with retinol, retinaldehyde, 13Z-RA
and all E-RA [2]. The latter has selective biological activity for normal
growth and epithelial differentiation. It has been shown that retinoic acid
receptors (RARs) have a high affinity for both all E retinoic acid and its
9Z isomer; which is the natural ligand of the retinoid X receptors (RXRs)
[3]. The six isoforms of these proteins (RARa, RARp, RARy and RXRa,
RXRp, RXRy) are transcription factors interacting with their target genes
through specific DNA-binding domains.
European researchers found recently that retinoic acid-induced
apoptosis in leukemia cells was mediated by paracrine action of tumor-
selective death ligand trail (tumor necrosis factor-related apoptosis-
inducing ligand) [4].
P-carotene
71
CHEMISTRY
Since its discovery in 1909, the elucidation of its structure by Karrer in

1931 [5] and its first total synthesis [6], vitamin A has represented a
challenging target molecule for chemists [7]. The first industrial
synthesis of retinol was performed at Hoffmann-La Roche (H-L R) [8],
followed by other approaches of the Baadische Anilin-& Soda Fabrik
(BASF AG) [9], and Rhone-Poulenc (R-P) (today: Aventis) [10]
Both companies possess today the economically most important
processes, and use p-ionone as starting material. Fig. (1).
6c^' OR •---
vitamine A
H-LR
PhjP NaOMe
P'^Phs, CI" vitamine A
OH HCl '
BASF AG
"OAc
OH
PhS02Na
^Y^^^^ /BuOK vitamine A
R-P
OAc
Fig. (1). Industrial syntheses of vitamin A.
The emphasis for the past twenty years has been the stereoselective
syntheses of vitamin A, and its derivatives, retinal and retinoic acid [11].
A range of recent conceptions, using tricarbonyliron complex [12,13],
Palladium-catalyzed cross-coupling reactions such as:
Negishi reaction [14] (organozinc reagents and organotriflates or
halides),
Suzuki coupling [15] (orgaboron reagents and organotriflates or
halides),
Stille reaction [16] (organostannate and electrophiles),
Heck reaction [17] (organotriflates or halides with olefins),
72
Sonogashira coupling [18] (copper (I) acetylenes with iodoalkenes)...

were described.
Each strategy could be achieved by two methods: e.g. for the
construction of the C(6)-C(7) bond or C(7)-C(8), either the coupling of C9
with Cii or of Cio with Cio synthons had been done, Fig. (2).
Fig. (2). Strategies
C(6)-C(7) strategies:
Using a Stille's reaction, De Lera et al connected a tetraenic stannate

with a triflate derived from the trimethylcyclohexanone [19,20]. First, the
tetraenylstannate was prepared by Horner-Wadsworth-Emmons
condensation of the known stannate [21] and the carbanion of the
phosphonate [22, 23], Fig (3).
(EtO)2(0)P> COOEt
CHO ^COOEt
Bu.Sn^
Fig (3).
Treatment of 2,2,6-trimethylcyclohexanone with lithium di-

isopropylamide (LDA) followed by phenyltriflimide (7V-phenylbis
(trifluoromethanesulphonimide) gave the corresponding triflate [24]. The
73
best coupling reaction could be achieved with Farina's 'soft' palladium

(Pd2(dba)3) with AsPhs as ligand and DMPU, Fig (4) [25].
OTf ^COOEt
a)LDA b) Bu3Sn"
phenyltriflimide Pd2(dba)3, NMP, DMPU, AsPh3
COOEt c) K:OH, EtOH, H2O ^^COOU
Fig. (4). Dominguez, Iglesias, and De Lera (1998, 2001).
As an extension of this procedure, they synthesized the side chain of

9Z-retinoate stereoselectively and attached it to the hydrophobic ring by a
high yielding thallium accelerated Suzuki cross-coupling reaction, Fig.
(5) [26]. The tetraenylstannate used for the coupling reaction was
obtained by Mn02 oxidation of the known stannyldienol [27], to the
corresponding aldehyde (86%), followed by condensation with the
phosphonate (52%) and reaction of the tetraenylstannate with a solution
of iodine. The product was immediately added to the organoborane, in
the presence of Pd(PPh3)4 then TIOH was added, to provide the 9Z-
retinoate in 84% yield. The organoborane was freshly prepared from the
cyclohexanone, via its hydrazone, which was transformed into the iodide.
COOEt
a) BuLi, 1 b) Mn02
BuSn ^ ^ K2C03^ BuSn''
-^:^ ^ OEt
BuSnH, CuCN [ ^j^Q c) BuLi, DMPU
OH4 ""OH
BuSn •
COOEt COOEt
e) H2N>fH2 I J) /BuLi, B(0Me)3 B(0H)2

J ^ ^ ^
I2, Et3N, DBN " Pd(PPh3)4 *" kjl\ COOEt
Fig. (5). Pazos, and De Lera (1999).

74
In this exhaustive work De Lera et al described the syntheses of the

retinoid skeleton via the Stille coupUng for the formation of side-chain
single bonds [28].
C(7)-C(8) strategies:
A stereoselective synthesis of all E retinal, via a condensation of a Cio

chloroacetal with p-cyclogeranylsulfone was described by Julia et al.
[29]. The chloroacetal was reacted with the silylenol ether, using
TiCl4/Ti(OMe)4, to give in 63% yield, the chloromethoxyacetal
derivative as a mixture of ElZ isomers (80/20). The aldehyde was
converted in 97% yield into the corresponding acetal with HC(0Me)3
and camphorsulfonic acid in methanol, Fig. (6).
OMe OMe OMe

a) Ti(0Me)4 b) HC(0Me)3
TiCl4 CHO OMe
OMe
01 CI
OMe
CI
Fig. (6).
This building block was condensed with the anion of (i-

cyclogeranylsulfone. During flash-chromatography the intermediate was
hydrolyzed to the sulfone-aldehyde, as a mixture of three isomers in 95%
yield. Retinal was obtained from this sulfone by treatment with MeONa,
for 10 days, in the dark (90%), Fig. (7).
OMe I OMe
SO2 I OMe I
OMe
CHO
Fig. (7). Chemla, Julia, and Ugen (1993).

75
Chabardes developed a process for the preparation of vitamin A and

its intermediates, from cyclogeranylsulfone and Cio aldehyde-acetals
[30], For example, chlorocitral reacted with ethylene glycol, HC(0Me)3
and pyridinium tosylate to provide the chloroacetal (40%), as a mixture
of two isomers. Reaction of this allylchloride with A^-methylmorpholine
oxide (NMO) and Nal furnished the aldehyde, as a mixture of four
isomers. These compounds underwent condensation with p-
cyclogeranylsulfone. Further chlorination of the sulfone-alkoxide salts,
led to a mixture of sulfone-chloride acetals and their products of
hydrolysis in 45-50% yield. Double elimination of the chloride and the
sulfone, followed by hydrolysis with pyridinium tosylate (PPTS) gave
retinal, as a mixture of all E and 13Z isomers (78/22). The overall yield
from the chloroacetal was 18%. In another 'one-pot' example, retinal was
obtained in 52% yield from the aldehyde, and was then isomerised and
reduced to retinol (all E: 95.5, 13Z: 4, 9Z: 0.5) Fig. (8).
s
a) NMO I I O-^ ^) L A ^2
CI Nal, DMF /PrMgCl
c) SOCI2, pyridine
d) MeOK j^^^^'-'^^::^.-'^^
e) PPTS
Fig. (8). Chabardes (1994).
Honda et al described a highly Z stereoselective [2,3]-sigmatropic

rearrangement that provided trisubstituted E,Z synthons, starting from A^-
tiglyl-p-methallyldimethylammonium salts [31]. The application of this
key triene synthon to the stereoselective synthesis of 13Z-retinol was
reported from a trieneester. Thus, prenylbenzyl ether was converted via
ene-type chlorination followed by amination into internal allylamine.
This was reacted with ethyl 3-bromotyglate in acetonitrile to give the
76
quaternary salt. Treatment of the latter with EtOK in ethanol resulted in

the formation of an ylide. This latter underwent [2,3]-sigmatropic
rearrangement to furnish the diene that possessed a newly formed Z and
tiglyl-origin E stereochemistry, Fig. (9).
a) Br COOEt
OSi/BuMeo OSi/BuMeo
h) EtOK
NMeo
"^ EtOOC
0SirBuMe2
COOEt
Fig. (9).
Treatment of this synthon with peracetic acid resulted in the formation

of a A^-oxide intermediate. A Cope elimination gave the triene, Fig. (10).
EtOOC c) AcOOH EtOOC
0Si/BuMe2 0Si/BuMe2
^0°C
EtOOC
0Si/BuMe2
Fig. (10).
?BuMe2Si was then replaced by rBuPh2Si and the transformation of

the ester group to formyl group was carried out by treatment with
aluminium hydride (AIH3), followed by manganese dioxide oxidation.
This triene aldehyde was reacted with the anion of p-cyclogeranylsulfone
and quenched with AC2O. Desilylation to the acetoxysulfone (80%), and
77
reductive cleavage with sodium amalgam gave the desired 13Z-retinol

(63%), Fig. (11).
e)Bu4NF,y)TBDPSCl
EtOOC »• OHC ^
g) AlCl3/LiAlH4, h) Mn02
OSi/BuMeo OSi/BuPh,
OSi/BuPho
Fig. (11). Honda, Yoshii, and Inoue (1996).
A one-pot procedure was developed by Otera et al from p-

cyclogeranylsulfone [32]. Its lithium salt reacted with 3,7-dimethyl-8-
oxo-2,6-octadienyl acetate to the sulfone-alcohol. The hydroxyl group
was protected to the MOM ether with MeOCH2Cl. Double elimination
could be achieved with potassium MeOK to provide vitamin A in 50%
yield. Fig. (12).
.o 9
o. ^ ^ so.
OHC OAc OAc
a) Nal, BuLi OH
c) MeOK
b)MOM-ci y^^^Yi^^^^ ^^^^^^^^^^^V^ÔAc
^ \ X \ OMOM
Fig. (12). Orita, Yamashita, Toh, and Otera (1997).

78
A similar synthesis was patented by Odera [33].
Two patents by Takahashi et al reported the synthesis of vitamin A

via a Cio dihalogeno derivative [34,35]. In one procedure the
halogenodiene was prepared by bromination of 3,7-dimethyl-2,5,7-
octatrien-1-yl acetate. Addition of the latter and /BuOK in DMF to the
Cio sulfone provided the retinol sulfone (34%). Again, double
elimination (MeOK), gave vitamin A acetate, Fig. (13).
a) Br2 Brv
OAc
b) /BuOK, DMF
SOo I Br
PY c) MeOK -^::s^^^^ OAc
Fig. (13). Takahashi, Furutani, and Seko (2000).
They also developed a second process via other dihalo-compounds

[36]. Treatment of the 1,2-bromo-hydroxy chain with TiCU in DME,
gave mainly the l-bromo-4-chloro unit. Condensation with the Cio
sulfone in DMF, in the presence of /BuOK gave the retinyl-
sulfoneacetate. Elimination of the tolylsulfmate with KOH in DMF
produced vitamin A acetate in 87% yield. Fig. (14).
Fig. (14). Takahashi, and Seko (2001).

79
In a similar route, Takahashi et al made use of non-halogenated

sulfones [37].
Similar processes were related. TiCU was added to a solution of the
diol to give a crude mixture of isomers in which the 5-chlorosulfone was
the main compound in 95% yield. The mixture was treated with MeOK
to produce crude retinol. Acetylation with acetic anhydride (AC2O) in
pyridine, in the presence of DMAP, provided the retinyl acetate in 70%
from the diol [38,39], Fig. (15).
OH
c) AC2O, DMAP
Fig. (15). Takahashi, Furutani, and Seko (2000).
C(io)-C(ii) strategies:
Mestres et al [40] published a regioselective addition of a lithium

trienediolate (generated from hexa-2,4-dienoic acid or dihydropyran-2-
one) to p-ionone. Dehydration of the hydroxyacid, afforded a mixture of
9EIZ, 13£'/Zretinoic acids which, isomerised in the presence of I2, led to
all E retinoic acid in 35% and 30% yield, starting from dienic acid and
pyranone, respectively, Fig. (16).
COOH
COOH
Fig. (16). Aurell, Parra, Tortajada, Gil, and Mestres (1990); Aurell,
Came, Clar, Gil, Mestres, Parra, and Tortajada (1993); Aurell, Ceita,
Mestres, Parra, and Tortajada (1995).
80
A concise preparation of retinoids via new enaminodiesters synthons

was described by Valla et al [41]. For example, all £-retinoic acid was
synthesized within one day by a 'one-pot' process. The enaminodiester
synthon was prepared from methyl isopropylidenemalonate and
dimethylformamide dimethylacetal (DMF-DMA) and then condensed
with the lithium enolate of p-ionone. A Grignard reaction with the
obtained ketodiester led to the retro carbomethoxyretinoate.
Saponification and concomitant decarboxylation, provided mainly all E
retinoic acid {all E/UZ: 90/10, 72% from (J-ionone), Fig. (17).
a)LDA COOMe
b) MeMgBr
^v
l^^^J^ COOMe
COOMe
COOMe
COOMe c)K0H,Me0H,H20 COOH

^
COOMe ^HCllM
Fig. (17). Valla, Cartier, Labia, and Potier (2001).
A short synthesis of retinal was described by Taylor et al. [42] based

on the addition of a Cn vinylalane to a methylpyrylium salt. The 13Z-
retinal (48%) was isomerised to all E retinal by a previous procedure
[43]. p-Ionone was first converted into the alkyne and then into the
vinylalane, using the Negishi methodology [44]. Addition of an excess of
this alane to 4-methylpyrilium tetrafluoroborate [45] gave 13Z-retinal,
being isomerized to the all E isomer (I2 in benzene/ether). Fig. (18).
a) MejAl, ZrClz AlMejBuLi

^
(TI-C5H5)2, BuLi
c)l2 CHO
CHO
Fig. (18). Hemming, De Meideros, and Taylor, (1994); Taylor,

Hemming, and De Meideros (1995).
81
Through two successive Stille reactions, Parrain et al [44] realized a

stereo selective synthesis of all E, 13Zand 9-A2or-retinoic acids. First, the
coupling of £'-l,2-bis(tributylstannyl)ethene and Z- or E-tributylstannyl-
3-iodoalk-2-enoates was performed, followed by iododestannylation. The
second step involved another vinyltin which was synthesized by
stannylation of the Negishi dienyne, derived from p-ionone [47]. To
obtain the substituted vinylstannate, the dienyne was treated with lithium
butyltributylstannylcyanocuprate (Lipshutz reagent) [48] to yield the
intermediate vinylcuprate, which was trapped with an excess of Mel in
the presence of hexamethylphosphoramide (HMPA). The reaction
occurred to the advantage of the terminal vinylstannate (up to 92%). The
coupling partner was obtained from tetrolic acid, which was converted
into E vinyliodide by stannylcupration of the generated stannate [49].
The Z vinyliodide was more classically obtained by hydroiodination [50].
Stille coupling of the P-iodovinylic acids (protected as the corresponding
tributyltin esters) with £'-l,2-bis(tributylstannyl)ethene, catalyzed by
dichlorobis(acetonitrile)palladium provided dienyltins with retention of
the configuration of the two double bonds in fair yields.
Iododestannylation yielded quantitatively the dienic acids, Fig. (19).
a) Bu3SnBuCuLi, j
yr-—— T- C00HBu3Sn^ ^
^COOH
d) BuSnOMe, PdClzCMeCN);
^^ V c)HI e)l2./)KF,HC\ j
SnBu3 g)PdCl2(MeCN)2,DMF I ^ O ^ : ^ ^ ^ ^
COOH I ^ \^-!v
COOH
Fig. (19). Thibonnet, Abarbri, Duchene, and Parrain (1999).
The first palladium-catalyzed cross-coupling reaction used in the

synthesis of retinoids was described by Negishi and Owczarczyk from a
Ci4 alkenylzinc [51]. The synthesis was carried out via a Pd(PPh3)4
82
catalyzed coupling of the C14 alkenylzinc (obtained from the iodide) with
the Ce iodide (derived from 3-methyl-2£'-penten-4-yn-l-ol), followed by
further deprotection with BU4NF. Vitamin A was obtained in 38% yield
based on p-ionone, with complete control of stereo- and regiochemistry,
Fig. (20).
Q a) LDA, Cl-P(0)(0Et)2, LDA
b) MesAl, Cl2ZrCp2,12,
c) DIBAL-H, I2
ClSiPh2/Bu, EtsN, DMAP
d) /BuLi, ZnBr2
P(i(PPh3)4 e) BU4NF
Fig. (20). Negishi, and Owczarczyk (1991).
A highly stereoselective synthesis of retinol vz^ a CM + C6 route was

depicted by De Lera et al [52]. A Suzuki reaction of a C14 alkenyliodide
with a C6 alkenylboronic acid afforded retinol in 83% yield, with
retention of the geometries of the coupling partners. The alkenyliodide
was obtained by a zirconium-mediated methylalumination and a
subsequent Al/I exchange by slow addition of ICN. Coupling with the C6
boronic acid (12 hrs to reach completion), afforded retinol in 83% yield
[53], Fig. (21).
Fig. (21). Torrado, Iglesias, Lopez, and De Lera (1995).

83
C(ii)-C(i2) strategies:
Stereoselective syntheses of all E, 9Z-retinoic-acids and llZ-retinal were

developed from p-ionone-tricarbonyliron complex [12]. Treatment of the
complex (prepared from p-ionone and dodecacarbonyliron, (Fe3(CO)i2)),
with the lithium salt of acetonitrile, Wada et al obtained the nitrile, in
88% yield, Fig. (22).
O
a) LDA, MeCN CHO
^
Fe(C0)3 Q
(EtO)2P''''~V^COOEt
^
c) BuLi
COOEt ,)j^30H COOH
MeOH, HjG
Fig. (22).
Contrarily, the reaction of the lithium enolate of ethyl acetate with

subsequent dehydration gave predominantly the ethyl 9Z-
ionylideneacetate in 89% yield, Fig. (23).
O a) LDA, MeCOOEt
^
Fe(C0)3
/'-p^ CHO ^)BuUTHF

(C0)3
g) NaOH,
^
^ MeOH, H2O
COOEt COOH
Fig. (23). Wada, Hiraishi, Takamura, Date,

COOEtAoe, and Ito (1997); Wada,
(2000).
84
These compounds were converted to the corresponding all E and 9Z-

retinoic acids via P-ionylideneacetaldehydes. Thus, the reaction with the
Uthium sah of (EtO)2P(0)CH2C(Me)=CHCOOEt in THF made possible
the C20 ester-complex. The complex was removed by CUCI2 in EtOH
(98%) and saponification of the ethyl retinoate, the retinoic acids could
be obtained {all E: 89%, 13Z: 8% and 9Z: 59%, 9Z,13Z: 12%,
respectively).
The Peterson reaction of the chlorovinyl-complex with ethyl

trimethylsilylacetate provided the HZ isomer preferentially (77%), and
the 1 IJE" isomer as a secondary product (15%). The ester was transformed
into the Cig ketone (Ph3SnCH2l, BuLi, Et20, 79%). Reaction with
(/PrO)2P(0)CH2CN afforded the llZ-retinonitrile in 73% yield. The
complex was removed by CuCb (72%) and DIBAL-H reduction led
quantitatively to llZ-retinal, Fig. (24).
EtOOC
Fig. (24). Wada, Hiraishi, Takamura, Date, Aoe, and Ito (1997); Wada
(2000).
Wada et al. [13] have previously reported similar syntheses of all E,

9Z-retinoic acids and 1 IZ-retinal.
A short access to retinal was reported by Duhamel et al. [54,55] via

the enolate of prenal, prepared from the corresponding silyl enol ether or
enol acetate. The diene reacted with p-ionylideneacetaldehyde to give the
dihydropyranol as the single reaction product. The dihydropyranol was
85
easily converted into retinal (43% yield) by dehydration, ring opening

and further dehydration in the presence of a catalytic amount of
pyridinium chloride or boric acid, Fig. (25).
OAc
a) MeLi
J^„." t ^
OSiMe,
pyridinium chloride, DMF
Fig. (25). Duhamel, Guillemont, and Poirier (1991); Cahard, Duhamel,

Lecomte, and Poirier (1998).
These authors also described a three-step synthesis of 13Z-retinoic

acid [56]. The obtained hydroxydihydropyrane (66%) was oxidized either
by Jones's reagent (CrOs, water, H2SO4, 90%) or Corey's reagent
(pyridinium chlorochromate (PCC), 65%). Finally, the dihydropyranone
was transformed into retinoic acid (as a mixture of9E, 13Z, and 9Z,13Z),
by /BuOK, according to a known procedure [57], Fig. (26).
or PCC GOGH
Fig. (26). Cahard, Mammeri, Poirier, and Duhamel (2000); Cahard,

Duhamel, Lecomte, and Poirier (1998).
This French group patented a process for the preparation of vitamin A

from vinyl-P-ionol, by BF3-Et20 catalyzed condensation with a C5
sulphide (50% yield) [58].
The phenylthioretinal was reduced with NaBH4 to give the
corresponding alcohol (99.5%), which was acetylated (AC2O, -100%).
86
The resulting sulphide-acetate was oxidized with w-chloroperbenzoic

acid (MCPBA) and the sulfoxide was eliminated by heating in CCI4 to
supply vitamin A acetate in 76% yield. Fig. (27).
SPh
OMe ^Ô 6)NaBH4
OH
a) BF3-Et20
•Qll c) Bi^N, AC2O ^Y^vA.^^

SPh
d) MCPBA ^Y^^Â-^^^^ ecu ^ OAc

kA. SOPh reflux
Fig. (27). Ancel, Bienayme, Duhamel, and Duhamel (1992).
Another work of Duhamel and Ancel [59] related this synthesis of

retinal via p-ionylideneacetaldehyde. Condensation of methallyl-
magnesium chloride with diethyl phenyl orthoformate (Et02CH0Ph) led
after bromination of the ene-acetal, deshydrohalogenation (NaOH 50%),
ethanol elimination with hexamethyldisilazane (HMDS) and ISiMes, to
the bromo-dienol ether. This latter was submitted to bromine lithium
exchange and the lithio enol ether was then condensed with p-
ionylideneacetaldehyde to give retinal. Fig. (28).
GEt GEt GEt
MgCl (Et0)2CH0Ph Br,
NaGH
GEt GEt GEt
Br
HMDS /BuLi CHO

». <^^GEt
ISiMe3 Br >Ov-^CHG
Fig. (28). Duhamel, and Ancel (1992).
In a similar approach, Duhamel et al [60] studied the catalyzed

condensation (BF3-Et20 or ZnCb) of vinyl-P-ionol with a chloro-
enolether. The intermediary aldehyde {all EI9Z\ 65/35) had been
87
dehydrohalogenated (l,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 86% or

LiCl, 75%), to a mixture of retinals. This mixture had been isomerized to
all E retinal, according to literature procedures, [61] Fig. (29).
a) BF3-Et20 or ZnCl2^
CHO
orLiCl, DMF
Fig. (29). Duhamel, Duhamel, and Ancel (1994).
In connection with a work related to the syntheses of C5 building

blocks, Quintard et al [62] described a synthesis of retinal from P-
cyclocitral. This aldehyde was condensed with the vinyl lithium salt of
the C5 acetal.
The lithiated compound was obtained via the vinyltin derivative which
was first converted into the vinyl iodide before doing the halogen-metal
exchange. Fig. (30 and 31).
OEt OEt
J^^^^ .;Bu3SnMgMe,CuCN i ^ J . ^ ^ ^ ^ c)BuUort^.U u^J.^^^
OEt
b)\2
Fig. (30).
In an iterative fashion, the hydroxyacetal (intermediately formed by

condensation of vinyllithium salt with p-cyclocitral) was dehydrated with
aqueous HBr. This allowed the simultaneous hydrolysis into p-
ionylideneacetaldehyde, as a mixture of7E,9E (80%) and 7£,9Z (20%).
The reaction had been repeated with the same C5 unit and finally,
retinal could be obtained as a mixture of isomers, containing 68% of all
E isomer (47%) yield from P-cyclocitral), Fig. (31).
>CcCHO
Fig. (31). Beaudet, Launay, Parrain, and Quintard, (1995); Launay,

Beaudet, and Quintard (1997).
Bienayme and Yezeguelian [63] described a new synthesis of retinal

via a Heck vinylation of a C15 tertiary allylic alcohol with a C5
iodoacetal. Thus, the bromo acetal was prepared by a known procedure
[64], by a bromination-dehydrobromination reaction sequence (E and Z
isomers: 40/60).
The iodo acetal could be easily obtained (as a mixture of E and Z
isomers, 40/60), by a nickel catalyzed iodine-bromine exchange. This
synthon reacted smoothly with the C15 tertiary allylic alcohol in the
presence of a catalytic amount of palladium acetate and a stoechiometric
amount of either a silver or a thallium salt. The C20 hydroxy-acetal was
obtained in 38% yield, as a mixture of E and Z isomers (48/52). Finally
retinal was obtained by treatment with dilute HBr in refluxing acetone, as
a mixture of £" and Z isomers (C(9)=C(io) and C(i3)=C(i4)), Fig. (32).
I GEt I OEt I OEt I OH
""^^ 1 ^ 3 ' c)lK,NiBrJn ^ ^^^ ^Pd(OAc),
Fig. (32). Bienayme, and Yezeguelian (1994)
In another study, Bienayme [65] obtained retinal in three steps from p-

ionone, involving a Pd-catalyzed rearrangement of a mixed carbonate,
derived from ethynyl-retro-ionol.
89
Thus, the P-ionone was smoothly deconjugated and ethynylated to

give ethynyl-retro-ionol as a mixture of ElZ stereoisomers. Formation of
the carbonate and its Pd-catalyzed rearrangement produced
straightforward a mixture of aldehydes and a allene compound. After
silica-gel chromatography, the allenic-aldehyde was conjugated with a
catalytic amount of HBr in acetone. Retinal was obtained as a mixture of
E and Z isomers (75/25), which could be converted into the all E isomer
by simple equilibration. Fig. (33).
uC^^ ^ QC —"
/^^^N.x^^ fl)MeONa,NMP r^y^^^^'''''^^ ^^ Ê~MgCl
o
T^ ?$^
...^^ ^ " ^ 6)Pd(dba)3,P(napht)3
c) Pd(0Ac)2 ^HBr CHO

Y^
^. CHO
Pd(dba)3, P(napht)3
Fig. (33). Bienayme (1994, 1995).

A similar route was patented by Ancel and Meilland [66]. The
ethynyl-retro-ionol was acetylated (Ac20-DMAP-Et3N) and this
propargylic acetate was reacted with methyl butadiene acetate in the
presence of BF3-Et20. The allenic-retinal, obtained in 61% yield was
isomerised in retinal by HBr in acetone (yield: 50%), Fig. (34).
CHO
Fig. (34). Ancel, and Meilland (2000).

90
Salman et al [67] described a process for the preparation of 13Z-

retinoic acid (isotretinoin) in a single step from p-
iony lideneacetaldehy de.
Thus, isotretinoin was obtained by treating methyl-3,3-
dimethylacrylate with LDA, followed by addition of p-
ionylideneacetaldehyde and further hydrolysis with 10% sulphuric acid.
The pH had to be adjusted to 2.8 ±0.5, Fig. (35).
' b)
MeO
a) LDA c)H2S04,pH=2.8 \ X \ COOH
Fig. (35). Salman, Kaul, Babu, and Kumar (2001).
Recently Valla et al showed that new 'P-methylenealdehydes'

synthons could be substituted to 7jE',9£'-ionylideneacetaldehydes (derived
from a and P-ionones) in a Stobbe reaction [68,69].
Regioselective isomerization of these P-methylenaldehydes in Et2NH
produce the compound {EIZ\ 97/3). These synthons were synthesized by
formylation of ionones and concomitant acetalysation of the sodium salts
of the hydroxymethylenic compounds. Wittig reaction and acidic
hydrolysis of the p-methyleneacetals produced the p-
methy lenealdehydes.
Hence, Stobbe-like condensation with dimethyl-isopropylidene
malonate and saponification of malonic acid, half-esters afforded the
corresponding 14-carboxyretinoic acids, as a mixture of all E and 9Z
isomers (80/20). The all E diacid was easily removed by crystallization
from MeCN or ether, Fig. (36). A stereospecific decarboxylation in 2,6-
dimethylpyridine led to isotretinoin.
91
O OMe
>Q a) MeONa, HCOOMe "OMe c) ?h^?CH2
b) H2SO4, MeOH
OMe COOMe
OMe d) HCOOH
COOMe y)NaOH
COOH '
g) ether COOH h) 2,6.dimethyl
COOH pyridine COOH
Fig. (36). Valla, Andriamialisoa, Prat, Giraud, Laurent, and Potier

(1999); Giraud, Potier, Andriamialisoa, and Valla (1999).
A related stereoselective synthesis of all E retinoic acid was also

performed by Valla et al [70] from the 14-carboxyretinoic acid, derived
from p-ionone, using pyridine (2 eq.) at room temperature for 20 hrs. The
crude retinoic acid mixture {all E/13Z: 97/3) was crystallized in MeCN or
AcOEt to provide pure all E retinoic acid. Fig. (37).
COOH a) pyridine COOH

^
COOH
Fig. (37). Valla, Andriamialisoa, Prat, Laurent, Giraud, and Potier

(2000).
A new preparation of the Cig ketone, an important synthon for the

synthesis of vitamin A had also been published by Valla et al [71].
Hence P-ionone and acetonitrile were condensed in the presence of KOH,
to afford the nitrile (80%, ElZ isomers: 80/20). A Reformatsky reaction
of ethyl bromoacetate with the nitrile provided the ethyl P-
ionylideneacetoacetate in 70% yield. Subsequent reduction with NaBH4,
followed by esterification (MeS02Cl) and desulfonation of the unstable
92
ester, led to the acid {ElZ isomers, 80/20) in 80% yield. Reaction of the
latter with MeLi afforded the Cig ketone in 70% yield, as a mixture of
9£/Z isomers (80/20), Fig. (38).
^V-'^Ô ci) MeCN CN b) Zn, BrCHsCOOEt
KOH
V^^^^^^/J^^^^ c) NaBH4 COOH
d) MeS02Cl-Me3N
Fig. (38). Andriamialisoa, Valla, Zenache, Giraud, and Potier (1993).
In addition, these researchers described a series of 9- and 13-

methylene analogues. The synthesis of 9 and 13-methylene isomers of
retinal has also been reported [72]. Hence, the above described P-
methylenealdehyde was condensed with the carbanion of diethyl 2-
oxopropylphosphonate, to give the methylene ketone in 51% yield.
Condensation of the ketone with A^-ethylidenecyclohexylamine afforded
the 9-methylene isomer of retinal, as a 13£/13Z mixture (80/20), Fig.
(39).
CHO a) (EtO)2POCH2COMe
^)MeCH=NC6Hii
c) (C00H)2
Fig. (39). Laurent, Prat, Valla, Andriamialisoa, Giraud, Labia, and Potier
(2000).
93
The synthesis of the 13-methylene isomer was performed from P-

ionylideneacetaldehyde {ElZ: 80/20). Condensation with acetone
provided the conjugated ketone which, after formylation
(MeONa/HCOOEt) and ketalisation (H2SO4/CH3OH), produced the p-
ketoacetal {9EIZ\ 80/20). A Wittig reaction with methyltriphenyl
phosphorane (/BuOK/PhaP^CHs, Br") followed by hydrolysis of the p-
methyleneketal, produced the 13-methylene isomer of retinal, as a 9E and
9Z mixture (80/20), Fig. (40).
O
P><C^x-v^^,.^^ a) MeCOMe b) HCOOMe/MeONa
^^^^V^^V
Ijl MeONa c) H2S04/MeOH
O OMe I II OMe
OMe ^Ph3PCH2 X^^^:Â,.^^%AAoMe
e)HCOOH /K.^--:^:^^^^
pentane
Fig. (40). Laurent, Prat, Valla, Andriamialisoa, Giraud, Labia, and Potier
(2000).
In a comparable approach, Valla et al [73] described the synthesis of

9-methylene analogues of retinol, retinal, retinonitrile and retinoic acid,
using the p-methylenealdehyde derived from p-ionone. Homer-Emmons
condensation with ethyl 4-(diethoxyphosphoryl)-3-methylbut-2-enoate
carbanion afforded the ester in 55% yield, as a mixture of 13£'/13Z
isomers (50/50). This ethyl 9-methylene-retinoate was saponified with
ethanolic NaOH to give the corresponding 9-methylene-retinoic acid in
55% yield (13£/13Z: 50/50). The retinol analogue was obtained by
DIBAL-H reduction of the ethyl ester (75%, 13£/13Z isomers: 65/35).
In the same way, the anion of ethyl 3-cyano-2-methylprop-2-enyl-
phosphonate was reacted with the P-methylenealdehyde to give the 9-
methylene-retinonitrile in 50% yield, as a mixture of 13£'/13Z isomers
(65/35). DIBAL-H reduction of the latter compound provided the related
retinal (70%, 13£/13Z: 65/35). Alternatively, Mn02 oxidation of the
94
alcohol gave retinal in 75% yield, as a mixture of isomers (13E/13Z:

65/35), Fig. (41).
b) NaOH - " \ ^ - ^ ^ \ ^ ^ .--^-.^ COOH
jCOOEt
^ - " ^ ^ |c)DIBAL-H r^^^'^Y^''^^:-^^
O
a) (EtO)2P>. i. .COOEt y)Mn02
CHO
v^^^^^CHO
O
^ (EtO)2P'xA^CN
x ^ e) DIBAL-H
CN
Fig. (41). Valla, Prat, Laurent, Andriamialisoa, Giraud, Labia, and Potier
(2001).
These French chemists described a synthesis of ethyl 9-methylene-

13£ and 13Z-retinoates via the Julia strategy [74]. The required new C15
sulfone was prepared by O-silylation of p-ionone, followed by catalytic
condensation (ZnBr2) of the enol with PhSCH2Cl.
A Peterson olefmation of the ketosulphide led to the methylenic
sulphide. Oxidation (using bis(trimethylsilyl) peroxide [75]), gave the
Ci5 9-methylenesulphone, without any detectable oxidation of the double
bonds.
Thus, condensation with ethyl 4-bromo-3-methyl-2-butenoate (2£/2Z:
50/50) provided the sulphone-ester, as a mixture of isomers (13£'/13Z:
50/50). Elimination to the ethyl 9-methylene-retinoate (2£/2Z: 50/50)
was done by treating the crude mixture with EtONa in cyclohexane. Fig.
(42).
95
OSiMe
Q a) LDA, Me3SiCl b) PhSCHsCl, ZnBr2
l^'^^^Y'^^ ^) Me3SiCH2MgCl
peroxyde
X^^^^^Â^ SOoPh
Br- COOEt
r^^^Y^^^/^^^ COOEt
I H
e) BuLi
Fig. (42). Valla, Laurent, Prat, Andriamialisoa, Cartier, Giraud, Labia,

and Potier (2001).
These researchers also described further syntheses of modified

retinoids such as: 9-demethyl-14-carboxyretinoic acid [76], 9-methylene-
13-demethyl analogues of natural retinoids [77], aromatic 9-methylene
and 13-demethyl-retinol, retinal, and ethyl 13-demethyl-9-methylene
retinoate [78], Fig. (43).
COOH
COOH
R = CH20H;CH0; COOEt
Fig (43). Giraud, Andriamialisoa, Valla, Zennache, and Potier (1994);

Valla, Prat, Laurent, Andriamialisoa, Cartier, Labia, and Potier (2001).
96
The Wittig reaction of lithium a-(dimethylamino)-alkoxydes and a

Ci5 alkyltriphenylphosphonium salt was used by Wang et al to elaborate
the ethylenic linkage of retinol [79]. This in situ method offers the unique
advantage in its application to labile aldehydes, which otherwise would
become isomerised or self-condensed, Fig. (44).
P"Ph3,Br- ^> /Bir^^'^0

/BuLi, /BuOK
Fig. (44). Wang, Wei, and Schlosser, (1999).
Three analogous processes involved the reaction of the C15

phosphonium salt with the 5-hydroxy-4-methyl-2(5/i/)-furanone, in the
presence of a base, as described below.
To generate the phosphorane, Magnone [80,81], Wang et al [82] and
John and Paust [83] used respectively sodium methoxide,
triethylamine/MgCb in A^,A^-dimethylacetamide and LiOH in A^,A^-
dimethylformamide. For the isomerization step, the two first authors
emploied rose Bengal as photosensitizer and the latter Erythrosine B, to
give isotretinoin. Fig. (45).
a) BrMg"^ b) PhsP, HCl, EtOH

OH *^
c) NaOMe or
Et3N, MgClj, AcNMe2
or LiOH, DMF
e) KOH, rose Bengal

or Erythrosine B
Fig. (45). Magnone (1996,1999); Wang, Bhatia, Hossain, and Towne

(1999); John, and Paust (1994).
97
White et al. developed a stereospecific synthesis of Z-olefins,

including isotretinoin [84]. Thus, isotretinoin was obtained by a
Reformatsky reaction of p-cyclocitral with the C5 bromoester, followed
by DIBAL-H lactone reduction, lactol ring opening, selective olefin bond
formation with ethyl 4-diethoxyphosphoryl-3-methyl-2-butenoate and
further saponification, Fig. (46).
0 OH
'A
1
•rS
t^
\ / II \ > \ /
>C^"« .)EtO^^^^
Uk zii " L-
> ^ b) DIBAL-H r^^
r^
^
0 °
d) KOH, EtOH, H2O I JC" COOH
Fig. (46). White, Hwang, and Winn (1996).
Tanaka et al reported a synthesis of vitamin A derivatives from C15

phosphonates [85]. Vitamin A acetate was prepared in 92% yield by
reaction of the C15 phosphonate with 2-methyl-4-acetoxy-2-butenal, Fig.
(47).
a) /BuONa, DMF, PhMe
Fig. (47). Tanaka, Hanakoa, and Takanohashi (1994).
Babler and Schlidt [86] described a route to a versatile C15

phosphonate, used for a stereoselective synthesis of all E retinoic acid
and p-carotene. Base-catalyzed isomerization of the vinyl-phosphonate
afforded the corresponding allyl-phosphonate as the sole product.
Horner-Emmons olefination with ethyl 3-methyl-4-oxo-2-butenoate
concluded the facile synthesis of all E ethyl retinoate. The C15
phosphonate was synthesized starting from the epoxide of p-ionone.
Subsequent isomerization with MgBr2, afforded the C14 aldehyde in 93%
98
from p-ionone. A modified Homer-Emmons olefmation with tetraethyl

methylenediphosphonate led to the vinyl phosphonate in 93% yield.
Isomerization to the allylic phosphonate was perfomied with /BuOK. The
synthesis of ethyl retinoate was carried out via Homer-Emmons
olefination with ethyl 3-methyl-4-oxo-2£-butenoate (61%), Fig. (48).
>Q fl)Me2S=CH2 Z>)MgBr2 / ^ ^ ^ ^ C H O

^v
c)CH2(P(OEt)2)2 r^V^V-^^^-<^P(0Et)2 ^
Q) tBuOK
Fig. (48). Babler, and Schlidt (1992).
Ancel et al patented a procedure for the preparation of vitamin A

from p-ionol and C5 synthons [87]. For example, condensation of 4-
chloro-1,1 -dimethoxy-3 -methy l-2£'-butene with benzenethiol and
distillation of the crude acetal gave 80% of the sulphide enolether which,
condensed with vinyl-p-ionol to phenylthioretinal (50%). Retinal was
obtained in 92% yield from the corresponding sulfoxide (obtained by
MCPBA oxidation) and heating in CCI4.
In a similar fashion, the retinyl acetate (76%) could be synthezised:
quantitative borohydride reduction of phenylthioretinal to the alcohol,
followed by its acetylation gave the acetate, which was oxidized to the
sulfoxide by MCPBA and then eliminated by heating in CCI4, Fig. (49).
99
.OH ÔMe
SPh^
^^
a) BF3-Et20
d) NaBH4.
e)Ac20, Et^N
^^Y^""^^ CHO
J) MCPBA
CHO
L^^Â^ SOPh
g)CCl4 A
OAc
Fig. (49). Ancel, Bienayme, Duhamel, and Duhamel (1993).
C(i2)-C(i3) strategies:
Wada et al described a new strategy for the stereoselective syntheses of

llZ-retinal by palladium-catalyzed cross coupling of a Ci6 tetraenyl
stannate with a C4 £" or Z-vinyl triflate [88]. P-Ionylideneacetaldehyde
was dibromomethylenylated at 0°C with CBr4 and triphenylphosphine.
Stereoselective hydrogenolysis of the dibromo compound with tributyltin
hydride in the presence of a catalytic amount of Pd(PPh3)4 proceeded
cleanly to the C16 bromotetraene in 86% yield.
The synthesis of llZ-retinal required the boronic-partner, which was
prepared from 2-butyn-l-ol by addition of the tributylstannyl cuprate
(83%), followed by protection of the alcohol with /BuMeiSiCl
(TBDMSCl) (93%). The tributylstannyl group was substituted with
boronic acid in three steps: lithiation, quenching alkenyllithium with
triisopropyl boronate and hydrolysis to the boronic acid. The Suzuki
coupling of the C16 tetraene with the boronic compound was carried out
in THF at room temperature, in the presence of a catalytic amount of
100
Pd(PPh3)4, Ag2C03, and an aqueous solution of KOH (77%). Removal of

the silyl ether with tetrabutylammonium fluoride (TBAF) and successive
oxidation with BaMn04 provided the 1 IZ-retinal in 85% yield, Fig. (50).
^ ^ ^ a)CBr4,PPh3
/ K ^ ^ ^ c)Pd(PPh3)4,KOH,Ag2C03
L^Jls, Br B(0H)2
OTBDMS
OTBDMS
f) BU4NF, BaMn04
e) TBDMSCl, KH
BuLi, B(0/Pr)3, aq. HCl
SnBu
d) (Bu3Sn)2CuCNLi2
CHO
OH
Fig. (50). Uenishi, Kawahama, Yonemitsu, Wada, and Ito, (1998).
A similar strategy had been developed for the preparation of all E, 9Z

and 13Z-retinoic acids [89]. P-Ionylideneacetaldehydes {E or Z) were
converted to the corresponding alkynes (ICHiF'^Phs, Y and NaN(TMS)2)
in 70% yield. Stannylcupration of the alkynes with
butyltributylstannylcyanocuprate (BusSnCuBuCNLii) afforded the
tetraenylstannates, which were coupled with the vinyl triflates, using
tris(dibenzylideneacetone)dipalladium (Pd2(dba)3) and triphenylarsine
(AsPPhs) as a ligand. Thus, 9£-stannate and ^-triflate furnished ethyl all
E'-retinoate in 60% yield. Under the same conditions, the coupling
reactions of 9£'-stannate and 9Z-triflate and £'-triflate led to ethyl 13Z-
retinoate and 9Z-retinoates in 43 and 39% yields, respectively. The
coupling of E tetraenyl-stannate with Z-triflate and Z-tetraenyl-stannate
with £-triflate afforded ethyl 13Z- and 9Z-retinoates in 43 and 39%
yields, respectively. Fig. (51).
101
^ " O a) ICHzPhj?""!- | - ' ' ^ [ t ^ ' " ^ = > ' - " " W ^ b) BujSnCuBuCNLij
NaN(TMS)2
COOEt
j^^K-^^V-^^
nBu, c)Pd2(dba)3,AsPh3
COOEt
EoY Z
Zstannate
COOEt
Fig. (51). Wada, Fukunaga, and Ito, (2001).
Conclusions
The retinoid activities are influenced by the complex interactions of their

nuclear receptors that mediate their effects.
Free vitamin A, its esters and the retinoic acids, ATRA, 9Z-RA and
13Z-RA, currently are the most widely tested in clinical studies. These
naturally occurring retinoids tend to be in vivo pan-activators of receptors
(non-receptor specific). Since these retinoic acids are readily
interconverted in vivo, each can activate a wide spectrum of retinoid
receptors, signalling pathways, and biological effects. The receptors are
involved in regulating transcription of specific genes. They regulate cell
differentiation, proliferation, loss... The relationship between variations
in receptor expression by organ site and responsiveness to chemo
preventive agents is currently under study.
These established facts made that retinoid chemistry of today, more
than ever, a challenge for chemists.
This account, relative to retinoid chemistry, was in ad equation and
corroborated the older methodologies of retinoid syntheses, which
favored the C(ii)-C(i2) way. Significantly, since 1990, more than forty
references have linked P-ionylideneacetaldehyde as an intermediate in
the syntheses of retinoids (ref: CAS, Sci. Finder). This choice could be
102
attributed in parts to the fact, that this C15 aldehyde could be synthesized
in many ways from p-ionone, a relatively Cn inexpensive product.
ADCDP = 1, r (azodicarbony l)-dipiperidine

BASF AG = Badische Anilin-& Soda Fabrik AG
CRBPII = cellular retinol-binding protein type II
dba = dibenzylideneacetone
DBN = l,5-diazabicyclo[4.3.0]non-5-ene
DBU = 1,8-diazabicyclo[5.4.0]undec-7-ene
DIBAL-H = diisobutylaluminium hydride
DMAP = 4-dimethylaminopyridine
DMF = A^,A^-dimethylformamide
DMF-DMA = A^//-dimethylformamide, dimethylacetal
DMPU = 1,3-dimethyl-3,4,5,6-tetrahydro-2(l^-pyrimidone
HMDS = hexamethyldisilazane
HMPA = hexamethylphosphoramide
HMPT = hexamethylphosphorous triamide
H-LR = Hoffmann-La Roche
LDA = lithium diisopropylamide
LDE = lithium diethylamide
LRAT = lecithin:retinol acyltransferase
MCPBA = /w-chloroperbenzoic acid
MOM == methoxymethyl
NMO = A^-methylmorpholine oxide
NMP = 1 -methyl-2-pyrrolidinone
PCC = pyridinium chlorochromate
PhH = benzene
phenyltriflimide = A^-phenylbis(trifluoromethanesulphonimide)
PhSH = benzenethiol
PTSA = para toluene sulfonic acid
PPTS = pyridinium paratoluenesulfonate
RA = retinoic acid
RARs = retinoic acid receptors
R-P = Rhone-Poulenc
RBP = retinol-binding protein
103
RXRs = retinoid X receptors

TBAF = tetrabutylammonium fluoride
THF = tetrahydrofurane
TMS = trimethylsilyl
Tf = trifluoromethanesulfonyl (triflyl)
TBDPSCl = /butyldimethylchlorosilane
DEDICATE
This review is dedicated to Professor Pierre Potier, Member of the

Sciences Academy of France, for his 68th birthday.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge Pr B. Corbel (UBO Brest) for

helpful discussions. Also, we are indebted to Pr J.-J. Le Yeuc'h (UBO
Quimper) and Dr N. Boeker for assistance in the revision of this
manuscript.
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 28
BIOACTIVE TETRAMIC ACID METABOLITES
EMILIO L. GHISALBERTI
Department of Chemistry^ University of Western Australia, Nedlands

6009 WA. Australia
ABSTRACT: Secondary metabolites containing the tetramic acid (2,4-pyiTolidine-2,4-

dione) ring system have been known for almost half a century, and well before the par-
ent system was synthesised. Although the first naturally occurring tetramic acids were
identified because of their activity as antibiotics and/or mycotoxins, more recently
tetramic acid-containing compounds have been found to display a remarkable diversity
of biological activities. The often unusual and intricate substituents modifying the tetra-
mic acid structural unit make the synthesis of these metabolites a challenging target. Re-
cent studies has confirmed that these metabolites have a wide distribution and play a
significant role in ecological interactions. They have been isolated from marine mol-
lusks, sponges and cyanobacteria, terrestrial and marine microorganisms, particularly
endophytic fungi. In an attempt to bring all these strands together, this review will con-
sider the structure, chemistry, biosynthesis, bioactivity, distribution and ecology of this
diverse group of metabolites.
INTRODUCTION
The term ^tetramic acid_ was coined in 1901 to refer to the heterocycUc
system l,5-dihydro-4-hydroxy-2H-pyrrol-2-one (1), a tautomer of 2,4-
pyrrolidinedione (2) which is the predominant species in solution [1].
Although tetramic acid was apparently synthesised only in 1972 [2], a
number of natural substances had been identified as derivatives of tetra-
mic acids well before that time. Secondary metabolites containing the
tetramic acid ring constitute an increasingly important class of bioactive
natural products. Compounds containing this structural unit, sometimes
intriguingly camouflaged, exhibit a diverse range of biological activities
and have attracted the interest not only of natural products chemists, but
also of chemical ecologists, medicinal and synthetic chemists. The tetra-
mic acid moiety, in most cases, is present as a 3-acyl derivative with the
acyl group varying from the simple acetyl group to structurally complex
entities incorporating several stereogenic centres. The increasing activity
in this area, stimulated by an increasing number of bioassays and a larger
resource pool, makes periodic reviews of the field useful.
no
It is somewhat surprising to find that there have been only three pub-
lished reviews on tetramic acids. The first reports on the advances in
tetramic acid chemistry up to 1993 [1], another describes the synthesis of
tetramic acid antibiotics [3], and a third, dealing with the structure, isola-
tion and synthesis of naturally occurring tetramic acids, was published in
1995 [4]. The present review is an attempt to cover the field of tetramic
acid metabolites with particular emphasis on the structure, biosynthesis
and biological activity of these compounds.
For the purpose of this review, it has been necessary to circumscribe

the type of tetramic acid metabolites to be considered. Thus, the large
group of cytochalasins, chaetoglobosins and penchalasins, e.g. 3, will not
be included here as they have been covered in detail elsewhere [5-7], For
these metabolites, a tetramic acid species has been postulated as the puta-
tive precursor 4 that undergoes a Diels-Alder reaction [8-10].
From a biosynthetic perspective, naturally occurring tetramic acids can

be regarded to arise from the assembly of an amino acid and an activated
acyl entity derived from an acetyl group or a more complex activated es-
ter. Fig. (1). Alternatively, the simple tetramic acid formed can undergo
substitution at C-3 with a second acyl group. On this basis, compounds
such as lactacystin (5) have not been considered [11]. The carboxylic acid
Ill
group of the a-amino acid (from L-leucine) is not involved in the hetero-
cyclic ring in (5), Fig. (2).
Fig, (1). Biosynthetic pathways to 3-acyltetramic acids
STRUCTURE AND SPECTRAL PROPERTIES
Aspects of the structure and properties of tetramic acids have been pre-
sented in detail [1,3] and only a summary will be given here. Tetramic
acid is a much weaker acid (pK^ 6.4 in water) than its oxygen counterpart
and exists mainly in the 2,4-diketone form. The presence of an acyl group
at C3 results in an increase in acidity (pK^ 3.0-3.5) and proton NMR
spectra indicate complete enolisation and the presence of tautomeric
forms. As shown in Fig. (3), these can be separated into two sets of _ii-
temaL tautomers (A/B, C/D), which are rapidly interconverting, and two
pairs of __externaL isomers that interconvert slowly on the NMR time
scale and often give rise to separate NMR signals [1,3]- ^^C-NMR spec-
troscopy is more useful in determining the predominant tautomeric spe-
cies and it indicates that the one represented by D is more important. On
the other hand, if the nitrogen is acylated, the preferred form is A since
the lone pair on nitrogen does not enhance the proton acceptor ability of
the lactam carbonyl at C2 [3,1]. Selected examples of differently substi-
tuted tetramic acids and their spectral parameters are given in Fig. (4).
112
Of some interest is the ability of 3-acyltetramic acid to form stable

complexes with ions of transition metals, e.g. Cu^^, La^"", Sm^"^, Eu?'^, Gd^^,
and boron complexes [1]. Indeed, examples are available in which the
tetramic acid occurs naturally as magnesium or calcium salts. Methods of
isolation or purification involving acidic solvents generate conditions that
Fig, (2). Biosynthetic assembly of lactacystin (5)
SLOW
^N^â FAST
R C
Fig. (3). Tautomeric forms for 3-acyltetramic acids

113
are capable of converting salts to the conjugate acid [13-15]. In one case,
it was found that the free acid derived from the magnesium salt was un-
stable and prone to oxidation [16]. For tetramic acids embedded in a mac-
rocyclic lactam ring, it was found that all prior isolations of these type of
compounds involved acidification during extraction or chromatography
[16]. On the other hand, it is also possible for the tetramic acid to seques-
ter metal cations as observed for the case of corresponding tetronic acid
that formed the calcium salt on chromatography with silica gel 60 [17].
^^C-NMR (CD^CN)
IR (film) 1726.1657-1617 cm"''(several bands) TAUTOMER
UV (CH3CN) 238. 286 nm
f^3 H,C^-''
HaC^ 27.5 178.0
26.3 172.8
i^C-NMR (CDCI3) ^^C-NMR (CD^CN)

IR(KBr), 1713,1620 cm-'' IR(ZnSe), 1690,1664 cm-''
UV (CH3OH) 245, 285 nm UV (CH3OH) 235, 290 nm
Fig. (4). ^^C-NMR, IR and UV spectral parameters of typical

tetramic acids
114
ACYLTETRAMIC ACIDS
In this section, tetramic acids with an acyl group substituent at C-3 are
discussed. The simplest of the naturally occurring 3-acyl tetramic acids,
tenuazonic acid (6), was first isolated from the culture filtrate of Alter-
naria tenuis [18] and, subsequently, from other fungal species (A. alter-
nata, A. longipes, Pyricularia oryzae) [19,20]. Species of Altemaria are
known to produce more than 70 secondary metabolites, many of which,
particularly those from the Altemaria altemata complex, are mycotoxins
[19]. The absolute stereochemistry of 6 (55,65) was deduced from the
formation of L-isoleucine on ozonolysis followed by acid hydrolysis [21].
Tenuazonic acid has also been isolated as a mixture of calcium and
magnesium complexes from Phoma sorghina, the fungus implicated in
the aetiology of onyalai, a haematologic disorder affecting Black African
populations south of the Sahara [22]. Tenuazonic acid (TA) readily forms
complexes with magnesium" to yield Mg"TA2, and with transition metals
(Cu" TAj, Ni"TA2 ^^^ Fe"'TA3) [23]. An X-ray crystallographic study of
the copper-fc/5-tenuazonate monohydrate, Cu(TA)2.H20, showed that the
chelate is formed between the enolic oxygen of the acyl group and the
amide oxygen [24]. With this in mind, and considering that the isolation
of tenuazonic acid from the liquid cultures of A. tenuis required acidifica-
tion, it is highly probable that these covalent complexes are the __naturaL
form of the metabolite [22].
The biosynthesis of tenuazonic acid was studied using [l-^'^C]-labeled

acetate. N-acetoacetyl-L-isoleucine (7) was detected by radioactive trap-
ping, indicating that amide formation, rather than carbon-carbon bond
formation is probably the first step. None of the simple tetramic acid (8)
115
was detected [16,25]. It was also found that the analogues of tenuazonic
acid (9-11) were obtained when the medium was supplemented with L-
valine [27] L-leucine [27] or L-norvaline [20].
T ° 9 R = i-Pr
10R = i-Bu
11 R = n-Pr
Tenuazonic acid has a broad toxicity spectrum and is regarded as a

mycotoxin [28]. It was first detected as a growth inhibitor of tumour cells
(human adenocarcinoma), and was later shown to have weak antibacterial
and, at high dose levels (100-500 fig/ml), antiviral activity towards polio-
virus MEF-1, enterovirus (ECHO-9), respiratory viruses (parainfluenza-
3), vaccinia and Herpes simplex (HF). It has been shown to be an inhibi-
tor of peptide bond formation in preventing substrate binding to acceptor
site of peptidyltransferase in human ribosomes [29].
Tenuazonic acid is also produced by the rice fungal pathogen Pyricu-
laria oryzae and exhibits a conspicuous stunting effect on rice seedlings.
In plant cells, it also inhibits growth by interfering with protein synthesis
at the ribosome level [30]. Tenuazonic acid induces a defence reaction
(browning) on the leaves of varieties of rice resistant to P. oryzae. When
sprayed on rice seedlings at 1000 ppm, it inhibits infection by 98.5-99.7%
after 7 days from application [31]. Alternaria alternata isolated from
field-grown Beta vulgaris produced 150 mg/L of the acid in the sodium
form. Tenuazonic acid was toxic to 5. vulgaris in the 10"^ M range [15].
The first recorded example of a naturally occurring tetramic acid mag-
nesium salt was isolated from a new Pseudomonas species, P. magne-
siorubra, obtained from the surface washings of the marine green alga
Caulerpa peltata [33]. Magnesidin is an inseparable mixture (--1:1) of the
magnesium chelates of the 3-hexanoyl (12) and 3-octanoyl (13) tetramic
acid derivatives; the exact proportions of the mixture vary according to
methods of cultivation and isolation. They were rediscovered 20 years
later as the antibiotic principles from the gram-negative bacterium Vibrio
gazogenes isolated from marine mud [34]. Magnesidin inhibits various
116
gram-positive bacteria (MIC 2-7 |Lig/ml), particularly spore bearers, and

prevents the decay of foodstuff caused by spore germinating organisms.
The toxicity of magnesidin in mice were LD50 50 (ip) and 1000 mg/kg
(orally and s.c). Replacing magnesium by other metal cations, Li^, Na^,
K"", Cu^^ and H^, resulted only in reducing the activity towards Bacillus
subtilis (MIC 2.5-30 îg/ml) [35,36].
Mg',2+
12 n = 3
13 n = 5
J 2
Ras oncogenes play an important part in cell growth and differentia-

tion. Agents that reverse the transformed phenotype caused by ras to
normal may provide selective anti-cancer drugs. In a search for such
compounds, two tetramic acids (14, 15) were isolated from the marine
sponge Melophlus sarassinorum collected at Spermonde Islands in Indo-
nesia [37]. The gross structures were deduced from spectral data, al-
though the NMR spectra were complicated by the fact that the compounds
exist as tautomers, exo and endo, in the ratio -9:1.
H3C(CH2W ^ . ^ > ^ A
HdC(CH2)ir A ^N—CH3 ^ ^ ^^^ "Y N—CH3
14 15
Hcp(CH2)l3
N—CH3
16
AcO
117
This complication could be overcome by acetylation which provided

the C-4-O-acetyl derivative, e.g.l6. For melophlin B (15), the configura-
tion at C-5 was determined by classical methods. Thus, treatment with
NaI04 and KMn04, followed by hydrolysis with HCl, gave N-methyl
alanine which was derivatised with N-a-(2,4-dinitro-5-fluorophenyl)-L-
leucinamide. HPLC analysis of the derivative showed it to correspond to
that prepared from L-alanine. The melophlins exhibited moderate cyto-
toxic activity against HL60 cells at 0.2 and 0.4 îg/ml respectively, and
reversed the morphology of H-ra^ transformed NIH3T3 fibroblasts at 5
|LXg/ml. At a concentration of 1 |Lig/ml, they arrested the NIH3T3 cells in
the Gl phase of the cell cycle, an indication that they act on the ras-
mediated signal transduction pathway.
The P-triketone (17) was isolated from the fermentation broth of an
isolate of the bacterium Apiosordaria ejfusa. The structure of 17 was de-
termined from NMR spectral studies [38]. If the broth was acid treated
prior to extraction, a mixture of the two isomers, a- and p-apiodionen
(18, 19) were isolated and were found to be in equilibrium in solution
[39]. The absolute stereochemistry of these compounds has not been as-
signed. Apiodionen exhibited weak activity as inhibitor of topoisomerase
I and II [40].
A similar compound, bripiodionen (20), has been isolated from a

Streptomyces sp [41]. NMR spectral data determined for a fresh sample of
the metabolite indicated predominantly a single isomer, probably that
with 5(7)£'-configuration. If a MeOH or DMSO solution was left for a
few days, a 1:1 mixture of two isomers (20, 21) became evident, as found
118
for apiodionen. Bripiodionen was discovered in a screen for identifying

inhibitors of human cytomegalovirus protease, a P-herpes DNA virus.
This virus is an opportunistic pathogen in immunocompromised or im-
munosuppressed individuals. Bripiodionen displayed inhibitory activity
against the protease with IC50 30 |LIM, and also showed low cytotoxicity
(IC50 34 |LiM) on the murine lung carcinoma-derived cell line.
Cultures of Streptomyces rimosus var paromomycinus characteristi-

cally develop UV absorption at 240 and 278 nm due to formation of
malonomicin (22), a compound that shows antiprotozoal activity towards
Trypanosoma congolense, the causative agent of sleping sickness in cattle
[42]. Malonomicin contains an unique aminomalonic acid unit that, on
brief heating in water, undergoes decarboxylation and results in a com-
pound devoid of biological activity [43]. Hydrolysis of the compound
yielded L-serine and racemic aspartic acid. The structure was elucidated
by chemical and spectroscopic methods [43,44] and was confirmed by
total synthesis [45].
OH OH
HN ^ ^ °°^"
HoN 23
Aspects of the biosynthesis have been resolved. The amino acid (see
23) involved in the heterocyclic ring arises from L-2,3-diaminopropionic
119
acid [46]. Evidence for the involvement of 3-oxoadipic acid to generate

firstly the amide and then the heterocyclicringhas been obtained [47,48].
Cyclopiazonic acid (24) is pehaps the most studied of the tetramic acid
group of compounds, a consequence of its intriguing structure, range of
biological activity and its environmental importance. Originally isolated
from Penicillium cyclopium, it was later found as a metabolite from sev-
eral species of Penicillium and Aspergillus that infect a number of agri-
cultural commodities such as groundnuts, com, cheese, meat [49]. The
structure was established by chemical degradation and from NMR spec-
troscopy [50], and was confirmed by X-ray crystallographic studies,
which also established the absolute stereochemistry [51]. The bisseco-
dehydrocyclopiazonic acid (25) and the imino derivative (26) were also
isolated [52]. The imino derivative (26) accumulates later in the fermen-
tation and may be an artefact, whereas (25) accumulates in the early
stages of fermentation, but decreases as cyclopiazonic acid is formed. The
biosynthetic pathway to (24) has been shown to involve tryptophan, me-
valonic acid and acetate as precursors of P-cyclopiazonic acid (25) which
undergoes ring closure to (24).
O OH
24 R = OH
26 R = NH2
Cyclopiazonic acid is a potent inhibitor of calcium uptake and acts as a

selective inhibitor of the sarco-endoplasmic reticulum Ca^'ÂTPases
(SERCAs) [53], it induces charge alteration in plasma membranes and
mitochondria and it can function as an antioxidant [54].
A number of tetramic acid derivatives acylated with a liposaccharide
unit have recently been discovered in marine sponges. The first example
was uncovered in the sponge Ancorina sp using a bioassay to detect inhi-
bition of blastulation in starfish embryos. The structure of the compound
(27), named ancorinoside A, was determined by spectroscopic techniques
120
[55]. Interpretation of the NMR parameters was difficult since, in solu-

tion, this compound exists as a mixture of 4 tautomers (see Fig. (3)). A
full analysis of the NMR parameters required spectra for pyridine and
methanol solutions to be analysed.
The absolute configuration of the p-glucose and |3-galactouronic acid
groups was confirmed as D by GC analysis of the trimethylsilyl ethers of
l(L-a-methylbenzyl-amino)-l-deoxyalditols derived from the water-
soluble fraction from acid hydrolysis of (27). The stereochemistry at C-5
was secured by the usual method (hydrolysis and Lemieux degradation of
OH
^ CO2H
27 Ancorinoside A o^^
28 Ancorinoside A
Mg salt C02® Mg'*
HO\.--V^|- CO2H
OH
R o^N.
29 R = H Ancorinoside B
30R = CH3 Ancorinoside C
CO2H
31 Ancorinoside D
the chloroform-soluble fraction). This afforded N-methyl-D-aspartic acid

which translates to a 5/?-configuration. Moreover, the CD spectrum of
(27) showed positive Cotton effects at 285 (Ae +0.79) and 240 nm (Ae
+0.76), opposite to those observed for tenuazonic acid (6) and equisetin
(see below) which have the 55-configuration. Interestingly, the corre-
sponding water-soluble magnesium salt (28) was isolated from the same
sponge [56]. When fertilized starfish eggs were cultured from fertilization
in the presence of 27, or the magnesium salt (0.4 |ig/ml), the development
proceeded normally to 256-512 cell stage at which point, the embryos
ceased to develop further without any sign of blastulation [55,56].
121
A suite of similar compounds, ancorinosides B-D (29-31), was subse-

quently found in another marine sponge from Japan, Penares sollasi, to-
gether with ancorinoside A [57]. These compounds, which were obtained
in 0.004, 0.003, 0.001 and 0.0006% of the wet weight of the sponge,
showed some activity as inhibitors of recombinant MTl-MMP. The ma-
trix metallo-proteinases (MMPs) belong to the proteases which contain a
catalytic zinc-binding domain. They are linked to a range of physiological
and pathological processes including wound healing, angiogenesis, in-
flammation, tumor progression and metastasis. Interestingly, the ancori-
nosides are much less active than tenuazonic acid in which the complex
architecture is reduced to the basic tetramic acid group.
Ph.
"J^^^^Hs
^fr »/wvvrvAA/v»
33
HsC^
32R, =CH20H;R2 = H
3 4 R i = H ; R 2 = CH20H
Equisetin (32) was first isolated in 1974 as a metabolite of the white

mould Fusarium equiseti [58,59]. It was found to exhibit a range of bio-
activities, including antibiotic and HIV inhibitory activity, cytotoxicity
and mammalian DNA binding [59-61]. Equisetin was reisolated in 1989
[60] following the implication of this fungus as a promoter of chronic en-
vironmental diseases, including leukemia [62]. The elucidation of the
structure was complicated by the presence of an equilibrium mixture of
tautomers which precluded detailed NMR analysis. This was reduced to a
single tautomer by formation of the phenylboronate (33), the spectral
properties of which facilitated the assignment of structure. NOE experi-
ments and molecular mechanics calculations provided the relative stero-
chemistry of the decalin system. The absolute configuration of the serine-
derived tetramic domain was established as 5 by application of CD meth-
ods and comparison with the CD spectrum obtained for tenuazonic acid
122
(6). The assigned absolute configuration is supported by two elegant

syntheses [63,64].
Fusarium equiseti and F. pallidoroseum are frequently reported as
secondary colonisers of mono- and dicotyledons and cause disease on
many crop plants such as snapbean, tomato, banana, cantaloup and
muskmelon [65]. A numberof isolates of the two species were observed to
produce equisetin (32) and epZ-equisetin (34). The two are related via
epimerisation at C-5 of the tetramic unit, a reaction that in vitro can be
observed under pyridine catalysis and leads to an equimolar mixture of
the epimers. Both were found to suppress germination and inhibit the
growth of seeds at concentrations of 2.5-10 |ig/ml. In addition, they ad-
versely affect growth of young sedlings, cause necrotic lesions on the
roots, cotyledons and coleoptiles of tested plants.
Phomasetin (35), from a Phoma sp, has been shown to be the enanti-
omeric bishomologue of equisetin from NMR and chiroptical comparison
between the two [66]. Using an in vitro biochemical assay designed to
identify inhibitors of integrase-catalysed strand transfer, equisetin recov-
ered from the fungus Fusarium heterosporum and phomasetin from
Phoma sp. were isolated as inhibitors of human immunodeficiency virus
type 1 (HIV-1) integrase in vitro [67]. Equisetin and related compounds
inhibit 3' end-processing and strand transfer as well as disintegration
catalysed by either the full-length enzyme or the truncated integrase core
domain (amino acids 50-212). These compounds also inhibit strand trans-
fer reactions catalysed by stable complexes assembled in vitro and inte-
gration reactions catalysed by pre-integration complexes isolated from
HIV-1-infected cells and are mechanistically distinct from many previ-
ously described inhibitors of HIV-1 integrase. Equisetin specifically in-
hibits the substrate ion carriers of the mitochondrial inner membrane. It is
a potent inhibitor (IC50 8 nM) of DNP-stimulated ATPase activity of liver
mitochondria and mitoplast [68].
Cryptosporiopsis cf quercina is an endophytic fungus first isolated
from the plant Tryptergyium wilfordii. It causes no discemable external
disease symptoms on T, wilfordii or Quercus sp from which it has also
been isolated. It has been suggested that it may function as a symbiot; the
plant providing support and nutrition to the fungus which in turn partici-
pates in the association by producing antifungal metabolites. The fungus
was grown on oat seeds in water (800 ml) for 4 weeks, and the dichloro-
methane soluble portion of the culture filtrate was obtained. Repeated
123
chromatography and bioassay-guided fractionation, using the phytopatho-

gen Sclerotinia sclerotiorum, led to the isolation of cryptocin (36) (63
nig) [69]. As observed for equisetin, the NMR spectra were complicated
by the existence of a mixture of tautomers. Fortunately, cryptocin crys-
talised from a mixture of ethyl acetate-methanol as rhomboidal crystals
that were amenable to an X-ray crystallographic study. Cryptocin crystal-
lised as a sodium salt with sodium coordinating each of the oxygen atom
in the molecule. The absolute stereochemistry remains to be determined.
»AAA/\AAA/»
H3C
36 37 38
Cryptocin (36) exhibits potent activity against certain phytopathogenic

fungi; the oomycetes Pythium ultimum (0.78 _g/ml), Phytophthora cin-
namoni (0.78 _g/ml), P. citrophthora (1.56 _g/ml), the ascomycetes Scle-
rotinia sclerotiorum (0.78 _g/ml), Pyricularia oryzae (0.39 _g/ml), the
basidomycete Rhizoctonia solani (6.25 _g/ml), the fungi imperfecti
Geotrichum candidum (1.56 _g/ml) and Fusarium oxysporum (1.56
_g/ml). However, it showed little activity (> 80 _g/ml) against human
pathogenic fungi such as Candida albicans and Aspergillus fumigatus,
and in this respect does not share the activity of certain other tetramic acid
metabolites such as the aurantosides that are active against C albicans.
Interestingly, P. oryzae is the most sensitive pathogen to cryptocin. This
fungus, which causes rice blast and is responsible for significant crop
losses, is one of the five targeted diseases in the development of fungi-
cides [70]. Cryptocin is also active against R. solani, a representative of
the basidiomycetes that cause cankers, heart and stem rots, root rots, and
blights of woody and viney plants.
A metabolite (CJ-17,572) from a strain of the fungus Pezicula sp ap-
pears to be identical to cryptocin, although the possible identity of the two
was not mooted [71]. The lack of reported details, NMR parameters for
124
cryptocin and m.p. for the Pezicula metabolite, makes comparison diffi-
cult. Of some interest is the observation that attempted acetylation of the
Pezicula metabolite yielded a derivative (37) in which the secondary al-
cohol had been eliminated and the enol oxygen at C4 acetylated. The
metabolite (CJ-17,572) inhibited the growth of multi-drug resistant strains
of Staphyllococcus aureus (MIC 10 |ig/ml) and Enterococcus faecalis
(MIC 20 |ig/ml) and exhibited cytotoxicity against HeLa cells (ICg^ 7.1
Jig/ml).
NH2
39 40
Yet another analogue (CJ-21,058) (38) of equisetin was isolated from

an unidentified soil fungus found at Nagasaki, Japan [72]. It showed mar-
ginally greater activity than CJ-17,572 against S. aureus (MIC 5 |ig/ml)
and E. faecalis (MIC 5 ^g/ml). Interestingly, CJ-21,058 was discovered
using an assay for SecA inhibiting activity. Sec A is a dimer of 102 kDa
subunits found in the cytoplasm and bound to the inner membrane and is
the peripheral domain of a core containing an integral domain comprising
SecY, SecE and SecG proteins. SecA couples the energy from ATP
binding and hydrolysis to protein translocation through repeated cycles of
insertion and deinsertion of SecA. Compounds that inhibit association of
the enzyme complex or of ATPase activity of SecA could provide a new
class of antibiotics. CJ-21,058 showed an IC50 of 15 |ig/ml.
Other examples in which the decalin system has been modified have
been described. The epoxide (39) (PF1052) has been reported as a me-
tabolite from an isolate of a Phoma sp. It showed good activity against
Staphylococcus aureus (MIC 3.13 |ig/ml). Streptococcus parvulus (0.78
|Lig/ml) and Clostridium perfringens (0.39 |Lig/ml) [73].
A Microtetraspora sp isolate recovered at Andhra Pradesh in India,
produced a metabolite BU-4514N assigned structure (40) from NMR data
125
[74]. It has been claimed to be active against Gram-positive bacteria and

to be effective as a nerve growth factor (NGF) mimic. NGF is a protein
known to be essential for the development and maintenance of certain
sympathetic and sensory neurons in the peripheral nervous system. NGF
appears to have functions in the cholinergic neurons in the basal fore-
brain. BU-4514N is useful for treating neurodegenerative disorders such
as Alzheimer_s disease by mimicking the effect of NGF. Cultures of
PCI2 rat pheochromocytoma cells respond to NGF by differentiating into
sympathetic neuron-like cells. The cells stop dividing, produce nurite-
like structures and produce increased levels of neurotransmitters and neu-
rotransmitter receptors [75].
N—r^^
CO^Hs
o»»'
42
Vermisporin (41) is produced by the fungus Ophiobolus vermisporis

[76]. Its structure was determined by chemical degradation to the deriva-
tive (42) which was studied by X-ray crystallography and provided the
absolute configuration [77]. Vermisporin exhibits antimicrobial activity
towards Bacteroides spp (0.25-2 |ig/ml), Clostridium perfringens (0.25-2
fig/ml) and methicillin-resistant Staphylococcus aureus (0.12-0.5 |Lig/ml).
A metabolite of Ophiobolus rubellus produces the tetramic acid (43) that
has been claimed to be an inhibitor of proline hydroxylase (IC5019|LiM)
[78].
Three related tetramic acids have been reported from Chaetomium
globosum. Two (44, 45) differ in the stereochemistry of the amino acid
component, and the third is the methyl ester of 44 [79]. It is claimed that
these compounds are chemokine receptor antagonists and can be used to
treat HIV-1 infections.
126
44Ri = C02H;R2=OH
4 5 R i = OH; R2 = C02F
An isolate of Streptomyces lydicus gave lydicamycin (46), a metabolite

that showed activity against gram-positive bacteria, Bacillus subtilis
(MIC<L5 fig/ml) and Staphylococcus aureus (MIC 6.2 îg/ml), and the
yeast, Cryptococcus neoformans, but was inactive towards gram-negative
bacteria [80], The structure and relative configuration were determined
from NMR spectral studies. Given the c/^-fusion of the bicyclic ring, the
tetramic acid portion and the long chain substituents are anti, but the ab-
solute configuration is not known [81,82].
The next tetramic acid to be considered was isolated from lactic acid
bacteria and is unusual in that, while it includes an acetyl group at C-3, it
contains an a,P-unsaturated fatty acid as a substituent on N-1. Lactic acid
bacteria have long been used to make various dairy products, sauerkraut
or also sourdough for bread. Antibiotics from Lactobacillus protect
against infections of Salmonella and Helicobacter, a causative agent of
stomach ulcers [83]. One of these lactobacilli, L. reuteri, is a constituent
of the naturally occurring intestinal bacteria in humans and animals. In a
sourdough that is used in the production of a commercial baking product,
a strain of L. reuteri was found to have an inhibitory effect against gram-
127
positive bacteria [84]. The antibiotic, named reutericyclin (47), was ob-
tained from cell extracts and culture filtrate in a yield of -Img/L [85].
The gross structural features, presence of a tetramic acid and E-
decenoyl side chain, could be inferred from NMR studies. Methanolysis
(HCl/MeOH) of 47 and pentane extraction of the quenched reaction mix-
ture gave two compounds that were determined to be the methyl esters of
decenoic acid and N-(2-decenoyl)leucine. The nature of the 3-acyl tetra-
mic acid was deduced from the identification of 48 and 49 in the aqueous
portion of the methanolysis reaction mixture following treatment with
trifluoroacetic acid anhydride. The unusual C-C bond fragmentation un-
der acidic conditions, and the structure of the antibiotic was confirmed by
synthesis of racemic 47 [86]. The configuration at the lone chiral centre
was established as R by chiral GC. The carbon NMR spectrum of 47 indi-
cated an equilibrium between three tautomers in which the A^-pyrrolin-4-
one form is preferred (60%) and the two internal tautomers (50, 51) make
equal contributions (20% each).
k OCH3
47 49
i'^-^
50
ENOYLTETRAMIC ACIDS
Blasticidin (52) is an antibiotic isolated from Streptomyces griseochro-

mogenes in 1955 [87]. Detailed physicochemical properties were re-
ported in 1968, but the structure remained undefined [88]. Interest in this
metabolite was renewed recently with the isolation of the homologous
compounds aflastatin A and B (53, 54), metabolites of 5. griseochro-
128
mogenes that exibit the unusual property of inhibiting aflatoxin formation

by strains of Aspergillus [89].
HO, 9H0H
?H QH QH QH QH QH QH QH QH OH OH V ^ N ^ ^ ^ ^ H
OH ' ' ' OH OH OH
53 R = CH3
54R = H
HO. 9HQH
?HQH OH OH OH OHOH OH OH OH OH OHVA^^^QH
OHOH OH
Aflatoxins are a group of mycotoxins produced by some strains of As-

pergillus, e.g. A.flavusÂ. nomius.A. parasiticus, and A. tamarii. These
fungi do not always produce aflatoxins, but under certain environmental
conditions, high temperature and humidity, they infect crops such as pea-
nuts or com and produce the toxins which have been recognised as potent
carcinogens towards mammals and a risk factor of liver cancer in humans
[90]. Strains of Streptomycetes whose culture broths inhibited aflatoxin
production in A. parasiticus, but not the growth of the fungus, were un-
covered and investigated [91]. Of these, Streptomyces griseochro-
mogenes was found to produce an inhibitor in the mycelium that was ex-
tracted into methanol. Fractionation and chromatography yielded aflas-
tatin A (12 mg/100 ml) and B (0.14 mg/100 ml). The determination of
the structure of aflastatin A (53), C62Hji5N024, relied largely on NMR
analysis of the metabolite and degradation products. The ^^C-NMR of the
free acid contained broad signals due to the presence of tautomers. The
signals sharpened considerably in the spectrum of the diethylamine salt
that was formed in the last HPLC separation in which the amine was
added to the mobile phase. Fig. (5) illustrates the degradation sequences
that revealed the structural components of 53. Perhaps, the most chal-
lenging task was the determination of the configuration of the 29 stereo-
centres and one double bond. The strategy adopted is shown in Fig. (5).
129
stereochemistry
from optical rotation
1.Q3;Me2S
2. LiAIH4
OH
3. BzCN, EtgN
Nal04
1. Nal04
2. NaBH
QH
.OH
OH OH OH OH OH OH OH OH OH OH OH 0
?«V^ OH
1. Nal04
2. NaBH4
3. 3N HCI
4. BzCI
relative configuratfon by 5.O3
J-based method I.O3
2. NaBH4
3. AC2O
4. NaOMe OBz
BzO'
5.H^
comparison with I
authentic sample I
I OH OH OH OH OH OH OH OH OH OH
OH = • = • -" OH OH
relative configuration by
J-based method
Fig. (5). Degradation scheme to determine structure and stereochemistry of aflastatin A (53)
With the structures of aflastatin A (53) and B (N-demethyl aflastatin

A, 54) secured, the structure of the related blasticidin A (52) was deter-
mined in a similar manner. Studies on the biosynthesis of aflastatin A
130
have shown that C-7, 27,29,33 and 35 arise from glycolic acid whereas
all the others are as expected [6]. All the three compounds inhibit pro-
duction of aflatoxins in A. parasiticus at 0.5 jxg/nil [90,92,93]. Aflastatin
A inhibits the formation of norsolorinic acid, an anthraquinone precursor
of aflatoxin biosynthesis [94], and melanin production in Colletotrichum
lagenarium [95]. In both cases, indications are that inhibition occurs at an
early step of the biosynthesis. The effect of removing the tetramic acid
group has ben investigated. Thus, compound (55) and the corresponding
methyl glycoside were found to significantly reduce expression of genes
encoding enzymes involved in the aflatoxin pathway, and did not show
antifungal activity [96].
DIENOYLTETRAMIC ACIDS
A number of tetramic acids bearing a 1-oxopentadienyl substituent at C-3

have been discovered. The first of these was streptolygidin (56) isolated
from the actinomycete Streptomyces lydicus in 1955 [97]. This metabo-
lite showed strong antibiotic activity against gram-positive bacteria and is
a potent inhibitor of terminal DNA transferase and bacterial RNA po-
lymerase [98,99]. The dienoyl functionality at C-3 is crucial for its activ-
ity and the presence of 1 and 5-substituents, although not essential, does
improve activity [100]. A metabolite named afragilimycin was identified
as the sodium salt of streptolygidin [101].
c CONHMe
H
Elucidation of the structure, first assigned a molecular formula

C32H46N2O9 and later revised to C32H44N2O9 [97], was achieved by a com-
bination of classical degradation methods, spectroscopic methods and by
comparison of data from synthesised model subunits [97,102, 103]. The
structure of the 2,3,6-trideoxyaldohexose forming the N-glycoside was
determined by synthesis [104] thus establishing the absolute stereo-
131
chemistry of the glycoside. A complete stereochemical assignment was

achieved in conjunction with work carried out in defining the structure of
a related antibiotic, tirandamycin A (57) isolated from 5. tirandis [105].
The biological activity of 56 and 57 are similar [106,107], as is the mode
of action [108,109], although streptolydigin is the more potent.
OH o
Tirandamycin A (57) on periodate oxidation afforded tirandamycic

acid (58) which was converted to the corresponding p-bromo phenacyl
ester. X-ray cystallographic studies on this heavy atom derivative re-
vealed the structure and absolute stereochemistry of the dienoyl portion
of tirandamycin A [110]. Structural correlation between tirandamycic
acid and the corresponding acid (59) derived from streptolydigin was
achieved as shown below. The stereochemistry of the P-methylaspartic
acid portion of streptolydigin had been assigned the L-threo configura-
tion [102] and the configuration at the anomeric carbon of the N-
glycoside as p- from NMR studies.
COgH
CHgOH
S.flaveolusprovided tirandamycin B (60) which contains an extra hy-

droxy 1 group on the 2,9-dioxabicyclononane skeleton [111]. Tiranda-
lydigin (61) incorporates structural features of 56 and 57 and is also pro-
132
duced by a Streptomyces strain [112] and showed activity (MIC 0.5-32

mg/ml) towards a number of pathogenic anaerobes, streptococci, entero-
cocci and legionellae [113].
OH o
Two related antibiotics have been isolated from an unidentified

actinomycete strain [114,115]. The two metabolites, 62 and 63, differ
only in the presence or absence of the methyl group on the nitrogen in the
tetramic ring [116]. Although both are broad spectrum antibiotics with
activity against anaerobic and aerobic bacteria, the N-demethyl analogue
(63) is almost twice as potent [115]. The structures of these metabolites
rests on NMR spectral data and on X-ray crystal structure of the p-
bromphenacyl ester of the acid obtained from the acyl portion of the
compound [114]. The last representative of this group of antibiotics is
nocamycin II which was isolated from Norcardiopsis syringae [117-119].
This is the dihydro analogue of 63 in which the ketone in the dioxabicy-
clononane system is replaced by an a-hydroxyl. The structure originally
assigned to a congener, nocamycin I, was revised to 63 [118].
62R = CH3
63R=:H
POLYENOYLTETRAMIC ACIDS
Plasmodial slime molds (Myxomycetes) are eukaryotic bacteriovores

usually occurring in terrestrial ecosystems. Although a few species appear
to be confined to the tropics or subtropics, the majority are cosmopolitan
and approximately 1000 species have been recognised. In the assimilative
133
part of their life cycle, they form a free-living multinucleate, acellular

mobile mass of protoplasm (plasmodium) which feeds on living bacteria.
The Plasmodium undergoes sporulation to develop into a small fungus-
like fruiting bodies with unique structure and colour. Myxomycetes are
particularly abundant in temperate forests where they are found on the
bark of living trees,on leaf litter, soil, and dung of herbivorous animals.
The chemical nature and role of the yellow plasmoidal pigments of Phy-
sarum and Fuligo species have been studied because of their involvement
in phototaxis and induction of sporulation. Apart from their activity as
photoreceptors, these pigments may also protect vulnerable plasmodia
from microbial attack and be useful because of their metal chelating prop-
erties
The Plasmodia of the wild type of Physarum polycephalum are bright
yellow and produce an orange-red pigment that is also present in a white
mutant [120]. This compound, named physarorubinic acid (64), binds cal-
cium and other metals very well and this made acquisition of the ^H-NMR
spectrum difficult (line broadening) unless the sample was washed with
aq. EDTA solution. The structure contains a decapentaene system and
signals at 5 102.4 (C-3), 172.6 (C-7), 174.5 (C-2) and 194.0 (C-4) are
characteristic of C-3 acylated tetramic acid unit.
OH O
N
\
CH3
The stereochemistry was determined by comparison of CD data of the
decahydroderivative of compound (64) to that of the synthetic chiral
analogue (65). This established the 5-configuration at C-5. Interestingly,
fuligorubin (66), present as the yellow calcium salt in the aethelia of the
closely related myxomycete Fuligo septica [121], incorporates /?-
glutamic acid whereas P. polycephalum uses ^-serine. Leocarpus fragi-
lis, Plasmodia of which are found in autumn attached to dead conifer nee-
dles or grass, produces the group of tetramic acid pigments (67-70) that
have incorporated iS-tyrosine [122].
134
.CO2H
'OH H3C
CH.I3 65
^ 68 n = 3 O CH3 ^"
69 n = 4 70
P. polycephalum has an unusual life cycle. Young plasmodia live in-

side decaying trees and move away from light, whereas older plasmodia,
at the end of their growth phase, move towards the light and sporulate.
This behaviour is mediated by photoreceptors in the UV-A or blue light
range which contains maxima at X^^^ 350 nm and 460 nm (blue light). It
has been shown that light stimulates the formation of two orange-red me-
tabolites (0.003 and 0.005% yield) that can be extracted into chloroform
and show UV absorption at - 250 (In e 4.14) and 390 (In 8 4.74) [123].
The structures of these compounds, polycephalin C (71) and B (72), has
been secured.
HO 0 _
71 R = CH3
72R= H
A more elaborate C-3-polyenoyl side chain is presented by

erythroskyrine (73), a pigment first isolated in 1949 from Penicillium is-
landicum. It was reisolated in 1954, but the gross structure was elucidated
only in 1965 [124]. At that time, the only stereochemical point resolved
was the assignment of the 5-configuration at C-5, deduced from the for-
mation of L-N-methylvaline on degradation of the pigment with ozone
[125,126]. Detailed NMR studies of 73, including NOE measurements.
135
and analysis by the Mosher-Trost chiral ester (esterification of the C21

hydroxyl group with /?-and 5-0-methyl mandelic acid) indicated the /?-
configuration [127]. This has been conclusively confirmed by synthesis
[128]. Erythroslcyrine is considered to be a mycotoxin and is also active
against Staphylococcus species [129].
Oleficin (74), a polyenoyltetramic acid characterised by the presence
of the desoxy sugar D-digitoxose on the C-3 hexenoyl side chain, is an
antibiotic isolated from a strain related to Streptomyces parvulus [130].
The pentenoyl analogue, a-lipomycin (75) and the aglycone P-

lipomycin (76) are metabolites of 5. aureofaciens [131] which, together
with oleficin, inhibit gram-positive bacteria but are inactive to fungi
[131,132]. Oleficin exhibited an LD50 40 mg/kg (i.v.) in mice and was ef-
fective against Yashida s.c. sarcoma in mice [133]. Oleficin depleted
Mg^^ and Ca^"^ ions in isolated rat liver mitochondria and increases mem-
brane permeability [134] It also induced disintegration of the mitochon-
drial genome in Saccharomyces cerevisiae [135]. Altamycin (77), isolated
from a variety of Actinomyces pneumonicus, also belongs to this group
containing a tetraene system in the side chain [136].
OH
' OH
n^\ 74n = 5 I I I ^^^H

^ \ 75n = 4 O \
^"3 77 n = 3 76 ^^3
Trichoderma spp are a well known fungal group that produce a wide
range of metabolites with diverse activities [137]. The only tetramic acid
derivative isolated so far is harzianic acid (78) which was isolated from a
136
Strain of T. harzianum collected from a water sample at Hiroshima in Ja-

pan. The structure and the configuration of the double bonds were deter-
mined from spectral parameters [138]. The tetramic acid portion of
harzianic acid includes an unusual amino acid residue. Harzianic acid ex-
hibited only weak antibiotic activity against Pasteurella piscidida (MIC
12.5 |Lig/ml) and Proteus mirabilis (MIC 25 |ig/ml).
OH 0
The orange pigments aurantoside A and B have been isolated from the
marine sponge Theonella swinhoei [139]. The structures contain a di-
chlorohexaene chain and a glutamine-derived tetramic acid that is modi-
fied by an N-trisaccharide unit. The structure and absolute sterochemistry
of the trisaccharide group was established as D-xylo-D-arabino-D-arabino
by GC analysis of the hydrolysis product. The configuration at C4 was S
since L-aspartic acid was obtained on Lemieux oxidation (periodate/ per-
manganate) followed by hydrolysis. The original structure was revised to
include an ^-geometry of the terminal double bond [140]. The auranto-
sides were first isolated as the antifungal and cytotoxic constituents and
were later shown to inhibit binding of interleukin-6 to its receptors. Au-
rantoside C was obtained from a sponge, Homophymia conferta
(Theonellidae) (Phillipines) and was mildly toxic to brine shrimp [141].
Resonances for C-1 and C-2 were not observed in ^^C-NMR spectrum but
were detected in the hexylacetate derivative (5^ 174, C-1, and 5^ 100.3,
C-2). Aurantoside D, E and F from the sponge Siliquariaspongia japonica
(Theonellidae) were obtained by bioassay-guided fractionation [140]. Au-
rantoside F was four times more toxic (IC5Q 0.05 |ig/ml) than D or E (IC50
0.2 |xg/ml), whereas A and B did not show activity at 5 îg/ml against P-
388 murine leukemia cells. On the other hand, aurantoside F was inactive
towards C albicans and A. fumigatus, which were sensitive to the others,
in particular E (MIC 0.16 and 0.04 |ig/ml with inhibitory zones of 9.7 and
13.6 mm) [140].
137
The rubrosides are a group of eight related compounds, similar to the au-
rantosides, that have been obtained from Siliquariaspongia japonica
[141]. The methanol extract from the sponge exhibited significant activity
in the 3Y1 rat fibroblasts assay and bioassay-guided fractionation resulted
in the isolation of the metabolites, e.g. 85. Compared to the aurantosides,
OH
O' HO OH
H2N- OH
79 R = CH3 Aurantoside A O,, ^Q

Y)"CH3
80 R = H Aurontoside B
or V\ HO OH
84 Aurantoside F
•3^" 'OH
they present a slightly more elaborate polyene unit at C-3 that terminates
138
in a 4-chloro-2-methyltetrahydrofuran ring. The rubrosides induced nu-

merous intracellular vacuoles in 3Y1 rat fibroblasts at 0.5-1.0 fxg/ml.
Rubrosides A, C, D and E were cytotoxic against P388 murine lukemia
cell with IC50 0.046-0.21 fig/ml and were active against Aspergillus fumi-
gatus and Candida albicans.
H O
MACROCYCLIC LACTAMS
This section covers a growing group of metabolites that contain a tetramic

group, or a modified tetramic acid, embedded in a macrocyclic lactam.
The first example of this class was ikarugamycin (86) which was isolated
from the culture medium of Streptomyces phaechromogenes var ikaru-
ganensin. This metabolite showed strong antiprotozoal, in vitro amoebic,
and activity towards gram-positive bacteria [142]. The structure was de-
termined using classical oxidative degradation methods and NMR spec-
troscopy [143]. The amino acid incorporated into the tetramic acid ring
was shown to be L-ornithine which was obtained from 86 by acid hy-
drolysis. Ikarugamycin encapsulates sodium ions very strongly and can
sequester them from solution of siUca gel [144]. Capsimycin (87), a re-
lated compound produced by a Streptomyces strain, showed antifungal
activity against the phytopathogens Phytophthora capsicii and Pythium
debaryanum [145]. Its structure was deduced by NMR spectral compari-
son with ikarugamycin and was established by X-ray diffraction studies,
which also revealed the absolute stereochemistry.
A collection of the deep-sea sponge Discodermia dissoluta from Grand
Bahaman Island provided another example; discodermide (88) [146]. The
139
0 H^ \,.o.H ^^ Ikarugamycin
87 5,6-p-epoxy
structure and stereochemistry, with the exception of C-16 and C-17, were
assigned on the basis of spectral analysis. Discodermide exhibited cyto-
toxic (P388; IC50 0.3 [xg/ml) and antifungal activities (C albicans; MIC
12.5 jxg/ml). A number of related metabolites have been uncovered. Al-
teramide A (89) was isolated from the bacterium Altermonas sp. recov-
ered from the marine sponge Halichondria okadai [147]. An incompletely
characterised congener, the 25-desoxy analogue, alteramide B, is also
produced. Alteramide A shows cytotoxic activity to P388 (IC50 0.1
|Lig/ml), murine lymphoma LI210 (IC50 ^-^ ^g/iÔ and human epidermoid
carcinoma (IC50 5.0 îg/ml) cell lines. Interestingly, altemamide A on ex-
posure to light undergoes a [4+4]cycloaddition to yield a compound de-
void of theactivity associated with the parent compound.
Cylindramide (90) is a cytotoxic compound isolated from Halichon-
dria cylindrata (7.10'^% wet wt.), but very likely of bacterial origin
[148]. The structure was deduced from spectroscopic analysis, the relative
stereochemistry (only) of the bicyclo[3.3.0]octene unit by NOESY tech-
niques, and the absolute stereochemistry of the amino acid of the tetramic
acid ring (25,3S) from the recovery of erythtro-L-p-hydroxyornithine
from oxidative degradation of 90. It showed cytotoxicity to B16 mela-
noma cells (IC50 0.8 |Lig/ml).
Aburatubolactam A (91) and C (92) were isolated from the culture
broth of Streptomyces sp recovered from a mollusk. X-ray diffraction
studies established the structure of aburatubolactam A which was reported
to inhibit TPA-induced superoxide anion generation by human neutro-
phils [149]. Aburatubolactam C appears to be cytotoxic for various prolif-
erating tumor cells of human and murine origin (0.3-5.8 M'g/inl) via
apoptotic DNA fragmentation [150,151].
140
"OH
88 Discodermide 89 Aiteramide
Stenotrophomonas maltophilia (formerly Pseudomonas maltophilia),

isolated from the rhizosphere of rape plants (Brassica napus), yielded the
antifungal agent maltophilin, for which only the plane structure has been
described [152]. This compound inhibited the growth of various sapro-
phytic, human-pathogenic and phytopathogenic fungi, but was inactive
towards gram-positive and gram-negative bacteria. The corresponding
compound in which the cyclohexanone carbonyl is reduced to the alcohol
(dihydromaltophilin) cooccurs with maltophilin in an isolate of Strepto-
myces sp [153]
A Stenotrophomonas strain SB-K88, isolated from the fibrous root sur-
face of sugar beet cultivated in a field infested with Polymyxa betae, sup-
pressed rhizomania and seedling damping-off of sugar beet [154]. The
bacterium is therefore a member of the competitive rhizosphere micro-
flora.. Three fungitoxic metabolites, xanthobaccin A-C (93-95), were
produced by this organism grown in liquid cultures, and all inhibited the
growth of several fungal phytopathogens, particularly Pythium ultimum
(MIC 1 |Lig/ml) [155,156]. Although the plane structure assigned to xan-
thobaccin A (93) corresponds to that of maltophilin, more details have
been obtained for the former, including the configuration of the amino
acid group and relative configuration of the tricyclo[7.3.0.0^'^]dodecane
ring system [155]. It is useful to note that dihydromaltophilin may corre-
spond to xanthobaccin B [156]. Interestingly, the xanthobaccins and
maltophilin exhibit antifungal activity in vitro but no antibacterial activ-
ity. On the other hand, ikarugamycin (86) shows antibacterial activity
against gram-positive bacteria.
141
90 Cylindramlde ^ ^ O
OH
93 Xanthobaccin A
94 27-dihydro
9516-deoxy
An ethanol extract from Geodia sp., a marine sponge from the Great
Australian Bight, showed activity in inhibiting larval development of the
nematode Haemonchus contortus (LD99 14|Lig/ml) [16]. The active mate-
rial (LD991 |Lig/ml) was soluble in BuOH. Energy dispersive spectroscopy
(EDS) indicated the presence of magnesium and HR-ESIMS(-ve) and AA
spectroscopy was consistent with the formula [C27H3iN205]2Mg. The
structure of geodin A magnesium salt (96) was deduced from spectro-
scopic analyses, but the relative stereochemistry of the amino acid com-
ponent of the tetramic acid and the absolute stereochemistry remain to be
assigned [16]. Attempts to convert the salt to its conjugate acid with either
HCl or TEA. gave an unstable compound that could not be characterised.
Mg2^
96
J 2
142
N.ACYL.4.METHOXY-3.PYRROLIN.2-ONES
Compounds containing the 4-0-methyl ether of N-acylated tetramic acids

form a significant group of derivatives that, in the main, are found in ma-
rine organisms and, more specifically, are biosynthesised by marine cya-
nobacteria. The first example of this class was dysidin (97), isolated from
an Australian variety of the marine sponge Dysidea herbacea [157]. The
structure was determined by X-ray diffraction studies which also dis-
closed the 5, S absolute configuration of the two stereocentres [157].
Blue-green algae are frequently associated symbiotically with D. herba-
cea, and often constitute 50% of cellular material of the sponge [158].
Although terrestrial cyanobacteria are well-recognized producers of a
wide range of bioactive compounds, marine species have received less
attention until recently [159]. One of the most abundant and studied ma-
rine cyanobacteria is the pantropic Lyngbya majuscula (Oscillatoriaceae).
A prolific producer of metabolites, it has so far yielded more than 110
secondary metabolites including compounds that exhibit antiproliferative,
immunosuppressants, antifeedant and moUuscidal activities [159,160].
Shallow water varieties of the cyanophyte contain N-substituted amides
of 75'-methoxytetradec-4£'-enoic acid and of 75-methoxy-9-methylhexa-
dec-4£'-enoic acid called malyngamides, a sub-class of which contains the
4-methoxy-3-pyrrolin-2-one system [158].
The structure of the first example of this type, malyngamide A (98),
was determined by spectral and chemical means. Hydrolysis led to 4-
methoxy-A^-pyrrolin-2-one, which is also a component of the alga. Mild
acid hydrolysis yielded a P-ketoamide, indicating the presence of an
acyclic P-methoxyenamide, the E-configuration of the alkenyl chloride
functionality was determined from NOE measurements [161]. Isomalyn-
gamide A (99), from a Hawaiian sample of L. majuscula, differs in the
configuration of the chloromethylene group [162]. A number of malyn-
gamides have been identified more recently. A sample of L. majuscula
from Madagascar produces malyngamides Q and R (100,101) which dif-
fer from other types in that the pyrrolidinone ring is derived from glycine
instead of serine [159]. The S-configuration at C-4 in the pyrrolidone ring
was established by chiral GC-MS analysis of serine (as the pentafluoro-
propyl serine methyl ester) released on ozonolysis and acid hydrolysis.
143
CH3{CH2)5
^^ 0CH3
9CH3 n Y " l
100 R = H CH3(CH2)
101R = CH3 R I) OCH3
From another shallow-water variety of L majuscula found in Hawaii, a

new crystalline metabolite pukeleimide C (102) was isolated. The struc-
ture was secured by X-ray studies [163]. Chiroptical studies showed the
compound to be racemic and, probably, an artefact. A separate collection
of the cyanophyte did not contain pukeleimide C but, instead, yielded a
group of related metabolites, e.g. 103, whose structures were assigned
from spectral data [164]. Unfortunately, no information is available on the
bioactivity of these metabolites.
€H3
HaCa
102
Althiomycin (104) was isolated in 1957 from Streptomyces althioticus

[165] and showed a broad spectrum of activity towards gram-positive and
gram-negative bacteria through inhibition of protein synthesis at the pep-
tidy Itransf erase stage (cf tenuazonic acid) [166]. It has been characterised
as an agent with low toxicity and good selectivity towards prokaryotes.
Althiomycin has also been isolated from Streptomyces matensis, Cysto-
144
bacter fuscus, Myxococcus xanthus and strains of M. virescens, gram-

negative bacteria usually found in soil [167]. The structure of 104 was
determined by a combination of spectroscopic methods, partial synthesis
and X-ray crystallographic studies [168,169]. X-ray studies yielded the 5-
configuration of the stereogenic centre in the thiazoline ring [169]. The
remaining stereocentre could not be determined since althiomycin is al-
ways isolated as a mixture of diastereomers with both R and 5-
configurations at this carbon. Synthetic approaches to althiomycin and
analogues and biological studies of the interaction with prokaryotic
ribosomes have been undertaken [170].
104
The terrestrial cyanophyte Scytonema mirabile yielded mirabimide E

(105) which possesses an unusual tetrachlorinated ethylene group [171].
Detailed spectral analysis, including NMR analysis of a sample of 105
that had been uniformly enriched in ^^C and ^^N yielded the gross struc-
ture. Lemieux degradation, prior to acid hydrolysis, afforded alanine
which was shown to have the 5-configuration by chiral GC-MS of the
isopropyl ester of N-trifluoroacetylalanine. Reaction of 105 with nitrosyl-
sulfuric acid followed by methanolysis yielded the p-ketol (106) which
afforded the methyl ester (107) on treatment with magnesium methoxide.
CH3 107
The stereochemistry of this compound was determined by the Mosher

method which, together with the fact that J2.3 = 7 Hz is consistent with an
145
anti-airangement of the P-hydroxy-a-methyl group, indicated the 2J?,

3J?-stereochemistry. Mirabimide E possesses selective toxicity on solid
tumours. In contrast, the congeneric mirabimide A-D, e.g. 108, are
moderately cytotoxic but did not exhibit selective cytotoxicity [172].
OCH3
The soft-bodied and slow-moving sea hare Dolabella auricularia pro-

duces potent defence compounds. This nudibranch has been recognised
since 60 AD and its toxic extract has been used by some, particularly in
Roman times, to dispatch political rivals [173]. Of the many metabolites
produced by this organism, a group of remarkable cytotoxic peptides
known as the dolastatins have attracted considerable attention. Of these,
dolastatin 10 and 15 (109) are endowed with potent antiproliferative ac-
tivity with dolastatin 10 having the greater activity. These compounds are
present in extremely small quantities; from 1600 kg of £>. auricularia
28.7 mg (1.8.10-6%) of dolastatin 10 and 6.2 mg (3.9.10-7%) of dolas-
tatin 15 were obtained [174]. The exceedingly low yields of dolastatins
recovered from D. auricularia have suggested that the moUusk is not the
real producer of these compounds [175]. Moreover, some dolastatins and
analogues have been found in certain strains of Lyngbya majuscula and
assemblages of L. majuscula and Schizothrix calcicola, implying that at
least some metabolites isolated from D, auricularia, a generalist herbi-
vore, have a cyanobacterial origin [175].
Dolastatin 15 is a lipophilic pentapeptide esterified with N-acylpyrroli-
done containing seven stereogenic centres all with iS-configuration. The
structural determination was achieved primarily from NMR studies and
comparison with analogues, and the absolute configuration was estab-
lished from X-ray analysis and by synthesis [176]. Dolastatin 15 is also
highly cytostatic affecting dividing cells but does not interfere with the
146
viability of resting cells.and shows low toxicity to proliferating nonleu-

kemic cells.
Sorangium cellulosum is an ubiquitous soil bacterium that belongs to
the order Myxococcales. It has the ability to glide over solid surfaces, to
live in a biofilm and form fruiting bodies. Members of this taxon are a
particularly rich source of metabolites with remarkable biological activi-
ties [177]. From one strain, another example of N-acyl-4-methoxy-3-
pyrrolin-2-ones has been isolated. Eliamid (110) has been claimed to have
cytostatic, nematocidal and fungicidal activities [178].
109
110
O CH3
MODIFIED TETRAMIC ACIDS
There are several examples of metabolites where the tetramic acid domain
is teasingly disguised. In these cases, it is difficult to be definitive about
the nature of the apparent modification unless this is substantiated by bio-
synthetic studies. The case of lactacystin (5), Fig. (2), has already been
considered. Another example is presented by the oxazolomycin group of
antibiotics, e.g. (Ill), found in strains of Streptomyces [179]. Biosyn-
thetic studies indicate that the carboxylic acid of the amino acid required
to form tetramic acids contributes to the formation of the P-lactone [180].
In this section, metabolites that probably have a tetramic acid origin are
presented.
The neurotoxic lipophilic tripeptide, janolusimide (112), was isolated
from the Mediterranean nudibranch Janolus cristatus. The structure was
determined by spectroscopic and classical chemical methods [181] and
confirmed by synthesis [182]. It has been suggested that the pyrrolidine-
2,4-dione may arise from condensation of valine and isobutyrate [181].
147
N-CH3
Integramycin (113) is a novel hexacyclic acid isolated from the culture

broth of an Actinoplanes sp. It was detected from its activity (IC50 ^ M^^)
in an in vitro assay to evaluate inhibition of HIV-integrase (strand trans-
fer) [183]. The structure and relative stereochemistry were deduced from
spectral data. In methanol, an epimeric mixture of the C-2 methoxy
ethers are formed. Acetylation with acetic anhydride/pyridine generated a
separable 1:1 mixture of the C-2 epimeric pyridinium tetracetates (114).
Interestingly, both epimers maintained activity in the coupled assay (IC5Q
6 jxM), but were much less active in the strand transfer assays (IC50 30-46
|LlM).
A 3-deoxy tetramic acid heterocyclic system is contained in oteromy-
cin (115), a metabolite produced by an unidentified fungus (possibly an
Ascomycete). Oteromycin was uncovered using a bioassay to identify
antagonists of the endothelin receptor subtype B that is implicated in both
vasoconstriction and vasodilation [184]. Oteromycin showed IC5Q 2.5 |LIM
but had little effect in the angiotensin II receptor binding assay (IC50 >25
|LiM). The structure and relative stereochemistry rests on detailed spectral
analysis.
The obligate marine fungus Ascochyta salicorniae, obtained from the
marine alga Ulva sp. from the North sea off Germany, is considered to be
an algal endophyte. The fungus was mass cultivated on a solid medium,
the ethyl acetate extract of which showed antimicrobial activity [185].
148
Ascosalipyrrolidinone A and B (116,117) were isolated and their struc-

tures determined by spectroscopic techniques. The two metabolites differ
N—H
114 115
only in the substituent at C21; butoxy or methoxy. Given that during the
isolation n-BuOH and MeOH were used, the possibility that these sub-
stituents are artefactual cannot be discounted. Ascosalipyrrolidinone A
exhibited antibacterial activity against Bacillus megaterium, and the fungi
Mycotypha microsporum and Mycobotrium microsporum at 50 |ig/disk.
Moderate activity towards chloroquine-resistant Plasmodium falciparum
(IC50 736 ng/ml) and significant activity towards Trypanosoma cruzi and
r. brucei subsp. Rhodesiense. The corresponding analogue (118) with ty-
rosine as the contributed amino acid has been isolated as an inhibitor of
platelet-activating factor acetyltransferase from Penicillium rubrum [186].
116R=(CH2)^H3
117R = CI^ H3C CH3 118
CH3
A diastereoisomer of this compound, its 5-dehydro derivative (119),

and two artifacts, 120 and the 4,5-acetonide, have been isolated from the
149
ascomata of Talaromyces convolutus [187]. This fungus produces yellow

pigments on the ascomata that contain azaphilones, pre-anthraquinones
and the modified tetramic acids. Compounds 118 and its diastereoisomer
inhibited growth of the filamentous pathogenic fungi Aspergillus fumi-
gatus, A. niger and Candida albicans, with an activity comparable to that
of the known antifungal agent amphotericin B.
HOHQ
Pramanicin (121) was isolated as the antifungal agent from a fungus of

the genus Stagonospora [188]. It exhibited modest antifungal activity
against a range of microbes including the human opportunistic pathogen
Cryptococcus neoformans, the agent responsible for cryptococcal menin-
gitis. It showed an effect on vascular endothelial cells and may act by in-
creasing calcium permeability in cells [189]. The structure and relative
configuration were determined by spectroscopic analysis [188]. In later
work, pramanicin (121) was found to co-occur with the diene precursor
(122) [190].
\
OH 121
\ 122
OH
150
In perhaps the most thorough study of its type, the biosynthesis of

pramanicin has recently been investigated [190]. A series of experiments
using acetate, malonate and serine precursors, labelled with ^H, ^^C, ^^N
and ^Ô revealed the following set of events. The hydrophobic tail arises
from a starter acetate unit that is extended with six malonate units. The
possibility of malonate acting as a starter unit was eliminated by the ob-
servation that incorporation of diethyl[2-^^C]malonate, which acts as a
convenient source of malonyl CoA by hydrolysis and thioesterification,
labels the extender units in preference to the stater unit by a factor of two.
Moreover the incorporation of [1-^^C, ^H3]acetate occurs with significant
retention of three deuterium atoms. A decanoyl chain is elaborated by a
fatty acid synthase (FAS), but the transfer of this onto a polyketide syn-
thase could not be demonstrated. Attempts to show the incorporation of
decanoyl N-acetylcysteamine failed. In any event, the decanoyl interme-
diate is further extended by addition of two intact acetate derived
malonate units, the extension probably being directed in a PKS-like man-
ner.
NHAc
124
The formation of the pyrrolidone group involves condensation of an
acetate with L-serine. Since a significant proportion of L-[1,2,3-^^C,
^^N]serine was incorporated into pramanicin, the carbon skeleton of ser-
ine is incorporated intact. Acylation with the 14-carbon moiety then en-
sues leading to the conjugated dieneone tetramic acid intermediate 122.
In fact, this compound co-occurs with 121 and, interestingly, is almost
exclusively produced when 123 and 124 are used as precursors. The re-
maining steps involve formation of the trans-diol at C-3 and C-4 and ep-
oxidation of the terminal alkene in the dienone chain.
Vancoresmycin (125) is a metabolite extacted from the mycelium of an
actinomycete strain of the genus Amycolatopsin sp. It exhibited potent
activity against gram-positive bacteria, including vancomycin-resistant
strains such as Enterococcus spp. [191], but was inactive towards gram-
negative bacteria and showed no fungal activity. The plane structure was
derived from MS and NMR studies which also revealed the stereochem-
151
istry of the 4 trisubstituted double bonds. The configurations of the 26

stereocenters remain to be determined [191].
OH OH
HaC-N
HoC
The pseurotins are a class of metabolites that are structurally charac-

terised by an l-oxa-7-aza-spiro[4.4]non-2-ene-4,6-dione skeleton. The
first compounds of this type, e.g. 126, were isolated from Pseudeurotium
ovalis [192,193], but were also obtained from Cordyceps sophioglossoU
des [194], Diheterospora chlamydosporia [195] and Aspergillus flavus
[196]. The pseurotins have been shown to exhibit diverse activities, in-
cluding inhibition of chitin synthase [196], neuritogenic activity by in-
ducing cell differentiation [197] and amorphine antagonism [198]. The
biosynthesis of pseurotin A has been studied in some detail [199]. In
summary, a propanoyl starter unit is condensed in four successive cycles
with the malonyl extender unit. The resulting pentalcetide (127) combines
with phenylalanine, probably first with formation of the amide (128) and
then with closure of the lactam ring to form the tetramic acid (129) [199].
152
QH O
O 127
129 O O 128
CONCLUDING REMARKS
The structures of over 120 naturally occurring compounds recognisable as

tetramic acid derivatives have been described in the literature so far.
Comparable numbers have been derived from fungi (35) and bacteria
(34). A similar number have been isolated from cyanobacteria and/or ma-
rine sponges. These can be grouped together since, as pointed out previ-
ously, marine microorganisms are frequently involved in the production
of metabolites isolated from marine macroorganisms.
A substantial number of the metabolites isolated still require resolution
of stereochemical ambiguities, particularly those examples in which the
C-3 acyl groups contain multiple stereogenic centres. A point of interest
relates to the isolation of tetramic acid metabolites as salts and whether or
not these salts represent the 'natural' form of the metabolites.
The role of the tetramic acid group in determining the bioactivity of
the metabolites is another question of significant interest. In this respect,
it might be important to distinguish between an in vivo role and an in vitro
effect. The natural function of the tetramic acid group, particularly as the
salt, might be related to the translocation of metal ions or transport of the
molecule. Metal ions such as Mg (II) can act as transmembrane transports
and influence the fluidity and permeability of membranes [200]. On the
other hand, in some bioassays it might not matter whether the compound
is present as the salt or as the conjugate acid. The nature of the bioassay
might be such that transport of the metabolite is not a problem.
The function of the often extravagant acyl substituent is also puzzling.
The activity of the ancorinosides, e.g. (27), as membrane type 1 matrix
metalloproteinase inhibitors does not seem to depend on the liposaccha-
153
ride substituent at C-3. In fact, tenuazonic acid which does not have fiinc-
tionality apart from the 3-acyltetramic acid is considerably more active
[57]. Some of the questions that will require an answer are illustrated by a
few observations on the activity of blasticidin (52) and the aflastatins (53,
54). Removal of the tetramic acid group did not eliminate the inhibitory
effect on aflatoxin production by Aspergillus flavus or render the modi-
fied blasticidins toxic to the fungus [96]. A fragment of aflastatin A (53),
containing the tetramic acid portion and an eight carbon chain at C-3,
showed no inhibitory activity on production of the toxins [92]. It is clear
that tetramic acid metabolites, because of their intricate structures and di-
verse bioactivity, will continue to attract attention for some time yet.
154
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CHEMISTRY AND BIOLOGICALACnVITIES OF GINKGO BILOBA
K. SASAKI, K. WADA and M HAGA
Department of Hygienic Chemistry, Faculty of Pharmaceutical Sciences, Health Sciences

University of Hokkaido, Ishikari-Tobetsu, Hokkaido 061-0293, Japan,
ABSTRACT: Ginkgo biloba L is the solo member of the Ginkgoaceae famfly, and its leaf
extract (EGb) has been clinically used for the treatment of cerd)ral insufficiency. Several
experimental studies have demonstrated that EGb has neuroprotective properties against various
age-related physiological dianges and cerebral injuries induced by ischemia, edema and
apoptosis. The major bioactive prindples of EGb arc recognized to be flavonoids and
terpenoids, ginkgolides and bilobalide. Pharmacological actions of EGb and its constituents
indude platelet-activating factor antagonism, anti-oxidant and free radical scavenging properties,
as well as modulation of neurotransmission, cerebral glucose and lipid metabolism and cerebral
membrane fluidity. These effects are involved in the mechanism related to the protective effect
of the extract in the central nervous system.
INTRODUCTION
Ginkgo biloba L , a dioecious plant, is called a living fossil, because it is the single extant
spedes of the family Ginkgoaceae, which made its appearance in the Permian age [1]. Thus
far, a great variety of compounds have been isolated from the leaves, seeds and root bark of
Ginkgo biloba, induding alcohols, aldehydes, ketones, steroids, catechines, flavonoids,
terpenes and organic adds [2, 3]. After the year 1965, for instance, a standardized Gingko
biloba leaf extract (EGb) termed EGb761 has been used chiefly in Europe for the treatment of
peripheral vascular disease or cerebral insuffidency induding difficulties of concentration and of
memory, absent mindedness, confusion, lade of energy, tiredness, decreased physical
performance, depressive mood, anxiety, dizziness, tinnitus, and headache [4, 5].
Furthermore, in a placebo-controlled, double-blind randomized trial, it has been suggested that
EGb761 is capable of stabilizing and, in a substantial number of cases, improving the cognitive
performance and the social functioning of demented patients tested using the Alzheimer's
Disease Assessment Scale-Cognitive subscale (ADAS-Cog) and Geriatric Evaluation by
Relative's Rating Instrument (GERRI) [6, 7]. A more extensive discussion on dinical trials
will be found in the book by van Beek [8], and reviews [9, 10]. Despite the accumulation of
considerable evidence in recent years to support the clinical efficacy of EGb [1, 8, 11], the
predse medianisms of action of EGb have not yet been fully eluddated; moreover, there is only
partial agreement concerning which constituents of EGb are involved in its mechanism of the
action in the central nervous system (CNS). Conceivably, however, benefidal effects of EGb
could be explained, at least in part, by its neuroprotective and/or neurotrophic properties against
various symptoms induced by impaired brain functions in advanced age [12].
EGb761 is made up of 24% flavonoid glycosides and 6% terpenes, which are accepted as
active prindples. Many undesirable constituents of Ginkgo biloba leaves (e.g., ginkgoUc add
due to its allergic action) are eliminated by the extraction procedures used to prepare the extract
166
The extraction prooedures and oomponents present in £Gb761 are described in detail elsewhere
[11, 13].
In this article, we present the principal constituents isolated from leaves and seeds of
Ginkgo biloba, and deal with the pharmacological properties of standardized EGb and its
constituents mainly in CNS functions.
CONSTITUENTS OF GINKGO BILOBA L. A N D ANALYTICAL METHODS
Tlie represoitative unique constitu^ts so far reported for Ginkgo biloba L. are stated briefly
below. Adetailed description of this field has been presented in the book of van Beek [8].
Flavonoids
Flavonoids are major constituents of Gingko biloba leaves, and present as biflavones, flavones,
flavonols and associated glycosides (Table 1). Biflavones, in particular, are characteristic
constituents of Ginkgo biloba. They are of the amentoflavone (1) type, and differ from each
other in the number and position of the methoxy groups. Bilobetin (2), ginkgetin (3) and
isoginkgetin (4) were diaracterized by Baker et d, [14] and Nakazawa et al. [15]. Moreover,
sdadopitysin (5) was isolated by Miura et d, [16] and 5' -methoxybilobetin (6) was isolated by
Joly et d, [17]. Briangon-Scheid et d, [18] rqwrted a normal-phase high-performance liquid
chromatography (HPLC) system for the identiHcation and the quantitative determination of these
biflavones by a two-step gradient of isocratic elution. Furthermore, they also showed the
presence of amentoflavone. Pietta et d, [19] reported a reversed-phase HPLC system using
tetrahydrofuran-l-piopanol-water as the eluent for the analysis of biflavones.
No. Ri R2 R3 R4
(1) OH OH OH H
(2) OMe OH OH H
(3) OMe OMe OH H
(4) OMe OH OMe H
(5) OMe OMe OMe H
(6) OMe OH OH OMe
^mmmmmmmmmmmmmmmmmammammmmmmmmmd
167
Table 1. Flavonoids of Ginkgo biloba Leave
No. Compound
Biflavones
(1) Amcntoflavone
(2) Bilobetin
(3) Ginkgetin
(4) Isoginkgetin
(5) Sciadopitysin
(6) 5'-Methoxybilobetin
Flavonois
(7) Kaempferol
(8) Quercetin
(9) Isorhamnetin
(10) Myricetin
(11) Kaempferol-3-O-glucoside
(12) Kaempferol-7-O-glucoside
(13) Kaempferol-3-O-retinoside
(14) Quercetm-3-O-glucoside
(15) Quercetin-3-O-rhamnoside
(16) Quercetm-3-O-rutinoside (Rutin)
(17) Isorhamnetw-3-O-glucoside
(18) Isorhamnetin-3-O-rutinoside
(19) Myricetin-3-O-rutinoside
(20) 3'-0-methylmyricetin-3-0-rutinoside
(21) Kaenipferol-3-0-(2"-0-glucosyl)rhaninoside
(22) Quercetin-3-0-(2"-0-glucosyl)rhamnoside
(23) Kaempferol-3-0-[6'"-0-{p-(7""-0-giucosyl)coumaroyl}-2"-0-glua)syl]rhamnoside
(24) Quercetin-3-0-[6"'-0-{p-(7""-0-glucosyl)coumaroyl}-2''-0-glucosyl]rhamnosi^^
(25) Quercetin-3-0-(6"'-0-/7-coumaroyl-2"-0-glucosyl)rhamnosyl-7-0-glucoside
(26) Kaempferol-3-0-(6"'-0-/?-coumaroyl-2"-0-glucosyl)rhaninoside
(27) Quercetin-3-0-(6"'-0-/>-coumaroyl-2"-0-glucosyl)rhamnoside
(28) Kaempferol-3-0-[ a -rhamnosyl-(l-»-2)- a -rhaninosyl-(l--^6)]- P -glucoside
(29) Quercctin-3-(9-[ a -rhamnosyl-(l-»-2)- a -rhamnosyl-(l->6)]- j8 -glucoside
Flavones
(30) Apigenin
(31) Delphidenon
(32) Luteolin
(33) Apigenin-7-O-glucoside
(34) Luteolin-3'-0-glucx)side
168
._
c—
No. Rl R2 Fl3 R4
R3
(7) H H H H
ÔH (8) H H OH H
1 \ (9) H H OMe H
RaO^
r^V"°^
T )^} '^R4 •
(10)
(11)
H
glucose
H
H
OH
H
OH
H
il^^JLj
(12) H glucose H H
ORi (13) rutinose H H H
OH 0 (14) glucose H OH H
(15) rhamnose H OH H
(16) rutinose H OH H
(17) glucose H OMe H
(18) rutinose H OMe H
(19) rutinose H OH OH
(20) rutinose H OMe OH
\,„^ ^^^^û
•••••1
V
Ri
f^^^*"
^^0^^'^^^^Y^^
>^^^^^>r^ No. Ri 1
OH 0 J (21) H 1
H^CT—0 ^ (22) OH 1
OH
mmmmmS
More than 20 flavonol glycosides have been isolated from ginkgo leaves. The major
aglycones of ginkgo flavonoid glycosides are kaempferol (7), quercetin (8) and isorhamnetin
(9), and the minor one is myricetin (10). They were determined by reversed-phase HPLC and
diode array detection after hydrolysis of flavonol glycosides [20]. Two new coumaric esters
of flavonol diglyoosides were isolated by Nasr et d. [21, 22]. Their structures were
determined from the spectrum data to be kaempferol/quercetin-3-0-(6"' -0-/7-coumaroyl-2"-0
glucosyl)rhamnoside (26 and 27). Moreover, Hasler et d, [23] isolated new flavonol
diglycosides from the leaves of Ginkgo biloba and determined their structures to be
kaempferol/quercetin-3-0-(2"-Oglucosyl)rhaninoside (21 and 22), kaempferol/quercetin-3-0-
[6"'-0{p-(7""-glucosyl)coumaroyl}-2"-0-glucosyl]rhamnoside(23 and 24), and quercetin-3-
0-(6'"-0-/?-coumaroyl-2"-0-glucosyl)rhanmosyl-7-Ogluooside (25). A procedure that
combined counter-current chromatography (CCC) and HPLC was developed for the isolation
and the purification of flavonol glycosides, and two novel flavonol triglyoosides (28 and 29)
169
were isolated [24]. Fiavones (30-34) also can be identified in ginkgo leaves [25]. The
methods for the analysis of flavonoid glycosides using micellar electrokinetic capillary
chromatography [26], HPLC with diode-array UV detection [27] and thermospray liquid
chromatography mass spectrometry [28] were developed. Lobstein et al, separated flavonoids
using gradient elution with acetonitrile and O.IN phosphoric add within 50 min [29]. Hasler
et d, [25] described fingerprint HPLC separation of Ginkgo biloba, and 33 flavonoids were
assigned. Recently, a multidimensional counter-current diromatography system for the
preparative isolation of isorhamnetin, kaempferol and quercetin from crude extract was
described by Yang et d. [30]. Flavonoid metabolites after oral administration of EGb to rats
and humans were analyzed by reversed-phase liquid diromatography-<iiode array detection, and
the results indicated that more extensive metabolism of flavonoids takes place in humans than in
rats [31, 32].
R2O OR3
OH o J
HaC-Ô-V
No. R1 R2 R3
OH
(23) H H glucose
(24) OH H glucose
(25) OH glucose H
(26) H H H
(27) OH H H
OH ,CH3
OH O I OH
OH No. Ri
(28) H
OH-^^^^^^-V (29) OH
HO I
OH
170
No. Ri R2 R3
(30) H OH OH
RiO (31) H H H
(32) H OH H
(33) glucose H H
(34) H OH glucose
Terpenes
Ginkgolides, the bitter principles of Ginkgo biloba U were isolated from the leaves by
Furukawa [33] for the first time and their chemical structures deteraiined by Maruyama et al.
[34] and Nakanishi et d. [35]. The five known ginkgolides, ginkgolides A (35), B (36), C
(37), J (38) and M (39) have a cage-like molecule structure with six five-membered rings and a
tert'hwtyl group, and differ in the positk)n and number of substituted hydroxyl groups on the
spirononane framework. Ginkgolide M is isolated only from the root of the gingko tree. The
diemistry and pharmacological properties of ginkgolides were reviewed by Braquet et al. [36].
From molecular electrostatic potential (MEP) and molecular lipophilidty potential (MLP)
studies, ginkgolides, are considered to be two-pole molecules. Negative electrostatic MEP
C(CH3)3
O' -0-
CH3 R2 0-^0
(40) Bilobalide
Ri R2 R3
(35) Ginkgolide A H OH H
(36) Gini<golide B H OH OH
(37) Ginkgolide C OH OH OH
(38) Ginkgolide J OH OH H
(39) Ginkgolide M OH H OH
171
areas are generated around the lactonic cycles. Tliree MEP areas are present in ginkgolldes B,
Q and M, while in ginkgolides A and J, diere are two NfEP areas due to the fusion of the
negative MEP area as the cage is relatively dosed. Furthermore^ the existence of a marked
hydrophobic zone surrounding the tert-hutyl lqx)philic moiety indicated diat it might lodge in a
hydrophobic pocket of the membrane. Furthermore, Corey et d. [37, 38] carried out the total
syndiesis of ginkgolides. Further experiments demonstrated that ginkgolide M can be
syndiesized from ginkgolide C [39]. Extoisive and oonq)lete NMR analysis has been
performed [40].
Bitobalkie (40) was isolated from the leaves by Wsinges et d. [41], and is a sesquiterpene
lactone, whidi has a tert-bMtyl group and two hydroxyl groups in its chomcal structure [42].
Tlie ginkgo terpenes described above (bilobalide and ginkgolides) seem to be unique
constituents to Ginkgo biloha because they have nev^ been found in any other plants.
GinkgoUdes A, B, C and bilobalide have b e ^ analyzed by reversed-phase HPLC with dther
UVdetectwn at 220 nm [43, 44] or refractive index detection [45]. Furthermore, bilobalide
and ginkgohdes A and B were also analyzed by axillary dectrophoresis with U V detection at
1S5 nriL Reasonable separation was accomplished by a phosphate and sodium dodecyl sulfate
buffer [46]. Chauret et d. [47] developed an analytical method coupling a gas chromatograph
to a high-resolution mass spectrometo: operated in die selected-ion monitoring mode, and
confirmed the production of ginkgolides in Gingko biloba cells cultured in vitro. Recently, a
method based on liquid chromatography coupled with dectrospray mass spectrometry was
developed for the analysis of terpenes in EGb [48].
Total and individual terpene (ginkgoHdes A, B, C, J and bilobalide) contents wore evaluated
in leaves, shoots and roots of a young Ginkgo biloba (three years old) cultivated in a
greenhouse in natural light [49]. Tlie leaves accumulate more terpenes than the roots and
shoots. The contents of ginkgolide A and bilobalide readi a maximum value at the end of
summer or at the begiiming of autumn. Recently Carrier et d, [50] detomined terpene contents
in leaves of the terminal buds, rosettes and side branches, stem and bark, root and root meristem
of the three-year-old Ginkgo biloba. Bilobalide was absent from underground parts, whereas
it was the major constituent in aerial parts. Ginkgolide A occurred at the highest concentration,
followed by relatively equal amounts of ginkgolides B and C, and a small amount of ginkgolide
J. Laurain et d. [51] found that production of ginkgolides A, B, Q and J and bilobalide
occurred in two cell cultures derived from a female prothallus and from putativdy transformed
embryos, by transfection wiaiAgrobacterium rhizogenes agropine type strain CFBP2409 (A4)
Other Constituents
Ibataer d. [52] isolated long-chain betulaprôl-typepolyprenols containing 14 to 22 isoprene

units (41) from the ginkgo leaves and determined their structures by mass spectroscopy, ^H-
,. ^
r >
"-^ ^^ OH
n
0)-terminal trans trans cis
a-terminal
(41) n=10-18
172
NMR and ^^C-NMR. The concentrations of these polypienols increased from 0.04 to 2.0% of
dry weight with maturation of the leaves. A supercritical fluid chromatographic procedure for
the quantitation of polyprenols in the ginkgo leaves was developed by Huh et d, [53].
6-Hydroxykynurenic add was isolated from ginkgo leaves; the first time it was found in a
gymnosperm. The content reached a maximum value of 0.24% in autumn [54].
To darify the taxonomical dassification, Kraus et d. [55] isolated the water-soluble
polysaccharides from Ginkgo biloba leaves. The polysacdiaride mixture could be separated
into GFl (MW 23,000), GF2a (MW 500,000), GF2b (MW 24,000) and GF3 (MW 40,000).
GFl and GF3 are mainly composed of arabinose and galacturonic add, respectively, and they
are common polysacdiarides of higher plants. On the otiier hand, GE2a and GF2b are
composed of large amounts of mannose, rhamnose and glucuromc add, and seem to be unique
to Ginkgo biloba.
Seven k)ng-diain phenols (42a-g) were isolated from the sarootesta of ginkgo seeds, and
showed antitumor activity against sarcoma 180 asdtes in mice [56]. They indude anacardic
add, bilobol and cardanol, whidi are major allergemc substances of the ginkgo sarcotesta. In
additk)n, the structure-activity relationship for antitumor activity against Qiinese hamster V-79
was investigated [57]. A specific method for the quantitative analysis of ginkgolic adds in the
leaves and seeds of Ginkgo biloba using the HPLC-electrospray ionization-mass spectrometry
technique was developed [58].
Following removal of the outer fleshy layer and roasting or boiling, the albumen of ginkgo
seeds is used as a food in Asia. 4-OMethylpyridoxine (MPN) (43), whidi has been known to
have antivitamin Be activity was isolated from the seed of Ginkgo biloba (the Jsânese word:
gin-nan) as the toxic prindple of gin-nan food poisoning [59]. Synthetic MPN is known to
induce oonvulsk)ns through a reduction of tiie brain GABAlevels in humans and in a variety of
experimental animals. Furthermore, MPN is present in not only the ginkgo seed but also in its
leaves. However, the amount of MPN in the extract of ginkgo leaves is much lower than the
concentration that causes the detrimental effects [60]. Fiehe et d. demonstrated biosynthesis of
MPN in cell-suspension cultures of Ginkgo biloba [61]. Recentiy, Wada et d, wrote a review
of the literature concerning the relationship between gin-nan food poisoning and MPN [62, 63].
Kimura et d, [64] purified and diaracterized a 30 kDa Ginkgo glycoprotein from ginkgo
seeds, using an antiserum againstj81-*2 xylose-containiag //-glycans.
Ri R2 R3 1
R2
^RiJ; Ginkgoic acid (CH2)12CH3 COOH H 1
HO-1 (CH2)7CH=CH(CH2)5CH3 GOGH H 1
KJ c. (CH2)9CH=CH(CH2)5CH3 GOGH
H 1
TR3 d.
e.
Bilobol (CH2)7CH=CH(CH2)5CH3
(CH2)9CH=CH(CH2)5CH3
H
H
GH
GH
1
1
f. Ginkgol (CH2)7CH=CH{CH2)5CH3 H H 1
(42) g. (CH2)9GH=CH(CH2)5CH3 H
H 1
173
CH20CH3
.CH20H
H 3 C ^ N ^
(43) 4-0-Methylpyridoxine
NEUROPROTECTIVE PROPERTIES OF EGB AND ITS CONSTITUENTS ON

THE BRAIN
There is oonvindng evidence that EGb and its constituents have neuroprotective actions under
experimental conditions sudi as hypoxia/isdiemia, seizure activity and nerve damage.
Cerebral Hypoxia and Cerebral Ischemia
With regard to beneficial effects of EGb in situations such as hypoxia and isdicmia, Karcher et
d. [65] reported that the survival time induced by hypobaric hypoxia was gready prolonged in
rats treated with EGb (100 mg/kg, Lp.) before 30 min hypoxia. It was shown that the non-
flavonefi-actionof EGb was responsible for the prolonged effects on the survival time of mice
under lethal hypoxia [66]. EGb also retards the breakdown of the brain energy metabolism in
hypoxic artificially ventilated rats [66]. Spinncwyn [67], using a geibil model of bilateral
forebrain ischemia induced by occluding the conmion carotid arteries, showed that pretreatmait
with EGb (30 and 60 mg/kg/day, p.o., for 14 days) produced an increase in the area of
surviving hippocampal CAl neurons. Furthermore, the treatment with EGb reduced ischemic
brain damage with middle cerebral artery ocdusion [68]. The teipenefiraction(ginkgolides A
and B, and bilobalide) of EGb seems to be related to these effects, Le., ginkgolides A and B,
and bilobalide are able to reduce the infarct volume after focal ischemia in mice and rats, and the
number of damaged neurons in cultures after glutamate exdtotoxidty and hypoxia is also
reduced by ginkgolide B and bilobalide [69].
Cerebral Edema
Gabard and Chatterjee [70] were the first to demonstrate the protective and curative effects of
EGb (100 mg/kg, p.o., twice a day for 15 days) against cerebral edema induced by triethyltin
(TET, 0.002% solution in drinking water) in the rat Subsequently, Otani et d. [71] showed
that concurrent administration of EGb (100 mg/kg, p.o.) and TET (0.002% in drinking water)
to rats for 14 days reduced the development of a cytotoxic edema in the white matter of the
brain, as well as the abnormal levels of water and sodium contents induced by TET alone.
Odier experiments by Sancesario and Kreutzberg [72] showed that EGb therapy accelerated the
reabsorption of TET-induced cerebral edema and improved the astroglial reaction. As regards
174
active constituents, datterjee et d, [73] indicated that bilobalide might be responsible for the
anti-edema effects of EGb. Bilobalide (10 mg/kg p.o., for 6 days) and EGb (100 mg/kg p.o.
for 6 days) act protectively and curatively against TET-induced dianges of neuropathobgical
parameters, including body weight, consumption of food and water, and pain reaction time in a
hot-plate test
The TET-induced inhibitory influence on cyclic 3',5'-AMP phosphodiestCTase (PDE)
activities precedes edema formation in the rat brain [74]. To darify the medianism of the
protective action of EGb against TET-toxidty in rats, in vitro and ex vivo effects of EGb on
PDE activities of cerebral tissue were investigated [75]. Higher concentrations of EGb (5-250
mg/L) inhibited the PDE activity in the brain in normal rats, whereas lower concentrations
(0.25-4.0 mg/L) of EGb enhanced the activity of the enzyme. The inhibitory effect of TET on
the high affinity PDE activity (measured with 0.25 //M cydic AMP) of the brain was diminished
in the presence of low EGb concentrations. Furthermore, preventive and curative treatment of
TET-poisoned rats with EGb (100 mg/kg, p.o., for 7 days) prevented both the formation of
edema and the fall of PDE activity induced by TET alone. These results suggested the anti-
edema action of EGb might be partiy assodated with its modulating influences on cellular cyclic
AMP levels via activation of membrane-bound PDE
Neurotrophic Activity in Neural Iiyuiy
Barkats et d. [76] showed the effects of dironic treatmoit with EGb on age-dqpendent
structural dianges in the hippocampi of three strains of 15-month-old inbred mice (C57BIV6J,
BALB/cJ and DBA/2J). Treatment with EGb (50 mg/kg/day, in the drinking water for 7
months) significantly inoreased the projection field of intra- and infira-pyramidal mossy fibers
(iipMF) in the CA3 of the hippocampus as compared with control mice, although there was no
difference in the sensitivity to EGb among the mouse strains. Since it has been considered that
the size of the projection field of iipMF correlates strongly with spatial learning [77], this
neuroprotective and/or neurotrophic action of EGb on the hippocampal iipMF might be useful in
explainiag the benefidal effect of EGb on spatial learning. This hypothesis was supported by
the findings that bilateral frontal cortex-lesioned rats treated with EGb (100 mg/kg, i.p. for 30
days) had improved retention of a delayed-spatial alternation task compared to subjects with
lesions treated with saline, and that the treatment with EGb reduced the extent of brain swelling
in histological examination [78]. Furthermore, EGb inaeased protein synthesis in many brain
regions after unilateral labyrinthectomy in the adult rat, and the most stimulated brain regions
were assodated with sensory, behavioral and learning systems [79]. EGb also protects
against oxidative damage to DNA and oxidation of mitodiondrial glutathione, and prevents age-
related morphological changes in mitochondria in the brain and liver in old rats [80, 81].
Anumber of studies have been made concerning the effects of EGb on vestibular compensation.
Administration of EGb (50 mg/kg, Lp. for 30 days) accelerated vestibular compensation
induced by unilateral vestibular neurectomy in rats [82, 83]. The results obtained by perfusion
of EGb into vestibular nuclei of alert guinea pigs suggest that EGb has a direct excitatory effect
on the neuronal level [84]. Further experiments indicated that the extract without the terpenes
was most effective in this experimental model of CNS plastidty involved in vestibular
compensation [85]. On the other hand, Sasaki et d, investigated the effects of bilobaUde, a
terpene constituent of EGb, fi-om the viewpoint of electrophysiology with the use of a rat
hippocampal slice preparation [86]. Bilobalide (10-500 ^M), increases the amplitude of
175
population spikes evoked by dectrical stimuladon, indicating an enhancement of excitability of

hippocampal CAl pyramidal neurons. Moreover, in an experimental model of perq)heral
neuropathy, it has been shown, by means of electiophysiotogical and histological tedmiques,
that bilobalide exerts trophic and protective effects on motor nerves. Hie reinnervation of the
extensor digitorum longus musde following traumatk: nerve damage occurs more rîdly in
bilobalide-treated rats [87].
Apoptosis
Apoptosis is associated with cerd^ral ischemia and scversl neurodegenerative diseases, such as
Alzheimo-'s and Parkinson* s diseases [88]. In die study described by Dtdier et al. [89],
apoptosis was induced by sectioning of the olfactory nave in adult rats, and was evaluated by
measuring either the thidmess of the q)ithelium in relation to neuronal death or DNA
fragmentation. These workers showed that pretreatment with EGb (50 or 100 mg/kg/day)
reduced the rate of lesion-induced apoptosis of olfactory neurons in the olfactory mucosa of
adult rats. In addition, EGb also prevents apoptosis induced by treatmoit with hydrogen
peroxide and ferrous sulfate in cerebellar neuronal cells dissociated from rats [90], and protects
PC12 nerve cells in a dose-depradent manner against die B amyloid (AB)-induced apoptosis and
neurotoxidty measured using the 3-(4,5-dimetiiylthia2Dl-2-yl)-2,5-diphenyl tetrazolium
bromide and trypan blue assays [91]. With regard to the anti-apoptotic effect of EGb and its
terpene constituents, Ahlemeyer et al. [92] compared their effects on apoptosis induced by
serum dq)rivation and staurosporine. In cultured chick embryonic neurons, EGb (10 mg/L),
ginkgolide B (10 ;<N^, ginkgolide J (100 fiM) and bilobalide (1 /^M) reduced q)optotk: damage
induced by serum- deprivation. In mixed neuronal/glial cultures from neonatal rat
hippocampus, EGb (100 mg/L) rescued rat neurons from apoptosis caused by serum
deprivation, ginkgolide B (100 /^M) reduced staurosporine-induced apoptotic damage, and
bilobalide possessed anti-apoptotic effects in botfi serum-deprived and staurosporine-treated
neurons. Furthermore, bilobalide (25-100 fiM) attenuates the reactive oxygen, spedes (ROS)-
induced apoptosis in PC12 cells, and also reduces ROS-induced elevation of c-Myc, p53, and
Bax and activation of caspase-3 [93]. Recentiy, 4-hydroxy-4-rcft-butyl-2,3,5,6-
tetrahydrothiopyran-1-oxide (10-100 nM), a bibbalide-derived compound, has been shown to
protect diidc and rat neurons against serum dq)rivatk)n- and staurosporine-induced apoptosis
[94]. On the other hand, Rapin et al. [95] showed that the addition of EGb (5-20 mg/ml) or
ginkgolide B (0.2 or 0.4 mg^fil) to the culture medium of rat hippocampal cells led to an
increase in cdl viability and a diminution in the number of apoptotic cells induced by the
peroxyl radical-generator, 2,2'-azobis-2-amidinopropane (AAPH, 20 or 50 mM), whereas
additk)n of bilobalide (0.1-1.0 mg/ml) was ineffective. Moreover, oral administratk)n of EGb
(50 mg/kg/day) and ginkgolide B (2 mg/kg/day, p.o.) to rats caused a significant inaease in cell
viability and a highly significant decrease in the numbers of damaged cells in both
spontaneously occurring and AAPH-induced apoptosis, whereas, bilobalide (2 mg/kg/day, p.o.)
was ineffective. Recently, it was demonstrated that the flavonoid component of EGb, rutin,
quercetin, and a mixture of flavonoids and terpenes protect cerebellar granule cells from
oxidative damage and apoptosis induced by hydroxyl radicals, while terpenes of EGb do not
protect against apoptosis [96, 97]. Moreover, pretreatmoit of PC12 nerve cells with
ginkgolide B or ginkgolide C does not rescue the cells from AB-induced apoptosis and cell
death, although these constituents prevent the AB-induced increase of free radical production
176
measured using 2,7-dicfaloFofluorescem diaceTatc^, and inhibit die AB-induced 4-hydioxy-2-

nonenal modification of spedfic mitochondrial target proteins [98]. Therefore, there is some
disagreement as to which constituents of EGb are diiefly responsible for its anti-apoptotic
action. It is probable that tiie animal spedes and the model used for the induction of apoptosis
are involved. It needs further study before any condusions can be drawn.
Anticonvulsant Activity
Bilobalide, a main constituent of the teipene fraction of the EGb, possesses anticonvulsant
activity against convulsions induced by pentylenetetrazol, isoniazid, 4-0-methylpyridoxine, and
electroshock. Reduced durations of convulsions and prolonged onset time of convulsions
induced by chonical oonvulsants and dectroshock were observed in mice treated orally with
bilobalide (30 mg/kg/day, for 4 days). However, bilobalide has no protective effect against
bicuculline- and strychnine-induced convulsions [99,100].
Learning and Memory
Winter [101] demonstrated the effects of EGb in aperitive opoant conditioning of mice. EGb
was administered orally at a dose of 100 mg/kg for 4 or 8 weeks prior to the training and was
maintained for 10 weeks sftci training. Treatment with EGb increased the number of correct
responses and reduced incorrect responses. Furthermore, the learning- and memory-
in^roving effects of EGb were confirmed using some conditioned-reflex methods (shutde-box,
step-down, stq)-through, and water maze), when the extract was administered orally for 7 days
before training attiireedoses of 10, 30, and 90 mg/kg to young and old rats [102].
The radial maze has been widely employed in studies concerning learning and memory, and
it has been shown to be sensitive to the aging process. In a trial using an eight-arm radial
maze, effects of EGb (30 and 60 mg/kg/day) administered for 3 weeks before testing and
throughout the testing period were investigated. Parameters of learning (the number of arms
visited, the number of errors and time spent to complete the test) were improved in EGb-treated
rats compared to controls [103]. Recendy, anotho^ trial using the eight-arm radial maze was
performed in aged male Fisch^ 344 rats (20 months old). Chronic pretreatment with EGb
showed a significant positive effect on continuous learning and on delayed nonmatching to
position tasks in aged rats [104].
StoU et d. [105] investigated the effects of chrome treatment with EGb on age-related
cognitive dianges using passive avoidance learning. Aged nuoe treated daily with 100 mg/kg
EGb for three weeks had signifkantly improved short-term memory measured by the avoidance
latency 60 seconds after shock, but long-term memory measured by the avoidance latency 24
hours after shodc did not improve. These results were supported by the findings that dironic
(30 days) and acute treatments with EGb (60 mg/kg) oihanced short-term memory on olfactory
recognition in young and aged rats [106].
Administration of scopolamine to rats induces amnesia in passive avoidance learning.
EGb administered intraperitonealiy 30 min before the initial trial at doses of 150-500 mg/kg
significantly attûated the amnesic effects of scopolamine on step-through latendes in a
retention trial 4 hours after training for the passive avoidance test in rats [107]. Hoyo* et d,
showed that, using an animal model of intracêbroventricular streptozotodn treatment, EGb
treatment compensated for deterioration in working memory, referrace memory and passive
177
avoidance bdiavior and restored a defikit in co-ebral energy metabolism [108]. Furthennore,
pretreatment with EGb (50 and 100 mg/kg, p.o.) ameliorated the impairment of memory
induced by bilateral occlusion of the carotid arteries in mice when the passive avoidance test was
carried out 48 hours after ischemia [109]. These results lead to the condusion that EGb
possesses cognition-enhandng properties.
Anti-Stress and Antidepressant Effects
Porsolt et al. investigated the effects of repeated oral administration of EGb (SO and 100 mg/kg)
on various behavioral models of stress in rodents [110, 111]. The bdiavioral models induded
learned he^lessness", shock-suppressed licking (\bgel conflict test), forced swimming-
induced immobility C^havioral despair^, shock-suppressed expiration (four-plates test),
spontaneous exploration (staircase test) and food consumption (emotional hypophagia). EGb
inaeased the amount of food consumption in the emotbnal hypophagia test and reduced the
acquisition of behavbral defidts induced by dectric shocks in the learned helplessness"
paradigm in both young and old rodents. However, EGb had no marked effects in other
models tested. These results indicated that EGb has anti-stress propoties; however, it cannot
be assimilated into either classical antidepressant or anxiolytic activity.
In addition, it was shown that treatmoit with EGb (100 mg/kg, in 5% ethanol) reduces the
development of the polydipsia induced by the stress of daily handling, anesthetization with
ether and oral intubation [112]. Rapin et al. [113] have reported that oral treatment with EGb
(50 or 100 mg/kg/day, for 20 days) suppresses auditory stress-induced alterations of
discrimination learning in both young and old rats; EGb was espedally effective in decreasing
the number of inefSdent lever presses and in reducing the reaction time in older animals.
Furthermore, treatment with EGb counteracted the auditory stress-induced inaeases in the
plasma concentrations of epinq)hrine, norepinephrine and corticosterone in both young and old
rats.
Wada et al. revealed that feeding a diet containing a 5% powdered dried Ginkgo biloha
leaves or an aqueous extract of these leaves to 4-week-old male ddY mice for 7 days shortened
the sleeping times induced by anesthetics (hexobarbital, a-chloralose plus urethane). A similar
effect was obtained in animals treated orally widi bilobalide or ginkgolide A (both at 10 mg/kg)
[114]. Subsequently, Brochet et al. [115] showed effects of single intraperitoneal injections of
EGb and ginkgolide B and bilobalide on barbital-induced narcosis in the mouse. Single
injections of EGb (25 and 50 mg/kg) ginkgolide B (1 mg/kg) and bilobalide (2 and 5 mg/kg), 60
min prior to sodium barbital (180 mg/kg, i.p.), significantly shortened barbital-induced sleeping
time and increased the latency to onset of sleep. From these data, they suggested that EGb
indudes constituents that have CNS-stimulatory activity.
MECHANISMS OF ACTION OF EGB AND ITS ACTIVE CONSTITUENTS
It has been proposed that possible mechanisms undo'lying the neuroprotective and/or
neurotrophic propolies of EGb in the CNS indude influences on neurotransmitters, oiergy
metabolism and membrane functions, as well as antioxidant activities and inhibition of platelet-
activation factor (PAF).
178
Improvement in Enei^gy Metabolism
As shown in Figure (1), injected [**C]2-deoxy-D-glucose ( f ^CJEXj) is transported toward

cerebral tissue through the blood braio barrier and then phosphorylated into [^^C]£)G-6-
phosphate ([^^C]DG-6P), whicfa accumulates in the cells. Measuring radioactivity in brain
sections by autoradiography, it is possible to calculate local cerebral glucose utilization (LCGU)
[116].
Krieglstein et d. [117] investigated effects of EGb on LCGU by means of the [^^C]DG
tedmique and on local cerebral blood flow (LCBF) using [^^Cjiodoantipyrine. Pretreatment
with EGb (130 mg/kg, Lv.) significantly increased LCBF and blood glucose levels, although
there was no influence of EGb on LCGU. On the other hand, in the awake adult rat, Lamour
et d. [118] found significant and widespread decreases (range 3-26% reduction) of LCGU in 21
brain regions of rats injected with EGb (150 mg/kg, i.p.), but not ginkgolide B (10 mg/kg, i.p.).
TTic largest dedines of LCGU were observed in the hq)pocampal formation, cerebral cortex,
globus pallidus, caudate-putamen, ventral thalamus, infmor oolliculus and superior olive.
Similar results were obtained when rats received rq)eated treatment with EGb (50 mg/kg/day,
for 15 days) [119]. In regard to LUGU, this discrepancy between these data and those of
Krieglstein et d. may be due to different methods of drug administration (L v. vs. Lp., acute vs.
dironic).
BBB
rPlasman I Brain
Glu - • Glu
[^^C]DG ^[i4c]DG —•[^^C]DG-6P
Fig. (1). Representative model of cêbral glucose (Glu) consumption.

The labeled 2-deoxyglucose ([^^C]DG) injected enters into competition
with the circulating glucose through the blood-brain barrier (BBB), and
stops at the 2-deoxygluoose-6-phosphate ([^^C]DG-6P).
When ischemia or hypoxia impairs the oxygen supply, mitochondrial respiration is

deaeased and consequently aiergy production is damaged. Glycolysis is enhanced to
compensate for the decrease in ATP regeneration. TWo experimental rat ischemia models
(normobaric hypoxia and carotid clamping) induced a diminution in glucose uptake and LCGU.
EGb (100 mg/kg p.o. for 5 days) administered preventively increased deoxyglucose uptake and
179
LCGU in the cortex, hippocampus, and caudate nudei of rats subjected to normobaric hypoxia.
Treatmoit with EGb significantly increased LCGU in rats with bilateral ligature of the carotid
arteries, but did not modify the deoxyglucose uptake [120]. Kardier et al. [65] estimated the
brain energy metabolism in rats treated with EGb (100 mg/kg Lp. 30 min before hypoxia) under
various experimental conditions. Before as well as after hypobaric hypoxia, the brain glucose
level and glucose-6-phosphate level were elevated by EGb, probably due to enhanced cerebral
blood flow. In the situations of hypobaric or hypoxk hypoxia, the cerebral lactate level of
EGb-treated rats was slighdy lower, whereas its pyruvate level was elevated as compared with
controls, and tiierefore the lactate^yruvate ratk) was markedly deaeased. In addition, the
levels of cerebral aeatine-P and ATP were less severely deaeased by EGb treatment after 7.5
min of hypobaric hypoxia
Janssens et d. [121] showed that EGb and bilobalide inhibited the hypoxia-induced
decrease in ATP content of endothelial cells in vitro. In addition, these compounds delayed the
onset of glycolytic activation, as evidenced by increased glucose transport, as well as by
inaeased lactate production under hypoxia. Tbe respiratory control ratio of mitochondria
isolated from livers of rats treated orally with EGb and bilobaUde increased, suggesting the
protection of ATP content These results indicated that EGb and bilobalide prevented tfie
uncoupling of oxidative phosphorylation in mitochondrial respiration. Further investigations
showed that in vivo (8 mg/kg) and in vitro (0.8 /nM) treatments with bilobalide markedly
inaease the mitochondrial respiratory control ratio, probably by lowaing oxygen consumption
during state 4. Bilobalide protects both complexes I and in from inhibition induced by Amytal
or antimydn A [122]. Further studies demonstrated that bilobalide prevents the deaease in the
state 3 respiration rate induced by ischemia in the liver and brain [123]. Hierefore, bilobalide
may protect mitochondrial respiratory activity under ischemic conditions by maintaining
complex I and III activities, thus preserving ATP regeneration as long as oxygen is present
These medianisms appear useful in explaining the protective effect of EGb on the onset of
ischemia-induced damage.
Furthermore, Bruel et d. [124] found that EGb (0.25 //g/ml) inaeases glucose transport
and glycogen synthesis in cultured vascular smooth musde cells. Essentially similar results
were obtained by oral treatment with EGb in liver and skeletal musdes [125], and in
erythrocytes [126], Vasseur et d, [127], using intracellular mkaroelectrodes, indksited that
administration of EGb (200 mg/kg/day, p.o.) or bilobalide (8 mg/kg day, i.p.) to mice increases
the in vitro sensitivity of their pancreatic 6 cells to glucose. In addition, EGb and bilobalide
correct the impaired glycogen synthesis in liver and muscle cells of the diabetic rats injected with
alloxan or strq)tozotocin [125, 127]. Taken together, these findings indicate that
administration of EGb causes an increase in glucose uptake and glycogen synthesis in various
tissues in part via its bilobalide constituent, and EGb could be useful in treating non-insulin-
dependent diabetes mellitus.
Antioxidant Effects
In dissodated rat cerebellar neurons, the effect of EGb on oxidative metabolism was studied
using a flow-cytometer and 2',7-dichlorofluorescin (DCFH), which is oxidized to a highly
fluorescent compound by intracellular hydrogen peroxide [128]. Preincubation with EGb
dose-dependently decreases DCFH fluorescence, and reduces the isonomydn-induced increase
of DCFH fluorescence, suggesting that EGb reduces oxidative metabolism in both resting and
180
Ca^Moaded brain neurons [129, 130]. Further experiments indicated that myrioetin and
quôetin, tiie flavonoid constituents of EGb, reduce oxidation of DCFH in both resting and
Ca^Moaded brain neurons [131]. EGb also gready delays the time-dependent increase in the
number of dead cerebellar neurons during exposure to hydrogen peroxide [132]. Taken
together, these results indicate that EGb and its flavone constituents protect the brain neurons
against oxidative stress induced by hydrogen peroxide, whidi is involved in ischemic brain
damage.
An antioxidant action of EGb has been reported against peroxyl radicals, hydroxyl radicals
and superoxide anions [133, 134, 135, 136]. Production of activated oxygen species (Oj*,
H2O2, OH') in human neutrophils stimulated with phoibol myristate acetate is significantly
decreased in the presence of EGb [134, 135]. The free radical scavenging activities of ginkgo
flavonoids against the diphenyl picryl hydrazyl radical are as follows: myricetin > quercetin >
kaempferol > luteolin. As for biflavones, the best radical scavengo* is amentoflavone,
followed by bilobetin, ginkgetin, isoginkgetin, and sdadopitysin [137]. Recently, free radical
scavenging activities of terpene-free EGb and quercetin wê revealed by means of an in vitro
electro-spin resonance assay [138]. Additionally, the in vivo experiments showed that
terpene-free EGb inhibits cutaneous blood flux, whidi reflects the skin inflammatory level
[138]. In regard to ginkgo terpenes, it has been revealed by means of electron paramagnetic
resonance and U\7VIS spectroscopy that ginkgolides B, C, J and M, as well as bilobalide but
not ginkgolide A, scavenge superoxide and hydroperoxyl radicals in dimethyl sulfoxide as an
aprotic solvent [139].
Akiba et d. showed that EGb prevents the platelet aggregation induced by a combination of
100 f4M terr-butyl hydroperoxide and Fe^*. However, ginkgolides A, B and C, which are
known to be PAF-antagonists, have no influence on this aggregation. Therefore, it was
suggested that free radicals, but not FAF, might be involved in platelet aggregatk)n induced by
oxidative stress [140].
Serotonin (5-HT) produces a rapid elevation of superoxide that stimulates the mitogenesis of
bovine pulmonary artery smooth muscle ceUs (SMCs). EGb scavenges superoxide elevated
by 5-HT, hence preventing 5-HT-induced mitogenesis on both SMCs and Chinese hamster lung
fibroblasts. These results indicate that EGb inhibits the cellular transduction signaling process
that leads to mitogenesis, as a result of its antioxidant activity [141].
In addition to radical scavenging properties, it has been reported that EGb reacts with nitric
oxide (NO) in in vitro systems [136], and inhibits NO production induced by lipopolysaccharide
plus intoferon-Y in maaophage cell Une RAW 264.7 [142]. Fre-treatment with oral
administration of EGb reduced nitric oxide overproduction after transient brain ischemia in the
MongoHan gerbil [143]. Further experiments showed that EGb inhibits NO production by
attenuating the level of iNOS mRNA in a human endothelial cell line (ECV304) [144], also
inhibits the activation of protein kinase C (PKC) induced by sodium nitroprusside (SNP), NO
generator, and that its flavonoid constituents have protective properties against toxicity induced
by SNP on cells of the hippocampus [145]. Recently, it was shown that ginkgolide A,
ginkgolide B and bilobalide inhibit NO production in macrophages derived from a human
monocytic cell line through attenuation of iNOS mRNA expression. However, these
components have no effect on the eNOS-mediated NO production in endothelial ceUs [146].
181
Influences on the Neurotransmitters
Numerous studies have demonstrated age-related changes in levels of neurotransmitters and

their recq>tors in certain areas of the brain. There is a decrease in the levels of acetylcholine
and in the numbers of muscarinic receptors and 6-adrenocqptors in the cêbral cortex and
hippocampus of the brain in patients suffering from Alzheimer's disease and in the brains of
aging rodents, diaracterized behaviorally by a sevore impairment ia cognitive functions [147,
148, 149]. Numbers of 5-HT recq)tors and levels of dopamine and noradrcnalin and 5-HT
have also shown age-related diminution [150, 151, 152], and are known to be involved in the
regulation of mood [153]. Furthermore, it has been demonstrated that the activity of
monoanune oxidase (NfAO), which rûlates the brain concentrations of 5-HX norq)inephrine
and other biogenic amines, inaeases with advancing age [154]. Hius, the inhibition of NfAO
has been shown to produce antidepressant or anxiolytic responses in animal models and in man
[155].
Brain Levels of Biogenic Monoamines

Nforier-Teissier et d. [156] determined that administration of EGb alters the levels of
catecholamines, indolamines and their metabolites in some brain areas of young rats and mice.
Marked changes in the EGb-treated brain were found for norepinephrine, 5-HT, and its
metabotite, 5-hydroxyindole-3-acetic add, whereas it was less effective for dopamine and its
mâbolite 3,4-dihydroxy-phenylacetic add. EGb-induced changes depend on the route of
administration (p. o. or L p.), dose and duration of treatment (acute or dironic).
In old rats (26 months old), oral administration of EGb (10 mg/kg and 30 mg/kg, for 7
days) produces elevations of 5-HT in the frontal cortex, hippocampus, striatum and
hypothalamus, and of dopamine levels in the hippocampus and hypothalamus compared with
controls. On the other hand, EGb decreases the 5-HT level in the pons, and those of
norepinephrine in the hippocampus and hypothalamus [157]. In this connection, Racagni et al,
[158] showed that the O-methylated amine metaboUte of norepinephnne, normetanq)hrine, was
markedly elevated (+500%) in the cerdjral cortex by du:onic oral administration of EGb (100
mg/kg, for 14 days), suggesting an increase of norq)inephrine turnover. In additbn, treatment
with EGb (50 or 100 mg/kg/day, for 20 days) diminished the inareased plasma levels of
epiDq)hrine, norepinephrine, and corticosterone induced by acute auditory stress in young and
old rats [113].
GABA is the major inhibitory neurotransmitter in the CNS and acts to counter glutamate-
induced exdtatk)n. Bilobalide (30 mg/kg/day, p.o., for 4 days) elevates GABA levels in the
hippocampus and cerebral cortex in mice. These effects of bilobalide are due to a potentiation
in glutamic add decarboxylase activity and an enhancement in the protein amount of 67 kDa
glutamate decarboxylase. Furthermore, isoniazid and 4-O-methylpyridoxine, pot^t
convulsants, induce reductk)ns in brain GABA levels, whereas bilobalide counteracts these
effects. These results indicate that potentiation of GABAergic transmission induced by
bilobalide might explain its anticonvulsant activity against isoniazid and 4-Omethylpyridoxine
[159,160].
Monoamine Oxidase Activity

White et al, [161] explored in rat brain mitodiondrial extracts the effect of EGb on MAO activity
in vitro. MAOA and MAOB activities wore assayed using [^H]5-HT and [^*C]B-
182
phenetfaylamme as substrates, respectively. EGb inhibited both NfAOAand MAOB activities

of rats and mice in vitro to similar extents. These results have suggested that the inhibition of
MAO may be a mechanism underlying antidq)ressant or anxiolytk: responses of this extract
obtained in animal models and man. Similar observations using a fluorimetric method were
shown for EGb, but not for ginkgolide A and ginkgolide B [162]. Sloley et d. showed that
kaempferol is a primal in vivo, but not ex vivo, rat brain MAQ-inhibitor in EGb [163].
Besides, the effects of long-term treatment with EGb (500 mg^g/day, for 7 months) on cêbral
MAO activity were investigated in mice subjected to a chronic mild stress. EGb induced
reductions in basal MAO activity in 18-month-old mice. Hie age-related inaease in brain
MAO activity was lower in die untreated mice subjected to stress and EGb potentiated this effect
[164]. Recently, the effects of EGb on aggression woe investigated using MAO-A knockout
nuce. EGb reduced their aggressive behavior in resident-intruder confrontations to levels seen
in wild types, and decreased their [^H]ketanserin binding to 5-HT2A reoq)tors in the frontal
cortex [165].
On the other hand. Fowler et d, recently measured MAO-A and MAO-B activities in the
human brain using positron emission tomography and ["C]dorgyline and ["C]Ir<ieprenyl-D2,
and showed that administration of EGb (120 mg/day, for 1 month) to 10 subjects had no
influence on brain MAO-A and MAO-B [166]. Moreover, for the same dosages at which EGb
(25, 50, and 100 mg/kg, p.o., for 5 days) exerted antistress propolies, EGb did not exhibit any
activity indicative of MAO inhibition in mice [167]. Therefore, fliere is uncertainty about
whether MAO inhibition is the medianism responsible for the anti-stress activity of EGb. This
will be the subject of further study.
Acetylcholine Receptors
Taylor [168] examined the effects of chronic administration of EGb (100 mg^g/day, for 28
days in drinking water) on the binding of [^H]quinudyidinyl benzilate ([^H]QNB) to the
muscarinic cholinergic receptors of die hippocampus of young (3 months old) and old (24
months old) male Fisher 344 rats. Asignificant diminution in the number (B^^) of muscarinic
receptors was observed in the old rats compared to the young rats. In contrast, EGb produced
a maiiced iacrease of the B,^ value io the hippocampus of old rats in comparison to controls of
the same age, and also a slight increase of the B,^ in the young rats.
Rapin et d, [11] have reported an increase of the acetylcholine synthesis rate constant
evaluated by a bolus injection of [^H]choline in the hqipocampus of 4-month-old rats after acute
administration of EGb (100 mg/kg Lp.). Similar resuhs were obtained in the frontal cortex,
hippocampus and corpus striatum after dironic treatment with EGb (100 mg^g/day p.o. for 21
days). On the other hand, the acetylcholine turnover rate was not modified by either acute or
chronic administration of EGb. These results indicate that EGb might increase acetylcholine
release.
In the cholinergic nerve terminals of the hippocampus of 24-month-old Wistar rats,
Kristofikova et d, [169] showed that both in vivo administration with EGb (50 mg/kg/day for
30 days in drinking water) and in vitro applicatbn of EGb to synaptosomes (15-30 /^g/ml, i.e.
50-100 //g/mg proteio) cause significant increases in high-affinity dioline uptake (HACU)
levels. Subsequently, these workers showed that EGb (100 figfal) markedly elevates the
specific binding of [^H]hemidiolinium-3 ([^H]HCh-3) (to 306%) in hippocampal synaptosomes
from young Wistar rats, suggesting that EGb causes an ino-ease in the number of dioline
183
cairiers [170]. Taken together, these results suggest tiiat EGb might enhance the cholinergic
neurotransmitter syston on presynaptic nerve terminals.
Adrenoceptors
With regard to the number ( B ^ ) of 6-adrôceptors measured with pH]dihydroa^)renolol

([^HjDHA) in rat oerd)ral cortex, Taylor [168] demonstrated that chronic administration of EGb
(100 mg/kg/d for 28 days) had no effect on fi-adroiergic binding in either young or old rats,
although tiie B ^ value for B-adrenocq)tors was significantly reduced with advance of age.
However, Brunello et d. [171] and Racagni et d. [158] rq)orted that the afGnity (K^) and B ^
of [^H]ra[A binding to B-adrenocqptors decreased with diromc treatment with EGb
(lOOmg/kg/day) for 2 months or 30 days.
In aged rats (24 months old), the B ^ of [^H]rauwolscinc binding to aa-adrenoceptors in
the hippocampus and cerebral cortex is significantly reduced as compared with young rats (4
months old). Chronic treatment with EGb (5 mg/kg/day, Lp. for 21 days) does not alter the
BB„ value for Cj-adrenoccptors binding measured with [^HJrauwolsdne in the hippocampus of
young rats, but significantly increases the B , ^ of [^H]rauwolscine binding in aged rats [172].
These results suggest that a^-adrenoceptors involved in the control of noradrenergic activity are
more susceptible to EGb treatment in aged rats.
[^HJAmine Uptake
EGb inhibits the uptake of [^H]norepinq)hrine ([^H]NE) and [^H]dopamine and [^H]5-
hydroxytryptamine^^[^H]5-HT) into in vitro synaptosomes prepared from the striatum and
cortex in a concentration-dq)endent mann^. Tlie rank order of potency for the inhibition of
amine uptake is NE > dopamine > 5-HT [173]. Similar results were obtained by Ramassamy
er d, [174]. These workers showed that EGb deaeased the specific uptakes of [^H]dopamine,
[^H]5-HT and [^H]choline by synaptosomes prepared from tiie striatum of mice in a
concentration-dependent manner. Tlie IQ^ values were 637 figfiol for [^H]dopamine uptake,
803 /ig/ml for [^H]5-HT uptake, >2000 //gAnl for [^H]choline uptake. However, they
conduded that the inhibition of amine uptake caused by EGb appears to be non-specific, since
EGb also prevents the specific binding of the dopamine uptake inhibitor [^H]GBR12783 to
membranes prq)ared from striatum.
EGb in vitro modifies the [^H]5-HT uptake by synaptosomes prepared firom nuce cerebral
cortex in a biphasic manner. As mentbned above, the uptake of [^H]5-HT is inhibited by a
high concentration of EGb [174]. On the other hand, low concentrations of EGb (4-16 //g/ml)
significantly inaease [^H]5-HT uptake. A similar inaease was also obtained when
synaptosomes were prepared from the cortk:es of mice treated orally with EGb, either acutely
(100 mg/kg, 14 hours and 2 hours before death) or semi-dironically (2 x 100 mg/kg/day, for 4
days). Furthermore, such an inaement in the [^H]5-HT uptake is attributed to the flavonoid
constituents of EGb [175], and may be associated with the mechanism of its antidq)ressant
activity.
5'HT Receptors
Adeaeased (22%) number of 5-HTi^ recq)tor binding sites labeled by [^H]8-hydroxy-2(di-/i-

184
propylainino)tetralin (pHJS-OH-DPAT), a S-HTi^ receptor agonist, in cerebral cortex

membranes of Wistar rats was observed in aged (24 months old) rats as compared with young
(4 months old) animals. Chronic treatment with EGb (5 mg/kg/day, for 21 days) did not alter
the B ^ value in young rats, whereas it significantly inaeased it in aged rats (33%) [176]. On
the other hand, Bolanos-Jimîez et d, showed that chronic treatment with EGb (50 mg/kg/day,
14 days) produced a relatively small diminution in pHJS-OH-DPAT binding to hippocampal
5-HTi;^ receptors in 18-month-old rats [177]. There is at the moment no dear explanation for
this disaepancy.
An inhibitory effect of S-OH-DPAT on forskolin-stimulated adenylyl cyclase activity is
observed in hippocampal membranes of the guinea pig and rat, and has been used as an index of
the functional activities of S-HTj^ receptors [178]. Q)ld stress induces a reduction of the
inhibitory effect of S-OH-DPAT in the hippocampus isolated from 18-month-old rats, although
it has no influence on either the affinity or number of [^H]8-0H-DPAr binding sites. The
administration of EGb (50 mg/kg p.o. for 14 days) prevents the cold stress-induced reduction
in the inhibitory effect of 8-(Xl-DPAr on forskolin-stimulated adenylyl cyclase activity in old
rats. These results indicate that EGb prevents the stress-induced desensitization of
hippocampal 5-HTu^ receptors; thus, its effects might explain anti-stress and antidepressant
properties of EGb [177].
NMDA Receptor
Taylor [173] showed that EGb acts in vitro as an inhibitor of radioligand binding to the
competitive and non-competitive sites of ^-methyl-i>-aspartate (NMDA) receptors. In
addition, the most potent inhibition (K^ = 0.5 mg/ml) is observed for non-competitive
NMDAsites labeled by ['H]MK-801.
MPTP-Induced Dopaminergic Neurotoxicity
It is known that l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) selectively causes

degeneration of the nigrostriatal dopaminergic neuronal pathway in several animal species,
which is considered an animal model of Parkinson's disease [179]. As shown in Figure (2),
MPTP administered systematically crosses the blood brain barrier, and is oxidized by MAOB
into MPP*. This metaboUte is concentrated into dopaminergic neurons and consequently
destroys such neurons by generation of ifree radicals [180, 181]. In mice implanted
subcutaneously with osmotic minipumps releasing MPTP for 7 days (105 /^g/h/mouse)
(approximately 100 mg/kg/day), a decrease in [^H]dopamine uptake by a synaptosomal fraction
prepared from striatum was observed. This neurotoxic effect was prevented by the chronic
injection of EGb (approximately 100 mg/kg/day in drinking tap water) for 17 days. Such a
protective activity of EGb against MPTP neurotoxicity is unlikely to depend on inhibition of the
MPTP uptake by dopamine neurons, since the concentration at which EGb prevents
[^H]dopamine uptake is too high to reach the brain under in vivo experimental conditions [174,
182]. Therefore, it appears likely that a possible explanation lies in thefree-radicalscavenging
property of EGb, which neutralizes free radicals generated from MPP* in the dopaminergic
neurons.
In this regard, another study showed that protective and curative treatments with EGb
prevented the reduction of striatal dopamine levels induced by MPTP. MPTP (30 mg/kg/day.
185
Nigrostriatal
dopamine neuron
BBB
I iÂstrocyte
MPTP • ' - > MPTP
injection
Fig. (2). Hypothesized mechanisms of neurotoxicity of MPTP. After injection

of MPTP, its native fomi crosses the blood brain barrier (BBB) and is oxidized by
monoamine oxidase B (MAO-B) into MPP^. This metabolite is transported and
concentrated into nigrostriatal dopamine and exerts a neurotoxic effect
i.p. for 6 days) sigtdficantly reduced striatal dopamine levels in C57 mice. On the other hand,
when C57 mice were pretreated with EGb (20, 50, 100 mg/kg/day, Lp.) for 7 days and then
treated with the same extract 30 min before MPTP injection for 6 days, the neurotoxic effect of
MPTP was antagonized in a dose-dependent manner. Moreover, in mice treated with EGb (50
mg/kg/day, Lp.) for 2 weeks after MPTP-lesion, the recovery of striatal dopamine levels was
accelerated. MPTP is oxidized by MAO-B into MPP^ a positively charged species, whereas
EGb, but not ginkgolides A and B, inhibits MAO-B activity. Therefore, another possible
explanation for this protectk)n might lie in the inhibition of MAO activity caused by EGb to
prevent the oxidization of MPTP into MPP* [162].
Effect of EGb on the Neuroendocrine System
It is well known that neuro^docrine dianges with advancing age provide information about
CNS functions [183]. The serum prolactin (PRL) level inaeases in old rats (26 months old)
compared with young rats (3 months old). Administration of EGb (10 mg/kg/day, p.o., for 7
days) deaeases the blood PRL level, while greatly inaeasing adrenocorticotrophic hormone
(ACTH) in old rats compared with age-matched controls. On the other hand, EGb, at a dose
of 30 mg/kg, deaeases the serum level of growth hormone (GH) and ACTH in young rats
compared with age-matdied controls [157].
Glucocorticoids are also essential for many aspects of normal brain development; however,
hyperseaetion induces pathological states such as damage to the hippocampus [184].
Treatment with EGb (100 mg/kg/day, for 8 days) causes a 50% reduction of the plasma
corticosterone level This reduction is probably due to deaeases in the number (B,^), mRNA
186
expression and protein levels of adrenal mitochondrial peripheral-type benzodiazepine receptors

(PBR), whidi are a key element in the rûlation of cholesterol transport Similar results have
been observed in the chronic administration of ginkgolides A and B (2 mg/kg/day, i.p. for 8
days)[185]. Further study demonstrated that EGb and ginkgolide B also decreased PBR
expression and cell proliferatk)n in the highly aggressive human breast cancer cell line
MDA-231, which is ridi in PBR [186]. With regard to steroidogenesis, in addition to PBR,
an ACTH-dependent process is also responsible for its regulatfon. Using cultured
adrenocortical cells, Amri et al, [187] have shown that ex vivo treatment with EGb (100
mg/kg/day, p.o., for 8 days) and ginkgolide B (2 mg/kg/day, i p . , for 8 days) reduces ACTH-
stimulated cortkx)Sterone production by 50% and 80%, respectively. Moreover, Mardlhac
et al. [188] showed that administration of EGb (50 or 100 mg/kg p.o., for 14 days) reduces
basal cortkx)sterone seaetion and the subsequent inaease in oortkx>tropin-releasing hormone
(CRH) and arginine vasopressin {/^/?) gene expression. Ginkgolide B (2 mg/kg/day i.p., for
14 days) reduces basal corticosterone seaetion without alteration in the subsequent CRH and
/WF inorease. However, the stimulation of CRH gene expression by insulin-induced
hypoglycemia is attenuated by ginkgolide B. These results indkrate that EGb and ginkgolide B
are also able to affect the hypothalamic-pituitary-adrenal axis at the hypothalamic level
Corticosteroids play a pivotal role for the development of behavbral sensitization to
an^hetamine through the type II glucocorticoid receptor [189], Trovero et d. [190] showed
that EGb reduces D-amphetamine (0.5 mg/kg, i.p., for 12-24 days)-induced behavioral
sensitization as estimated by increasing values of locomotor activity, although EGb itself has no
locomotor effect Furthermore, chronic administration of D-amphetamine reduces the density
of [^H]dexamethasone binding sites of type II glucocorticoid recq)tors in the dentate gyrus and
the CAl hippocampal regions of D-amphetamine-treated animals, indkating down-regulation of
type II glucocorticoid receptors. On the other hand, pretreatment with EGb (50 or 100
mg/kg/day, p.o., for 20-24 days) prevents this down-regulation of type II glucocorticoid
receptors, suggesting that EGb restores the density of these receptors.
Taken together, these studies indicate that EGb is able to modukte both stress-induced and
age-related behavioral sensitizatk>n by regulating alterations of the neuroendocrine system.
Effect of EGb on the Phospholipid Metabolism
Effects of EGb on ischemia-induced principal changes in the cerebral lipid metabolism were
reviewed by Robin et al. [191]. Figure (3) shows diagram of cerebral lipid metabolism
following ischemia. Pretreatment witii EGb could normalize the increased mitodiondrial lipid
peroxide content and cytosolic lactase dehydrogoiase activity and the deaeased mitochondrial
phospholipid contents and superoxide dismutase activity in rat brain after occlusion of common
carotid arteries [192]. Rogue et d. [193] have shown that EGb is a potent inhibitor of
phospholipase C (PKQ in vitro (ICJQ: 82 //g/ml). Furthermore, in isdiemic rats pretreated
with a single injection of EGb (100 mg/kg, i.p.), a deaease in PKC activity is observed as
compared with untreated ischemic animals.
Impairment of membrane-bound Na,K-ArPase, whidi is responsible for maintaining and
restoring membrane potential, and an increased level of malondialdehyde (MDA), which is a
known as an index of lipid peroxidation, are seen aft^ unilateral focal cerebral ischemia in the
mouse. Pretreatment witii EGb (100 mg/kg/day, p.o. for 10 days) preserves the Na,K-ArPase
activity during cerebral ischemia and prev«its the kiaeased MDA levels caused by cerebral
187
Hydroperoxide
Prostaglandin
Leukotriene
Fig. (3). Diagram of cerebral lipid metabolism following ischemia. During ischemia,
AIT-synthesis is disturbed by deficiencies in oxygen and glucose supplies. The
subsequent energy failure stimulates phospholipase C (PLC) and Aj/Aj (PIAj/Ai), and
thereby leads to formation of diacylglycerol (DAG), which is converted to free fatty adds
(FFA) by Upasc, and leads to accumulation FFA and lysophospholipids. Among FFA,
especially, the peroxidation of arachidonic add (AA) initiates a cascade leading to
lq)oxygenase and cydooxygenase metabolites (prostaglandins and leukotrienes) and
hydroperoxide, which are augmented during reperfusion following ischemia.
Aoetylation of lyso-platelet-activating factor (lyso-PAF) leads to PAF, a mediator of
inflammation.
ischemia [194]. Similar inhibitory effects of EGb on MDA production induced by hydrogen
peroxide have been shown in erythrocyte membranes [195, 196].
Electroconvulsive shock (ECS), as well as ischemia, induces inaeases in free fatty add
(FFA) and diacylglycerol (DAG) in the rat brain, probably due to the breakdown of membrane
phospholipids through the activation of phospholipases (PLC, PLAj/Aj). EGb treatment (100
mg/kg/day, p.o. for 14 days) selectively decreases endogenous FFA levels and increases
endogenous DAG levels in the hippocampus. Therefore, ECS-induced accumulation of FFAis
prevented in the hippocampus of EGb-treated rats during clonic seizures (30 sec to 2 min after
188
ECS). Furthermore, the inaeased DAG levels induced by ECS are delayed by EGb treatment
and the subsequent decrease in DAG levels is accelerated by EGb treatment in both the
hippocampus and cortex [197].
Hypoxic or ischemic conditions led to an immediate release of free dioline via the
breakdown of choline-containing phospholipids in rat hippocampus slices. Klein et d. [198]
showed that bilobalide inhibited the hypoxia-induced dioline release in a dose-dependent
manner both in vitro (ECjo*. 0.38 /^M) and ex vivo (2-20 mgAcg, p.o.). Asimilar reduction of
dioline release was confirmed after administration of EGb (200 mg/kg, p.o.). Bilobalide also
inhibits the ^-methyl-D-aspartate-induced, PLAj-dependent release of dioline from
hippocampal phosphol4)ids both in vitro (10-100;^M) and in vivo (20 mg/kg, Lp.) [199].
Rabin et d. [200] investigated the effect of EGb on the rate of FFAreincorporation into brain
phospholipids during reperfiision following ischemia in the gerbil brain by means of quantitative
autoradiography and biodiemical analysis. Isdiemia-reperfusk>n selectively reincorporated
arachidonic add (AA) into brain phospholipids. Pretreatmait with EGb (50 or 150 mg/kg/day,
for 14 days) accelerated AA reincorporation following isdiemia, suggesting that EGb
ameliorates the neurotoxic reaction caused by prolonged ^posure of the brain to high
concentrations of AAand its metabolites, and stabilizes the membrane bilayer.
Taken together, these results showed that EGb can prevent isdiemia-induced Na,K-ArPase
injury, and suppress hypoxia- and ECS-induced membrane phospholipid breakdown in the
brain, and bilobalide might be associated with its protective action. In addition, EGb reduces
AA-induced neuronal damage as a consequence of the increase in reincorporation of A A
Therefore, these medianisms might provide a possible explanation for neuroprotective
properties of EGb and bilobalide against oxidative damage.
Anti-PAF
Platelet-activating factor (PAF), a potent phospholipid inflammatory mediator, enhances

glutamatergic exdtatory synaptic transmission in the h^>pocampus [201]. Braquet et d, [202,
203] showed that ginkgolides, mainly ginkgolide B, act as potent antagonists of PAF in various
cell types. Pretreatment of ginkgolides (10 mg/kg/day, p.o., for 7 days) ameliorated
behavioral impairments assessed by the MacGraw stroke index, and inq)roved the
mitodiondrial respiration evaluated by the respiratory control ratio (RCR) following cêbral
ischemia obtained by bilatâl ligature of the common carotid arteries in Mongolian g^bils
[204]. The order of these effects in cerebral isdiemia was ginkgolide B > ginkgolide A >
ginkgolide C > ginkgolide J, and was correlated with that of their PAF antagonistic properties
described by Braquet et d, [203]. Consistent with this finding, liu et d. showed that both pre-
and post-hypoxic treatment with ginkgolide B (25 mg/kg/dose, two serial doses) decreased the
inddence of cerebral infarction in hypoxic ischemic brain injury of immature rats [205].
Moreover, Akisu et d, [206] found that endogenous PAF concentrations in brain tissue
markedly inaeased in the hypoxic-ischemic brain in immature rats. Pretreatment with EGb
reduces endogenous PAF concentrations in cerebral hypoxic-ischemic brain injury of immature
rats as compared with controls. These results indicate that the PAF-antagonistic activity of the
ginkgolides contributes to the neuroprotective effect against brain injury associated with an
episode of the post-isdiemic phase. This idea was supported further by the observation that in
primary neuronal cultures isolated from onbryonic rat cerebral cortex, ginkgolide B
demonstrates protective effects against glutamate neurotoxidty involving PAF [207].
189
It has been demonstrated that ginkgolide B prevents bng-tenn potentiation (LIP) induced
by PAF in the h^}pocampus [208] and in the voitral part of the medial vestibular nudei [209].
These results suggest that PAF might act as a retrograde messenger in ITP, which activates the
presynaptic mechanisms enhancing the glutamate release. Izqui^do et d, found that pre- or
immediate post-training intrahippocampai or intraamygdala infusion of the PAF antagonist,
ginkgolide B, produces amnesia for avoidance tasks. These results support the idea that PAF
may play a role in memory formatfon [210].
Amine Uptake and Membrane Fluidity
Protonged incubatbn of synaptosomes prqiared from the striatum in the presence of ascorbk:
add (10^ M) decreases the ability of synaptosomes to take up [^H]dopamiae. Similar
inhibition is also observed in the ability of cortical synaptosomes to take up [^H]5-HT.
Furthermore, this decrease is potaitiated by addition of Fe^* tons. EGb (4-16 /igAni), in
particular its flavonoid fraction, prevents the reduction in the ability of synaptosomes to take up
either 5-HT or dopamine [211]. Moreover, EGb (10 //g/ml) prevents a decrease in binding of
[^H]GBR12783, the dopamine uptake inhibitor, to dopamine recq)tors in the presence of the
combination of ascorbic add/Fe^* ions. These results suggest that EGb-induced prevention of
the impainnent of the ability of synaptosomes to take up [^H]amine by the ascorbic add/Fe^*
ions is related its inhibitory properties in generation of free radicals. However, EGb does not
modify the inaeased [^H]dopamine release that is triggered by high potassium concentrations in
the presence of the combination of ascorbate/Fe^*, suggesting that the vesicular exocytotic
dopamine release does not seem to depend upon peroxidation [212].
Furthermore, Ramassamy et d. [213] showed that the combination of ascorbic acid/Fe^*
ions could deaease synaptosomal membrane fluidity measured by fluorescence polarization
using 1,6-diphenyl 1,3,5-hexatriene in a concentration-dq)endent manner. Free radical
generation by ascorbic add/Fe^* results in a decrease of membrane fluidity through the
peroxidation of neuronal membrane Upids. These membrane altCTations were prevented by
either EGb (2-16 ;<g/ml) or quercetin (0.1 fiM). Regarding neuronal membrane fluidity, Stoll
et d, [105] assessed the effects of EGb in vivo in young (3 months old), middle-aged (12
months old) and aged (22 to 24 months old) female NMRI mice. There was a significant
improvement in membrane fluidity in the aged animals treated with EGb at a dose of 100 mg/kg
for three weeks.
Taken together, these results reveal that EGb prevents not only the ascorbic add/Fe^*-
induced impairment in amine uptake and membrane fluidity by generating free radicals, but also
the impairment in age-related membrane fluidity. This preventive effect of EGb is probably
due to itsradicalscavenging properties.
CONCLUSION
The convincing evidence from the experimental work presented hare supports the conclusion
that EGb is effective for the treatment of dementia and cerebral insuffidency. To sum up the
major characteristics of the pharmacological activities of EGb, particularly in the CNS, the
following conclusions can be reached. EGb improves hypoxic tolerance and protects cerebral
neurons against s^ptosis-, edema- and ischemia-induced injury. In addition, EGb inhibits
age-related impairment of cerebral neurotransmitters, mduding muscarinic receptors, and 2-
190
adreooo^tors and 5-HT,A leocptois, increases the uptake of cboiine, and improves
deterioration in cognitive functions, induding learning and memory, with advancing age. It
is worth noting that EGb demonstrates evid^ce of neuroprotective effects against various age-
related physiological changes and injuries, although EGb has hardly any influence under normal
conditions; that is, EGb may fadlitate a spontaneous cure of damage occurring in the brain with
advancing age. Hierefore, such attributes appear to be associated with the observation that
EGb is effective in mild to moderately affected patients with ALdieimer's disease and
multi-^nfarct donentia.
It is accepted that flavonoids and teq)enes, composing 24% and 6% of the standardized
extract, respectively, contribute to the pharmacological activities of EGb as active constituents.
Ginkgolides and bilobalide are especially unique constituents in Ginkgo biloba^ and have been
found in no other plants. It has been generally considered diat the anti-oxidant activity of
flavonoids and the platelet-activating factor antagonism of teipenes are usefiil for explaining the
greater part of the restorative and/or neuroprotective properties of EGb as described above.
On the other hand, as far as we know, there seem to be no publications concerning clinical
trials demonstrating the effectiveness of other antioxidants or anti-PAF antagonists on dementia.
Therefore, the possibility of a separate yet unidentified mechanism remains to be clarified. For
example the activities of bilobalide on membrane phospholipids and mitochondria may
contribute to other defense medianisms of EGb. Furthermore, a comprehensive
understanding of the greater part of EGb constituents, Le., those other than flavonoids and
terpenes, is still lacking. FurthCT elucidation of the constituents of EGb is thus necessary.
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[213] Ramassamy, C ; Girbe, F.; Christen, Y; Costentin, J. Free RacL Res. Commun.,
1993,19,341.
Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol 28
CHEMISTRY AND BIOLOGICAL ACTIVITIES OF

ISOPRENYLATED FLAVONOIDS FROM MEDICINAL
PLANTS (MORACEOUS PLANTS AND
GLYCYRRHIZA SPECIES)
TARO NOMURA, TOSHIO FUKAI, and YOSHIO HANO
School of Pharmaceutical ScienceSy Toho University, 2-2-1 Miyama,
Funabashiy Chiba 274-8510, Japan
ABSTRACT: Among a large number of phenolic compounds isolated

from natural source, various isoprenoid-substituted phenolic compounds
have often been found in plants. Moraceous plants and licorice (Glycyrrhiza
species) are rich sources of the isoprenoid-substituted phenolic compounds,
including flavonoids. Some of the Morus flavonoids, such as kuwanons G
and H, have been regarded as optically active Diels-Alder type adducts.
Furthermore, some of the isoprenylated-flavonoids from the moraceous
plants and licorice showed the interesting biological activities. This article
reviews the biological activities of the isoprenylated-flavonoids from the root
barks and/or barks of moraceous plants and from Glycyrrhiza species by
our group. The chemical studies conceming the biological activities of these
compounds are also described briefly.
200
I. INTRODUCTION (SURVEY OF ISOPRENYLATED

FLAVONOIDS FROM THE MORACEOUS PLANTS AND
GLYCYRRHIZA SPECIES)
Moraceous plants
Moraceae comprise a large family of sixty genera and nearly 1400

species, including popular species such as Artocarpus, Morus, and Ficus,
which are found in temperate, subtropical, and tropical regions of the
world. Mulberry tree, a typical plant of genus Morus, has been widely
cultivated for its leaves, which serve as indispensable food for silkworm.
In addition, the root bark of the mulberry tree (Mori Cortex, Morus alba
L. and other of genus Morus, "Sang-Bai-Pi" in Chinese, "Sohakuhi" in
Japanese) has been used as a material of traditional Chinese medicine for
an anti-inflammatory, diuretic, antitussive, expectorant, and antipyretic
purposes [1-3]. The earliest written reference to the use of Mori Cortex
is contained in the "Shen Nong Ben Cao Jing" (SNBCJ, shin-no hon-zo
kyo in Japanese), the first Chinese dispensatory whose original
anonymous volumes probably appeared by the end of the third century
[4,5]. In the Chinese book, 365 crude drugs are classified into three
classes (upper: plants with lowest side-effects and nontoxic, useful for
health care; middle: plats that are nontoxic or possess only weak toxicity
in whose use care must be exercised; lower: toxic and only for clinical
use. Mori Cortex is described as belonging to the middle class. The
crude drug is used as a component in traditional Chinese medicinal
prescriptions, such as "Wuhu Tang (Gokotou in Japanese)" and
"Mahuang Lianqiao Chixiaodou Tang (Maou-rensho-shakushozu-tou)",
which are applied clinically as a therapy for bronchitis and for nephritis,
respectively [6]. On the other hand, a few pharmacological studies on
the mulberry tree had demonstrated a hypotensive effect of the extract in
rodents [7,8]. Considering the above and reports described later, it was
suggested that the hypotensive constituents would be a mixture of many
phenolic compounds. Our interests were focused on the phenolic
constituents of the mulberry tree. So, we have studied phenolic
compounds of the mulberry tree and the related plants [8].
About seventy kinds of new phenolic compounds could be isolated
from Japanese cultivated mulberry tree {Morus alba, M. bombycis, and M,
Ihou) and Chinese crude drug "Sang-Bai-Pi" (the root bark of Chinese
mulberry tree). Most of them are isoprenylated flavonoids. Among
them, kuwanon G (1) was the first isolation of the active substance
exhibiting the hypotensive effect firom the Japanese Morus alba root bark
[9]. Furthermore, kuwanon G (1) and its isoprenylated derivative
kuwanon H (2) [10] are considered to be formed through an enzymatic
Diels-Alder type reaction of a chalcone and a dehydrokuwanon C or its
201
sanggenon A
morusin (3) : R = H
(4): Ri = CH2CH=CMe2. R2 = OH
artonin E (7): R = OH
(4'): Ri = OH, R2 = CH2CH=CMe2
kuwanonG(1):R = H
kuwanon H (2): R = CH2CH=CMe2
OH O
artobiloxanthone (8)
OH
sanggenon C OH 0
(5): Ri = CH2CH=CMe2, R2 = OH sanggenon 0 (6)
cycloartobiloxanthone (9)
(5'): Ri = OH, R2 = CH2CH=CMe2
OH
brosimoneA(13) soroceal (15)
Fig. (1). Structures of compounds 1 - 1 5 from moraceous plants.

202
equivalent, Fig. (1). Since that time, about forty kinds of Diels-Alder
type adducts, structurally similar to that of 1 have been isolated from
Morus species. These Diels-Alder type flavonoids are characteristic
constituents oiMorus species [8,11-14].
Morusin (3), a flavone derivative, isolated from the root bark oi Morus
alba L., as a main isoprenylated flavonoid, has a structure bearing an
isoprenoid moiety at the C-3 position and a 2',4'-dioxygenated pattem in
the B ring [15]. These features are one of the characteristics of the
isoprenylated flavonoids of Morus root bark.
Furthermore, from the Chinese crude drug "Sang-Bai-Pi" purchased in
Japanese market, our group reported a series of isoprenylated flavonoids,
such as sanggenons A (4) [16] and C (5) [17]. Recently, the structure of
sanggenons A and C were revised from 4' and 5' to 4 and 5, respectively,
[18], Fig. (1). Sanggenon C (5) seems to be a Diels-Alder type adduct
of a chalcone derivative and a dehydroprenyl (=3-methyl-1,3-butadienyl)-
phenol having a sanggenon A type partial structure. From the root bark
of one of the Chinese mulberry tree, Morus cathayana, a series of
isoprenylated flavonoids could be isolated [19-21]. Some of the
flavonoids are the sanggenon A type flavanones (SATF), 3-hydroxy-
flavanone having a prenyl (=3-methyl-2-butenyl) group at 2 position and
an ether linkage between C-3 and C-2' positions, such as 4 and 5. Most
SATFs are (27?,55)-flavanones, sanggenons A (4), C (5), L, M (100),
sanggenols F, G, and J, and soroceins D and F [22], but sanggenon O (6)
is (2iS',ii?)-flavanone [23]. The stereochemistry at C-2 and C-3 of
sanggenons B (45), Fig. (6), D (33), E, P (sorocein H), S, sorocein E, and
sanggenols H and I is still unclear. Earlier studies of flavonoids and
stilbenes with one or more isoprenoid groups (prenyl group,
2,2-dimethylpyran ring, geranyl group, famesyl group, etc.) from Morus
species have been summarized in review articles [8,11-13,24-26].
On the other hand, the plants of Artocarpus species distribute over the
tropical and subtropical regions, and have been used as traditional folk
medicine so called "Jamu" in Indonesia against inflammation, malarial
fever and so on. Many kinds of isoprenylated flavonoids have also been
isolated from Artocarpus species by Venkataraman's group and other
several groups [27-30]. Our group also studied the constituents of
Indonesian Artocarpus species, such as A. heterophyllus, A, communis, A.
rigida, A. venenosa (^Paratocarpus venenosa), and A. altilis, a
moraceous plant from Sri Lanka [30-32]. About seventy kinds of
isoprenylated flavonoids have been isolated from these Artocarpus
species. The compounds, except some ones, have a characteristic
structure bearing an isoprenoid side chain at the C-3 position of flavone
skeleton, and the B ring has a 2',4',5'-trioxygenated pattem, such as
artonin E (7) corresponds to 5'-hydroxymorusin [31]. In addition to the
feature, some of the flavone, such as artobiloxanthone (8) and
203
cycloartobiloxanthone (9) have a unique structure having the C-C linkage

between an isoprenoid side chain at the C-3 position and the 6'-carbon of
the B ring of flavone skeleton. This C-C linkage was considered to be
synthesized biogenetically through a phenol oxidation in the plants
[29,30]. These flavonoids are characteristic constituents oi Artocarpus
species.
Some of the Artocarpus flavonoids, such as artonin I (10), have been
regarded as intermolecular [4+2] cyclo-addition product from the
isoprenyl portion of a dehydroprenylphenol, as a diene, and the a,p-
unsubstituted bond of a chalcone skeleton, as a dienophile. As artonin I
(10) was considered to be formed through the Diels-Alder type reaction
of a chalcone derivative, morachalcone A (11) and artocarpesin (12), the
precursor artocarpesin (12) was added to the Morus alba cell cultures to
produce artonin I (10) [32]. Earlier studies of the isoprenylated phenols
from Artocarpus species have been summarized in review articles
[27,28,30].
Most Diels-Alder type adducts with a dehydroprenylflavonoid and a
chalcone (DADCs) have been isolated from Asian Morus and Artocarpus
species. However, some DADCs have also isolated from American
moraceous plants. From Brosimopsis oblongifolia, a Brazilian
moraceous plant, a series of DADCs, brosimone-family, were isolated.
One of them, brosimone A (13) is a unique adduct, and it is likely to form
through an intra-molecular [4+2] cycloaddition reaction between the
dehydroprenyl moiety at the A' ring, as the diene, and the a,P-double
bond of the chalcone skeleton (A ring-Ca-B ring), as the dienophile, of
brosimone D (14) [33], Fig. (1). From a Brazilian moraceous plant,
Sorocea bonplandii, ketalized Diels-Alder type adducts, such as soroceal
(15), have been isolated [34]. On the other hand, from Paraguayan
moraceous plants, Sorocea bonplandii, our group isolated the similar
ketalized Diels-Alder type adducts along with a unique adducts,
sorocenol B (16) [35], Fig. (2), which may be a derivative induced from
the Diels-Alder type adducts between a chalcone derivative and a de-
hydroprenylated resorcinol through the oxidative reaction.
A series of isoprenylated flavonoids has been isolated from the
following moraceous plants: Brosimopsis oblongifolia, a Brazilian plant
[36], Chinese Cudrania tricuspidata [37], and Taiwanese and Chinese
Cudrania cochinchinensis [38]. Many xanthones with one or two
isoprenoid groups have also been isolated from these Cudrania species
[11]. Latex from the wood of Antiaris toxicaria, a toxic Indonesian
plant, has been used for arrow poison. From the root bark of the plants,
antiarones A (17) and B (18) [39], the first examples of isoprenylated
aurone derivative, have been isolated along with the unique
dihydrochalcone derivatives, antiarones J (19) and K (20) [40], Fig. (2).
These compounds (19 and 20) are biogenetically considered to be formed
204
through a cyclization accompanied by hydration of the isoprenyl group

attached to the B ring of chalcone derivatives such as antiarone E (21)
[41]. Two new cyclomonoterpene-substituted isoflavones, ficusins A
(22) and B were isolated from the Indonesian moraceous plant, Ficus
septica Barm F [42]. Earlier studies of phenolic compounds (flavonoids,
xanthones, benzaldehydes) have been summarized in review articles
[8,11,30].
OH 0
antiarone A (17) antiarone B (18)

sorocenol B (16)
OH O
antiarone J (19): Ri = OH. R2 = CH2CH=CMe2
antiarone K (20): Ri = OMe, R2 = H antiarone E (21)
Fig. (2). Structures of compounds 1 6 - 2 2 from moraceous plants.
Glycyrrhiza species
Licorice (liquorice, kanzoh in Japanese, gancao in Chinese) is the name

applied to the roots and stolons of some Glycyrrhiza species
(Leguminosae or Fabaceae) and has been used by human beings from
ancient times. The genus Glycyrrhiza consists of about 30 species and
chemical studies have so far been carried out on 15 of them. Glycyrrhizic
acid (110) is the major triterpenoid saponin in licorice root and the main
sweetener of the herb. The saponin has been isolated from G. glabra^ G.
uralensis, G. inflata, G. aspera, G. korshinskyi, and G. eurycarpa, and
thus, these plants are generally accepted as licorice. In European
countries, G. glabra is chiefly used as licorice. On the other hand, in
Asian countries, G. glabra, G. uralensis, G. inflata, and G. eurycarpa are
used as licorice. Following extraction, the herb yields the licorice
products of commerce which are used as sweetening agents, flavoring for
American type tobaccos, chewing gums, candies, etc., as a depigmenta-
tion agent in cosmetic, and as pharmaceutical products, e.g., anti-ulcer.
205
anti-hepatitis medicines, antitussives, etc. Among them the most

important industrial use of the herb is in the production of additives as
flavor and sweetening agents [43].
In SNBCJ, licorice is described as belonging to the upper class and is
recommended for lengthen one's life span, for improving health, for
cures for injury and swelling, and for its detoxification effect. One
hundred ten prescriptions are recorded in the earlier Chinese medicinal
book "Shang Han Lun", where seventy prescriptions include licorice [43].
In the Japanese market, Chinese licorice is classified by its place of
production, e.g., Northeastem licorice (Touhoku kanzoh in Japanese),
Northwestem licorice (Seihoku kanzoh), Xinjiang licorice (Shinkyou
kanzoh), etc. Among these licorice, Northeastem licorice had been
identified as G. uralensis, but the original plants of the others had been
unidentified. We investigated the phenolic constituents of certain
Glycyrrhiza species identified by authorities, and many phenolic
compounds were isolated from these plants [43].
The main phenols of licorice are glycosides of liquiritigenin (100) and
isoliquiritigenin (70), e.g., liquiritin (113), isoliquiritin, liquiritin apioside,
licuraside, etc. [43]. As minor phenolic compounds, many isoprenoid-
substituted flavonoids, chromenes, dihydrophenanthrenes, and dihydro-
stilbenes were isolated from Glycyrrhiza species. Some of them
characterized each plant [43^5]. The 5- and 6-positions of most
flavonoids from European licorice are unsubstituted, but the 5-position of
flavonoids from Chinese licorices is generally substituted with a
methoxyl group or a hydroxy1 group (the 5-OH of some compounds
forms ether linkage with an isoprenoid group at the 6-position). For
example, the main isoprenoid-substituted flavonoid of G. glabra var.
typica, Russian licorice, is a pyranoisoflavan, glabridin (23). The
5-position of most flavonoids from the plants is unsubstituted, e.g., 23,
glabrene (24), glabrol (25), 3-hydroxyglabrol (26), etc.. Fig. (3).
glabridin (23) giabrene (24) glabrol (25): R = H

3-hydroxyglabrol (26): 3*-(Y,y-dimethylallyl)-
R = OH kievitone (27)
Fig. (3). Structures of compounds 23 - 26 from licorice {Glycyrrhiza glabra).

206
On the other hand, from Chinese and Kyrghiz G. glabra, both

5-unsubstituted flavonoids (e.g., 24) and 5-oxygenated flavonoids, e.g.,
3'-(y,y-diniethylallyl)-kievitone (27), have been isolated. Nevertheless,
5-position of the most flavonoids with one or two isoprenoid groups from
these plants is substituted with a hydroxyl or a methoxyl group. The
main isoprenoid-substituted flavonoid of the Kirghiz licorice is
compound 27, but the isoflavan (23) has not been isolated [46-49],
Isoprenoid-substituted flavonoids isolated from commercial Kyrghiz
licorice and European licorice that was cultivated in Japan were
summarized in Table 1 [49-51].
Table 1, Flavonoids isolated from Kyrgyz and European Glycyrrhiza glabra
Kirghiz [49] European [50,51 ]
3'-(Y,Y-Dimethylallyl)-kievitone (27) +-H-¥

Glisoflavanone (3',6-diprenyl-2',4',5,7-tetrahydroxyisoflavanone) +++ -
Glyasperin A (77) ++
Glyasperin C (61) ++
Glyasperin D (62) +++
Isoderone(DMP;4',3']-5,7-dihydroxyisoflavone)'' +
Semilicoisoflavone B (68) ++
8-(Y,Y-Dimethylallyl)-wighteone (58) ++
Gancaonin G (60) +
Gancaonin H (DMP;4',5']-6-prenyl-3',5,7-trihydroxyisoflavone) ++
1-Methoxyphaseollidin (125) +++
Edudiol (3,9-dihydroxy-l-methoxy-2-prenylpterocarpan) ++
Glabrene (24) ++ +++
Glabridin (23) - ++++
4'-C>-Methylglabridin (123) - ++
Hispaglabridin A (3'-prenylglabridin) - ++-H-
Glabrol (25) - +++
3-Hydroxyglabrol (26)* - +++
Glabrone (DMP;4',3']-2',7-dihydroxyisoflavone) - ++
Medicarpin (3-hydroxy-9-niethoxypterocarpan) - ++++
Shinpterocarpin (DMP;3,4]-9-hydroxypterocarpan) - +++
Euchrenone as (DMP;4',3']-7-hydroxy-8-prenylflavanone) - ++
Glyinflanin K (2DMP;7,8, ;2',3']-isoflavan) - ++
Glyinflanin G (2DMP;4,5, ;4',3']-2',3-dihydroxychalcone) - ++
Kanzonol U (DMP;2',3']-4',6-dihydroxy-2-arylbenzofuran] - ++
Kanzonol V (DMP;2',3']-4',6-dihydroxy5-prenyl-2-arylbenzofuran) - +++
Kanzonol W (DMP;7,8]-2',4'-dihydroxy-3-arylcoumarin) - +++
Kanzonol X (3',8-diprenyl-2',4',7-trihydroxyisoflavan) - +++
Kanzonol Y (3,5'-diprenyl-a,2',4,4'-tetrahydroxy-dihydrochalcone) - +++
Kanzonol Z (DMP;7,8]-3,4'-dihydroxy-3'-prenylflavanone) - +++
3-Hydroxyparatocarpin C** - +++
Yields from dried licorice roots: -H-i-H=more than 0.01%; +-H-=between 0.01 and 0.001%; ++=0.001-0.0001%;
+=1-0.1 ppm. • The compound was obtained from the stolons. ° 2,2-dimethylpyrano[b=DMP. ** Tentative
name used here (DMP;4,5]-3'-prenyl-2',3,4'-trihydroxychalcone).
The difference of the substituents at C-5 is expected that European

and Chinese licorices exhibit different actions in therapeutically use.
For example, 5,6-disubstituted isoflavans do not showed a potency of
207
anti-HIV activity in vitro, but two isoflavans with no substituent at both

5- and 6-positions obtained from Erythrina lysistemon (Leguminosae)
have the activity as described later [52].
As described the above, moraceous plants and Glycyrrhiza species are
rich sources of isoprenylated phenolic compounds. The phenolic nuclei
having the isoprenoid-derived substituents, e.g., simple isoprene or a
monoterpenoid, vary over a wide range from a simple phenol to
complicated ones. Some of the moraceous plants studied by our group
have been used as traditional herbal medicines in the native countries. It
is interesting to clarify the relationship between the usage and biological
activities of the isoprenylated phenolic compounds. So we studied some
of the biological activities of these compounds. This article reviews the
biological activities of the isoprenylated flavonoids isolated from the
moraceous plants and isoprenoid-substituted phenols (flavonoids,
xanthones, dihydrostilbenes, and dihydrophenanthrenes) from
Glycyrrhiza species by our group and other several groups.
11. HYPOTENSIVE ACTIVITY OF ISOPRENYLATED FLAVO-

NOIDS FROM THE ROOT BARK OF MORUS SPECIES
The first report for the hypotensive effect of the mulberry tree was
presented by Fukutome in 1938, who asserted that oral administration of
the hot water extract of the mulberry tree showed a remarkable
hypotensive effect in rabbits [53]. Ohishi reported the hypotensive
effect of the ethanol extract of mulberry root bark [54]. Suzuki and
Sakuma reported that the hypotensive activity seemed to be due to
phenolic substances, and that the effect disappeared on acetylation [55].
Later, Katayanagi, et aL reported that the ether extract of the root bark
gives to rabbit (6 mg/kg, i.v.) showed a marked hypotensive effect and
that the active constituents seemed to be a mixture of unstable phenolic
compounds [56]. Tanemura ascribed the activity of mulberry root bark
to acetylcholine and its analogous presumably contained in the alcohol
soluble fraction, and that the hypotensive constituents produced a
yellowish-brown precipitate on treatment with Dragendorff reagent [57].
Yamatake, et al reported that n-butanol- and water-soluble fractions of
mulberry root bark had similar effect except for those on the
cardiovascular system. Both fractions showed cathartic, analgesic,
diuretic, antitussive, anti-edema, sedative, anticonvulsant, and
hypotensive actions in mice, rats, guinea pigs and dogs [7]. On the
beginning of our study of mulberry tree, the hypotensive constituents had
not been identified. In view of the reports, we assumed that the
hypotensive compounds of the plant would be a mixture of unstable
208
phenolic compounds and therefore undertook a study of the phenolic

constituents of the root bark of the cultivated mulberry tree.
The root bark of the cultivated mulberry tree was extracted
successively with n-hexane, benzene, and methanol. The methanol
extract, 1-20 mg, showed a dose-dependent decrease in arterial blood
pressure in pentobarbital-anesthetized rabbit, Fig. (4). The extract was
fractionated successively by silica gel column chromatography (C.C.),
polyamide C.C, silica gel preparative (p.) TLC, and p. HPLC leading to
isolated of kuwanons G (1, 0.2% yield) [9] and H (2, 0.13% yield) [10].
The root bark of Moms alba
n-Hexane
Residue Extract
Benzene
Residue Extract
I Methanol C.C, p. TLC
-T
Residue extract Morusin (3), kuwanons C (42), D, E (43), F,
Ethyl acetate soluble portion oxydihydromorusin (46),
I C.C, p. TLC, p. HPLC mulberroftiran A (47)
Kbwanons G (1), H (2), L (44), M (35), albanol B (97)

mulberrofurans C (28), F (29), and G (30)
Fig. (4). Isolation procedure of flavonoids from the root bark of Morus alba.
mmHg
PN n > < l i < M H H H H M M ( H i t i i
kuwanon G 1 mg/kg i.v.

mmHg
tsmmm |ioo
50
iilSliSirJ'^^
10 s
kuwanon H I mg/kg i.v. '
Fig. (5). Effects of kuwanon G (1) and kuwanon H (2) on blood pressure. Electrocardiogram (ECG),
phrenic nerve discharge (PN), and electroencephalogram (EEG) in a gallamine-immobilized rabbit.
209
Both compounds (1 and 2) almost equally caused decrease of arterial

blood pressure in a dose dependent and reversible manner at the dose of
between 0.1 and 3 mg/kg, i.v. in pentobarbital-anesthetized as well as in
un-anesthetized, gallamine-immobilized rabbits. Fig. (5), [58].
These hypotensive actions of kuwanons G (1) and H (2) were not
modified by atropine or eserine, suggesting the non-cholinergic nature
origin. Furthermore, neither propranol nor diphenhydramine affected
their actions on the arterial blood pressure. Although they produced no
significant change in both electrocardiogram (ECG) and respiration when
administered intravenously in rabbits. The hypotensive effects of kuwa-
non G (1) and H (2) did not accompany with heart rate change [58]. In
pentobarbital-anesthetized pithed dogs, kuwanons G (1) and H (2) also
significantly decrees of femoral arterial blood pressure. These effects
suggested that mechanism of hypotensive effects of kuwanons G (1) and
H (2) mediated through peripheral system.
Mulberrofurans C (28) [59], F (29) [60], and G (30) [60], Fig. (6),
were also isolated as hypotensive components from the mulberry tree.
Mulberrofuran C (28) is considered to be formed by a Diels-Alder type of
enzymatic reaction process of a chalcone derivative and dehydromoracin
C (31) or its equivalent. Furthermore, mulberrofurans F (29) and G (30)
seems to be Diels-Alder type adducts derived from chalcomoracin (32)
and mulberrofuran C (28), respectively, by the intra-molecular ketali-
zation reaction of the carbonyl group with the two adjoining hydroxyl
groups, 3'(5')-OH and 2"-0H. Intravenous injection of mulberrofuran C
(28, 1 mg/kg) produced a significant hypotension (37 mmHg fall) in
rabbit (male, 3.3 kg) anesthetized with pentabarbital sodium (30 mg/kg).
Single intravenous injection of mulberrofurans F (29) and G (30) (both
0.1 mg/kg) caused a marked depressor effect in rabbit by 26 mm Hg and
16 mm Hg, respectively.
On the other hand, in Japan, "Sang-Bai-Pi" (the root bark of Chinese
mulberry tree) imported from China has been used as an herbal medicine,
hence a study of the components of this crude drug purchased in the
Japanese market was undertaken. Its phenolic components are different
from those of Japanese mulberry tree. For example, morusin (3) and
kuwanon G (1) are the main phenolic components of Japanese mulberry
tree, in the case of "Sang-Bai-Pi", these components are minor ones,
while sanggenons A (4) [16], C (5) [17], and D (33) [61] are the main
components [24]. Sanggenons C (5) and D (33) showed the hypotensive
effects as follows: Sanggenon C (5) caused transient decrease in arterial
blood pressure at the doses of 1 mg/kg in pentobarbital-anesthetized
rabbit by 15 mm Hg, while at the doses of 5 mg/kg the compound (5)
caused a transient decrease by 100 mm Hg, which continued for more
210
mulberrofuran C (28): R = H
mulberrofuran F (29): R = CH2CH=CMe2
chalcomoracin (32): R = CH2CH=CMe2
mulberrofuran G (30): R = H
kuwanon E (43)
sanggenon B (45)
Fig. (6). Structures of flavonoids (28 - 44) from moraceous plants.

211
than one hour by 15 mm Hg [17,62]. Sanggenon D (33) caused a

transient decrease at the dose of 1 mg/kg in pentobarbital and urethane
anesthetized male Wister strain rat by 35 mm Hg, while the compound
(33) caused a decrease by 80 mmHg at the doses of 1 mg^g in
spontaneously hypertensive rat [61,63].
III. ANTI-TUMOR PROMOTING ACTIVITY OF MORUSIN (3)
Cancer chemoprevention is the most important subjects in cancer

research at present and is a new medical strategy for cancer prevention,
which was established by recent understanding of molecular multistage
carcinogenesis in humans. To find nontoxic cancer preventive agents,
Fujiki and his coworker studied natural products derived from marine and
plant sources [64,65]. In 1987, Yoshizawa, et aL reported that (-)-
epigallocatechin gallate (EGCG), which is a main constituent of green tea,
inhibited tumor promotion by teleocidin in mouse skin [66]. In 1988,
Fujita, et aL reported the inhibitory effect of EGCG on carcinogenesis
with 7V-ethyl-A^-nitro-A^-nitrosoguanidine in mouse duodenum [67]. On
the other hand, in the course of our examination the constituents of the
Morus root bark, we found the following novel photo-oxidative
cyclization. When a solution of morusin (3) in chloroform (CHCI3) was
irradiated using high-pressure mercury lamp, morusin hydroperoxide (34),
Fig. (6), was obtained in ca, 80% yield [68]. The reaction did not occur
in the dark and was depend on the solvent; the reaction occurred in low
polar or nonpolar solvent such as CHCI3 and benzene, but not in protic
solvent. The reaction mechanism was suggested as follows [69]:
morusin (3) in the ground state interacts with an oxygen molecular to
form a contact charge transfer complex [3 O2] (CCTC). On
irradiation, the CCTC gives an excited charge transfer state that
presumably leads to reactive species such as free radicals as described in
Fig. (7). Recently, the proof of presence of the CCTC was provided by
laser desorption/ionization time-of-flight mass spectrometry of 3 [70].
The hydroperoxide (34) was also obtained with the oxidation of morusin
(3) with singlet oxygen or radical initiator [71].
HO^^s^ÔH
hv
34
•OOH
Fig. (7). Reaction mechanism of photo-oxidative cyclization of morusin (3).

212
This photoreaction and the relative reaction of morusin (3) along with
the anti-tumor promoting activity of EGCG encouraged us to examine the
anti-tumor promoting activities of a series of isoprenylated flavonoids
isolated from Morus species. First we examined the inhibition against
three biochemical effects; the specific binding of ^H-12-O-tetra-
decanolylphorbol-13-acetate (TPA) to mouse particulate fraction, the
activation of Ca^'^-activated phospholipid-dependent protein kinase
(protein kinase C) with teleocidin, and induction of ornithine
decarboxylase (ODC) with teleocidin in mouse skin [72]. Interestingly,
of the eight isoprenylated flavonoids, morusin (3), kuwanons G (1) and M
(35), mulberroforan G (30), and sanggenon D (33) gave similar results in
these biochemical tests as described in Table 2.
Table 2. Effects oi Morus flavonoids on biological and biochemical activities
Inhibiting of Inhibition of Inhibition of

specific [^H]TPA activation of ODC induction
binding protein kinase C (%)
(ED50 jimol/L) (ED50 fimol/L)
Morusin (3) 57 80 43
Kuwanon G (1) 99 40 34
Kuwanon H (2) 100 80 -35
Kuwanon M (35) 85 22 25
Mulberrofuran G (30) 34 46 10
Sanggenon A (4) 62 80 -62
Sanggenon C (5) 48 46 -17
Sanggenon D (33) 60 42 17
100
^ o
Concentration (mol/L) of morusin (3)

Fig. (8). Effects of morusin (3) on specific binding of [^H]TPA to a mouse skin particulate fi-action.
Various concentrations of morusin (•) or TPA (o) were incubated with a particulate fi-action of mouse skin in
the presence of 4 nmol/L [^H]TPA for 2 h at 4°C, and the assay mixture was filtered on glass filter membrane
with acetone cooled in a dry ice-ethanol bath. Non-specific bindings were measured in the presence of
500-fold excess of unlabelled TPA.
213
Of these five compounds, morusin (3) is the least toxic and can be
isolated as one of the main phenolic compounds from the root bark.
The more detailed data for the above these biochemical tests of
morusin (3) were as follows [73]. As shown in Fig. (8), morusin (3)
caused dose-dependent inhibition of the specific binding pHJTPA to a
mouse skin particulate fraction. The concentration of morusin (3) for
50% inhibition (ED50) was 57 |amol/L, whereas that of unlabelled TPA
was 4 nmol/L.
As morusin (3) was assumed to interact with the phorbol ester receptor,
we examined whether it inhibited the activation of protein kinase C by
teleocidin in vitro [73]. Fig. (9) shows that morusin (3) inhibited the
phosphorylation of histone type III-S by protein kinase C dose-dependent
and that 80 |imol/L morusin caused 50% inhibition.
100 VA
o
50
a
0.
VA 10- 10-*
\o-
Concentration (mol/L) of morusin (3)
Fig. (9). Inhibition by morusin (3) of activation of protein kinase C by teleocidin in vitro.
The assay mixture (0.25 mL) contained 20 jimol/L CaCh, 7.5 |ag of phosphatidylserine, 2.3 (^mol/L teleocidin,
and various concentrations of morusin (3) with 0.05 units of partially purified enzyme. Enzyme activity was
measured as the incorporation of ^^P from [7-^^P]ATP into histone type III-S during incubation for 3 min. at
30^.
Furthermore, we examined the inhibition of the induction of ODC

induction by teleocidin in mouse skin. Application of 11.4 nmol
morusin (3) caused 43% inhibition of the induction of ODC by 11.4 nmol
teleocidin [73]. From the results of these three tests, morusin (3) might
inhibit the tumor-promoting activity of teleocidin on mouse skin.
As shown in Figs. (10) and (11), the percentage of tumor bearing mice
in the group treated with 7,12-dimethylbenz[a]anthracene (DMBA) plus
teleocidin reached 100% by week 15, o in Fig. (10). In contrast, the
onset of tumor formation was delayed 5 weeks by treatment with morusin
(3), • in Fig. (10), and the percentage of tumor-bearing mice in the group
treated with DMBA plus teleocidin and morusin (3) was 60% at week 20.
The average number of tumors per mouse in week 20 was also reduced
from 5.3, o in Fig. (11), to 1.1, • in Fig. (11), by morusin (3) treatment.
214
On the other hand, morusin (3) itself did not show a tumor promoting
activity on mouse skin, x in Figs. (10) and (11). From these results,
morusin (3) is an anti-tumor promoter judging from its ability to inhibit
the short-term effects induced by tumor promoters.
100
to
10 20 10 20
Weeks of promotion Weeks of promotion
Figs. (10) and (11). Inhibition by morusin (3) of tumor promotion by teleocidin in a two-stage
carcinogenesis experiment on mouse skin. Inhibition was achieved by a single application of 100 ^g of
DMBA, and teleocidin (2.5 }ig) and morusin (1 mg) were applied twice a week throughout the experiments.
As mentioned the above, morusin (3), kuwanon G (1), kuwanon M

(35), mulberroforan G (30), and sanggenon D (33) showed inhibitory
effects in the three biochemical tests. The anti-tumor promoting
activities of later four flavonoids with one or two isoprenoid groups have
not been tested in a two-stage carcinogenesis experiments, due to
limitations of their amounts available, but their inhibitory potencies to the
three biochemical tests were almost similar to that of morusin (3).
Furthermore, the twelve isoprenylated flavonoids from the moraceous
plants and two flavonol glycosides (48 and 49) from Epimedium species
(Berberidacaceae) [74] along with quercetin (50) were tested for
inhibitory effects on carcinogenesis by a test for inhibition of specific
binding of [^H]TPA to a mouse skin particulate fraction.
While the other biochemical tests and the inhibition of tumor
promotion of teleocidin in a two-stage carcinogenesis experiment have
not been carried out, due to limitation in their amounts available, some of
isoprenylated flavonoids from the moraceous plants showed the similar
inhibitory potencies to those of morusin (3) and the related compounds,
Figs. (6) and (12), as shown in Table 3.
On the other hand, EGCG and green tea extract are acknowledged
cancer-preventive agents in Japan [75,76]. Natural products with anti-
tumor promotion activity isolated from foodstuff and medicinal plants
have been summarized by Konoshima and his co-worker and Akihisa and
215
his co-worker [77,78]. Considering these results as well as the results of

biochemical tests and anti-tumor promoting activity of the isoprenylated
flavonoids from the moraceous plants in a two-stage carcinogenesis
experiment with teleocidin, the isoprenylated poly-phenolic compound
seems to be interesting compounds for finding cancer preventive agents
and the more detailed experiments should be carried out.
Table 3. Effects of the isoprenylated flavonoids on inhibition of specific [^H]TPA binding (ID50, ^mol/L)
Kazinol C (36) 80 Kuwanon L (44) 80

Kazinol E (37) 70 Sanggenon B(45) 95
Kazinol F (38) 98 Oxydihydromorusin (46) 95
Kazinol J (39) 90 Mulberroftiran A (47) >100
Kazinol M (40) 100 Ikarisoside A (48) >100
Kazinol N (41) >100 Ikarisoside B (49) >100
Kuwanon C (42) 80 Quercetin (50) >100
Kuwanon E (43) 83
OMe
oxydihydromorusin (46)
ikarisoside A (48): R = Rha
mulberrofuran A (47) ikarisoside B (49): R = Glu(1 ^ 2)Rha
OCH3
OH O
quercetin (50):
Ri = OH,R2=R3 = H
antiarone L (57)
cirsilioi (51):
Ri = H, R2 = 0Me, R3 = Me artonin H (56)
Fig. (12). Structures of flavonoids (46 - 57) from moraceous plants, Epimedium species, and test reagents
(50 and 51).
IV. INHIBITION OF ARTONIN E (7) AND RELATED

COMPOUNDS ON 5-LIPOXYGENASE
Previously, we reported the effects of Morus flavonoids on arachidonate

metabolism in rat platelet homogenates, such as inhibition of 12-hydroxy-
5,8,10-heptadecatrienoic acid (HHT), thromboxane B2, and 12-hydroxy-
5,8,10,14-eicosatetraenoic acid (12-HETE) [79,80]. As described in the
216
introduction, Artocarpus plants (Moraceae) have been used as traditional

medicine in Indonesia for swelling and malarial fever. This usage
seems to be expecting for effect of anti inflammation. As leukotrienes
are known to be chemical mediators of anaphylaxis and inflammation, a
number of compounds have been studied and developed as selective
inhibitors of 5-lipoxygenase, the enzyme initiating leukotriene bio-
synthesis from arachidonic acid. So the inhibitory effect of the
Artocarpus flavonoids against arachidonate 5-lipoxygenase was
examined [81]. Yamamoto, et aL screened various flavonoids, and
found that cirsiliol (51), Fig. (12), potently inhibited 5-lipoxygenase and
proposed two structural factors of the flavonoids for the specific
inhibitory activity, one is catechol type of the B ring and the other is the
presence of an alkyl-like side chain at the C-3 position [82,83]. We had
interesting for the inhibitory effects of a series of Artocarpus flavones on
the 5-lipoxygenase activity.
Seven Artocarpus flavonoids and morusin (3) were tested for their
inhibitory actions on arachidonate-5-lipoxygenase purified from porcine
leukocyte [84]. As shown in Fig. (13), the IC50 values varied depending
on the structural modification of the compound. The compounds having
three hydroxyl groups at positions 2\ 4\ and 5' on the B ring (compounds
7, 8, 52 and 55) were more potent inhibitors. Thus, the vicinal diol
partial structure was important for 5-lipoxygenase inhibition.
OH 0
OH o
artonin A (54)
heterophyllin (52) cycloheterophyllin (53)
Inhibitory effects (IC50 ± SD, N=3, îmol/L) on

arachidonate 5-lipoxygenase activity 1
Morusin (3) 2.9 ± 0.4

Artonin E (7) 0.36 db 0.03
Artobiloxanthone (8) 0.55 db 0.20
j Cycloartobiloxanthone (9) 1.3 ±0.2
Heterophyllin (52) 0.73 ±0.21 '
OH 0
Cycloheterophyllin (53) 1.6±1.0
artonin B (55) Artonin A (54) 4.3 ± 0.5
Artonin B (55) 1.0 ±0.1
Fig. (13). The inhibitory effect (IC50 ± SD) on arachidonate 5-lipoxygenase activity.
As shown in Fig. (14), 5-lipoxygenase was inhibited depending on the

concentration of artonin E (7), which gave the lowest IC50 (0.36 |Limol/L)
of all the eight compounds. On the other hand, morusin (3), which
217
lacked the 5'-hydroxyl group of artonin E (7), was a less potent 5-

lipoxygenase inhibitor (IC5o=2.9 |Limol/L). Artonin E (7) was
significantly more potent than cirsiliol (51, Fig. (12), IC5o=1.3 |Limol/L),
which was reported as a 5-lipoxygenase inhibitor. This finding was
consistent with the report that the inhibitory activity of cirsiliol (51) with
5-lipoxygenase was enhanced by introducing a lipophilic alkyl group at
the C-3 position of theflavoneskeleton.
Inhibitory actions of artonin E (7) and morusin (3) on other
mammalian arachidonate oxygenases were examined. Artonin E (7)
inhibited two 12-lipoxygenase from porcine leukocytes and human
platelets, 15-lipoxygenase from rabbit reticulocytes, and fatty acid
cyclooxygenase from bovine vesicular glands (IC5o=2.3, 11, 5.2, and 2.5
|amol/L, respectively). However, IC50 values for these oxygenases were
higher by one order of magnitude than that for 5-lipoxygenase. Morusin
(3) also inhibited these enzymes (except for human platelet 12-
lipoxygenase) with IC50 values of micro molar order as follows: two 12-
lipoxygenase from porcine leukocytes and human platelets, 15-
lipoxygenase from rabbit reticulocytes, and fatty acid cyclooxygenase
from bovine vesicular glands; IC5o=3.4, > 30, 3.3 and 1.6 |imol/l,
respectively. These results indicated that artonin E (7) was a relatively
specific inhibitor for 5-lipoxygenase. Thus, which the selectivity for
5-lipoxygenase was not observed with morusin (3). Significant
differences of IC50 values of artonin E (7) and morusin (3) between
porcine leukocyte 12-lipoxygenase and the human platelet
12-lipoxygenase should be noted since the leukocyte and platelet
12-lipoxygenase were distinct both catalytically and immunologically.
Concentration (|imol/L)
Fig. (14). Dose-dependent inhibition of 5-lipoxygenase by artonin E (7, • ) , morusin (3, o), and cirsiliol (51,
A).
V. INHIBITION OF ARTONIN E (7) AND RELATED

COMPOUNDS ON MOUSE TNF-a RELEASE AND THEIR
CYTOTOXIC ACTIVITIES
218
As described in Chapter III, morusin (3) has been found to be anti-tumor

promoter in a two-stage carcinogenesis experiment with teleocidin.
Considering the similarity of the structures between morusin (3) and
artonin E (7), artonin E (7) was expected to be an anti-tumor promoter.
Furthermore we found a novel photo-oxidative cyclization of artonin E
(7) as follow: photo-reaction of artonin E (7) in CHCI3 containing 4%
ethanol solution with high-pressure mercury lamp produced
artobiloxanthone (8) and cycloartobiloxanthone (9), and the treatment of
artonin E (7) with radical reagent (2,2-diphenyl-l-picrylhydrazyl: DPPH)
resulted in the same products, Fig. (15), [84].
OH 0
(±)-artobiloxanthone (8) (±) -cycloartobitoxanthone (9)
artonin E (7)
8 9
hv, 24 h. CHCI3 34% 3%

DPPH, 24 h, CHCI3 (in the dark) 70% 4%
Fig. (15). Photoreaction of artonin E (7) and the reaction with radical reagent.
As described in Chapter III, we have reported the photo-oxidative

cyclization on morusin (3). These results suggested that the photo-
OH 0 OH 0
(±) -cycloartobiloxanthone (9) (±)-artobiloxanthone (8)
Fig. (16). Plausible mechanism for the formation of artobiloxanthone (8) and cycloartobiloxanthone (9) from
artonin E (7).
219
oxidative cyciization of artonin E (7) may proceed through phenol

oxidation via the semiquinone radicals described in Fig. (16). This
chemical reactivity and the similarity of the structures between morusin
(3) and artonin E (7) encourage us to examine the anti-tumor promoting
activity of artonin E (7).
Recently, Fujiki, et al. proposed a new tumor promotion mechanism
applicable to human cancer development on the basis of experiment with
okadaic acid. They described that tumor necrosis factor-a (TNF-a)
induced by okadaic acid acts as a mediator of human carcinogenesis [65].
As briefly summarized in Fig. (17), okadaic acid inhibits the action of
protein phosphatase type 1 and 2A, resulting in the accumulation of
phosphorylated protein. Fujiki's group has shown that TNF-a acts as a
timior promoter in BALB/3T3 cell transformation in vitro. The results
of the studies on the okadaic acid class tumor promoters suggest that
inflammatory stimuli or chemical tumor promoters induce TNF-a release
from target tissues, and TNF-a gene expression in the initiated cells.
This released TNF-a acts as a tumor promoter in the autocrine and
paracrine system. According to the assumption that TNF-a is an
endogenous tumor promoter associated with inflammatory potential,
many historical puzzles of tumor promotion, such as its relationship to
inflammation, can be solved. Based on this new tumor-promotion
pathway, inhibition of TNF-a production leads to inhibition of tumor
promotion. Furthermore, recent investigation has revealed that TNF-a
is involved in various diseased, such as rheumatoid arthritis, Crohn's
disease, multiple sclerosis, graft-versus-host disease, HIV, malaria, sepsis,
and cachexia associated with cancer [85-90]. So, specific inhibitions of
TNF-a production will almost certainly be effective not only in cancer
prevention but also in the therapy and prevention of these other diseases.
{jene expression
—1 protein i ^ phosphorylated t - c-fos
okadac acid proteins 1
j — ' phosphatase 1 ojun
NF-KB
ODC
TNF-a - • p
~3
phosporylated
proteins
t
' _
TNF-a — •
V _
Fig. (17). Mechanism of tumor promotion with okadaic acid.
Based on the above descriptions, we examined the inhibitory effect of

the Artocarpus flavonoids on TNF-a release stimulated by okadaic acid
using BALB/3T3 cells. This experiment was carried out in co-operation
with Dr. Fujiki's group (Saitama Cancer Center Research Institute, Japan).
All the compounds tested inhibit the TNF-a release stimulated by
220
okadaic acid at suitable lower concentration. This result suggests that

several Artocarpus flavonoids act as anti-tumor promoter against to the
okadaic acid type promotion. However, the detail mechanism is not
clear at present, Fig. (17). The comparison of the inhibitory effects of
the Artocarpus flavonoids against the TNF-a release (Table 4) and
arachidonate 5-lipoxygenase, Fig. (13), was carried out. Artonin E (7)
was the most potent inhibitor on both tests and the other compounds,
artobiloxanthone (8) and heterophyllin (52), inhibited stronger than
cycloartobiloxanthone (9), cycloheterophyllin (53), and morusin (3).
The compounds showing stronger activity, all have three hydroxyl groups
in the B ring. This characteristic feature might be important factor for
both biological activities [91,92]. It is also noteworthy that the
bioactivities of these flavonoids may reflect the use of Artocarpus species
to the treatment for inflammation and malarial fever in Jamu medicines as
is stated above.
Table 4. Inhibitory effects (IC50, Mmol/L) of six flavonoids for the release of TNF-a from BALB/3T3
cells by treatment of okadaic acid
Morusin (3) 1.76 Artonin E (7) 0.43

Artobiloxanthone (8) 0.94 Cycloartobiloxanthone (9) 1.94
Heterophyllin (52) 0.48 Cycloheterophyllin (53) 7.8
We also examined the cytotoxic activities of the Artocarpus flavonoids,

artonins A (54), B (55), E (7), H (56), heterophyllin (52), and cyclo-
heterophyllin (53), against cancer cells, mouse L-1210 and colon 38.
All compounds tested showed the cytotoxic activities against both cancer
cells (Table 5) [93]. Among them, cytotoxicity of heterophyllin (52),
artonins B (55) and E (7) wêre stronger than critical drug, l-(2-tetra-
hydrofuryl)-5-fluorouracil (TFFU). While we examined the cytotoxic
activities of three dihydrochalcone derivatives isolated from Antiaris
toxicaria (Moraceae), antiarones J (19), K (20), and L (57), against the
two cancer cells [94]. All the compounds showed the weak cytotoxic
activities against both cancer cells. Artonin E (7) also exhibited
Table 5. Cytotoxic activities (IC50, |ig/mL) of Artocarpus and Antiaris flavonoids against L-12i0 and
Colon 38 cells
L-1210 Colon 38 L-1210 Colon 38
Artonin A (54) 8.8 14.3 Cycloheterophyllin (53) 4.7 4.6

Artonin B (55) 23 1.4 Antiarone J (19) 77.0 70.4
Artonin E (7) 2.2 1.9 Antiarone K (20) 81.3 46.3
Artonin H (56) 8.8 3.5 Antiarone L (57) 80.4 >100
Heterophyllin (52) 2.3 1.3 TFFU* 2.9 3.9
' Positive control.

221
cytotoxic activities against human oral cells and MT4-cells as shown in

Chapter VII (Table 7).
VI. BOMBESIN RECEPTOR ANTAGONISTS, KUWANONS G

(1) AND H (2), ISOLATED FROM MORUS SPECIES
Bombesin and its mammalian counterparts, gastrin-releasing peptide

(GRP) and neuromedin B (NMB), have been shown to have a wide range
of physiological and pharmacological functions [95]. Ligand-binding
and molecular cloning studies have revealed two pharmacologically
distinct G-protein-coupled receptor subtypes for mammalian bombesin-
like peptides; a GRP-preferring (GRP-R) and an NMB-preferring
bombesin receptor (NMB-R) [96].
A series of observations indicates that the mammalian bombesin-like
peptides may act autocrine growth factors in human small cell lung
carcinoma (SCLC) and other cancers. First, many human SCLC cell
lines have been shown to express bombesin-like peptides [97]. Second,
peptide bombesin receptor antagonists or anti-bombesin antibodies inhibit
SCLC cell growth in vitro and in vivo [98,99]. These data suggested
that the bombesin receptor antagonists might be useful for the treatment
of some kinds of SCLC and other cancers. Because most antagonists
reported thus far are peptides except for CP-70,030 and CP-75,998 (first
synthetic non-peptide antagonists) [100-102], so, Fujimoto's group
(Shionogi Research Laboratories, Shionogi & Co. Ltd., Osaka, Japan)
screened the four hundred plant extract samples to search for non-peptide
bombesin receptor antagonists. The methanol extract of the under-
ground part of cultivated mulberry tree, Morus bombycis, was found to
potently inhibit [^^Î]GRP binding to Swiss 3T3 cells. Bioassay-directed
fractionation led to the isolation of two known flavone derivatives,
kuwanons G (1) and H (2), which were identified by direct comparison
with the authentic samples [103].
The antagonistic profiles of kuwanons G (1) and H (2) were
characterized from the following results [103]. Kuwanon H (2)
inhibited specific binding of [^^Î]GRP to GRP-referring receptors in
murine Swiss 3T3 fibroblasts with K{ value of 290±50 nmol/L, which is
more potent than that of kuwanon G (1), K\ value=470±60 nmol/L. The
Ki value of 2 was about one order of magnitude more potent than those of
CP-70,030 and CP-75,998, but had no effect on endothelin-1 or
neuropeptide Y binding. While kuwanon H (2) inhibited specific
binding of [^^Î]bombesin to rat esophagus membranes, the Ki value was
about one order of magnitude less potent, Ki value of 2=6,500±2,000,
than that of [^^Î]GRP toSwiss 3T3 cells.
While bombesin (10 ^ mol/L) increased intracellular Ca^"^ levels in
Swiss 3T3 cells, kuwanon H (2, 500 nmol/L) attenuated the bombesin-
222
induced increase in cytosolic free Ca^"^ concentration ([Ca^"^]!) by 60%,

but not bradykinin- or endothelin-1-induced increase in [Ca^"^]}, Fig. (18).
r\ t\
r
808
215 < \ A^ Y1
f t t t t t t t t t t
BK S BK V ET-1 S ET-l
V BOM S BOM
Fig. (18). Effect of kuwanon H (2) on agonist-induced increases in [Ca^^\ in Swiss 3T3 cells. Cells were
stimulated by 10"* mol/L bombesin (BOM), 10"* mol/L endothelin-1 (ET) or 10"* mol/L bradykinin (BK).
Kuwanon H (S, 500 nmol/L at the final concentration) or dimethyl sulfoxide (V) was added 1 min before
stimulation.
In Swiss 3T3 cells, GRP stimulates ["^H]thymidine incorporation in a

concentration-dependent manner. Kuwanon H (2) inhibited GRP-
induced DNA synthesis in Swiss 3T3 cells. The IC50 value was around
100 nmol/L, close to its K, value for [^^Î]GRP binding to Swiss 3T3 cells,
Fig. (19). Kuwanon H (2) demonstrated selectivity toward GRP, as
concentration of 10"^ mol/L uninfluenced basal and 5% serum-induced
[ HJthymidine incorporation. From above results, kuwanon H (2)
appears to be a selective antagonist for GRP-R.
B
o.
35000
B
o
25000
15000
-log (2) mol/L

Fig. (19). Dose-dependent effects of kuwanon H (2) on basal (o) and GRP (10" mol/L)-induced DNA
syntheses in Swiss 3T3 cells (•). Values are the mean ± S.E. for four determinations.
As bombesin family peptides are thought to be autocrine growth

factors for SCLC, the results described above suggested that kuwanon H
223
(2) might be useful against SCLC. Unfortunately, however, kuwanon H

(2) had no effect on the growth of two human SCLC lines, Lu-134 and
NCI-HI 28.
At the time, kuwanon H (2) was the most potent of non-peptide
bombesin receptor antagonists (NPBRA) that had been reported. Its
affinity might be too low to determine whether the non-peptide antagonist
is effective against human lung cancers. However, kuwanon H (2), and
possibly kuwanon G (1) also, can serve as lead compounds for more
rational drug design in the synthesis of more potent antagonists.
Furthermore, these compounds may be useful tools on the study of
GRP-R. Recently, it was reported that NPBRA, PD 176252, with high
binding affinity which was developed via the application of a peptoid
drug design strategy [104].
VIL EFFECTS OF PHENOLS AGAINST BACILLUS SUBTILIS

(M45) (REC-ASSAY), HUMAN ORAL CELLS, AND
HIV-INFECTED MT-4 CELLS
Rec-assay was developed by Kada et al. for screenings chemical and

enveloped mutagens. Recombination less mutant strain of Bacillus
subtilis (M45) is more sensitive to the cell-killing action of chemical
mutagens, e.g., mytomycin C, A^-nitroso-A/-methylurethane, etc., than the
wild-type bacteria (HI7) [105]. This assay was also useful for pre-
screening of anticancer drugs, such as enediyne-family antibiotics [106].
For the constituents of plants, the assay was modified and used
exclusively for the detection of anti-mutagen compounds [107]. Since
the sensitivity of the rec-assay to chemicals having induction activity of
DNA damage is higher than from other screening technique, such as
Ames test, this method may be useful for pre-screening of anticancer
agents in crude drugs. Furthermore, the antibacterial compounds
against the wild-type strain (HI7) may be expected that these anti-
bacterial compounds have another bioactive potency. We tried the
application of the rec-assay (unmodified) for the detection of bioactive
phenolic compounds obtained from Glycyrrhiza species [51], and spore
rec-assay [108,109] was used for moraceous flavonoids as shown in
Table 7. Sixty-nine Glycyrrhiza phenols out of a total 108 compounds
showed inhibitory activity against the growth of both HI7 and M45
strains. Cytotoxic activities of these antibacterial compounds
{Glycyrrhiza phenols and moraceous phenols) against human oral
squamous cell carcinoma (HSC-2) and human T-lymphoblastoid cell line
MT-4 cells were also shown in Table 7 [110-113] along with other
biological activities reported until the middle of 2002. In the Table,
relatively strong-cytotoxic compounds against HSC-2 (CC5o<25 |ig/mL)
224
that showed no activity against these B. subtilis strains were also shown
in Table 7. On the comparison between these bioactivities of the
phenolic compounds, relationship between the antibacterial activity
against B, subtilis and cytotoxicity against HSC-2 or MT-4 cells was not
found.
Hatano et al. reported antibacterial effect of licorice flavonoids
against methicillin-resistant Staphylococcus aureus (MRSA) [114].
8-(y,Y-Di-methylallyl)-wighteone (58) and 3'-(Y,Y-dimethylallyl)-kievi-
tone (27) showed relatively strong activity against clinically isolated
MRSAs (MIC=8 lag/mL). Licochalcone A (59), gancaonin G (60),
glyasperins C (61) and D (62), glabridin (23), licoricidin (63),
licocoumarone (64), and isoangustone A (65), Fig. (20), showed slightly
weak activity against the bacteria (MIC=16 |ig/mL) [114].
glyasperin C (61): Ri = R2 = H
8-(Y,Y-dimethylallyl)-wighteone (58): licochalcone A (59) glyasperin D (62): Ri = Me, R2 = H
Ri = R3 = R4 = H, R2 = CH2CH=CMe2 licoricidin (63):
gancaonin G (60): ^^^ Ri = H, R2 = CH2CH=CMe2
Ri = Me, R2 = R3 = R4 = H
isoangustone A (65):
Ri = R2 = H, R3 = 0H,
R4 =CH2CH=CMe2 i licocoumarone (64)
Fig. (20). Structures of licorice flavonoids (58 - 65).
Table 6. Antimicrobial activity of licorice flavonoids (MIC, ng/mL)

MSSA MSSA MRSA MRSA M luteus E. coli
FDA 209P Smith K3 ST 28 ATCC9341 NIHJ JC-2
Glabridin (23) 12.5 12.5 12.5 12.5 12.5 >100
Glabrene (24) 12.5 12.5 12.5 12.5 25 >100
Licochalcone A (59) 3.13 6.25 6.25 6.25 6.25 >100
Licochalcone B (82) 25 100 100 100 >100 >100
Liquiritigenin (101) >100 >100 >100 >100 >100 >100
Liquiritin (113) >50 >50 >50 >50 >50 >50
Licoisoflavone B (67) 12.5 12.5 12.5 12.5 ND ND
Formononetin (116) >25 >25 >25 >25 >100 >100
Licoricidin (63) 3.13 3.13 6.25 6.25 6.25 >100
Glycyrol (76) >100 >100 >100 >100 >100 >100
Isoglycyrol (117) >25 >25 >25 >25 >100 >100
3-0-Methylglycyrol(118) >16 >16 >16 >16 >16 >16
Vestitol(119) >50 >50 >50 >50 >50 >50
Licoricone (120) 25 >50 >50 >50 >50 >50
Glycyrin (121), >50 >50 >50 >50 >50 >50
Isolicoflavonol (122) 12.5 12.5 25 25 25 >100
GancaonolB(123) >32 >32 >32 >32 >32 >32
Glyasperin D (62) 6.25 6.25 6.25 6.25 12.5 >50
Gancaonin I (126) 3.13 1.56 1.56 3.13 3.13 >50
AMOX 0.1-0.2 0.20 25-50 50 0.025 3.13
MSSA means methicillin-sensitive Staphylococcus aureus. M. and E. mean Micrococcus and Escherichia,
respectively. A positive control used was amoxicillin (AMOX). ND, not determined.
225
We also screened anti-MRSA flavonoids [115] in the course of the

study of anti'Helicobacter pylori flavonoids from licorice described in
Chapter X. In our screening (Table 6), glabrene (24), licoisoflavone B
(67), and gancaonin I (126) also exhibited anti-MRSA effect. Among
these compounds, 23, 24, 27, 59, 61, 62, 63, 67, and 126 exhibited
relatively high anti-bacterial activities against B. subtilis (HI7), Table 7.
Rec-assay
Seven compounds, licoisoflavanone (66), licoisoflavone B (67),

semilicoisoflavone B (68), gancaonin C (69), isoliquiritigenin (70), 6-
prenyleriodictyol (71), and 8-prenyleriodictyol (72), Fig. (21), showed
positive results in the rec-assay. Isoliquiritigenin (70) was most potent
of the seven compounds (Table 7).
The simple chalcone 70 has distributed widely in Glycyrrhizin plants
as a minor constituent, and its glycosides are mainflavonoidsof licorice
as described in Chapter 1. Recently, Okuyama et al. reported inhibition
effect of the chalcone (70) on azoxymethane-induced murine colon
aberrant crypt foucus formation and carcinogenesis [116].
licoisoflavanone (66)
""OH
6-prenyleriodictyol (71): Ri = CH2CH=CMe2. R2 =H

8-prenyleriodictyol (72): Ri = H, R2 = CH2CH=CMe2
gancaonin C (69) isoliquiritigenin (70)
Fig. (21). Structures o f licorice flavonoids (66 - 72).
Anti-HIV activity
Anti-HIV activity of prenylflavones from mulberry tree, kuwanon H (2),

morusin (3) and its derivatives, was reported by Luo et al. [117]. We
studied the effect of Morus flavones on HIV-1 me infected MT-4 cells,
but no flavone showed anti-HIV activity in our screening system [111].
These discrepant results might be due to multiple acting sites of
226
flavonoids. We also screened anti-HIV flavonoids from moraceous

plants and Glycyrrhiza species, however, only two flavonoids from
Glycyrrhiza species and a 2-arylbenzofuran from Morus species showed
weak anti-HIV activity with selective index (SI=50% cytotoxic
concentration (CC50) for MT-4 cells / 50% effective concentration (EC50)
for HIV-infected MT-4cells): 3-hydroxyglabrol (26, SI=10), kumatake-
nin (73, SI=20), and moracin C (74, SI=12), Figs. (3) and (22), [111].
Manfredi et al. reported the isolation of an anti-HIV diprenylated
bibenzyl from G. lepidota, but its therapeutic index (TI=EC5o/CC5o) was
small [118]. McKee et al. investigated anti-HIV activity of isoprenoid-
substituted isoflavans from Erythrina lysistemon (Leguminosae). They
concluded that both a free 4'-hydroxy1 group and a lack of substituents at
positions C-5 and C-6 are necessary for even minimal in vitro anti-HIV
activity [52].
MeO.
OMe
norartocarpetin (83) wjghteone (84)
Fig. (22). Structures offlavonoids73 - 84 from Glycyrrhiza species and Moraceous plants.
227
Cytotoxic activity against human oral squamous cell carcinoma

(HSC-2)
Most potent isoprenoid-substituted phenols against HSC-2 cells (CC5o<

10 |ig/mL) were gancaonin R (75), glycyrol (76), glyasperin A (77),
licoricidin (63), antiarone I (78), artonin E (7), broussoflavonols B (79)
and C (80), kazinol B (81), and morusin (3), Table 7. Structure-activity
relationship of isoprenoid-substituted phenols for the cytotoxic activity
against HSC-2 cells could not be clear. Nevertheless, most compounds
having higher cytotoxic activity have two sets of a hydrophilic group
(hydroxyl group) in the vicinity of hydrophobic group (isoprenoid group)
at two different sites on a same plain in the molecule. We also
investigated the cytotoxic activity of these compounds against normal
human gingival fibroblasts (HGF) and compared with activity against
HSC-2 cells. Licochalcone B (82) exhibited significant tumor
selectivity: index of tumor specificity (ITS=CC5o for HGF/CC50 for
HSC-2) was 42 [111]. Norartocarpetin (83) was also showed tumor
selectivity (ITS=11) [99]. ITSs of 23 compounds of 63 isoprenoid-
substituted phenols were 2'-^4 [110-112].
Apoptosis is a normal physiological process that occurs during
embryonic development as well as during the maintenance of tissue
homeostasis. It can be induced by a variety of treatments, such as UV
irradiation, cytotoxic chemotherapy, etc. Cells, which die by apoptosis
usually, suffer similar morphological change, including nuclear
condensation, cytoplasmic blebbing, and DNA fragmentation. Wang et
al. reported induction of apoptosis by apigenin (4',5,7-trihydroxyflavone)
and related flavonoids through cytochrom c release and activation
caspase-9 and caspase-3 in leukaemia HL-60 cells [119]. We found that
glabrol (25) and wighteone (84) induced intemucleosomal DNA
fragmentation in HL-60 cells, but not in HSC-2 cells [111]. This is
consistent with the report by Yanagisawa-Shirota et al.; induction of
intemucleosomal DNA fragmentation depends on the cell types, rather
than apoptosis inducers (ascorbic acid, H2O2, tumor necrosis factor,
etoposide, hyperthermia, and UV irradiation) [120].
VIII. ESTROGEN-LIKE ACTIVITY OF PHENOLS FROM THE

MORACEOUS PLANTS AND GLYCYRRHIZA SPECIES
Traditional Japanese women with their high soy intake, a rich source of
228
Table 7. Inhibitory activity against Bacillus subtilis HI7 and rec-assay (disk division method) of
isoprenoid-substituted phenols (75 |ig/disk), their cytotoxic activities against HSC-2 and MT-4 cells, and
other biological activities
Trivial name Inhibition Rec-assay* HSC-2^ MT-4'' other activity^

forHH" CC50 CC50
(Hg/mL) (Hg/mL)
(Licorice phenols/
Angustone B + + 14 6 AFE, FDC

Bavachalcone -H- - 12 2
(broussochalcone B)
Dehydroglyasperin C -H- - 31 10
3'-(y,y-Dimethylallyl)-
kievitone (27) ++ - 20 10 ABM
8-(Y,Y-Dimethylallyl)-
wighteone (58) + - 22 11 ABM, EBV, LAT
Edudiol +-I- ± ND ND
Echinatin _ ± 45 12 ABE
Formononetin (116) - - 20 100 ABH, APA, EBV, ODD
Gancaonin C (69) + + 125 9
Gancaonin E - - 13 55 ALR, 5LG
Gancaonin G (60) + - 19 28 ABM
Gancaonin H + - 97 14
Gancaonin I (126) ++ - 42 >50 ABH, ABM
Gancaonin O + - 20 55
Gancaonin P ND ND 14 22
Gancaonin Q - - 11 6
Gancaonin R (75) ++ ± 8 18 ALR, ICO, 5LG, NKA,
Gancaonin S -H- ± 10 2 IC0,5LG,NKA
Gancaonin U + ± 12 2 ALR, IOC, 5LG, NKA
Gancaonin V -H- - 34 10
Gancaonin Y + - ND ND
Glabranin ND ND 14 >100 AIA, IPP
(glabranine)
Glabrene (24) -H- ± 12 10 ABE, ABH, ABM, AFE,
AMA, AOA, EAA
Glabridin (23) +++ ± 13 4 ABE, ABH, ABM, AFE,
AOA, EAA, EAB, ETA,
ICO, IMI, PSO, SAE
Glabrol (25) 4-+ ± 18 >100 ABE
Glycycoumarin -f+ - 32 13 ABC, ABE, AOA, CPH
Glycyrin - - 14 11 ABC, ABE, ABH
Glycyrol (76) - - <4 14 ABC, 5LG
(neoglycyrol)
Glyasperin A (77) + - <8 53
Glyasperin B -H- - 46 9
Glyasperin C (61) -H- - 31 34 ABM
Glyasperin D (62) -H- ± 48 >10 ABM, ABH
Glyasperin J ++ ± ND ND
Glyasperin K + - ND ND
Glisoflavanone ++ - 21 46
Glyinflanin A ++ - ND ND
(glycyrdione A)
Glyinflanin B ++ - 31 27
Glyinflanin C ++ - 19 8
(glycyrdione C)
Hispaglabridin A (124) + - 14 >100 ABE, AOA, MOS
3-Hydroxyglabrol (26) -H- - 31 12 ABE
3-Hydroxy-
paratocharpin C^ + - 38 12 ALA
Isoderrone ++ - ND ND
Isoglycyrol(117) - - 16 >100 ABC, ALR
Isoliquiritigenin (70) ++ ++ 22 14 ABE, ATP, EAA, MCC,
MGA, TFV, UFI
229
Table 7 (continued)
Kanzonol B (81) + ± ND ND SOA

Kanzonol G + - ND ND
Kanzonol H + - 46 >10
Kanzonol P + - ND ND
Kanzonol R ++ - ND ND
Kanzonol S -I-+ - ND ND
Kanzonol U + - ND ND
(glabrocoumarone A)
Kanzonol V + - ND ND
Kanzonol W + - ND ND
Kanzonol X -H- - ND ND
(tenuifolin B)
Kanzonol Y + - ND ND
Kumatakenin (73) — — 375 51 AFE, APV, AVC, SST,
TCD
Licochalcone A (59) -H- — 20 15 ABE, ABH, ABM, ALA,
ATP, CPH, LFP
Licochalcone B (82) - - 4 16 ABE, CPH, LFP
Licoflavonol -H- - 22 13 ABE
Licoisoflavanone (66) ++ + 72 40
Licoisoflavone A + - 55 21 AFE, AOA, TD
(phaseoluteone)
Licoisoflavone B (67) -H- + 43 7 ABM, AFE, TD
Licoricidin (63) -H- ~ 8 15 ABE, ABH, ABM, AOA,
5LG, NKA, LAT
Licoricone (120) + - 45 64
Licorisoflavan A - - 14 47 ABH, BDB
Medicarpin + - 45 53 AOA, FDC, QRI
1-Methoxyphaseollidin (125) -H- - 28 12 ABH, LAT
1 -Methoxyficifolinol + - 11 8
3-(9-Methylgancaonin P ++ - ND ND
4'-(9-Methylglabridin (123) -H- ± ND ND ABE, ABH, AOA, MOS
Naringenin + ± ND ND ARI
Paratocarpin L ++ - 24 14
(macarangaflavanone B)
Pinocembrin -I-+ ± 105 >100 AIA
6-PrenyleriodictyoF (71) ++ + ND ND
S-PrenyleriodictyoF (72) ++ + 35 60
6-Prenylnaringenin (90) ++ - 29 32
Semilicoisoflavone B (68) ++ + 78 22
Shinpterocarpin -H- - ND ND
Sigmoidin A ND ND 20 29 ABE, AFE, ESEC, IMS
Sigmoidin B (99) ++ - 43 26 ABE, AFE, ESEC, IMS
Topazolin + - 19 4
Wighteone (84) ++ - 20 12 ABE, AFE, CTK, FDC,
(erythrinin B) HPE
(moraceous phenols)*
Albanin D _ _ 17 >8
Albanol B (97) ND ND <8 9
Alvaxanthone ND ND 9 ND
Antiarone B (18) ++ - ND ND
Antiarone F ++ - 30 >100
Antiarone G ++ - 13 11
Antiarone H - - 20 >100
Antiarone I (78) -H- - <8 11
Antiarone J (19) ++ - ND ND CTC, CTL
Artobiloxanthone (8) +-I-+ + ND ND 5LG, TAR
(KB-l)
Artonin E (7) -hH- ± <8 4 CTC, CTL, 5LG, RAR
(KB 3)
Broussoflavonol B (79) ND ND <8 >100
Broussoflavonol C (80) - - <8 20
230
Table 7 (c ontinued)
Broussoflavonol E ND ND 12 10
Cudraphenone B ND ND 13 ND
Cudraphenone D ND ND 20
Cycloartobiloxanthone (9) -H- ± ND ND CTC, CTL, 5LG, TAR
Heterophyllin + - ND ND CTC, CTL, 5LG, TAR
Isoalvaxanthone ND ND 14 ND
Kazinol B (81) + - 19 12 ARP,ICO,5LG,NOP
Kazinol E (37) - - <8 9 AOA, ETA, TPA
Kazinol F (38) -H- - 10 9 ETA, TPA
Kazinol N (41) ++ ~ 20 10
Kuwanon C (42) -HH- - 15 44 ABE, AFE, ARP, CTM,
(mulberrin) 5LG, 12LG, NOP, SPB,
TPA
Kuwanon G (1) -H- - 54 66 BRA, FHA, FTB, HPA,
(albanin F, moracein B) ODC, PKC, TPA
Kuwanon M (30) - - 24 7 HPA, ODC, PKC, TPA
Kuwanon R + - ND ND
Moracin C (74) ND ND 17 19 AFA
Morusin (3) ++ - <8 9 ABE, AFE, ANC, AOA,
(mulberrochromene) ARI, ARP, ATP, CTM,
FEA, FHA, FTB, ICO,
5LG, NOP, ODC, PKC,
PSO, SPB, TPA, TAR
Mulberrofiiran B ND ND 23 ND
Mulberrofliran G (25) ND ND <8 2 ARI, FEA, FHA, FTB,
(Albanol A) ODC, PA, PKC, TPA,
Norartocarpetin (83) - - 45 15 ETA, ICO
Oxydihydromorusin (46) ++ + 12 >100 AVR, FEA, FHA, FTB,
(morusinol) SPB, TPA
Sanggenol C ND ND 25 >10
Sanggenol M ND ND 10 ND
Sanggenon A (4) ND ND 23 ND
Sanggenon B (45) -hH- ± 22 ND AFE, ABE, ICO, 5LG,
NOP, TPA
Sanggenon C (5) +++ ± 13 ND ABC, ABE, AFE, FHA,
FTB, HPA, PKC, TPA
Sanggenon M (100) ND ND 21 ND
Sorocein F ND ND 24 ND
"Diameter of inhibition zone (8 mm paper disk was used), +=less than 11 mm, -H-=between 11 and 18 mm,
-H-+=more than 18 mm. Diameter of inhibition zone for kanamicin (10 |ig/disk) is 22 mm.
* Difference in diameter of inhibition zone between Bacillus subtilis M45 (rec: - ) and HI 7 (rec: +), diameter
of inhibition zone on M45 minus that on HI7; -^diameters are same, ±=less than 2 mm, +=between 2 and 5
mm, -H-=more than 5 mm. A positive control is mitomycin C (0.75 îg/disk): the difference of inhibition zone
is 6 mm. Inhibition zones of a negative control (kanamicin) were same for the both strains.
^CCso of doxorubicin was 2 |ag/mL.
'^ 50% Cytotoxic concentration against human T-lymphoblastoid cell line MT-4 cells without HIV infection.
" ABC = antibacterial action against a cariogenic bacterium, Streptococcus mutans [121,122];
ABE = antibacterial effect [8,123-132];
ABH = antibacterial effect against Helicobacter pylori [see the text, 126,127],
ABM = antibacterial effect against MRSA [see the text, 114, 115,133];
AFA = anti-feedant activity against silkworm [134];
AFE = antifungal effect [112,134-140];
AIA = anti-inflammatory activity [141,142];
ALA = anti-Leishmania activity [143,144];
ALR = inhibition on aldose reductase [43];
AMA = anti-mutagenic activity [145];
ANE = anti-nociceptive effects in mice [146];
AOA = antioxidant activity [123,125,147-154];
APA = anti-protozoa activity [155];
APV = anti-picomavirus activity [156];
ARI = aromatase inhibitory activity [157];
ARP = inhibition of aggregation of rabbit platelets [158,159];
ATP = anti-tumor promoting activity [see the text, 160,161];
AVC = antiviral activity against Coxsackie virus [162];
231
AVR = antiviral activity against rhinovirus type 2 [8];

BDB = benzodiazepin-binding stimulator [163];
BRA = bombesin receptor antagonist [see the text];
CPH = inhibition of the cytopathic activity of HIV [164];
CTC = cytotoxic activity against colon 38 [see the text];
CTK = cytotoxic activity against KB cells [165];
CTL = cytotoxic activity against L1210 [see the text, 166];
CTM = cytotoxic activity against mouse macrophage cell line (RAW 264.7) [167];
EAA = estrogen agonist activity [168];
EAB = estrogenic and anti-proliferate properties in human breast cancer cell [169];
EBV = inhibitory effect on TPA-induced Epstein-Barr virus early antigen [170];
ESEC = effect on sea-urchin egg cleavage [171];
ETA = effect on tyrosinase activity [153,172-174];
FDC = feeding deterrents for Costelytra zealandica (white) [135,175];
FEA = inhibition of formation of 12-HET from [l-'*C]arachidonic acid [see the text, 79,80];
FHA = inhibition of formation of 12-HHT from [l-^'^CJarachidonic acid [see the text, 79,80];
FTB = inhibition of formation of thromboxane Bi from [l-''*C]arachidonic acid [79,80];
HPA = hypotensive activity [see the text];
HFE = hepato-protective effect [176];
ICO = inhibition on cyclooxygenase [43,172,177,178];
IFF = inhibition of photo-phosphorylation [179];
IMI = inhibitory effect on melano-genesis and inflammation [172];
IMS = inhibition of macrophage superoxide production [180];
LAT = inhibitory effect on lysoFAF (platelet-activating factor) acetyltransferase [181];
LFF = effect on leukotriene formation in human polymorpho-nuclear neutrophils [182];
5LG = inhibition on 5-lipoxygenase [43,177],
12LG = inhibition on 12-lipoxygenase [177];
MCC = inhibition effect on murine colon carcinogenesis [116];
MGA = mutagenic activity [183];
MOS = protection of mitocondrial fractions against oxidative stresses [184];
NKA = inhibition on Na^ K^-ATPase [43];
NOP = inhibitory activity on NO production from lipopolysaccharide-induced nitric oxide (NO) production
from mouse macrophage cell line (RAW 264.7) [167];
ODC = inhibitory activity on induction of ODC [see the text];
ODD = reducing of endogenous oxidative DNA damage [185];
PKC = inhibition of activation of protein kinase C [see the text];
PSO == inhibitory effect on production of superoxide anions [172];
QRI = quinone reductase-inducing activity [186];
SAE = synergistic anti-oxidative effect with lycopene [187];
SPB = substrate for PCB-degrading bacterium, Burkholderia sp. [188];
SOA = stimulation of superoxide anion generation in rat neutrophils [189,190];
SST = suppression of SOS-inducing activity of Trp-P-l {umu test) [191];
TAR = inhibitory effect on TNF-a release [see the text];
TCD = inhibitory activity on TNF-a-induced cell death in mouse hepatocytes [192];
TFV = tube formation from vascular endothelial cells of rats [193];
TPA = inhibiting of ^H-TFA binding [see the text];
UFI = preventive effect on ulcer formation induced by severe necrotizing agents in rats [194].
/Eriodictyole, liquiritigenin (101), sophoraflavanone B (86, AFA), isobavachin (94), kanzonol Z, euchrenone
as, ovaliflavanone B, prenyllicoflavone A, glepidotin A, 3,3'-di-0-methylquercetin, 3,4'-di-0-methylquercetin,
paratocarpins B and C, glyinflanin G, and licuraside exhibited no effect against both M45 and HI7 strains.
^ No name: names in the table are tentative name used here.
* Antiarones A and K, brosimone L, cudraflavanone A, and cyclohetrophyllin (53) showed no effect against
both M45 and HI 7 strains. These data were obtained with spore rec-assay (38 îg of sample/disk).
ND means not determined. The structures of all phenolic compounds isolated from Gfycyrrhiza species until
1996 were reviewed in the reference [43], new compounds from the plants since the time to 2000 were
summarized in the proceeding [44].
plant-derived estrogens (phytoestrogens [195]), have a low incidence of

breast cancer and few menopausal symptoms. This has led to the
hypothesis that at the menopause phytoestrogens might act as natural
selective estrogen receptor modulators, tweaking estrogenic responses in
the cardiovascular system, bone, and brain, but dampening responses in
232
the breast and uterus. The finding that the soy-derived phytoestrogen
genistein (85), Fig. (23), preferentially binds to the form of the estrogen
receptor found mainly in the cardiovascular system lends some credence
to that belief [196]. It is expected by recent many evidence that phyto-
estrogens exert beneficent actions to chronic diseases, e.g., heartattacks
and other cardiovascular problems, osteoporosis, Alzheimer's disease, etc.
[197]. Nevertheless, the isoflavonoids and Ugnans bind wîth low^
affinity to estrogen receptors, and thus, it is also suggested that they may
induce production of sex hormone binding globulin in the liver and in this
way influence sex hormone metabolism and biological effects [198].
Recently, Cooke et al. reported that genistein (85) decreased mouse
thymocyte numbers and doubled apoptosis, indicating that the mechanism
of the genistein effect on loss of thymocytes is caused in part by
increased apoptosis [199]. In addition, genistein (85) produced
suppression of humoral immunity. These data indicate that use of soy-
based infant formulas and soy/isoflavone supplements has aroused
concern: genistein (85) and daidzein (102) may be capable of producing
thymic and immune abnormalities. Therefore, the screening of phyto-
estrogen from medicinal plants may be important.
genistein ( 8 5 ) : R = OH
daidzein ( 1 0 2 ) : R = H OH 0
sophorafiavanone B ( 8 6 ) : R = OH licoflavone C (87): R^ = R2 = H
isobavachin ( 9 4 ) : R = H 8-prenylquercetin (88): R i = R2 = OH
noranhydroicaritin (93): Ri = OH, R2 = H
lupiwighteone (89)
6-prenylnaringenin ( 9 0 ) : R = H
17p-estradiol(92)
lonchocarpd A ( 9 1 ) : R = CH2CH=CMe2
Fig. (23). Structures of phytoestrogens (85, 87, 88, 93, and 102) and related compounds having no
estrogenic effect (89-91).
Numerous phytoestrogens with a diversity of structures have now been

recognized [168,169,200-210]. Akiyama et al. reported that
sophorafiavanone B (86), Fig. (23), isolated from a Thai crude drug,
Anaxagorea luzonensis (Annonaceae), showed about two-fold higher
affinity for the bovine uterine estrogen receptor than that of genistein (85),
Table 8 [197]. They also reported that synthetic 8-prenylated flavonoids,
licoflavone C (87) and 8-prenylquercetin (88), also exhibited the binding
233
affinity but their binding affinities were weaker than that of 86. In the
report [197], it was also reported that a synthetic 6-prenylated isoflavone,
lupiwighteone (89), and 6-prenylated and 6,8-diprenylated flavanones, 6-
prenylnaringenin (90) and lonchocarpol A (91), Fig. (23), did not exhibit
the binding affinity against the receptor. This study indicated that the
position of the steric hydrophobic bulky group (prenyl group) is
important for the binding affinity of prenylated flavonoids.
We examined whether other types of phenols with two or more
aromatic rings bind to the estrogen receptor [45,211]. About one
hundred phenols with isoprenoid groups or without side chains from
moraceous plants and Glycyrrhiza species and synthetic flavonoids were
evaluated with the estradiol receptor-binding assay [204]. Among them,
13 compoimds exhibited weak binding affinities (1C50<1 |ig/mL) in
which three compounds were isolated from moraceous plants (95, 97, and
100), and six were constituents oi Glycyrrhiza species (24, 75, 76, 94, 99,
and 101), Fig. (24).
HOJ
tetrahydrogiabrene (96) * bH
alband B (97)
OH
sigmoidin B (99): Ri = OH, R2 = CH2CH=CMe2 sanggenon M (100)

8-geranylapigenin (98) liquiritigenin (101): Ri = R2 = H
Fig. (24). Estrogenic compounds from moraceous plants (95, 97, and 100) and Glycyrrhiza species (99 and
101) and synthetic estrogenic flavonoids with a isoprenoid group (96 and 98).
Relative binding affinities of these compounds against 17p-estradiol

(92) (RBA=[IC5o of 92 (nmol/L)] / [IC50 of test sample (nmol/L)]) values
are shown in Table 8. The affinity of gancaonin R (75) was stronger
than those of genistein (85, RBA=0.004) and daidzein (102,
RBA=0.00035) [205]. The affinities of the other 12 compounds were
similar to those of the isoflavones (85 and 102) in dietary foods.
Recently, Vaya et al. reported binding affinities of glabrene (24),
isoliquiritigenin (70), and glabridin (23) for human estrogen receptor
[168].
234
Table 8. Relative binding affinities of phenolic compounds for the bovine uterine estrogen receptor
Compound RBA Sources
Gancaonin R (75) 0.016 G. uralensis (aerial part)

Noranhydroicaritin (93) 0.0064 synthesis
8-Prenylquercetin (88) 0.0057 synthesis
Isobavachin (94) 0.0054 G. pallidiflora (root)
Isobavachalcone (95) 0.0045 M cathayana (root)
Glabrene (24) 0.0022 G. glabra (root)
Tetrahydroglabrene (96) 0.0016 synthesis
Glycyrol (76) 0.0012 G. uralensis (underground part), G. aspera (root)
Albanol B (97) 0.0011 M alba (root bark)
8-Geranylapigenin (98) 0.00095 synthesis
Sigmoidin B (99) 0.00077 G. uralensis (aerial part), G. eurycarpa (aerial part)
Sanggenon M (100) 0.00048 M cathayana (root bark)
Liquiritigenin (101) 0.00038 Glycyrrhiza species (root and aerial part)
Compound RBA Sources [ref.]

Sophoraflavanone B (86) 0.0071 Anaxagorea luzonensis [197]
Sophoraflavanone B (86) 0.0091 synthesis [197]
Licoflavone C (87) 0.0063 synthesis [197]
8-Prenylquercetin (88) 0.0015 synthesis [197]
Hinokiresinol 0.00083 Chamaecyparis obtusa [209]
Nyasol (c/5-hinokiresinol) 0.00017 Anemarrhena asphodeloides [209]
(3i?)-nyasol 0.0010 Anemarrhena asphodeloides [209]
(35)-nyasol 0.0067 Anemarrhena asphodeloides [209]
Neoflavone dimer T (0.028)* Pistacia chinensis [210]
Neoflavone dimer 2° (0.0028)* Pistacia chinensis [210]
Dihydrochalcone* 0.0010 Dracaena loureiri [204]
Homoisoflavone*" 0.0024 Draceana loureiri [204]
10-Hydroxycoronaridine (0.003)* Tabernaemontana penduliflora [208]
Coronaridin (0.0005)* (not reported) [208]
" 3,3"-Dimer of 3,4-dihydro-4-(4'-hydroxyphenyl)-7-hydroxycoumarin. * No data of the positive control (92)

was reported. * 4,4'-Dihydroxy-2,6-dimethoxydhihydrochalcone. *" 5,7-Dihydroxy-3-(4-hydroxybenzyl)-4-
chromone.
On the molecular modeling examination of gancaonin R (75), the

prenyl groups did not lie into the lipophilic pocket of estrogen receptor
that is illustrated in Fig. (27). These groups existed near the B and C
rings of 17p-estradiol (92) as shown in Fig. (25). On the molecular
models, the volume of 17p-estradiol (92) was 266.9 A^ (based on van der
Waals radius of atoms) and that of compound 75 was 374.5 A^, and the
distance between 3-0- and 17-0- of 92 was 10.9 A and 4'-0- and 3(5)-0-
of 75 was 11.0 A. The overlapping volume of these two models of 75
and 92 was 204.2 A l
We also studied the structure-activity relationships with computation
modeling of these estrogenic phenols (Table 8) and non-estrogenic
phenols [45]. For the examination of molecular modeling of the iso-
prenoid-substituted phenols, we made a space model of estrogen receptor
(SMER) with an imaginary compound (103), Fig. (26), that built up with
235
estrogenic steroids (104-108) having higher binding affinities than that of

17p.estradiol(92)[212].
Fig. (25). Molecular models of gancaonin R (75, ball and stick) and 17p-estradiol (92, stick): overlay of B
ring of 75 and A ring of 92. These molecular models were minimized with KfM2, and then calculated with
Mopac6.
104
SMERmodd(103)
106 107 108
Fig. (26). Structures of an imaginary compound (103) that built up with estrogenic steroids (104 - 108)
having higher binding affmities than that of 17p-estradiol (92).
The SMER, Fig. (27), is similar model to estrogen receptor excluded

volume (RExV) postulated by Kym et al. [212]. The volume of the
SMER was 424.7 A^. Most flavonoid skeletons (A-C rings) lay into the
SMER, however, the prenyl (geranyl) groups existed out of the SMER as
shown in Fig. (27).
By our examination of the modeling as illustrated in Fig. (27), it was
indicated that orientations of these estrogenic compounds in tiie binding
site of the estrogen receptor relative to 17P-estradiol (92) depend on tiieir
236
SMER
A ring of 76 and 103
Prenyl group of 76 Lipophilic pocket
Fig. (27). Overlapping of the SMER and molecular model of glycyrol (76).
skeletal structures as shown in Fig. (28). The binding sites of 17p-

estradiol in estrogen receptor-a and -(5 have been determined by X-ray
crystallographic study [213,214]. In Fig. (28), the amino acid residues
beside the phenols mean hydrogen-bonding binding site of the estrogen
receptor-a [213]. The orientations of these prenylated phenols against
binding site of estrogen receptor-p [214] may be similar to against
estrogen receptor-a. Fig. (28). The binding sites of albanol B (97), a 2-
arylbenzofuran derivative wîth three additional aromatic rings, in
estrogen receptor-a may be different from those of prenylated phenols.
Fig. (28): the binding sites of 97 may be similar to those of raloxifene.
Asp 351, Glu 353, Arg 394, and His 524 [213], but the binding of 97 is
not completely because of lacking of the binding to His 524, and the A, B,
and C rings of 97 may lie in the lipophilic pocket of the receptor as
shown in Fig. (29). A and E rings of sanggenon M (100) may also
locate in the lipophilic pocket. Fig. (29).
From the molecular modeling analyses of isoprenoid-substituted
phenols that did not showed binding affinity for the estrogen receptor, it
was indicated that the binding sites at C-3 and C-17 of 17p-estradiol are
rigid, but the lipophilic pocket near C-4~C-7 is flexible.
IX. AKll'HELICOBACTER PYLORI ACTIVITY OF LICORICE

FLAVONOIDS
Helicobacter pylori is a bacterium that lives in the human stomach and

duodenum. The bacterium is generally recognized as one of the
etiological agents of peptic ulcer. Therefore, it is generally accepted
that ulcer patients with H. pylori infection require treatment with
antimicrobial agents in addition to anti-secretory drugs, whether on first
237
[Glu 3531
H----[His 524]
[Glu 353]
[H2O]-
IH2O] [Arg 394]

17p-estradioi(92)
[Arg394] isoflavones (Ri = O. A^-^ : 85 (R2 = OH), 102 (R2 = H)
lsoflav-3-ene (R, = R2 = H, A^"*): 24
Isoflavan (Ri = H2. R2 = H, no A^'^= ^•^): 96
[Glu 3531 H - - . [His 524]

....-[His 524]
[Glu 353].,
[H2O]'''
[H2O]-
[Arg 394]
dihydrostilbene :75
[Arg 394]
coumestan: 76
[Asp 351],^
[His 524]
[Glu 353]»
[H2O]- [Glu 353],
[Arg 394] [H2O]-'
[Arg 394]
flavones (R = H, A^^): 87,98

flavonols (R = OH. A^^): 88,93 albanol B (97)
flavanones (R = H, no A^'^): 86,94,99,101
[His 524]
[Glu 353]
[H2O]'' /
[Arg 394]
[Arg 394] isobavachalcone (95)
sanggenon M (100)
Fig. (28). Orientations that flavonoids and a dihydrostilbene (75) may adopt in the binding site of the
estrogen receptor relative to 17p-eatradiol (92): the amino acid residues mean binding site of estrogen
receptor-a. A-D mean the positions of the A-D rings of 17p-estradiol but not the rings of these phenols.
presentation with the illness or on recurrence [215]. On the other hand,

gastric cancer is one of the most frequent cancers on a worMwide and the
leading cause of death from cancer. Since the discovery of H. pylori
[216,217], an association between the bacterium and gastric cancer has
238
A ring
Ertng
(SMERandlOO)
(SMERand97)
Fig. (29). Overiapping of the SMER (stick) and molecular model of albanol B (97, ball and stick) and
sanggenon M (100, ball and stick).
been suspected. Descriptive epidemiological data indicate that gastric

cancer occxirs morefrequentlyin some populations that have higher rates
of H. pylori infection. Furtheraiore, rates of both K pylori infection
and gastric cancer are correlated inversely with socioeconomic status and
increase as a ftmction of age and/or intake of dietary salt [218,219].
Recently, Uemura et al. reported the first evidence of the association
between K pylori and gastric cancer with a long-term study [220].
They studied a large group of Japanese patients with duodenal ulcer,
gastric hyperplasia, or non-ulcer dyspepsia. During the study, gastric
cancer developed in 2.9% of the patients infected with K pylori but in
none of the uninfected patients.
However, most people with chronic H. pylori infection have no
symptoms of peptic ulcer or gastric cancer, which raises questions
regarding preventive agents against these diseases; infected individuals
without disease symptoms may be protected by anti-bacterial compounds
in the diet and/or medicinal plants used frequently [221-239]. Many
anti-//. pylori agents with a diversity of structures have been isolated
from plant sources [240-252]. However, their antibacterial activities
against the bacterium in stomach are unclear [253] as the bacterium in
the narrow interface between the gastric epithelial cell surface and the
overlying mucus gel [215,217]. Among these antibacterial compounds,
flavonoids could be expected to show anti-//. pylori effects in vivo,
because kaempferol (109) exhibited antibacterial action in K pylori-
infected Mongolian gerbils [243]. As the biological activities of
239
flavonoids are generally weak, the phenolic compounds may act as

bacterial suppressors in the stomach.
Most elderly Japanese favor Kampo-medicines (traditional Chinese
medicines modified in Japan) rather than synthetic medicines.
Generally, traditional Chinese medicines consist of mixtures of crude
drugs and require extraction with boiling water for lengthy periods.
Sasaki et al. reported that aqueous solutions of some kinds of licorice
saponins solubilize water-insoluble substances such as a-tocopherol and
oleanolic acid [254]. The solubilizing effect of the saponins is expected
to result in licorice extract containing lipophilic compounds such as the
isoprenoid-substituted flavonoids. Licorice extract is frequently used in
Japanese over-the-counter (OTC) drugs that can be purchased without a
doctor's prescription, e.g. stomachic, cough medicines etc. [255].
Therefore, we studied anti-K pylori activities of the flavonoids from
licorice [256].
Licorice is for the most part derived from Glycyrrhiza glabra, G.
uralensis and G. inflata [43]. G. glabra is used worldwide but the other
two species are mainly consumed in Asian countries as described in
Chapter 1. The common constituents of these licorices are triterpenoid
saponins, glycyrrhizic acid (110) and licorice-saponin G2 (112), Fig. (30),
a flavanone, liquiritigenin (101), Fig. (24), its 4'-0-glucoside, liquiritin
(113), and an isoflavone, formononetin (116). These saponins (110 and
112) exhibited no anti-//. pylori activity and the aglycone of 110,
glycyrrhetic acid (111), showed weak activity (Table 9) as reported
previously [257]. The flavanone glucoside (113) also exhibited no
inhibitory activity against the bacterium, and its aglycone (101) showed
weak activity (Table 9). The main flavanone of the licorices is
compound 113 but the aglycone (101) is a minor component in these
plants. Kim et al. reported the anti-//. pylori activity of the metabolites
of poncirin (114) from Poncirus trifoliate (Rutaceae) by human intestinal
bacteria [246]. They speculated that one of the metabolites,
isosakuranetin (115), contributes to the prevention of gastritis to some
degree because of its anti-if. pylori activity (MIC=10 |ig/mL against H,
pylori ATCC 43504, 5x10^ cfu). In the case of licorice, the intestinal
bacteria would be expected to hydrolyze these glycosides (110 and 113).
However, it may be difficult that these hydrolysates (111 and 101) to be
transferred from the intestine to the stomach. Formononetin (116) is
frequently isolated from leguminous plants. The compound exhibited
weak anti-//. pylori activity when bacterial concentration was 2x10^ cfu.
The compound also showed weak antibacterial activity against a
clarithromycin (CLAR, a macrocyclic antibiotic)-resistant strain, GP98,
at higher bacterial concentration (2x10^ cfu) but not against
CLAR-sensitive strains (Table 9). Strain GP98 is also an amoxicillin
(AMOX, a penicillin class antibiotic)-resistant strain. The compound
240
may be a bacteriostatic agent for these CLAR (AMOX)-sensitive strains.

Nevertheless, the isoflavone (116) is a minor constituent of licorice.
These observations suggested that anti-//. pylori agents in licorice are
isoprenoid-substituted flavonoids
OOH H00<
= A
OH 0
kaempferoi (109)
glycyrrtiizic acid (110): Ri = A, R2 = CH3

glycyrrtietlc acid (111): Ri = H, R2 = CH3
licorice-saponin G2 (112): Ri = A, R2 = CH2OH
a OCHa
OH 0
OMe
liquiritin (113): R = p-D-gtucopyranosyl poncirin(114):
R = 2-0-a-L-rhamnopyranosyl-
p-D-glucopyranosyl
isosakuranetin (115): R = H
Fig. (30). Structures of compounds 103 - 109.
We selected eight flavonoids from licorices (G. glabra^ G, inflata,

and G, uralensis) to test for anti-//. pylori activity (Table 9). A
pyranoisoflavan, glabridin (23), and a pyranoisoflav-3-ene, glabrene (24),
are characteristic flavonoids of European G. glabra as described in
Chapter 1. These compounds exhibited weak anti-//. pylori activity
against these four strains. The characteristic flavonoids of G. inflata are
licochalcones A (59) and B (82). Compound 82 did not exhibit anti-^.
pylori activity. However, compound 59 showed weak bioactivity. The
characteristic flavonoids of G. uralensis are an isoflavan with two prenyl
groups, licoricidin (63), a prenylated coumestan, glycyrol (76), a
coumestan with a dihydropyran ring, isoglycyrol (117), and a pyrano-
isoflavone, licoisoflavone B (67). These compounds were also isolated
from G aspera but this licorice is commercially unimportant because of
its small plant size [43]. No anti-//. pylori activity of the coumestans
(76 and 117) could be detected against the strains examined, but the
isoflavan (63) and the isoflavone (67) exhibited antibacterial activity.
The inhibitory activity of 67 against the growth of CLAR and AMOX-
resistant strain GP98 is of considerable value: its minimum inhibitory
concentration (MIC) was 3.13 |ig/mL against 2x10^ cfu of this strain.
Nevertheless, this compound is a minor component of licorice, and thus
241
the main anti-//. pylori agent of G. uralensis may be licoricidin (63).
Tabic 9. AnXi'Helicobacter pylori activities (MIC, ng/mL) of the characteristic compounds of licorices
(Glycyrrhiza glabra, G. inflata, and G. uralensis)
ATCC 43504 ATCC 43526 ZLM 1007 GP98 (cfii)'* source
Glycyrrhizic acid (110) >100 >100 >100 >100 (a)

>100 >100 >100 >100 (b)
Glycyrrhetic acid (111) 50 50 50 50 (a)
25 25 25 25 (b)
Licorice-saponin G2 (112) >100 >100 >100 >100 (a)
>100 >100 >100 >100 (b)
Liquiritigenin (101) 50 50 50 50 (a)
50 50 50 50 (b)
Liquiritin (113) >50 >50 >50 >50 (a)
>50 >50 >50 >50 (b)
Formononetin (116) >100 >100 >100 12.5 (a)
12.5 12.5 12.5 12.5 (b)
Glabridin (23) 12.5 12.5 25 12.5 (a) G. glabra
12.5 12.5 12.5 12.5 (b)
Glabrene (24) 12.5 12.5 12.5 12.5 (a) G. glabra
12.5 12.5 12.5 12.5 (b)
Licochalcone A (59) 25 25 25 25 (a) G. inflata
25 25 12.5 12.5 (b)
Licochalcone B (82) >50 >50 >50 >50 (a) G. inflata
>50 >50 >50 >50 (b)
Licoricidin (63) 12.5 12.5 6.25 12.5 (a) G. uralensis
12.5 12.5 6.25 6.25 (b)
Licoisoflavone B (67) 6.25 6.25 6.25 6.25 (a) G. uralensis
6.25 6.25 6.25 3.13 (b)
Glycyrol (76) >50 >50 >50 >50 (a) G. uralensis
>50 >50 >50 >50 (b)
Isoglycyrol (117) >100 >100 >100 >100 (a) G. uralensis
>100 >100 >100 >100 (b)
AMOX** 0.05 0.05 0.05 0.20 (a)
0.025 0.025 0.025; 0.10 (b)
* (a): 2x10^ colony forming units (=cfti), (b): 2x10^ cfii. ** Positive control; amoxicillin (=AMOX).
ATCC 43504, ATCC 43526, and ZLM 1007 are CLAR-sensitive strains.
Next, we attempted to isolate further flavonoids exhibiting anti-^.

pylori activity from the extract of G. uralensis. In 1967, Takagi and
Ishii reported that one of the flavonoid-rich fractions of G. uralensis
(FMIOO), which also included about 15% glycyrrhizic acid (110), is
effective in prevention of digestive gastric ulcer by suppressing gastric
secretion [258,259]. The fraction was developed as an anti-ulcer drug
and ten similar medicines containing licorice extract have been also
supplied as prescribed drugs for treatment of gastric ulcer, duodenal ulcer,
and gastritis [255]. Our study of FMIOO showed that the medicine
exhibited anti-/f. pylori activity but did not contain licoricidin (63),
which is the main isoprenoid-substituted flavonoid in G. uralensis
[259,260] and exhibited anti-//. pylori activity as described above. The
other antibacterial agent 67 was not detected in FMIOO on TLC analysis.
The above investigations indicated strongly that licorice extract contains
some anti-//. pylori flavonoids.
242
H3CO.
3-0-methylglycyrol (118)
OH 0
glycyrin (121) isdicofiavonol (122)
6,8-diprenylorobol (124) 1-methoxyphaseollidin (125)
HO^ ^..^ ^ 0 ^
OH O _ . ^ ^
0CH3
gancaonin I (126) gancaond C (127) dihydroisoflavone A (128)
OCH3
4'-0-methylglabriclin (129) hispagiabridin A (130) shinflavanone(131)
Fig. (31). Structures of compounds 117-128 isolated from the active fractions of the methanol extract of
G. uralensis and compounds 129 -131 from the dichloromethane extract of G. glabra (Russian licorice).
The isolation of flavonoids from the methanol extract of G. uralensis

was carried out under non-basic conditions, because some flavonoids
isomerize under basic conditions, e.g. racemization of flavanones and
isoflavanones, ring-open reaction of flavanones etc. Bioactive fractions
were separated by some chromatographic methods and each step was
monitored with anti-//. pylori activity with the paper disk method.
Eighteen compounds were isolated from these bioactive fractions and
243
their anti-H. pylori activities were shown in Table 10. The MICs of the
growth of H. pylori of vestitol (119), licoricone (120), 1-methoxy-
phaseollidin (125), and gancaonol C (127), Fig. (31), were similar to that
of licoricidin (63). The activities of the other flavonoids were weak and
similar to those of glycyrrhetic acid (111) and liquiritigenin (101). All
the compounds investigated here had weaker anti-//. pylori activity;
however, these compounds may be chemopreventive agent agents the H.
pylori infection. Furthermore, these compounds may be bacteriostatic
agents for the bacteria in the stomach and prevent peptic ulcer or gastric
cancer disease in H. pylori-mfQCtcd people. However, further pharma-
cological and clinical studies including the antibacterial effect in liquid
medium are required for confirmation of this hypothesis.
Imakiire et al. also reported antibacterial activities of compound 23,
4'-0-methylglabridin (129), hispaglabridin A (130), glabrol (25) and
shinflavanone (131), Fig. (31), from the lipophilic extract of Russian
licorice, G. glabra; Maruzen P-TH® that is a material of medicines and
cosmetics [126,127].
Table 10. Anti-Helicobacter pylori activities (MIC, |ig/mL) of the flavonoids from Glycyrrhiza uralensis
ATCC 43504 ATCC 43526 ZLM 1007 ZLM 1200 GP98 (cfu)*
Glyasperin D (62) 25 25 12.5 25 12.5 (a)

25 25 12.5 25 6.25 (b)
3-0-Methylglycyrol (118) >16 >16 >16 >16 >16 (a)
>16 >16 >16 >16 >16 (b)
Vestitol (119) 12.5 12.5 12.5 12.5 12.5 (a)
12.5 12.5 12.5 12.5 6.25 (b)
Licoricone (120) 12.5 12.5 12.5 25 12.5 (a)
12.5 12.5 12.5 12.5 12.5 (b)
Glycyrin (121) 50 50 50 50 25 (a)
50 50 25 50 25 (b)
Isolicoflavonol (122) 50 25 25 50 25 (a)
25 25 25 25 12.5 (b)
Gancaonol B (123) >32 32 32 32 16 (a)
32 16 32 16 16 (b)
6,8-Diprenylorobol (124) >50 >50 50 >50 50 (a)
50 50 50 50 50 (b)
l-MethoxyphaseoUidin (125) 16 16 16 16 16 (a)
16 8 16 16 8 (b)
Gancaonin I (126) 50 50 50 >50 50 (a)
50 50 50 50 50 (b)
Gancaonol C (127) 16 16 32 32 16 (a)
16 8 16 16 16 (b)
Dihydrolicoiso- >25 >25 25 >25 25 (a)
flavoneA(128)^ >25 >25 25 >25 25 (b)
CLAR** 0.025 0.0125 < 0.0063 0.0125 50 (a)
0.0125 < 0.0063 < 0.0063 < 0.0063 12.5 (b)
AMOX** 0.05 0.05 0.05 0.025 0.2 (a)
0.025 0.025 0.025 0.125 0.1 (b)
* (a): 2x10^ cfu, (b): 2x10^ cfu. ^ Tentative name used here.
** Positive control; clarithromycin (=CLAR) and amoxicillin (=AMOX).
244
Protonpump inhibitor-based triple therapy is now the most

commonly accepted eradication regimen for peptic ulcer patients with H.
pylori infection. However, CLAR resistance is an increasing problem
as its use has become more common in recent years [234,261]. It is
interesting that licorice flavonoids exhibited anti-//. pylori activity
against not only CLAR and AMOX-sensitive strains but also CLAR and
AMOX-resistant strain CP98: Although licorice has been used as a crude
drug in Japan from more than 1200 years [262], these strains have not
developed resistance to the licorice flavonoids. These compounds may
be useful as lead compounds in the development of a new class of anti-//.
pylori agents.
X. EFFECTS OF ISOPRENYLATED FLAVONOIDS FROM

MORUS SPECIES ON TESTOSTERONE 5a-REDUCTASE
In Japan, the extracts of mulberry tree have been used for promotion of
hair growth and prevention of baldness [263]. Testosterone
5a-reductase catalyses the reduction of testosterone to its active form,
5a-dihydrotestosterone (5a-DHT). 5a-DHT has been implicated in
certain androgen-dependent conditions such as benign prostatic
hyperplasia, acne, and male pattern boldness [264]. And 5a-reductase
activity is high in situ. Inhibitions of 5a-reductase may be usefuU for
the treatment of these diseases. Therefore we studied on 5a-reductase
inhibitory activity of some isoprenylated flavonoids isolated from the
root bark of Japanese mulberry tree [265]. Table 11 shows the
5a-reductase inhibitory activity of flavonoids isolated from the root bark
of Morus species. Most of the flavonoids had inhibitory activities
against 5a-redactase, and showed the activity in the range of 10^ - lO"''
mol/L. Kuwanon E (43) had the most potent activity of these
compounds and its IC50 value is 6.9x10"^ mol/L, while kuwanon G (1)
had no effect at 10"^ mol/L. Fig. (32) shows the effects of kuwanon E
(43) on the Lineweaver-Burk plots of rat prostate 5a-reductase activity
using testosterone as a substrate. The addition of 3x10"^ mol/L
kuwanon E (43) produced a parallel shift indicating un-competitive
inhibitor. And the apparent K\ value is 7.6x 10~^ mol/L.
Enzyme kinetic studies of inhibitor are very important for
considering as a therapeutic agent. It is interesting to note that
isoprenoid-substituted flavonoids having non-steroidal structures are
potent un-competitive inhibitors of 5a-reductase. So, it would be
expected that the isoprenoid-substituted flavonoid derivertive would be
an interesting lead compounds for testosterone 5a-reductase inhibitor.
245
Table 11. Effects of Morus flavonoids on testosterone 5a-reductase
Inhibition (%)* IC50 (mol/L)
Morusin (3) 59.6

Oxydihydromorusin (46) 35.6
Kuwanon C (42) 63.0 8.2x10"^
Kuwanon E (43) 94.0 6.9x10"'
Kuwanon G (1) 0
Kuwanon H (2) 100 1.8x10"^
Kuwanon L (44) 53.0 4.4x10"^
Mulberrofiiran A (47) 24.2
Mulberrofuran G (30) 37.0
' Final concentration at 100 |imol/L.
lA'estosterone (1/10^ mol/L)

Fig. (32). Lineweaver-Burk plots of inhibition of prostatic 5a-reductase by kuwanon E (43). The assay
was carried out at varied concentration of [4-*'*C]testosterone in the absence (o) or in the presence of 0.3
Hmol/L kuwanon E (•).
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PLANT POLYPHENOLS:
STRUCTURE, OCCURRENCE AND BIOACTIVITY
PffiRGIORGIO PIETTA, MARKUS MINOGGIO, LORENZO

BRAMATI
ITB-CNR, Via FJli Cervi, 93- 20090 (MI), Italy
ABSTRACT: Main dietary plant polyphenols are grouped into structural types and their
occurrence in most common foods and beverages is briefly described. The major groups
of plant polyphenols which are examined include flavonols, flavones, flavanones,
isoflavones, anthocyanins, proanthocyanidins and flavanols. The current evidence on the
absorption and the metabolism of each group is discussed. The biochemical and
pharmacological activities of plant polyphenols are summarized, including antioxidant
and anti-radical activity, chelation of metal ions, modulation of some enzymes activity,
anticarcinogenic, antiatherosclerotic, anti-inflammatory, spasmolytic, hepatoprotective,
antiviral, antimicrobial and oestrogenic activity, and inhibition of histamine release. An
overview on the epidemiological evidence linking the intake of plant polyphenols and
diminished risk of chronic diseases is also included.
INTRODUCTION
Polyphenols constitute one of the most and widely distributed groups of

substances in the plant kingdom, with more than 8000 phenoUc structures
currently knovra. They can be divided into at least 10 different classes
based upon their chemical structure, ranging from simple molecules, such
as phenolic acids, to highly polymerized compounds, such as taimins.
Flavonoids constitute the most important group with a common
structure of diphenylpropanes (C6-C3-C6), consisting of two aromatic
rings linked through three carbons that usually form an oxygenated
heterocycle. Based upon the variations in the heterocyclic ring ,
flavonoids can be subdivided into eight major subclasses, including
flavonols, flavones, flavanones, isoflavones, flavanols, anthocyanins,
proanthocyanidins and tannins.
Plant polyphenols have been of interest for long time owing to their
role in plant pigmentation, reproduction and protection against predators
and pathogens. Recently, interest in the biological effects of plant
polyphenols (particularly flavonoids) has increased, because of the the
potential health benefits associated with some dietary polyphenols. This
258
contribution provides a brief description of the chemistry of plant

polyphenols, their occurrence, bioavailability and bioactivity.
CLASSES OF PLANT POLYPHENOLS
I. Hydroxy benzoic acids derivatives
The hydroxybenzoic acid derivatives (HBAs) are phenolic compounds

with a general structure Ce-Ci. The related C6-C2 acids (phenylacetic
acids) occur occasionally as minor components of foods.
Variations in the basic structure of HBAs include hydroxylation and
methoxylation of the aromatic ring, Fig. (1), Table 1.
Fig. (1). Basic structure of hydroxybenzoic acids
Although HBAs can be detected as free acids in some fruits (e.g.,

gallic acid in persimmons) or after being released during fruit and
vegetable processing, they occur mainly as conjugates. For example,
gallic acid and its dimer ellagic acid may be esterified with a sugar,
usually glucose to produce the so-called hydrolysable tannins. In addition,
gallic acid may esterify condensed tannins (i.e., derivatives of flavanols
like those present in tea) or quinic acid [1,2]. Four HBAs, namely 4-
hydroxybenzoic acid (4-HBA), vanillic acid (3-methoxy-4-hydroxy),
syringic acid (3,5-dimethoxy-4-hydroxy) and protocatechuic acid (3,4-
dihydroxy), are constituents of lignin [3]. These acids occur also as esters
of glucose.
259
Table 1. Structure of the most common hydroxybenzoic acids
Trivialname MW Position of Position of

(Da) OH groups OCH3 groups
Benzoic acid 122 /

Salicylic acid 138 2
(2-Hydroxybenzoic acid)
4-Hydroxybenzoic acid 138 3
Protocatecliuic acid 154 3,4

(3,4-Dihydroxybenzoic)
Gallic acid 170 3,4,5

(3,4,5-Trihydroxy benzoic)
Vanillic acid 168 4 3

(4-Hydroxy-3-methoxybenzoic
Isovanillic acid 168 3 4

(3-Hydroxy-4-methoxybenzoic)
Syringic acid 198 4 3,5

(4-Hydroxy-3,5-dimethoxybenzoic)
Dietary occurrence
Some herbs and spices are comparatively rich in various HBAs. After
hydrolysis, protocatechuic acid is the dominant HBA in cinnamon bark
(23-27 mg/kg), accompanied by saUcylic acid (7 mg/kg) and syringic acid
(8 mg/kg). Gallic acid dominates in clove buds (175 mg/kg) and is
accompanied by protocatechuic acid (10 mg/kg), genistic acid/4-HBA (7
mg/kg) and syringic acid (8 mg/kg) [4]. The fruit of anise {Pimpinella
anisum) contains 730-1080 mg/kg of the glucoside of 4-HBA [3].
The skin of potato tubers contains protocatechuic acid (100-400 mg/kg
FW) and vanillic acid (20-200 mg/kg) along with up to 30 mg/kg of
gallic, syringic and salicylic acids [5].
Cereals contain also different HBAs. Canadian wheat flours were
found to contain vanillic acid (up to 16 mg/kg) and syringic acid (up to 7
mg/kg). Oats contain vanillic acid, 4-HBA and salicylic acid, particularly
in the hulls [6].
Alcoholic beverages (wine and beer) have a different content of HBAs.
The gaUic acid content of French wines and spirits can reach 31-38 mg/1
260
[7]. White wines contain less HBAs than red wines, namely 16-46 and
65-126 mg/1 for white and red Califomian wines [8]. Barley contains
vanillic acid (6-17 mg/kg) and syringic acid (1-22 mg/kg), and both are
found in malt (12 mg/kg each) and hops (59 and 30 mg/kg). These two
acids are foimd in stout, ale and lager beers in the range from 0-2 mg/1)
accompanied by gaUic, protocatechuic and 4-HBA (0.1-1.8 mg/1 each)
[9].
Table 2 gives an overview of the occurrence of some HBAs in foods
[10,11] and Table 3 shows the content of the major HBAs in selected
foods [12].
Table 2. Occurrence of some HBAs in different dietary sources (10,11]
HBAs Dietary source

Benzoic acid universal component of angiosperms, csp. of berries
Salicylic acid anise, dill, white mustard, allspice,rosemary,thyme, majoram
4-Hydroxy benzoic acid raspbcny, gooseberry, pecans, anise, fennel
Vanillic acid vanilla, garden cress, paprika
Syringic acid rosemary, basil, thyme, garden cress
Protocatechuic acid tarragon, clove, anise, cinnamon, blackberry, blueberry
Gallic acid tea, nuts, olive oil
Table 3. Content of some HBAs in fruits (mg/kg) [12]
4-Hydroxy benzoic acid Protocatechuic Gallic acid

Food acid
Blackberry 6-16 68-189 8-67
Blackcurrant 0-6 10-52 30-62
Raspberry 15-27 25-37 19-38
Redcurrant 10-23 3-8 -
Strawberry 10-36 - 11-44
White currant 5-19 - 3-38
2. Hydroxycinnamic acid derivatives
Cinnamic acids are ^ra«5'-phenyl-3-propenoic acids differing in their ring

substitution, Fig. (2).
COOH
I II
%^
Fig. (2). Basic structure of cinnamic acids
261
The most common cimiamic acids are caffeic (3,4-dihydroxycimiamic

acid), ferulic (3-methoxy-4-hydroxy), sinapic (3,5-dimethoxy-4-hydroxy)
and p-coimiaric (4-hydroxy) acid, Table 4 [13].
These compoimds are widely distributed as conjugates, mainly as
esters of quinic acid (chlorogenic acids, CGA).
Table 4. Structure of the most common cinnamic acids
Trivialname MW Position of Position of

(Da) OH groups OCH3 groups
Cinnamic acid 148 /
p-Coumaric acid 164 4

(4-Hydroxycinnamic)
Cafleic acid 180 3,4

(3,4-Dihydroxycinnamic)
Ferulic acid 194 4 3

(4-Hydroxy-3-metlioxycinnamic)
Sinapic acid 224 4 3,5

(3,5-Dimetlioxy-4-iiydroxycinnamic)
Depending on the identity, number and position of the acyl residues,

these acids may be divided into the following groups: mono-esters of
caffeic, p-coumaric and ferulic acid; di-, tri- and tetra-esters of caffeic
acid [14,15]; mixed di-esters of caffeic and ferulic acid or caffeic and
sinapic acid [16]; mixed esters of caffeic acid with dibasic aliphatic acids
(e.g., oxalic, succinic) [17].
Cinnamic acids may condense with molecules other than quinic acid,
including rosmarinic, malic and tartaric acid, aromatic amino acids,
choline, mono- and polysaccharides, glycerol, myo-inositol, and different
glycosides (anthocyanins, flavonols and diterpenes) [13].
Dietary occurrence
There is no doubt that caffeic acid is the cinnamate that occurs most
extensively, and the various caffeoylquinic acids (CQA) and
dicaffeoylqumic acids (diCQA) are the most ubiquitous conjugates.
Usually the 5-isomers dominates, but in some fruit and brassicas the 3-
isomer is prevalent. Because of the quantity commonly consumed, coffee
262
beverage must top the list, with 200 ml instant brew (2% w/v) supplying
50-150 mg CQA, mg. Blueberries, aubergines, apples, cider and green
mate are good sources in some populations [13].
However, in view of quantities consumed by other populations, wines
could make a significant contribution for tartaric acid conjugates and
grapes and grape juice for caftaric acid, respectively. Lettuce is the major
source of chicoric (dicaffeoyltartaric) and caffeoylmalic acid (up to 3
mg/100 g), but endive may have twice the concentration. Spinach is
ahnost certainly therichestsource of conjugated p-coumaric acid at some
30-35 mg/100 g [18].
Broccoli florets and leafy cruciferous vegetables will be the major
source of sugar esters and of conjugated sinapic acid (10 mg/100 g).
Tomato and tomato products are likely to be the major source of
glucosides at up to 13 mg/100 g in total, and possibly the second richest
source of conjugate/7-coumaric acid (3 mg/100 g).
Cereal bran and bran-enriched products are the most important source
of wall-bound cinnamates with up to 30 and 7 mg ferulate/10 g in maize
and wheat bran, respectively. This would make these products the richest
dietary source of ferulic acid. However, coffee brew could supply up to
10 mg ferulate (as feruloylquinic acid, FQA) per 200 ml cup [13], and it
is the first for conjugated ferulic acid , followed by Citrus juices.
Table 5. Dietary sources of individual cinnamates and each major class of conjugate [13,18]
Name Dietary source

Cinnamates
CafTeic acid Coffee beverage, blueberries, apples, ciders
p-Coumaric acid Spinach, sugar beet fibre, cereals brans
Ferulic acid Coffee, citrus juices, sugar beet fibre, cereal brans
Sinapic acid Broccoli, kale, other leafy brassicas, citrus juices
Conjugates
Caffeoylquinic acids Coffee beverages, blueberries, apples, ciders
p-Coumaroylquinic acids Sweet cherries
Tartaric conjugates Coffee
Malic conjugates Spinach, lettuce, grapes, wines
Rosmarinic acid 1 Culinary herbs, mixed herbs, possibly stuffing
Cell wall conjugates Spinach, sugar beet fibre, cereal brans
263
3. 4-oxo-flavonoids: flavonols, flavones, isoflavones, flavanones,

chalcones, dihydrochalcones
The term 4-oxo-flavonoids includes a group of complex polyphenols that

share a common structure of diphenylpropanes (C6-C3-C6) characterized
by two aromatic rings and an oxygenated heterocycle, Fig. (3). The three
rings are referred to as the A, B, and C (or pyrane) rings.
J'
11 B 1
.<^\ ^?\,/^\e^^
1 A c II
% / ^
II
0
Fig. (3). Basic structure of 4-oxo-flavonoids
Their biosynthesis derives from the condensation of three acetyl units

and of a derivative of hydroxycinnamic acid leading to the formation of a
conmion intermediate, tetrahydroxychalcone. This chalcone is precursor
of several compounds, the most important being the 4-oxo-flavonoids
[19].
The 4-oxo-flavonoids can be distinguished based upon the
modifications of the nucleus, including the pyronic cycle saturation, the
number and the position of hydroxyl groups and the degree of
methylation and glycosylation. In plants,flavonoidsoccasionally occur as
aglycones, the most commonly forms being 0-glycoside derivatives.
Flavones may also occur as C-glycosides. The bond between the aglycone
and sugar moieties is generally located at 7- (flavone, flavanone), 3-
(flavonols), or 4'-position. The sugars are mono-, di-, tri- and even
tetrasaccharides, being D-glucose, L-rhamnose, glucorhamnose,
galactose, and arabinose the most frequent [19,20].
Depending on their aglycone structure, at least five different groups of
4-oxo-flavonoids are distinguishable: flavonols, flavones, flavanones,
isoflavones and chalcones.
264
Dietary occurrence
The concentration of 4-oxo-flavonoids depends on the plant,

environmental conditions, the part of the plant consumed, the degree of
ripeness as well as on the food processing. Flavonoids are preferentially
located in the epidermis: solubilized in the vacuolar sap (especially
flavones and flavonols glycosides) or in the epicuticular zone [20]. More
detailed information about the occurrence of the different compounds is
given in each subchapter.
3.1 Flavonols
Flavonols (about 380 aglycones) are characterized by the presence of a

hydroxyl group at position 3, Fig. (4). About 90% of the flavonols have
additional hydroxyl at positions 5 and 7.
r^'^4/^
II B 1
l < ^ \ /^^/^'^^K
1 M1 ^ 1
% / ^ S " " ÔH
1 0II
Fig. (4). Basic structure of Flavonols
Flavonols occur mainly as O-glycosides and the diversity of the

glycoside moiety in this group is noteworthy with about 200 different
quercetin and kaempferol glycosides described to date [19]. Table 6
reports the most common flavonols.
265
Table 6. Structure of the most connnon flavonols

MW Position of Substituents Position of tiie
Trivialname (Da) OH groups substituents
Aglycones
Fisetin 286.24 3,7,3',4'
Galangin 270.24 3,5,7
Kaempferol 286.24 3,5,7,4'
Morin 302.24 3,5,7,2\4'
Myricetin 318.24 3,5,7,3\4\5'
Quercetin 302.24 3,5,7,3',4'
Rhamnetin 316.27 3,5,3',4' OCHj 7
Glycosides
Quercltrin 448.38 5,7,3\4' 0-Rh 3
Rutin 610.53 5,7,3',4' 0-Ru 3
Rh = rhamnose = 6-dcoxy-L-mannose (C6H12O5); Ru = nitinose == 6-0-D-glucose
Dietary occurrence
Flavonols are present in plant foods mainly in the leaves and in the outer
parts of plants with quercetin and kaempferol the most common ones.
Quercetin and its glycoside are ubiquitous in fruits and vegetables.
Conversely, kaempferol and myricetin are less distributed (Table 7) [21-
23].
Specific quercetin glycosides have been detected in onions (quercetin-
4'-glucoside and quercetin-3,4'-diglucoside), broccoli (quercetin-3-0-
sophoroside, kaempferol-3-(9-sophoroside), green beans (quercetin-3-0-
glucuronide) and tomatoes (rutin = quercetin-3-O-rhamnosyl-glucoside)
[24,25], red wine (rutin) and tea (rutin, quercetin-3-(9-glucoside and
quercetin-3-O-galactoside) [26].
Preparation of fruits and vegetables for consumption (for example
peeling, skinning and cooking) can decrease quercetin and kaempferol
content significantly. For example, boiling, microwave cooking and
frying of onions or tomatoes involves a decrease in the content of
flavonols by 30 to 80% [2].
266
Table 7. Content of flavonois (expressed in mg/kg or mg/1) in foods determined by HPLC metliods
after liydrolysis of tlieir glycosides [21-23]
Food Quercetin Kaempferol Myricetin

Apple 20-36 - -
Apricot 25 - -
Bean, French 39 <12 -
French processed 17 <3.8 -
Green 16 - -
Broccoli 30-37 60-72 -
Currrant, black 37 1 -
Red 8-13 - -
Endive <1.3 46 -
Grape, black 15-37 - 4.5
White 2-12 - 4.5
Grape juice 4.4 - 6.2
Grape fruit juice (fresh) 4.4 - -
Kale 110-120 210-470 -
Leek - 30 -
Lettuce 14-79 - -
Onion 340-347 -
Orange juice 5.7 - -
Strawberry 6-8.6 5-12 -
Tea, black 14-17 14-16 3.0
Tomato 2-14 - -
Cherry tomato 63 -
Wine, red i 8.3 7.9
3.2 Flavones
Flavones differ from flavonois since the hyciroxyl group at position 3 of

the C-ring is missing, Fig. (5).
Fig. (5). Basic structure of flavones

267
About 300 different aglycones have been identified, and the most
frequently are luteolin, apigenin (especially in parsley) and diosmetin (in
Citrusfruits).Among glycosides, the 7-0- and C-forais are very conunon,
and are characterized by a carbon-carbon bond between the anomeric
carbon of a sugar molecule and the Ce or Cg carbon of the flavone
nucleus. Table 8 describes the most common flavones [19].
Table 8. Structure of the most common flavones
MW Position of Substituents Position of the

Aglycones
Apigenin 270.24 5,7,4'
Chrysin 254.24 5,7
Diosmetin 300.27 5,7,3* OCH3 4'
Luteolin 286.24 5,7,3\4'
Pinocembrin 256.24 5,7
Glycosides
Isoorientin 448.36 5,7,3',4' Glue 6
Isovitexin 432.36 5,7,4' Glue 6
Orientin 448.36 5,7,3\4' Glue 8
Vitexin 432.36 5.7,4' Glue 8
Glue = glueosc
Flavones contribute to plant tissue color provided that they occur in

high concentrations or are complexed with metal ions. Some flavones
participate in taste; for example, the highly methoxylated aglycones
nobiletin, sinensetin and tangeretin are responsible for the bitter taste of
citrus peel. On the other hand, some glycosylated flavones (for instance
neodiosmin and rhoifolin) reduce the bittemess of some substances
(limonin, naringin, caffeine, quinine) [2].
Dietary occurrence
Flavones are found mainly in grains and herbs and not frequently in fruits.
Apigenin and chrysoeriol have been detected in parsley, while cereal
grains and herbs contain apigenin and its glycosides as well as luteolin
[21,27].
268
Table 9. Content offlavones(expressed in mg/kg or mg/1) in foods determined by HPLC metliods after
hydrolysis of tiieir glycosides [21,28]
Food Luteolin Apigenin

Celery leaf 200 750
Stalk 5-20 16-61
Sweet peper, red 5-11 -
3.3 Isoflavones
Isoflavones are a distinct class of flavonoids which stracturally differ

from the commonflavonoidsin B-ring orientation, Fig.(6).
Fig. (6). Basic structure of isoflavones
The isoflavones have a chemical stracture similar to that of

mammalian oestrogen 17P-estradiol [28]: the phenoUc ring and a pair of
hydroxyl groups separated by a distance comparable to that occurring in
mammalian oestrogens are key structural elements of most compounds
that bind to oestrogen receptors [29,30]. Thus, isoflavones have recently
become best known for their oestrogenic activity, hence the name
"phytoestrogens". However, small differences in structures of the
individual phytoestrogens can dramatically alter their activity. For
instance, daidzein and genistein share identical structures except for an
additional hydroxyl group on the A-ring of genistein, but this favours up
to five- or six-fold oestrogenic activity of genistein in some assay systems
[31].
269
Table 10. Structure of the most common isoflavones

Aglycones
Daidzein 254.23 7,4' / /
Genistein 270.23 5,7,4' / /
Glycosides
Acetyldaidzein 430.23 7,4' Acetyl-gluc 6"
Acetylgeiiistin 446.23 5,7,4' Acetyl-gluc 6"
Acetyl-gluc = Acetyl-glucoside
Dietary occurrence
Isoflavones are present in plant foods either as aglycones (genistein or

daidzein) or - predominantly - as different highly polar and water-soluble
glycosides, including acetyl and malonyl glucosides and p-glucosides of
daidzein and genistein [32]. Legumes are the main source of isoflavones.
Soybeans are particularly rich in daidzein and genistein. Table 11 shows
the wide range in total isoflavone concentrations in soya products and
other legumes [32,33]. The concentration of isoflavones in soy foods is in
the range of 0.1-3.0 mg/g. However, theseflavonoidshave been detected
also in black beans, green split peas and clover [34]. Otherflavonoidsof
this group, including biochanin A and formononetin, have been found in
chick peas, green beans, and sunflower seeds.
Table 11. Content of isoflavones (expressed in ^g/g) in foods [3233]
Isoflavones (total) (fig/g) |xg per average portion size (g)

Food
Soya bean 579-3812 34740-228720 (60)
Tofu 79-674 10270-87620(130)
Soy flour 833-1778 16660-35560(20)
Textured soya protein 701-1184 28040-47360 (40)
Soya milic 34-175 3400-17500(100)
Miso 256-890 4608-16020(18)
Soy clieese 34-47 1360-1880(40)
Tofu yoghurt 151 18120(120)
Soy sauce 13-75 65-375 (5)
Green split peas 73 2920 (40)
3.4 Flavanones, chalcones, dihydrochalcones

270
Flavanones arise from flavones after reduction of the double bond in the
heterocycle (position C2/C3), Fig. (7).
Fig. (7). Basic structure of flavanones
Among aglycones, the best known are naringenin and hesperidin. Their
glycosylated forms occur commonly as O- or C-glycosides, usually as
rutinosides (6-0-a-L-rhamnosyl-D-glucosides) and neohesperidosides (2-
0-a-L-rhamnosyl-D-glucosides) attached at position 7. Flavanones
contribute to the flavour of citrus [19]. Table 12 reports the structures of
some common flavanones.
Table 12. Structure of some common flavanones

Aglycones
Eriodictyol 288.26 5,7,3*,4'
Hesperetin 302.28 5,7,3' OCH3 4'
Naringenin 272.26 5,7,4'
Glycosides
Hesperidin 610.57 5,3' Rli-Gluc; OCH3 7;4'
Naringin 580.54 5,4' O-Rh-Gluc 7
Rh = rhamnose = 6-dcoxy-L-mannosc (CeHnOs); Glue = glucose
Flavanones can be easily converted to isomeric chalcones in alkali (or

vice versa in acidic media) provided that there is a hydroxyl substituent at
position 2' (or 6') of the chalcone.
Chalcones are unsaturated and, along with dihydrochalcones, contain
an open pyronic cycle and a carbon skeleton numbered in a way different
from other flavonoids, Fig. (8, 9). Native chalcone glycosides tend to
transform into flavanone glycosides during extraction procedures.
Chalcones per se are therefore of restricted occurence in foods [35].
271
OH OH
, ^ ^ ^ 4 ^ rf^^^y
^==^,
OH OH
Fig. (8). Isosaiipiupurin (Chalcone) Fig, (9). Phloretin (Dihydrochalconc)
Dietary occurrence
Flavanones are found in a small number of foods. Chick peas (with the
flavanone garbanzol), cimiin, pepperaiint (both with hesperidin),
hawthom berry, licorice, rowanberry and citrus fruits are among those
fews containing molecules of this group. Naringenin and narirutin
glycosides are present in hawthomberry and rowanberry; liquoritigenin in
licorice roots. Flavanones neohesperidose (such as naringin) are found in
grapefruit and are usually bitter; the tasteless flavanone rutinosides (such
as hesperidin) are present in oranges [35,36]. Flavanone glucosides are
comparatively rare in species but are found for instance in different herbs
[37]. Hesperidin and the aglycones naringenin, eriodictyol and hesperitin
have been reported in the herbal tea (Honeybush tea) prepared from the
legume Cyclopia intermedia [38]. Naringenin and eriodictyol have been
reported in potato [5].
Citrusfiruitsand associated products (fiuit juices, peeled freshfruit)are
a major dietary source of flavanones (Table 13) [35]. However, the
distribution is quite scattered, and much higher concentrations are found
in the solid tissues compared to the juice. For example, an individual
drinking orange juice (250 ml) will have a daily flavone intake (as
aglycones) in the range of 25-60 mg; eating the flesh of a whole orange
(200 g) will provide about 125-375 mg.
Chalcones are comparatively rare in foods. Naringenin chalcone is
present in tomato skin and may be present in juice, paste and ketchup.
Acid hydrolysis, commonly applied prior to HPLC, converts the chalcone
to the corresponding flavanone (naringenin), which is naturally present
only in trace amounts (2-15 mg/kg) in the tomato [23].
Dihydrochalcones (DHCs) are characteristic of apples and derived
products (apple juice, cider, pomace etc.), and their content depends on
272
the cultivar. Phloretin 2'-glucoside (phloridzin), phloretin 2'(2"-xylosyl-

glucoside) and 3-hydroxyphloridzin have been identified unequivocally
and some investigators have reported phloretin 2' (2"-xylosyl-
galactoside). They are present in the skin, pulp and especially in tiie
seeds, where they account for up to 60% of the total phenols (compared
with less than 3% in the epidermis and parenchyma zones). When eating
an apple, the seeds and core of the fhiit are usually discarded and thus
some of the apple dihydrochalcones are not mgested. Whole apple fruits
are processed industrially to produce juices and ciders, and therefore the
contribution of these processed products to the intake of DHCs can be
higher than that of fresh apples (250 ml of apple juice or cider supply
about 1-5 mg phloretin. By contrast, a dessert apple of about 100 g
supplies less than 1 mg [39,40].
The dihydrochalcones aspalathin and nothofagin have been identified
as the main flavonoids in the South-Afiican Rooibos tea {Aspalathus
linearis) [41].
Table 13. Flavanones characteristic of common citrus fruits
Sweet orange Sour orange Lemon Grapefruit Lime Mandarin

(Citrus (Citrus (Citrus (Citrus (Citrus (Citrus
Flavanones sinensis) aurantium) limon) paradisi) aurantifolia) reticulata)
Eriocitrin - - ++ - - -
Narirutin + - - •H- - ++
Hesperidin +++ - +++ Trace +++ +++
Naringin - -H^ - 4-H- - -
Neohesperidin - ++ - Trace - -
4. Flavanols (FIavan-3-ols)
Flavanols have a C-ring structure similar to that of 4-oxo flavonoids, but

they are characterized by the lack of the double bond at the 2-3 position
and of the 4-oxo-group, Fig. (10) [19].
Fig. (10). Basic structure of flavanols

273
The flavan-3-ols most occurring in nature are (-f-)-catechin and (-)-

epicatechin (EC), although gallocatechin and epigallocatechin have also
been identified [42], Proanthocyanidins (or condensed tannins) include
oligo- and polymeric forms of the monomeric flavanols and will be
examined later. Polymerization of monomeric flavanols can occur as a
result of auto-oxidation, but more often it is catalyzed by
polyphenoloxidase (PPO), an enzyme that is present in most plant tissues
[43].
4.1 Catechins - Dietary occurence
Catechins are widely distributed in plants; however, they are rich only in
tea leaves, where catechins may constitute up to 25% of dry leaf weight.
Catechins of green tea include the flavanols epicatechin, epigallocatechin,
and their gallate esters (Table 14).
Table 14. Structure of the most common catechins

Trivialname OH groups substituents
(+).Catechin (C) 290.3 3(^-0H)
(-)-Epicatechin (EC) (cis form) 290.3 3 ( ^»M^»NOH\
Epigallocatechin (EGC) 306.4 3 ( VWSAAVOH)^ 5 »
Epicatechin-gallate (ECg) 442.4 3
OH
Epigallocatechin-gallate (EGCg) 458.4 5' 3
OH
The most abundant monomeric flavanols of black tea are (-)-

epicatechin gallate (ECg) and (-)-epigallocatechin (EGC) and (-)-
epigallocatechin gallate (EGCg) [44]. Indeed, quantitative analyses of
black tea reports levels of 31-79 mg/1 for EC, 5-91 mg/1 for EGC, 18-229
mg/1 for EGCg and 8-110 mg/1 for ECg; for green tea levels from 10-94
274
mg/1 for EC, 20-287 mg/1 for EGC and 60-408 mg/1 for EGCg are found
[45].
During fermentation in the preparation of black tea, oxidative
polymerization of flavanols occurs with the formation of theaflavin,
theaflavingallates, thearubigins, and epitheaflavic acid [44].
Flavanols have been determined in apples, apricots, pears, cherries,
peaches and plums [47,48].
The contents of (+)-catechin and (-)-epicatechin in red wine are
relatively high (up to 208 mg/1 for catechin and 90 mg/1 for EC) [49].
Low levels of (+)-catechin (-5 mg/1) and (-)-epicatechin (-1 mg/1) have
been reported for lager beers [50].
Data on grapes are limited; qualitative studies show the presence of
(+)-catechin, (-)-epicatechin and ECg in black and white grape seeds and
skins [51].
Recently, chocolate and cocoa have gained interest because of their
contents of catechins and related polymers (procyanidin oligomers) [52].
Fruit juices processing may seriously affect flavanol content. For
example, the preparation of commercial apple juice decreased the flavanol
content in a stepwise manner. In particular, crushing and pressing, storage
of the concentrated juice at room temperature and decolorization by
treatment with activated carbon destroy theflavanolsalmost entirely [46].
5. Anthocyanidins, anthocyanins, proanthocyanidins, tannins
5.1 Anthocyanidins and anthocyanins
The fundamental nucleus in anthocyanidins (aglycones) is flavylium

chloride. Most of the anthocyanidins are derivatives of 3,5,7-
trihydroxyflavylium chloride. Thus, the hydroxylation pattems in the
natural anthocyanidins fall into the three basic groups of pelargonidin,
cyanidin and delphinidin. Anthocyanidins are rarely found in fresh plant
material because of their instability [19].
On the other hand, anthocyanins, i.e. the glycosylated anthocyanidins,
are an important group of water-soluble pigments occuring in 27 families
of food plants (mainly red fruits and vegetables). Fig. (11) [53]. Table 15
shows the most common anthocyanidins and anthocyanins.
275
Fig. (11) Basic structure of anthocyanidins and anthocyanins
Table 15. Structure of the most common anthocyanidins and anthocyanins

Trivialname OH substituents
Anthocyanidins
JM
Pelargonidin 271.70 3,5,7,4'
Cyanidin 287.70 3,5,7,3*,4'
Delphinidin 303.70 3,5,7,3\4',5'
Malvidin 331.75 3,5,7,4* OCH3 3*,5'
Anthocyanins
Pelargonidin-3-glucoside 432.70 5,7,4' OGluc 3
Cyanidin-3-glucoside 448.70 5,7,3\4' OGluc 3
DeIphinidin-3-glucoside 499.70 5,7,3',4',5' OGluc 3
Malvidin-3-glucoside 492.70 5,7,4' OGluc; OCH3, 3;3',5'
The natural anthocyanins vary in:

• the basic anthocyanidin skeleton, i.e. the number and position of
hydroxyl and methoxyl substituents;
• the identity, number and positions(s) at which sugars are
attached to the skeleton; the most common sugars are glucose,
galactose, rhamnose and arabinose ( as 3-glycosides or 3,5-
diglycosides).
• the extent of sugar acylation and the identity of the acylating
agent(s); the most common acylating agents include ciimamic
acids (caffeic, p-coumaric, ferulic and sinapic), which may
themselves be glycosylated, and a range of aliphatic (for
example acetic, malic, malonic, oxalic and succinic acid) and
aromatic acids.
The anthocyanins occur in the vacuole as an equilibrium of four

molecular species: the coloured basic flavylium cation and three
276
secondary structures (the quinoidal base, the carinol pseuodobase and the
chalcone pseudobase) [54]. The pH changes the colour intensity of the
anthocyanins (for example, by the addition of vinegar or other acids while
cooking or processing). Commonly, anthocyanins are red in acid, violet in
neutral, and blue in alkaline solution. In fact, when cooking a food that is
red, such as red cabbage, it may be helpful to add an acidic substance
such as vinegar (or tomato juice or lemon juice) to prevent the food from
tuming purple. Anthocyanins contribute significantly to the red purple,
and blue color of flowers, many fruits of higher plants, vegetables and
associated products, beverages and preserves. Anthocyanins and
polymeric pigments derived from anthocyanins by condensation with
other flavonoids, are responsible for the color of red wine. It has been
recognized that anthocyanin-rich extracts might be used as food additives.
Many factors influence the stability of anthocyanins. Heat and light can
destroy sensitive anthocyanins during processing of fruits and vegetables.
In particular, anthocyanins are rapidly destroyed in the presence of a high
sugar concentration; thus processed foods containing large amounts of
sugar or syrup would not have the same amount of anthocyanins as their
improcessed counterparts [55],
Dietary occurrence
Anthocyanins are widespread in food plants, with an estimated worldwide

consumption of 10000 tonnes from black grapes alone [53]. The
anthocyanin content of many fruits and vegetables has been estimated by
various methods (Table 16) [56-58]. The main sources of these plant
pigments are fresh fruits such as cherries, plums, strawberries,
raspberries, blackberries, grapes, red currants and black currants.
277
Table 16. Content of anthocyanins in foods (expressed in mg/l or mg/lcg) [56-58]
Food Anthocyanins
Blackberry 1150
Blueberry 825-4200
Cherry 20-4500
Cliolceberry 5060-10000
Cranberry 600-2000
Currant (blacic) 1300-4000
Grape (red) 300-7500
Orange, Blood O'uice) 2000
Raspberry, black 1700-4200
Raspberry, red 100-600
Strawberry 150-350
Cabbage, red 250
Onion up to 250
Wines, red 240-350
Wines, Porto + Marsala 140-1100
5.2 Proanthocyanidins
Proanthocyanidins (PAs, syn condensed tannins) are polymeric flavan-3-

ols whose elementary units are linked by C-C and occasionally C-O-C
bonds (polymerization degree between 3 and 11), Fig. (12) [19].
Oxidative condensation occurs usually between carbon C4 of the
heterocycle and carbons Ce or Cg [59].
A characteristic of PAs is that they yield anthocyanins upon heating in
acidic media, hence their name, they yield anthocyanidins (hence their
name).
Two main types of PAs can be distinguished according to the
substitution pattern of their B-ring:
• Procyanidins: main constituents are catechin and epicatechin,
and are characterized by the presence of two hydroxyl groups
(3\ 4') in the B-ring.
• Prodelphinidins: main constituten is epigallocatechin, which has
three hydroxyl groups (3', A\ 5') in the B-ring.
278
^<^°" r
YY^^K'^^^ÔH HO^ ^«^ .0^ ^1* ^^5-^
" \ ^ - \ / ° ^ ^ v ^ V , OH
./'x
OH OH
»°\,^'-v^?\^'v^'
.1
% 4^ ÔH
Dimers:
Procyanidin B3: R3' = H Trimer.
Prodelphinidin B3: RS' = OH
Fig. (12). Basic structure of proanthocyanidins
Dietary occurence
Common sources of PAs are fruits, such as apple, strawberry, pear and
grape, beverages such as red wme and tea, and chocolate (Table 17) [59].
PAs complex protems, and are responsible for the astringency of foods
and beverages (e.g. grape skin and seeds, cider, wine) [19].
279
Table 17. Content of proanthocyanidins in foods (expressed in mg/1 or mg/lOOg) [59]
Food Proanthocyanidins
Apple 17-50
Apple juice nd-298
Barley 64-126
Beer 3.5-19,5
Blackberry 9-11
Cacao bean 260-1200
Cherry 10-23
Grape 1-160
Grape juice 3.546
Lentil 316-1040
Pear 0.7-12
Pear juice 11-74
Raspberry, red 2-48
Strawberry 2-50
Wines, red nd-500
5.3 Tannins
Tannins are compounds of intermediate to high molecular weight that

distinguish them from the groups of low molecular weight plant
phenolics. Tannins with a molecular weight up to 30000 DA have been
found in certain Leguminosae. One of their main characteristics of tannins
is the formation of insoluble complexes with proteins leading to the
astringency of taimin-rich foods (for instance tea) because of the
precipitation of salivary proteins [59].
Plant tannins are subdivided into two major groups (Table 18) [60]:
1. Hydrolyzable tannins: they consist of a central glucose molecule
linked to molecules of gallic acid (gallotannins) or
hexahydroxydiphenic acid (ellagitannins), Fig (13). They are
readily hydrolyzed, hence their name. The most common
hydrolyzable tannin is tannic acid, Fig. (14), which is a
gallotannin formed by a pentagalloyl glucose molecule esterified
by five gallic acid units.
280
OH
HO. OH
COOH O OH
Fig. (13). Structures of gallic (a) and ellagic (b) acid
OH
HO. X. .OH
CO
/
OH2C
OH
oc—o I
oc
HO" ^ "OH
OH
Fig. (14). Structure of tannic acid
Condensed tannins (= proanthocyanidins): unlike hydrolysable

tannins, condensed tannins are polymeric flavans that are not
readily hydrolysable. They often consist of molecules of catechin
and epicatechin joined by carbon-carbon bonds. Hence catechin
and epicatechin are referred to as monomers; oligomers
containing 2-4 (epi)catechin units are referred to as oligomeric
procyanidins (OPC).
281
Table 18. Classification of tannins
Type of tannin Example
1. Hydrolyzable tannins:
• Gallotannins Pentagalloylglucose
• EUagitannins and metabolites Geranin, Corilagin
• Ellagitannin oligomers Agrimoniin
2. Condensed tannins:
• Proanthocyanidlns: Procyanidlns Epicatechin oligomers
Prodelphinidins Epigallocatechin oligomers
• Galloylated proanthocyanidlns
ABSORPTION AND METABOLISM OF SELECTED PLANT

POLYPHENOLS IN HUMANS
Gut absorption
The absorption and the metabolism of dietary polyphenols arte

determined primarily by their chemical structure, with glycosylation
playing an important role. In fact, glycosylation influences the
bioavailability of the polyphenols. It is generally stated that flavonoid
glycosides are hydrolyzed before being absorbed [61]. Therefore, the first
step of metabolism should involve the removal of the sugar moiety by
enzymes-(glycosidases). Glycosidases activity can occur in the food itself
(endogenous or added during process) or in the cells of the
gastrointestinal mucosa or can be secreted by the colon microflora. Non-
enzymatic deglycosylation in the hiraian body, such as in the acid
conditions of the stomach, does not occur [62]. The absorption of
polyphenols should therefore be controlled by enzyme specificity and
distribution.
Polyphenols with attached glucose are potential substrates for
endogenous human enzymes, while attached rhamnose is not a substrate
for human p-glucosidases and so is only cleaved by colon microflora a-
rhamnosidases [63].
Deconjugation and reconjugation reactions in metabolism
After the hydrolysis of a polyphenol glycoside to the free aglycone,

polyphenols are conjugated by methylation, sulfation, glucuronidation or
a combination. However, there are exceptions to this sequence, as
282
supported by different studies [64-68] reporting the absorption of intact

polyphenols glycosides. This is a critical point, since the formation of
conjugates dramatically alters the biological properties of the circulating
metabolites. Furthemiore, it should be reminded that significant
differences between the administration of drugs (usually in himdreds of
milligrams in one concentrated dose) and the consumption of dietary
polyphenols (usually <100 mg in a diluted dose) exist. These differences
imply that drugs can readily saturate the metabolic pathways that rely on
the supply of cofactors such as UDP-glucuronic acid. Hence,
unconjugated drugs are often found in the blood. On the other hand,
polyphenols found in food are not expected to saturate the metabolic
pathways, therefore being in circulation in the conjugated forms [63].
Metabolism by the gut flora
Polyphenols that are not absorbed in the stomach or small bowel will be
carried to the colon. Polyphenols that are absorbed, metabolized in the
liver and secreted with bile back to the small intestine will also reach the
colon. Here, microorganisms degrade both unabsorbed and absorbed
flavonoids. Indeed, colonic bacteria produce glycosidases,
glucuronidases, sulfatases that can strip flavonoid conjugates of their
sugar moieties, glucuronic acids and sulfates [61]. Human intestinal
bacteria are able to hydrolyse 6>-glycosides [69] as well as C-glycosides
[70]. In addition, the degradation involves the splitting of the heterocyclic
oxygen-containing C-ring. Degradation products can be absorbed [71],
and subsequently metabolized by enzymes present mainly in the liver,
where 3'-0-methylation by catechol-0-methyltransferase,
dehydroxylation, p-oxidation, and conjugation with glucuronic acid,
sulfate, and glycine occurs [72]. These metabolites are considered to
contribute to the biological effects of dietary flavonoids (antioxidants).
In general, the metabolism of dietary flavonoids may be sununarized as

shown in Fig. (15).
283
INTESTINE
Flavoaoid (agUcones
and glycosilated
fonns)
and products of the
microbic metabolism
FECES
Fig. (15). Metabolism of dietary flavonoids. GlcA = glucuronic acid; UGT = uridine 5'-
diphospoglucuronosyl transferase; Met = methyl; Sulf = sulfate; COMT = catechol-O-methyl
transferase; PST = phenol sulfo transferase
1. Flavonols
Absorption of flavonols from the diet was long considered to be

negligible, because flavonols are present in plants bound to sugars as >8-
glycosides. Only aglycones were supposed to be absorbable, whereas
glycosides were thought to be non- or only marginally absorbable [61].
One of the main flavonols studied is quercetin. Indirect evidence for
the presence of quercetin conjugates in humans was obtained by Manach
et al [73], who found the presence of quercetin in plasma after the
consumption of a complex meal rich in plant products only after p-
glucuronidase and sulfatase treatment. Furthermore, the authors reported
284
the presence of a methylated derivative of quercetin, isorhamnetin, in

three out of the ten subjects. These results are in good accordance with
data obtained by Conquer et al [74], where the concentration of quercetin
in fasting plasma of a quercetin-supplemented group was 23-fold higher
than that of the placebo-group.
Urinary excretion of quercetin increased significantly with dose and
time after the consimiption of fruit juice (blackcurrant and apple juice in a
1:1 mixture) in humans [75]. Ranges from 0.29-0.47% of ingested
quercetin were found in the urine. The presence of quercetin in urine
shows that it was absorbed by the gut, but the urinary content does not
necessarily reflect absolute absorptive efficiency because absorbed
quercetin may be metabolized (conjugated), stored and excreted through
other routes such as the biliary tract. However, since quercetin is present
in a variety of fruit and vegetables, plasma concentrations or urinary
excretion of quercetin may potentially be useful as biomarkers of habitual
intake of these foods.
The absorption of intact quercetin glycosides has been demonstrated
by some authors [64,65,76]. Holhnann demonstrated in ileostomy
subjects (who lack colon with the bacterial flora, thus circumventing the
problem of microbial degradation), that the quercetin glycosides from
regular foods (onions, tea) were far better absorbed than pure aglycone
(52%vs24%).
LC-ESI-MS analyses allowed the detection of intact flavonol
glucosides (rutin) in plasma of healthy volunteers after the consumption
of tomato extract [65].
Glycosides of flavonols from onions, such as quercetin-4'-0-glucoside
and quercetin-3'-0-methyl-4'-0-glucoside, have been found in the
plasma of volunteers with a peak of absorption of 0.5 - 4 h [64].
On the other hand, a diet-controlled cross-over-study [77] with
supplementation of quercetin and its glycoside rutin showed that rutin
occurred only as conjugated form (with glucuronic acid and/or sulfate
groups) in plasma suggesting that this glycoside was not absorbed in its
original form. Conversely, quercetin was detected in conjugated as well
as unconjugated (aglycone) forms. Interindividual differences in the
pharmacokinetics of both compounds were considerable. Sesnik et al [78]
found also no intact quercetin glucosides and only traces of aglycone in
human plasma after the administration of quercetin-3-glucoside or
quercetin-4'-glucoside as an oral solution, while quercetin glucuronides
were the major metabolites in plasma.
285
Interestingly, different studies demonstrated that the sugar moiety of

quercetin glycosides is an important detemiinant of their absorption and
bioavailibility [79-82]. Quercetin-3-rutinoside and quercetin-4'-glucoside
are important forms of quercetin in foods. The first one accounts for about
40% of quercetin in black tea [83] and the second one for about 45% of
quercetin in onions. [84]. Although the intake of quercetin-3-rutinoside is
twice that of quercetin-4'-glucoside, the absorption of quercetin-3-
rutinoside is only 17% of ingested dose, whereas the absorption of
quercetin-4'-glucoside is 52% of ingested dose [76]. Furthermore the
bioavailibilty of quercetin-3-rutinoside is only 20% of that of quercetin-
4'-glucoside [81].
Olthof et al [82] have tested the bioavailibilty of quercetin-3-glucoside
in comparison to that of quercetin-4'-glucoside and concluded that both
quercetin glucosides are rapidly absorbed in humans, irrespective of the
position of the glucose moiety. In addition, this study provided
information on the metabohsm of quercetin into isorhamnetin (3'-
methoxyquercetin). Of the ingested quercetin glucosides, --50% is
absorbed in the small intestine and subsequently converted into
isorhanmetin, in the liver and in other organs. The 50% of ingested
quercetin that is not absorbed in the small intestine is metabolized by the
colonic microflora into quercetin aglycone and phenolic acids, which
might be absorbed from the colon [73].
The bioavailibility of quercetin-glycosides from onions, containing
mainly quercetin-p-glucosides, was superior to that of various quercetin
glycosides from apples (containing a mixture of quercetin-p-galactosides
and p-xylosides) and of pure quercetin-3-rutinoside (major species in tea).
The possible matrix effect of the foods remains unclear.
It seems that overall percentage of absorption, determined by
measuring plasma levels of flavonols after enzymatic hydrolysis, does not
exceed 2-3% of the ingested dose. It is also likely that, as with other
micronutrients, the existence of a steady-state concentration of these
compounds could result in diminished absorption. Thus, it is conceivable
that the major parts of these flavonoids are either degraded to phenolic
acids in the large intestine or excreted in the faeces [72].
2. Flavones
Data on the absorption of flavones (namely luteolin) are limited. Shimoi

286
et al [85] investigated the intestinal absorption of iuteolin and iuteolin-7-

0-P-glucoside in rats and humans. The absorption analysis using the rat-
everted small intestine demonstrated that Iuteolin was converted to
glucuronides during passing through the intestinal mucosa and that
luteolin-7-O-P-glucoside was absorbed after hydrolysis to Iuteolin. In
plasma, either free Iuteolin as well as its monoglucuronide and methylated
conjugates were present, while luteolin-7-O-p-glucoside was not detected.
This indicates that glucosides may be first hydrolyzed to Iuteolin by the
microbacteria. The same authors reported that in humans free Iuteolin and
its monoglucuronide have been detected in plasma after oral
administration of Iuteolin.
3. Isoflavones
Intestinal microflora plays a key role in the metabolism and

bioavailibility of isoflavones [86]. After ingestion, soybean isoflavones
are hydrolyzed by intestinal glucosidases, which release the aglycones,
daidzein and genistein, Fig. (16).
demethylation
dehydroxylation
intestinal glucosidases reduction
ring cleavage
equol
malonylglucosides daidzein dihydrodaidzein
acetylglucosides genistein 0-desmethylangolensin
p-glucosides p-ethylphenol
absorption
hepatic conjugation-enterohepatic cycling

urinary excretion
Fig. (16). Metabolic fate of soybean isoflavones in humans (Setchell 1999)

287
These aglycones may be absorbed or further metabolized to different

metaboUtes, including equol, 0-<iesmethylangolensin, and p-ethylphenol
[87]. This metabolism seems to be highly variable in individuals. Setchell
et al [88] showed that after ingestion of 40 g/day soya meal over 5 days,
only four of six subjects excreted equol in urine (3-7 mg/day). After the
consumption of 40 g of whole soya flour for 2 days higher levels of
daidzein compared with genistein were recovered in the urine despite the
soya contained higher levels of genistein. Equol and O-
desmethylangolensin were detected in all individuals; in all cases, the
levels peaked on the first day post-challenge and then fell slowly
remaining above pre-challenge levels on the third day post-challenge [89].
Furthermore, tiie metabolism of isoflavones is influenced by different
components of the diet. A high fiber diet may increase the growth and/or
activity of bacteria responsible for equol production in the colon [90].
This is relevant since equol has an oestrogenic potency higher than the
precursor daidzein [91].
According to Rowland et al [92] dietary fat decreases the capacity of
gut microbioflora to synthesize equol. Additionally, the subjects may be
poor or good equol excretors, confirming that extensive interindividual
variation in isoflavone metabolism exists.
Knowledge of the pharmacokinetics of isoflavones in soy foods is
essential for making recommendations regarding efficacy in clinical
studies, rhe plasma half-life of daidzein and genistein, measured from
their plasma appearance and disappearance curves, is 7.9 h in adults; peak
concentrations occur 6-8 h after administration of the pure compounds
[93] with peak concentrations ranging firom 80 to 800 ng/ml (after a
single oral dose of 50 mg of pure isoflavones).
Similar plasma concentrations were detected for daidzein, genistein
and equol in adults consuming modest quantities of soy foods containing
about 50 mg/d of total isoflavones [91].
Few studies have been carried out in infants fed on soya formula.
Absorption of isoflavones by the infant was demonstrated firom the
appearance of daidzein and genistein in the urine of 4-month-old infants
fed soy formulas. Equol was not detected in the urine [94]. A later study
by Setchell et al [95] did not confrnn these results because equol was not
detectable or present only in traces in the serum of 4-month-old infants
fed soya infant formulas. Isoflavone concentration in human breast milk
increased after the consumption of a soya-rich diet, but their contribution
seems trivial in comparison to that from soy infant formulas [93].
288
4. Flavanones, chalcones, dihydrochalcones
The flavanones have received less attention in comparison to flavonols

and isoflavones, although their intake from the diet can be high and they
exhibit promising biological activity. Little infomiation is available about
the absorption or the kinetic behavior of the flavanones naringenm,
hesperetin and their glycosylated fomis naringin, hesperidin, and
narirutin.
Studies conceming urinary excretion of these compounds have
confimied their bioavailibility from fruits and that they are excreted, at
least to some extent, into the urine. The renal excretion of naringin,
naringenin and its glucuronides after the consumption of grapefruit juice
(20 ml/kg body weight) was investigated by Fuhr et al [96]. Only
naringenin and its glucuronides appeared in urine after an average lag-
time of 2 h. Neither naringin nor its glucuronides were found. The data
suggest that cleavage of the sugar moiety, presumably by intestinal
bacteria, is the first step of naringin metabolism. These data were
confirmed by Lee et al [97] who detected naringenin glucuronide in urine
samples after the administration of grapefruit juice (containing about 214
mg naringin).
However, a recent study reported that the glycoside naringin was
recovered (0.02% of the administered dose) in urine as unchanged
molecule, hence confirming those glycosides are absorbable [66].
The influence of glycosylation on the metabolism of naringenin-7-
glucoside and its aglycone in the conscious rat model was examined by
Choudhury et al [98]. It resulted that via oral route the glycoside group is
cleaved by an intestinal enzyme and then the aglycone is glucuronated
within the epithelium. By contrast, after intravenous dosing the majority
was detected as native glucoside in the urine.
Bioavailibility and kinetics of naringenin and hesperetin from orange
and grapefiiiit juices was also investigated by Erlimd et al [99]. Both
flavonoids were absorbed from the juices with great interindividual
variations. The authors hypothesized that these variations were caused by
differences in gastrointestinal microflora. Peak plasma concentrations of
naringenin and hesperetin were reached between 4.8 and 5.5 h, indicating
that an absorption takes place in the distal parts of the small intestine or
the colon (where enzymes capable of cleaving theflavonoidglycosides in
question are present).
289
5. Flavanols - Catechins
Early studies (in the 1970s) on the pharmacokinetics of (+)-catechin

revealed that tiiis flavanol is absorbed from the gastromtestinal tract
following administration to healthy volunteers (4.2 g in the form of
gelatin capsules) [100]. (•f)-Catechin was excreted in the urine together
with several imidentified metabolites, and the amount excreted within 24
h was about 7.5% of the administered dose.
Other authors suggested that catechins are converted to glucuronyl
derivatives in the intestinal mucosa and are further metabolized by
methylation, sulfation and conjugation with glucuronic acid, sulfate and
glycine [101].
Indeed, Lee et al [102] determined flavanol conjugates in human
plasma after the ingestion of green tea. EGCg was mainly present as a
sulfate conjugate (65%), followed by the free form (20%) and the
glucuronide (15%). EGC on the other hand was mainly present as
glucuronide (60%), followed by sulfate (30%). About 10% was detected
as imconjugated EGC aglycone.
More recently. Da Silva et al [103] detected the presence of
glucuronides, O-methylglucuronide sulfate, and glucuronide sulfate of
epicatechin (EC) in plasma of rats fed epicatechin.
Absorption of (-)-epicatechin from chocolate has been studied by
different authors [104-106]. Baba et al [104] found maximum levels of
total EC metabolites in plasma after 2 h of chocolate or cocoa intake.
Sulfate, glucuronide and sulfoglucuronide conjugates of non-methylated
EC were the main metabolites present rather than methylated forms. In
urine samples, excretion of total EC metabolites within 24 h was about
30% of total EC intake after chocolate and 25 % after cocoa consumption.
A 12-fold increase in plasma epicatechin concentration from 22 to 257
nmol/L was reported by Rein et al [105] after consmnption of 80 g
semisweet (procyanidin rich) chocolate within 2 h after ingestion. The
total antioxidant capacity of plasma increases of 31% within the same
time, and plasma 2-thiobarbituric acid reactive substances decreased up to
40%. These data support that consimiption of chocolate increases plasma
epicatechin concentrations and decreases plasma baseline oxidation
products. These results have been confirmed in another study by Wang et
al [106].
The bioavailability and metabolism of catechins was studied in humans
after consumption of black tea containing 15.48 mg of EGC, 36.54 mg of
290
EC, 16.74 mg of EGCg and 31.1 mg of ECg [107]. Plasma concentrations

of EGC, EC and EGCg increased significantly reaching peak plateau
between 5 and 8 h (peak levels of 145,174 and 20.1 nmol/L respectively).
ECg on the other hand increased linearly over the 24h-period, peaking at
50.6 nmol/L. Urinary excretion of EGC and EC paralleled the rise in
plasma levels. EGC, EC and ECg peaked at 5 h whereas EGCg at Ih.
Fecal catechin excretion varied widely from subject to subject, but it was
significantly different from baseline for all catechins. Furthermore the
authors calculated the percentage of ingested catechins. Only 1.68% of
the total catechins consumed (400 mg) was found in the plasma, urine and
feces, providing evidence that catechins undergo considerable metabolism
and/or degradation either in the gastrointestinal tract or in the body after
absorption.
After ingestion of green tea infiision (400 mg of total catechins),
epigallocatechin gallate (EGCg) and epicatechin gallate (ECg) were
detected in human plasma with an significant increase of these two free
catechins after enzymatic hydrolysis with glucuronidase/sulfatase,
indicating their presence in plasma mainly in the conjugated form [108].
At the same time, detectable amounts of final 60 mg catechin metabolites
were found in plasma and urine, including 4-hydroxybenzoic acid, 3,4-
dihydroxybenzoic acid, 3-methoxy-4-hydroxy-hippuric acid and 3-
methoxy-4-hydroxybenzoic acid.
LC/ESI-MS analyses were applied to determine urinary glucuronidated
and sulfated tea catechins after the administration of green tea to humans,
mouse and rats [109]. The major conjugates were identified as
monoglucuronides and monosulfates of EGC and EC. Besides these
metabolites, also 0-methyl-EGC-O-glucuronides, 0-sulfates and O-
methyl-EC-0-sulfates in human urine were detected. Furthermore, the
ring-fission metabolites of EGC and (-)-epicatechin, 5-(3',4',5'-
trihydroxyphenyl)-x-valerolactone and 5-(3 ',4'-dihydroxyphenyl)-y-
valerolactone respectively, have been detected in the monoglucuronide
and monosulfate forms.
It is not known whether tea catechin conjugates possess any biological
activities. In a study by Manach et al [110], the glucuronic/sulfate
conjugates were shown to have the same electrochemical behaviour as the
parent drug. Considering that the oxidation potential of chemicals may
represent their antioxidant capacity, the electrochemical behaviour of the
conjugates suggests that they are effective antioxidants. Nevertheless,
because the glucuronic acid/sulfate conjugates are generally more
291
hydrophilic than the parent compound, the tissue distributions of these

metabolites are likely to be more limited than those of the parent
catechins [111].
6. Hydroxycinnamates, hydroxybenzoic acids
Non-ruminants possess several intestinal Na'^^-dependent saturable

transport systems. These include the well-known sodium-glucose co-
transporter (SGLTl), responsible for the active uptake of glucose, and it
appears to be specific for cinnamic and ferulic acid and possibly for other
hydroxy-cinammic acids [112].
Healthy volunteers have shown to excrete caffeic, p-coumaric and
ferulic acid in the urine after the consumption of various fruits [113]. The
excretion of free ferulic acid in urine peaked after 7 h at a concentration
of-'T jiM after the consumption of tomatoes (36-73 g containing 21-44
mg ferulic acid). The concentration of free ferulic acid plus glucuronide
(and possibly sulfate) conjugates exceeded 20 |aM and accounted for
some 11-25% of the dose [114].
Simonetti et al [115] studied the plasma levels of caffeic acid after
consumption of 100,200, 300 ml of red wine (caffeic acid content of 9.01
mg/L) that provided about 0.9, 1.8, and 2.7 mg of caffeic acid,
respectively. The highest plasma levels of caffeic acid was reached 60
min after ingestion and decreased to basal values within 180 min (for 100
and 200 ml) and within 240 min (for 300 ml). Both, the absence of caffeic
acid in plasma before the trial and its significant, dose-dependent
increment after red wine ingestion suggest that it may be a possible
marker of consimiption of beverages containing this acid. Furthermore,
200 and 300 ml red wine intake produced a significant increase in plasma
total antioxidant capacity (TRAP).
Conceming HBAs, their metabolism involves conjugation with sulfate,
glucuronate and glycine. Methylation may also occur, as may
demethylation, dehydroxylation and decarboxylation (this only if there is
a 4-hydroxyl) [3].
292
7. Anthocyanins, proanthocyanidins
a. Anthocyanins
Dietary anthocyanins have gained much attention based on the

recognition of the "French paradox'* which led to the suggestion that
some components of red wine (in particular anthocyanins) may protect
against coronary heart disease.
Limited evidence on the absorption of intact anthocyanins exist until
today.
There are reports, based upon spectral properties from DAD-HPLC of
anthocyanin-like substances in plasma [116] and urine after acidification
[117]. However, the ^max observed for the components in urine was at 430
nm. Anthocyanins should not have an absorption peak around this
wavelength. Thus, the detected compounds appeared to be anthocyanin
metabolites [68].
These spectroscopic data do not provide conclusive evidence for the
absorption of intact anthocyanins. Conversely, anthocyanins from
elderberry were detected in human plasma using a more selective
approach based on HPLC coupled to UV detection (512 nm) [67]. These
findings have been confirmed by a recent study on the detection of
anthocyanins in their unchanged glycosylated form in urine and plasma
after ingestion of 720 mg anthocyanins [68].
b. Proanthocyanidins (PA)
The complexity and the lack of commercial pure standards of PA make

their analysis difficult. Their absorption depends on their degree of
polymerization. In some in vitro experiments, only PA dimers and
trimers, but not polymers with an average polymerization degree of 7,
were absorbed through an intestinal epithelium cell monolayer [118].
Experiments in chicken and sheep showed that polymeric PAs were not
absorbed through gut barrier [119,120].
Evidence occurred that polymeric proanthocyanidins could be
degraded by the colonic microflora into low-molecular-weight
compounds, which would be subsequently absorbed. The group of Deprez
[118] investigated their metabolism by human colonic microflora
incubated in vitro in anoxic conditions using non-labeled and ^"^C-labeled
293
purified proanthocyanidin polymers. Degradation occurred almost totally

after 48 h of incubation; metabolites identified were low-molecular-
weight phenolic acids: phenylacetic, phenylpropionic and phenylvaleric
acids, monohydroxylated mainly in meta or para-position.
It is supposed that, once fermentation products have crossed the
intestinal barrier, they reach the liver through the portal vein, where they
are further metabolized by dehydroxylation, methylation or conjugation to
sulfate esters or glucuronides as it has been shown for other flavonoids
[59].
POLYPHENOLS: BIOCHEMICAL AND PHARMACOLOGICAL

PROPERTIES
Polyphenols are endowed with different biological activities, including:
1. Antioxidant/anti-radical activity and chelation of metal ions

2. Modulation of some enzymes activity
3. Anticarcinogenic activity
4. Antiatherosclerotic activity
5. Anti-inflammatory activity
6. Inhibition of histamine release and spasmolytic activity
7. Hepatoprotective activity
8. Antiviral and antimicrobial activity
9. Oestrogenic activity
1. Antioxidant and anti-radical activity, chelation of metal ion
Polyphenols can act as antioxidants by a number of potential pathways.

The most important is likely to be by free radical scavenging, in which
the polyphenol can break the radical chain reaction. Polyphenols are
effective antioxidants in a wide range of chemical oxidation systems,
being capable of scavenging peroxyl radicals, alkyl peroxyl radicals,
superoxide, hydroxyl radicals, nitric oxide and peroxynitrate in aqueous
and organic environments [121]. This activity is due to the ability of
donating an H atom from an aromatic hydroxyl group to a free radical,
and the major ability of an aromatic structure to support an unpaired
electron by delocalization around the 7i-electron system. Phenolic acids
294
and flavonoids may be good antioxidants, particularly those possessing

the catechol-type structure [122-125].
Phenolic acids and flavonoids (PP-H) can also act as free radicalchain
(ROO*) reaction terminator, as follow:
ROO* + PP-H -> ROO-H -f PP*
The PP* radical is relatively stable and could react in another reaction
as terminator, as follow:
ROOVPP*->ROO-PP
The interaction between flavonoids and phenolic acids with other

physiologic antioxidants, such as ascorbate or tocopherol, is another
possible antioxidant pathway for these compounds [72,126,127].
Nevertheless, like must other antioxidants, flavonoids may also act as
prooxidant in particular circumstances [128,129].
Phenolic acids and flavonoids can also act as chelating agents,
complexing transition metals that are responsible of the initiation of
peroxidative processes (Fenton and Haber-Weiss reactions). This property
is much stronger in phenolics having a catechol, pyrogallol, or 3-hydroxy-
4-carbonyl group [130].
2. Modulation of some enzymes activity
Interaction of low molecular weight molecules, such as phenolic acids

andflavonoids,with macromolecules can modify their chemical-physical
properties. Different studies confirm the ability of phenolics in
modulating some enzymes, such as hydrolases, transferases, kinases,
oxidases, hydroxylases, glutathione S-transferase, nitric-oxide synthase,
cytochrome P450 systems, ATPases, lipases, phospholipases, adenylate
cyclase, RNA and DNA polymerase, human DNA ligase I, ribonuclease,
reverse transcriptase, topoisomerase, aromatase, hyaluronidase, elastase,
HIV-1 proteinase and integrase, aldose reductase [131,132]. Specifically,
enzymes such as xantine-oxidase, nitric oxide synthase, phospholipase,
cyclooxygenase and lipoxygenase, involved in the production of free
radicals in biologic systems, are also inhibited, in vitro, from different
polyphenols [132,133].
295
Overall, these activities may explain the correlation between

polyphenols and their anticancer, antithrombotic, anti-atherogenetic and
anti-inflammatory effects. However, more research is needed to determine
which of these activities can realistically be translated into clinical effects.
3. Anticarcinogenic activity
The antioxidant effect, the modulation of some enzymes and induction of

apoptosis are at the root of the anticancer mechanism. Carcinogenesis is a
multi stage process of genetic change that may be initiated by increased
and persistent damage to DNA causing permanent alterations in the
genetic message when the cell replicates its DNA and divides. ROS
(Reactive Oxygen Species) are potential carcinogens because they can
induce structural damage to DNA by oxidation, methylation, depurination
and deamination reactions.
The ability of some polyphenols to reduce or inhibit the oxidative
damage to DNA is well documented. For example chlorogenic acid
inhibits DNA damage in vitro caused by peroxynitrite [134], coumaric
acids are good free radical scavengers [135], caffeic and
dihydroxybenzoic acids are able to inhibit iron induced DNA damage
[135,136]. Chlorogenic acid can inhibit DNA damage caused by
monochloramine [137]. Many kind of flavonoids are able to scavenge free
radicals protecting DNA from oxidation, including the isoflavone
genistein [138,139], theflavonesluteolin and apigenin [140], the flavanol
epigallocatechin gallate [141], the flavonols myricetin and kaempferol
[140], quercetin [140-142], and its glycosylated derivatives, quercetin-3-
glycoside, quercitrin and rutin [140]. DNA damage may be also reduced
by metal binding properties of flavonoids, as demonstrated for rutin and
quercetin [143].
Another mechanism in reducing carcinogenesis by polyphenols is the
modulation of the enzymatic system that is in charge of the
metabolization of carcinogenic molecules. The cytochrome P450 family
of enzymes metabolizes a large number of procarcinogens to reactive
intermediates, which bind covalently to DNA and can induce mutation.
Severalflavonoidsare able to inhibit the activity of this family enzymes,
as reported by different authors [144-149]. The inhibition of this activity
byflavonoidsis directly correlated to their antimutagenic properties.
The glutathione transferases (GST), together with the tripeptide
glutathione (GSH), conjugate the highly reactive and potentially
296
carcinogenic substances, making such molecules more polar, thereby

facilitating their excretion. Flavanones, flavones, flavonols increase
hepatic GST levels and activity in rats [148,150].
Furthermore, by modification of gene expression, some polyphenols
may prevent or reverse carcinogenesis, inducing apoptosis or inhibiting
neoplastic transformation. Gallic acid induces selective cell death in
cancer cell [151], and hamster fed caffeic acid with the diet were shown
to be less susceptible to the effect of methylazoxymethanol, an initiator of
colon carcinogenesis [152]. Caffeic acid phenethyl ester from propolis,
given to mice bearing a germline mutation in the Ape gene and
spontaneously developing numerous intestinal adenomas by 15 weeks of
age, decreased tumor formation by 63% at a dietary level of 0.15% and
the examination of intestinal tissue from treated animals showed that
tumor prevention was associated with increased enterocytes apoptosis
[153]. Protocatechuic acid from Hibiscus sahdariffa induced apoptosis in
human leukemia cells [154]. Green tea polyphenols and epigallocatechin
gallate increase fragmentation in several human and rodent carcinoma
cells, but not in normal epidermal keratinocytes [155]. Epigallocatechin
gallate also induces apoptosis in transformed fibroblast [156] and in
human histiolytic lymphoma U937 cells [157]; theasinensin D, theaflavin,
theaflavin digallate induced apoptosis in the same lymphoma cells [157].
Quercetin, rutin, morin, gallic acid and tannic acid inhibited the growth of
human prostate cancer cell (LNCaP) at different concentrations, and
induced apoptosis [158].
4. Antiatherosclerotic activity
Cardiovascular heart diseases (CHD) are considered as the clinical

expression of advanced atherosclerosis. One of the initial steps in
atherogenesis is the oxidative modification of LDL and the uptake of the
modified lipoprotein particles by macrophages, which in tum become
lipid laden cholesterol-rich cells, so-called foam cells [159]. An
accumulation of foam cells in the arterial wall is the fu-st visible sign of
atherosclerosis and is termed fatty streak, the precursor to the
development of the occlusive plaque [160]. It is well known that
oxidation of LDL can be initiated in vitro by incubating isolated LDL
particles with cells (macrophages, lymphocytes, smooth muscle cells, or
endothelial cells), metal ions (copper or iron), enzymes, oxygen radicals,
or UV-light. However less is known about the mechanisms by which
297
LDL becomes oxidized in vivo. There is evidence that LDL is protected

against oxidation in plasma by water-soluble antioxidative substances,
such as ascorbic acid, uric acid, or bilirubin. Thus, it is likely that the
majority of oxidative modification of LDL occurs in the artery wall,
where LDL is largely isolated from the plasmatic antioxidants. Recent
evidence suggests that metal ions (copper or iron) and the enzymes
myeloperoxidase and lipoxygenase play major parts in the modification
of LDL [161].
In in vitro studies the oxidation of LDL by endotheUal cells,
macrophage and Cu"*^ can be inhibited by a wide range of polyphenols
and polyphenol-rich extracts [162-164]. Such effects may be due to
polyphenols by direct scavenging of the oxidizing species, by
regeneration of a-tocopherol in LDL [165], by their ability in binding
metal ion and LDL protein [166].
In addition to in vitro studies, several animal models and trials with
human subjects indicate that ingestion of polyphenols increases the
resistance of LDL oxidation ex vivo [167,168].
Some polyphenols inhibit platelet aggregation reducing the risk of
thrombosis [171-173]. This effect may be due to a series of interaction
of flavonoids in different biochemical pathways, such as by inhibition of
cyclooxygenase and lipoxygenase, that are involved in the arachidonic
acid metabolism in the platelets, or by inhibition of the formation of
tromboxane and of the receptor function of the same [173-176]. Regular
consumption of wine, tea and chocolate has been associated to the
reduction of platelet aggregation, cardio-vascular diseases and
thrombosis [171,177-179].
5. Anti-inflammatory activity
Inflammation is a highly complex biochemical protective response to

cellular injury. It is important in the maintenance of homeostasis when
the organism is challenged by noxious agents or by tissue mechanical
injury. Inflammation is associated with a drastic rise in the number of
polymorphonuclear leukocytes and monocytes in the affected tissue and
with the release of inflanunatory mediators such as prostaglandins and
cytokines. Under normal conditions, inflammation results in the
complete recovery of the integrity of the affected tissue, but if the
response to the triggering stimulus is not subjected to tight regulation.
298
cellular and extracellular components of the organism adjacent to the

inflammation site can be injured, inducing a condition known as chronic
inflammatory disease. A chronic inflammatory like environment
characterizes the pathogenesis of various diseases such as
atherosclerosis, arthritis, and Crohn's disease, and it is thought to be
among the causative factors of more than 30% of human cancers. For
these reasons it is very important to localize and reduce the
inflammatory response. In this scenario, polyphenols are involved as
immunomodulatory and anti-inflammatory agents, scavenging ROS and
modulating the activity of key enzymes of the inflammatory response
[172,180482].
6. Inhibition of histamine release and spasmolytic activity
The flavonoids quercetin, hyperoside and isoquercetin, present in the

ethanolic extract of Drosera madagascariensis, are inducers of
spasmolytic and anti-inflammatory effects in guinea-pig ileum by
affecting cholinergic M3 and histamine HI receptors [183].
In an in vitro study by Yamada et al [184], triphenols, such as
pyrogallol and gallic acid, and among flavonols, myricetin, inhibited
histamine release from rat peritonei cells. Pyrogallol and gallic acid, and
also o- and /?- diphenols, such as catechol and hydroquinone and all
flavonols tested, strongly suppressed leukotriene B4 release in the same
cells. Another in vitro study on rat basophilc leukemia cells
demonstrated that, among tea polyphenols, (-)-epigallocatechin gallate
(EGCg), (-)-epigallocatechin (EGC) and (-)-epicatechin gallate (ECg)
have different inhibitory effects on histamine release induced by a
calcium ionophore, with the following magnitude: EGCg>ECg>EGC
[185]. In the same study was also demonstrated that pyrogallol and gallic
acid exert inhibitory activity and a mixture of these two compounds
inhibited histamine release as strongly as EGCg.
7. Hepatoprotective activity
Polyphenols are also endowed to have hepatoprotective effects. For

example, quercetin reduces liver oxidative damage, ductural
proliferation and fibrosis in biliary-ostructed rats, suggesting that it may
299
be a useful liver protective agent in patients with biliary obstruction

[186].
8. Antiviral and antimicrobial activity
Polyphenols may act as antimicrobial and antiviral agents as

demonstrated by several studies in vitro. Polyphenol-rich extracts from
various plants, such as Betula pubescens^ Epilobium angustifolium,
Perillafrutescens, Pinus sylvestris, Rubus chamaemorus, Rubus idaeus.
Solarium tuberosum, propolis and pure compounds, were tested to
evaluate their antimicrobial activity against different bacteria and yeasts
species, such as Bacillus subtilis, Escherichia coli, Mycobacterium
tuberculosis H37Rv, Pseudomonas aeruginosa. Salmonella spp,
Staphylococcus aureus. Streptococcus piogenes, Aspergillus niger,
Candida albicans, Saccharomyces cerevisiae and showed growth
inhibitory and bactericidal effect at different concentrations [187-192].
Naturally occurring flavonoids with antiviral activity have been
recognized since the 1940s [193]. Quercetin, morin, rutin, taxifolin,
dihydrofisetin, leucocyanidin, pelargonidin chloride, apigenin, catechin,
hesperidin, and naringin have been reported to possess antiviral activity
against some of 11 types of viruses [193]. (-)-Epigallocatechin gallate and
theaflavin digallate inhibited the infectivity of both influenza A virus and
influenza B virus in Madin-Darby canine kidney cells in vitro [194].
9. Oestrogenic activity
Plant-derived oestrogens may exert both oestrogenic and anti-

oestrogenic effects, depending on several factors, including their
concentration, the concentrations of endogenous oestrogens, and
individual characteristics, such as gender and menopausal status
[195,196]. The anti-oestrogenic activity of phytoestrogens may be
partially explained by their competition with endogenous 17p-estradiol
for oestrogen receptors [197]. Many of the potential health benefits of
phytoestrogens may be attributable to features that do not involve
oestrogen receptors, such as their influence on enzymes, protein
synthesis, cell proliferation, angiogenesis, calcium transport, Na"^/K"*"
adenosine triphosphatase, growth factor action, vascular smooth muscle
cells, lipid oxidation, and cell differentiation. Phytoestrogens may have
300
favorable effects on the risk of cardiovascular disease and are thought to

be hypocholesterolemic, anticarcinogenic, antiproliferative,
antiosteoporotic, and hormone altering [195,196,198,199].
Finally, flavonoids can bind to structural proteins and this feature
could explain their ability to enhance the integrity of connective tissue.
EPIDEMIOLOGIC EVIDENCE OF PLANT POLYPHENOL

HEALTH BENEFITS
1. Risk of CHD diseases
Several epidemiological studies have reported inverse relation between

intakes of flavonols and flavones and cardiovascular heart diseases
(CHD).
In a prospective study of 3454 men and women (age 55 years and
older), a significant inverse association between the intake of catechin-
rich tea and radiographically quantified aortic atherosclerosis was found
[200]. Similarly, inverse association between the consumption of red wine
and CHD mortality (French paradox) have been suggested [201]. This
beneficial effect of red wine may be due to the antioxidant ability of the
wine phenolics to inhibit the oxidation of LDL to an atherogenic form
[202],
In the Zupthen Elderly Study [203] flavonol and flavone intake at
baseline in 1985 of approximately 800 men (aged 65-85 years) was
determined using the cross-check dietary history method. Men were
divided into tertiles of flavonol and flavone intake. After five years of
follow-up 43 men died from heart disease in this period. Flavonol and
flavone intake, expressed as tertiles, was inversely associated with
mortality from coronary heart disease and to a lesser extent with the
incidence of first myocardial infarction. Furthermore, the association
between long-term flavonol and flavone intake and risk of stroke in a
cohort of 552 middle-aged Dutch men free fi"om history of stroke at
baseline was also investigated within this study. Men were divided into
quartiles of flavonol and flavone intake, and followed for 15 years.
During this period 42 men had a first stroke event. Flavonol and flavone
intake was strongly inversely associated with stroke risk. In both studies,
the men in the highest category of flavonol and flavone intake
(>30mg/day) had about one-third the risk of getting the disease compared
301
with men in the lowest category. The major sources of dietary quercetin
and other flavonols were revealed as tea and onions (fruits and vegetables
had minor importance).
The same authors [204] confirmed these results in the Seven Country
Study. The contribution of flavonols and flavones in explaining the
variance in coronary heart disease mortaUty rates across 16 cohorts from
seven countries was studied. Flavonol and flavone intake was inversely
correlated with mortality from coronary heart disease. Thesefindingare
in line with the results of a cohort study in Finnland [205], where a
significant inverse gradient was observed between dietary intake of
flavonoids and total and coronary mortality.
A modest but not significant inverse correlation between the intake of
flavonols and flavones and subsequent mortality rates was found in a
prospective cohort study of US Health Professionals by Rimm et al [206].
The authors do not exclude thatflavonoidshave a protective effect in men
with established coronary heart disease although strong evidence was
missing. Also other studies failed to demonstrate a significant statistical
association between the intake of polyphenols and CHD. In Great Britain
for instance coronary and total mortality even rose with the intake of the
majorflavonolsource, tea [207]. The most likely explanation for the latter
observation is that in this study tea consumption merely acted as a marker
for a lifestyle that favours the development of cardiovascular disease.
Indeed, men with the highest intake of tea and flavonols tended to be
manual workers, and they smoked more and ate more fat [208].
2. Risk of cancer
The epidemiological evidence for a beneficial support of polyphenols in

cancer disease is contradictory and less clear than its role in CHD.
The Zupthen Elderly Study found a weak inverse association between
flavonoid intake from fruit and vegetables sources and cancer of the
alimentary and respiratory tracts combined [209]. The same authors
observed no independent association with mortality from other causes
between flavonoid intake and cancer mortality in the Seven Country
Study [204].
ICnekt et al [210] studied the relation between the intake of flavonoids
and subsequent cancer among 9959 finnish men and women during a
follow-up in 1967-1991, An inverse association was observed between
the intake offlavonoidsand incidence of all sites of cancer combined. Of
302
the major flavonoid sources, the consumption of apples showed an

inverse association with lung cancer incidence.
The cancer protective effects of black and green tea consiraiption,
important sources of flavonol in specific countries, have been investigated
mainly in case-control studies. Kohlmeier et al [211] evaluated the
epidemiologic literature about tea and cancer prevention, concluding that
cohort studies do not suggest a protective role for tea drinking in the total
risk of cancer. Site-specific studies give a more complex picture. For
example, a protective effect of green tea on the development of colon
cancer is suggested. On the other hand, evidence for black tea is less
clear, with some indication of a risk of colon or rectal cancer associated
with regular use of black tea.
In another cohort study of a Japanese population, researcher surveyed
more than 8000 individuals over 40 years of age on their living habits,
including daily consumption of green tea. Results found a negative
association between green tea consumption and cancer incidence,
especially among females drinking more than 10 cups per day [212].
3, Vasoprotective effects (Hypertension)
Experimental studies have shown that the administration of green tea-

enriched water to laboratory animals is associated with a reduction in
blood pressure [213].
Different epidemiologic studies have suggested that drinking either
green or black tea may lower cholesterol concentration and blood pressure
[214,215].
In a epidemiological study of Japanese women, a history of stroke was
less common among those who drank more green tea. There was no
statistically significant reduction in blood pressure alone among those
women who drank more tea [206].
4. Oestrogenic effects
Phytoestrogens represent a family of plant compounds that have been

shown to have both oestrogenic and anti-oestrogenic properties.
Accumulating evidence from molecular and cellular biology experiments,
animal studies and, to a limited extent, human clinical trials suggests that
phytoestrogens may potentially confer health benefits related to
303
cardiovascular diseases, cancer, osteoporosis, and menopausal symptoms.

These potential health benefits are consistent with the epidemiological
evidence that the risk of heart disease, various cancers, osteoporotic
fractures, and menopausal symptoms is lower among populations that
consume plant-based diets, particularly among cultures with diets that are
traditionally high in soy products.
One study over 9 months noted a significant reduction in total
cholesterol in premenopausal women when they consumed soy products
with 45 mg conjugated isoflavones/day in comparison to levels during a
control period when they were fed isoflavone-free soy products. The
treatment group difference was significant despite the small sample size
and the selection of healthy, normocholesterolemic women who had
limited room for detectable improvements [216]. The pattem of soy
intake and its association with blood lipid concentrations in the Hong
Kong Chinese population was studied in a total of 500 men and 510
women with an age range of 24-74 years by Ho et al [217]. In men, soy
intake and total plasma cholesterol were negatively correlated (r = 20.09,
P = 0.04), as were soy intake and LDL cholesterol fr = 20.11, P = 0.02).
The respective values in women <50 y old were r = 20.11, P = 0.04 and r
= 20.11,P = 0.05.
In the Framingham Offspring Study a group of 939 postmenopausal
women was studied to correlate the association between dietary
phytoestrogen intake and metabolic cardiovascular risk factors. Mean
blood pressure, waist-hip ratio (WHR) and lipoprotein levels were
determined in quartile categories of dietary phytoestrogen (isoflavones
and lignans) intake. In the highest quartile of intake of isoflavones,
plasma triglyceride levels were 0.16 mmol/L lower (95% CI, -0.30 to -
0.02) compared with the lowest quartile of isoflavones and the mean
cardiovascularriskfactor metabolic score was 0.43 points lower (95% CI,
-0.70 to -0.16) than the lowest quartile [218].
Hormone-related cancers of the breast, ovary, endometrium, and
prostate have been reported to vary by as much as 5 to 20-fold between
populations. Migrant studies indicate that the difference is largely
attributable to environmental factors rather than genetics [219,220]. The
highest rates of these cancers are typically observed in populations with
Westem lifestyles that include relatively high fat, meat-based, low fiber
diets, whereas the lowest rates are typically observed in Asian populations
with Eastem lifestyles that include plant-based diets with a high content
of phytoestrogens [219,221].
304
In a case-control study Ingram et al [222] reported a significant

reduction in breast cancer risk among both premenopausal and
postmenopausal women who consumed phytoestrogens. In a study of
Asian-Americans of Chinese, Japanese, and Filipino heritage, it was
reported that tofii consumption was significantly and inversely associated
with breast cancer [223].
Similar findings were reported from a case-control study of women in
Singapore in which soy intake was inversely, and animal products intake
was positively, associated with breast cancer, although these findings
were significant only among premenopausal, not postmenopausal women
[224]. Soy and fiber consumptions were both associated with a decreased
risk of endometrial cancer among the multiethnic population of Hawaii, a
finding that was limited to women who had never used oestrogens and
had never been pregnant [225].
In a study conducted in Boston and Helsinki, it was demonstrated that
the lowest excretion of enterolactone and equol was found in a group of
postmenopausal breast cancer patients compared to healthy omnivorous
and vegetarian women [226].
The continual loss of bone mass in the elderly is a natural process of
aging. Women have a higher incidence of osteoporotic fractures than men
due to their lower peak bone mass, but in addition, the abrupt decrease in
oestrogen secretion in postmenopausal women accelerates bone loss.
Currently, osteoporosis-related fractures are lower in Asia than in most
Westem communities, possibly due to the phytoestrogen-rich soybeans
and vegetables consumed in large quantities in the Asian diet [227].
Investigation about rates of hip fracture in Hong Kong and the U.S.
reported that for men and women 85 yr of age or more, the rates in Hong
Kong were roughly one third the rates in the U.S. [228].
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PROMISING PHARMACOLOGICAL ACTIONS OF

CROCIN IN CROCUS SATIVUS ON THE CENTRAL
NERVOUS SYSTEM
SHINJI SOEDAi, TAKASHI OCHIAIi, H I R O S H I SHIMENOi,

HIROSHI SArr02, KAZUO ABE2, MINORU SUGIURA^,
HIROYUKI TANAKA^ FUTOSHI TAURA^ SATOSHI
MORIMOTO^ a n d YUKIHIRO SHOYAMA^ *
^Facidty ofPharmaceutical Sciences, Fukuoka University, Fukuoka814-

0180, Japan
^Graduate School ofPharmaceutical Sciences, The University of Tokyo,
Tokyo 113'0033, Japan
^Graduate School ofPharmaceutical Sciences, Kyushu University,
ABSTRACT: Information on clinically available activities in both peripheral and

neuronal systems dF crocin in saffron has been accumulated. The LTP-blocldng effect erf
ethanol was significantly improved by oral-, intravenous-, and intracerebroventricular-
administraticm of crocin, respectively. We investigated the effects of ethanol and CTOcin
on synaptic potentials mediated by N-methyl-D-aspartate (NMDA) receptors in tfie
dentate gyrus of rat hippocampal slices. Crocin alone did not affect synaptic potentials
mediated by non-NMDA (x NMDA recq>tors. Crocin did not affect the inhibiticm of
non-NMDA response by 100 mM ethanol, but significantly blocked the inhibition of
NMDA response by 10-50 mM etiianol. We perfcnmed whole-cell patch receding with
primary cultured rat hippocampal neurons, and confirmed tiiat crocin blocked etfianol
inhibition of inward currents evoked by the application of NMDA. We also demonstrated
that crocin suppresses the effect of tumor necrosis factor (TNF)-a on neuronally
differentiated PC-12 cells. The modulating effects of crocin on the expression of Bcl-2
family proteins led to a marked reduction of a TNF-a~induced release of cytochrome c
from the mitochondoria. Crocin also blocked the cyotochrome c-induced activation of
caspase-3. We found that crocin inhibited the effect of daunorubicin as well. The present
paper focuses on the pharmacological actions of crocin on the central nervous system
and reviews briefly the findings of such studies on the prevention of neuronal
progranmaed cell death (apoptosis).
INTRODUCTION
Saffron {Crocus sativus L. ; Iridaceae)findsuse in medicine as well as a

flavoring and coloring agent. It has three main chemical compounds. The
bright red coloring carotenoids; a bitter taste, picrocrocin; and a spicy
314
aroma, safranal. The carotenoid pigments consist of oooelin (JKP4>^Jiioo^^

estei; crooetin<^B-gentiobk)fiylK^^ and crocetin-di-(P-D-
digentiobiosyl)-ester (crocin) as indicated in Fig. (1).
COOH
Hocx:
Crocctin
COO
OOC
R1-OH2C
R2-OH2C
Ri=Glc R2=Glc Crocin
Ri=Glc R2=H Crocctiii-(fi-D-gentiobiose)-(B-D-glucosyl)-ester

Ri=H R2=:H Crocetin-di-(6-D-glucosyi)-estcr
Fig.(l). Structures of crocetin and its glycosides
Previously, we showed that crocetin glucose esters increased from the

period before bloonung and reached maximum in the full blooming
period [1]. They are sensitive for the presence of o^gen, light irradiation
because of the polyene structure, and to an indigenous p-glucosidase
which hydrolyzes crocin to crocetin di-(p-D-glucose)-ester [1]. The
artificial pathway is indicated in Fig. (2). Moreover, it is evident that
storage of saffron at -20 °C promotes the constant supply of saffron with a
homogîeous pharmacological activity [1]. In order to check the quantity
of saffi'on, we have already prepared a monoclonal antibody (MAb)
against crocin, and established a competitive ELISA using the anti-crocin
MAb [2].
In the peripheral blood system, crocetin derivatives prevent an
elevation in bilirubin levels [3] and also reduce elevated levels of serum
cholesterol and triglyceride [4]. Anti-tumor activity of saffron is observed
in mice transplanted with several types of tumor cell lines including
sarcoma 180, Ehrlich ascites carcinoma, and Dalton's lymphoma ascites
[5]. Saffron shows an inhibitory effect on chemical carcinogenesis in mice
[6], and the effect of crocetin on skin papilloma and Rous sarcoma has
been reported [7]. Escribano et al. [8] have recently demonstrated that
crocin inhibits tiie growth of HeLa cells and suggested pro-apoptotic
315
COO
QOC
HOH2C /ÔHa'
O^^ÔH O^' OH
Crocetin-(B-D-gcntiobiose)-(6-D-glucosyi)-cstcr
Crocetin-di-(fi-D-glucosyl)-ester
Fig.(2). Artificial pathway of crocin
properties of the compound. More recently, we have reported that orally

administered ethanol extract of saffron and crocin exhibit inhibitory
effects on the two-stage carcinogenesis of mouse skin papillomas [9].
These results suggested that crocetin and/or crocetin glucose esters
contributed to the anti-tumor activities of saffron.
The development of natural products having alleviation properties for
the symptoms of learning and memory impairments has been chnically
expected. In the brain, the hippocampus is a very important region in the
learning and memory processes, and the long-term potentiation (LTP)
induced from the brain tissue is closely related to leaming and memory
[10].
We reported the effects of ethanol extract of C sativus and its purified
components on the central nervous system in terms of leaming behaviors
in mice and the LTP in the dentate gyrus of hippocampus in anesthetized
rats and in the CAl region of rat hippocampal slices [11-13]. This review
also discusses the values of folk medicines in modulating apoptotic cell
death, together with our recent data of crocin's effect on neuronal cell
death.
Neuronal cell death is required for the development of the nervous
system. However, recent studies suggest that neurons die from
programmed cell death (apoptosis) in brains deprived of oxygen by stroke
[14] and trauma [15], and in the brains of Alzheimer's patients [16].
Therefore, prevention of neuronal apoptosis has been considered to be a
desirable therapeutic strategy for treating such neurodegenerative
diseases, although the value of this approach is not yet evident. We have
recently reported that crocin suppresses tumor necrosis factor (TNF)-a-
316
induced apoptosis of PC-12 cells by modulating the mRNA expression of

Bcl-2 family proteins, which trigger downstream signals culminating in
caspase-3 activation followed by cell death [17].
Effect of crocin on LTP
We already indicated that intravenous injection of ethanol blocked the
LTP induced by tetanic stimulation [18]. However, when saflBron crude
extracts were injected intracerebroventricularly, the blocking effect of
ethanol on the LTP decreased dose-dependently [19]. Moreover, crocin
prevents the ethanol-induced impairment of memory acquisition in ST
and SD tests [20]. From these results it is easily suggested that crocin
antagonized the blocking effect of ethanol on the induction of LTP.
Crocin of 50 mg/kg ameliorated the blocking effect of ethanol on the
LTP at approximately 84% compared to the control as indicated in Fig.
(3).
3.00D
Control EtOH 10 50 50 100 50 100 (mg/kg)

alone Crocin Ctocctin Crocctin
gentioblose di-glucose ester
glucose ester
Fig.(3). Effects of crocin and its analogues on the LTP-blocking effect of ethanol.
The vehicle or drug was intraceiebroventricularly injected 20 min before tetanus, and saline or 30% ethanol
was intravenously injected at a volumn of 2 ml/kg 15 min before tetanus. The AUC from 5 to 60 min after
appUcation of tetanus was calculated and defined as an index of magnitude of LTP in each group. The data are
represented as the means ± SEM of the number of observations shown in parentheses. ••p<0.01 vs. control
group. -Hp<0.05, -HpO.Ol vs. ethanol group in Duncan's multiple range test.
317
Effects of crocin on the induction of LTP in the CAl region of rat

hippocampal slices
In the control experiments, strong tetanic stimulation induced robust LTP.
Ethôl (30%; 10-15 ml/kg) did not show any significant effect on the
baseline synaptic responses, but suppressed the induction of LTP
following strong tetanic stimulation in a concentration-dependent manner
(Fig. (4)).
The effect of crocin on the LTP-suppressing effect of ethanol was
investigated (Fig. (4)). The potentiation induced by strong tetanic
stimulation in the presence of 20 mg/kg crocin and 30% of ethanol (15
ml/kg) was significantly larger than that in the presence of 30% of ethanol
(15 ml/kg) alone, indicating that crocin clearly attenuates the action of
ethanol [21].
Fig.(4). Effects of ethanol and crocin on

LTP induced by strong tetanic stimulation in
the CAl region of rat hippocampal slices.
The inset in A is a representative evoked
potential recorded from the CAl pyramidal
cell layer. Calibration hards: vertical 2 mV,
horizontal 10 msec. The population spike
an^litude was defmed as an average of the
ampUtude from the first positive peak 1 to the
succeeding negative peak 2 and the ampHtude
Time(iiiln) from the negative peak 2 to the second
positive peak 3. A Time-course of
(23)
potentiation induced by strong tetanic
stimulation in the control stices (O, n=23)
and in the slices treated with 30% of ethanol
si
1 i1
(15 ml/kg) ( • , n=27 ) and in the shces
(7) treated with 30% of ethanol (15 ml/kg) and
(16) # 20 mg/kg crocin ( A , n=7). (1) and (2)
OLJE (27) indicated 30% ethanol of 15 ml/kg and 10
(7)
ml/kg, respective^. Ethanol or crocin was
added in the perfusing ACSF from 15 or 20
Co«t
( 1 ) ( 2 ) 10 20 30 min, respectively, before tetanic stimulation.
Crocin (mg/kg) The ordinate indicates the population spike
+ 30% EtOH(15 ml/kg) anÛtude expressed as a percentage of the
baseline values immediately before tetanic
stimulation.
B: Summary of the effects of ethanol and crocin on the induction of LTP. The magnitude of LTP was evaluated
with the population spike an^)litude 30 min after tetanic stimulation. The numbers of observations in each
groiq) are shown in parentheses. All data are represented as the mean ± SEM *^p<0.01 vs. control, ^p<0.05 vs.
30% of ethanol (15 ml/kg) alone. Duncan's multiple range test.
318
Effects of ethanol and crocin on non-NMDA receptor-mediated

synaptic potentials in hippocampal slices
The synapic potential mediated by non-NMDA receptors was recorded in
normal ACSF. When ethanol (10-50 mM) was added to the perfusmg
medium, no significant change in non-NMDA receptor-mediated synaptic
potential was observed. However, the addition of ethanol at a mghCT
concentration (100 mM) induced a small reduction m non-NMDA
receptor-mediated synaptic potential (Fig.(5)A). The reduction m non-
NMDA response rapidly occurred after the addition of 100 mM etiianol
and reached a steady state within 10 min. After washing out the ethanol,
the response gradually returned to the normal level. When 10 \iM crocin
was added 10 min prior to the ethanol, the non-NMDA response was
similarly reduced in the presence of 100 mM ethanol (Fig-(5)B) Crocin
(10 nM) did not significantly affect the inhibitory effect of 100 mM
ethanol on non-NMDA response (Fig.(5)C) [22].
Ii1g.(5). Effects of ethanol and crocin on non-

NMDA receptor-mediated synaptic potential
evoked in nonnal ACSF in rat hippocampal
slices.
E, lOOtnMQ
to
(A) Representative experiment showing the effect
ethanol
a. I i L of ethanol on non-NMDA reseptor-mediated
0 5 10 15 20 25 30 35 response. (B) Representative experiment showing
Vme (min) the influence of crocin on ethanol-induced
B inhibition of non-NMDA response. Crocin (10
fiM) was applied 10 min prior to ethanol (white
bar). (C) Concentration-effect curves for ethanol
inhibition of non-NMDA response in the absence
(O) or presence ( • ) of 10 MM crocia
lOOmMethanola
CO L... —
10 MM crocin
a. —1 i L. J L
0 5 10 15 20 25 30 35
Tvne (min)
Ettianoi (mM)
319
Effects of ethanol and crocin on NMDA-induced currents in single

hippocampal neurons
In order to confinn the possible interaction of ethanol and crocin on
NMDA receptors, we also performed whole-cell patch recording with
primary cultured hippocampal neurons and measured membrane currents
induced by the application of NMDA in a voltage-clamped condition.
Application of 100 fiM NMDA induced an inward current of 100.2 ±
9.8 pA (n=10) at a holding potential of -60 mV. The NMDA-induced
inward current was not affected by 10 \iM CNQX (data not shown), but
was completely abolished by 30 \iM APV, supporting the fact that the
response was mediated by NMDA receptors. Etiianol inhibited NMDA-
induced currents in a concentration-dependent manner. Crocin (10 |iM)
had no effect on NMDA-induced currents by itself (data not shown), but
attenuated the inhibitory effect of ethanol on NMDA-induced currents.
The concentration-effect curve for ethanol was shifted to the right by the
presence of crocin [22].
Effect of crocin on TNF-a-induced morphological changes and DNA
fragmentation in the nucleus of PC-12 cells
The effect of crocin on the TNF-a-induced cell death of PC-12 cells is
shown in Fig. (6). In serum-free GIT medium conditions, PC-12 control
cell morphology remained intact at 24 h (panel A). However, the cells
treated for 24 h with TNF-a (500 units/ml) appeared rounded and showed
the characteristics of necrotic and/or apoptotic cells (panel B). In the
combination with 10 fxM crocin, PC-12 cell morphology retained intact
neuronal cell morphology at 24 h (panel C). Crocin alone had no effect
on the morphology of PC-12 cells (data not shown).
We next analyzed DNAfragmentationin the nuclei. Treatment of PC-
12 cells with TNF-a (500 units/ml) for 24 h caused DNA fragmentation
(lane 2 in panel D), while the nuclei in control (lane 1) and inlO fiM
crocin-treated cells (lane 5) were intact. Lanes 3 and 4 show that 1 and
10 jiM crocin blocked TNF-a-induced DNA fragmentation. This data
suggests that crocin prevents TNF-a-induced cell death of PC-12 cells at
a concentration range of 1-10 jiM.
The TNF-a-induced DNAfragmentationmay have been caused by
caspase-activated deoxyribonuclease (CAD) in PC-12 cells. In non-
apovtotic cells, CAD is present as an inactive complex with the inhibitor
r [23,24]. During apoptosis, caspase-3 inactivates I^"^, leaving CAD
free to f\mction as a nuclease [25]. Therefore, we used the fluorogenic
substrate, Ac-DEVD-MCA, to determine whether the caspase-3 in PC-12
cells was activated by treatment with TNF-a and/or crocin. As shown in
320
Fig. (7), TNF-a treatment resulted in an elevation of caspase - 3 activity

in the cells at 6 h (a 3.9-fold increase), and the elevation lasted for 24 h.
Crocin (0.1-10 jiM) suppressed the TNF-a-induced activation of caspase-
3 in a concentration-dependent manner. Caspase activity in the co-
presence of 10 (AM crocin was near the control level, while the crocin (10
\xM) alone had no effect on caspase activity in cells imtreated with TNF-a.
These results suggest that crocin can suppress the TNF-a-induced cell
death of PC-12 cells by blocking the activation of caspase-3. It is possible
that the crocin treatment may also suppress upstream signals for caspase-
3 activation.
Lane 1: cx>flliol
99 î !#• Lane 2: TNF adone
5000-
3000- Lane 3: TNF+1JJL M crocta
Lane 4: TNF+10/zM crocin
1500-
1000- Lane 5:10/i M crocin alone
1 2 3 4 5
Fig.(6). Morphological appearance of PC-12 cells treated for 24 h with vehicle alone (panel A), TNF-a
(500 units/ml) alone (panel B), or TNF-a (500 units/ml) plus 10 \»M crocin (panel C). In panel D,
inlemucleosomal DNAfragmentationwas detected by agarose gel electrophoresis of DNA extracted from cells
treated for 24 h.
321
control -i»~TNF.a(500 U)
IOMM crodn -•><-TNF +0.1 MM crodn
TNF +l/iM crocin -A^TNF +10/1M crocin
Time (h)
Flg.(7). Effect of crocin on TNF-a-induced activation of caspase-3 in PC-12 cells. Cells were incubated
for the indicated periods in GIT medium supplemented with vehicle alone, TNF-a alone, crocin alone, or their
combination. After lysis of the cells, caspase-3 activity was assayed as described in Materials and methods.
Each bar represents the mean ± S.D. of three independent experiments. M P<0.01; ###P<0.001, compared to
TNF-a alone.
Crocin blocks the release of cytochrome c from mitochondria by

modulating the expression of Bcl-2 family protein mRNAs
Bcl-2 and related cytoplasmic proteins are key regulators of apoptosis
[26]. Anti-apoptotic proteins such as Bcl-2 and BC1-XL prevent apoptosis
in response to numerous stimuli. During the apoptotic process,
cytochrome c is released from mitochondria, but the release can be
inhibited by the presence of Bcl-2 on the organelles [27]. The released
cytochrome c forms an essential part of Sie apoptosome, which is
composed of cytochrome c, Apaf-1, and procaspase-9 [28]. The complex
formation results in activation of caspase-9, which leads to the stimulation
of caspase-3. BC1-XL has recently been reported to bind to Apaf-1 [29]. It
may inhibit the association of Apaf-1 with procaspase-9 and tiiereby
prevent caspase activation.
Fig. (8) shows the effects of TNF-a, crocin or their combination on
Bcl-2 and BC1-XL mRNA levels in PC-12 cells. TNF-a treatment had no
effect on the BC1-XL mRNA levels at 3 h but significantly decreased the
mRNA expression at 18 h, compared to the control (panel A). Crocin (10
fxM) alone or its combination with TNF-a appeared to rather enhance the
expression of BC1-XL mRNA at 3 h. At 18 h, the TNF-a-induced decrease
322
in BC1-XL mRNA levels was reversed by 1-10 jiM crocin in a

concentration-dependent manner. On the other hand, crocin alone, TNF-a
alone or their combination had little or no eflfect on Bcl-2 mRNA levels at
3 h (panel B). The Bcl-2 mRNA levels at 18 h were slightly decreased by
TNF-a treatment, but significantly increased by the presence of crocin,
compared to the control. These results suggest that crocin alone has the
ability to increase the mRNA expression of BC1-XL, but not of Bcl-2, in
PC-12 cells at an early point (3 h).
Bax, Bcl-Xs and LICE are known as pro-apoptotic proteins [26].
Therefore, we next determined the effects of TNF-a and/or crocin on
these mRNA expressions in PC-12 cells. Fig. (9) shows that a 3-h
treatment with TNF-a increased the mRNA levels of Bax (panel A), Bcl-
Xs (panel B) and LICE (panel C) in the cells 2.6-, 1.8-, and 1.6-fold,
respectively, compared with the control. The Bax mRNA expression in
TNF-a-treated cells at 3 h was similar to that in TNF-a plus crocin-
treated cells. However, TNF-a-induced expression of Bcl-Xs and LICE
mRNAs at 3 h was significantly reduced by treatment with 1-10 jiM
crocin. Furthermore, the TNF-a-induced enhancement of Bcl-Xs and
LICE mRNAs at 18 h was reduced in a concentration-dependent manner
by the presence of crocin. At 3 h, crocin selectively modulated the
expression of Bcl-Xs and LICE mRNAs. Together with the increase in the
anti-apoptotic BC1-XL mRNA, the crocin-induced decrease in the two pro-
apoptotic mRNAs may greatly decrease the subsequent TNF-a death
singals. Since the enhancement of caspase-3 activity in TNF-a-treated
cells was observed at 6 h (Fig. (9)), the modulation of the mRNA levels
by crocin at 3 h may be important in determining the activation of
caspase-3 following tiie release of cytochrome c from mitochondria.
However, it is not known how crocin changed the balance between the
mRNA expressions of anti- and pro-apoptotic Bcl-2 family proteins. The
balance between the competing activities of these proteins may greatly
affect the maintenance oftiieirdownstream target, mitochondria.
Measurement of cytochrome c revealed that TNF-a significantly
increased the cytosolic cytochrome c levels in PC-12 cells at 6 h (Fig.
(10)). The increase in cytochrome c was suppressed to near the control
level by the presence of 1 or 10 |iM crocin. Therefore, our present results
show the possibility that crocin has a pharmacological effect that prevents
apoptosis of neuronal cells [17].
323
a control
BIOMM crodn
fife "rNF-a (500U)
0TNF+lAiM crocin
OTNF+5/xM crodn
DTNF+IOMM crocin
3 18
TlmeCh)
3 18
Time (h)
Fig.(8). RT-PCR analyses of TNF-a and/or crocin-treated PC-12 cell lysates for the mRNA expression of
anti-apoptotic proteins, BC1-XL and Bcl-2. The experimental procedures were those described in Materials and
methods. The bands in agarose gel were visualized and photographed under UVradiation.The iôtographs
were scanned and quantified by using an NIH Imager. The expression of GAPDH mRNA was used as a control.
Each bar represents the mean ± S.D. of three independent experiments. *P<0.05; *• P<0.01; ***P<0.001,
compared to the control. #P<0.05; ## P<0.01; ###P<0.001, compared to TNF- a alone.
324
o control
"^lO/iMcrocin
•TNF-a (500U)
OTNF+lMMcrodn
OTNF+5MMcrodn
aiNF+lO/xMcroclD
3 18
Time(h)
Fig.(9). RT-PCR analyses of TNF-a and/or crocin-treated PC-12 cell lysates for the mRNA exjwession of
pro-apoptotic proteins, Bax, Bcl-Xs and LICE. The experimental procedures were those described in Materials
and methods. The bands in agarose gel were visualized and photographed under UV radiation. The
photographs were scanned and quantified by using an NIH Imager. The exjwession of GAPDH mRNA was
used as a control. Each bar represents the mean ± S.D, of three independent experiments. • • P<0.01;
••*P<0.001, compared to the control #P<0.05; ## P<0.01; ###P<0.001, compared to TNF-a alone.
325
*
66 r *
l - ^ 50
\ 1B
^
n *
«:
5|20
10
0 i^ 1 "
m
TNF(ir) - 500 500 500
crocin(MM) 1 — —
— 1 10
Fig.(10). ELISA of cytosolic cytochrome c levels in PC-12 cells, treated for 6 h with TNF-a and/or ciocin.
The experimental procedures were those described in Materials and methods. Each bar represents the mean ±
S.D. of three independent experiments. ** P<0.01, compared to the control. M P<0.01, compared to TNF-a
alone.
DISCUSSION
It is well-known that the hippocampus is very important in the learning

and memory processes. The appearance of LTP by high-frequency
afferent stimulation is suggested to be closely related to the cellular basis
of learning and memory [10]. The relation between LTP phenomenon
and its inhibition by ethanol using rat hippocampal slices has been
reported by several groups [30-32].
An oral administration of crocin had no effect on memory acquisition
in normal mice but improved the ethanol-induced impairment of learning
behaviors of mice in passive avoidance performance tasks. This
phenomenon resembled that of crude extract of saffron as reported
previously. The tendencies of the effect between CSE and crocin are
similar to each other. From these results it can be easily speculated that
crocin is the most important principle in crude extract of saffron. Other
crocetin glucoside esters weakly antagonized the blocking effect of
ethanol on the LTP compared to crocin.
The LTP-suppressing action of ethanol is reduced by the presence of
crocin in rat hippocampal slices in in vitro experiment. Crocin alone does
not affect the basehne responses or the potentiation induced by weak
tetanic stimulation. It becomes evident that crocin attenuates the action of
ethanol within the hippocampus supporting in vivo experiments as
326
already documented. Although impairment of ethanol for brain functions

including learning and memory is well known, the mechanism is still
obscure. Several groups reported the LTP-blocking effect of ethanol using
rat hippocampal slices [30-32]. We confirmed that crocin prevents the
LTP-suppressing effect of ethanol in the CAl region of rat hippocampal
slices [21].
We demonstrated for the first time that crocin selectively antagonizes
the inhibitory effect of ethanol on NMDA-receptor-mediated responses in
hippocampal neurons. This action of crocin may underlie the antagonism
against ethanol-induced memory impairment. Crocin should be useful as
a new pharmacological tool for studying the mechanism of ethanol
inhibition of NMDA receptor fimctions.
We have also demonstrated that crocin prevents TNF-a-induced
apoptotic morphological changes and DNA fragmentation of PC-12cells.
Moreover, crocin suppressed the TNF-a-induced expression of Bcl-Xs
and LICE mRNAs and simultaneously restored the cytokine-induced
reduction of BC1-XL mRNA expression. The modulating effects of crocin
on the expression of Bcl-2 family proteins led to a marked reduction of a
TNF-a-induced release of cytochrome c from the mitochondria. Crocin
also blocked the cytochrome c-induced activation of caspase-3. To
confirm how crocin exhibits these anti-apoptotic actions in PC-12 cells,
we tested the effect of crocin on PC-12 cell death induced by
daunorubicin. The anthracycline antibiotic, daunorubicin, has been
proven to have a therapeutic benefit in the treatment of a variety of
neoplasia. Recently, daunorubicin was shown to induce apoptosis in cells
by the activation of ceramide synthase [33] or of sphingomyelinase
(SMase) [34]. Therefore, in contrast to TNF-a signaling through the
membrane receptors, daunorubicin is capable of triggering cell death
within PC-12 cells. We found that crocin inhibited the effect of
daunorubicin as well (data not shown). Taken together, these results
indicate that crocin can block PC-12 cell death induced by both outer and
inner apoptotic stimuli. Our recent data confirm that crocin has the ability
to reduce both TNF-a- and daunorubicin-induced increase in intracellular
ceramide levels of PC-12 cells (unpublished data). Daunorubicin can also
stimulate the generation of reactive oxygen species (ROS) [35]. In PC-12
cells, the generation of ROS activates neutral SMase to generate ceramide,
which, in tum, induces cell death [36]. Glutathione directly inhibits the
activation of the SMase [37]. Crocin is reported to have antioxidant
properties [39], and it is conceivable that the antioxidant action of crocin
contributes to the blocking of PC-12 cell death, induced by TNF-a and/or
daimorubicin. In the final process of apoptosis, caspase-3 is activated to
induce the activation of deoxyribonuclease. Of the crocetin derivatives
(crocin, aooet]bdi(P-I>ûoo6yl)«tei;aood^
tested, crocin was the most potent inhibitor for tike TNF-a-induced
327
activation of caspase-3 in PC-12 cells (unpublished data).

Many medicinal plants and their purified components modulate
programmed cell death in vitro and in vivo. However, the ability of
medicinal plants to induce apoptotic cell death in cancerous cells, but not
to prevent apoptosis in normal cells, has been the topic of much research.
Our recent studies showed that crocin also had the abilities to inhibit skin
tumor promotion in mice [9] and to prevent TNF-a-induced apoptosis in
PC-12 cells [17]. The pharmacological action of crocin to prevent both
cell proliferation and apoptosis suggests that the two biological processes
share a common patitiway at some point, where crocin may act. Apoptotic
signaling by TNF-a involves recruitment of specific proteins to the death
domain of the p55 TNF-a receptor (TNFRl) including TRADD, TNFKX
receptor-associated factors, receptor-interacting protein, leoqAr-iitecactirg
ptotanas80ciatedIch-l/CED-3 homologous protein with a death domain,
and Fas-associated death domain. The formation of this TNFRl complex
leads to tiie activation of a number of signahng intermediates such as c-
Jun N-terminal kinases (JNK), phospholipase A2, sphingomyelinase, NF-
KB, caspases. Reports have implicated these pathways in both promotion
and suppression of apoptosis by TNF-a as well as by other agents
including anti-cancer drugs, radiation, and Fas. The magnitude and types
of signaling events activated as well as their coordinate interaction with
other pro-/anti-apoptotic pathways determine the response of a cell to
TNF-a and other inducers. Therefore, the sum of these signals and their
cross-regulation to each other determine survival and death outcomes.
Phosphatidylinositol 3-kinase (PI3K) is reported to be a critical signaling
molecule involved in regulating cell survival and proliferation pa&ways.
PI3K can regulate cellular ceramide levels induced by TNF-a, suggesting
the existence of cross-talk between the cell survival and death patiiways.
It is of particular interest to note that crocin suppresses ceramide
generation followed by TNF-a-induced activation of SMase in PC-12
cells.
In this review, we confirmed the structure-activity relationship
between crocin and other crocetin glucose esters. The elimination of
glucose moiety from the crocin molecule greatly decreased its
pharmacologic^ activity, while neither gentiobiose nor glucose moiety
alone mimicked the effect of intact crocin (data not shown). Therefore,
these results clearly suggest that the two gentiobiose moieties attached to
crocetin play an important role in the exhibition of the biological
activities. This tendency was observed in the studies on the inhibitory
effect of crocetin derivatives on two-stage carcinogenesis of mouse skin
papillomas [9].
328
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SYNTHESIS AND MODIFICATION OF MARCFORTINE

AND PARAHERQUAMIDE CLASS OF
ANTHELMINTICS
B YUNG H. LEE
Preclinical Development, Pharmacia Animal Health, 7000 Portage Road,

Kalamazoo, MI 49001, USA
ABSTRACT: Three distinct chemical classes for the control of

gastrointestinal nematodes are available: benzimidazoles,
imidazothiazoles, and macrocyclic lactones. The relentless development
of drug resistance has severely limited the usefulness of such drugs and
the search for a new class of compounds - preferably with a different
mode of action - is an important endeavor. Marcfortine A (1), a
metabolite of Penicillium roqueforti, is structurally related to
paraherquamide A (2), originally isolated from Penicillium paraherqueL
Chemically the two compounds differ only in one ring; in marcfortine A,
ring G is six-membered and carries no substituents, while in
paraherquamide A, ring G is five-membered with methyl and hydroxyl
substituents at C14. Paraherquamide A (2) is superior to marcfortine A as
a nematocide. 2-Desoxoparaherquamide A (PNU-141962, 53) has
excellent nematocidal activity, a superior safely profile, and is the first
semi-synthetic member of this totally new class of nematocides that is a
legitimate candidate for development. In this review, total synthesis of
paraherquamides was also described.
INTRODUCTION
Helminths, especially parasitic nematodes, cause severe health problems

in humans and domestic animals. Although several classes of
anthelmintics are commercially available, their suboptimal therapeutic
spectrum and diminished activity due to the development of drug resistant
332
Strains of parasites [la,b] necessitates the unending search for compounds,

particularly for veterinary medicine.
0-4—CH^
MarcfortineA (1) Paraherquamide A (2)
Fig. (1). Structures of Marcfortine A and Paraherquamide A
The discovery of a new class of anthelmintic having a broad activity

spectrum and acceptable safety profile is a rare event. Despite intensive
efforts, only three distinct classes of anthelmintics have been
commercialized over the past 40 years: the benzimidazoles, the
imidazolides and the macrocyclic lactones (MCL) [2]. Pharmacia Animal
Health is committed to the discovery of a new class of anthelmintics with
a unique mode of action.
Marcfortine A (MFA), isolated at Pharmacia from a fermentation broth
in the early 90's, was shown to be active against the free-living nematode
C elegans in a relatively high throughput screen. MFA (1) is a fungal
metabolite of Penicillium roqueforti first reported by Polonsky et al. [3].
Paraherquamide A (2, PHA), a metabolite of Penicillium paraherquei [4],
is structurally similar to MFA, the only difference being in ring G, which
in PHA is 5-membered with a methyl and a hydroxyl substituent at Cu,
while in MFA ring G is six membered and has no substituents. The
antiparasitic activity of both MFA and PHA has been previously described
by workers at Merck [5].
MODIFICATION OF MFA AND PHA
Synthesis of Paraherquamides from MFA
PHA is a more potent nematocide than MFA, against some species,

based on a literature review. Our attempts to improve the potency of
333
MFA by simple chemical modifications, such as N-1 derivatization,

oxidation, reduction etc., were not successful.
QH3
Br~A ON I
\ \ HgC CHo f^^^^'^ CH3
)
(70-98 %; VN -NH
CH,
1 (MFA)
1. PhSe-SePh, NaBH4 (70-90%)
2. Nal04, Heat (80%)
CH3 . CN CH,
Y "^
H3C CH3 ,.^V°-y^^3^J;jaOH_ > \^'\^^'f\}^^'
(50%
I.OSO4/NMO
2. Nal04
3. NaBH4
^
(50%)
6 (PHB)
Less active than MFA
Fig. (2). Conversion of MFA to PHB
As mentioned earlier, the only structural difference between PHA and

MFA resides in ring G, and therefore our analog program centered on this
ring. An added reason was that the envisaged synthetic pathway could also
provide PHA, a compound not in our possession at the time, and essential
for comparative assays. Removal of the methyl and hydroxyl groups at
C14 in PHA would yield paraherquamide B (PHB), a compound identical
to MFA except for the size of ring G (PHB five versus MFA six). The
planned synthetic sequence required the opening of ring G and an
334
oxidative removal of one carbon atom, to be followed by ring closure as

shown in Fig. (2).
Treatment of MFA (1) with cyanogen bromide [6] opened ring G to
yield the bromo derivative 3 [7]. Attempts to dehydrobrominate 3 in one
step via a base-catalyzed elimination with DBU/CH3CN, KOH/MeOH, or
r^rr-BuOK/DMSO were unsuccessful. However, the required methylene
entity could be introduced by converting 3 first to a selenide, then
oxidation with periodate, followed by thermolysis in benzene to provide
compound 4. Hydrolysis of the cyano group with NaOH in ethylene
glycol [8] produced 5 (50% yield). Osmium catalyzed oxidation of 5 in
the presence of 4-methylmorpholine A^-oxide (NMO) gave a diol, which
was cleaved to an aldehyde upon treatment with periodate. Treatment of
the aldehyde with sodium cyanoborohydride resulted in an intramolecular
reductive amination to yield the desired product PHB (6).
The seven step conversion of PHB to PHA is shown in Fig. (3).
Oxidation of PHB (6) in THF/H2O with iodine in the presence of
bicarbonate [9] gave 16-oxo-paraherquamide B (7, 40%). Treatment of 7
with LDA and phenylselenyl chloride followed by oxidation of the
resulting selenide with H2O2 gave the a,p-unsaturated lactam 8.
Attempted epoxidation of 8 with H202/NaOH, m-CPBA,
isovaleraldehydeAÔ(acac)2, or n-BuLi/H202 failed to give the epoxide 9.
However, when 8 in THF was treated with r^rf-butylperoxide [10] in the
presence of triton B, the epoxide 9 (58%) was obtained. It was assumed
that attack on the double bond by peroxide would occur from the least
hindered side to yield an a-epoxide; proof was obtained from the
stereochemistry of alcohol 11 (vide infra). In any case, the
stereochemistry of these chiral centers is of little consequence, since they
are removed at a later stage of the synthesis.
While standard methods utilizing NaBHj, BH3-THF, or superhydride
failed to open the epoxide ring, samarium iodide [11] provided the
required 14a-hydroxy compound 10 in a good yield (85%). Although this
single electron transfer reagent has been shown to cleave epoxy-ketones,
to our knowledge this is the first instance of its application to amides.
Reduction of 10 with LAH/AICI3 gave 11 in a modest yield (24%). The
stereochemistry of 11 was established by comparison of its ^H NMR
spectrum with that reported by Blizzard et al. in their synthesis of 11 by an
altemative synthetic route [5j]. Swem oxidation of 11 produced ketone 12
(71%). Reaction of 12 with methyl magnesium bromide in THF gave
PHA in a 50% yield (based on recovered starting material) with the
335
formation of only a trace of the related a-methyl epimer. Semisynthetic

PHA proved to be identical by TLC, ^H NMR and HRMS to the natural
product kindly provided by Professor Yamazaki. We were able to then
confirm that the nematocidal activity of PHA is superior to MFA, which in
turn is superior to that of PHB.
1.LDA/PhSeCI
,
2. H2O2
(58%)
6 (PHB)
Y- OH
triton B
(58%)
Swern
Oxidation MeiVlgBr
LAH/AICI3
• 1
(24%) HO (71%) (50%)
CH3
Hr,CCH,,^v04-^H3
2 (PHA)
H3C fif \ Q
More active than l\^FA
° CH3'-'
Fig. (3). Conversion of PHB to PHA

336
Hydroxylation of MFA at C14, C15, C16 via a Novel Cyanogen Iodide

Reaction
While the reaction of MFA with cyanogen bromide (BrCN) in refluxing
chloroform caused ring fission to yield 3 [Fig. (4)], under the same
conditions cyanogen iodide did not provide the iodo analog 13.
CH^
14B
reflux 1 h
15A 15B
Fig. (4). Reaction of MFA with CNI
Cyanogen Iodide (ICN) has been used extensively for the cyanation of
alkenes and aromatic compounds [12], iodination of aromatic compounds
[13], formation of disulfide bonds in peptides [14], conversion of
dithioacetals to cyanothioacetals [15], formation of rran^-olefins from
dialkylvinylboranes [16], lactonization of alkene esters [17], formation of
guanidines [18], lactamization [19], formation of a-thioethter nitriles [20],
iodocyanation of alkenes [21], conversion of alkynes to alkyl-iodo alkenes
[22], cyanation/iodination of p-diketones [23], and formation of alkynyl
iodides [24]. The products obtained from the reaction of ICN with MFA
in refluxing chloroform were fran^-16-iodo-17-cyanomarcfortine A (14)
337
and 17-cyanomarcfoitine A (15) in 90% and 10% yields, respectively

[25]. The likely mechanism of the formation of these products is shown in
Fig. (5).
CH3
HoC C H o x ; ^ ^ 0 4 - - C K
Fig. (5). A plausible mechanism of the reaction of MFA with CNI
The iminium ion intemiediate 16 is generated by a free radical

oxidation of 1 and is in equilibrium with the enamine intermediate 17;
cyanide ion addition to 16 gives a low yield of compound 15 while the
favored trans addition provides the main product 14. Although generation
of an iminium intermediate with chlorine dioxide [26] or bromine [27] has
been reported, these reagents did not produce compounds such as 14,
suggesting that equilibration with the enamine did not occur. The
Polonovski-Potier reaction applied to aspidospermane generated an
enamine intermediate, which upon treatment with CNBr gave a product
similar to 14 in three steps [28].
The trans adduct 14 upon treatment with 45% aqueous KOH in MeOH
for 3 h at room temperature gave the 16,17-dehydro derivative 18 in 90%
yield. The utility of this novel cyanogen iodide reaction was further
demonstrated on MFA by affording a procedure for the regiospecific
introduction of a single hydroxyl group at C14, C15, and C16. These
analogs were required to determine the significance of the hydroxyl group
338
on anthelmintic activity. Compound 18 thus became the common

intermediate for the synthesis of 14a-hydroxymarcfortine A (23), 15a-
hydroxymarcfortine A (27), and 16a-hydroxymarcfortine A (29).
I.LDA/PhSeCI
2. HgOg/NaOH
(65%)
More active than MFA
Fig. (6). Synthesis of 14a-hydroxymarcfortine A from 18
Preparation of 23 from 18 was achieved in five steps [Fig. (6)].

Hydrolysis of 18 with a catalytic amount of p-toluenesulfonic acid in 95%
methanol at room temperature for 1 h gave 19 (90%). The C15-C16
double bond was then introduced by selenation [Lithium diisoproylamide
(LDA) and phenylselenenyl chloride] at C16 followed by hydrogen
peroxide oxidation and subsequent elimination of phenylselenenic acid by
an aqueous alkaline workup ( I N NaOH) to give the C15-C16 dehydro
analog 20 (65%). Compound 20 underwent allylic oxidation with Se02in
refluxing dioxane (1.3 equiv, 1 h) to provide in a modest yield (35%) the
desired a-hydroxyl derivative 21. Reduction of the double bond with
lithium triethylborohydride (7 equiv/THF, 0 ""C, 0.5 h) gave 22 (86%).
The regiospecific reduction of the C17 amide in 22 was achieved by
treatment with BH3-DMS (10 equiv/THF) to provide 14a-
hydroxymarcfortine A (23, 75%). The couphng constant between the C14
hydrogen and the CI5 hydrogen is 2 Hz, indicating the hydroxyl group is
axial.
339
3 1.LDA
18
Inactive
18
NMO
(80%)
inactive
Fig. (7). Synthesis of 15 a- and 16a-hydroxyniarcfortine A from 18
The preparation of 15a-hydroxymarcfortine A (27) and 16a-

hydroxymarcfortine A (29) is shown in Fig. (7). Treatment of 18 with
LDA (4 equiv/THF, -78 ""C) followed by 1.5 equiv of Davis's reagent 24
[(2-benzenesulfonyl)-3-phenyloxaziridine] [29] gave the desired C15-
hydroxlated material 25 (y-hydroxylation, 30%) along with the 17-oxo
derivative 19 (a-hydroxylation, 10%). ^H NMR of 25 indicated a single
stereoisomer in which the C15 hydrogen has two pseudoaxial and two
pseudoequatorial couplings. Reaction of 25 with 2.6 equiv of Se02 in
aqueous ethanol at ambient temperature for 16 h furnished 26 (50%),
which was reduced with LAH (2.75 equiv/THF, 0 ""C, 0.5 h) to yield 27
(25%). Treatment of 18 with a catalytic amount of OSO4 and 4.2 equiv of
NMO gave 28 (80%); the carbonyl at C17 was reduced with BH3-DMS (6
340
equiv/THF, 0 ""C, 1 h) to yield 16a-hydroxy-MFA 29 (60% yield based on

recovered starting material).
While compounds 27 and 29 lack nematocidal activity, we found 23 to
be more active than MFA.
Synthesis of 14p-hydroxy-MFA (31) and 14p-methyl-14a-hydroxy-

MFA (32)
CH^
HoC CHq Ô-ÛcHg Swern

/) Oxidation
CHg^ 30
MeMgBr
NaBH.
CH^
HX ^-.^04-CH3
32 as active as PHA 31 (50%) + 23 (3%)
Fig. (8). Synthesis of 14P-hydroxy-MFA and 32
Because of the enhanced biological activity of 14a-hydroxy-MFA (23),

the synthesis of its antipode was carried out as shown in Fig. (8).
Swern oxidation (oxalyl chloride, DMSO, NEts, -78 ''C, 1 h) of 23
provided 14-oxo-MFA (30, 74%). Reduction of 30 with NaBHU (6
equiv/THF, 0 ''C, 1 h) gave M^-hydroxy-MFA 31, 50%) and 14a-
hydroxy-MFA (23, 3%). Compound 31 was inactive, thus demonstrating
the need for correct stereochemistry of the hydroxyl group at C14. To
assess the effect of the C14 methyl group on biological activity, 30 was
reacted with methylmagnesium bromide to yield 32 (50% based on
recovered starting material), the six membered homologue of PHA. The
341
related a-methyl epimer was present only in trace amounts. Compound 32

is the first MFA analog with nematocidal activity comparable to that of
PHA.
An Improved and Practical Synthesis of 14a-hydroxy-MFA (23).
SPh
1.LDA \ 4
2. PhS-SPh V-N
(60%) CHo
33
1.,x;s^COOOMg
CI, "COQ- (76%)
2. heat
COOOMg f^'ô
23 (70 g)
Fig. (9). An Improved and Practical Synthesis of 14a-hydroxy-MFA
To provide additional material for clinical trials, and to eliminate the use
of hazardous reagents such as ICN, PhSeCl, and Se02, an improved
synthesis of 23 was devised [Fig. (9)].
Treatment of MFA 1 with sodium bicarbonate and iodine [30] in
refluxing aqueous THF produced 19 in one step (76%, 350 g scale),
eliminating the use of ICN and Se02. Compound 19 was reacted with
LDA and phenyl disulfide to give 33 (65%). Oxidation with the
magnesium salt of perphthalic acid followed by refluxing in toluene
provided 34 (76%). Repeated reaction with the magnesium salt of
perphthalic acid and diethylamine gave the rearranged product 22 (61%)
[31]. The carbonyl group at C17 in compound 22 was reduced with BH3-
DMS (10 equiv/THF) to provide 14a-hydroxy-MFA 23 (70 g, 50%). The
stereospecific rearrangement of 34 was previously described [32].
342
Synthesis of 15a-Methyl-14a-Hydroxy-MFA (36) and ISp-Methyl-

14a-Hydroxy-MFA (41)
Due to the enhanced nematocidal activity observed following the

introduction of a methyl group at C14 [32, Fig. (8)], its effect on C15 was
next investigated.
Me2CuLI 11 Q BH3-DMS HoC--<^N—\X
(60 %) (50 %)
HO
CH,
36 very active
1.LDA
35
2. MeSOgSMe (37)
(57 %)
BHq-DMS
(33 %)
40
Fig. (10). Synthesis of 15a-MethyM4a-Hydroxy-MFA (36) and 15p-MethyM4a-Hydroxy-MFA (41)
Conjugate addition of lithium dimethylcopper to 21 [33] gave 35 in

60% yield [Fig. (10)]. The cis addition of the methyl group is likely due
to the bulky copper-oxygen intermediate [34]. Subsequent treatment of 35
in THF with the BH3-DMS complex gave compound 36 (50%) which, in
our laboratory, had good activity against Haemonchus contortus and
Trichostrongylus colubriformis. Compound 41 (the epimer of 36) was
343
prepared from 35 in four steps [35]. Sufenylation of 35 with methyl

methanethiol sulfonate 37 and LDA gave 38 in 57% yield. Oxidation with
m-CPBA in methylene chloride at -78 °C gave a sulfone intermediate
which on heating under reflux in toluene/ethylene chloride yielded 39
(70%) with elimination of methanesulfonic acid. Reduction with lithium
triethyl borohydride gave 40 in a 60% yield. A second reduction with the
BH3-DMS complex provided the desired 41 in a 45% yield.
9-BBN
>/ NaOH/HgOg
1.MsCI/NEt3
2. DBN
(70%)
45 Inactive
Fig. (11). Synthesis of 45, a C14,15-fused ring analog.
While compound 36 demonstrated excellent nematocidal activity, the

epimer 41 at the same concentration was totally inactive. Perhaps the
conformation of the G-ring relative to the hydroxyl group is more
important than the chirality of the methyl group at CI5. To further
344
investigate this hypothesis, compound 45, having a C14-C15 fused ring,

was synthesized. In addition, 45 could also define the importance of
hydrogen bonding at C14 [Fig. (11)].
Synthesis of 45 , a C14,15-Fused Ring Analog
Compound 21 [Fig. (11)] was reacted with vinyl magnesium bromide and
copper iodide in THF at 0 ^C to give the 1,4 addition product 42 in 48%
yield [35]. Hydroboration with 9-BBN followed by NaOH and H2O2
workup provided a mixture of the desired alcohol 43 (20%), recovered
starting material 42 (20%), and the C17 reduced alcohol 44 (5%). Further
reduction of 43 with the BH3-DMS complex gave 44 in 35% yield. The
two combined samples of 44 were mesylated (CH3S02Cl/NEt3) and then
treated with excess DBN to give the furan-containing MFA analog 45 in
70% yield. Compound 45 is inactive, thus demonstrating that hydrogen
bonding of the C14 hydroxyl group is indeed important for anthelmintic
activity. The significance of the geometry of ring G on anthelmintic
activity was next investigated.
Fission of ring-G: The Synthesis of Analog 48
CH, C;H3
O4-CH3 COCI2 U
//
QJJ Pyridine
/toluene
^
H^C-
O
J
^^" O' bH O CH, 46
LiBEtgH 40%
HaCsâC^ H3C CH3 ^^^YO-lr-CHa ^^^^ H3C H H3C CH3 ^?vô4-CH3
HO-4—(•-v4J> NaBHgCN H O
-NH
80% H,
48
Inactive
Fig. (12). Synthesis of 48.

345
Compound 2 [Fig. (12)] in pyridine was treated with phosgene in toluene

[5j] to give the carbamate 46, which on reduction with lithium triethyl
borohydride gave 47 in a 40% yield. Subsequently, compound 47 was
treated with formaldehyde and sodium cyanoborohydride to yield 48
(80%). Compound 48 is biologically inactive, and in our computer model
(minimum energy conformation) it resembles 14p-hydroxy-MFA 31,
which is also inactive. It can be concluded that the naturally occurring
conformation of ring G is essential for biological activity.
Economic Viability
The biological profile of compounds 32 and 36 points towards their use

in large food animals. The cost of these compounds, however, is
prohibitive if synthesized by the procedures outlined in Fig. (8) and Fig.
(9). Therefore, an alternative scheme had to be developed, as outlined in
Fig. (13).
The envisaged route required the selective introduction of the crucial
14-hydroxyl group into MFA via a biotransformation procedure. The
resulting 23 would then be a common intermediate for the synthesis of 32
(two chemical steps) and 36 (three chemical steps).
Primary Fermentation - • IVIFA

BJotransformation
. m Q^
VN/
O CH,
23
Three
Chemical Steps
Tvw)
Chemical Steps
HoC
36 32
Fig. (13). Economic viability

346
Microbial Hydroxylation (Biotransformation) at Eight Individual

Carbon Atoms of MFA
Many reviews and hundreds of papers have been published on the use
of mono-oxygenases for the introduction of oxygen atoms onto various
substrates [36]. We were especially interested in nonactivated
stereospecific carbon atom hydroxylation. Therefore, a large number of
biotransformation experiments were carried out utilizing cultures reported
in the literature, and random samples from the Pharmacia culture
collection. Screening was performed by adding a solution of 1 (10 mg)
[37] in DMF (0.4 mL) to vigorously growing culture fermentations in 500
mL wide-mouth flasks. Incubation was conducted at 28 °C, and shaking
was continued for 1-3 days depending on the culture. The mixture was
thoroughly extracted with chloroform, centrifuged, and the solvent
removed at 35 °C under reduced pressure. The residue was analyzed by
TLC (three solvent systems) and HPLC. Promising samples were scaled
up 10- to 100-fold and refermented in a Labraferm fermentation tank.
Purification was achieved by various chromatographic techniques, and the
structure determined with the aid of NMR and MS. Whenever
semisynthetic samples were available, a side by side comparison was also
performed.
27
CH3
l>^0-4-CH.
o I to
N-Demethylatlon/^ ^ " s
Arrows indicate position of hydroxylation
Fig. (14). Biotransformation products
The great majority of cultures either totally metabolized 1 or left it

unchanged. However, a few cultures did provide hyroxylated products.
347
Extensive efforts were undertaken to increase the yields of

biotransformation products by changing the media, temperature, time of
fermentation, etc. The highest yields obtained were for compounds 23, 27
and 29, which on scale-up gave a 10-15% yield, in addition to recovered
starting material (30%).
In summary, utilizing biotransformation techniques, eight out of the
28 carbon atoms in MFA were successfully hydroxylated. In Fig. (14),
arrows indicate the sites of hydroxylation. It is noteworthy that despite the
hindered nature of C14, cultures UC 5059, UC 11141, and UC 11144 were
able to introduce a hydroxyl to give 23 in a 10-15% yield.
Discovery of 2-Desoxo-15a-Methyl-14a-Hydroxy-MFA (49)
Scale up studies of a second potential candidate yielded the desired 36

and a small amount of a less polar compound, characterized as the 2-
desoxo derivative 49 [Fig. (15)]. The excellent nematocidal activity of 49
encouraged us to attempt the selective reduction of the C-ring carbonyl in
MFA (1), to yield 52. Reduction of 1 with three or ten equivalents of
LAH yielded 50 (carbonyl reduced in ring F) and 51 (carbonyls reduced in
rings C and F) respectively, both lacking nematocidal activity; we were
unable to synthesize 52.
Discovery of 2-Desoxo-PHA (PNU-141962,53)
To follow up the excellent nematocidal activity of 49, PHA was

selectively reduced with the alane-NMe2Et complex to furnish 53, albeit in
only a 5% yield [Fig. (16)].
Attempts to improve the yield of this one step procedure with various
reducing agents, such as LAH, LAH/AICI3, NaBEU/Acetic acid,
NaBIVCFsCOOH, Red-Al, Super-hydride, Li-9-BBN-hydride, BH3-THF,
Li-tri-r-butoxyaluminum hydride and LiBHU, were unsatisfactory. The
highest yield obtained was with LiBEU (10-20%). A lengthier but higher
yielding synthesis of 53 was developed (4 steps 60-70%) by using our
previously described process [38]. Compound 2 was reacted with 9-
fluorenylmethyl chloroformate (Fmoc-Cl, 1.5 equiv) in the presence of
NaH (3 equiv) at 0 °C to give a quantitative yield of 54 [39]. Reduction of
54 with NaBHU in MeOH at 0 °C gave 55, which was deprotected with
piperidine in THF to give the imine intermediate 56.
348
HO ;HN '/>^NH
35 (20 grams) ^"3
BH3-DMS
CH3 CH3
O-4-CH3 04-CHo
oi +
36 (10 grams) 49 (10 miligrams) as active as 36
CH3
04-CH,
-//-
LAH(3or10equiv) 0 CK
52
(3 equiv)
O-UCH,
51 Inactive
5Q Inactive
Fig. (15). Synthesis of 49,50, and 51
This was further reduced with NaBKU in MeOH at 0 °C to give 53.

Compound 53 displayed excellent activity in our jird and sheep models.
Indeed, in our hands, this compound was two to four times more potent in
sheep than the parent compound (PHA) against the important
349
gastrointestinal nematodes, Haemonchus contortus and Trichostrongylus

colubriformis.
^ O CH
53 PNU-141962
CHo
CH3 0-4-CHo
O-V-CH3
K)—>"
J, I- NaBH. Hqi
N-Fmoc ^ 3 O CH3 OH
H3C Jl'X Overall yield 60-70% ^ CH, 56

^ CHq ^'^
Fig. (16). Discovery of 2-Desoxo-PHA (PNU-141962,53)
It should be noted that workers at Merck found PHA to be considerably

more potent than we did [40a], a difference which may be due to the use
of different vehicles. While this was an exciting development, we were
concerned about the toxicity of this PHA analog, because Merck workers
reported [40b] that PHA is quite toxic to mice, with an estimated LD50 of
< 15 mg/kg. In dogs, the toxicity is even greater, with death seen at doses
as small as 0.5 mg/kg [40b], reducing chances for commercialization.
Interestingly, PHA is relatively safe in jirds, sheep and rats. Structure
activity relationship studies performed by Merck and Pfizer workers did
not yield analogs with lower toxicity. In contrast, compound 53 was not
toxic to mice at doses up to 50 mg/kg. Furthermore, dogs treated with 53
at 20 mg/kg experienced no toxic effects besides mild and reversible
350
mydriasis. The exceptional improvement in selective biological activity

due to the removal of a single oxygen atom in ring C is noteworthy
indeed.
In conclusion, rational drug design led to the synthesis of two highly
active compounds, 32 and 36. Scale up studies of 36 yielded a minor
product, the 2-desoxo-MFA derivative 49, which in turn led to the
discovery of 2-desoxo-PHA (PNU-141962), a compound that is currently
under development.
Efficacy of MFA, PHA and PNU-141962
Drug evaluations were conducted in Mongolian gerbils (jirds)

concurrently infected with the gastrointestinal nematodes H, contortus and
T, colubriformiSr or monospecifically infected with O. ostertagU using
established techniques [41a,b]. This model permits assessment of the
activity of experimental compounds directly against target parasites in
vivo, using very little drug. Treatments were given on day 10 or day 6
post-inoculation in the K contortus and T. colubriformis or O. ostertagi
models, respectively.
Table 1. Efficacies of MFA, PHA and PNU-141962 against gastrointestinal nematodes in experimentally
infected jirds following oral dosing.
95% Effective Dose (mg/jird)

Compound
H. contortus T. colubriformis O. ostertagi
MFA 0.33 0.11 4.0
PHA 0.33 0.11 0.5
PNU-141962 0.33 0.11 1.0
Drugs were administered orally in 0.2 ml vehicle (17% DMSO: 83%

vehicle #98). Animals were examined for worm burdens on day 13 {H.
contortus and T. colubriformis) or day 8 (O. ostertagi) post-inoculation (3
days post-treatment).
Approximate ED95 values for MFA, PHA and PNU-141962 against 3
nematode species in jirds are shown in Table 1. Against H, contortus, the
approximate ED95 for MFA, PHA and PNU-141962 was 0.33 mg/jird
351
(roughly 10 mg/kg). These 3 compounds were also approximately

equipotent against T. colubriformis in the jird model, with an EDc^^ of 0.11
mg/jird. Against O. ostertagU the approximate ED95 for each compound
was higher than that observed for the other nematodes. Against this
species, the ED95 values for PHA and PNU-141962 were 0.5 and 1.0
mg/jird, respectively; these values were 4- to 8-fold lower than those for
MFA (-4 mg/jird).
Table 2. Efficacy of Marcfortines, PHA and PNU-141962 in sheep experimentally infected with H.
contortus following oral dosing of conqiounds in 60:40 propyleneglycol/glycerol formal vehicle.
95 % Effective Dose (mg/kg)

Compound H. contortus
MFA 12.5
23 7 ,
32 2
PHA 1-2
PNU-141962 0.5-1
36 4-5
The compounds were also tested in sheep experimentally infected with

Haemonchus contortus. The treatments were given orally in propylene
glycol/glycerol formal (60:40 v:v) vehicle on day 35 post-inoculation.
Animals were necropsied 7 days post-treatment and examined for worm
burdens. Approximate ED95 for the marcfortine compounds, PHA and
PNU-141962 are shown in Table 2. Against K contortus, the approximate
95% effective dose for PNU-141962 was 0.5-1.0 mg/kg; ED95 for the
marcfortines ranged from 1-12.5 mg/kg and for PHA 1-2 mg/kg. It should
be noted that formulation has not been optimized for ruminants, and
subsequent preliminary pharmacokinetic studies have shown that the
bioavailability of PNU-141962 in sheep is low (10-15%) in the vehicle
used, when dosed orally or subcutaneously (unpublished observations).
352
Mode of Action of PNU-141962, MFA and PHA

Similarities in structure and anthelmintic spectra of the
paraherquamides and marcfortines suggest that these compounds share an
anthelmintic mechanism. This concept is supported by observations that
PHA and PNU-141962 displaced [^H]marcfortine A in competition
binding assays using membranes prepared from adult H, contortus;
competition was also observed in binding assays using membranes
prepared from the free-living nematode Panagrellus redivivus and
[^H]PNU-141962 as ligand (our unpublished observations).
PHA, PNU-141962, and related compounds rapidly induce flaccid
paralysis of parasitic nematodes in vitro, without affecting ATP levels
[42]. While the mechanism of action of these new anthelmintic agents
was initially poorly understood, they appear to share a binding site with
phenothiazines in membranes prepared from C elegans [43]. Recent
observations in insects suggest that PHA binds to invertebrate nicotinic
acetylcholine receptors (nAChR) and is an antagonist of acetylcholine
(ACh) at these receptors [44]. To determine if a similar mechanism of
action is found for these compounds in nematodes, we investigated the
mechanism of action of this anthelmintic class using muscle tension and
microelectrode recording techniques in isolated body wall segments of
Ascaris suum [45a]. Our findings can be summarized as follows. None of
the compounds significantly altered A. suum muscle tension or membrane
potential when given alone. However, paraherquamides blocked (when
applied before) or reversed (when applied after) depolarizing contractions
induced by ACh and nicotinic agonists, including the anthelmintics
levamisole and morantel. These effects were mimicked by the nicotinic
ganglionic blocker mecamylamine, suggesting that the anthelmintic action
of PNU-141962 and related paraherquamides and marcfortines is due to
blockade of cholinergic neuromuscular transmission. To further test that
concept, we examined the effects of these compounds on three subtypes of
human nAChR. In these studies, a Ca^"^ flux assay was used to measure the
function of nAChR expressed in cultured mammalian cells. PNU-141962
blocked nicotinic stimulation of cells expressing a3 ganglionic (IC50 = 6
\\M) and muscle-type (IC50 = 3 |LIM) receptors, but was inactive at 100 |xM
vs the a7 CNS subtype. This compound also paralyzed the parasite H.
contortus in culture with an IC50 value of approx. 0.1 uM, thus
demonstrating the basis for host vs. parasite selectivity. It is noteworthy
that PHA is more effective in blocking mammalian nAChR than is PNU-
141962, perhaps explaining the greater mammalian toxicity observed with
the prototype drug.
353
Isotopic Labeling of PNU-141962 (53) with Deuterium [45b]
2. NaBHaCN
(preferred) (500/^
Fig. (17). Isotopic Ubeling of PNU-141962 (53) with Deuterium
Modifications of MFA and PHA led to the discovery of 2-

desoxoparaherquamide A (PNU-141962, 53) which is as active as PHA
and has an improved safety profile. In order to do preclinical studies, we
354
wished to synthesize radio-labeled PNU-141962. In this reason, we

prepared [CD3]-2-desoxoparaherquamide A (62).
Although the synthesis of [C24"^H]-PHA has been reported [45c], the
labile nature of position-24 with respect to acid hydrolysis rendered such
labeling unsuitable for our preclinical studies. Using a deuterium labeled
reagent, we have developed a synthetic strategy that is suitable for the
introduction of '"C and ^H into the 14-methyl group of PNU-141962
through the appropriate choice of labeling in the reagent [Fig. (17)]. The
dehydration of PNU-141962 to exo-olefin 57 through the use of DAST
was readily accomplished. A method has been reported for the conversion
of an exo-olefin derivative of PHA to its ketone by sequential reactions
involving bromination, ozonolysis and debromination with zinc [5j].
However, these procedures were long, low yielding and, worse, the
benzene ring of PHA was also brominated. In our hands, glycolation of
the exo-olefin with osmium tetroxide followed by oxidation of the glycol
to the ketone with sodium periodate proved more suitable to our purposes.
Earlier, we reported a method for the stereospecific addition of MeMgl to
14-oxoparaherquamide B [7]. Using this methodology we successfully
methylated the carbonyl at position-14 of ketones 60 and 61 in a highly
stereoselective manner.
Treatment of 53 with DAST [(diethylamino)sulfur trifluoride] in
methylene chloride provided 57 in 50% yield. Compound 57 was treated
with osmium tetroxide at 5 °C in the presence of NMO (4-
methylmopholine iV-oxide) for 18 h to provide 58 and 59, which were
separated by silica-gel chromatography. Compounds 58 and 59 were
treated with sodium periodate at 5 °C for 18 h to provide 60 and 61
respectively. Compounds 60 and 61 were treated with CDsMgl followed
by NaBHsCN to give 62 in 20% and 50% yields, respectively.
In conclusion, isotopic labeling of 2-desoxoparaherquamide A (PNU-
141962) with deuterium was achieved from PNU-141962 in four steps in
anticipation of using its method of synthesis for the preparation of the
corresponding ^^C and ^H labeled products.
Semi-Synthesis of 3-Epi-paraherquamide A (65)
To investigate the significance of the chirality of the C3 position on

anthelmintic activity, we prepared 3-epi-PHA [65, Fig. (18)].
355
CH«
0-4-CH3 H3C CH3 ^ o^
= \=. C
y" 3
CHo
^ 0 bH,
O CH, 63
56
f-BuOCI
NEtg
CH2CI2,0 °C
rt 16h HgCfâ o '^=\P^3

^y
^-kteCXJ
0 CH3
-3 :T "°-. V-v^,
CH3 AcOH H3C Jt-^^ CI
65
64
Fig. (18). Semi-Synthesis of 3-Epi-paraherquamide A (65)
Compound 56 [39] was heated under refluxing in xylene to give

rearranged product 63 in good yield. Compound 63 was subjected to
conditions described by Williams [46] to provide 65 in low yield.
Compound 65 did not show ant anthelmintic activity .
€26 Dialkyl and Spiroalkyl Analogs of MFA
HCOOH
MFA •
Fig. (19). Outlined synthesis of 67

356
To investigate the effect of changes at the C26 position of MFA on

anthelmintic activity, several of C26-dialkyl and spiroalkyl analogs were
prepared [47]. The analogs were synthesized in four steps starting with
cathecol 66 [Fig. (19)], which was prepared from MFA in 80% yield by
stirring in formic acid for 16 h.
K2CO3
Br
"V\—'——^
R Kl m-CPBA
R 66
68a: R, R == cyclobutyl
68b: R, R == cyclohexyl
68c: R, R == diethyl
68d: R. R =: ethyl-methyl
68e: R, R =: dimethyl
C[ r\ SnCU
Ô-^ R •
MTPI
Fig. (20). Synthesis of 67
The general route outlined below, follows the modified method of

Williams [48] used his preparation of the g^m-dimethyl dioxepin ring of
PHB. By this method we were able to prepare the four dioxepin-ring
anaologs in which the geminal methyl groups at C26 of MFA were
replaced by a cyclobutyl 67a, cyclohexyl 67b, diethyl 67c, and ethyl-
methyl 67d groups [Fig. (20)].
357
The catechol 66 was coupled with the appropriate bromo reagent 68a-d
in the presence of K2CO3 and KI in acetone/water to give mono-alkylated
products 69a-d in 29-82% yield.Epoxidation with m-CPBA in CH2CI2
followed by workup with sodium bisulfite [49] (to remove the N-oxide)
gave epoxides 70a-d in 40-100% yield. Ring closure using SnCUin THF
provides alcohols 71a-d (50-85% yield) which were dehydrated with
methyltriphenoxyphosphonium iodide (MTPI) in THF/DMF to provided
the final products 67a-d in 20-30% yield.
To verify the regiochemistry of the alkylation described above we
reduced MFA with borane-methyl sulfide complex to provide 72 in 40%
yield [Fig. (21)]. This compound was identical to the one prepared from
the catechol 66 and 4-bromo-2-methyl-2-butene (73) using the chemistry
reported in step 1 of Fig. (20), thereby conforming the assigned
regiochemistry of compounds 68a-d.
The biological activity of compounds 67a-d was evaluated in our
standard anthelmintic assay which uses immunosuppressed Mongolian
gerbils inoculated with Haemonchus contortus and Trichostrongylus
colubriformis [41]. The compounds were administrated orally at a dosage
rate of 0.33 mg/gerbil. Unlike MFA, none of these compounds gave the
95% clearance of helminthes we use as a criteria for determining activity,
and were deemed inactive.
BH3-DMS /—\ âS. P^;

MFA •( N-^K^ Ji^jJ^ I ^ ^ 66
K2CO3/KI
Acetone/HgO
Fig. (21). Reaction of MFA with BH3-DMS
C24 and C25 Substituted MFA Derivatives
To investigate the effect on anthelmintic activity by changing the

substituent pattern at C24/C25, we synthesized a number of analogs [Fig.
(22)] [50].
358
Swem oxidation of 71e provided the ketone 74, which was subjected to
Wittig olefination with methyltriphenylphosphonium bromide and n-BuLi
to give the exocyclic methylene containing compound 75 in 50% yield.
The versatile ketone 74 was also epoxidized with trimethylsulfoxium
iodide and potassium r-butoxide in DMSO to give 76 in 35% yield,
whereas Grignard chemistry (MeMgBr) gave a quantitative yield of 77.
Treatment of 77 with DAST afforded a 35% yield of the desired analog
78.
V-OH ^
Fig. (22). C24 and C25 Substituted MFA Derivatives
We reasoned apriori that both compounds 75 and 78 would have

anthelmintic activity since they contained the crucial C26 dimehtyl moiety
and likewise retained the proper geometry of the A ring found in the
parent compound MFA based on modeling studies. Furthermore, these
structures should be less hydrolyzed under acidic conditions.
359
With the exception of compound 75 none of these compounds were

active. Since the exocyclic methylene analog 75 had activity we chose to
prepare a simplified analog [Fig. (23)]. Thus, the catechol 66 was reacted
with 79, K2CO3 and Nal in DMF for 16 h to give the exocyclic methylene
compound 80 which lacked the C26 dimethyls. Compound 80 was
inactive, which further emphasizes the importance of C26 dimethyls.
KgCOg/Nal
CI
79 ^'
STEREOCONTROLLED TOTAL SYNTHESIS OF (+)-PHB

As part of on going efforts of Williams and his coworkers [46] to
elucidate the biosynthesis of the core bicyclo[2.2.2] ring system of the
related alkaloids the brevianamides [51], they have applied methodology
originally developed for the stereocontrolled total synthesis of (-)-
brevianamide B [52] to complete the first stereocontrolled total synthesis
of(+)-PHB.
Retrosynthetic analysis
As outlined in Fig. (24), a convergent synthesis of the enantiomer of

the natural PHB was envisioned to contain four key carbon-carbon bond-
forming reactions.
The first task would involve the construction of a suitably a-alkylated
proline derivative [52]. The second important coupling would be the
Somei/Kaetani-type alkylation [53] of the suitably protected gramine
derivative 86 and the requisite piperazinedione 85. The third and most
crucial C-C bond-forming reaction was a stereofacially controlled
intramolecular SN2' cyclization reaction and concomitantly installs the
isopropenyl group that will be utilized in the fourth C-C bond-forming
reaction. Standard procedures to effect this transformation involve strong
protic acids [52], and there was reason for concern about the reactivity of
360
the more highly oxygenated indole 83 as a practical synthetic precursor to

82.
î" 84
(R)N
MeOgC—'N<^^^
Y 86
85 O
Fig. (24) A conversion synthesis of (+)-PHB
Construction of the Dioxepinooxindole Ring System
The known pyruvic acid 87 [Fig. (25)] [54] was oxidatively

decarboxylated [55] to afford the phenylacetic acid 88, which was
reductively cyclized to give the required oxindole 89 in nearly quantitative
yield.
361
OH Ho
OH NaOH O ^-^
H2O2 Pd/C HO (99%)
HO NO2 OMe
(81-93%) (92%)
OMe
89
88
f y y , ^ pren^ bromide^ f ^ T V o ^•"^"^^^^. f V V o
OH (52%) OR (640/^) / y ^ ^
90 91 R = prenyl HO gj
r^
NaBH. 1. protection
^
BFgOEtg
2. Mannich
(46%)
HO
RO
93 94 R = f-BuMe2Si
Fig. (25). Construction of the Dioxepinooxindole Ring System
Oxindole 89 was cleanly demethylated upon treatment with boron

tribromide. The resulting oxindole 90 was subjected to the prenylation
conditions, and the desired alkylated product 91 was obtained in 52%
yield. The epoxidation/Lewis acid-mediated cyclization proved to be
successful on this substrate. The epoxide product was directly treated
with SnCU in THF to provided the desired 92. When oxindole 92 was
treated with NaBHt (1.6 equiv)and BFs'OEta (3.5 equiv) in THF, the
desired 93 was obtained. The indole 93 was treated with TBDMSCl and
imidazole in DMF, to provide the required 0-silylated indole, which was
easily converted to the gramine 94 through the well known Mannich
procedure.
362
OR'
o H
95
R = COOMe
R' = f-BuPhSi R-o R" = f-BuMegSi
HO
Reagents: (a) 94, 0.5 equiv of PBuj, CH3CN, reflux, 73%; (b) LiCl, HMPA, 100 °C, 89%; (c) Me30BF4,
Na2C03. CH2CI2. 62-71%; (c) (i) BOC2O, DMAP, EtsN, CH2CI2; (ii) /1-BU4NF, THF, 85-90%; (d) NCS,
Me2S, 74-86%.
Construction of the Diketopiperazines Containing a Dioxepinoindole

Ring
The epimers 96 (prepared in twelve steps from 95) were condensed

with the gramine 94 providing the indole 97 in 73% yield as a mixture of
two diatereomers. The indole 97 was converted to 98 with four steps. [Fig.
(26)]
363
Construction of the Bicyclo[2.2.2] Ring System
Allylic chloride 98 was reprotected with r-BuPhaSiOTf to provide 99 in

77-82% yield.
NaH, benzene
N
^
î *BOC (syn, 93%;
anti, 85%)
ization ^
ENDO Cyclization f-^
RO
100
N
BOC
99b
RO
The stage now set to effect the SN2' reaction. [Fig. (27)]
Compound 99 was refluxed in benzene with 20 equiv of NaH, resulting in
a very clean and high-yielding cyclization reaction furnishing the desired
product 100, and the undesired anri-diastereomer was not detected.
It is generally accepted that SN2' reactions favor a syn orientation [56]
(i.e., the incoming nucleophile attacks the 7C-electrons from the same face
as the departing leaving group, polarizing the 7i-system in the proper
orientation for a "backside" displacement on the C-Cl bond).
Alternatively, a frontier molecular orbital analysis has indicated [56a] that
the stabilization imparted by a HOMONuc-LUMOaiiyiic interaction is greater
for the syn overlap. While the greatest level of diastereoselectivity was
observed with a nonpolar aprotic solvent (benzene), a fairly significant
change in the relative amounts of the syn- and anri-diastereomers can be
realized by simply changing the solvent to a more polar solvent such as
DMF. In the present system, additional stabilization for the endo transition
364
State may be due to the formation of a tight contact ion pair between the
chlorine atom and sodium atom of the enolate species in the transition
state for the formation of the C-C bond. [57] The poor ligating solvent
benzene is not capable of effectively solvating the enolate cation nor the
developing chloride anion in the transition state. It is reasonable that this
type of association favors the rotamer that positions the allylic chloride
moiety over the enolate, resulting in the desired syn stereochemistry.
The Final C-C Bond-Forming Reaction on the Indole
With the bicyclo[2.2.2] ring system constructed in a reliable and high-

yielding sequence, attention was turned to the final C-C bond-forming
reaction on the indole. [Fig. (28)]
A search of the literature revealed a 1982 Trost and Fortunak paper
[58] wherein PdCl2 and AgBF4 were utilized to effect the Heck-type
cyclialkylations of various isoquinuclidine model compounds. Compound
100 was exposed to these conditions, affording the heptacycle 101 in 63-
82% yields.
There are several reports of methods that will selectively reduce a

tertiary amide in the presence of a secondary amide[59]. The secondary
lactam of 101 was protected as the lactim ether 107 and treated with
diborane; however, the spectral characteristics of the major products
isolated were consistent with reduction of both the tertiary amide and the
lactim ether. In 1991 Martin et ai [60] successfully used alane to reduce a
tertiary amide in the presence of an oxindole (secondary amide) relying on
the known rate difference for reduction between these two groups [61].
However, initial experiments with this reagent gave poor results, with
the secondary amide undergoing reduction along with the tertiary amide.
Compound 101 [and 107, Fig. (29)] is sufficiently twisted such that the
g^m-dimethyl groups effectively block the P-face of the tertiary amide,
leaving the a-face relatively unencumbered. However, a modification of
the alane procedure [60], proved satisfactory for this transformation. The
piperazinedione 101 was pretreated with AlEts, with the expectation that
this Lewis acid would form a complex with the more exposed secondary
lactam [106, Fig.(29).] and leave the tertiary lactam accessible for
reduction.
365
102 ; R = f-BuMegSi, R' = H, R" = BOC 105

103 ; R = f-BuMegSi, R' = Me, R" = BOC
104 ; R = f-BuMepSi. R' = H, R" = H
Reagents: (a) PdClz, AgBF4. MeCN; (b) NaBH4 (63-82% from 100); (c) 1.1 equiv of EtaAl, 5.0 equiv of
AIH3-DMEA, THF, toluene; (d) 2.0 equiv of NaCNBHa. AcOH, MeOH (65% from 101); (e) 2.5 equiv of NaH,
2.0 equiv of Mel, DMF (98%); (f) 80 equiv of TEA, CH2CI2 (96%); (g) /-BuOCl, pyridine, -15 °C; (h) 90%
THF, 10% H2O, pTsOH (76%); (i) MTPI, DMPU (79%).
Fig. (28). Synthesis of (+)-PHB
O \ ^
R = f-BuMe2Si
Fig. (29). Structures of 106 and 107
Following 10 min of precomplexation with AlEts, 5 equiv of AIH3-

Me2NEt complex was added, followed by quenching with NaCNBHs,
366
acetic acid, and methanol to provide the desired amine 102 in 63% yield.
Compound 102 was smoothly alkylated with methyl iodide, affording the
N-methylated product 103 in 95-98% yield. Compound 103 was
subsequently deblocked with 80 equiv of TFA in CH2CI2 to yield the
penultimate heptacycle 104 in 97% yield.
The stage was now set for the final transformations involving the
oxidative pinacol-type rearrangement and dehydration. Thus, treatment of
105 with r-BuOCl and EtsN in CH2CI2 provided the two chloroindolenines
108 and 109 (2.25:1 ratio, respectively, Figure 30). The solvent was
removed, and the crude reaction mixture was refluxed with a solution of
95% THF, 4% H2O, and 1% TFA, giving a 62% yield of oxindole
products (43% of the desired 105 and 19% the epi product 110). The C-3-
epi-isomer (110) was easily distinguishable from the desired isomer (105)
by the upfield shift of the g^m-dimethyl signals in the ^H NMR spectrum.
The relative amounts of products (105 and 110) indicate that the
cyclization was stereospecific under these conditions. It was thus deduced
that an increase in the ratio of the desired oxindole 105 to the undesired
isomer 110 could be achieved simply by finding a method that would
increase the ratio of chloroindolenines (108:109).
108
Fig. (30). structures of 108,109 and 110
The a-face of 104 is considerably more hindered than the p-face, a

supposition that was supported by the difficulties encountered in the
reduction of 101 and 107. Increasing the steric bulk of the chlorinating
agent should favor attack on the p-face of 104, thus providing a greater
relative amount of chloroindolenine 108. When 104 was treated with t-
BuOCl in pyridine instead of triethylamine, the desired chloroindolenine
367
108 was produced in a much more stereoselective fashion. It can be

speculated that r^rr-butyl hypochlorite forms a bulky complex with
pyridine, delivering the chlorine more selectively to the least hindered a-
face of 104 (only a small amount, 5%, of the undesired isomer 109 was
formed under these conditions.
Employing a minor modification of the solvent system, the crude
mixture of 108/109 was refluxed with a solution of 90% tetrahydrofuran,
10% H2O containing 15 equiv of p-toluenesulfonic acid to give the desired
oxindole 105 in 76% yield (from 104), with only 4% of the undesired 110
being formed.
The final dehydration reaction (MTPI, DMPU, 18 h) on the

alcohol 105 produced (+)-PHB (81) in 79% yield. This substance proved
to be identical to the natural product by comparison of the ^H and ^^C
NMR spectra, mobility on TLC, IR spectra, mass spectra, and UV spectra.
Comparison of the CD spectra of the natural (-)-PHB (6) and the synthetic
(+)-PHB (81) confirmed the expected enantiomeric relationship between
these two products.
ASYMMETRIC, STEREOCONTROLLED TOTAL SYNTHESIS

OFPHA[62]
Williams and his coworkers have previously described as symmetric

synthesis of the simplest paraherquamide derivative, PHB from (5)-
proline. In approaching the synthesis of PHA which contains the unusal P-
hydroxy-|3-methylproline residue, a new method needed to be developed
to generate a suitably functionalized a-alkyl-P-hydroxyproline moiety that
could be conscripted for a multistep construction of PHA.
Construction of the a-Alkyl-p-Hydroxyproline Moiety
As described previously [63], p-ketoester 111 [Fig. (31)] was subjected to

Baker's yeast reduction to afford the optically active P-hydroxyester 112
(60-80% yield). Dianion alkylation of 112 with (E)-3-methyl-4-(0-/^rr-
butyldimehtylsilyl)-2-butene afforded the desired a-alkyl product 113 in
58-70% isolated yield.
368
Boc Boc Boc

I I I
N>^COOEt ^N cOOEt ^N cOOEt
Q: O
(60-80%)
OH
U^-^YÔTBS
^" 113
111 112
(86%) c.d.e
^Y^NH f,g
^N cOOEt
V - - ^ v ^ ^ OTBS (93%) ^ . . ^ - ^ ^ OTBS (79%)
OTBS
OMOM
114
Reagents: (a) Baker's yeast; (b) LDA, THF, HMPA, (E)-ICH2CH=C(Me)CH20TBS; (c) 5.7 equiv
MOMCl, (/Pr)2NEt, CH2CI2; (d) 2.7 equiv ZnBr2, CH2CI2; 0 °C (e) K2CO3. 2 equiv BrCHiCOBr, CH2CI2; (f)
NH3 in MeOH (5.7 M), 25 °C; (g) 3 equiv NaH, toluene, HMPA, 25 °C; (h) 1.3 equiv n-BuU, THF, 11.1 equiv
ClCOOMe, - 78 °C; then 4 equiv ClCOOMe, 5 equiv LiN(TMS)2. - 78 °C.
Protection of the secondary alcohol as the corresponding methoxy

methyl (MOM) ether, followed by removal of the Boc group with ZnBr2 in
dichloromethane and acylation of the incipient secondary amine with
bromoacetyl bromide in the presence of K2CO3 afforded the
bromoacetamide 114 in 86% yield from 113. Treatment of 114 with
methanolic ammonia afforded the corresponding glycinamide which was
directly subjected to cyclization in the presence of NaH in toluene/HMPA
to afford the bicyclic compound 115 in 79% overall yield from 114. Next,
a one-pot double carbomethoxylation reaction was performed by the
sequencial addition of n-BuLi in THF followed by addition of
methylchloroformate, that carbomethoxylated the amide nitrogen atom.
Subsequent addition of four equiv of methyl chloroformate followed by
the addition of 5 equiv of LiN(TMS)2 afforded 116 as a mixture of
diastereomers in 93% yield that were taken on directly without separation.
369
Construction of the Tryptophan Derivative 120
Somei-Kamatani coupling of 116 [Fig. (32)] with the gramine derivative

94 in the presence of tri(n-butyl)phosphine gave the tryptophan derivative
117 as a 3:1 mixture of diastereomers in 70% yield.
TBS*
n O br"*"*^' R' = COOMe,R" = MOM
:MOM
94 (70%)
OTBS
OMOM
OTBS
116 R' = COOMe
• /--p^N
(68 -71%) V - N , ^
OTBS
120
Boc
Boc
Reagents: (a) 0.7 equiv («-Bu)3P, MeCN; (b) 5 equiv LiCl, H2O, HMPA, 105 °C, 5 h; (c) 2.5 equiv
Me30BF4, CS2CO3. CH2CI2.25 °C; (d) DMAP, 3 equiv (Boc2)0, CH2CI2; 0 °C (e) 3.3 equiv TBAF, THE, 25 °C;
(f) 1.1 equiv Msci coUidine, CH2CI2, 0 °C; (g) 3.3 equiv TBSOTf, 2,6-lutidine, CH2CI2,0 °C.
Decarboxylation of 117 was effected by treatment of 117 with LiCl in

hot, aqueous HMPA at 105 °C providing 118 as a mixture of
diastereomers that were separated and carried forward individually.
Protection of the secondary amide group as the corresponding methyl
lactim ether was accomplished by treating 118 with trimethyloxonium
tetrafluoroborate in dichloromethane that contained cesium carbonate.
Next, the indole nitrogen atom was protected as the corresponding Boc
derivative by treatment with dicarbonic acid bis(f^rr-butyl)ester in the
presence of DMAP and the silyl ether was removed with
tetrabutylammonium fluoride to provide diol 119 in 52-78% overall yield
from 118. Selective conversion of the allylic alcohol to the corresponding
370
allylic chloride was accomplished by mesylation in the presence of

coUidine. Silylation of the secondary alcohol with rerr-butyldimethylsilyl
triflate in the presence of 2,6-lutidine afforded the key allylic chloride 120
in 68-71% yield over the two steps.
Construction of the Bicyclo[2.2.2] Ring System and the Final C-C

Bond-Forming Reaction on the Indole
OMOM,
h (85%),
i (97%)
OH
Reagents: (a) 20 equiv NaH, TOP, reflux 30 h; (b) 3.1 equiv AgODp4, 4.68 equlv PdCh. MeCN, propylene
oxide; then NaBH4, EtOH; (c) 0.1 M HCl, THF; (d) 2-hydroxypyridine, toluene, 120 °C, 2 h; (e) 5 equiv
(iBu)2AlH, CH2CI2. 0 °C; (f) NaH, Mel, DMF, 0 °C; (g) 6 equiv B-bromocatecholborane, CH2CI2, 0 °C; (h)
Sequiv l,l,l-triacetoxy-l,l-dihydro-l,2-benziodoxol-3(7f/)-one (Dess-Martin periodinane), CH2CI2, 25 °C; (i)
TFA, CH2CI2, 25 °C.
The stage now set to effect the SN2' reaction. [Fig. (33)] Compound
120 was refluxed in THF with 20 equiv of NaH, resulting in a very clean
371
and high-yielding cyclization reaction furnishing the desired product 121,

and the undesired anr/-diastereomer was not detected.
Closure of the seventh ring was effected by treatment of 121 with 4.68
equiv of PdCl2 and 3.1 equiv of AgBF4 [58] in acetonitrile containing
propylene oxide as an acid scavenger. The incipient heptacyclic a-
pallladium adduct was worked up immediately by the addition of the
ethanol and NaBH4to afford the desired indole 122.
Cleavage of the lactim ether 122 was effected with 0.1 M HCl to give
the corresponding ring-opened amine methyl ester that was recyclized by
treatment of this material with 2-hydroxypyridine in hot toluene.
Chemoselective reduction of the secondary amide was effected by
treatment of of the product obtained from the previous step with excess
diisobutylaluminum hydride in CH2CI2 to furnish 123 (50-72% yield)
[64]. Methylation of the secondary amide 123 proceeded in 96% yield.
Cleavage of the MOM ether with bromocatecholborane [65] (91% yield)
followed by oxidation of the secondary alcohol with Dess-Martin
periodinane [66] (85% yield) and cleavage of the Boc group and TBS
ether with TFA (97% yield) gave the ketone 124.
Formation of the Spiro Oxindole
The final, critical oxidative spirocyclization of the 2,3-disubstituted

indole to the spiro oxindole was effected by treatment of 124 with tert-
butyl hypochlorite in pyridine to provide the labile 125 [Fig. (34)]. The
Pinacol-type rearrangement was conducted by treating compound 125
with p-toluenesulfonic acid in THF/water. It is assumed that the
chlorination of 124 proceeds from the least hindered face of the indole, to
give the a-chloroindolene 125. The hydration of the imine functionality
must also occur from the same a-face that is syn to the relatively large
chlorine atom furnishing the ^yn-chlorohydrin 126, that subsequently
rearranges stereospecifically to the desired spiro oxindole 127.
The dioxepin ring was then formed by dehydration of the secondary
alcohol 127 with MTPI in DMPU to afford 14-oxo-PHB 12 [7]. This
material has been previously described by a Pharmacia group (obtained
semi-synthetically from MFA) and comparison of the authentic and
synthetic materials [^H and ^^C NMR, IR, exact mass, mobility on thin-
layer chromatography (TLC)] conformed the identity of this substance.
Treatment of the synthetic ketone with MeMgBr gave (-)-PHA (2) in 42%
yield that was identical in all respects [^H and ^^C NMR, IR, exact mass,
372
mobility on TLC, m.p. (250 °C (dec)), [ajo = -22 (C= 0.2 MeOH)] to the
natural PHA [4].
OH
TsOH
O f-BuOCI
THF/HgO
OH
CH3
^*^LNx^xkÔ •
(55%)
O CH3O
54 % from 125 127
3 MeMgBr
(42%)
12
Fig. (34). Formation of the spiro oxindole
CONVERSION OF MFA TO PARAHERQUAMIDES VIA A

NOVEL PLATINUM-OXYGEN-MEDIATED RING
CONTRACTING REACTION [33]
Our earlier conversion of MFA to PHA required 13 steps [7]. By
employing a ring contracting reaction utilizing platinum and oxygen
(Pt/Oi) at a key point in the synthesis, we were able to directly convert
marcfortine A to the intermediate 16-oxoparaherquamide B (7), thereby
eliminating six steps in our earlier synthesis.
373
Pt/C A CH«
H3C Ch
dioxane/water
o CH,
7 (14-23%)
o p
\J/ H3C CH,
06^
CH^
128 (2-3%) 129 (9-19%) 130 (14-19%)
MCPBA
130 7
(80%)
1
4
MCPBA
H
o P
p^JiiU/M^c CH3 p<j^^H^C CH3
CH-,
Fig. (35). Reaction of MFA with oxygen
Although the Pt/02 reaction has been used for oxidation of primary and
secondary alcohols [67], hydroxylation of 12a-deoxytetracyclines [68],
oxygenation of cholesterol [69], and oxidation of tertiary amines [5f], this
374
reaction has not been used extensively compared to singlet oxygen

reactions [70].
When marcfortine A was treated with Pt on carbon (10%) in
dioxane/water under an oxygen atmosphere (balloon pressure, room
temperature, 2-4 days), four products (7, 128-130) were isolated [Fig.
(35)]. The reaction was not accelerated by heating. Performing the
reaction under oxygen at 1000 psi resulted in acceleration with very little
effect on the product ratios. Yields varied depending on the activity of the
platinum.
^3^. p^3 o p
%J/ H3C CHo
Pt/Og followed
by MCPBA HO H >-N
23 10 (43%)
131 (3%)
HQC CH<3
Pt/0« J\. HoC CHQ
HO-f-^4v> HO-
H3C >-N
H,C
° CH3
32
132 (10%)
133 (10%)
o o
V ^ H3C CH3 , ,,^
M:^40<J (3%)
H3C Y\
CH^
132 PHA
AIHg-NMegEt
Fig. (36). Reaction of 23,32 with oxygen

375
Dioxo compound 130 was converted to 7 in 80% yield by treatment

with m-chloroperbenzoic acid (m-CPBA). According to the literature, six-
membered rings containing a 1,2-dicarbonyl moiety are converted to five-
membered ring hydroxy acids only in the presence of a strong base [71].
By contrast, our method is performed under neutral conditions and is more
efficient.
Subsequently, we examined several MFA derivatives as substrates for
the Pt/02 reaction. When 32 was subjected to Pt/Oa chemistry, three
products (132-134) were isolated in poor yield [Fig. (36)]. In the case of
23, the Pt/02 reaction mixture was treated straightaway with m-CPBA
without isolation of any intermediate products, giving the desired
compound 10 (43% yield). Compound 10 can be converted to PHA (2) in
three steps. Compound 132 was converted to PHA (2) by treatment with
alane-dimethylethyl amine complex in THF (30% yield).
HqC CHo Pt/0« HO^ O
36
135 (6%)
Fig. (37). Reaction of 36 with oxygen

376
When 36 was subjected to Pt/02 chemistry [Fig. (37)], four products

(35, 135-137) were isolated. Although the Pt/Oi chemistry performed on
36 does give 137 (the desired product) in trace amounts, it can be more
readily prepared by oxidation of 136 with m-CPBA (80% yield).
Subsequently, 137 was reduced with LAH/AICI3 in THF to give 138 (40%
yield).
CH«
Pt/C
0-0 \
[O]
B \
Partial aiiyiiic
oxidation
o CH3
C: X = H,H
D: X = H,OHorO
[0] [H2O2]
130 *• 7
oo
[0] [H2O2]
128+ 129
o CH3
E: X = H,OHorO >
Fig. (38). A plausible mechanism for the Pt/O^ meddiated ring contracting reaction
377
A plausible mechanism for the Pt/Oi-mediated ring contracting reaction

is shown in [Fig. (38)]. In this mechanism, A exists in equilibrium with B
(one can speculate about the formation of A). Intermediate B undergoes
molecular oxidation accompanied by partial allylic hydroxylation to give
intermediates C and D, Each of these is further oxidized to give putative
intermediate E and product 130. A final oxidative step, probably involving
hydrogen peroxide generated in situ and proceeding by a mechanism
paralleling that described for the m-CPBA reaction, leads to products 7,
128, and 129.
ACKNOWLEDGEMENTS
We are grateful to Professor Yamazaki at Chiba University for furnishing

a sample of natural paraherquamide A.
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ACARICIDES OF NATURAL ORIGIN,

PERSONAL EXPERIENCES AND REVIEW
OF LITERATURE (1990-2001)*
GUIDO FLAMINI
Dipartimento di Chimica Bioorganica e Biofarmacia, Via Bonanno 33,
56126 Pisa, Italy
ABSTRACT: Acari are responsible for millions of dollars worth of damages each year
as a result of infestations in animals, plants and man. They affect our health directly
and prosperity as animal and plant parasites, vectors of disease, and producers of
allergens. The indiscriminate use of pesticides has destroyed many of their natural
enemies of hitherto harmless species and quickly induced resistance in many parasites.
At present, the control of acarid parasitic diseases in agriculture, human and veterinary
medicine is mainly based on the use of drugs; for this reason the lack of effective drugs
often prevents the control of some parasitic diseases, making them more serious and
important. The use of commercial drugs involves many problems; besides the drug-
resistance shown by the most important parasites, the environmental damage and the
toxicity of many synthetic drugs, represent the main problems that strongly limit their
use. In addition, drug residues in plant and animal food products are important reasons
forfiirthereconomic losses for farmers and must be regarded as potentially hazardous to
man and envh-onment. Plant-derived compounds are generally more easily degradable
and could show a smaller negative environmental impact with respect to synthetic
drugs. For these reasons, the evaluation of the antiacarid activity of plant extracts is
increasingly being investigated in order to obtain new leads, as demonstrated by recent
studies that have evaluated and confirmed the effectiveness of many plant compounds
on bacteria,fimgi,protozoa, hehnints and arthropods. This review will be limited to the
class Arachnida, sub-class Acaridi, particularly to their control in agriculture, veterinary
and human medicine with natural methods.
INTRODUCTION
Mites and ticks, collectively known as the Acari, constitute the second
most diverse group of animals on the planet today and are of interest to
humans for a variety of reasons. They affect our health and weel being
directly as plant, animal and human parasites, vectors of disease, and
producers of allergens. They are responsible for millions of dollars worth
Dedicated to the memory of Prof Serena Catalano

382
of economic losses each year as a result of infestations of animals, man

and plants. The acari are ubiquitous, being members of every major
ecosystem on earth, and their specific habitats run the gamut from the
familiar to the truly bizarre. From our backyards to the geothermal
springs of the Yellowstone caldera and from the subcutaneous tissue of
turtles to our very own hair follicles, mites carry out a nearly invisible
existence. While awareness of the acari dates back to ancient Egypt (1550
BC) and was further demonstrated through the writings of the major
Greek scholars, the science of acarology originated in 18th century in
Europe. Linnaeus described the first mite species, Acarus siro, in 1758,
thus laying the groundwork for the field of systematic acarology. Today,
over 48000 described species of mites and ticks have been described
worldwide, while current estimates place total diversity at over a half-
million species.
The Class Arachnida, to which the order Acari belongs, together with
the Class Insecta, the Class Crustacea and others, constitute the Phylum
Arthropoda. All the classes contain species useful to man, but also many
pests that can cause economic losses and/or diseases. This review will be
limited to the order Acari, particularly to their control with natural
methods in agriculture, veterinary and human medicine.
The indiscriminate use of inorganic pesticides, destroyed many
harmless species including natural enemies of these mites and ticks [1].
From 1940 onwards, organochlorine and organophosphate pesticides
were introduced, but tresistance was quickly acquired in many arthropod
parasites, including acari; fortunately many useful predatory mites
became resistant too. The emergence of resistance to parasiticides is one
of the most serious challenges faced by modem farmers. Perhaps it is the
simplicity of treating parasite attacks with very effective drugs or
pesticides on a routine basis and the proven cost-effective gains in
productivity that will accrue in the short term, that has led to the
predominance of synthetic pesticides [2]. Broadly speaking, resistance is
the ability of the parasites to survive doses of drugs that would normally
kill parasites of the same species and stage. It is inherited and selected for
because the survivors of the pesticide treatment pass genes for resistance
on to their offspring. These genes are initially rare in the population or
arise as rare mutations in genes but as selection continues, the proportion
of resistance genes in the population increases and so does the proportion
383
of resistant parasites. Drug susceptibility is a resource that needs to be

preserved using appropriate techniques of parasite management. In many
cases susceptibility to certain pesticides in some parasites has been lost
forever. One approach to this problem is to develop new susceptibilities
by using novel drugs. However, the application of synthetic chemical
substances is still the common method to control or eradicate parasites of
plant and animals. Unfortunately, many acaricides have non-specific
properties, affecting other organisms (crops, non-vertebrates and
vertebrates) to varying degrees. The damage to these non-target organisms
may be reflected by different effects, like direct mortality, long term
population reductions, bioaccumulation within the food web. The
indiscriminate dispersion of these substances must be regarded as
potentially hazardous to man and environment.
Plants are the richest source of organic compounds on Earth. Many of
these natural chemicals are endowed with pesticide properties. It is well
known that some pesticides of plant origin have been in use before the
time of the Romans (i.e. pyrethrum, obtained from the flower heads of
Chrysanthemum cinerarifolium). In 1959, the term Integrated Pest
Management (IPM) was coined [3]. IPM consist in the application of the
best mix of control tactics for a given pest problem in comparison with
the yield, profit and safety of the alternative mixes [4]; these control
methods ought to be environmentally acceptable and sustainable [5].
However, many of these studies have little bearing on what is actually
happening in the field.
In veterinary medicine, the control of ectoparasites is of great
importance due to their effects on livestock profitability and the health
status of animals. Infestations on livestock can cause intense irritation
leading to poor condition, weight loss, reduced milk yield, and hide or
fleece damage. Furthermore, many species of acari are responsible for
transmission of diseases to the host animals themselves or act as vectors
of a number of diseases to humans [6].
APICULTURE
During the 90s, several cases of resistance to common acaricides
employed in beekeeping by Varroa mites (Acari: Varroidae) were
reported from different countries [7-10]. In Italy, the consequences of the
resistance were disastrous colony losses. Available statistics show that in
384
certain districts, losses often exceeded 70% and in some locations even
reached 90% [11]. Varroa mites originated in southeast Asia, where they
parasitized the Asian honeybee. Gradually they have spread and now^
afflict most of the European honeybees. Though the Asian honeybee has
been able to tolerate the mite: it was a pest, but not a fatal one, European
honeybees have almost no resistance, and the mite has proved fatal to
millions of the colonies in Europe and America. Varroa mites suck the
body fluids from adults and brood, prefering the latter, especially drone
brood. The problem of developing suitable treatments was difficult in the
case of the Varroa mites because most substances effective against the
parasites have unacceptable side-effects on bees. Towards the end of the
80s products based on pyrethroids were used. These substances were
very effective on mites, and without any appreciable effects on bees.
Pyrethroids are synthetic analogs of pyrethrins, secondary metabolites
obtained from the flower heads of Chrysanthemum cinerarifolium.
Synthetic pyrethroids replaced the natural ones because they apparently
exhibited no negative effect on plants, animals and humans.
Unfortunately, the limits of this "magic bullet" soon became evident, with
serious impact on beekeeping because of the resistance developed by
Varroa. Since the creation of EU Varroa experts' group, several lines of
research in alternative control measures have been explored: apicultural
techniques for reducing the number of mites, increasing bee resistance, and
searching for acaricidal products that are generally recognized as safe for
humans, such as some natural derivatives [12]. Among these compounds
many simple carboxylic acids have been used, such as formic, oxalic and
lactic acids. The first report on the use of formic acid as miticide was
published in 1980 [13]. Five years later Wachendorfer et al. [14]
developed and carried out field trials on the "lUertisser Milbenplatte"
(=mite plate), an adsorbent cardboard soaked in formic acid on which the
bees alighted before entering in the hive. Other methods of treatment
included dispensation of formic acid by means of evaporators, wood
shavings, or use of liquid reservoirs with wicks [15-27]. The mortality
rate of Varroa mites was however quite variable, with percentages ranging
from 12% [24] to 98.8% [21], with a prevalence of intermediate values
(47%-56%) [18,21,26]. A strong dependence upon the administration
method was observed: in western Switzerland the percentage of mites
killed was 80% when formic acid was applied on the plates, but 90%
385
when applied on viscose cloth [16]. Many of the above techniques

involve removal of one or more frames from the hive, and most involve
handling of liquid acid by the beekeeper, with the risks associated with
dispersion and the corrosive nature of this substance. To resolve these
problems, recently a gel formulation of formic acid has been developed.
Kochansky and Shimanuki [28] experimented with various gelling agents,
obtaining the desired rheological characteristics and releasing behavior of
the active principle, with fumed silicas and Carbopols 934 and 941. The
fumed silicas gave the best results, with optimum evaporation speed for
application to bee hives (2-3 weeks). A gel formulation tested in
Argentina under autumnal climatic conditions showed a 92% mortality in
Varroa jacohsoni with a low variability, indicating that this kind of
formulation could be the best one [29]. Formic acid was evaluated for its
effect on the honey bee colony; Westcott and Winston [30] examined
various parameters such as colony weight gain, brood survival, sealed-
brood area, emerged-bee weight, number of retumed foragers, pollen-load
weight and worker longevity in treated and control colonies. Only sealed-
brood area was lower in formic acid treated colonies; furthermore, also the
honey production was lower in treated colonies, but this difference from
control was not statistically significant. Another study confirmed that the
use of formic acid can be considered safe, in fact the average length of life
of the treated bees was 24.4 days compared with 23.8 days of controls
[31]. Also during heavy Varroa Jacobsoni infestations and hives
weakened by the dry summer conditions, it was demonstrated that 8
consecutive treatments with formic acid during the month of August gave
satisfactory mite control without harming or irritating the bees [15].
However, these conditions are not sufficient for a proper use of formic
acid in apiculture, but it is necessary to evaluate whether this substance
can accumulate in honey, wax and propolis. Various HPLC methods have
been developed [32,33], but only one [34] compared treated honey
samples and controls, conclusively showing that the former contained
higher levels of formic acid (25-51 mg/kg) than the latter (20 mg/kg).
Recently, the influence of formic acid on honey taste has been evaluated
by means of an expert test panel [35]. The acid was artificially added to
different honeys and the testing persons remarked an increased acid taste
in the honeys adulterated with acid quantities above the taste threshold,
that is the lowest correctly distinguishable concentration of an additive in
386
honey. The taste threshold for acacia honey was about half of the one for
honeydew honey, because the latter has a much stronger aroma and can
whitstand more acid than acacia honey, which has only a weak aroma.
The taste thresholds of formic acid were 150-300 mg/kg in acacia honey
and 300-600 mg/kg in honeydew honey; in pure water it was 10 mg/kg,
about 20 to 50 times below that in honey. Similar thresholds were also
found in Italy [36]; Italian authors also noted that after application in
autumn at the end of the bee season, the formic acid content increases
significantly and may exceed the natural content. Later on, the formic acid
content decreases slowly due to evaporation, to reach the original level in
the following spring. Therefore, formic acid treatments should be
performed in autumn to avoid adverse consequences to the taste of
honey, harvested the following spring.
Further to formic acid treatment, oxalic acid [21,21,37-40] and lactic
acid [17,18,20,21,41-46] have been evaluated for their activity against
Varroa jacobsoni and V. destructor. The application of oxalic acid has
been studied both in different periods and by means of different methods.
It seems that the best period is autumn-winter, the broodless period
(average efficacy 99.44% vs. 52.25% in the presence of brood) [40].
These data have been confirmed in Italy, where a spray treatment of
oxalic acid in water was compared with a topical application of the acid in
a sucrose-water solution; the best result was obtained using the former
method. In general, for both treatments, the highest mite mortality
coincided with the treatment applied with no sealed brood [38]. With
regard to the toxicity of oxalic acid, two contrasting studies are present in
literature. The first one claims that bee mortality in treated hives was not
significantly different from the bee mortality in controls [37], while the
other paper suggests negative long-term effects, with a statistically
significant negative effect on brood development and the death of some
queens [39]. On the presence of oxalic acid in honey, it seems that its
content in samples taken from the nest after treatment did not differ
significantly between treated and control ones [37,38]. If the application
is performed in autumn, the honey content of oxalic acid is comparable to
its natural amount, that is below its taste threshold, determined in 300-
400 mg/kg for acacia honey and 700-900 mg/kg for honeydew honey [35].
Lactic acid was less active than the previous organic acids [18,21] and
again it showed a greater effectiveness during late summer-winter
387
treatments [18]. When its activity was compared with some commercial
acaricides, results show it is less effective than coumaphos, a synthetic
coumarin (umbelliferone) derivative, but also less toxic [42,45].
Rademacher [47] prepared a new formulation containing a synthetic
acaricide, cymiazole hydrochloride, and lactic acid as active ingredients;
furthermore, the content of cymiazole was 50% less than in a commercial
preparation. The mixture resulted highly toxic to Varroajacobsoni, with
the tolerance of honey bees a 100 times greater than that of the mites;
field tests have shown that the mixture killed 83% and over 90% of mites
in colonies with and without brood, respectively. Some authors reported
an increased bee mortality after treatments with lactic acid [18,41].
Immediately after applications in autumn, the lactic acid content of honey
increased up to 1500 mg/kg, but four weeks later it decreased to about
500 mg/kg: this was below the taste threshold of 800-1600 mg/kg in rape
honey [35].
Formic acid was also found effective against another bee parasite,
Tropilaelaps clareae (Acari: Laelapidae). This mite develops in brood
cells and causes disturbances in metamorphosis, resulting in abnormal,
incompletely developed individuals; in the case of a severe attack, bee
larvae are killed. It caused substantial losses of Apis mellifera colonies in
Pakistan, but a single application of formic acid (20 ml) to infested
colonies gave complete control of the mite population, however, it also
destroyed all the brood and some adult bees. Two treatments of 15 ml
also gave complete control of the mite population, but caused only
minimal destruction of brood [48]. The same findings were obtained in
another study [49]. A following study confirmed these results and also
demonstrated the effectiveness of sulfur (450 mg/frame) for T, clareae
control [50]. Furthermore, sulfur was not toxic for bees [31].
Tracheal mites were also killed by formic acid [51]. This parasite,
Acarapis woodi (Acari: Tarsonemidae), lives in the tracheal tubes of adult
honey bees. The bees die because of the disruption to respiration caused
by the mites clogging the tracheae.
In summary, although these organic acids are natural honey
constituents, the international food legislation prohibits honey additives
adulterating its taste. Therefore, the residues of these substances in honey
have to remain below their taste threshold [35].
Another natural substance tested against bee mites was the oil obtained
388
from neem tree, Azadirachta indica, kernels. Its first use in beekeeping as
acaricide dates back to 1995, when it was tested against the tracheal mite
Acarapis woodi [52]. A commercial preparation of neem oil containing
3000 ppm was used to prepare a syrup containing 3 or 6 ml/1 of the drug.
The colonies of Apis mellifera were treated in mid-May, and in August no
mites were found in the colonies treated with 3 ml/1, while they were still
high in controls. Furthermore, the treatment not only killed adult mites
but also reduced the number of mite eggs. Treated colonies collected more
pollen and produced more honey. Topical applications of neem oil in
laboratory to infested bee were highly effective against both Varroa
jacobsoni dead Acarapis woodi [53]. Approximately 50-90% V.jacobsoni
mortality was observed 48 h after treatment with neem oil, with
associated bee mortality lower than 10%. Although topically applied
neem oil did not result in direct A. woodi mortality, it offered significant
protection of bees from reinfestation. Significantly, azadirachtin-rich
extract was ineffective at controlling both the mites, suggesting that
azadirachtin. Fig. (1), one of the main insecticide compounds of neem
tree, has no significant miticidal properties; in fact, honey bees were also
deterred from feeding on a sucrose syrup containing more than 0.01 mg/ml
of azadirachtin-rich extract.
MeOO£.
1 ÔH
MeOOC
MeOOC î o
Fig. (1). Structure of azadirachtin
The same research group evaluated neem oil in the field [54]. They
sprayed a 5% solution of the oil on infested honey bee colonies, killing
about 90% Varroa mites but obtaining only a slight but not statistically
significant decrease in tracheal mite infestation levels. Unfortunately this
treatment caused 50% queen loss and the treated colonies showed one-
third as many adult bees and one-sixth as much brood as untreated
389
colonies. The authors hope that these negative effects on bees could be
remedied by changes in the formulation, the application technology, and
the season of application. In another trial the same researchers compared
the spray applications of neem oil with feeding oil or azadirachtin-rich
extract in a sucrose syrup [55]. They concluded that spray application
was more effective than the other methods only on Varroa. They also
reported that spray treatments had no effect on adult honey bee
populations, however treatments reduced the amount of sealed brood in
colonies by 50% and caused queen loss at higher doses. Besides neem oil,
Majeed also tested Azadirachta indica dust obtaining an effective level of
control and increased honey production with both the formulations [56].
Also pure azadirachtin, both commercially formulated and purified, was
tested for its effects on Varroa mites, infested bees and healthy bees [57].
Azadirachtin was administered in 50% sucrose syrup at various
concentrations, ranging from 6 to 162 |i<g/ml to infested and non-infested
worker bees: in both the experiments this compound caused a dose-
dependent reduction in syrup consumption; mite mortality was
significantly lower than their host bees at all test concentrations. Similar
results were obtained using a topical application of azadirachtin in water.
Feeding experiments with worker larvae however, gave unsatisfactory
results, with high larval mortality and abnormal pigmentation.
Other vegetable oils have been shown to be effective in the control of
tracheal mites. The oils were administered as vegetable shortening mixed
in a sugar patty. The oils did not kill the mite directly, but instead made
the bees unattractive or unrecognizable to the mite. Vegetable oils have
been used to control Varroa as well, and several commercial formulations
have been developed that are claimed to kill good percentages of mites.
However, research is somewhat contradictory about their effects [58,59].
Danish researchers performed a trial with a formulation of rapeseed oil
combined to an emulsifier, as well as soyabean oil also with different
emulsifiers. The oils were either sprayed on bees or administered in sugar
patties. While the oils with high concentration of emulsifier killed high
levels of mites (up to 97%), the side-effect was significant bee deaths
(over 50%). Oil mixtures with less emulsifier were not effective in killing
mites. Oil patties similar to those used with tracheal mites did not
significantly reduce Varroa levels. The researchers' conclusion is that
vegetable oils do not seem a realistic altemative for Varroa control [60].
390
Perhaps essential oils are the most studied natural substances as

alternative acaricides in apiculture. In fact these substances are already
present in hive products, i.e. Lejeune et al. asserted that propolis contains
5 to 10% of essential oils [61]. Furthermore, essential oils are well-known
parasiticides: they are being used as fungicides, bactericides and
insecticides for many years. Furthermore, the acaricidal properties of
essential oil constituents have already been proved since the beginning of
last century [62]. More than 150 essential oils and components of
essential oils have been evaluated as miticidal substances in laboratory
screening tests. However, very few of them proved successful when
tested in field trials. Thymol and thymol blended with essential oils or
essential oil components offer a promising exception [63]. For the twelve
year period employed in this review, many papers have appeared in
literature. In 1990 Colin tested the essential oils of two Labiatae species.
Thymus vulgaris L. and Salvia officinalis L., against Varroa jacobsoni
[64]. He also developed a formula to calculate the efficiency of the
applications. He treated bee colonies with aerosols from aqueous
emulsions ( 1 % for thyme and 0.5% for sage) and compared the results
with those obtained from a positive control treated with amitraz. The
compared treatments showed similar efficiencies. However, a major
drawback is that essential oils must be used in the absence of brood or at
least with a small brood area. Consequently, the most suitable time for
treatment is not winter, but after a large summer honey flow, which
reduces queen egg laying. In this case the great majority of female mites
are exposed to the aerosol action. Concerning the honey, sensory tests did
not give any evidence of thyme or sage after essential oil application.
However, traces of some essential oil components were revealed by GC.
The miticidal properties of menthol against the tracheal mite Acarapis
woodi were first reportede in 1968 [65]. Duff and Furgala [66] determined
the effectiveness and proper timing for menthol application as a control
for A. woodi infestations in Minnesota. They found that menthol is
somewhat difficult to deliver equally in a field study, depending on
outdoor temperature, bee population, size of the hive and amount of
direct sunshine on the colony that can affect menthol vaporization.
However, they concluded that, in Minnesota, spring application was the
best treatment, as it reduced the percentage of honey bees infested and
precluded winter mortality. Later, in 1993, the same authors substantially
391
confirmed their previous results [67]. In 1992, the same authors again
evaluated the effects of menthol on healthy honey bees. They stated that
the use of menthol in spring reduced brood production and confined the
bees spatially away from the menthol bag; however, menthol did not
affect honey production [68]. A similar study about the effect of Thymus
vulgaris and Salvia officinalis essential oils (2% water emulsions) on
mite-free honey bees was conducted by Marceau [22]. The colonies,
treated with these essential oils, both by microdiffusion and evaporation,
were not negatively affected. The same results were obtained in a
following study as well [23]. A more accurate study on the effect of
essential oils as miticides was performed by Kevan et al. [69], which
determined the following LD50 of some essential oils and pure
constituents administered to bees as feeding solutions: cinnamon oil 150
ppm, clove oil 200 ppm, wintergreen oil 500 ppm, pinene 1500 ppm,
thymol 100 ppm. In another paper the same researchers reported that
menthol was ineffective at causing mortality of mites with the highest
doses adminesterable [70]. Thymol seems to be a very promising natural
acaricide in apiculture, but has an inconsistent efficacy against Varroa
[17,64,71-75]. During the 90s, some commercial preparations against
Varroa appeared. They contained natural products, mainly thymol. The
first one was Apilife VAR® (Chemicals LAIF, Italy), a vermiculite tablet
impregnated with a 20 g mixture of thymol (76%), 1,8-cineole (16.4%),
menthol (3.8%) and camphor (3.8%). Various research groups tested this
preparation both in laboratory and field conditions [76-79]. All the
authors reported high efficacy (74-95%), particularly if employed under
optimal temperatures (>13°C). A formulation without camphor showed
the same positive results as well [78]. Apilife VAR was not toxic for
honey bees and the residues (only thymol) found in honey and wax were
innocuous to humans.
Another registered product, Thymovar®, consists of a sponge cloth
that functions as a vehicle for the drug thymol (15 g). The effects of this
preparation were similar to that of Apilife VAR [80]. Thymol was also
employed as "Frakno thymol frame". It consists of thymol crystals
placed into an evaporation box built into a brood frame and hung next to
the brood nest, to be replaced about 2-3 times a year. However, its
efficacy was judged insufficient for long-term treatment [81]. Finally, the
latest thymol-based acaricide is Apiguard®; a gel formulation designed for
392
a more controlled release. It was tested both against Varroajacobsoni and

Acarapis woodi [82]. Varroa mortality in treated groups was about 4
times higher than natural mortality, whereas in treated Acarapis infested
groups it was 6-8 times higher. Mattila et al. found this preparation not
toxic for adult bees or for 4-5 days old larvae, but it was quite toxic for
younger larvae [83]. Honey production during treatment was significantly
reduced in colonies treated with Apiguard, though the yield of the entire
season was not significantly different from controls [84].
A patent application has been presented for Varroa control by means
of acyclic and cyclic terpenes, mainly linalool, linalyl acetate, eugenol and
anethole; total control of the mite was observed when honey bees were
fed 50% sugar syrup containing 1% linalool [85].
Besides thymol, other terpenes have been tested for their toxicity
against Varroajacobsoni. Imdorf et al. determined in vitro the effective
miticidal air concentrations, but with minimal effects on the bees as
follows: 5-15 |ig/litre air for thymol, 50-150 jig/litre for camphor and 20-
60 |iig/litre for menthol; 1,8-cineole was too toxic for honey bees [86].
Another interesting paper considered the efficacy of different isomers of
menthol on Acarapis woodi [87]. The natural crystals obtained from the
plant, synthetic crystals and the L-form gave more than 96% mite
mortality, while the D-form crystals only a 37% mortality.
Colin et al. proposed a different method for characterizing the
biological activity of essential oils on Varroa mites [12]. Starting from the
supposition that all the field or laboratory experiments were based only
on counting the dead mites after therapeutic administration, they affirmed
that to describe a more real pattem of the biological activity of these
compounds, lethality tests had to be complemented by behavioural tests.
Authors used four essential oils: Thymus vulgaris (containing 30% of
thymol). Salvia officinalis (with thujanols as major components),
Chenopodium spp. (with ascaridole as main constituent), and Anona spp.
(with geraniol and linalool among the principal components). They first
determined the lethal dose, in order to choose the proper doses for the
following behavioural tests. These consisted in repellency test with
choice and without choice. They concluded that the essential oils of
Thymus, Salvia and Chenopodium not only exhibited an acute toxicity in
direct contact tests, but they prevented also the treated bee pupae from
being parasitized: the repellent effect was so strong that a majority of
393
mites do not move close to the treated pupae and probably died from
starvation [12].
A recent survey about essential oils and their pure constituents used to
control Varroajacobsoni, contained three interesting tables that reported
the toxicity of essential oils for V. jacobsoni and Apis mellifera after 24,
48 and 72 hours in a topical application and in an evaporation test, and
the effects of essential oils on behavior and reproduction of V. jacobsoni
and on the bee brood [63]. The most interesting oils were those of
cinnamon and clove, with 100% mite mortality after 24 h and no
significant toxicity on honey bees. Furthermore, clove essential oil
produced small brood mortality, and it was an inhibitor of mite
reproduction. Other effective oils were anise, fennel, lavender, rosemary
and wintergreen, which killed 100% mites after 48-72 hours. On the
contrary, the oils obtained from garlic, onion, oregano and thyme, were
found to be very toxic for honey bees. Among pure constituents,
camphor, linalool, linalyl acetate and pinene resulted small brood
mortality and inhibited mite reproduction.
The variable responses observed are probably the main drawback for
the practical use of essential oil as miticides. It must be pointed out that
the same plant species often produces essential oils with variable
composition because of environmental and/or genetic factors; many
species have varieties, the so called chemotypes; for instance at least
seven chemotypes are known for Thymus vulgaris [88,89]. Also the
extraction process influences the composition of the essential oils. For
these reasons, it is advisable that authors report the composition of the
essential oils used in the biological investigations. Unfortunately, only
one paper reported this important information [64].
Summarizing, the use of natural products as miticides in apiculture,
with the exception of some substances, is not widespread. In extensive
laboratory tests many compounds showed significant acaricidal
properties. However, very few of them have proven to be effective when
applied in field trials. Considerable variations in local environmental and
colony conditions can affect efficacy. In case of mixtures, such as
essential oils, the difficulty in obtaining standardized compounds also
affects treatment predictability. Nevertheless, identifying new acaricide
compounds with low toxicity to honey bees is fundamental for providing
candidate compounds for field trials. Furthermore, the development of
394
effective delivery systems could greatly contribute to the effectiveness of

some promising molecules infieldconditions.
VETERINARY AND HUMAN MEDICINE
a) Ticks
In many areas of the world, particularly the tropics, arthropod-borne
diseases are among the major limiting factors to the efficient production of
livestock and poultry. These diseases result in weakening, lameness,
blindness, wasting, congenital defects, abortions, sterility, and death of
the infested animals. Some exotic arthropod-borne diseases of livestock
are zoonotic and affect humans as well as animals. Some of the most
devastating of all animal diseases caused by arthropod-borne blood
protozoa, include babesiosis of cattle, sheep, goats, horses, and swine;
theileriosis, the East Coast fever syndrome, and Mediterranean fever; the
trypanosomiases causing illness in cattle, sheep and goats, camels, pigs,
dogs, and many wild game species; as well as several arthropod-borne
protozoa that cause diseases of birds. The most prominent groups of
arthropods that transmit etiological agents pathogenic to livestock are
those that are blood-feeding (hematophagous), such as ticks. They are the
most versatile vectors, for they parasitize all vertebrate groups, except
fishes. The tick-borne diseases they transmit are among the most
significant animal health deterrents to efficient livestock production.
Ticks are obligate ectoparasites of vertebrates. The family Ixodidae
comprises approximately 80% of all tick species, with the most
economically important ixodid ticks attacking livestock in tropical regions
belonging to the genera Amblyomma, Boophilus, Rhipicephalus and
Hyalomma,
Amblyomma sp. (Acari: Ixodidae) is a three-host hard tick, commonly
found in cattle, sheep and goats in Asia and Africa. The African species
are of the greatest economic significance because they transmit the
rickettsial pathogens Cowdria ruminantium, which causes Heart-Water
(Nairobi sheep disease) and Coxiella burnetii in cattle and sheep.
Furthermore, it is associated with streptothricosis, the actinomycete
infection of the skin of cattle, caused by Dermatophilus congolensis
[90,91]. It also causes theileriosis in cattle [92]. Infected manraials bite
the sites of infection or rub their bodies against hard objects damaging
their skin. As a result, they do not fetch a good prices in livestock
395
markets and their skin can be worthless for use in the manufacture of
leather goods. Furthermore, because of the blood sucking ticks, the
animals become weak and anemic, resulting in reduced milk and meat
production; severe infestations usually leads to premature death. Many
synthetic acaricides have been used to control this tick, including
organochlorine derivatives, organo-phosphorous compounds and
carbamates. However, besides resistance problems, these compounds are
expensive, especially for third world countries, and sometimes they cause
toxicity problems in animals and farmers [93,94].
Ndumu et al. evaluated the effectiveness of Azadirachta indica seed oil
against the larvae of this parasite [95]. They administered the oil as
hydroalcoholic solutions ranging 4.2-100% and computed the mortality
within 60 hours. Authors observed that the mortality of larvae was
concentration and time dependent; 100% mortality was observed with
100% pure neem oil after 48 h. The LD50 of different concentrations were
33.3% (56 h) and 66.7% (48 h). Author also observed little or no adverse
effects on treated animals. Furthermore, they stated that the open wound
caused by tick bites and therefore exposed to potential fungal and
bacterial attacks, could be protected by the microbicidal properties of the
neem oil. Previously, the effectiveness of neem oil was also observed by
Williams and Mansingh against another tick species of the same genus. A,
cajennense, another cattle tick [96].
Malonza et al. observed that the leaves of a Capparidaceous plant,
Gynandropsis gynandra, exhibited repellent properties against all stages
of Amblyomma variegatum [97]. In field conditions the ticks were not
found up to 2-5 m from the plant in areas where this species was
predominant, while, in the laboratory, ticks which were continuously
exposed to its leaves, died. The effectiveness of the plant was most
pronounced on juvenile stages and least pronounced on adults. Another
plant, snakeweed {Gutierrezia sarothrae and G. microcephala,
Asteraceae) was found useful against A. americanum [98]. Domestic
rabbits that had received diets containing 5% or 10% of this plant leaves
showed 39% and 33% reduction of tick attachment, respectively.
Dichloromethane extracts of the leaves, topically administered on sheep
skin, significantly reduced tick attachment with respect to control. A
third species, Margaritaria discoidea (Euphorbiaceae), showed acaricidal
properties against A, variegatum [99]. Its water extract was effective on
396
nymphs, but not on adults. The hexane extract at 6.25% was found more
effective, causing 100% mortality of adults. Its application at a 50%
concentration on the ears of rabbits prevented the attachment of adult
ticks for at least 4 days, while its direct application on engorging ticks
induced mortality of 70% adults on rabbits and 50% adults on cattle in
the field.
Ivermectin is a macrolide antibiotic produced from a fungus first
isolated from a soil sample in Japan, Streptomyces avermitilis. In the mid-
80s, ivermectin was introduced as probably the most broad-spectrum
anti-parasite medication ever. It is effective against most common
intestinal worms (except tapeworms), most mites, and some lice. It is also
effective against larval heartworms (the "microfilariae" that circulate in the
blood), but not against adult heartworms (that live in the heart and
pulmonary arteries). Avermectins, to which ivermectin belongs, are
agonist for the neurotransmitter y-aminobutyric acid (GABA). GABA is
a major inhibitory neurotransmitter. In mammals, GABA-containing
neurons and receptors are found in the Central Nervous System; while in
arthropods and nematodes GABA is found primarily in the Peripheral
Nervous System (neuromuscular junction). This difference in the
localization of the GABA receptors, could be the reason for the large
margin of safety of avermectins-containing products in mammals. The
binding of avermectins to a neuronal membrane increases the release of
GABA. GABA binds to the GABA receptor-chloride channel complex of
postsynaptic neuronal membranes, causing an influx of chloride ions. This
influx hyperpolarizes the neuronal membranes, making them less
excitatory and decreasing nerve transmission. The hyperpolarization of
neuronal membranes mediate a flaccid paralysis in arthropods and
nematodes [100-101]. Ivermectin has been used in different way of
administration for the control of Amblyomma. Soil et al. experimented an
intraruminal bolus against natural infestations of five different African
tick species on cattle, among which Amhlyomma hebraeum (the other
species were Boophilus decoloratus, Hyalomma spp., Rhipicephalus
appendiculatus ^ndi Rhipicephalus evertsi evertsi) [102]. Unfortunately,
the observed reduction in the number of engorged female ticks was not
statistically significant. An experiment aimed at controlling A.
americanum in free-living white-tailed deer, Pound et al. prepared whole
kernel com treated with 10 mg of ivermectin per 0.45 kg corn to use as
397
bait [103]. Monitoring of the free tick populations through two years of
study, showed 83.4% fewer adults, 92.4% fewer nymphs, and 100.0%
fewer larval masses in the treatment versus control. In 1997, Miller et al.
compared the treatment of pasture cattle with ivermectin (administered
orally at 200 |ig/kg or by injection at 40 |ig/kg) against A. americanum
[104]. They observed no significant difference in the number of
unengorged, small, and medium sized female ticks to the untreated control
compared with those treated orally. However, the number of large female
ticks was reduced. Significantly, fewer small, medium, and large female
ticks were found on the injection treated cattle, compared with the
untreated controls. There were also significantly more unengorged females
on the animals treated by injection than on those treated orally or left
untreated. Wilson et al. evaluated the effects of ivermectin on the volume
of blood ingested by A, americanum [105]. Adult females were collected
from Bos taurus hosts, treated and untreated with ivermectin, and they
observed that the mites feeding on treated animals contained smaller
quantities of blood. Recently, a bioabsorbable injectable microsphere
formulation has been developed to provide long-lasting delivery of the
drug [106].
Some researchers noticed that some chemicals attract adult of
Amblyomma [107,108]. Among these substances many were of natural
origin, such as nonanoic acid, methyl salicylate, benzyl alcohol,
benzaldehyde, heptadecane and squalene. Other authors exploited this
feature to prepare some drugs in which the acaricides were associated to
the pheromone-like chemicals to control Amblyomma [109,110].
Another "natural" way to control Amblyomma is to use its
hyperparasitic fungi, such as Beauveria bassiana and Metarhizium
anisopliae [111,112]. In adult of ^. variegatum, M. anisopliae induced a
mortality of 37%, while B, bassiana induced no mortality. However, both
fiingi induced significant reduction in engorgement weights, egg masses
and egg hatchability; B, bassiana completely inhibited egg hatchability.
Authors concluded that these fimgal species were capable of inducing high
mortalities, decreased fecundity and egg hatchability, and they could
represent a great potential for tick control. Their ability to reduce
fecundity and egg hatchability is of greater importance than adult
mortality, in fact a reduction of egg hatchability by 99-100% may mean a
very significant reduction in the next generation of ticks, and has a greater
398
impact on tick population than the direct mortaUty on engorging females,

which may destroy only a few dozen ticks.
Boophilus sp. are one-host ticks, which occur in all tropical and sub-
tropical regions of the world, where they feed preferably on cattle. They
are the main vectors of Babesia species, B, bovis and B. bigemina, causing
babesiosis in cattle. Boophilus ticks, together with many other tick
species, also transmit Anaplasma marginale, the rickettsia that causes
anaplasmosis of cattle on all continents. Many natural remedies have been
tested against this tick. Among them we can find essential oils and their
purified constituents. Brazilian researchers evaluated the effectiveness of
the essential oil and of its components a- and p-pinene, obtained from
the grass Melinis minutiflora (Poaceae) [113]. All the chemicals showed
lethal effect on Boophilus microplus larvae. Later, the same authors tested
1,8-cineole and «-hexanal, from the same oil, that showed individually
100% lethal effect on cattle-tick larvae within 10 min [114]. It must be
pointed out that these two papers are among the few articles that report
the composition of the tested essential oil. Two further essential oils,
Cymbopogon citratus and C nardus (Poaceae), have been examined by
Chungsamamyart and Jiwajinda [115]. They found that the oils extracted
from fresh leaves, diluted in EtOH, exhibited a higher activity against
adults and larvae of J5. microplus than the oils extracted from dried leaves.
Fresh C citratus volatile oil exhibited 85-100% mortality against engorged
female ticks at all the dilutions tested (up to 1:4), whereas fresh C.
nardus oil exhibited 85-90% mortality up to 1:3 dilutions. On larvae,
fresh C citratus volatile oil showed >90% mortality up to 1:16 dilution,
while fresh C. nardus oil was endowed with a similar activity up to 1:12
dilution. The same research group also evaluated the activity of the peel
oils and pure limonene of some cultivars and species of Citrus (Rutaceae)
against the same ectoparasite [116]. The oils from C reticulata and C.
maxima cv. Thong-dee showed a good acaricidal activity against engorged
female ticks at the 1:10 dilution, activity being 2 times higher than that of
limonene. C sinensis and C. maxima peel oils, diluted 1:10, exhibited high
larvicidal activity, while the oils of C. hystrix, C. reticulata, C. suncris and
C. maxima (immature fruits) showed moderate larvicidal activity, about
1.5 times stronger than that of limonene. The essential oils extracted from
berries, bark, leaves and twigs of Pimenta dioica (Myrtaceae) were
compared for their effectiveness against Boophilus microplus with that of
399
eugenol, isoeugenol and four commercial synthetic acaricides [300]. The

berry essential oil was more effective at inhibiting oviposition (no egg
laying at 3 mg/g body weight) and causing mortality of the ticks (100% at
3 mg/g) than all the other extracts, the synthetic products and methyl
eugenol. Authors hypothesized that the activity of the berry essential oil
could be attributed to eugenol (100% toxicity at 3 mg/g and no egg laying
at 2 mg/g), which accounted for more than 65% of the whole oil.
Korpraditkul et al. conducted an experiment using a vetiver {Vetiveria
zizanioides, Poaceae) extract to control cattle tick [118]. Three ecotypes
of vetiver grass were used, 'Si Sa Kef, 'Uthai Thani', and Thetchabun'.
Two methods of essential oil extraction were employed, steam distillation
and solvent extraction (using two solvents, ethanol and dichloromethane).
When adjusting the oil's concentration at 10%, applied to treat dairy cow
tick at larval and adult stages as well as egg-laying stage, the results
indicated that the chemicals extracted from the roots of different ecotypes
possessed different efficiency in controlling ticks. The extract obtained by
steam distillation of the dried 'Uthai Thani' vetiver root killed ticks at
both stages at the highest rates, with mortality rate of larvae and adults of
50.7 and 20.0%, respectively. In addition, the condition of the root also
played a role in controlling ticks; extract from dry vetiver root was able to
control larval stage ticks better than adult stage, while extract from fresh
root was able to control adult stage of ticks better than larval stage. It was
also found that the oil extracted from vetiver root showed no significant
difference in controlling ticks when compared with citronella. The ethanol
extract from dried roots of Thetchabun' ecotype showed very good
results, giving 99.4% mortality of larvae and inhibiting adults from laying
egg at 46.7%. The results indicated that the extract from vetiver roots was
able to control the growth of ticks during larval and adult stage, and
including egg-laying stage of ticks. The ethanol extract had the highest
potential for controlling cow ticks. According to these authors, if the
extraction method is improved, or its concentration is adjusted to the
optimum level, the experiment may give better resuhs.
Many non-volatile extracts have been studied for their effectiveness
against Boophilus sp. Those obtained from legumes plants seem to be
particularly promising. Cruz-Vazquez et al. evaluated the tropical species
Stylosanthes humilis and S. hamata (Fabaceae) against the larvae of
Boophilus microplus in plots experimentally infested [119]. They
400
observed that after four weeks the percentage larval survival was 5.1% for
S, humilis, 7.5% for S. hamata, while in control plots it was 18.9%.
Khudrathulla and Jagannath used the methanolic extract of another
species of Stylosanthes, S. scabra, on different life stages of three tick
species, B, microplus, Haemaphysalis intermedia and Rhipicephalus
sanguineus [120]. In vitro trials, conducted by the tea bag method,
showed a dose-dependent activity both on larval and nymphal mortality,
with the exception of H. intermedia nymphs, which were not killed by
the extract. Finally, authors declared that, in preliminary assays, the
aqueous extract showed better acaricidal properties. Regassa reported the
results of a questionnaire survey about the traditional tick control
methods used in three provinces of Western Ethiopia [121]. The most
frequently employed drugs were the latexes of Euphorbia obovalifolia
(Euphorbiaceae) and Ficus brachypoda (Moraceae), the juice obtained
from the leaves of Phytolacca dodecandra (Phytolaccaceae) and Vernonia
amygdalina (Asteraceae), the fruit juice of Solanum incanum
(Solanaceae), the seeds of Lepidium sativum (Brassicaceae) mixed with
fresh cattle faeces, the juice of crushed leaves and bark of Calpurnea
aurea (Papilionaceae), and the commercially available spice of Capsicum
spp. (Solanaceae) mixed with butter. The same author tested in vitro the
activity of these preparations of Capsicum spp., E. obovalifolia, S.
incanum and F. brachypoda in vitro against Boophilus decoloratus,
obtaining 30-100% killing effects. Following in vivo treatments with the
same extracts ofE. obovalifolia and F. brachypoda on naturally infested
indigenous cattle, reduced infestation up to 70%. In another survey on the
activity on Boophilus microplus of several crude EtOH extracts of
Jamaican plants, the authors evaluated the ability of the drugs to kill
engorged ticks and inhibit the oviposition or embryogenesis [122]. They
associated an acaricidal index, ranging from 0 to 100, on the basis of the
effectiveness verified. The most active species were Quassia simarouba
(100) (Simaroubaceae), Symphytum officinale (99) (Boraginaceae),
Nicotiana tabacum (95) (Solanaceae), Hibiscus rosa-sinensis (93)
(Malvaceae), Ricinus communis (82) (Euphorbiaceae), Salvia serotina
(80) (Lamiaceae), Stachytarpheta jamaicensis (79) (Verbenaceae),
Ocimum micranthum (76) (Lamiaceae), and Spigelia anthelmia (75)
(Loganiaceae). Recently, Chungsamarnyart and Jansawan tested the
extracts in water or in 10% EtOH of the mature Tamarindus indicus
401
(Fabaceae) fruits, from which the seeds had been removed, on engorged
female of Boophilus microplus using the dipping method [123]. The pure
organic acids contained in the fruits, oxalic, malic, succinic, citric and
tartaric acids, were bioassayed at 0.5% and 1% concentrations. Among
the extracts, the most effective were the crude 1:2 ones. Among the
organic acids, 0.5% and 1% oxalic acid exhibited the highest acute
acaricidal activity, while 1% tartaric acid showed the highest delayed
activity. All the compounds caused patchy haemorrhagic swelling lesion
on the parasite skin, as documented by photos. The EtOH extracts of five
marine algae were assayed against Boophilus microplus [124]. The author
evaluated the effects of topical applications on mortality, oviposition and
embryogenesis in the ticks. The most toxic extracts were those of
Laurencia obtusa (Rhodomelaceae) and Liagora elongata (Liagoraceae),
whereas Liagora farinosa, Padina vickerisiae (Dictyotaceae) and
Stypopodium lobatum (Dictyotaceae) resulted barely effective. On
embryogenesis the results were different, with L obtusa, L farinosa and
S. lobatum as the most effective extracts.
Different preparations obtained from neem tree, Azadirachta indica,
were tested on Boophilus sp. Williams investigated the adverse effect of
EtOH extracts of neem (and of Artocarpus altilis, Moraceae) on egg laying
and hatching in B, microplus [125]. Egg laying was inhibited by 50% at an
extract concentration of 0.54 |ig/tick; the same dose also caused a 65%
hatching failure; the extracts of ^. altilis were more effective (0.46 |Lig/tick
and 80%, respectively). The activity of the extracts has been reported to
be due to the inhibition of protein and lipid sequestration by ovaries and
oocytes. Kalakumar et al. compared the activities in vitro and in vivo of
neem oil with those of Annona squamosa (Annonaceae) seed oil and
pyrethrins against B. microplus [126]. Neem oil was the least effective
substance: it was only 60-75% effective in infested cattle and buffaloes
and it was unable to inhibit oviposition, while the other two extracts were
100% effective both against infestation and oviposition. Neem oil was
also tested in association with Eucalyptus (Myrtaceae) and Pongamia
(Fabaceae) oils on Boophilus microplus infested cattle and goats [127].
The most effective mixture was neem/eucalyptus oils. Authors evidenced
a significant reduction of total proteins and total lipids in the treated
ticks. The herbal formulation AV/EPP/14, containing extracts of A cor us
calamus (Araceae), Azadirachta indica (Meliaceae), Pongamia pinnata
402
(Fabaceae), Cedrus deodara (Pinaceae) and Eucalyptus globulus

(Myrtaceae), and Pestoban, an Indian herbal solution containing extracts
of Cedrus deodara, Azadirachta indica and Embelia ribes (Myrsinaceae),
were tested by various researchers. AV/EPP/14 was 100% effective
against larvae and nymphs of Boophilus microplus within 24-48 h from
application; furthermore, it reduced 95% egg laying and hatchability
[128,129]. Pestoban was found effective in a single application in light
infestations in Buffaloes and cattle, while a second application was
required for heavy infestations [130]. Similar results were obtained by
Srivastava and Sinha, which observed that the larval and nymphal stages
of ticks were more susceptible than the adults [131]. On calves, this
herbal preparation did not induce inflammatory or other tissue changes,
contrary to synthetic products which caused severe tissue reactions
[132,133].
Many avermectin derivatives (see above) have been tested on
Boophilus spp. Maske et al. observed tick elimination within 72 hours
and prevention of reinfestation for 30-32 days with ivermectin [134].
Better results were obtained when ivermectin was administered as an
intraruminal sustained-release bolus: treated animals showed significant
less re-infestations of Boophilus annulatus for 90 days [102]. These
results were recently confirmed by Miller et al. [135]. In Brazil a
subcutaneous injection of 1% ivermectin (200 |lg/kg) showed a very good
efficacy for about one month against B. microplus [136]. Also doramectin
was highly effective in removing tick populations and in controlling re-
infestations under conditions of continuous field challenge [137-140].
Many purified natural compounds were assayed for acaricidal activity
against Boophilus sp. From the EtOH extract of the aerial parts oiBontia
daphnoides L. (Myoporaceae), Williams et al. isolated the sesquiterpene
furan epingaione. Fig. (2), that showed growth regulatory activities on
gravid adult female of Boophilus microplus [141]. It inhibited 50%) egg
hatching at 0.4 mg/g tick body weight. The activity was due to the
inhibition of the sequestration of protein into eggs; histological
examination of ovarian sections from the treated ticks revealed significant
degeneration of funicle cells and reduction in yolk content.
The dichloromethane extract of Hyptis verticillata (Lamiaceae) yielded the
sesquiterpene cadina-4,10(15)-dien-3-one. Fig. (2) [142]. Besides its
insecticidal activity against Cylas formicarius, this chemical inhibited the
403
metabolism of lipids during embryogenesis of 5. microplus eggs in a dose-

related manner. Thus, 69.78% lipid was metabolized in the control eggs,
compared to 59.61% and 35.93% in the eggs produced by ticks treated
with 0.9 and 1.8 mg/g of the cadinene derivative, respectively. Authors
speculated that the inhibition of oviposition could be due to the effect of
this substance on neuromuscular binding sites, since egg laying in Acarina
is a neuromuscular process.From the EtOH extract of the roots of the
herbaceous tropical plant Petiveria alliacea L. (Phytolaccaceae),
dibenzyltrisulfide. Fig. (2) was isolated [143]. This compound showed a
LD50 value of 0.92 |Lig per adult tick in topical treatments; the same
values for synthetic acaricides were 5-10 times higher. Dibenzyltrisulfide
was also effective as inhibitor of oviposition, with a IOD50 of 0.221
|lg/tick (the doses of commercial acaricides were 1.3-29 times higher). The
compound was capable in reducing the hatching success of eggs
oviposited by the treated ticks.
Epingaione
Cadina-4,10(15)-dien-3-one
Dibenzyltrisulfide
Fig. (2). Structures of epingaione, cadina-4,10(15)-dien-3-one, and dibenzyltrisulfide
Williams et al. tested the acaricidal activity of five pure natural

phenylpropanoid derivatives, the compounds that accounts for 80-85%
of the essential oil of Pimenta dioica [144]. Eugenol was the most
effective compound in killing the adult ticks and in reducing egg laying and
hatching at 3.2 mg/kg body weight. For mortality, the effectiveness was
100% for eugenol, 50% for isoeugenol, 40% for safrole, 30% for
methyleugenol, while benzo-l,3-dioxole was ineffective; famesynic acid, a
404
commercial insect growth regulator, was comparable to methyleugenol.

Similar results were observed for oviposition and egg hatchability.
Authors gave some consideration to the structure/activity relationships:
with respect to eugenol, which was the most effective compound. Benzo-
1,3-dioxole, which lacks the propenyl substitution, was not active;
substituting the hydroxyl by methoxyl reduced the activity, while
changing the olefin center from 1-2' to 2 - 3 ' in isoeugenol reduced the
killing efficacy by 50%. The methylene bridge in safrole was more
effective than the methoxy groups in methyleugenol.
The aqueous solutions of the monoterpenes piquerol A and B, purified
from Piqueria trinervia (Asteraceae), showed an acaricidal potential on
larvae of B. microplus, but neither compound prevented oviposition.
Piquerol A, Fig. (3), was also toxic for gravid female ticks [145].
Spinosyns are natural metabolites produced under fermentation
conditions by the actinomycete Saccharopolyspora spinosa. One such
product, with the proposed common name of spinosad, a mixture of
spinosyn A and spinosyn D, Fig. (3), has been developed by Dow-
Elanco and evaluated against B. microplus [146]. The results of this study
demonstrated that a single whole-body spray treatment with spinosad,
applied to cattle infested with all parasitic life stages of the tick, provided
85-90% control and almost complete protection against larval
reinfestation for 1-2 weeks.
Recently, many hyperparasites of the tick have been evaluated as
control methods, in particular the entomogenous fungi Metarhizium
anisopliae [147-149], Beauveria bassiana [112], Verticillium lecanii [150]
and the bacteria Cedecea lapagei 117 and Bacillus thuringiensis var.
kurstaki [151].
Other important tropical ixodid ticks species belong to the Rhipicephalus
and Hyalomma genera. They can be vectors of many pathogens, such as
the medically important Coxiella burnetii and Rickettsia conorii and the
canine ones Anaplasma, Babesia and Ehrlichia spp. These two ticks have
been also found on humans who venture into tick-infested caves and
burrows [152,153]. In India, the two parasite species infesting goats, have
been treated with an emulsion of tobacco leaf extract, mustard oil, DDT
and copper sulphate, securing their removal within 2 days and preventing
reinfestation for 33 days [154]. Comparison of the acaricide effectiveness
of Annona squamosa (Annonaceae), Azadirachta indica (Meliaceae) oils
405
and pyrethrins against Rhipicephalus haemaphysaloides and Hyalomma

anatolicum showed 100% efficacy for A, squamosa and pyrethrins,
whereas neem oil was only 60-75% effective [126].
Piquerol A
R
R=H spinosyn A
R=CH3 spinosyn B
Fig. (3). Structures of piquerol A and spinosad.
The Indian herbal preparation Pestoban (see above) have been

evaluated by many authors, obtaining 50-100% control on adults or larval
stages of the two ectoparasites [130-132,155]. Also, the herbal
ectoparasiticide AV/EPP/14 (see above) showed similar results on
different hosts [129,156-159]. An African ground mixture of natural
products, containing dried tobacco leaves and 'Magadi Soda' (principally
sodium bicarbonate), commonly sold in local markets in East, West and
Central Africa, prevented the completion of all feeding phases of
Rhipicephalus appendiculatus, suppressed the oviposition capacity of the
engorged ticks and drastically reduced the hatchability of the eggs. Larvae
and nymphs were killed within 24 h from the application of the
substance, while many adults were killed within 2-3 days. This product
could replace commercial acaricides among resource-poor farmers in
Africa [160]. An African plant, Gynandropsis gynandra (L.) Brig.
406
(Capparidaceae) showed repellent and acaricidal activities against all life

stages of Rhipicephalus appendiculatus, and was particularly effective on
nymphs. Field investigations indicated that ticks were not found up to 2-
5 m from the plant in areas where the plant was predominant; this species
could be introduced, as pasture plant, for tick control among resource-
poor farmers in Africa [97]. The same research group evaluated the tick-
repellent potential of the essential oil and its constituents obtained from
the same plant [161]. The repellency of the essential oil hydrodistilled
from the fresh aerial parts against R, appendiculatus was tested at four
different test concentrations (lO-'^-lO"! |LI1) using a climbing bioassay.
Apart from methyl isothiocyanate, all the identified constituents were
assayed and their effectiveness compared with the commercial arthropod
repellent DEBT (A^,A^-diethyl-toluamide). At the higher doses (0.1 and
0.01 ]\X) the percentage repellency of the oil was comparable to DEBT,
while at lower doses it was slightly less effective. The most active
components were m-cymene, nonanal, a-terpineol, a-cyclocitral, p-
cyclocitral, nerol, geraniol, carvacrol, a-ionone, (£')-geranylacetone,
nerolidol and cedrene (isomer not specified), all of which had repellencies
comparable to that of DEBT at the higher treatment levels; next in
hierarchy benzaldehyde, phenylacetaldehyde, p-ocimene, linalool,
phenylacetonitrile and methyl salicylate were found. The essential oil
obtained from the leaves of another African plant, Ocimum suave
(Lamiaceae), showed repellent and acaricidal properties against all the
stages of 7?. appendiculatus [162]. The LC50 of the essential oil, dissolved
in liquid paraffin, was 0.024% in vitro, while a 10% solution killed all the
immature forms and more than 70% of adults on rabbits, and further
protected them from reinfestation for at least five days. Another plant
species, Margaritaria discoidea (Euphorbiaceae) showed acaricidal
activity on R, appendiculatus [99]. Its water extract was effective both on
nymphs and adults. The hexane extract from dry wood was found even
more effective, causing 100% mortality of nymphs and adults at 6.25%.
Its application at a 50% concentration on rabbit ears prevented the
attachment of adults for at least 4 days, while its application directly on
engorging ticks induced mortality of 70% adults on rabbits and 50%
adults on cattle in the field, thus resulting as effective as the standard
concentration (0.05%) of the synthetic acaricide chlorfenvinphos.
Many papers report studies about the use of abamectins (see above)
407
against Rhipicephalus and Hyalomma spp. on various hosts, both as

acaricides and as reinfestation preventives. All the papers reported good
efficacy [102,134,155,163-165], with the exception of a case of
Hyalomma dromedarii on dromedaries treated with ivermectin [166].
Finally, also for these ticks, the biological control with the
entomogenous fungi Beauveria bassiana and Metarhizium anisopliae
[111,112] or different strains of the bacterium Bacillus thuringiensis
[167,168], showed different degrees, of effectiveness.
Other important ticks, mainly distributed in North America, are Ixodes
spp. (Acari: Ixodidae); certain species are vectors for Babesia spp. or
Anaplasma spp. of cattle; /. dammini is the vector for Lyme disease
(Borrelia burgdorferi). These bacteria are transmitted to humans by the
bite of infected ticks; individuals who live, play or work in residential
areas surrounded by tick-infested woods or overgrown brush are at risk of
getting Lyme disease. Rash and flu-like symptoms are present in early,
localized disease, while disseminated disease includes arthritis, carditis
and neurologic disorders; in the U.S. about 15000 cases are reported
annually. The control of the tick is performed mainly by means of
synthetic acaricides, and very few papers are present in the literature
about use of natural products or derivatives. The first one reports the
effectiveness of Urariapicta (Fabaceae) against /. ricinus [169], a plant
used in Nigerian folk medicine for control of ectoparasites. The aerial
parts were extracted with MeOH, and the residue partitioned with
EtOAc and water; the water-insoluble fraction was further partitioned
into alkaline-soluble and alkaline-insoluble fractions. Furthermore, another
aliquot of aerial parts was extracted with water. All the total and
fractionated extracts were assayed on non-engorged /. ricinus ticks. All
the extracts showed acaricidal activity. Water extract was the least
effective (35% mortality at 5% concentration). The MeOH extract was
very effective, showing 100% mortality at 1% concentration; its water-
insoluble fraction killed 98.86% of the ticks at 0.8%. The alkaline-
insoluble and the alkaline-soluble constituents of the above fraction
showed 80% and 97.76% acaricidal activity at 1% and 0.5%
concentrations, respectively. The effective fractions were analyzed for
the presence of different classes of compounds and authors suggested that
it was attributable to more than one class; in fact the phytochemical
screening indicated the presence of phenolic and flavonoid derivatives in
408
the alkaline-soluble fraction, while sterol and terpene derivatives were

detected in the alkaline-insoluble fraction. Panella et al determined the
acaricidal activity of the extracts of 13 plants on immature ticks (13-16
weeks old), using 10 different doses, ranging from 0.0001 to 2% [170].
Nine extracts resulted effective, in particular the essential oils obtained
from Chamaecyparis nootkatensis, C. lawsoniana (Cupressaceae)
(LC5o=0.151% and 0.487% w/v, resp.), Juniperus viriginiana and J.
occidenatalis (Cupressaceae) (LC5o=0.328% and 0.633%, resp.),
Calocedrus decurrens (Cupressaceae) (LC5o=0.343%), Thuja plicata
(Cupressaceae) (LC5o=0.821%), Artemisia tridentata (Asteraceae)
(LC5o=1.180%), Sequoia sempervirens (Taxodiaceae), (LC5o= 1.673%),
Foeniculum vulgare (Apiaceae) (LC5o=0.744). Authors also assayed
some pure terpenes, and four of them resulted effective, namely a-
cedrene (LC5o=1.524%), cedryl acetate (LC5o=1.556%), thujopsene
(LC5o=3.168%) and 4-terpineol (LC5o=3.860%). Another paper citing
use of natural chemicals against Ixodes ticks, reports the isolation and the
effectiveness against /. ricinus of two naphthoquinones from Calceolaria
andina (Scrophulariaceae), Fig. (4) [171]. The nymphal stages were
topically treated with 0.25 p.l of the compound in acetone, and the LD50
of the naphthoquinones resulted 120 and 50 ng, respectively. Highly
significant, the acute toxicity of the two compounds on mammals was
very low, 1366 and 1072 mg/kg respectively in oral tests, and >2000
mg/kg in dermal tests.
o
BTG505R=H
BTG504R=COCHb
Fig. (4). Structures of naphthoquinones from Calceolaria andina.
Other "natural" methods to control Ixodes sp. contemplate the use of

entomopathogenic nematodes belonging to the genera Steinernema and
Heterorhabditis [172], or flocks of free range helmeted guineafowl
(Numida meleagris) feedy on infested meadows [173].
409
b) Mange mites
Mange is a common skin disease of animals caused by different species of
mites. The most severe form of mange is caused by the mite Sarcoptes
scabiei, which is also the cause of human scabies. Some forms of mange
are known to all domesticated animals, no matter how well-taken care of
or pampered they are. The main genera implicated are Sarcoptes,
Notoedres (Acari: Sarcoptidae), Psoroptes, Chorioptes, Otodectes (Acari:
Psoroptidae), Demodex (Acari: Demodicidae), and Knemidocoptes (Acari:
Knemidocoptidae).
In the management of livestock, both for production and breeding
purposes, the mite genera mainly responsible for causing mange are
Psoroptes, Chorioptes and Sarcoptes mites. Modem animal husbandry
practices create favorable conditions for the multiplication of
ectoparasites, such as mange mites. The subclinical form of the
infestation, which reduces productivity considerably, is well recognized.
Mange mites are mostly transmitted from animal to animal by contact.
Since mange mites are able to survive outside the host, sheds, boxes and
pasture fencing must be considered as sources of reinfestation. The
increased incidence of mange and resistance to the usual forms of
treatment, with increasingly frequent therapeutic failures, has prompted
researchers to look for more efficient forms of acaricide therapy, which
are more acceptable to human and animal patients. Although traditional
topical treatments, including polysulfurous compounds, possess good
antiparasitic action, they have numerous undesiderable characteristics
including an unpleasant odour, require numerous applications, frequently
provoke local irritation and occasionally treated human and animal
patients develop signs of systemic toxicity. There are also other causes
for the failure in treatment of scabies: reinfestation after the end of
treatment, paucisymptomatic forms with rare mites (difficult to find),
possible resistance of these mites to some antiparasitic compounds at
atoxic concentrations, unmotivated human patients or with immune
deficiencies that are strongly prone to parasitosis, and patients who are
sensitive to the active ingredient or substances contained in commercial
preparations [174]. There is no synthetic acaricide yet with which the
eggs of the mange mites can be reliably destroyed. A second treatment is
always necessary to kill the larvae, which have hatched from the eggs in
the meantime. The mites are often concealed in the crusts and scabs.
410
where they are often not directly reached by the acaricide during
spraying; for this reason a mange remedy with vapour effect should be
favoured.
Psoroptes mites prefer hairy parts of the body; by piercing the skin
and sucking lymph fluid they cause pustules that spread rapidly, burst
and form typical yellowish-sticky scabs and crusts. They are particularly
active in the cold season and infest sheep, cattle (especially fattening
bulls), and horses. The animals suffer from intense itching, become
restless, bite and rub the affected parts and the hair or wool becomes
detached. In young animals growth is arrested at first, followed by severe
emaciation and death if the infestation is heavy. While in the past
psoroptic mange in cattle was considered rare, it is today one of the main
problems in bull fattening management.
Sarcopies mites, which are also the cause of human scabies, prefer
hairless parts of the body, which is why mange in the pig, for example, is
always sarcoptic mange. The mites burrow tunnels in the skin, suck
lymph and feed on young epidermal cells. At first acute symptoms, such
as skin reddening and pustules, are observed on the affected parts of the
skin, causing severe itching; even a few mites can cause severe clinical
symptoms. In horses and sheep Sarcoptes mites cause the so-called "head
mange", in susceptible horses this can spread to the neck and shoulder
region. In cattle sarcoptic mange can become a problem particularly in
dairy herds, the body areas most affected being the head, neck and udder.
Sarcoptes mites can also cause mange in dogs and cats. Sarcoptes mites
can survive for approx. 2 weeks off the host.
Chorioptes mites preferentially attack the fetlocks and the base of the
tail, where they chew at the skin surface, thus causing inflammations,
scaly lesions and a powdery coating. The so-called foot and rump or tail
mange can occur in the horse, sheep but especially in dairy cows.
Demodectic mange is caused by small lancet-shaped follicular mites,
belonging to Demodex genus, whose parasitism is not always
accompanied by pathological lesions. These mites are also encountered in
healthy animals and man, and need additional factors in order to produce
clinical demodicosis. The disease has greatest significance in dogs. In other
species, such as horse, cattle, pig, sheep, goat and rabbit, host-specific
Demodex species can occur, but they rarely cause disease.
The genera Psoroptes and Sarcoptes have been subjected to intense
411
Study, at least from the natural miticidal products point of view.

Historically, in 180 BC, Cato the Censor advocated the anointing of
sheep after shearing, with equal parts of olive oil dregs, water in which
lupines had been steeped and the lees of good wine to control the disease.
During the 19th Century, many internal cures appeared, including the
feeding of sulphur, but all failed. Dipping was developed in the 19th
Century, the method is still used today and it was the first to achieve any
real success. William Cooper in 1843 produced the first commercial dip.
Among natural products, many different materials were used, including
hellebore and turpentine. Sulphur, nicotine and arsenic were the most
commonly used and effective, however, they stained, damaged and
devalued fleeces and caused sheep to lose weight [175].
In the literature, among the natural substances, there are more than 350
reports about the use of avermectins (see above) in mange control, too
many for a complete survey (for the most recent ones, both in human and
veterinary medicine, see i.e. [176-179]). Despite the large use of these
microbial derivatives, no cases of resistant mites have been reported till
now. Our research group has evaluated various natural substances for the
control of sarcoptic mange. In 1994 we tested in vitro the essential oil of
the lamiaceous plant Lavandula angustifolia Miller (composition
reported in the paper) and of some of its main constituents (linalool,
linalyl acetate and camphor), against the adult stage of Psoroptes cuniculi
[180]. All the tested substances were placed in 6 cm airtight petri dishes
and resulted toxic for the mites, that were immobilized within 15 min to 1
h. Linalyl acetate and camphor were active only at the highest doses (6
|il/dish), with 96.7% and 30.0% mortality, respectively. The essential oil
showed 98.3% mortality at 0.50 |Lil (100% at 2 |il), while linalool was the
most effective compound (96.7% at 0.25 îl, 100% at 0.50 îl). In further
research on the essential oil of Z. angustifolia and its constituent linalool,
we evaluated the activity of this essential oil on the same parasite using
inhalation, rather than direct contact between mites and compounds
[181]. The mites were placed into 6 cm petri dishes covered with filter
paper, that enabled gas-exchange, and inserted then into 9 cm petri dishes
containing the test substance. The essential oil caused a mortality
significantly higher than the controls in the range 2.5-6.0 |il (59-100%,
respectively), while linalool showed a similar action down to 0.7 jil. To
verify if linalool was the only effective constituent, we prepared an
412
artificial mixture, in physiological saline, composed of 27% linalool, the

percentage found in the essential oil, and tested in the same way. From
the dose/mortality curves, linalool was the most active compound, while
the mixture was the least active. We obtained the ED50 and ED90 values
reported in Table (1).
Table 1. Acaricidal activity of essential oil, linalool and artificial mixture from Lavandula angustifolia on
Psoroptes cuniculL
Compounds EDsodil) ED9o(lll)
Linalool 1.633 3.505
Essential oil 2.387 3.350
Mixture 5.418 10.558
The static headspace analysis of the essential oil, showed that linalool
was the most volatile constituent, and indicated that this substance could
easily reach the mites in this kind of experiment. At higher dosages, the
miticidal activity of the essential oil cannot be ascribed only to linalool, in
fact the oil was more active than the artificial mixture. Moreover, the
computed ED50 and ED90 did not overlap, giving a clear indication that
these substances really had different activities, and demonstrating that
linalool was not the only active compound in the oil. We also submitted
to GC analyses the solution obtained by crushing the dead mites in Et20
after the bioassays with the essential oil. This examination revealed only
linalool, so it can be stated that the miticidal activity by inhalation of the
essential oil of L. angustifolia is mainly due to its linalool content.
Linalool was one of the most active compounds, so we have investigated
it effectiveness for the topical treatment of parasitic otitis caused by P.
cuniculi in the rabbit and the goat [182]. The naturally infested animals
were treated with 2.5 ml of three different linalool concentrations (10%,
5% and 3%) in a mixture composed of vaseline oil (2%) and physiological
saline (98%). Both rabbits and goats, treated with the solution containing
5% linalool, recovered completely; no animal presented signs related to
any toxicological effect of linalool, but in some rabbits, treated with higher
concentrations, a transitory erythema of the ear skin was evidenced. The
therapeutic efficacy of linalool was similar to the drugs commonly
413
employed for the therapy of ear mange (topical application of 2.5 ml of

Acacerulen®, or systemic administration of 200 |ig/kg of Ivomec®), with
the exception of a goat treated with Neguvon®, which remained positive
even though the number of treatments (six) was much higher than that
advised by the manufacturer (two). The same goat, treated later with 5%
linalool, completely recovered. Previously, we have already observed the
efficacy of linalool against rabbit psoroptic mange, but we also evaluated
the presence of linalool residues in euthanized animals 24, 72 hours, 5,10
and 21 days after treatment. An analysis for residues in the skin, adipose
tissue, skeletal muscles, liver, kidney, lung and in milk of pregnant
females was also carried out. Significant amounts of linalool were found
only in the skin and in the adipose tissue of animals sacrificied within 72
hours after the treatment. However, these residues were below the toxic
dose of linalool, so it should not represent a hazard to human health if the
meat of these rabbits was used as food [183]. In our opinion, a
comparative study of the activity of each compound, even if it does not
permit assessment of the potential synergy and antagonism among the
components of an essential oil, could enable a determination of the
necessary structures for their pharmacological action; this information
should also allow the prediction of the biological activity of other
structurally related chemicals, and the assessment of their possible modes
of action. For this reason, we have also performed a study about the
structure/activity relationships of some natural monoterpenes against P.
cuniculi, both in direct contact and vapour diffusion assays [184]. In the
former tests, all the hydrocarbons, either acyclic or cyclic (limonene,
myrcene and y-terpinene), did not show any miticidal activity at all the
doses tested (0.125-1% in physiological saline). Thus the double bond
position and/or number seems to be unimportant for this kind of activity.
In contrast, the terpene alcohols (linalool, geraniol, nerol, menthol, 4-
terpineol and a-terpineol) were able to kill nearly 100% of mites at the
doses tested. Therefore, it appears that oxygenated functional groups
potentiate the acaricidal properties among these compounds. Neither the
acyclic nor the cyclic nature of the compound appeared to influence
miticidal activity. Similarly, neither the site of linkage (to the ring or to a
side-chain), nor the nature of the hydroxyl group (primary, secondary or
tertiary), influenced the activity. The cis/trans isomerism (nerol and
geraniol) also appeared unimportant. Thymol and eugenol killed nearly
414
100% of the mites at all dosages used, indicating that a phenolic function
can enhance the miticidal properties of terpenes. The low susceptibility
of parasites to linalyl acetate, particularly at the lowest doses, could be
related to the esterification of the oxygenated function. Estragole,
structurally close to eugenol, but with a methylated phenolic group
exhibited, at 1% concentration, an activity comparable with the same dose
of eugenol, but at 0.25% this miticidal action decreased (63%) and fell to
zero at 0.125%. These results indicate that the best miticidal activity, in
direct contact tests, can be related to compounds with free alcoholic or
phenolic groups. In vapour diffusion tests, at 6 |Lil, the results were
comparable to the direct contact tests. Thus, while hydrocarbons were
ineffective, alcohols and phenols maintained almost 100% toxicity.
Lowering the dose to 3 |Lil, all the alcohols preserved nearly the same
acaricidal activity, except nerol (83.3%) and 4-terpineol (41.7%). At 1 |Ltl
geraniol, menthol and thymol maintained about 100% effectiveness,
whereas the activity of linalool, eugenol, a-terpineol and nerol was
diminished. Linalyl acetate and estragole, like hydrocarbons, were
partially or completely ineffective at all doses tested. We have evaluated
also the activity, in vitro and in vivo, against eggs, larvae, nymphs and
adults of P. cuniculi of the essential oil (10, 5, 2 and 1%) and two water
extracts (20% and 7.5%) oiArtemisia verlotorum (Asteraceae) [185]. The
in vitro studies indicated that the essential oil was highly effective against
this mite; it killed 100% of larvae, nymphs and adults at all
concentrations and inhibited 100% egg hatching at concentrations of 10, 5
and 2% and 95% mortality was registerd at 1%. Both the aqueous
extracts killed 100% of larvae, nymphs and adults, and the 20%
concentration strongly inhibited (94%) egg hatching. The in vivo efficacy
was evaluated for the oil diluted at 5% and the water extract at 20%. The
compounds were applied directly to the infected ears (2.5 ml). The
treatment with the essential oil resulted in a clinical and parasitological
recovery in all the treated rabbits: neither clinical symptoms nor mites in
ear cerumen were found in these rabbits seven days after the treatment.
This was not the case for the aqueous extract, which completely cured
only one of the treated rabbits; in the other ones clinical lesions
disappeared, but eggs and mites were still present in the ears. Many other
herbs or pure compounds have been tested against manges. Among them
the most studied are probably the extracts of Azadirachta indica. In
415
literature two contrasting studies are present: Dakshinkar et al. [186]

reported a positive response against eggs, nymphs and adults of P.
cuniculi, while O^Brian et al. [187] noticed a failure in the complete
elimination of P. ovis on sheep. Other papers referred to positive effects,
ranging from moderate to very promising, against Sarcoptes scabiei both
on human and animal patients [188-190]. The same researchers evaluated
also the activity of the tea tree {Melaleuca alternifolia, Myrtaceae) oil,
which was found to give far better results than neem. Another promising
extract against P, cuniculi seems to be the one obtained from garlic. Allium
sativum (Liliaceae). Two different papers reported its effectiveness, both
against adults or nymphs and eggs [186,191]. Dakshinkar et al. [186] also
reported the efficacy of Annona squamosa extract. An emulsion obtained
from tobacco leaves (Nicotiana tabacum, Solanaceae) and other synthetic
ingredients gave protection for 29 days, besides Sarcoptes and Psoroptes,
also against Demodex mites [154]. When used alone, tobacco was less
effective than synthetic drugs [192].
During our research, we have investigated the effect in vivo of Thymus
vulgaris essential oil (composition reported) in a group of budgerigars
(Melopsittacus undulatus) with a natural infestation caused by
Knemidocoptes pilae [193]. The animals were topically treated with 10%
and 5% essential oil (diluted in DMSO) on beak, vent, legs, wings and
aroimd the eyes. The results were compared with ivermectin and DMSO-
treated controls. The clinical and parasitological recovery was observed
only in the birds treated with 10% and 5% thyme essential oil and
ivermectin. The two concentrations showed the same effectiveness, but at
10% the essential oil caused death in two animals, while in birds treated
with 5% solution no adverse reaction was observed. No other
investigation with natural compounds (apart from avermectins) against
Knemidocoptes is present in literature.
In India, a comparative study between the essential oil of Cedrus
deodar a (Pinaceae) and benzyl benzoate have been performed [194]
against sarcoptic mange in sheep. The drugs were topically applied (doses
not given) and the essential oil, of unstated composition, was the most
effective, producing a complete recovery of the treated animals after the
fifth application, while treatment with benzyl benzoate gave only a
partial recovery; the essential oil gave also better results in haematological
responses. The general conditions of the animals improved after the first
416
application, and the itching and rubbing disappeared after the third one.
Benzyl benzoate is one of the most studied pure natural derivatives in
mange control, both in man and animals, but it can be unpleasant to use
because of its unpleasant smell, can cause itching, buming and stinging
[195-197]. Furthermore, in literature many conflicting reports may be
found about its real effectiveness. Positive results were described on
humans infested by Sarcopies scabiei [190,198], against Psoroptes and
Sarcopies mites in rabbits [199] and against Demodex folUculorum in man
[197]. In contrast, failures or incomplete recovery were obtained against
sarcoptic mites on pigs [200], Psoropies in rabbits [201] and Demodex in
dogs [202]. In Rwanda, scabies is the most important problem in parasitic
dermatology; in order to find new anti-scabies agents, Heyndrickx et al.
[203] tested a series of 15 plants used in the Rwandese traditional
medicine to treat this disease. The plants were extracted in a percolator
with EtOH and assayed at 30 mg/ml. The 100% active extracts were
assayed at lO-l, 10"^, 10"^ and 10'"^ mg/ml. The hexane, CHCI3, water and
EtOH extracts of the active plants were also bioassayed. Out of 15 plants
tested, four showed a 100% Psoropies mortality: Heieromorpha irifoliaia
leaves (Apiaceae), Neorauienenia miiis roots (Fabaceae), Penias
longiflora roots (Rubiaceae), and Psorospermum febrifugum roots
(Guttiferae). The EtOH extracts of A^. mitis and P. longiflora showed the
greatest activity, killing the mites up to 10"^ and 10"^ mg/ml, respectively.
Also the hexane and CHCI3 extracts of these two species showed good
acaricidal activity: both the extracts were effective up to 10'^ mg/ml in the
case of A^. miiis, and up to 10"^ mg/ml in the case of P. longiflora.
Several commercial herbal preparations have been tested, mainly in
India, against mange mites. Among these we can found Himax (M/s Indian
Herbs, Saharanpur), containing Cedrus deodara, Polyalihia longifolia and
P. excessa. This preparation always showed good acaricidal action against
psoroptic, sarcoptic and demodectic mites on dogs, goats, sheep, rabbits
[191,204-206]. Charmil, another herbal preparation (Dabur Ayurvet Ltd.,
India), containing extracts of Cedrus deodara and Pongamia pinnaia,
showed even better results against Sarcopies, Psoropies and Noioedris
mites on buffaloes, cattle, pigs, dromedaries, dogs and rabbits [207-212].
Other very effective commercial herbal preparations are Ectozee and
Pestoban (containing extracts of Cedrus deodara, Azadirachia indica,
Emhelia ribes) and AV/EPP/14 {Acorus calamus, Azadirachia indica,
417
Pongamia pinnata, Cedrus deodara. Eucalyptus globulus). They have

been tested against various mange mites associated with different hosts
(i.e. see [213-217]).
In comparison, for the genera Otodectes and Chorioptes, with the
exception of avermectins, no natural compound has been evaluated.
c) House Dust Mites
The term "house dust mites" is applied to a large number of mites found
in association with dust in dwellings. Unlike some other kinds of mites,
house dust mites are not parasites of living plants, animals, or humans.
House dust mites primarily live on dead skin cells regularly shed by
humans and their animal pets. Skin cells and squames, commonly called
dandruff, are often concentrated in parlour and sitting room, mattresses,
frequently used furniture and associated carpeted areas, and may harbour
large numbers of these microscopic mites. For most people, house dust
mites are not harmful. The medical significance of house dust mites arises
because their microscopic moulted skins and faeces being a major
constituent of house dust, induces allergic reactions in some individuals.
For those individuals, inhaling the house dust allergen triggers rhinitis or
bronchial asthma. Expert panel reports and position statements from the
European Union, the US National Heart, Lung and Blood Institute
(NHLBI), and the American Academy of Allergy, Asthma and
Immunology (AAAAI) have recommended dust mite allergen avoidance
as an integral part of asthma management [218-221]. House dust mites
belong to different genera and species, the main ones are
Dermatophagoides pteronyssinus, D. farinae and Euroglyphus maynei
(Acari: Pyroglyphidae). However, there is a great variation in the acarid
fauna among the different regions of the world. The diversity of mite
fauna in a given (micro)habitat is not only due to the direct influence of
environmental temperature and humidity upon the mite development and
survival, but ecological and evolutionary factors may also play a role in
mite diversity. The term 'house dust mites' is used originally to refer to
those mites belonging to Pyroglyphidae. At present, the term 'dust mites'
is more widely used, and this is in reference to all pyroglyphid and non-
pyroglyphidites that are implicated in dust-borne respiratory allergy.
Dermatophagoides pteronyssinus (literally "skin-eating mites") is
considered as the true house dust mite and has a cosmopolitan
418
distribution. Together with D. farinae (=flour, also infests stored food), it

accounts for 80-90% of the total mite population generally found in
houses. No pesticides are currently labeled for house dust mites.
However, some commercial products are available for treatment of house
dust mites and their allergens. The active ingredients are benzyl benzoate
and tannic acid. Benzoic acid esters, such as benzyl benzoate, are very
effective acaricides in both laboratory and field evaluations. Health risks
appear to be slight as benzoates are rapidly metabolized in the body to
hippuric acid, which is excreted in the urine. Most of the studies
evaluated the effectiveness of these compounds on house dust mites, and
all the authors agree about their effectiveness and safety, both in
laboratory tests and homes, i.e.: [222-226].
Other studies concerned the activity of some plant essential oils.
Among these, Chang et al. investigated the antimite activity of the
essential oil and their constituents obtained from the heartwood of
Taiwania cryptomerioides (Cupressaceae) against Dermatophagoides
pteronyssinus and D. farinae [227]. The tests were performed in resin
plates, using Et20 solutions containing various concentrations of the
essential oil or pure components; the mites were introduced after air
drying. The results showed that, at either high (12.6 [xg/cm^) or low
concentration (6.3 jiig/cm^), the activity in decreasing order was a-cadinol
(100%), T-muurolol (100% and 80% on D. pteronyssinus, 83.3% and
56.7% on D, farinae), ferruginol (80% and 56.7%, 68.1% and 36.7%,
respectively), and T-cadinol (70.0% and 4.7%, 20.4% and 14.1%,
respectively). The mortalities due to the treatments with 12.6 [ig/cm^ of
the whole essential oil were 67% and 36.7% on D, pteronyssinus and D.
farinae, respectively. Authors suggested some structure-activity
relationships, in particular an equatorial OH at C-9 (a-cadinol) seems to
be an important factor for antimite activity. In contrast, the type of ring
junction (C-5/C-10) was less important: whether in cis configuration (T-
cadinol) or trans (T-muurolol) with axial C-9 OH, Fig. (5), their antimite
activities were lower than a-cadinol.
Yatagai et al. studied the essential oils of the leaves of six Melaleuca
species (Myrtaceae), a well-known insect repellent plant [228]. The oil
obtained from M hracteata exhibited the strongest activity against D.
pteronyssinus, killing all mites after 24 hours at the two doses tested (0.13
and 1.28 jig/cm^). M argentea, M. dealbata and M saligna showed mild
419
activities, with mortalities greater than 50% at the lower dose after three
days. Although the essential oils of M symphyocarpa and M acacoides at
the higher dose killed all mites after three days, the mortalities at 1/10 of
that dose were only 13% and 0%, respectively. Other Japanese
researchers tested the activity of the essential oils obtained from the
leaves of Lauraceae trees {Cinnamomum camphora, C. japonicum, Persia
thunbergii, Actinodaphne lancifolia, Neolitsea sericea and Under a
umbellata) [229]. A^. sericea oil showed the greatest activity against both
D. pteronyssinus and D. farinae. The major active constituents were a-
cadinal, caryophyllene oxide and the rare furane sesquiterpene
isosericenine Fig. (5). D. farinae resulted more susceptible than D.
pteronyssinus.
a-cadinol T-cadinol
COOMe
T-muurolol isosericenine
Fig. (5). Sesquiterpenes from Taiwania cryptomerioides and Neolitsea sericea
House dust mites were of interest also for our research group. In
particular, we have evaluated the activity of the essential oils of four
plants, Lavandula angustifolia, L stoechas, Mentha x piperita
(Lamiaceae) and Eucalyptus globulus (Myrtaceae), against a mite of stored
food, Tyrophagus longior (Acari: Acaridae) [230,231]. We have analyzed
by GC-MS all the essential oils and applied two different methods to test
the activity of these compounds: one by direct contact and the other by
vapour diffusion. In the direct contact assays five different quantities of
420
each undiluted substance (6, 2, 1, 0.5 |Lil) were spread on the internal
surface of petri dishes. The activity by inhalation was tested using two
petri dishes of different sizes: the smaller one, containing the mites, was
covered with a filter-paper disk and enclosed in a bigger dish containing 6
or 2 |il of each undiluted substance. At the highest doses, the essential oils
of the two lavender species and of peppermint killed 100% of the mites,
both by direct contact and inhalation. Eucalyptus oil was the least active.
We have also tested the activity of the main constituents of the above
essential oils, specifically linalool (27.3% in L angustifolid), linalyl
acetate (32.1% in L. angustifolia), camphor (10.0% in L stoechas),
fenchone {A12% in L. stoechas), 1,8-cineole (76.0% in E, globulus),
menthone and menthol (31.8% and 21.7%, resp., in M. piperita). Among
these compounds, menthol showed the highest activity, killing 100% of
the mites at the lowest dose (0.25 |il) by direct contact and at 6 |il by
inhalation. Linalool, fenchone and menthone showed good acaricidal
activity (100% mite deaths at 2.0, 1.0, 0.5 |LI1 in direct contact and 6.0 |Lil
each by inhalation). 1,8-cineole was the least effective compound, killing
81.7% mites at 6.0 |Lil and 50% at 6.0 |LI1 in direct contact and inhalation
tests, respectively. At present, we are evaluating the acaricidal activity of
the essential oils and main pure components of the branches of four Pinus
species against another pest of stored food, Tyrophagus putrescentiae
(Acari: Acaridae) [301]. The species used in this study were P, pine a, P.
halepensiSy P. pinaster and P. nigra, their essential oils being characterized
by GC-MS. The acaricidal tests were performed against mites isolated
from samples of seasoned Parma ham, avoiding direct contact of the
substances with the mites, but evaluating only their volatile fractions.
Each compound was tested at 8 and 6 |LI1. All the essential oils, with the
exception of P. nigra, had a good acaricidal activity. P. pinea oil was the
most effective one, killing 100% mites at 8 |LI1 and 20% at 6 |LI1. P.
halepensis and P. pinaster oils killed 60% and 53% mites at 8 |Lil,
respectively, while at 6 |il they were ineffective. P. nigra oil was
completely ineffective, a-pinene, p-pinene, myrcene, limonene, 1,8-
cineole, P-caryophyllene and germacrene D, the main constituents of
these oils, were assayed in the same way. 1,8-cineole was the most active
compound, killing 100% mites at 8 and 6 |il, followed by limonene which
killed 100% mites at 8 |il and 32% at 6 |xl. All the other compounds were
completely ineffective. Thirteen terpenes were tested by Sanchez-Ramos
421
and Castanera against Tyrophagus putrescentiae [232]. The authors used

myrtanol, pulegone, pinene, valencene, 1,8-cineole, linalool, linalyl
acetate, fenchone, menthone, a-terpinene and y-terpinene, and unspecified
isomers of caryophyllene and terpineol (valencene and caryophyllene
were incorrectly considered monoterpenes). The miticidal assays were
performed in cylindrical plastic cages by inhalation. Seven monoterpenes
showed a high acaricidal activity, in particular pulegone, 1,8-cineole,
linalool, fenchone, menthone, a-terpinene and y-terpinene. Their LC50
(vapour concentration, |il/l) were 3.7, 14.9, 7.0, 9.0, 4.7, 32.3 and 33.2,
respectively. Other authors have concerned that the differences in our
results [230,231] may either be due to the smaller size of T. putrescentiae
in relation to T. longior and/or different specificity of the components on
these two species. Interestingly, larvae and males of T. putrescentiae had a
significantly higher mortality, about 2-fold, compared to females when
exposed to the same dose of monoterpenes. All the compounds were
ineffective on eggs. Essential oils can be also used as laundry additives for
killing house dust mites [224,233]. In fact, both bedding and clothing may
contain high populations of these mites and their allergens. Authors noted
that washing in warm water removes most accumulated allergens but has
little effect on mites, and effective long-term control requires killing of the
mites. Higher water temperatures cannot always be used, so miticidal
additives are required. Low concentrations of five essential oils have been
evaluated: citronella, eucalyptus, spearmint, tea tree and wintergreen oils.
They were mixed 4:1 with Tween 20 as dispersant and tested at 0.8%. All
the oils killed more than 80% of mites after 30 mins, and with the
exception of citronella, they killed more than 60% of mites after 10 mins.
For shorter exposure times, tea tree oil was the most effective, killing 79%
mites in 10 mins.
Recently, French researchers extracted the bark of Uvaria pauci-
ovulata (Annonaceae) with EtOH and with CH2CI2 and assayed the
extracts on D. pteronyssinus [226]. The alcohol extract was weakly
effective, killing less than 50% mites at 1.67 g/m^, while the CH2CI2
extract, at the same dose, killed 100% mites. Bioassay-guided
fractionation of the non-polar extract led to the isolation of two active
compounds, benzyl benzoate and a bis-tetrahydrofuran acetogenin,
squamocin, Fig. (6). The EC50 were 0.33 and 0.06 g/m^ for benzyl
benzoate after 1 and 24 hours, respectively, while for squamocin they
422
were 2.7 and 0.6 g/m^, respectively. From the bark of Neolitsea sericea
(Lauraceae) two acaricidal lanostane triterpenes, 24Z-ethylidenelanost-8-
en-3-one and 24-methylenelanost-8-en-3-one, Fig. (6), were isolated and
characterized [234].
Squamocin
R=CH-CI^ 24Z-ethylidenelanost-8-en-3-one
R=CH2 24-methylenelanost-8-en-3-one
Fig. (6). Structures of squamocin from Uvaria pauci-ovulata and lanostanes from Neolitsea sericea
When mites were exposed to 16 [ig/cm^ of 12 and 13 for 72 hours, the

percentages of immobilized mites were 17.8 and 31.4%, respectively, and
26.9 and 31.4% when exposed to 32 iLig/cm^. Authors affirmed that these
data suggested that triterpenes having a 24-methylene group in the side
chain were more powerful miticides than compounds having the 24Z-
ethylidene moiety. Since Nathanson demonstrated that caffeine and other
methylxanthines interfere with insect feeding and reproduction [235],
American researchers have investigated its acaricidal effect on D.
pteronyssinus cultivated in vitro and its main allergen levels [236]. There
was a significant inhibition in the growth of treated cultures and in the
production of allergens. However additional studies are necessary to
determine the safety of caffeine on humans and animals.
Other effective substances for control of house dust mites seem to be
fungicides because fungal digestion of skin scales is a prerequisite for mite
utilization [237]. Finally, an alternative to organophosphorus pesticide
treatment in stored grain has been found in the use of inert substances,
423
susch as diatomaceous earths [238,239]. These are dusts composed of

Si02 from fossihzed diatoms. They act by absorbing waxy elements of
parasite cuticle, causing desiccation. Diatomaceous earths have a very low^
mammalian toxicity and leave no chemical residues. These authors
demonstrated the effectiveness of three different diatomaceous earths and
an amorphous precipitated silica against two storage mites, Acarus siro
and Lepidoglyphus destructor. Field treatments did not exceed 5 g/kg, and
nearly 99% of any dust applied to the grain was removed during normal
milling processes for flour production.
AGRICULTURE
Plant-feeding mites play important roles as agricultural pests of timber,
fruits, vegetables, forage crops, and ornamentals. In many instances, lack
of information about the correct identity of mites, as well as inadequate
knowledge regarding their biology and ecology, have hampered our ability
to effectively combat these mite pests. Their small size and cryptic
appearance make mites difficult to detect, and thus infestations are often
overlooked. Once established in a new area, certain biological
characteristics allow rapid escalation to pest status. These include high
egg production, various modes of reproduction (parthenogenesis,
paedogenesis, and sexual), short life cycles, a myriad of dispersal
techniques, and adaptability to diverse ecological conditions. These traits,
combined with an exponential increase in world trade, have set the stage
for potentially devastating situations that may threaten the sustainability
of the world's agroecosy stems. Miticidal compounds, as in veterinary and
human medicine, cannot be toxic for the plant host and no harmful
residues must be found in foods: Furthermore, in agriculture an additional
feature is requested: they must be devoid of undesirable effects on useful
non-target organisms, like pollinators and predator arthropods [240-242].
There are several different species of mites that can cause damage to a
wide variety of plants. The main species are Tetranychus sp.,
Oligonychus sp. (Acari: Tetranychidae), Phyllocoptruta oleivora,
Tegolophus australis (Acari: Eriophyidae). Among these, the two-
spotted spider mite, Tetranychus urticae, a polyphagous pest, is
probably one of the most dangerous for crops and ornamentals,
particularly in glasshouse. Its high reproductive capacity enables it to
cause serious damage in a short period of time. Furthermore, this parasite
424
has developed resistance to many synthetic acaricides [see i.e.: 243-246],

apart from the fact that many of these substances are toxic to useful non-
target arthropods [see i.e.: 247-249].
Numerous papers about its control by mean of natural substances are
present in literature. Serbian investigators prepared the 1:1 EtOH fluid
extracts of the aerial parts of Taraxacum officinale (Asteraceae), flowers
of Sambucus nigra (Caprifoliaceae) and leaves of Juglans regia
(Juglandaceae) [250]. They assayed these extracts and their 50, 10 and
2% dilutions against Tetranychus urticae isolated from Lamium
purpureum plants. Taraxacum was the most active extract, killing 100%
of the mites at 50% and about 90% at 10% dilution; at 2% it killed 57%
of the mites. Sambucus showed a very similar effectiveness, with 96.4%
mites killed at 50% and 91% at 10%, but at 2% only 31.7% of the mites
were killed. Juglans extract was less active, killing 100% of the mites only
at 100% concentration, and only 73% at 10%. Hiremath et al. compared
the activity of MeOH extracts obtained from 21 different African plant
species against adults of T. urticae using the leaf-dipping method [251].
The most active ones were the extracts from the whole plant of Celosia
trigyna (Amaranthaceae) and Combretum micranthum (Combretaceae),
leaves of Combretum glutinosum, and leaves and fruits of Prosopis
chilensis (Fabaceae). The insecticidal properties of Meliaceae plants have
been known for a quite long time, so Ismail evaluated the relative toxicity
of Melia azedarach extracts and some synthetic acaricides against newly
hatched larvae of T. urticae and third-instar larvae of an useful arthropod,
its predator Stethorus gilvifrons [252]. The methanol extract of the plant
was the most effective among the tested extracts, followed by acetone and
petroleum ether extracts, respectively. The toxicity of plant materials was
far less against the predator compared with two-spotted spider mite,
whereas a synthetic acaricide was equally toxic to the pest and its
predator. The study of the joint action revealed a strong synergism in the
mixture of bromopropylate with the methanolic extract of M azedarach;
interestingly, this mixture showed no effect on the predator. Melia
azedarach extracts also greatly affected the fecundity of the parasite,
especially when mixed with synthetic acaricides. The author suggested
that M azedarach extracts could be used in integrated pest management
programs for mite control. The crude alkaloids, the EtOH extract and the
oil of the bulb of the omamental plant Pancratium maritimum, a member
425
of the Amaryllidaceae family with strongly scented, white, narcissus-like

flowers, was active against T. urticae. Their LC50 values were 0.2, 0.36
and 1.5%, respectively [253]. The lipophilic fraction of seeds, leaves and
roots of Glossostemon bruguieri (Sterculiaceae), specifically the
unsaponifiable part, was tested against T, urticae [254]. The leaves were
the most toxic plant part to both adult and egg stages of T. urticae, with
LC50 values against eggs of 1.7 mg/ml. At 1.25 mg/ml oviposition was
totally inhibited. Artemisia absinthium (Asteraceae) is a well-known
insecticide, and its water extract is used worldwide against aphids;
however, the same extract showed very weak acaricidal activity [255].
The same was true for the water extract of Pinus sylvestris. When mixed
1:1, a clear synergistic interaction between absinth and pine shoot extracts
was detected with the leaf dipping method against T. urticae, with 77.5%
and 92.3% mortality on immature and adult stages, respectively.
Synergism was also evidenced by mixing synthetic acaricides with jojoba
{Simmondsia chinensis, Buxaceae) seed oil [256]. Thus, it was possible to
reduce the dose of acaricides used for control of the two-spotted spider
mite Tetranychus arabicus.
Some correlations have been postulated about the natural concentration
of foliar essential oils in six strawberry (Fragaria sp., Rosaceae) cultivars
and their different degree of susceptibility towards T. urticae [257]. On
the basis of leaf damage, the cultivars were classified as highly
susceptible, intermediate to susceptible, intermediate to resistant and
resistant. It was observed that the first two classes had a lower linalool,
a-terpineol and p-cyclocitral content. Resistant plants could be used to
obtain effective acaricidal compounds, as demonstrated by Amer and
Rasmy [258]: when larvae of T. urticae were reared on excised leaves of
Conyza dioscoridis (Asteraceae) or Carina indica (Cannaceae), they did
not develop to the protonymphal stage, whereas when reared on
Trigonella foenum-graecum (Fabaceae) or Brassica rapa (Brassicaceae)
developed to the adult stage in a significantly longer period and the
resulting females laid fewer eggs compared with controls. Crude extracts
of C. dioscoridis and T. foenum-graecum leaves showed a remarkable
toxic effect on adults and eggs, while extracts of C. indica and B, rapa
showed less intense ovicidal action.
Many essential oils and their pure constituents have been tested
against Tetranychus mites. Egyptian authors tested Thymus vulgaris oil
426
and pure thymol against T. urticae and both compounds were found
effective [259]. Thymol was more potent than thyme oil as a deterrent
factor for reducing egg laying by the mite. Mortality percentage reached
100% with both materials used, however, at lower concentrations, the
effect was more pronounced with thymol than thyme oil. A very
interesting paper deals about the insecticidal and acaricidal activities of
many monoterpenes and their possible phytotoxicity on host plants
[260]. Twenty-nine compounds, belonging to different chemical classes,
were assayed against T. urticae by mean of the leaf-dip method. In
particular, the alcohols carveol, carvomenthenol, citronellol, geraniol, 10-
hydroxygeraniol, isopulegol, linalool, /-menthol, perillyl alcohol, a-
terpineol and verbenol, the phenols carvacrol, eugenol and thymol, the
ketones t/-carvone, /-carvone, /-fenchone, menthone, pulegone, thujone
and verbenone, the aldehydes citral and citronellal, the acid citronellic
acid, the ether 1,8-cineole and the hydrocarbons limonene, a-terpinene
and y-terpinene were used. All the compounds were tested, in water with
Triton X-100 as wetting agent, at 10000 and 1000 ppm, and the activity
was assessed 24, 48 and 72 hours after treatment. The toxicity differed
depending on the concentrations and the exposure times. The
monoterpenes tested, except for 1,8-cineole, 10-hydroxygeraniol, a-
terpineol, verbenol and verbenone, caused 100% mortality at the highest
concentration after 24 hours. Carvacrol was the most effective at the
lowest concentrations, followed by citronellol. Geraniol produced 100%
mortality, whereas its analog 10-hydroxy geraniol showed 0% mortality.
Longer exposure time increased acaricidal effects. The most effective
monoterpenoids (carvacrol, carvomenthenol, carvone, citronellol, eugenol,
geraniol, perillyl alcohol, 4-terpineol, thymol) were evaluated in more
detailed tests. Of these, carvomenthenol and 4-terpineol showed greater
acaricidal activity (LC5o= 59 and 96 ppm, respectively) than others.
Furthermore, authors examined the phytotoxicity of some compounds to
both com roots and leaves 3 and 10 days after treatments, /-carvone was
the most phytotoxic compound, while pulegone was the safest. A table
with detailed data is reported in the paper. Two species known for a long
time as potential pesticides, particularly as insecticides and insect
repellents, have also been investigated as acaricides against T. urticae
[261]. The essential oils of Artemisia absinthium and Tanacetum vulgare
(Asteraceae) were obtained from whole cultivated plants harvested in fiiU
427
bloom by three different methods of extraction: a microwave assisted

process (MAP), distillation in water (DW) and direct steam distillation
(DSD), and their relative toxicity assayed by direct contact. All the oils
were tested at 1, 2, 4 and 8% as emulsions prepared in water containing
9% of denatured EtOH and 0.32% of Alkamul EL-620 as emulsifier, and
mite mortality was assessed after 48 hours. All three oils of ^. absinthium
were lethal to T. urticae, however there were differences in the degree of
toxicity, depending on the extraction methods, as reported in a table in the
paper. For example, at 4%, oil extracted by the MAP and the DW
methods caused 52.7 and 51.1% mite mortality, whereas oil obtained by
DSD resulted in 83.2% mortality. Consequently, the LC50 of the oil
extracted by DSD was lower (0.043 mg/cm^) than those obtained by
MAP (0.134 mg/cm2) and by DW (0.130 mg/cm2). The extracts of T.
vulgare obtained by DW and DSD showed greater acaricidal activity than
the extract prepared by MAP. At 4% concentration they showed 60.4,
75.6 and 16.7% mortality, respectively. Chemical analysis of the T.
vulgare extracts indicated that p-thujone is by far the major compound of
the oil (>87.6%), and probably contributes significantly to the acaricidal
activity of the oil. The paper demonstrated the importance of a further
factor affecting the variability in the effectiveness of extracts obtained
from the same species, that is the technique used for the extraction. Once
again, I would like to underline the significance of knowing the
composition of the essential oils used in the tests; unfortunately, in the
case of the three absinth oils, authors were not able to identify its major
constituent; moreover, another sesquiterpene, present in the DSD oil and
absent in the other two, that could be responsible of the greater toxicity
of the former oil, was also unidentified. The toxicity of vapours of the
essential oils obtained from four plant species, seeds of Cuminum
cyminum and Pimpinella anisum (Apiaceae), leaves of Origanum
syriacum var. bevanii (Lamiaceae) and fruits of Eucalyptus camaldulensis
(Myrtaceae) against another species of the same genus, the carmine spider
mite, T. cinnabarinus, was investigated in Turkey [262]. This mite is a
major greenhouse pest in this country and throughout the world. It
attacks a large range of 100 cultivated crops and weeds. It is a serious
pest on beans, eggplant, pepper, tomatoes, cucurbits, and many other
vegetables. It is also a pest of papaya, passion fruit, and many other
fruits. The carmine spider mite also attacks many flowers and ornamental
428
plants such as carnation, chrysanthemum, cymbidium, gladiolus,

marigold, pikake, and rose. The oils were assayed at 0.25, 0.50, 1.00 and
2.00 |il/l of air. The phytotoxicity of the vapours of these essential oils
was estimated by exposing tomato, bean and cucumber seedlings to the
highest dose for 96 hours. All essential oils, except that of eucalyptus,
caused 100% mortality of the carmine spider mite at or below the
maximum dose after 2-3 days of exposure. When the essential oils were
compared on the basis of their LT50 and LT99 values, the order of
toxicity was oregano>cumin>anise>eucalyptus. Phytotoxicity to
seedlings was manifested as discoloration and eventual drying of the first
two leaves. Cumin and anise oils were toxic to all plants tested, oregano
was toxic only to tomato, whereas eucalyptus to none. Some terpenes,
commonly found in essential oils, are pheromones of some arthropods
species. Pheromones are volatile chemicals used for communication
within individuals of the same species and, occasionally, between
different species (usually the latter are known as allomones) [263]. Two
examples are represented by famesol and nerolidol. Fig. (7), two highly
attractive compounds to Tetranychus mites. These compounds have been
added to common synthetic acaricides to improve their effectiveness
against moving stages of mites [264,265].
nerolidol
CH.OH
famesol
Fig. (7). Nerolidol and famesol, two sesquiterpene pheromones
Also many non-volatile compounds have been found effective against

Tetranychus mites. Among these chemicals, particularly interesting as a
new class of acaricides, are naphthoquinones, in particular two derivatives
isolated from Calceolaria andina (Scrophulariaceae), protected by patent
application, and designated as BTG 505 and BTG 504, Fig, (4)
[171,266,267]. These chemicals were found to exhibit high activity, even
against strains of T, urticae that are most resistant to a wide range of
429
commercial acaricides. Furtheraiore, the levels of activity were low on

many beneficial species, both insects and acari {Phytoseiulus persimilis
and Typhlodromus piri). Naphthoquinones have long been known to
inhibit mitochondrial respiration, however the primary site of action may
vary, depending upon the nature of substituents. Complex III was found
to be the primary site of action for the two 2-hydroxy-l,4-
naphthoquinones isolated from C. andina. Complex III
(ubiquinol:cytochrome c oxidoreductase), is found in mitochondria,
photosynthetic bacteria and other prokaryotes; the general function of the
complex is electron transfer between two mobile redox carriers, ubiquinol
(QH2) and cytochrome c. Electron transfer is coupled with the
translocation of protons across the membrane thus generating an
electrochemical proton potential that can drive ATP synthesis by ATP
synthase. Since the primary loss mechanism of applied naphthoquinones
from leaf surfaces was proved to be volatilization (though some
degradation also occurred leading to a half life of about 20 hours), authors
have shown that different types of formulations (not reported, patent
pending) can increase the efficacy against parasites and reduce
phytotoxicity. Concerning the commercial potential of these products,
the authors observed that naphthoquinones can occur in concentrations
up to 5% w/w in dried aerial parts of C andina, but this herb would be a
poor source for commercial production of the natural products. However,
this genus is amenable to hybridization, and from preliminary studies it
seems that it could be possible to produce more vigorous and high-
yielding cultivars. Furthermore, these compounds can be synthesized in
2-3 steps. Other synthetic (and natural, see below) acaricides, i.e.
tebufenpyrad, pyridaben, fenazaquin, have been shown to be active by
inhibition of another electron transport system of the mitochondrial
respiratory chain, Complex I (NADH:ubiquinone oxidoreductase).
Because of their high activity against various mite species, these METI
(mitochondrial electron transport inhibitors) acaricides are in widespread
use, but some strains of T. urticae from different parts of the world have
been reported to exhibit resistance to these substances. Very recently, a
strain of T. urticae from hops in UK was confirmed to have cross-
resistance to all the METI acaricides, despite having only been exposed to
a single compound. Naphthoquinones inhibit Complex III in the
mitochondrial respiratory chain, a system distinct from the Complex I,
430
but given the unpredictability of cross-resistance patterns and the fact

that metabolic resistance can confer resistance between chemical groups,
it was important to evaluate whether METI resistance also protected
mites against the naphthoquinones [268]. Experiments with METI
acaricide resistant strains and the standard reference susceptible strain,
ascertained that the activity of naphthoquinones against T. urticae
remained uncompromised.
•Mil
Fig. (8). Acetogenins from Annona glabra
From the seeds of another species, Abrus precatorius (Fabaceae), other

classes of compounds have shown promising effects against T. urticae.
From the non-saponified fraction of a crude petroleum ether extract,
coumarin, p-amyrin and a mixture of sterols were isolated and tested
against females and eggs in laboratory conditions. P-amyrin was the most
effective compound against both stages. Spraying females with sub-lethal
doses of p-amyrin caused a significant reduction in fecundity and the
viability of resulting eggs [269]. From the seeds of Annona glabra
(Annonaceae), three acetogenins, squamocin. Fig. (6), desacetyluvaricin
and asimicin, Fig. (8), have been extracted and their toxicity against
insects and mites evaluated; they have shown good insecticidal activity,
but no acaricidal effect against T. urticae [270] while, recently, squamocin
was found effective against the house dust mite Dermatophagoides
pteronyssinus [226].
The genus Pimpinella (Apiaceae) produces rare phenylpropanoids
with an unusual substitution pattern at the phenyl ring: the (l^*)-
propenyl-2-hydroxy-5-methoxybenzene skeleton of these compounds
431
has been named pseudoisoeugenol [271], Fig (9). Also derivatives of (1£)-
propenyl-4-hydroxybenzene have been isolated in some Pimpinella
species. The activity in the contact assay of 100 ppm of eight Pimpinella
phenylpropanoids against the red spider mite, T. telarius, has been
evaluated [272]. Epoxy-anoltiglate was the most effective compound,
killing 100% of the mites, while four other substances, epoxy-
pseudoisoeugenolisobutyrate, epoxy-pseudoisoeugenoltiglate, pseudoiso-
eugenolisobutyrate and isoeugenolisobutyrate showed lesser
effectiveness, killing 80-90% of the mites.
X)
HoCO'
H3CO'
Epoxy-anoltiglate epoxy-pseudoisoeugenolisobutyrate isoeugenolisobutyrate
H3CO" "^^ H3CO"
epoxy-pseudoisoeugenoltiglate pseudoisoeugenolisobutyrate
Fig. (9). Phenylpropanoids from Pimpinella species
The other phenylpropanoids tested, anoltiglate, isoeugenolisobutyrate

and isoeugenol were completely ineffective.
Other uncommon natural derivatives are 2-acylcyclohexane-l,3-diones
or p-triketones, typical of hops {Humulus lupulus, Cannabinaceae). The
p-acids from hop belong to this class of chemicals and are by-products of
hop processing for brewing. The fraction containing these products has
been examined in a choice bioassay for its effect on the feeding behavior
432
of T. urticae [273]. The results showed that both the highest

concentrations of culupulone, Fig. (10), the chiefs-acid component of the
fraction, and the whole p-acid fraction repelled T. urticae and also
affected its survival. The greatest difference between the pure compound
and the crude fraction treatments was seen in the oviposition of the mites:
significantly fewer eggs were found in the whole p-acid fraction. This
suggested that culupulone was not the only active component in the p-
acid fraction. Some secondary metabolites, epitaondiol diacetate,
stypetriol triacetate, epitaondiol monoacetate and epitaondiol. Fig. (10),
isolated from the brown alga Stypopodium flabelliforme (Dictyotaceae)
have been tested on adults of T. urticae [274]. Only epitaondiol showed
little acaricidal activity at 500 ppm (14% mortality), while the other
compounds resulted inactive at 1000 ppm.
epitaondiol
culupulone
ajoene
stypetriol
Fig. (10). Structure of hop and algal metabolites
Ajoene, Fig. (10), an unsaturated sulfoxide disulfide, is the principal

chemical responsible for garlic's anticoagulant properties. It has been also
investigated for its acaricidal activity on T. urticae [275]. Complete
mortality (100%) by ajoene was observed at 0.075% after 14 h of
treatment, a dose comparable with other synthetic acaricides used in the
experiment. At lower concentrations (0.05%), it affected female fecundity
433
and only 31.5% of the juvenile stages. These results suggested that
ajoene, besides having direct acaricidal effect, could also control
resurgence of the pest. Glandular trichomes of wild tomato, Lycopersicon
hirsutum f. glabratum (Solanaceae), yielded two methyl ketones: 2-
tridecanone and 2-undecanone [276]. They are known to cause mortality
in several herbivorous insect species; it seems that this kind of chemicals
could be considered defensive substances against pollen-feeding animals,
as confirmed also by their abundance in wind-pollinated plants [277].
Dutch researchers investigated the effects of these compounds on two
strains of T. urticae, collected from tomato and cucumber crops in
greenhouses [276]. The two ketones were tested separately, in
combination in the ratio found in L hirsutum f glabratum and in several
other ratios to detect any synergistic interaction between them.
Synergistic effects were not detected. They measured both the direct
contact and residual toxicity, as well as the viability of the eggs produced
by ketone-treated females. Both compounds showed LC50 values
comparable to the formulated acaricide amitraz; 2-tridecanone was
slightly more toxic than 2-undecanone, but only against the tomato strain.
In the bioassays for the residual effects, no significant mortality occurred,
however the mites avoided feeding on the treated surface and the eggs
were laid almost exclusively on the untreated area. Furthermore, there was
no significant egg viability for most of the treatments. A new plant
species. Quassia sp. aff. bidwillii (Simaroubaceae) was discovered in
Australia and the MeOH extract of its aerial parts was tested against T,
urticae [278]. Because of its effectiveness, subsequent fractionation by
RP-chromatography gave the pure active quassinoid derivative
chapparinone, which showed a LC5o=47 ppm.
Neem extracts, pure constituents (i.e. azadirachtin) and formulated
products showed positive results against Tetranichus mites [279-283].
Less polar extracts were considerably more toxic than polar ones or cold-
pressed neem oil or commercial neem oil, and reduced the fecundity of the
mites on treated plants and the survival of nymphs hatched from treated
eggs; application of pentane extract or neem oil in sublethal
concentrations, caused growth disrupting effects on the nymphal stages
and ovicidal effects. Quantification of the insecticidal substance
azadirachtin in the extracts revealed that this compound was not the most
active principle against the mites [284].
434
Other promising control agents are microbial metabolites. Abamectin

was found much less toxic to the useful predatory mite Phytoseiulus
persimilis than to the parasite mite T, urticae [285]. A strain of the soil
bacterium Streptomyces platensis yielded three new substances having a
marked acaricidal activity against T. urticae, AB3217 A, B and C, Fig.
(11) [286,287], while another strain of Streptomyces, NKl 1687, produced
gualamycin. Fig. (11), that was able to kill 100% of dicofol-sensitive and
resistant mites (adults and larvae) at 250 |ig/ml [288].
AB3217A:R=H ^ ^^
O 11 I
AB3217 B:R= —c—(CH2)4—C-(CH3)2
(f
AB3217C:R= —C—(CH2)2—CH-(CH3)2
^ H N ^ ^
gualamycin
Fig. (11). Acaricidal agents of bacterial origin
Finally, as altematives to chemical compounds or as part of integrated

pest management programs, predatory arthropods or fungal pathogens
have been used. Among predators, the most used are phytoseiid mites
such as Phytoseiulus persimilis and Neoseiulus fallacis [289-293], while
the fungus Neozygites adjarica was tested as pathogen agent [294,295].
The acaricidal properties of some of the previous compounds were
435
attributed to the interference with mitochondrial electron transport due to

inhibition of Complex III. Some structurally diverse miticides are able to
inhibit Complex I (NADHiubiquinone oxidoreductase), a system distinct
from Complex III. Complex I is the first electron transport complex of the
mitochondrial respiratory chain. It oxidizes NADH and transfers the
electrons via a flavin mononucleotide cofactor and several iron-sulfur
clusters to ubiquinone (Q). So, Complex I contributes to the proton-
motive force that drives ATP synthesis. Besides many synthetic
products, some secondary products from microbial and plant sources
exhibit biological activity against agricultural pests because of their action
on Complex I. Rotenone and piericidin A, Fig. (12), were known for a
long time as high-affinity inhibitors of proton-translocating NADHiQ
oxidoreductase [296]. Rotenone is the most widely used inhibitor of
Complex I because of its high inhibitory potency and commercial
availability. It is the most potent member of the rotenoids, a family of
isoflavonoids extracted from Fabaceae plants. All known natural
rotenoids have been isolated in the thermodynamically stable cis-B/C ring
fusion; synthetic analogues in which B and C-rings planes are almost
coplanar were about 100-fold less active than natural rotenone, indicating
that the bent form is essential for the activity [297]. This conclusion is
supported by the observation that rotenol, in which the whole
conformation is not fixed due to opening of the C-ring, is about 200-fold
less active than rotenone. Another important feature for the activity is the
configuration of the isopropenyl group linked to the E-ring. Cube resin,
the roots extract of Lonchocarpus utilis and L. urucu (Fabaceae), is an
important acaricide. The four principal active constituents are rotenone,
rotenolone, deguelin and tephrosin. Fang and Casida identified further 25
minor rotenoids having variations in the B, D and E-rings, thereby
providing a new and unique set of compounds to elucidate structure-
activity relationships for the activity on Complex I [298].
The rotenone series and the deguelin series, with modifications in the
B, C and D-rings, followed similar overall substituent effects on activity.
In particular, the parent compounds rotenone and deguelin were more
potent than any of their derivatives. Hydroxylation or methoxylation in
the A-D ring system considerably reduced the potency. The trans
isomers were 7-100 fold less active than the corresponding cis isomers.
Authors affirmed that, considering the potency and the amounts, the four
436
major rotenoids accounted for more than 95% and probably almost all of
the biological activity of the cube resin as inhibitor of Complex I. Many
kinds of Streptomyces strains produce piericidin homologues. Piericidin A
is a very potent inhibitor of Complex I. The natural side chain of
piericidin A is not essential for the activity since piericidin B, C and D
analogues, in which the region from C-5 to C-13 differs, exhibit activity as
high as or only slightly less than piericidin A. On the basis of studies
with synthetic analogues, it was concluded that a branched methyl group
at C-3 and unsaturation between C-2 and C-3 are important for potent
activity [297].
OCH OCHo
OCHo OCHo
Rotenone R=H DeguelinR=H
Rotenolone R=OH Tephrosin R=OH
HoC
H3CO'
Piericidin A
HO* Capsaicin
Fig. (12). Complex I inhibitors
Capsaicin, Fig. (12), the pungent principle of Capsicum species

437
(Solanaceae), acts as competitive inhibitor for ubiquinone in Complex I.

Methyl capsaicin is more potent than capsaicin, indicating that the
phenolic OH is not essential for the activity [297]. Other natural
inhibitors of Complex I are annonaceous acetogenins. These compounds
belong to a wide group of natural products isolated from several species
of the Annonaceae family, which include more than 250 molecules with
diverse chemical structures. Among the various classes, it seems that
monotetrahydrofuranic derivatives are less potent than other acetogenins
[296,299].
CONCLUSIONS
The control of parasitic diseases is mainly based on the use of effective
drugs, both in agriculture or human and veterinary medicine; for this
reason the lack of effective drugs often prevents the control of some
parasitic diseases, making them more serious and important. At present,
however, the use of commercial drugs involves many problems that
strongly limit their use: foremost the drug-resistance problem shown by
the most important parasites, the environmental damage and the toxicity
of many synthetic drugs. In addition, drug residues in plant and animal
food products are important reasons of considerable economic losses for
farmers. The European Community's recent law (EC n. 1804/99)
regarding biological animal farming, limits the use of synthetic drugs,
while the use of homeopathic remedies and phytotherapies is allowed. All
these problems are stimulating the search for new and alternative control
methods, including the search of effective compounds characterized by
smaller environmental impacts in terms of residues and toxicity. Since
plant-derived compounds are generally more easily degradable and could
show a reduced environmental damage with respect to synthetic drugs, at
present the evaluation of the antiparasite activity of plant extracts is
being increasingly investigated, as demonstrated by the recent studies that
have evaluated and confirmed the effectiveness of many plant compounds
on bacteria, fungi, protozoa, helmints and arthropods. Much of present
day antiparasite chemotherapy is derived from practices and advances
made in the 19-20th Centuries, during which the antiparasite activity of
some plants has been scientifically confirmed. Nowadays higher plants
are still important sources of new active principles, among which the
antimalarial compound artemisinin is one of the most recently introduced.
438
Even so, the pharmacological control of some parasitic diseases is still

very difficult; among them we can find arthropod-related diseases.
Perhaps human and veterinary medicine are the most suitable fields for a
real application of natural drugs, in fact the treatment of these pathologies
is mostly topical, and particular drug-formulations are not required.
Furthermore, generally only a few treatments are necessary to kill all the
parasites. In agriculture, in spite of the studies performed to date, these
substances are perhaps still far from their effective use: their main usefiil
feature, that is their biodegradability, is also their weakness. Often, many
products are not able to persist in the environment for a period of time
sufficient for pests control. Further studies are necessary to prepare
better formulations that allow us to solve this problem. Other important
future research topics should concentrate on the evaluation of the toxicity
of these compounds, an unknovm feature for many natural compounds.
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PODOLACTONES: A GROUP OF BIOLOGICALLY

ACTIVE NORDITERPENOIDS
ALEJANDRO F. BARRERO, JOSE F. QUILEZ DEL MORAL and M.

MARHERRADOR
Department of Organic Chemistry, Institute ofBiotechnology, University

of Granada, Avda. Fuentenueva, 18071, Granada, Spain
ABSTRACT: More than seventy podolactones have been isolated mainly from
Podocarpus species. Some of these structures have also been found in filamentous fungi.
The different biosynthetic origin of these molecules considering their vegetal or fungic
source is discussed in this review. These molecules present a wide range of biological
activities: anti-tumor activity, anti-inflammatory activity, fungicide activity, insecticidal
activity and plant growth regulatory activity. These biological properties are analyzed in
detail, and in the light of their results, structure/activity relationships are discussed.
Finally, chemical reactivity, including interconversion reactions, and synthetic
approaches to these compounds are summarized.
INTRODUCTION
Podolactones are considered to be a group of natural products whose

basic skeleton contains a y-lactone between carbons 19-6 and a 6-lactone
between carbons 12-14, which are their characteristic functions Fig. (1)
[1]. The numbering of the podolactone skeleton has been assigned on the
basis of the totarane skeleton from which most of podolactones have been
proposed to be derived.
Fig.{l)
The podolactones with a nor- or bisnorditerpenoid structure are mainly

found in different species of the Podocarpus plant type [2-3]. Apart from
454
the Podocarpus species, five podolactones have also been isolated from a
New Zealand mistletoe, Ileostylus micranthus, which was parasiting P.
totara, from which it has been suggested that podolactones were
assimilated [4]. A small number of tetranorditerpenoid dilactones isolated
from the filamentous fungi Oidiodendron truncatum [5-6], O. griseum
[7], Aspergillus wentii [8] and a non-identified species of the
Acrostalagmus genus [9] have also been considered as fomiing a part of
this group.
These natural substances are of great interest due to both their unusual
structures and the potent wide-ranging spectrum of biological activities
that they possess: antitumor activity [10] in vitro and/or in vivo, anti-
inflammatory activity [7], fungicidal activity [11], herbivorous
manmialian antifeedant activity [12], insecticide activity against house-fly
larvae and other insects [13] and, lastly, a potent plant growth regulatory
activity, both as inhibitors and as stimulants [14-15].
BIOSYNTHETIC ORIGIN
All natural podolactones have been isolated from two types of natural
sources, plants related to the genus Podocarpus, from which the majority
of podolactones have been described, and the filamentous fungi. This
different natural source also possesses a different biosynthetic origin. The
first noticeable evidence supporting this difference is that, while
podolactones isolated from plants are nor- orfcwnorditerpenoids(i.e.
nagilactone C and nagilactone E), the dilactones isolated from fungi have
lost four carbons from a diterpene precursor (i.e. oidilactone C).
Nagilactone C Nagilactone E Oidiolactone 0
Appearance of podolactones in the extracts of Podocarpus species

along with various derivatives with a totarane skeleton (totarol, 12-
455
hydroxytotarol, 19-hydroxytotarol, totaral and 4p-carboxy-19-nortotarol)

[16] led the Hayashi's team to postulate the following biosynthetic
scheme for these substances (Scheme 1)* The pathway starts with 12-
hydroxytotarol, which suffers a meta-pyrochatecase-type fission to give a
hydroxy-acid, intemiediate which decarbonylates leading to the
corresponding a-pyrone, transformation already described in other
catechols [17],
Scheme 1. Proposed biogenetic pathway for podoactones isolated from plants.
On the other hand, the C-16 terpenoid dilactones isolated from fiingi
have been postulated to have a biosynthetic origin different from that
reported for podolactones from plants. Two possible biosynthetic ways
were envisaged, one supposing an oxidative loss of four carbons from a
diterpene precursor, and secondly, the addition of a C-1 unit to a
sesquiterpenoid [18]. The results obtained by administering isotopically
labelled acetic and mevalonic acids to an Acrostalamus fungi, together
with the isolation from this same fungus of acrostalidic acid, acrostalic
acid and isoacrostalidic acid let us to conclude an attribution of a
diterpenoid origin for these lactones with the following biogenetic
pathway proposal[19] (Scheme 2).
456
.CO2H COgH
-^ CO2H * CO2H
ciS' and frans-communic acid aerostatic acid
CO2H
'OMe
0 ^
"" CO2H ^^ 'CO2H
III acrostaiidic acid isoacrostalidic acid
Scheme 2. Proposed biogenetic pathway for podoactones isolated from fungi.
The route could start from the mixture of cis- and rrans-coimnunic
acids (or also from other diterpenes, such as isocupresic acid). The
isolation of hydroxyacid I as a natural product from Acrostalagmus
reinforces the possiblity of the existence of diene II as a key precursor,
not only of isoacrostalidic acid and acrostaiidic acid, but also of dilactone
III. The straightforward chemical conversion of I into III in a recent
publication by Barrero et al. [20] supports this hypothesis.
457
STRUCTURES OF NATURAL PODOLACTONES
Natural podolactones can be classified into three major stractural types

depending on the nature of the conjugated lactone system in the B/C ring
moiety [21], Fig. (2).
Type A: a-pirone [8(14),9(ll)-dienolide],

Type B: 7a,8a-epoxy-9(ll)-enolide,
Type C: 7,9(1 l)-dienolide.
Type A TypeB TypeC

Fig. (2).
Below we can see all the podolactones described up to date, distributed

according to the aforementioned classification with an indication of the
species from which they were identifed and their bibliographic reference.
First, the podolactones isolated from Podocarpus species are presented
and, later, the podolactones found in fungi will be listed.
458
Podolactones from plants
Type A
nagilactone A nagilactone B nagilactone C

1 2 3
P. nagi, P. macrophyllus P.nagi[\7\ P. nagi, P. nivalis, P. halii, P.
P. philippinensis, P. polystachyus macrophyllus, P. purdeanus,
[M, 22-22] lleostylus micranthus [4,17,25-27]
0 0 O
inumakilactone E
6
P. macrophyllus,
P. polystachyus [24, 26]
O
C02Me
1 -deoxy-2a-hydroxy- 15-methoxycarbonyl-
hallactone A nagilactone D
nagiiactone A
7
8 9
P. ham [30] P. nag/ [31]
P./lag/[31]
459
3p-hydroxy- 15-hydroxy-
10
nagiiactone A nagitactone D
P. nagi [32]
11 12
P. nagi [32] P. nag/ [32]
O R=p-D-glc
O 1-deoxy-2p,3p- urbalactone
epoxynagilactone A nagilactoside A
14 15
13
P. urbanii [34] P. nagi [35]
P. nagi [33]
O
3-deoxy- 1-deoxy-
nagilactone C 3-ephnagilactone C nagiiactone A
16 17 18
lleostylus micranthus [4] P. na^/ [36] P. nag/[16]
460
2,3-dehydro- R=p-D-glc
1-deoxy- 2,3-dehydro-
nagilactone A nagilactoside B
nagilactone A
20 21
19
P. na^/[16] P. nagi [37]
P. nag/[16]
R*,
epi-sellowin C R=p-D-glc-(1^^6)-
R=p-D-glc-(1 -•Sj-p-D-glc- p-D-glc-nagilactoside D
22
nagilactoside C 24
P. nagi [37]
23 P. nagi [38]
P. nagi [38]
R=p-D-glc-(1-^6)- R=p-D-glc-(1-*-3)- R=:p-D-glc-{1-#-6)-

p-D-glc-nagilactoside E p-D-glc-(1-^ 6)-p-D-glc- p-D-glc-(1-^ 3)-p-D-glc-
nagilactoside F nagilactoside G
25
26 27
P. nagi [38]
P. nagi [39] P. nag/ [39]
461
TypeB
OH
0 inumakilactone A O podolactone A O podolactone B

28 29 30
P. macrophyllus, P. neriifolius [42-43] P. neriifolius [42]
P. philippinensis [40-41,22]
O
SOMe
0 inumakilactone B ^ podolactone C
podolactone D
31 32
33
P. polystachyus, P. macrophyllus P. neriifolius
P. milanjianus [45-48] P. neriifolius [45-47]
P. neriifolius [23,44-45]
0
O O
'%^
O sellowin B nagilactone E
35 36
P. sellowii [29,47] P. nap/ [49]
462
SOaMe
::o I o;
0Glu(0H)4
inumakiiactone A hallactone B podolide

giucoside 38 39
37 P. hallii, P. sellowii P. gracilor [9]
P. macrophylus P. polystachyus [24,30,47]
P, philippinensis [40-41,22]
O
O nagilactone Q O 2,3-dihydro-
16-hydroxypodollde
16-hydroxypodolide
40 (salignone H)
P. sellowii 42
41
P. milanjianus [50] P. nagi [52]
P. saligna [51]
2p,3p-epoxypodolide milanjilactone A salignone I

43 44 45
P. nagi [52] P. milanjianus [53] P. saligna [54]
463
K^
O 16-hydroxy-
O 3-deoxy-2a-hydroxy- salignone M nagiiactone E
nagiiactone E
47 48
46
P. saligna [55] P. nagi [56]
P. nagi
lleostylus micranthus [4,36]
TypeC
Gluc-0'
O ponaiactone A O ponaiactone A podolactone E

glucoside
49 51
P. na/fa// [57] 50 lleostylus micranthus
P. r?a/fa// [57]
P. neriifolius [4,45]
-O
d ' ip-hydroxy- O 3p-hydroxy-
nagilactone F nubilactone A nagilactone F
52 53 54
P. nubigena [58]
P. nagi P. naflf/ [33]
464
2,3-dehydro-16-hldroxi-
nagilactone F miianjilactone B nagilactone F
55 56 57
P. nagi, P. milanjianus, P. sellowii P. milanjianus [53] P. nagi [56]
P. macrophilus [49-50,25]
Ha,
nagilactone I 2a-hydroxynagilactone F
58 59
P. nagi [66] P. nagi
lleostylus micranthus [4,36]
465
Podolactones from fungi
-O
0^
PR 1388 Oidiolactone C oidiolactone D
60 (Oidiodendroiide C) (oidiodendroiide A)
Oidiodendron truncatum 61
Oidiodendron griseum [6,7] 62
Oidiodendron truncatum Oidiodendron truncatum
Oidiodendron griseum [7,11] Oidiodendron griseum [7,11]
O
'''OMe
O LL-21271a 0 LL-Z1271Y wentilactone A

63 64
65
Acrostalagmus Acrostalagmus
Aspergillus wentii [8]
Oidiodendron griseum [7,9,59] Oidiodendron griseum [7,9,59]
0
O
wentilactone B oidiodendroiide B
66 67 Oidiodendron griseum [7]
Aspergillus wentii [8,20] Oidiodendron truncatum [11]
466
Miscellaneous
OH
'''OH HO'
inumakilactone C inumakilactone D saljgnone A

69 70 71
P. macrophyllus [44] P. macrophyllus [60] P.sa//flfna[51,61]
OH
0^
C02Me V ^ ^ x * \ ^ COaMe
"'^^xÔH
H /
—0
saiignone B saiignone J saiignone K
72 73 74
P. saligna [61] P. sa//flfna [61] P. saligna [55]
C02Me
O dlhydrodeoxy-
saiignone L nagilactone J nubilactone A
75 76 77
P. sa//p/7a [55] P. /lagf/ [62] P. saligna [63]
467
BIOLOGICAL ACTIVITY OF PODOLACTONES
Anti-tumoral Activity.
a) Yoshida Sarcoma.
The in vitro bioactivitivity of 29 podolactones, 15 of them natural

products, against cultured Yoshida Sarcoma cells [64-65] was
investigated by Hayashi's group during the period between 1975 and
1979.
H2O3PO'
468
Table 1 summarizes the obtained results. Podolactones were grouped

on the basis of the previously reported structural subgroups in which these
natural compounds had been classified.
Table 1. Citotoxidty of natural and synthetic podolactones against Yoshida Sarcoma
Type A TypeB TypeC
Lactones ICso Lactones ICso Lactones ICso

(x 10-^ M) (xlO^M) (XIQ-^ M)
Nagilactone A (1) 3l0 Nagilactone E (36) 336 Nagilactone F (55)
Nagilaaone B (2) 17.2 NagUactone G (40) 1.48 89 o
12.2
Nagilactone C (3) 22.5 82 3.72 90 16.1
Nagilactone D (4) 3.32 23-ciihydro-16- 91 18.9
1 -deoxy-2a-hydroxy- hydroxypodolide (42)
nagilactone A(8) 16.4 and 16-hydroxy 14.8
3p-hydroxynagilactone podolide (41)
A (11) 487.0 Inumakilactone A( 28) 10.4
15-methoxycarbonyl Inumakilactone B (31) 4.11
nagilactone D (9) 21.5 83 18.3
10 305.0 84 87.0
78 1460.0 85 607.0
79 1000.0 86 4,19
80 138.0 87 20.6
81 119.0 88 110.0
469
Synthetic derivatives 92 and 93, which can not be not included in any
of the three A-C subgroups, turned out to be inactive against this cell line.
On the basis of the reported data, the following structure-activity

relationships were proposed:
i) The dilactones with few or no polar substituents showed strong
activity, a factor that could be related to the permeability of these
substances through the cell membrane. Thus, the most active compounds
(ICso -- 0.015 jig/mL), nagilactone F (55) and 7,8-epoxy-nagilactone F
(91), do not have any hydroxyl group. The monohydroxylated dilactones
nagilactone D (4), inumakilactone B (29) and nagilactone E (34) are
slightly less active, but still maintain a substantial activity
(IC5o=0.11-0.14 M-g/mL), while the dihydroxylated lactones nagilactone
A (1), nagilactone C (3) and inumakilactone A (26), and the
trihydroxylated 10 and 3p-hydroxynagilactone A (11) are, respectively,
10 and 100 times less active than the non-hydroxylated ones.
ii) The dienic system conjugated with the y-lactone is one of the most
important functional groups for anti-tumor activity.
///) The y-lactone group in positions 4p and 6P is important for the
activity, but not essential.
iv) The acetylation of hydroxyl groups reduces the activity by 10-50
times, probably due to steric effects.
v) The 7,8-epoxy group of the type-B dilactone is crucial, since the
hydrogenolysis product of the epoxide ring is completely inactive.
vi) Activity decreases when the configuration of the substituent is
changed on Ci? from a to p.
vii) Oxidation of the hydroxyl groups to ketones or the introduction of
a p epoxide in the A ring do not affect activity, but the introduction of an
a epoxide in position 2,3 reduces activity up to ten times.
470
b) P-388 Mouse Linfome
Podolide was the first dilactone reported to have antitumor activity in vivo
against P-388 leukemia in mice and citotoxycity in vitro towards cells
derived from P-388 murine leukemia [9]. More detailed reports on the
antileukemic activity of nagilactone C (3) and nagilactone E (36) was
given by Hayashi in 1975 [64]. Both lactones were effective with a dose
of 20 mg/kg/day (T/C 125%). Podolactone C also showed antineoplasic
activity against P 388 cells in vivo at 20 mg/kg/day (151% T/C) [48].
Finally, Bloor and Molly reported the cytotoxic activity of five
podolactones isolated from a New Zealand mistletoe. The more active
lactones were 3-deoxy-2a-hydroxynagilactone E (46), 2a-
hydroxynagilactone F (59) and podolactone E (51) showing IC50 (jxg/mL)
values of 0.06, 0.06 and <0.01 [big/mL, respectively [4].
c) Humane Nasopharynx Carcinoma (9KB)
Podolactones have also been reported to be active against the tumoral cell
line 9KB. Nagilactone G (40) and nagilactone F (55) exhibit a remarkable
in vitro activity against 9KB tumoral cells (ED50 '-lO"^ fxg/ml).
Milanjilactone A (44) and milanjilactone B (45) also showed a
significative in vitro activity (ED50 = 4 jig/ml y ED50 = 0 . 1 \ig/wl,
respectively) [53].
d) Other Cell lines (9KB)
Recently, Barrero et al. [20] have tested the activity of natural

podolactones LL-Z1271a (63) and 68, and of synthetic derivatives 94-99,
the latter being a mixture of isomers, against four tumoral cell lines: P-
388, A-549 (human lung carcinome), HT-29 (human colon carcinome)
and MEL-28 (human melanome) (Table 2). The two natural compounds,
as well as 98 showed a potent activity (IC50<1 M^g/mL). Besides, 63 and
68 exhibited a certain selectivity towards the cell lines P-388 and A-549.
471
OMe
Table 2. Cytotoxic activity of different podolactones against four ceD lines
IC5o(|ig/ml)
Cellline G 68 94 95 96 97 98 99_
P-388 0.12 0.50 20 2.0 10 >10 0.10 >10
A-549 0.12 0.50 20 2.0 20 >10 0.10 >10
HT-29 0.25 1.0 >20 5.0 >20 >10 0.12 >10
MEL-28 0.25 1.0 >20 2.0 >20 >10 0.12 >10
From these data, certain structure-activity relationships can be inferred.

As expected, the presence of the structural moiety 7,9(ll)-dien-12,17-
olide moiety is necessary to achieve the maximum activity. The closure of
the Y-lactone increased the biological activity four times (68 against 95).
On the other hand, the presence of methoxy groups at C-14 on dienolides
remarkably stressed the activity (it increases from 4 to 5 times). Finally, it
is worth-mentioning that 99, (mixture containing natural compound 64)
possessing a hydroxy group at C-14, appeared to be inactive.
472
Anti-inflamatory Activity
Very recently, in 1999, an European patent was published involving the

description of a pharmaceutical composition, which includes terpenoid
dilactones isolated from a new strain of Oidiodendrum griseum
filamentous fungi, together with some semi-synthetic derivatives from the
isolated natural podolactones [7]. This pharaiaceutical composition was
reported to be useful for the treatment of IL-1 (interleukin-1) and TNF
(tumor necrosis factor)-mediated diseases.
IL-1 and TNF are biological substances produced by a number of cells,
such as monocytes and macrophages. These substances have been
demonstrated to mediate a variety of biological activities thought to be
important in immunoregulation and inflammation. A few of the diseases
reported to be exarcebated and/or caused by high or unregulated amounts
of IL-1 are arthritis, osteoarthritis, tuberculosis, atherosclerosis,
rheumatoid arthritis, gout and acute synovitis. Some examples of TNF
mediated deseases are rheumatoid arthritis, osteoarthritis, chronic
pulmonary inflammatory disease and silicosis.
Authors claimed that a therapeutically effective dose for the active
compound will range from 0.01 to 100 mg/kg, generally from about 1 to
about 5 mg/kg; although the dosage also depends on the illness to be
treated.
Among all compounds reported in this invention, the following are the
ones most useful for the treatment of inflammation.
'OMe
PR 1388 (60) LL-Z1271a(63) 68

473
Oidiolactone D (62) Oidiolactone C (61)

O O
OMe OlVIe
OMe
102
Lactones 100-102 were synthesized from natural PR1388 (60).
Antimicrobial Activity
LL-Z1271a (63) was the first podolactone-type compound reported to

exhibit in vitro and in vivo antifungal activity against several
experimental fungal infections [9]. Barrero et al. [20] evaluated the
minimum inhibitory concentration of this compound together with
another seven natural and synthetic podolactone-related structures against
selected Gram-positive and Gram-negative bacteria and yeasts (Table 3).
103
474
Table 3. Antiiiiicrobial activity of selected compounds. MIC ( lig^mL).
Gram-positive bacteria' Gram-negative bacteria^ Yeasts'"

Compounds A B c D E F G H I
63 >25 >25 >25 >100 >100 >100 <3.12 <3.12 <3.12
68 >25 >25 >25 >50 >50 >50 <12.5 <6.25 <3.12
94 25-50 25-50 25-50 >100 >100 >100 25-50 <25 <25
95 <3.12 <3.12 <3.12 50-100 50-100 50-100 <6.25 <3.12 <6.25
96 12-25 50-100 50-100 >100 >100 >100 >100 25-50 >100
98 >25 >25 >25 >100 >100 >100 <6.25 <6.25 <3.12
99 >25 >25 >25 J >100 >100 >100 >25 >25
t ^^
103 >100 >100 >100 >100 >100 >100 >100 - -
' A: Enterococcusfaecalis S 48; B: Bacillus subtilis CECT 397; C: Staphylococcus aureus ATCC 8. ^ D:
Salmonella typhymurium LT 2; E: Escherichia coli; F: Proteus s.'' G: Candida albicans CECT 1394; H:
Saccharomyces cerevisiae; I: Cryptococum neoformans.
An analysis of the results led to the following conclusions. With

respect to tricyclic lactones, maximum activity is shown by compound 95,
which showed itself to be selective against Gram-positive bacteria and
yeasts. On the other hand, three dienediolides (63, 68 and 98) showed a
relevant activity against yeasts, and comparatively (from four to eight
times) less effect against Gra/n-positive bacteria. This comparison
suggests certain structure-activity relationships. Thus, the presence of the
structural moiety 7,9(ll)-dien-12,17-olide moiety is necessary to show
the maximum activity against yeasts. For molecules possessing the above-
mentioned functionalities, the closure of the y-lactone ring results in a
remarkable lack of activity against Gram-positive bacteria
In 1991, Kubo et al. described that 2a-hydroxynagilactone F (59)
showed growth inhibition against Saccharomyces cerevisiae (MIC 800
|Lig/mL) [66]. This same group extended this study to nagilactone C (3)
and nagilactone E (36), the most abundant podolactone in P. nagi. These
three nagilactones, alone and in combination with a variety of
phenylpropanoids, were investigated against C. albicans, S. cerevisiae
and P. ovale. The results, listed in Table 4, showed that the activity of the
nagilactones was significantly increased by Vi MIC of anethole. For
example, the activity of nagilactone E was remarkably increased 128
times when tested against C. albicans [67].
475
Table 4. Antiliiiigal activity of podolactones atone or in combination with V^ MIC of anethoi
MIC (fJig/ml) Podolactone alone//plus anethoie
Compound C. albicans S, cerevisiae P. ovale
2-hydroxynagilactone F (59) Not tested 800//6.25 Not tested

Nagilactone E (36) 800//6.25 100//6.25 50//1.56
Nagilactone C (3) Not tested 1600//400 1600//50
MeO
anethoie
Four oidiolactones, podolactone-type metabolites isolated from

Oidiodendrum truncata, were tested for antifungal activities against
Ustilago violacea, Eurotium repens and Mycotypha microspora. All the
substances exerted activity, LL-Z1271a (63) being strongly fungicidal
against U. violacea [6].
Recently, Kwai et al. justified their research for antifungal substances
after noticing that the incidence of life-threatening fungal infections has
steadily increased in inmunocompromised hosts such as
immunodefficiency-virus (HIV) infected people and cancer patients [11].
In these cases, Candida albicans is the major oportunistic pathogen, and
its resistance to orally taken azoles, the most important antifungals
currently known, is attracting much attention. Taking all this into
consideration, new drugs capable of stopping the infectious process are
required. As a result of their research, Kawai's group isolated five
tetranorditerpenoids from Oidiodendrum cf. truncatum, and their activity
was tested against 11 strains of pathogenic yeasts and 4 strains of
filamentous fungal pathogens (Table 5).
The antifungal activity against the pathogenic yeast C. albicans,
showed MIC^s of 8 to 32 (xg/mL for LL-Z1271a (63), PR 1388 (60) and
oidiolactone C (61), whereas the other two components, oidiolactone D
(62) and oidiodendrolide B (68), showed no antifungal up to a
concentration of 64 jxg/mL. This trend was the same in other pathogenic
yeasts, although the activities differed. In contrast, the tested compounds
had almost no activity against the filamentous pathogens. Possibly , the
functional moiety responsible for the antifungal activity is the planar six
476
membered-lactone. When the 6-lactone ring is opened or non-planar, the

activity decreases. Out of the three tested compounds, LL-Z1271a (63)
appeared to be the most active.
TaUe 5. Aiitifiingal activity of tetranorditerpenoids isolated fkom O, Truncaium
MIC (fig/ml)
Test organism LL-Z1271a PR 1388 oidiolactone C
Candida albicans ATCC 90028 8 16 i 32

Catoica/isATCC 90029 8 16 1 32
C. albicans 1463D 8 16 32
C. tropicalis IFM 46816 32 64 64
C parapsibsis IFM 46863 8 16 32
C. dubliniensis CBS 7987 8 8 32
C.kefyrWM 46921 2 2 16
C. guilliermondu IFM 46823 8 32 32
Pichia anomala IFM 47182 2 4 16
Criptococcus neoformans ATCC 90112 4 8 16
Exophiala dermatitidis 16 32 >64
Trichophyton mentagrophytes KCH 1155 32 64 >64
Aspergillus fumigatus IFM 41243 >64 >64 >64
AyZawisIFM41934 64 64 >64
AmgerH7160B 64 64 >64
It is also worth mentioning that compounds LL-Z1271a (63) and PR

1388 (60) were also effective against Criptococcum neoformans, the
causal agent of cryptococcosis. On the other hand, PR 1388 clearly
inhibited the growth of Histoplasma capsulatum, a dimorphic fungus
causing histoplasmosis, one of the most severe mycoses. This fact is
worth underlining since the fungi responsible for the most severe
mycoses, such as Coccidioides immitis (causing cidioidomycosis),
Paracoccidioides brasiliensis (causing paracoccidioidomycosis) and
Blastomyces dermatitidis (causing blastomycosis), are also dimorphic
organisms. So, PR 1388 could be very useful in the search of more
efficient drugs against these fungal infections.
477
Insecticidal Activity
Species of the Podocarpus genus have been reported to be resistant to

many insects, and nor- and bisnorditerpene lactones isolated from these
plants, such as nagilactones, have been shown to be responsible for this
resistance. Different studies on the effects of naturally occurring lactones
on the development of the housefly (Musca domestica) have been
reported by Singh et al. [25, 68-69]. These investigations include larval
development, pupation and adult emergence and LD50 concentrations
(dose required to give 50% total mortality) under defined conditions.
Table 6 shows the estimated LD50 concentrations for the housefly, and
where sufficient material was available, for the codling moth
(Laspeyresia pomonella) and the light-brown apple moth (Epiphyas
postvittana).
TaMe 6. Toxicity of diffrent lactones to housefly, codling moth and lig^t-brown apple moth
LDsoCppm)
Light-brown
Compound Housefly Codling moth
apple moth
PodoIactoneA(29) <250
Nagilactone A ( l ) 135.0
Hallactone B (38) 48.2
Nagilactone E (36) 40.8
Podolide (39) 33.9
Sellowin A (34) 13.3
Nagilactone C (3) IZO <6.3 76.4
14-Epi-ponolactone A (104) 9.7
Podolactone C (32) 8.2
Hallactone A (7) 3.5
Podolactone E (51) <1.3 175.2
Nagilactone D (4) 0.7 7.3 63.8
Podolactone A (29) happened to be the less toxic lactone, and the most
active compounds are nagilactone D (4) and podolactone E (51). These
podolactones did not produce mortality when applied topically to larvae
or adults (5 |Lig per insect), which suggests that these compounds are not
478
toxic by cx)ntact. It appears that they are either toxic by oral ingestion or
are antifeedants.
0'
14-epi-ponalactone A (104)
The relationship between lactone structure and toxicity to housefly is

also discussed by these authors. All the tested compounds contained an
a,p-unsaturated-Y-lactone, which forms ring C, and an electron rich group
at C-8; features considered to be essential for plant-cell growth inhibition.
The most active compounds have a relatively small, non-polar side chain.
The exception is podolactone C (32), but in this case the sulphoxide
moiety may contribute to its toxicity. The other structural feature which is
reported to strongly influence activity is substitution of ring A. All the
more active lactones have a lp,2p-epoxide group, a 3P-hydroxyl and a y-
lactone joining C-4 and C-6. Apart from structural considerations, the
authors claim that the toxicity of the lactones can be considered in terms
of their overall polarity. In general, compounds with decreasing polarity
show increasing toxicity.
Wih respect to the insecticide activity, Podocarpus macrophyllus is
widely known in Japan for its resistance to termite attack. Saeki disclosed
in 1970 that the termiticidal activity is entirely due to inumakilactone A
(28) and another unidentified compound of similar structure, the former
being more active [70]. This second compound was later reported to be
nagilactone D (4).
The methanolic extract of the leaves of P. gracilior (Kenya) caused
mortality within 12 days after incorporation into a meridic artificial diet
of several lepidopterous pest species. The toxic and growth inhibitory
action of nagilactones C (3), D (4) and F (55) and podolide (39) towards
these species are shown in Table 7. All tested podolactones are relatively
potent growth inhibitors (ED50: 4-30 ppm), while the concentration of the
compounds that cause mortality was about two orders of magnitude
479
higher (LD50: 300-2000 ppm), which could be due to a deterrent effect on

these compounds [71].
Table 7. Activities of nagilactones C, D and F and podolide towards different lepidopterous agricultuinl
pest species.
Species Test compound LD90 (ppm) EDw (ppm))
Heliothis zea NagilactoneC(3) 1500 20

NagilactoneD(4) 800 4
NagilactoneF(55) 30
PodoHde(39) 12
Spodoptera frugiperda Nagilactone C (3) 2000 18
Nagilactone D (4) 2000 6
Nagilactone F (55) 12
PodoUde(39) 2000 7
Pectinophora gossypieUa Nagilactone C (3) 1500 14
Nagilactone F (55) 20
PodoUde(39) 300 9
In 1992, Kubo et al. [13] studied the effect of the four most abundant
nagilactones in Podocarpus nagi -l-deoxy-2p,3P-epoxynagilactone A
(13), l-deoxy-2a-hydroxynagilactone A (8), nagilactone C (3) and
nagilactone D (4)- on the feeding and growth of tobacco budwomi larvae,
Heliothis virescens. Nagilactone C and D exhibit strong inhibition to the
growth of the first instar larvae at 168 and 166 ppm respectively. At these
concentrations, none of the larvae fed with nagilactone D reached the
third instar, while, when nagilactone C was used, half of the larvae
reached the third instar but growth was delayed. Most first instar larvae
fed on diets containing l-deoxy-2p,3p-epoxynagilactone A or 1-deoxy-
2a-hydroxynagilactone A reached the third larval instar but development
took longer periods than the control. Finally, when fifth instar larvae were
fed on a diet containing nagilactone D at 160 ppm, most of the larvae
could not pupate. The three remaining tested compounds were much less
active than nagilactone D against H. virescens fifth instar larvae. On the
other hand, these investigations also concluded that growth inhibition is
caused by feeding deterrence, with nagilactone D being the most potent in
inhibiting the mouthpart sensory receptors.
480
Antifeedant Activity Against Mammals
The antifeedant activity of these compounds has been described not only
for insects, but these dilactones have also been reported as the active
principles causing anti-feedant activity for a herbivorous mammal, the
guinea pig. Thus, the antifeedant activity of these compounds has been
tested employing 1% acetone solutions of natural dilactones. Nagilactone
A (1), nagilactone C (3) and l-deoxy-2p,3p-epoxynagilactone A (13)
showed activity, whilst their acetate derivatives were inactive. On the
other hand, the dilactones were not active with deers, protected species in
Japanese forests, which do no eat Podocarpus nagi. The activity of the
dilactones seems not simply to have been repellent, but actually
poisonous or toxic to the animal, so, the animal supplied only with a
sample diet containing ca. 0.5% of a crude extract of the leaves
completely rejected the diet for two continuous weeks [12].
Plant Regulatory Activity
Since the discovery of the first podolactones, the capacity of these

substances to inhibit, not only plant growth, but also the expansion and
mytosis of plant cells became apparent [42]. The Galbraith group, which
systematically studied this type of activity in different podolactones,
published a study of the structure-inhibitory activity of podolactones
using a pea-stem growth system [72]. The inhibitory properties in the
expansion on hook segments from peas are listed in Table 8. From the
analysis of these data, some conclusions were presented. The
a,p unsaturated 8-lactone forming ring C seems to be the essential group
for the activity. Besides this requirement, a basic centre on Cg is also
necessary. All the active compounds possess a double bond or epoxide
between C7 and Cs, or a double bond A^^^^\ They also possess a relatively
small non-polar lateral chain. The other structural feature which was
reported to influence the inhibitory activity is the shape of A ring. The
most active lactone, podolactone E (45), has a 1,2-epoxy group and a y-
lactone joining C-4 and C-6. Finally, the presence of a hydroxy at C-3
does not seem to affect the activity.
481
Table 8. Inhibitoiy activity of naturally occiirriiig lactones on hook segments Crom etiolated dwarf peas
% Control growth at concentration given
Compound 10'M 10^ M 10^ M
Inumakilactone C (69) 102 (5 X 10^ M)

NagilactoneB(2) 81
Nagilactone A (1) 79
Podolactone C (32) 100 70
Podolactone D (33) 98 60
Podolactone B (30) 82 60
Nagilactone C (3) 89 53
Podolactone A (29) 88 37
Inumakilactone A (28) 66 34
Inumakilactone B (31) 47 10
Podolactone E (45) 82 38 0
In 1972, Hayashi's group noticed that podolactones belonging to each

of the three above-mentioned structural groups show fine differences in
their allelopathic activity [49]. Thus, nagilactones A-D (1-4) (all A type
dilactones) inhibited the elongation of the Avena coleoptiles section at 10"
^-10"^ M, however, at 10'^ M, all compounds tumed out to be stimulatory.
On the other hand, B type [inumakilactone A (28) and nagilactone E (3^]
and C type [ponalactone A (49)] podolactones were exclusively inhibitory
in the whole concentration range studied. Experiments with Jerusalem
artichoke slices supported the above findings.
The studies on the inhibitory effects of podolactones A (29) and E (51)
and nagilactone E (36) were extended to a range of bioassays [73]. In
some of these assays, the effects of the podolactones were compared to
those of lycoricidinol and harringtonolide, plant growth inhibitors (Table
9).
482
TaUe 9. Concentration of podolactones and related compounds causing significant

(/' = 0.05) inhibition
Bioassay Concentration for

significant effect (i&M)
Germination of radish seed Harringtonolide 10

Podolactone E (51) 10
Lycoricidinol 10
Grov/ih of Arabidopsis Nagilactone C (3) 22

Podolactone A (29) 22
Wheat embryo coieoptile Lycoricidinol 1
Harringtonolide 10
Nagilactone C (3) 100
PodolaaoneE(51) 10
Lettuce hypocotyl (+ GA) Podolaaone A (29) 27
Lettuce radicle) Podolactone A (29) 27
Pea tips Podolactone E (51) 1

Harringtonolide 10
Lycoricidinol 1
Auxin transport in bean petiole Podolactone E (51) 10

GA-induced amylase in barley seed Podolactone A (29) 27
Reduction of mature pikelets in Podolactone A (29)
Lolium temulentum 0.27
In the course of an investigation directed at isolating active chemical

components from Podocarpus species, the groups of Hayashi and Kubo
[74] noticed that the activity of the crude extract of P. nagi was lower in
seed gennination and growth tests could then be explained when the
amount of nagilactone E (36) present in the crude extract and strength of
the activity of pure nagilactone E was considered. This led to the
examination of other components of the crude extract, the known
flavonoid epicatechin. Both components were tested employing lettuce
and rice seed. When epicatechin was tested alone it caused little change,
whereas assays using nagilactone E showed an increase in growth
inhibition when the concentration was raised. However, when both
compounds were applied together, the co-presence of epicatechin
removed the inhibitory effect of nagilactone E, and in some cases even
caused growth stimulation. The effect is strongest in the case of rice. So,
the inhibitory activity of the root was observed even at 0.1 and 1.0 jxg
mL'^ of nagilactone. The presence of epicatechin removed the growth
483
inhibition at 3 \ig mU^ and when 30 \xg mL"^ was applied, a 41%
stimulation of root growth was caused.
Studying the effect of nagilactones on the growth of lettuce seedlings,
Kubo et al. [75] classified the active podolactones isolated from P. nagi
into two groups according to their bioactivity. Group I [l-deoxy-2p,3P-
epoxynagilactone A (13), l-deoxy-2a-hydroxynagilactone A (8),
nagilactone A (1), nagilactone C (3) and 15-methoxycarbonyl nagilactone
D (9)] consists of podolactones which stimulate lettuce radicle growth at
concentrations between 1 and 10 \ig mL"^ On the other hand, Group II
[nagilactone D (4) and nagilactone E (36)] is composed of podolactones
which show a significant inhibitory effect on the growth of the lettuce
radicle at less than 10 jxg mL"\ It is worth noting that dilactones
belonging to Group I were able to stimulate the growth of the radicle at
lower concentrations (<10 jxg mL"^) while they were shown to be
inhibitory at higher concentrations (>100 \ig mL"^). From the above
results, the relationship between the number of hydroxyl groups and the
inhibitory effects of podolactones can be inferred, resulting that the less
polar compounds possess higher activities. Since all Group I dilactones
are type A podolactones, these findings are in accordance with the
Hayashi conclusion on the different activity shown by each of the three
podolactone structural types.
In contrast with Hayashi's reports, findings described in different
Japanese patents [76] revealed that all three podolactone structural types
are able to stimulate plant growth. Thus, when cucumber seeds are soaked
in a 1 ppm type A nagilactone C (3) aqueous solution for 24 hours, and
then cultivated for 18 days, they experienced a 54% increase in dry
weight of stems and leaves when compared with untreated controls. When
type B podolactones were investigated, these authors claimed that the
cucumber seeds showed a 30% increase in dry weight of stems and leaves
after being soaked in a 1 ppm type B nagilactone E (36) aqueous solution
for 24 hours and cultivated for 18 days. Finally, type C dilactones also
proved to be stimulant. For instance, tomato seeds were soaked in a 1
ppm aqueous solution of type C nagilactone F (55) for 22 hours, and then
cultivated for 5 weeks to show a 31% increase in dry weight of stems and
leaves when compared with untreated controls.
The aforementioned capability of podolactones to inhibit or stimulate
plant growth was again verified in a study carried out by Macias and
Barrero's groups [14]. In this study the allelopathic activity of 11 natural
484
and synthetic podolactones on the dicotyledoneae Lactuca sativa,

Lepidium sativum^ Lycopersicum esculentum and the monocotyledoneae
Allium cepa, Hordeum vulgare and Triticum aestivum was investigated. A
selection of the best results is shown in Fig. (3).
Fig. (3). Effects of selected dilactones in the gemination, radical and shoot length oil. sativum^ A. cepa and H.
vulgare in comparison with the commercial herbicide LOGRAN
^\ l£pi&£^:îm^lt,' :
10^ M
10' M
n
i^^"=B
10 H I
'iBi:. 10-' M
f.^CJ.. 10-^ M
|f#>-v;i;;i'
10" M
i?^^.^ r^l^ga:feg«j^2^m^jL. , ^;^ Jc 'or^^hl- \: lO"" M
I 10-" M
14'
10' M
Ri~
10" M
«3
485
The strong inhibition produced by compounds LL-Z1271a (63), 68

and 98 is noticeable (at a concentration of 10"^ M) in the germination and
growth of all the species studied (-90% average). This activity is probably
due to the y-lactone ring located between C19 and Q . Likewise, the
presence of a metoxyl group at Ci? increases the inhibitory activity at low
concentrations. The stimulation effect shown by 68 with dilution over
geraiination and growth of monocotyledon species is also noteworthy.
These compounds showed the same activity as the commercial
herbicide LOGRAN®, and in some cases even greater, which means that
they are excellent models for the design of new natural herbicides.
In spite of the aforementioned results, the Ohmae group, which has
carried out exhaustive studies on the content in nagilactones in forests
where P. nagi grows, questions the allelopathic effects of nagilactones
[77].
486
CHEMICAL REACTIVITY
Before the work reported by Hayashi's group in 1982 [21], the study of
podolactones reactivities was limited to the preparation of analogs to
facilitate their structure determination. This limited study of reactivity
could be explained considering the poor content of podolactones in their
natural sources.
Thus, to confirm the location of the two hydroxyl groups in
inumakilactone A (28), this compound was selectively acetylated, and its
diacetate selectively saponificated. The thus obtained 15- and 3-
monoacetates were oxidized to the corresponding ketones with Jones
reagent (Scheme 3) [40].
^ % ^ mild *f '''''^^'Oones
:o I ^
'*^ OAc
HO'
Inumakilactone A (28) 105
AC2O I NaOAc
O
AcO'
Schemes
With respect to the side chain, although some standard reactions failed
[78], different correlation reactions assayed to confirm the structure of
new isolated members were achieved successfully, some examples
include oxidation of the podolactone C sulphoxide group to give the
corresponding sulphone (110), as well as the glycol oxidative cleavage of
487
podolactone B 3-acetate (111) with NaI04 to give the cx)rresponding

methyl ketone (112).
S02Me
mCPBA Qi
podoiactona C
32
On catalytic hydrogenation with Pt02 inumakilactone A (28) yielded

the corresponding dihydro derivative 113 [40]. In contrast, the C ring
double bond of inumakilactone B (31) was unaffected when this dilactone
was catalytically hydrogenated with Pd-C [44].
ROo
OH H2
inumakilactone A (28) 113

488
inumakilactone B (31) 114
In the earlier studies on the chemical reactivity of podolactones, it was

noticed that some standard chemical transforaiations were reported to
lead to unexpected results. Some of these outcomes were found in the
treatment of nagilactone A (1) and its diacetate (116) with chromic acid
and sodium borohydride, respectively [17] (Scheme 4).
Scheme 4
The very same kind of transformation was found upon treatment of

nagilactone C-7 acetate with sodium borohydride [57]. Both
transformations supposes the first interconversion from type A to type C
podolactone groups. In this sense, the particularly limited availability of
489
type C podolactones led Hayashi and Matsumo to focuss their reactivity

studies on the formation of these type C podolactones from the more
abundant A and B types. In their paper, published in 1982 [20], two main
ways were reported to convert type B podolactones into type C. The
reductive cleavage of the 7,8-epoxy group experienced by nagilactone C
(3) and closely related podolide 39 led, after the corresponding
dehydration, to obtain type C podolactones. The second way of
conversion of type B dilactones entailed the treatment of nagilactone C or
its acetate (119) with alumina to give the acidic derivatives 126 or 127
which upon reduction with sodium borohydride gave the type C 3p-
hydroxynagilactone F (54). Scheme 5 summarizes these results.
Type A to type C conversion could be achieved using the previously
mentioned transformation of type A 7-acetoxyderivatives with sodium
borohydride; unfortunately, the type C dilactones so obtained were
epimer at C-14 of the natural dilactones (i.e. 129). A similar type of
double bond migration from 8,14 to 7,8 position took place under
photolytic conditions with concomitant introduction of a hydroxyl (or a
methoxyl) group at C-14. So, the irradiation of nagilactone A diacetate
(116) in aqueous THF gave the corresponding 14-hydroxy analog 130,
which, after reduction, gave a mixture of type C dilactones, epimers at C-
14, including 131, the acetoxy derivative of the natural product Ip-
hydroxynagilactone F (52). Transformation of type A to type C dilactones
was finally achieved (Scheme 6).
490
0 Podolide (39)
TsCI
AcO^
AcO
Scheme 5
491
Scheme 6
Having described the correlation among the different podolactone

types, some other transformations were reported, involving mainly the A
ring.
For instance, the P-epoxide on ring A has been proved to undergo
acidic cleavage. However, although some epoxide openings have been
reported on inumakilactone A (28) and sellowin A (34) and B (35) [26,
78], these data are not shown here since the structures of these three
natural dilactones were corrected after the publication of these
transformations [43,47]. Lately, nagilactone D acetate (132) was reported
to give, after treatment with HCl, the mixture of chlorohydrins
corresponding to the attack of chloride at both extremes of the epoxide
(Scheme 7). The same la-chloro-2p-hydroxy chlorohydrin was formed
when nagilactone C (3) was heated with rhodium chloride [21].
492
HCI
AcO'
132
Scheme 7
Another unusual transformation detected in podolactones was the

simultaneous deoxigenation and reduction of nagilactone C (3) to give
sellowin C (5) when treated with chromous chloride or Zn-Cu [78]. This
result could not be obtained by Hayashi et al. using the complex
Cr(C104)2/ethylenediamine in DMF, the 1,2-unsaturated analog 133 being
obtained. Compound 133 experienced reduction to natural podolactone
19, transfomiation that entails a double bond migration [21] (Scheme 8).
Selective hydrogenation at either the 1,2- or 2,3-position has not been
achieved [79], almost surely due to undesired side-reactions, as the
above-mentioned saturation of the C ring double bond or the reductive
cleavage of the 7,8-epoxide.
0 0
CrClg
Zn-Cu
Nagilactone C (3) Sellowin C

ethylenediamine
Cr(CI04)2| DMF
0
Pd-C
^
Hz
Schemes
493
Podolide (39) could be obtained from nagilactone E (36) by treating

the alcohol with tosyl chloride in refluxing pyridine. The same
elimination was accomplished on heating nagilactone E with phosphorus
oxychloride in pyridine. Although the ring A double bond has been
reported to show no reactivity, the reaction of podolide and 16-
hydroxypodolide with mCPBA in the presence of a radical inhibitor takes
place stereoselectively, yielding the corresponding a-epoxides [79, 21].
no I TsCI/PyrA mCPBA
^
orPGCIa/PyrA 4,4'-thiobis-
(6-rt)utyl-mcresol)
Nagilactone E (36)
Scheme 9
Although the epoxidation of the A^ double bond of type C

podolactones has been reported as being unsuccessful after several
conditions, when C-14 position is not substituted, the epoxidation of this
double bond of dilactone 68 is achieved to synthesize oidiolactone C (61)
[80].
dimethyloxirane
O Oidiolactone C (61)
494
Ichikawa et al. patented in 1999 a pharmaceutical composition which

included natural dilactones from Oidiodedron griseum together with some
synthetic derivatives prepared from the more abundant natural
compounds [7]. The previously unreported chemical transformations
included in this invention are shown in Scheme 10.
495
Synthesis of Podolactones
Few routes for synthesizing podolactones are known. Only the syntheses
of LL-Z1271a (63), nagilactone F (55) and 3p-hydroxynagilactone F
have been described, which are the molecules structurally more simple
than their other congeneric types but which, however, are biologically
much more active.
These syntheses are summarized below.
Synthesis of LL-Z1271a
a) Starting from Marrubiine
In 1973, the group led by Prof. Adinolfi synthetized the antibiotic LL-
Z1271a (63) [81] starting from ketolactone 139, obtained, in turn, by
degradation of the diterpene marrabiin (9% overall yield). This synthesis
basically consists of the formation of the 8-lactone C ring by nucleophilic
addition to carbonyl group and subsequent lactonization (Scheme 11)..
Bromination of 139 in glacial acetic acid, followed by treatment with
1,5-diazabicycle [4.3.0]-5-nonene gives unsaturated ketone 140 in a yield
of 85%. The latter's reaction with lithium ethoxyacetylide in THF gives
ethynyl-carbynol 141 (90%), which, on being treated with concentrated
sulphuric acid in ethanol, gives conjugated ester 142 (65%). The
oxidation of 142 with Se02 (2 moles) in glacial acetic acid for between 20
and 40 hours produces a 1:3 mixture of a- and p-acetyllactols (143),
respectively (70%). Treatment of 143 with hydrogen chloride in absolute
methanol gives compounds 63 and 98 in a proportion of 1:2.5,
respectively.
COaEt
a,b
^
85%
139
496
OAc f '"OMe OMe

70%
(a) AcOH, HBr; (b) DBN; (c) U O COEt, THF; (d) H2SO4, EtOH; (e) SeOz, AcOH, reflujo; (f) MeOH, HCl.
Scheme 11.
b) StartingfromWieland-Miescher Ketone
The Welch group tackles stereoselective synthesis of LL-Z1271a using

Wieland-Miescher diketone 144 [82] (3% overall yield) (Scheme 4). This
synthesis includes, as key steps, the stereoselective introduction of the
methyl on carbon 4 in an equatorial position, the foraiation of the y-
lactone via bromolactonization and the construction of the 6-lactone C
ring through a Meyer-Schuster rearrangement.
P-Ketoester 145 was prepared starting from Wieland-Miescher ketone
in 3 stages with an overall yield of 50%. The reaction of 145 with NaH in
HMPA at room temperature for 2 hours, followed by addition of
chloromethyl methyl ether gives 146 in 91% yield.
Treatment of 146 with lithium in anhydrous liquid ammonia/1,2-
dimethoxyethane, followed by addition of methyl iodide gives ester 147
in 72% yield. This sequence of reactions allows the highly stereoselective
achievement of the metoxycarbonile group's axial stereochemistry in
position 4. The synthesis of intermediates 148 and 149 is carried out using
conventional methods.
Enone 149, after treatment with bromine and then with anhydrous
potassium carbonate gives unsaturated y-l^ctone 150 (73%). After
hydrogenation of the double bond A^ and reaction with NaH in the
presence of ethyl formiate, 151 is obtained in 91% yield. Then, the double
bond A^ is regenerated and the aldehyde is protected in the form of acetal
(54% yield).
497
Finally, the synthesis is completed by using the Meyer-Schuster

rearrangement of the Aren-van Dorp synthesis on enone acetal 152.
Treatment of 152 with lithium etoxyacetilide in THF gives an unstable
tertiary allylic-propargylic alcohol that, when dissolved in methanol with
a catalytic quantity of sulphuric acid produces 63 and its epimer 98 in a
proportion of 7:3, respectively, in 42% yield.
7:3
(a) NaH, HMPA; (b) CHaOCHzQ; (c) U, NH3, DME; (d) CH3I; (e) UPrS, HMPA; (f) MeOH, H3O"; (g)
MeOH, APTS; (h) Jones; (i) AcOH, Brz; 0) CaCOa, DMA; (k) DCM, Brz; (1) K2CO3, DMF; (m) H2,
(Ph3P)3RhCl, benzene; (n) NaH, HCOOEt; (0) Et3N, PhSeCl, THF; (p) AMCPB, THF; (q) ethylene glycol,
H2SO4 cat, THF, CaS04; (r) LiC-COEt, THF; (s) H2SO4 5%, MeOH.
Scheme 12
498
c) Startingfromcis- and trans-Communic Acids
Recently, Barrero et al. have synthesized 63 from cis- and trans-

communic acids (153) [14], natural substances present in the cones of
Juniperus communis L. Two of the four rings, and three asymmetric
carbons have the same configuration as the final molecules. These
syntheses were achieved in 10 steps with overall yields of 1% and
involves as key steps (Scheme 13):
-degradation of the conmaunic acids' side chain,

-formation of the 6-lactone by mercuriation-demercuriation, and
-allylic functionalization of C-17.
Degradation of the side chain of the communic methyl esters is

achieved by using metachloroperbenzoic acid and then treating with
HIO4. Oxidation of the resulting aldehyde with Jones reagent and
esterification with diazomethane gives diester 154 (66%). Treatment of
154 with mercury acetate in reflux toluene quantitatively gives the
mercurial derivatives 155. Reaction of this mixture with sodium
borohydride in the presence of oxygen and subsequent dehydrogenation
with DDQ and p-toluensulfonic acid leads to dienolide 157 (45%).
Hydrolysis of the methyl ester and subsequent y-lactonization with lead
tetraacetate/light leads to dilactone 6 in 45% yield. Exposure of 68 to
Se02 in refluxing dioxane furnishes lactol 99 (100%), which, after
treatment with MeOH-catalytic sulphuric acid yields compounds 63 and
98, respectively.
C02Me ^COaMe
HgOAc
499
(a) i-m-CPBA, ii-HI04 (73%); (b) Jones reagent (90%); (c) CH2N2 (100%); (d) Hg(0Ac)2, toluene, A (100%);
(e) NaBH4, O2; (f) DDQ, dioxane, A (45% yield for steps e and f); (g) H2SO4, H2O (100%); (h) Pb(0Ac)4, hv
(45%); (i) Se02, dioxane, A (100%); (j) MeOH, H2S04,(85%).
Scheme 13.
Synthesis of Nagilactone F
a) Starting from Podocarpic Acid
The first synthesis of nagilactone F was carried out by Hayashi et al. [83]
starting from (+)-0-methylpodocarpic acid 158 in 23 steps (2% overall
yield). The main characteristics of this synthesis include the
transformation of the aromatic ring into the 8-lactone ring, the a
arrangement (equatorial) of the isopropyl group by photochemical
cyclization, as well as the construction of the y-lactone using radical
conditions (Scheme 14).
500
(a) Li, NH3, r-BuOH; (b) U3O*; (c) CH2N2; (d) H2, Pd-C; (e) LDA; (f) PhSeQ; (g) H2O2; (h) (i-Pr)2CuU; (i)
O3, MciS; 0) Jones reagent; (k) BjHe, THF; (I) APIS; (m) r-BuOK, DMSO; (n) hv, EtOH; (o) DDQ, BF3,
dioxane; (p) H2SO4, H2O; (q) Pb(0Ac)4, hv.
Scheme 14.
Birch reduction, followed by acid treatment and addition of

diazomethane leads to the A^^^^^-enone 159 in 41% yield. Then, the double
bond is hydrogenated and, by using PhSeCl and hydrogen hydroperoxide,
the double bond A^^ is formed. Treatment of the enone with lithium
disopropylcuprate-dimethyl sulfide complex gives an intermediate enolate
that is trapped again using PhSeCl. Enone 160 is obtained via oxidative
elimination (62%).
Ozonolysis of 160, followed by oxidation with Jones reagent,
esterification with diazomethane, reduction of the ketone with diborane
501
and treatment with p-toluensulfonic acid gives lactones 161 in a yield of

61%. Then, the double bond A^^^^^ is formed via the
selenylation/oxidation protocol. Subsequent treatment with potassium
êrr-butoxide in dimethyl sulfoxide gives dienic acid 162 (84%), which is
converted into lactone 163 by a photochemical process (90%). The
equatorial arrangement of the isopropyl group is understandable if we
bear in mind the free rotation of the bond Cs-Cu during the lactonization
process and its tendency to avoid interaction with the angular methyl on
Cio. Dehydrogenation of 163 with dichlorodicyano-p-benzoquinone in the
presence of BF3 gives dienolide 164 (38%). Finally, acid hydrolysis of the
methyl ester and subsequent treatment with lead tetraacetate in benzene
under irradiation gives 55 in 55% yield.
b) Starting from cis- and trans-Communic Acids
The same strategy used by Barrero et al. for synthesis of the fungic
metabolite LL-Z1271a (63) was used to synthesize nagilactone F (55).
Thus, starting from the mixture of communic acids, lactol 99 is achieved,
an immediate precursor of both 55 and epimer 165, which are obtained by
treatment with isopropylmagnesium bromide. Thus, the synthesis of
nagilactone F (55) is completed, with an overall yield of 10%. (Scheme
15).
o
nagilactone F (55) 165

9:1
Scheme 15
502
c) Total Synthesis
Burke et al. [84] synthetised nagilactone F (55) by a polyenic cyclization

initiated with acetal and concluded with vinylsilane, giving an overall
yield of 6%. The key steps in this synthesis were the coupling of
substrates 166 and 167 with control of the absolute and relative
stereochemistry, the cationic biscyclization to form the intermediate
tricyclic trans-anti-trans 169 and the formation of the D ring by regio-
selective intramolecular remote functionalization.
MeaSi
(a) n-BuU; (b) (2-thienyI)Cu(CN)y; (c) BF3-Et20; d) TiCU, Ti(i-Pr0)4; (e) Swem oxidation; (f) AcOH,
piperidine; (g) RhCb•3H2O, CaCOa, i-PrOH; (h) PhI(0Ac)2,12, ciclohexane, hv; (i) H2, Pt02; Q) RuOsHaO,
NaI04, CH2N2; (k) PhNMeaBra, THF; 0) U2CO3, UBr; (m) DIBALH; (n) TPAP, NMO; (o) UHMDS, TMSQ,
PhSeQ; (p) Davis oxaziridine.
Scheme 17.
503
Transmetalation of the vinylmercurial derivative with n-BuLi,

followed by reaction with 2-lithium thienylcyanocuprate and subsequent
addition of BFs-OEta and 167, leads to lactone 168 in 86% yield. Cationic
biscyclization of vinylsilane 168 with TiCU and Ti(0i-Pr)4 (at a
proportion of 5:1), followed by the cleavage of the remaining acetal
(oxidization of the hydroxypropyl ether side chain and subsequent P-
elimination of piperidinium acetate) leads to secondary alcohol 169.
Isomerization of the double bond, catalyzed by rhodium, followed by
remote functionalization of the axial methyl on C4 according to the
photochemical conditions established by Suarez et al. [(PhI(0Ac)2, I2]
gives tetrahydrofuran 170 in 35% yield. Catalytic hydrogenation of the
double bond A^, followed by oxidization using RUO4, esterification with
diazomethane and introduction of the double bond A^ leads to compound
171 (67% yield). Treatment of 171 with DIBAL-H, followed by
oxidization with tetrapropylammonium perrutenate (TPAP) and NMO
and subsequent generation of the double bond A^^^^^ give rise to 55 in 75%
yield.
Synthesis of(±)'3j3'hidroxinagilactoneF(58)
De Groot et al. synthesized (±)-3p-hydroxynagilactone F (54) [85] from

compound 172 [86] with an overall yield of 0.2% (13 stages). The key
steps in this synthesis were the formation of the 8-lactone by nuclelophilic
addition to carbonyl and subsequent treatment with/7-toluensulfonic acid,
the introduction of the isopropyl in an a equatorial disposition following a
procedure similar to that described by Hayashi et al. and finally the
formation of the y-lactone via bromolactonization.
Protection of the secondary hydroxyl in 172 and then condensation
with 2-methylpropanal leads to enone 173 (65% yield). Addition of
dilitioacetate gives rise to compound 174 (78%), which on exposure to p-
toluensulfonic acid, and subsequent saponification and light irradiation
leads to lactone 175 (57%). Dehydrogenation of 175 with DDQ and 2-
mesitylensulfonic acid (AMS) gives dienolides 176 and 177 in a
proportion of 3:2, respectively (60% yield). These compounds cannot be
separated, which means that their thioester derivatives need to be formed,
separated and then hydrolyzed again. The overall yield of this process for
176 is 17%. Finally, the formation of the y-l^^tone ring by
504
bromolactonization and subsequent deprotection of the secondary alcohol

using boron tribromide gives 3P-hydroxynagilactone F (54).
^ C02CH2SPh ^ COaCHaSPh
178 52% 179 24%

505
MeO^
(a) BaO, Ba(0H)2, DMSO, Me2S04; (b) LDA, (CH3)2CHCHO; (c) UCH2CO2U; (d) TsOH; (e) n-PrSLi; (f) hv,
EtOH; (g) DDQ, MSA; (h) r-BuOK, PhSCH2a; (i) chromatographic separation; (j) CF3COOH; (k) piridine,
HBr, Br2, AcOH; (1) K2CO3; (m) BBrj.
Scheme 18.
Synthesis of Oidiolactone C Starting from Communic Acids
The main drawback of the first method described by Barrero et al. [14] for
the synthesis of podolactones, lies in the presence of a mixture of three
isomers called "communic acids" (153), which requires a
chromatographic separation over silicagel impregnated with 20% silver
nitrate to eliminate mirceO'Communic acid to allow use of the 2-
component mixture of cis- and trans-isomors as starting material.
Furthermore, some steps in this former synthetic strategy, such as the
closure of the 8-lactone or the formation of the dienolide system need
improvement.
Studying the content in communic acids of the arcestides of different
Juniperus and Cupressus sempervirens (Mediterranean cypress), we
found the presence of only rrans-communic acid (180) in the arcestides of
cypress. Furthermore, this acid could easily be isolated from the acid
fraction on the hexane extract by crystallization of its sodium salt.
Starting with trans-communic acid, two diffrent strategies were
developed, the first one using diene 186 as the key intermediate, which is
summarized in Scheme 19.
506
CHO ^COaMe
VfJ^^^^'^'OCOCFa (70%)
COaMe
184
^COaMe CO2H
186 103
(a) O3, MezS (60%); (b) Jones, (c) CH2N2; (d) SeOj, r-BuOOH (67%); (e) TFAA, DMAP (86%); (f) Pd(PPh3)4
(70%); (g) NaCH3(CH2)2S (85%), (h) Pd(PPh3)4 (72%); (i) LDA, PhSeCl, H2O2 (80%); (j) DMDO (45%).
Scheme 19
There are three major achievements in this synthesis [20]. The first
consists of the selective degradation of 180 to 181 in 60% yield realized
via ozonolysis at low temperature. The second one was the allylic
elimination of trifluoroacetate 184 after exposure to Pd(PPh3)4, which
resulted in the formation of diene 185 in an acceptable yield (72% based
507
on 70% conversion). The third, and to our judgement, most interesting

achievement was the Pd(II)-mediated double lactonization of diacid 186
to form key intemiediate 103 in 70% yield (based on 80% conversion).
Fig. (4). This reaction constitutes the first example described of
bislactonization of conjugated dienes.
Figure (4)
The second synthetic approach to oidiolactone C (61) is summarized in

Scheme 20. This route also conmiences with the ozonolysis of trans-
communic acid 180. Now, when this compound was exposed to ozone in
excess, keto aldehyde 187 was obtained in 76% yield. The key step in this
approach was the y-lactone closure via chemoselective reduction of the
lactone moiety on compound 189 through a SN2"mechanism. Compound
189 could be prepared by saponification of the corresponding methyl
ester with sodium propanethiolate. Once the primary alcohol is oxidized,
the completion of the synthesis of key lactone 103 only requires the
allylic oxidation of the C-17 methyl with concomitant closure of the 8-
lactone. This conversion was achieved with Se02 in refluxing acetic acid
to give 103 in 51% yield.
508
103
(a) O3, MezS (76%); (b) Jones, CH2N2; (c) PhSeCl, H2O2 (77% for three steps); (d)
MeMgBr (84%);(e) NaS(CH2)2CH3 (83%); (f) LAH (88%); (g) PDC, DMF, CH2N2
(70%); (h) Se02, N B U O O H (51%).
Scheme 20
Synthesis of 3p-hydroxy-13,14,15,16-tetranorlabda-7,9(ll)-dien-
(19,6p),(12,17)-diolide
As indicated in the section describing the stractures of natural dilactones,

wentilactone B was wrongly assigned when the stracture of 3p-hydroxy-
13,14,15,16-tetranoriabda«7,9(ll)-dien-(19,6p),(12,17)-diolide was
isolated. Our synthesis of this compound allows the reassignment of the
structure of wentilactone B. Thus, the hydroxyl group of this natural
podolactone should be relocated at C-2 with an a- configuration [87].
Two double cyclization steps were employed in this synthesis. The first
involves the construction of the bicyclic skeleton via a Mn(III)-mediated
509
radical cyclization, and the second involves the transformation of this

bicyclic intermediate into the tetracyclic podolactone skeleton through a
Pd (Il)-mediated bislactonization of the corresponding conjugate diene.
COOMe ^COOMe
192 193 194 195R=CH20H

196 R= OHO
.CH2OH
198-
197 E.E 199

198E,Z
CH2OH .COOMe
COOMe
AcO^
205
207 R= COOH
208 R= COOEt
510
-^208 +
(a) (i). PBra, r-BuOMe, CPC, 25 min; (ii) 2. KCN, DMSO, 2 h, 80%; (b) (i) KOH 25%,
MeOH, 90^C, 9 h; (ii) CH2N2, ether, QPC, 5 min, 68% of 195 and 196 (ratio 4:1) in two
steps; (c) Se02, ^BuOOH, CH2a2, 5 ^C, 6 h, 65%; (d) LAH, THF, 0°C-*rt, 12 h, 86%
of 197 and 198 (ratio 4:1); e) (i) NCS, DMS, CH2CI2, O^C, 2.5 h, 85%; (f)
CHjCOCHCHsCOOEt, NaH, n-BuLi, THF, 2.5 h, 79%; (g) Mn(OAc)3-2H20,
Cu(OAc)2-H20, HOAc, 68%. (h) (i) PDC, DMF, 24 h; (ii) CH2N2, ether, 75% in two
steps; (i) (i) NaBH4, MeOH, 0°C, 15 min, 91%; (ii) ClOAc, DMAP, 20 h, 95%; (j) (i).
O3, CH2CI2, -78°C, 20 min; (ii) PPhj, -78^C-^rt, 2 h., 91%; (k) (i) PhSeCl, EtOAc, 60 h,
(ii) H2O2, Py, CH2CI2, 0°C^rt, 15 min, 77%; (I) Tebbe's reagent, THF, 0°C, 17 min,
69%; (m) NaSCH2CH2CH3, DMF, 50°C, 26 h, 60%; (n) Pd(0Ac)2, p-benzoquinone,
HOAc, acetone, 18 h, 78% of 209 based on the amount of 207 in the mixture of 207 and
208; (0) (i) LDA, -78°C, THF, 20 min; (ii) TMSCl; (iii) PhSeCl; (iv) H2O2, Py, CH2CI2,
1 min, 40°C, 36%.
Scheme 21
The acyclic precursor 200 was obtained from commercially available

geraniol 192. Standard transformations on 192 led to a mixture of diols
197 and 198 in a 4:1 ratio favorable to the desired E,E isomer.
Regioselective chlorination of 197 yielded 199. Alkylation of the dianion
of ethyl 2-methylacetoacetate with 199 afforded 79% of 200. Oxidative
free-radical cyclization of 10 using a 2:1 molar ratio of Mn(0Ac)3 and
Cu(0Ac)2 provided 68% of the corresponding bicyclic intermediate.
Following a similar process to that described for the synthesis of
oidiolactone C, 203 was subjected to ozonolysis and the to the presence of
PhSeCl/H202 to give unsaturated ketone 205 which was homologated to
diene 206 using Tebbe reagent. Finally The Pd(II)-mediated
biscyclization of diacid 207 gave dilactone 209 which was transformed
into 210 again using the selenylation/oxidization protocol.
511
ABBREVATIONS
IC50 = A 50% growth inhibition concentration in vivo.

T/C = Ratio of life-span of the treted mice to that of the
control mice.
ED50 = dose required to be effective against 50% of treated
tumoral cells
IL-1 = interleukin-1.
TNF = tumor necrosis factor.
MIC = mininum inhibition concentration
LD50 = dose required to give 50% total mortality, larvae to
adults
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol, 28
ANTITUMORAL ACTIVITY OF LIPIDS A

STUDIES IN ANIMAL MODELS AND CANCER
PATIENTS
DANIELE REISSER, NOLWENN GAUTHIER, ALENA PANCE,

JEAN-FRANCOIS JEANNIN
Cancer Immunotherapy Research Laboratory, Ecole Pratique des

Hautes EtudesJNSERM U517, Faculty ofMedicine, 7 Bd Jeanne d 'Arc,
21079 Dijon, France,
ABSTRACT: LPS consist of an 0-specific chain, a core and a lipid A. It brgan to be

used as a tumor therapy since the XEX century, but it became clear in the 1980s that the
biological activity of LPS is due to their lipid A component. LPS and lipid A interact
with membrane receptors on target cells which initiate signal transduction. The end result
of the intracellular signaling is mainly the activation of N F K B and AP-1, which then
induce the expression of diverse genes such as cytokines, adhesion molecules, etc. Thus
the antitumoral effect of LPS and lipid A is indirect and relies on the induction of an
immune response, both innate and acquired. These components aflfect tumor
development mainly inhibiting tumor blood flow and inducing necrosis as well as
apoptosis of the tumor cells. Cancer treatments with LPS and lipid A have been tested in
animal models as well as at the clinical level, either alone or as adjuvants in therapeutic
vaccines. In animals, these treatments have achieved certain efficacy, which depends on
the type of molecule and the protocol used. In general, an increased survival has been
obtained, accompanied in some cases of tumor regression and cure. In clinical trials, the
induction of an immune response has been evidenced, but the results are so far too
preliminary to permit a definite conclusion. Nevertherless, these components represent a
hope for cancer patients and now it is necessary to extend the studies to clinical phase III
trials.
INTRODUCTION
Cancer has become the second cause of death in industrialized countries.

Numerous advances in molecular biology and immunology have
contributed to a better knowledge of carcinogenesis, tumor growth, and
tumor-host interactions. They have opened new therapeutic strategies,
such as gene therapy, even though a number of obstacles to their
successful application remain. Therefore the therapeutic advances in the
518
short term will probably consist in more conventional concepts. The

immunological unresponsiveness of patients to their growing tumors is
an important aspect, therefore strategies to overcome this
unresponsiveness are an important approach in cancer therapy. An
antitumoral immune response can be induced in vivo with endotoxins.
Important progress in lipid A chemistry has been achieved, which
together with preclinical studies done in the past decade, allowed clinical
assays that will be reviewed here. The chemistry of endotoxins and lipid
A will be briefly introduced from an historical point of view. The
biological properties of lipid A relevant to their antitumoral activities
will be reported with particular attention to specific receptors and their
signal transduction. Lipid A antitumoral activities as well as their
mechanisms of action in animal models and cancer patients will be
reviewed.
fflSTORICAL BACKGROUND
Richard Pfeiffer [1] introduced the term endotoxin, in contrast to toxins

or exotoxins, to name toxic bacterial components not excreted from the
living bacteria but released during bacteriolysis. The first indications
about their composition were given by Boivin in the years 1935-1945
[2,3]. He purified substances composed of polysaccharides and lipids
with only small amounts of proteins named lipopolysaccharides (LPS) by
Shear and Turner [4]. Now we know that LPS are constituents of the
outer membrane of Gram-negative bacteria. The chemical and biological
properties of LPS were extensively studied by Westphal and Liideritz [5].
They proposed in the sixties, that all LPS consist of 3 regions: O-specific
side chain, core and lipid A as a common architecture. The O-specific
chain, usually composed of a polymer of repeated oligosaccharide units,
is a structure specific for any given strain of bacteria, which is
responsible for the antigenic properties of LPS. The core is an
oligosaccharide structurally less variable, which contains at least one
Kdo (2-keto-3-deoxyoctulosonic acid). A majority of core regions are
characterized by the additional presence of heptoses (L-glycero-D-
mannose-heptose). Some of the cores have common segments, and
constitute the link between the O-specific chain and the lipid A. Lipid A
519
is the lipidic component and is the structurally most stable part of LPS.
Lipid A consists of a diglucosamine backbone acylated by ester-linked
and amide-linked long chain fatty acids. The nature of the backbone
glycosyl residues is the same for a large number of LPS. The precise
structure of the lipids AfromEscherichia coli (E. coli) and Salmonella
typhimurium (S, typhimurium) were obtained at the beginning of the
years 1980 by Takayama, Kusumoto, Galanos and Rietschel [6-8]. The
first synthetic lipids A were chemically synthesized by Kusumoto and
collaborators in 1985 [9]. Since then it became feasible to prepare non-toxic
derivatives of lipid A.
A link between bacteria and tumor ther^ was found early, at the
beginning of the XVni century [10]. By the end of the XIX century, Coley
[11] developed a treatment for cancer with a mixture of bacterial toxins.
In 1943 Shear and Tumer [4] found that the antitumor effect of Coley's
toxin was due to endotoxins, and after several decades it was shown that
the biological activity of LPS was due to the lipid A [5]. We investigated
the structures of lipids A with regard to their antitumor activities [12],
finding that the optimum in vivo activity is obtained with diglucosamines
acylated by 3 long chain fatty acids.
BIOLOGICAL PROPERTIES OF LIPID A
Lipid A in vivo
LPS and lipids A alike, when injected in vivo interact with diverse serum
proteins which can be divided in 3 groups, depending on their capacity to
modify their activities. The first group of proteins which decrease the
activities of LPS consist of high-density lipoprotein (HDL), low-density
lipoprotein (LDL), the bactericidal permeability-increasing (BPI) protein
[13], and the cationic protein CAP37 [14]. The proteins which do not
modify their activities are albumin, lactoferrin [13], transferrin,
hemoglobin [15], and lysozyme. Finally some proteins such as LPS
binding protein (LBP) augment their activities [16,17].
A few hours after its injection, circulating lipid A is found mainly in
liver, lung as well as in the spleen, adrenals and kidneys [18]. In these
520
organs and in the blood, lipid A will bind target cells via binding proteins
and receptors.
Lipid A signaling pathway
Different receptors for LPS have been identified, of which the scavenger
receptor and the CDl 1/CD18 receptor mediate uptake and detoxification
of LPS. In contrast CD14, a glycosylphosphatidylinositol (GPI)-linked
protein, initiates LPS signal transduction. Membrane-bound or soluble
CD 14 can bind LPS in the absence of LBP, however LBP increases this
binding. Lipids A also bind LBP, scavenger receptor [19], CD11/CD18
[20] and CD14 [21]. Of these, CD14 is likely to be the main pathway for
initiating downstream signaling events, although in some cases soluble
LPS-binding proteins permit lipid A signaling independently [22].
Membrane bound CD 14 receptors are found on the surface of
monocyte/macrophages. The soluble CD 14 is capable of binding to
CD14-negative cells such as endothelial [23] or smooth muscle cells [24]
and of inducing several signaling pathways. CD14 lacks transmembrane
and intracellular domains, probably acting through the binding of distinct
transmembrane proteins, such as the Toll Like Receptors (TLR), which
transduce the signal. A general agreement is that TLR4 mediates the lipid
A signaling pathway initiated by the binding of lipid A to CD 14, since
TLR4-deficient mice are unresponsive to LPS in vivo [25]. In contrast,
the role of TLR2 in lipid A (and LPS) transduction is debated [26].
TLR4 requires the presence of the MD-2 surface molecule to transduce
the lipid A signal and probably of one additional molecule. MD-2 is co-
expressed with TLR4 on the cell surface.
The TLRs have an intracellular domain (TIR) which binds to a
homologous domain in the adaptor protein, MyD88. This protein
contains a death domain which interacts with the death domain of the
mammalian Interleukine-1 Receptor- (IL-IR)-Associated kinase (IRAK).
IRAK binds another adaptor, TRAF6 which seems to bridge 2 signaling
pathways: a) through the adaptor TAB2 activates the nuclear factor
(NF)kB-inducing kinase (NIK) and IkB kinase (KK); b) through the
recently identified Evolutionarily-Conserved Signaling Intermediate in
the Toll pathway (ECSIT) protein, activates another cascade of kinases:
521
MEKK-1, ERK, p38, and JNK/SAPK. The results are the

phosphorylation of IkB and its dissociation from NFKB, thus permitting
the translocation of NFKB to the nucleus, as well as the activation of the
AP-1 transcription factors Jun and Fos.
Furthermore, the LPS signal transduction involves the activation of G
proteins, of phospholipases C and D, the formation of diacyl-glycerol
(DG) and inositol triphosphate (IPS). DG mediates the stimulation of
protein kinase C (PKC) and IPS induces an increase of cytosolic Ca^.
The LPS signaling pathway also involves tyrosine kinases, constitutive
nitric oxide (NO) synthase (cNOS), cGMP-dependent protein kinase,
Ca^ channels, calmodulin and calmodulin kinase [27,28], as well as the
MAP kinases [29] ERKl, ERK2 and pS8 [2S]. The intracellular events in
response to LPS are due to lipid A because they are inhibited by
polymyxin B which is known to bind lipid A [27] and they are
reproduced by lipids A [S0,S1].
The capacity of lipids A to activate a signaling pathway depends on
their structure and is species-dependent. Thus, the lipid A analog lipid
IVa and the Rhodobacter sphaeroides lipid A (RSLA) are LPS
antagonists in human macrophages [S2], while acting as LPS agonists in
hamster macrophages [SS]. On the contrary, in mouse macrophages, lipid
IVa is a LPS agonist and RSLA a LPS antagonist [21]. CD14 is not
involved in the species dependence [21] but TLR4 is responsible for the
species-specific recognition of the lipid A structure [SS].
Lipids A first induce the expression of early inflammatory genes such
as tumor necrosis factor- (TNF)-a, EL-lp, type 2 TNF receptor, IP-10,
DS, D8 and D2 genes [S4]. Then further genes are activated such as other
cytokines and receptors, adhesion molecules, acute-phase proteins, tissue
factors, as well as the inducible NOS (NOS H). These cascades of events
initiated by lipid A provoke in their target cells complex responses in
vivo, whose relevance in the host response to tumor growth is reviewed
below.
Lipid A tolerance
Lipid A can induce a state of hyporesponsiveness to its own effects (or to

LPS) in animals or humans. This effect is called LPS tolerance and by
522
analogy lipid A tolerance [35-37]. LPS tolerance can be divided in early

and late tolerance which is mediated by anti-LPS antibodies. However,
there is no late tolerance to lipids A because lipids A are not
immunogenic.
In vivo, after a sublethal dose of lipid A, a transient period of
hyporesponsiveness follows during which a lethal dose of lipid A or LPS
is well tolerated in mice, rats and humans [37-40]. The decrease of lipid
A toxicity e.g. death rate and weight loss, is probably due to stabilization
of blood pressure, coagulation and temperature, as well as a decrease of
cytokine concentrations in serum, such as TNF-a, IL-lp, IL-6, and
colony stimulating factors (CSF) [35,38,41-44]. Pulmonary and hepatic
levels of IL-ip, IL-6, TNF-a, G-CSF, IL-Ra, IL-10, interferon-(IFN).y,
macrophage-inducing protein (MlP)-la, MIP-ip, monocyte
chemoattractant protein (MCP)-l, and NOS11 mRNAs in mice submitted
to septic shock are lower when they have been pretreated with lipid A
[45]. Induction of lipid A tolerance is concomitant with corticosterone
production and does not occur in adrenalectomized mice [46,47].
Glucocorticoids down-regulate many genes including TNF-a, IL-lp, and
IL-6 [48,49], which is mediated by the inhibition of NFKB nuclear
translocation and activity. If some molecules are decreased in tolerant
animals, others are increased such as the TNF receptor 2 and IL-10,
which are able to antagonize the effects of TNF-a or to inhibit the
expression of numerous cytokine genes. Lipid A tolerance is due to
CD 14 positive cells, mainly monocytes/macrophages. We have shown
that when rats are injected with different lipids A, their peritoneal
macrophages produce high amounts of TNF-a, IL-ip, IL-6 and NO in
vitro after the first i.p. injection, but are no longer active after 2-5 i.p.
injections. Furthermore they do not respond in vitro to lipid A or LPS
stimulation for at least 2 weeks after the last injection, while normal
macrophages do (unpublished results). The production of CSF and IFN-y
also decreases during lipid A tolerance [42].
In vitro, mouse as well as human monocytes/macrophages can be
tolerized to lipid A [50-52]. The in vitro induced tolerance lasts one or
two days during which the CD 14 receptor is not down-regulated [52-54].
Uncoupling of the CD 14 receptor to downstream signaling pathways, or
a specific blockade of the transduction, is unlikely to occur as explained
523
below. We have shown that the tolerisation of Raw 264.7 murine

macrophages to LPS [52] and Hpid A (unpubHshed results), is induced at
the transcriptional level. An increased level of IxBa has been reported as
a mechanism for the down-regulation of IL-lp in tolerized monocytes
[55]. Diminished levels of TNF-a and NO have been ascribed to a
predominance of the p50 homodimeric form of NFKB, which represses
transcription in the nuclei of macrophages tolerized to LPS [50,52]. This
coincides with the fact that in tolerant cells the level of the pi05, which
is the proform of p50, is increased, and then processed [56]. In addition,
the secretory leukocyte protease inhibitor (SLPI) is a LPS inhibitor
induced by LPS in macrophages [55] which suppresses the LPS-induced
activation of NFKB. Thus SLPI inhibits the production of TNF-a and
NO, constituting another mechanism of LPS tolerance.
STUDIES IN ANIMAL MODELS
Animal models permit the investigation of the antitumoral effect of lipids

A and LPS [57]. We will first consider the acute effects observed after
the first injection of lipids A or LPS. In each case, their relevance to an
antitumoral activity will be discussed.
Acute effects of LPS and lipids A
Effect on general physiology
The hallmark of a bacterial infection is fever due to LPS pyrogenicity. It

may have some relevance in cancer as hyperthermia is tested as a cancer
treatment on its own, as reviewed by Christophi et al. [58]. This effect is
mediated by cytokines (mainly BL-lp) produced in response to LPS.
In himians, LPS can induce coagulation [59] which might involve
TNF-a [60]. The importance of coagulation for timior irrigation and
therefore its effect on tumor growth is difficult to evaluate. However,
Parr et al. [61] showed that it is involved in but not sufficient for tumor
regression induced by LPS in mice, since lymphocytes were also
necessary.
524
LPS are known to activate complement via the alternative pathway.

Regression of subcutaneous MH134 tumor in C3H/He mice was shown
to implicate the complement as part of the antitumoral effect of LPS [62]
but this effect has not been since documented.
LPS can provoke a multi-organ failure, due to the secretion of acute-
phase reactants such as platelet-activating factor (PAF), TNF-a, and
LBP. Multi-organ failure is also due to the secretion of prostaglandin E2
(PGE2) and inflammatory cytokines such as TNF-a, EL-ip, IL-6, whose
production may increase the secretion of the acute-phase reactant, LBP
[63], thus constituting an amplifying loop. The presence of a tumor may
modify this host response as will be discussed below.
LPS are toxic for rabbit endothelial cells in vivo [64] and Choi et al.
[65] showed that LPS in vitro toxicity for human endothelial cells was
Fas-Associated Death Domain (FADD)-dependent. LPS are also involved
in the activation of endothelial cells, increasing their expression of
adhesion molecules. Lipids A increase the expression of Intercellular
Adhesion Molecule (ICAM)-1 by human endothelial cells [66] which, by
enhancing the adhesion of leukocytes to the endothelium, plays a role in
the inflammatory process. In Long Evans rats, the increase in ICAM-1
expression is mediated by IL-lp and TNF-a after LPS instillation in the
airways [67]. Is adhesion an important phenomenon during the metastatic
process? Taichman et al. [68] showed that LPS facilitate the adhesion of
several human tumor cell lines on endothelial cells in vitro. In this
regard, LPS could be prometastatic by increasing the arrest of tumor cells
in capillaries. On the contrary, Jibu et al. [69] showed that LPS promote
the detachment of tumor cells from lung endothelium, thus having an
antimetastatic effect. LPS also increase the production of NO in bovine
endothelial cells, via a protein tyrosine kinase pathway [70]. The
antimetastatic effect of the NO secreted by endothelial cells has been
shown in vivo by Rocha et al. [71] in a model of murine lymphoma.
Finally, activated endothelial cells release cytokines, whose role will be
discussed later.
525
Effect on innate immunity
As components of bacterial cell walls, LPS and lipids A are first

recognized by the cells of the innate immune system and are able to
activate them.
The increased expression of adhesion molecules by the endothelium
may activate polymorphonuclear neutrophils (PMN) in rabbits [72].
During endotoxic shock, activated PMNs release their granule content
and secrete both proinflammatory and cytotoxic molecules. Pickaver et
al. [73] were the first to show PMN cytotoxicity against tumor cells. We
showed that PMNs are toxic for PROb colon tumor cells [74] in BDIX
rats. In vivo, PMNs have been implicated in the Schwartzman reaction
[75], and may be involved in LPS-induced tumor necrosis. PMNs, when
activated by LPS, synthesize and release NO. The role of NO in tumor
growth will be discussed later. The decrease in tumor growth after
intradermal injections of LPS is attributed to the induction of TNF-a
secretion by PMNs both in intradermal tumors (Meth A sarcoma in
BALB/c mice, MH-134 hepatoma in C3H/He mice, Lewis Lung
carcinomas in C57BL/6 mice) and pulmonary Meth A metastases
[76,77].
Historically, since the 1970s, the first cells considered as good
candidates in the fight against cancer were macrophages. Macrophages
were shown to destroy tumor cells non-specifically in vitro in the
presence of LPS [78]. Mannel et al. [79] considered them as the effector
cells in the regression induced by LPS of a fibrosarcoma in C3H mice. In
v/vo, the role of macrophages is controversial and difficult to prove: they
are considered either as friends [80] or foes as their tolerization is
concomitant with the antitumoral effect of lipid A [81].
However, we showed that no correlation exists between the capacity
of LPS and lipids A to activate macrophages from BDIX rats in vitro and
their in vivo antitumoral activity to PROb tumors except for ILl-p
production [12,82,83]. In any case, the importance of macrophages has
been emphasized because they are the main producer of TNF-a in
response to LPS.
Natural killer (NK) cells are mononuclear cells able to lyse tumor
cells without prior sensitization to specific antigens [84]. When NK cells
526
are activated in vitro, they differentiate into Lymphokine-activated killer

(LAK) cells with a broader panel of target cells. NK cells from mice
which had received an injection of LPS from E. coli or lipid A from
Porphyromona gingivalis (P. gingivalis) have an increased in vitro
activity [85]. In vivo, GLA-27 reduced metastasis in (C57BL/6X
DBA/2)F1 mice via NK cell activation when injected one day before
B16-F10 melanoma tumor cells [86]. Furthermore, pretreatment with
ONO-4007 (Ono Pharmaceutical Co, Osaka, Japan) increased NK
activity in the liver of C57BL/6 mice and decreased the nimiber of EL4
hepatic metastases via the secretion of the NK-activating cytokine IL-12,
byKuppfercells[87].
Effect on cytokine production
One of the most important effects of lipids A on tumor growth occurs

through the modulation of cytokine production.
The main cytokine whose production is induced by lipids A, is TNF-
a. Historically, this was the first mechanism described to explain the
efficacy of LPS on tumor regression in experimental models. Gratia and
Linz [88] in guinea pig liposarcoma, and Schwartzman and Michailovski
[87] in mouse sarcoma, observed that filtrates of bacteria cultures
induced hemorrhage and necrosis within the tumors. However,
Andervont [89] tested bacterial filtrates on several murine tumors, and
found that not all of them underwent haemorrhagic necrosis, while
Seligman et al. [91] concluded that in BALB/c mice bearing Meth A
sarcoma the in vivo effect of LPS is not direct, but can be attributed to a
circulating factor [92]. Similarly, necrosis of Meth A tumors in BALB/c
mice treated by lipids A may depend on TNF-a [93]. However the
phenomenon is not clearcut as injections of antibodies directed against
TNF-a did not prevent tumor regression [94]. Consistent with these
results, we have shown that lipid A induces regression of TNF-a-
insensitive cells [81]. For a complete regression, an acquired immune
response is necessary since tumor regression does not occur in T cell-
deficient mice [95] or nude rats [96]. Moreover, to achieve tumor
regression, several injections of the lipids A: SDZ MRL 953 (Sandoz,
Vienna, Austria), DT-5461 (Daiichi Pharmaceutical Co, Tokyo, Japan),
527
or OM-174 (OM Pharma, Geneva, Switzerland) are needed, and these

protocols induce tolerisation of rat macrophages which then no longer
produce TNF-a (unpublished results).
As is implied by its name, the first TNF-a-dependent mechanism
described was the induction of tumor necrosis in vivo through its role in
tumor vasculature. However the mechanisms of the in vitro toxicity of
TNF-a to tumor cells imply apoptosis rather than necrosis [97]. Tumor
necrosis in SCED (severe combined immuno-deficiency) mice treated
with LPS does not lead to the rejection of tumors [98]. Furthermore,
necrosis and tumor regression must be dissociated since anti-IFN-y
antibodies inhibit LPS-induced regression of Meth A sarcoma in mice,
but not the necrotic hemorrhage attributed to TNF-a. It is now accepted
that the antitumoral effect of TNF-a is indirect and dependent on
acquired immune response. Matsumoto et al. [99] reported that, while
TNF-a itself has no effect on hepatoma KDH-8 tumor cells in vitro, the
antitumoral effect of the lipid A ONO-4007 against KDH-8 tumors in
vivo is inhibited by anti-TNF-a antibodies in WKAH rat, showing an
indirect effect of TNF-a.
IFN-y is a cytokine produced by lymphocytes, whose production can
be induced by LPS. This effect may be indirect, as macrophages
activated by lipid A produce IL-12 and/or IL-18 that are IFN-y inducing
cytokines [100-101]. Furthermore, human endothelial cells treated in
vitro with LPS, induce the secretion of IFN-y by T lymphocytes [102]. Its
requirement for tumor regression was evaluated in vivo by Dighe et al.
[98] who showed that DFN-y-unresponsive Meth A fibrosarcoma is not
rejected by BALB/c mice treated with LPS. However, IFN-y might act in
combination with TNF-a, as shown in C57BL/6 mice bearing B16
melanoma [103]. Particularly in combination with IL-ip and TNF-a,
IFN-y induces the expression of NOS 11 and the production of NO [104].
Other IFNs may also be involved as the lipid A DT-5461 acts through
DFN-a/p as well as IFN-y [105].
Another cytokine whose secretion can be induced by LPS or lipid A is
EL-ip. It is principally secreted by macrophages, but a variety of other
cell types release it, such as dendritic cells, endothelial cells, NK cells
and lymphocytes. It may be directly cytotoxic to tumor cells on its own
528
[106] or in association with IFN-a, as is the case for B16 melanoma in

C57BL/6 mice in vivo [103].
Other cytokines are involved in the effect of lipid A on tumor growth.
OM-174 decreases TGF-P mRNA and protein in rat PROb tumors [107].
ONO-4007 increases TNF-a, IL-ip, IL-12, and 11-6 while decreasing
TGF-P levels [108]. The secretion of XL-12 by dendritic cells is also
induced by microbial products Uke LPS [109]. These cytokines have
further effects on other cell types, which could be considered as indirect
effects of LPS. In this respect, 11-6 and IL-12 increase LAK induction
[110],
Long term effects of LPS and lipids A
Effect on bloodflow and angiogenesis
Some long term effects of LPS in vivo are vasodilation and hypotension.
These phenomena are of great relevance in cancer since they modulate
tumor blood flow and consequently the availability of oxygen and
nutrients needed for tumor growth. They also modulate access of
circulating toxic cytokines and immune cells to the tumor. According to
MacPherson and North [111], LPS act at two levels: general by
decreasing cardiac output and intratumoral by affecting the endothelium.
Injection of DT-5461 decreases blood flow in Meth A fibrosarcoma
subcutaneous nodules in BALB/c mouse [112] and in VX2 carcinoma
liver nodules in the rabbit [113]. This decreased blood flow involves
TNF-a, IFN-a/p and IFN-Y [112], and cannot be reproduced by
intravenous (i.v.) injections of TNF-a [113].
Another effect of LPS may be the inhibition of tumor angiogenesis.
This was evidenced in melanoma B16- bearing C57BL/6 mice treated i.v.
with the lipid A GL-60, after an IFN-y injection [114]. It was confirmed
in the same model using DT-5461 alone injected i.v. [94]. This effect of
LPS is likely to be due to the production of TNF-a. This cytokine may be
toxic for the endothelium [64], inhibiting at the same time the motility
and proliferation of endothelial cells [59]. Sato et al. [115] showed that
TNF-a inhibits tumor-induced angiogenesis in a rabbit comeal pocket
assay. On the contrary, Vukanovic and Isaacs [116] concluded that TNF-
529
a secreted by intratumoral macrophages in response to LPS stimulates

angiogenesis in primary prostatic cancer in the rat. Furthermore, NO,
which is also induced by LPS, has been suspected of increasing
neovascularisation [117]. But LPS also induce the in vivo production of
IL-18 [118] which inhibits angiogenesis in mouse T241 fibrosarcoma
[119].
Effect on NO production
NO was recognized as a mediator of macrophage cytotoxicity in vitro

[120] but its role in tumor growth is not unequivocal. NO was shown to
be involved in tumor-induced immunosuppression [121,122]. Tumor
cells engineered to produce NO show a reduced proliferation in vitro but
the cells producing the highest levels of NO present the highest growth
rate in nude mice [117]. On the other hand, an inverse correlation was
found between NO production and melanoma K1735 lung metastasis
formation in C3H/HeN mice [123] or ESbL hepatic metastasis formation
in the DBA/2 mouse [71]. The antitumoral effect of IL-12 in the MCA
207 subcutaneous fibrosarcoma in C57BL/6 mice [124] and of IL-10 in
the mammary carcinoma 66-1 in BALB/c mice [125] involves NO
production. The effect of the NO produced by human tumor cells on
tumor growth in nude mice may depend on the p53 status of these cells
[126]. All these controversial results could be due to the utilisation of
different models. Using the same model, we have also found opposing
effects of NO on PROb tumor growth in BDIX rats, but they depend on
the NO-producing cells. Without treatment, the NO produced by
macrophages upon activation by tumor cells inhibits T cell proliferation,
thereby promoting tumor growth [122]. Treatment with lipid A induces
the tolerance of macrophages, causing them to produce no further NO.
On the contrary the NO produced by the tumor cells is autotoxic and its
production upon activation by the lipid A treatment is concomitant with
tumor regression [81,104].
Effect on acquired immunity
LPS can also have a role in the acquired immunity against cancer.
530
Recent studies emphasize the role of dendritic cells in the onset of an

efficient primary immune response [127]. On one hand, tissue or blood
dendritic cells phagocytose and process soluble or particulate antigens
such as apoptotic bodies. After maturation they efficiently present
cognate antigens to naive lymphocytes. In this respect, by inducing the
maturation of dendritic cells in BALB/c and CBA/J mice [128], LPS
injected i.v. can induce an immune response against tumors. For an
efficient immune response, T cells must not only encounter their cognate
antigen, but also receive simultaneous costimulation. According to
Matzinger [129], costimulatory factors are released by antigen-presenting
cells (APC) in the presence of a danger signal frequently delivered by
bacterial products. Therefore LPS or lipid A may allow specific
lymphocytes to differentiate and kill tumor cells they would have
otherwise ignored.
LPS can be directly mitogenic for T cells [130], but the antitumoral
activity of lymphocytes depends on antigen recognition by their TCR in
the context of the major histocompatibility complex (MHC) class I or II.
Though LPS enhance it, T lymphocyte activity requires APC [131]. The
effect of LPS on T lymphocytes has been shown to depend on monocytes
independent of MHC, but to be due to the secretion of costimulatory
signals and IL-12 in humans [132]. In vivo, LPS induces principally the
proliferation of CD8+ T lymphocytes, but also that of CD4+ T and B
lymphocytes through the activation of APC and secretion of IFN a/p in
C57BL/6 mice [133].
The importance of lymphocytes in cancer treatments based on LPS
had been evoked by Parr et al. [61]. Their role was later emphasized in
A/J mice bearing SA-1 sarcoma or BALB/c mice bearing Meth A
fibrosarcoma, treated with LPS [95], and in BDDC rats bearing PROb
colon carcinoma, treated with bacterial extracts [96]. Furthermore, SCID
mice treated with intraperitoneal (i.p.) injections of LPS are unable to
reject Meth A fibrosarcoma [96]. However, lipid A treatment was
efficient in nude mice bearing human pancreatic tumors [134].
A well-known effect of LPS in mice is mitogenicity to B lymphocytes.
Lipid A from P. gingivalis is shovm to be mitogenic in vitro even to
splenic B cellsfi-omLPS-unresponsive C3H/HeJ mice [31]. The lipid A
ONO-4007 is mitogenic to spleen cells from BALB/c mice [135]. The
role of antibodies in tumor regression has solely been documented in the
531
Studies of Sinkovics et al. [136] implicating antibodies in leukemia

regression in LPS-treated mice. The relevance of the antibody response
in the antitumoral effect of LPS remains controversial, as will be
discussed later concerning data obtained in clinical studies.
Tumor effect on the host response to LPS and lipid A
The importance of tumor-induced immunosuppression as a limitation of

the efficacy of immunotherapy has been demonstrated [137]. In tumor-
bearing animals, macrophages produce suppressive molecules:
macrophages from sarcoma-bearing BALB/c mice secrete TGF-P, IL-10
and PGE2 [121], and macrophages from BDIX rats bearing colon
carcinoma produce NO which inhibits lymphocyte proliferation [122].
The presence of a cancer may increase the susceptibility of its host to
LPS as was shown in AB6F1 mice bearing SA-1 sarcoma [138]. This
effect was attributed to the activation of macrophages in a model of
fibrosarcoma in BALB/c mice [139]. Compared to macrophages from
normal mice, these macrophages produce less GM-CSF [140], and more
TNF-a [141] in the presence of LPS in vitro. Furthermore, macrophages
from C57BL/6 mice bearing Lewis lung carcinoma produce less NO, and
more PGE2 [142] in the same conditions. In rats, adherent splenic cells
from WKAH rats bearing KDH8 hepatoma produce more TNF-a than
cells from normal rats when treated by ONO-4007 [135]. Finally, Dalton
lymphoma cells impair the PKC metabolism of C3H/He mouse
macrophages treated with LPS [143].
In vivo, TNF-a production in spleen and liver as higher in C3H/He
mice bearing MM46 or MH134-tumor, after an i.v. injection of ONO-
4007 [144]. In Sprague Dawley rats, the presence of Ward colon
carcinoma worsens LPS damage of the kidney (measured as blood urea),
liver (measured as blood alanine aminotransferase) and lung (measured
as blood partial oxygen pressure), decreases blood albumin (a witness of
leakage), increases plasma levels of nitrite-nitrate and, consequently,
LPS lethality [145]. On the contrary, an LPS treatment with low effect on
normal Dark agouti rats normalizes metabolic parameters in the blood of
mammary carcinoma-bearing animals [146].
The effects of lipid A on tumor are summed up in Fig. (1).
532
LYMPHOCYTE!
Fig. (1). Effect of lipid A on tumor

Lipid A acts ( ^ > ) on distinct cell typesfrominnate immunity (monocytes/macrophages, NK
cells, PMNs, dendritic cells) and adaptive immunity (lymphocytes).
These cells produce cytokines or NO ( C 3 ) which target ( • ) other cells or directly the tumor,
resulting in toxicity ( • « ^ ) .
Lipid A can also inhibit angiogenesis, bloodflowin the tumor and the secretion of
immunosuppressive TGF-p by the tumor cells.
533
Treatments in animal models
LPS and lipids A treatments
LPS treatments
In animals, the first in vivo experiments were performed with bacterial
extracts in a guinea pig sarcoma model by Gratia and Linz [88], and with
LPS in mouse primary subcutaneous tumors by Shear and Turner [4].
The antitumoral effect of LPS on the growth of subcutaneous or
intramuscular tumors has been extensively investigated [61,147-153]. On
ascitic tumors, treatment with LPS was shown to be efficient in some
cases [153-155] while failing in others [61,156].
We tested the effect of LPS in a model of peritoneal carcinomatosis
(solid tumor) induced by PROb colon cancer cells in syngeneic BDIX
rats. We showed that i.p. injections of LPS from E. coli can cure 20 % of
the rats [157]. Comparing the effect of LPS from different strains in this
model, we found that the efficacy depends on the bacterial strain and on
the structure of the lipid A used. Whatever the lipid A used, we have
shown a correlation with in vitro macrophage secretion of IL-ip but not
with NO, TNF-a or IL-6 [83].
Lipids A treatments
Here, we will emphasize on treatments with lipid A, considering only
curative treatments beginning after tumor cell injection. After a review of
the literature, we will detail the effects and mechanisms of DT-5461,
ONO-4007 and OM-174, the three lipids A which have been mostly
documented.
Parr et al. [61] showed that lipid A has the same antitumoral effect
as whole endotoxin preparations on murine L5178Y lymphoma. The
effects of LPS and synthetic lipid A treatments were compared by
Shimizu et al. [158-161] on Meth A fibrosarcoma in BALB/c mouse. The
antitumoral activity of different lipids A has also been investigated. Ribi
et al. [162] used an extract from S, typhimurium containing lipid A,
which when injected directly into hepatocarcinoma line 10 tumors in
guinea pigs shows an antitumoral effect. This activity is attributed to a
monophosphoryl diglucosamine derivative of lipid A [163]. Synthetic
lipid A analogs also proved to be active in this system [164], as well as
534
when injected i.p. in Meth A fibrosarcoma-bearing BALB/c mice

presensitized with Propionibacterium acnes. The i.v. injection of the
monoglucosamine GLA-27 slows the growth rate of RL-1 lymphoma and
Meth-A sarcoma in BALB/c mice [165]. Shimizu et al. [160] compared
several lipid A analogs with regards to their antitumoral activity using
Meth A tumors in BALB/c mice. Antitumoral activity is not correlated
with mitogenicity of C3H/He mice splenocytes and NO production in
Swiss mice macrophages, but is correlated with macrophage TNF-a
production. A synthetic lipid A was shovm to inhibit the growth of
tumors induced in nude mice by the injection of ML\ paca-2 or Panc-1
human pancreatic tumor cells, likely through TNF-a secretion by
macrophages [134].
Association of lipids A with other immunomodulators

Treatments with lipids A were tested in association with diverse
immunomodulators. Intravenous injections of GLA-60 in association
with IFN-Y were found to reduce B16 melanoma lung metastases in
C57BL/6 mice [114]. DT-5461 injected i.v. in association with
indomethacin increases the survival of BALB/c mice bearing peritoneal,
liver and lung C26 colon carcinoma [166] through the inhibition of
angiogenesis. MDP (muramyl dipeptide), a Mycobacteria derivative,
potentiates the antitumoral effect on Meth A fibrosarcoma in BALB/c
mice, of several lipids A (A-171, A-172, 56, A-606, A-607, A-608)
injected i.v., but the associations were less efficient than LPS alone
[160,167]. MDP also increases the efficacy of DT 5461 in the same
model [158]. The increase was correlated with an in vitro mitogenic
effect of DT 5461 on spleen cells, as well as the production of NO and
TNF-a by macrophages. In 1996, the same team tested several lipid A
analogues, finding that the association with MDP showed no better
efficacy than LPS on Meth A sarcoma in BALB/c mice. In these mice,
cyclophosphamide injected 7 days prior an ONO-4007 treatment in order
to inhibit the immunosuppressive response, enhanced the efficacy of the
lipid A [108].
Treatment with the lipid A DT 5461

The synthetic diglucosamine compound DT-5461 has been reported to
reduce the weight of various tumors including murine Meth A
535
fibrosarcoma, MH134 hepatoma, MM46 mammary carcinoma, Lewis

Lung carcinoma, and C38 colon carcinoma, but not that of C26 colon
carcinoma [168] or L5178Y lymphoma [169]. The tumor size was
reduced by a necrotic process. DT-5461 i.v. injections induced TNF-a
secretion in subcutaneous Meth A tumors in BALB/c mice [112] as well
as in B16-BL6 tumors in C57BL/6 mice, decreased angiogenesis, and
reduced the number of spontaneous metastases [94]. The effect of DT-
5461 was accompanied by a reduced blood flow in the tumor which
could be reversed by antisera directed against TNF-a, IFN-oc/|3, and IFN-
Y [105,112]. No effect on the life span of animals bearing ascitic tumors
was observed. The antitumoral effect of i.v. injections of DT-5461 in a
rabbit hepatic carcinoma [113] was associated with blood flow reduction
in the tumor area. In vitro, it was shown that in the murine macrophage
cell line J774, DT-5461 enhanced cytokine production using LPS
receptors [30], and that in the murine macrophage cell line RAW 264,
signal transduction involved phosphorylation of MAP kinases [170].
Treatment with the lipid A ONO-4007

The monoglucosamine compound ONO-4007, injected i.v. slowed the
growth of subcutaneous MM46 murine mammary carcinoma in C3H/He
mice, increasing TNF-a secretion by intratumoral macrophages [80]. It
induced TNF-a production by spleen cells from BALB/c mice bearing
intradermic murine Meth-A fibrosarcoma, as well as their proliferation in
vitro [135]. Intravenous injections increased the survival of WKAH rats
bearing KDH-8 hepatocarcinoma subcutaneous tumors, but had no effect
on rats bearing KMT-17 fibrosarcoma, or SST-2 mammary
adenocarcinoma [171]. In WKAH rats, ONO-4007 acted by inducing the
production of TNF-a [99], as was confirmed in C3H/He mice bearing
MM46 mammary carcinoma or MH134 hepatoma [144]. The efficacy of
this lipid A may be limited to TNF-a-sensitive tumors in WKAH rats
[172]. While 3 i.v. injections every 7 days inhibited TNF-a production in
liver and blood, no tolerization was found in tumors [99]. A similar
effect was seen in hamsters bearing pancreatic carcinoma [173]. The
prolongation of survival of WKAH rats bearing c-WRT-7
myelomonocytic leukemia by i.v. injections of ONO-4007 can be
explained by a differentiating effect [174] that could not be reproduced
536
by a cytokine (BL-la, IL-6 or TNF-a) treatment. Recently, Mizushima et

al. [175] showed that subcutaneous 13762NF mammary tumors, but not
peritoneal or lung tumors, were cured by i.v. injections of ONO-4007 in
F-344 rats. The efficacy of ONO-4007 was enhanced in mice bearing
Meth A fibrosarcoma when cyclophosphamide was injected 7 days prior
treatment in order to inhibit an immunosuppressive response [106].
Subcutaneous injections of ONO-4007 increased vascular permeability in
the skin of normal mice by increasing the production of TNF-a and IL-
ip [176]. In the same conditions, i.v. injections induced NO production
in the lungs [177] but these effects were not studied in the context of
tumor growth.
Treatment with the lipid A OM-174

We investigated the antitumoral activity of OM-174 in a model of
peritoneal carcinomatosis induced by PROb colon cancer cells in
syngeneic BDIX rats. These cells are chemoresistant [178], NK-resistant
[179] and TNF-a-resistant [81]. Without treatment, all rats die of their
tumors. Treatment of peritoneal carcinomatosis (solid tumors) always
started after the formation of macroscopic nodules up to 3 mm. The
cumulative volume of these numerous nodules corresponds to the volume
of a large tumor. An equivalent stage of tumors in himians cannot be
resected and have always a fatal evolution. OM-174, which is a
triacylated diglucosamine, has a partial structure ofE, coli lipid A [180].
Repeated i.v. injections of OM-174, every 2 days, cured 90 - 100 % of
the rats. Such a success has never been obtained with a treatment of this
kind. Tumor disappeared via the apoptotic pathway without an
inflammatory reaction. OM-174 is not toxic to tumor cells in vitro^
therefore it does not induce tumor cell apoptosis directly. The
establishment of a specific immune response was evidenced with a
Winn-type assay, e.g. the protection of naive rats against a tumor by the
injection of spleen cell from rats cured of the same tumor. Treatment
efficacy depended on the number and frequency of injections which
indeed induced the tolerance of macrophages to OM-174 decreasing their
TNF-a production [81]. After a peak following the 2 first injections,
TNF-a in tumors returned to basal levels. Moreover, PROb cells are
resistant to TNF-a in vitro, in consequence in our model, TNF-a is
probably not involved in tumor regression. On the contrary, the efficacy
537
of both DT-5461 in CDFl mice [169], and ONO-4007 in BALB/c mice

[99], depended on TNF-a. In our model, during lipid A-induced txmior
regression, NOS II mRNA and protein levels were induced in tumor cells
with the concomitant production of NO [104]. Neither OM-174 nor TNF-
a induced NO production by tumor cells in vitro, whereas NOSII
expression is induced by IFN-y and IL-ip in these cells [122]. Therefore,
the in vivo NOS II induction may be indirectly due to the presence of
IFN-y and IL-ip in the tumors of treated animals [96]. Accordingly, we
determined that the treatment with OM-174 causes IFN-y and IL-ip
accumulation in tumors, at the mRNA and proteins levels (unpublished
results). The NO thus produced is autotoxic for tumor cells provoking
their apoptosis. Moreover, treatment with OM-174 inhibited the synthesis
of TGF-pi by PROb tumor cells [107], therefore abrogating its
immunosupressive role [181]. Furthermore the inhibition of TGF-pl
enhanced the synthesis of NOSII [107], thus increasing the autotoxic
effect of NO on tumor cells.
Therapeutic vaccination
Lipids A are also used in therapeutic cancer vaccination to cure tumors.

In this case, lipids A are used as adjuvants, e.g. administered
simultaneously with tumor extracts or tumor antigens, to increase the
immunogenicity of the vaccine or to inhibit the tumor-induced tolerance.
Therapeutic vaccines were tested in BALB/c mice bearing TA3-Ha
mammary carcinoma. The treatment consisted of 4 subcutaneous
injections, at 3-6 days intervals, of Detox [a commercial preparation of
cell wall skeletons from Mycobacterium phlei and non-toxic
monophosphoryl lipid A from Salmonella minnesota (S. minnesota) in
squalane oil and Tween 80 from Ribi Immunochemical research,
Montana, USA] mixed with Thomsen-Friedenreich (TF) antigen coupled
with KLH (Keyhole Limpet Hemocyanin) performed 5 days after the
tumor cell injection. This vaccination achieved the survival of 25 % of
the mice. Pretreatment of mice with cyclophosphamide in order to inhibit
any suppressive response, increased survival to 50 % when the treatment
began 5 days after tumor cell injection, and to 90 % when the treatment
began 2 days after tumor cell injection. Both antibody as well as delayed-
538
type hypersensivity (DTH) responses were obtained. Moreover, lymph

node cells were protective in a Winn-type assay [182],
In C3H/HeN mice bearing MH134 hepatoma, monophosphoryl lipids
A from P. gingivalis or S, minnesota Re 595 increased the survival of
mice when administered in combination with timior cell lysates and
Freund*s incomplete adjuvant [31].
Conciusion
In conclusion, various lipid A have been tested as treatments for tumor-

bearing animals, using different routes (intratumoral, intraperitoneal,
intravenous, intradermic). While the intradermic route permits the use of
greater doses without toxicity [76,77], most of the studies were
performed using several i.v. injections of lipid A. Optimum doses range
from 1 to 5 mg/kg for the 3 lipids A DT 5461, ONO 4007 and OM-174 in
rats and mice. In our model of colon carcinoma in rats, we showed that
the i.v. treatment is more efficient than an intraperitoneal one [81]. In
general, most studies showed that the treatments increase survival, or
slow the growth of established tumors in mice
[80,93,94,99,105,112,135,144,160,168], rats [12,81,96,107,171-
174,181], and rabbits [113]. To our knowledge, only two laboratories
reported the total cure of established tumors. Mizushima et al. [175]
showed that ONO-4007 cures subcutaneous tumors but not
intraperitoneal or lung ones. Onier et al. [81] reported that OM-174 cures
90-100 % of rats bearing peritoneal carcinomatosis consisting of a large
number of nodules between 1 and 3 mm, while all untreated rats die of
their cancer.
Lipids A are generally considered to act through TNF-a secretion
[112]. For instance ONO-4007 was shown to be efficient only on TNF-a-
sensitive tumors [171,172], therefore, only well vascularized timiors can
be affected. However, in our model, we showed that TNF-a is probably
not involved since this cytokine peakes after the first two injections and
then retums to basal levels before tumor regression. The efficacy of OM-
174 relies on an indirect induction of an autotoxic production of NO by
the tumor cells [104]. Perhaps a more important aspect is the
immunogenicity of tumor cells. After apoptosis or necrosis of tumor
539
cells, apoptotic bodies or debris can be phagocytosed by macrophages

and dendritic cells. These cells will then present the immunogenic
peptides to helper T lymphocytes which will induce a specific immune
response.
CLINICAL STUDIES
The aim of phase I trials is to determine the toxicity of potential drugs.

The following phase II trials are designed to study the pharmacological
properties and the potential effectiveness of a drug. The aim of a phase
III trial is to study the efficacy and safety of a particular protocol.
Treatments with LPS
Several phase I trials have been performed with LPS from Salmonella
abortus equi administered i.v. in patients who suffered from
disseminated cancer. White blood cell number decreased after each
injection and retumed to basal level by 24 hours. There were no changes
in coagulation parameters, and no disseminated intravascular coagulation
was observed. After the first injection of LPS, increases in TNF-a
concentration and IL-6 activity in serum were detected. However LPS
tolerance which is accompanied by a decrease in TNF-a and IL-6
production depended on the intervals between repeated injections, but it
was not determined whether it was a benefit or a draw-back. Injections of
IFN-y prevented this decrease in TNF-a and IL-6, and ibuprofen
attenuated LPS toxicity [183,186].
In a phase II trial, patients with colorectal cancer or non-small cell
lung cancer, LPS showed a low grade toxicity and induced a reduction of
TNF-a concentration in serum. One complete remission, stable for 36
months, was achieved [187].
In another phase I trial, i.d. injections of LPS from Pantoea
agglomerans were given to patients who suffered from disseminated
cancer and received cyclophosphamide, and ibuprofen, to attenuate
fever. Increases in the serum concentrations of TNF-a, IL-6 and G-CSF
were observed, without tolerance [188].
540
The low response of cancer patients to LPS treatments may be due to

low maximal tolerated dose (MTD), 4 ng/kg. In order to avoid this
problem, several trials were performed with lipids A.
Treatments with lipids A
A phase I trial was conducted with monophosphoryl lipid A (MPLA)

prepared from S, typhimurium or S, minnesota injected i.v. in patients
with disseminated cancer. Fever, chills andfetiguewae Ihe most OMnmoti
side eflFects and the dose of 250 \x^jr^ i.e. (250x1.7): 65=6 jugl^ was estimated
acceptable [189].
Another trial used i.v. injections of the synthetic lipid A SDZ MRL
953 in patients with disseminated cancer who received ibuprofen. The
most common toxicity was fever, and the MTD was not reached. The
lipid A had no significant effect on the serum concentrations of TNF-a,
EL-ip, IL-8, G-CSF and IL-6. White cell number increased within 12
hours after the first injection, mainly due to PMNs, and then retumed to
normal after 48 hours [37].
In a recent phase I trial the synthetic lipid A analog ONO-4007 was
given by i.v. injections to patients with cancer unresponsive to the
standard therapy. The limited systemic toxicity disappeared within 24
hours. The MTD was defined as 125 mg/patient [e.g. (125:65=2 mg/kg].
The lipid A increased serum concentrations of TNF-a and IL-6, without
affecting the concentrations of GM-CSF, IFN-y and neopterin. There was
a significant drop in lymphocyte counts after injections, but no effect on
clotting parameters [190].
The results of phase I trials of LPS and lipids A in cancer patients

show that the tested lipids A are approximately 30,000 to 500,000 fold
less toxic than the LPS (table 1). The MTD in humans ranging from 6
|Lig/kg to 2 mg/kg, is lower than or close to the optimal doses of lipids A
observed in rodents. Humans are more sensitive to lipid A than rodents,
therefore it is possible that similarly to the toxic dose, the effective dose
for humans is lower than for rodents. To this day the very few existing
phase n trials cannot answer this question.
These trials are summed up in Table 1.
541
Table 1. List of tfie clinical tiiab performed with LPS or lipids A injected to cancer patients.
Phase Product Tumor Route MTD Tested 1

Parameters
Vosika et al. I Lipid A Diverse i.v. 250 jig/m^ CC
Cancer Immunol Immunother (Salmonella) 6^g/kg
1984
Engelhardt et al I LPS Diverse i.v. 4ng/kg CC, CK
JBiolRespModif (Salmonella)
1990
1 Engelhardt et al. LPS Diverse i.v. 8ng/mg CC, CK
Cancer Res (Salmonella)
1991
1 Mackensen et al. I LPS Diverse i.v. CK
Blood (Salmonella)
1991
1 Mackensen et al. LPS Diverse i.v. CC,CK
Eur Cytokine Netw (Salmonella)
1992
Ottoetal. II LPS Diverse i.v. CC, CK
Eur J Cancer (Salmonella)
1996
1 Goto et al. I LPS Diverse s.c. >1800ng/kg CK
Cancer Immunol Immunother (Pantoea)
1996
1 Kiani et al. Lipid A Diverse > 39.6 ng/kg CC, CK
Blood SDZMRL
1997 953
1 DeBonoetal, I Lipid A 1 Diverse I.v. 125 1 CK '
Clin Cancer Res ONO-4007 mg/patient
1 2000 2mg/kg
CC = cell count, CK = cytokines, i.v. = intravenously, s.c. = subcutaneously, MTD = maximum

tolerated dose
Therapeutic vaccines
Several trials of therapeutic vaccination used the adjuvant property of

lipid A to enhance the vaccination efficacy against human tumors, which
are often considered as weakly immunogenic, or even tolerogenic.
The first trials of therapeutic vaccination against cancer using lipid A
as adjuvant were performed on melanoma patients with 0.25 ml Detox.
The composition of commercial vaccines used as therapeutic vaccines in
humans is given in Table 2. Some trials used a pretreatment vsdth 300
542
mg/m of cyclophosphamide to inhibit an eventual suppressive response

to the vaccine.
Table 2 . Composition of commercial vaccines used as therapeutic vaccines in cancer patients.
DETOX (Ribi Immunochem Research, Inc., Hamilton, Montana, USA)

0.25 ml: 250 mg cell wall skeleton from Mycobacterium phlei, 25 mg monophosphoiyl lipid A from
Salmonella minnesota R595 prepared as an oil-in-water emulsion with 2% squalane and 0.4% Tween 80 in 2X
noraaal saline.
Melacine (Ribi Immunochem Research, Inc., Hamilton, Montana, USA)

Homogenates of melanoma tumor cell lines mixed with Detox.
THERATOPE sTn-KLH (Biomira Inc., Edmonton,AB, Canada)

100 mg sTn-KLH emulsified in 0.25 ml Detox. (sTn = sialyl-Tn = DAcNeu2-6aGalNAc-0-Ser/Thr).
OncoVax-P (Jenner Biotherapies, Inc, San Ramon, CA, USA)

1.2 ml: 100 mg/ml recombinant PSA + liposomes of dimyristoyl phosphatidylcholine, dimyristoyl
phosphatidylglycerol, cholesterol + 200 mg /ml monophosphoiyl lipid A from Salmonella minnesota R595.
Melacine
The first phase I trial, performed by Mitchell et al. [191] and several
further phase I and II trials by the same group, studying different
parameters [192,193] are summed up by Mitchell et al. [194].
Homogenates of 2 melanoma cell lines, mixed with Detox were injected
s.c. to melanoma patients. Toxicity was minimal and local. There was no
correlation between the antibody response against melanoma
determinants and the clinical response. The remissions were correlated
with the presence of cytotoxic T lymphocyte (CTL) precursors. Most of
the CD8+ and CD4+ isolated clones lysed MHC-matched melanoma
target cells. Histological studies of regressing lesions showed the
presence of CD3+ lymphocytes, mostly CD4H-, perivascularly and at the
periphery of the tumor as well as the presence of macrophages
throughout the lesions. Cyclophosphamide did not improve the number
of responding patients, however lESf-a given to non-responding patients
led to a clinical response.
The vaccine, referred to as Melacine (Ribi Immunochemical
Research, Inc.), contains homogenates of melanoma cells mixed with
0.25 ml Detox. In a multicenter phase II trial, with patients with
543
disseminated melanoma, the toxicity was moderate, mostly local. Pre-

CTL number increased, and the expansion of CD8+ T cells was
correlated with increased survival Clinical responses (remissions and
disease stabilizations) were obtained [195].
The efficacy of Detox was contrasted with that of other adjuvants.
Helling et al. [196], treated melanoma patients with cyclophosphamide,
comparing Detox, BCG and the saponin QS-21 in a vaccine containing
the ganglioside GM2, conjugated with KLH. Detox toxicity was only
local. The antibody response was not increased by Detox, in contrast to
the other adjuvants. Schultz et al. [197] used vaccines made of materials
shed from melanoma cell lines, mixed with Detox or alum. Local side
effects occurred in the Detox group. In this trial, an antibody response
was present and more frequent in the Detox group than in the alum group
while there was no difference in DTH. However, the disease-free
survival was lower in the Detox groups than the alum group.
Eton et al. [198] mixed irradiated melanoma cells with Detox.
Toxicity was only local. Peripheral blood mononuclear cell cytotoxicity
against autologous melanoma cells was correlated with survival, but no
NK cell cytotoxicity occured. Two major responses were obtained, not
correlated with DTH response.
Jheratope
Detox was also used in immunotherapy directed against other types of

cancers.
Vaccines generally used sialylated (s)Tn, which are mucin epitopes
expressed on epithelial tumors, conjugated with KLH. This vaccine was
commercialized as Theratope (Biomira Inc., Edmonton, Canada). In a
phase I study O'Boyle et al. [199] injected the vaccine to colorectal
cancer patients. Toxicity was only local, and an antibody response was
observed.
Several trials were performed on breast cancer patients treated with
cyclophosphamide. Little local toxicity was found, and an antibody
response was evidenced. Partial clinical responses and disease
stabilizations were obtained. [200-202]. Adluri et al. [203] compared
vaccines using Detox or QS-21 in an adjuvant therapy for colorectal
cancer patients. Toxicity was mostly local. An antibody response was
544
induced against synthetic epitopes but not against natural antigens. No

DTH response was detected.
A phase II trial was performed on metastatic breast cancer patients
with or without cyclophosphamide [204]. The results showed a local
toxicity. Cyclophosphamide was not efficient. An antibody response was
evidenced. No complete remission was obtained.
On the other hand, a randomized trial with breast, ovarian and
colorectal cancer patients, showed an increase in the antibody response
by cyclophosphamide. This antibody response was correlated with
increased survival, when antibody levels to mucins were low before
immunotherapy. The beneficial role of this response might be due to a
blockage of immunosuppressive mucins [205-207].
In another study involving patients with metastatic breast, colorectal
and ovarian cancer, increased anti-sTn titers were correlated with better
survival. Even if before treatment, elevated titers of antibodies against
the mucin MUCl, were correlated with a poor response to
immunotherapy, CTL precursors to the MUCl were detected in
carcinoma patients [208]. A vaccine using 10 |Lig/ml MUCl-KLH mixed
with Detox was injected s.c. in breast cancer patients treated with
cyclophosphamide. A weak antibody response, and an ex vivo CTL
response against HLA-matched adenocarcinoma cell lines were seen. No
correlation with the clinical outcome was available [209].
Theratope was given to patients with breast or ovarian cancer who
received peripheral blood stem cell rescue after chemotherapy. Toxicity
was mostly local. In vitro, NK activity which was low before
immunization returned to normal values, toxicity against cells bearing
sTn antigen appeared, and lymphocytes responded to sTn, by
proliferation and IFN-y production. Antibodies against sTn were detected
in 16 patients, while the anti-MUC-1 antibody titer decreased [210]. The
remissions were longer in treated patients and there was a tendency to a
decreased risk of relapse [211].
Another vaccine, formulated by mixing irradiated cells from colon
carcinoma cell lines with Detox was injected to patients with colorectal
metastatic adenocarcinoma, with or without DL-la [212]. The vaccine
induced local toxicity, and fatigue which was increased in the group
treated with IL-la. DTH occurred in both groups. No clinical response
was available.
545
Since point mutations of the ras proto-oncogene are often found in

cancer, a vaccine was made with mutated ras peptides mixed with Detox.
In a phase I study, CD4+ proliferation and CD8+ cytotoxicity specific to
the mutated peptide, were observed. The side effects were minimal and
one patient showed a stabilisation of the disease [213].
OncoVax
Trials of therapeutic vaccination against prostate cancer used OncoVax-P

(Jenner Biotherapies, Inc, San Ramon, California). OncoVax-P consists
of 200 ng monophosphoryl lipid A (similar to that used in Detox) added
to 1 ml liposomes and 100 ^g PSA (prostate-specific antigen). Patients
received injections by different routes (intramuscular, intravenous or
subcutaneous) according to the trial, with or without GM-CSF, IL-2 or
BCG and cyclophosphamide pretreatment. No serious side effects were
seen. DTH and antibody responses were achieved. Vaccination increased
the PSA-reactive T cell frequency as determined by IFN-y secretion, but
no toxicity against PSA-expressing target cells was detected. The most
effective strategy could not be determined, and no conclusion about the
clinical efficacy of the treatment was possible [214,215].
Conclusion
The phase I and phase II trials of therapeutic vaccines (Table 3.) show a
weak toxicity of lipids A, furthermore they show a stimulation of the
acquired antitumoral immune response. CTL responses correlate better
than antibody responses to clinical outcomes, in agreement with current
concepts of antitumor immunity. Phase HI trials are now necessary to
determine the effective protocol.
546
Table 3. List of clmical trials performed with lipids A as adjuvant of therapeutic vaccines
administered to cancer patients.
Phase 1 Adjuvant 1 Antigen Tumor Tested i

parameters
Mitchell et al. 1 I Detox Cell material 1 Melanoma AR,DTH,
Cancer Res, 1988 CTL
Mitchell etal., 1 Detox Cell material Melanoma AR,CTL
AfmNYAcadSci,\99^
II Detox Cell material 1 Melanoma AR,CTL
Schultzetal. I Detox 1 Cell material Melanoma AR,DTH
Vaccine, 1995 |
Elhot et al. II Detox Cell material] Melanoma
Semin Surg Oncol, 1993
1 Eton et al. I Detox Cell material Melanoma CTL,DHT
Clin Cancer Res, 1998
Longenecker et al. Detox sTn-KLH j Breast AR
Ann NY Acad Sci, 1993
Adluri et al. I Detox sTn-KLH Colorectal AR,DTH
Cancer Immunol Immunother, 1995
1 Miles et al. II Detox sTn-KLH Breast AR
Brit J Cancer, 1996
Reddish et al. Detox sTn-KLH Breast, ovarian, AR
Cancer Immunol Immunother, 1996 colorectal
MacLean et al. Detox sTn-KLH Breast, ovarian, AR
J Immunother, 1996 colorectal
MacLean et al. Theratope sTn-KLH Breast AR
J Immunother, 1996
1 MacLean et al. Theratope J sTn-KLH Breast, ovarian AR
J Immunother, 1997
Sandmaier et al. I Theratope sTn-KLH Breast, ovarian AR,CTL,
J Immunother,\999 1 PR
Holmberg et al. Theratope sTn-KLH 1 Breast, ovarian CTL
Bone Marrow transplant, 2000
1 Reddish et al. Detox MUCl-KLH 1 Breast AR, CTL
Int J Cancer, 1998
Kleif et al. I Detox Ras Colorectal, CTL, PR
J Immunother, 1999 pancreas, lung
Woodlock et al. I Detox Cell material 1 Colorectal DTH
J Immunother, 1999
1 Harris et al. m\ QncoVax-P PSA 1 Prostate AR,DTH
1 Semin Oncol, \999 1
AR = antibody response, CTL = cytotoxic T lymphocytes, DTH = delayed type hypersensitivity,

PR = proliferative response
547
CONCLUSION
LPS immunotherapy was the first immunotherapy for cancers assayed in

patients in spite of its toxicity. The standardisation of animal models of
cancer, the discovery of the LPS composition and of lipid A activity, the
discovery of lipid A structure leading to its chemical synthesis, and the
synthesis of lipid A derivatives far less toxic than the natural lipids A,
restarted research in this field. At the same time, advances in
immunology allowed a better understanding of the mechanisms of action
of LPS and lipids A in whole organisms.
Most of the articles report a significant enhancement of the life span
of treated animals. However recent results show that it is now possible to
definitely cure animals bearing large tumors while the untreated
counterparts die of their cancer. The most effective structure so far
consists of diglucosamine acylated by 3 long chain fatty acids, and the
substitution of the diglucosamine backbone is now under investigation.
The best treatments consist of repeated i.v. injections, where frequency is
an important parameter, and the optimal dose is not necessarily the
maximal one. With the same lipid A, the best treatment schedule changes
from one animal model to the other. Comparative studies have not been
performed to elucidate if species and tumor origin and/or the
immunogenicity of tumor cells and their immunosuppressive effect are
important parameters at the origin of these differences.
Three lipids A have been more intensively studied in animal models,
all of them having indirect effects, mediated in vivo by the immune
system. For two of them, DT-5461 and ONO-4007, TNF-a is an
important mediator acting at the vascular level that provokes tumor
necrosis. For the third one, OM-174, the treatment induces the
accumulation of IFN-y and DL-lp in tumors, which activate NOS II
transcription in tumor cells that produce autotoxic NO, which then
provokes the apoptosis of tumor cells. At the same time this treatment
inhibits the production of TGF-pl by tumor cells which reduces the TGF-
pi induced immunosuppression and enhances NO production. Acquired
immune response, probably completes the tumor regression started by the
apoptosis process and, most probably induces specific memory.
Important questions have to be answered to facilitate the definition of
protocols for humans. For example, is the lipid A tolerance of
548
macrophages an important parameter for treatment effectiveness ? The

answer will influence the choice of doses and frequency of injections, in
order to determine whether to increase progressively the dose of lipid A
injected to each patient or not.
Several lipids A have been tested in cancer patients: MPLA, SDZ
MRL 953, and ONO-4007 were injected i.v. in phase I trials. The
maximal tolerated dose found is lower than or close to the optimal dose
defined in animals. Humans are more sensitive to lipid A than rodents so
it is possible that similarly to the toxic dose, the effective dose is lower in
humans than in animals.
Because it requires small amounts of lipid A generally injected s.c,
the adjuvant effect of lipid A has been largely investigated in cancer
patients, but only with MPLA. Phase I and phase II trials show weak
toxicity of different vaccines with MPLA and the development of an
immune response. Phase III are now necessary to find an effective
protocol.
Therefore it is now to soon to know or to predict whether the lipids A
will become an antitimioral medicine. A great deal of data are now
available, which justify and impose the necessity of phase III trials. New
efforts have to be made quickly because of the thousands of patients who
will die in the near future.
ABBREVIATIONS
APC = antigen-presenting cells; BPI = bactericidal permeability-

increasing protein; CSF = colony stimulating factor; CTL: cytotoxic T
lymphocytes; DG = diacyl-glycerol; DTH = delayed-type hypersensivity;
ECSIT = Evolutionarily-Conserved Signaling Intermediate in Toll
pathway; FADD = Fas associated death domain; G-CSF = granulocyte
colony stimulating factor; GPI = glycosylphosphatidylinositol; HDL =
high density lipoprotein; ICAM = intercellular adhesion molecule; i.d.:
intradermal; IKK == IkB-inducing kinase; IL-lp = interleukine-lp; IFN-y
= interferon-y; IP3 = inositol triphosphate; IL-IR = IL-1 receptor; IRAK
= IL-lR-associated kinase; i.p. = intraperitoneal; i.v. = intravenous; KLH
= Keyhole Limpet Hemocyanin; LAK = Lymphokine-activated killer;
LBP = LPS binding protein; LDL = low density lipoprotein; LPS ==
549
lipopolysaccharide; MCP = monocyte chemoattractant protein; MDP =

muramyl dipeptide; MIP = macrophage inflammatory protein; MHC =
major histocompatibility complex; MPLA = monophosphorylated lipid
A; MTD = maximum tolerated dose; NIK = K-inducing kinase; NFKB =
nuclear factor KB; NK = natural killer; NO == nitric oxide; NOS == nitric
oxide synthase; PAF = platelet-activating factor; PGE2 = prostaglandin
E2; PKC = protein kinase C; PMN = polymorphonuclear neutrophils;
PSA = prostate-specific antigen; RSLA = Rhodobacter sphaeroides lipid
A; s.c. = subcutaneous; SCID == severe combined immuno deficiency;
SLPI = secretory leukocyte protease inhibitor; TF = Thomsen-
Friedenreich; TLR = Toll Like Receptor; TNF-a = Tumor necrosis
factor-a.
ACKNOWLEDGEMENTS
The authors thank Conseil Regional de Bourgogne, association pour la

Recherche contre le Cancer (ARC), Ligues contre le Cancer de
Bourgogne et de Haute-Mame and Fondation pour la Recherche
Medicale for theirfinancialsupport.
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistryy Vol. 28
Prevention of Cancer Chemotherapy Drug-Induced Adverse

Reaction, Antitumor and Antimetastatic Activities by
Natural Products
YOSHIYUKI KIMURA
Second Department of Medical Biochemistry, School of Medicine, Ehime

University, Shigenobu-cho, Onsen-gun, Ehime 791-0295, Japan.
ABSTRACT: Although it has recently been thought that a number of medicinal plants
and foodstuffs have antitumor and antimetastatic activities, the basis for this hearsay is
unclear. Therefore, to clarify whether natural products have antitumor and antimtastatic
actions, I have been using biochemical and pharmacological approaches to study the
natural products isolated from various medicinal plants and foodstuffs. In the review, we
will introduce the biological and pharmacological actions of various components isolated
from some medicinal plants and foodstuffs on tumor growth and metastasis in
tumor-bearing mice. Chitosan and fish oils prevented the adverse reactions such as
gastrointestinal toxicity and myelotoxicity caused by cancer chemotherapy drugs without
interfering the antitumor activity of chemotherapy drugs. Stilbenes derivatives isolated
from Cassia or Polygonum species inhibited the tumor growth and metastasis to the lung
in highly metastaic tumor-bearing mice. Furthermore, I found that stilbenes inhibited the
angiogenesis in in vivo and in vitro models.
INTRODUCTION
Cancer is the largest single cause of death in both men and women,
claiming over 6 million lives each year vôrldwide. Cancer
chemotherapy drugs such as 5-fluorouracil (5-FU) derivatives, cisplatin
(CDDP), mitomycin, doxorubicin, taxisol, etc. have been used extensively
for the treatment of certain types of cancer. However, with these
treatments, severe gastrointestinal toxicity with diarrhea and mucosis, and
hematologic toxicity with leukopenia and immunosuppression, appear to
be dose-limiting factors. Furthermore, the removal of malignant tumor by
surgical operation, radiation therapy and/or adjuvant therapy with cancer
chemotherapy drugs may be curative. However, the removal of certain
cancers, for example, breast carcinoma, colon carcinoma and osteogenic
sarcoma, may be followed by the rapid growth of distant metastases to
lung, liver etc. Therefore, efforts are underway to develop new modulators
that inhibit the adverse reactions without loss of antitumor activity and
new drugs having antitumor and antimetastatic activities without adverse
560
reactions. In this review, I describe the following articles.
1) Prevention by Natural Products of Adverse Reactions Induced by

Cancer Chemotherapy Drug without Loss of Antitumor Activity.
a) Prevention by Chitosan of Myelotoxicity, Gastrointestinal Toxicity and

Immunocompentent Organic Toxicity Induced by S-Fluorouracil (5-FU)
[1] or Doxorubicin [2] without Loss of Antitumor Activity in
Tumor-Bearing Mice,
Chitin and chitosan are polymers with molecular weight of about 1000
kDa, and contain more than 5000 acetylglucosamine and glucosamine
units, respectively. Chitin is widely distributed in natural products such as
the protective cuticles of crustaceans and insects, as well as being found in
the cell walls of some fungi and microorganisms, and is usually prepared
from the shells of crabs and shrimps. Chitin is converted to chitosan by
deacetylation with 45% NaOH at lOO'^C for 2 h. Though chitosan is
reported to augment the natural killer activity, the antitumor activity of
chitosan is not clear yet. First, I examined the antitumor effects of
chitosan, but it had no effect. Gastrointestinal toxicity and myelotoxicity
are caused by the 5-FU after the phosphorylation in the digestive tract and
bone marrow tissue. To clarify whether chitosan enhances the antitumor
activity of 5-FU or doxorubicin and prevents the adverse reactions induced
by 5-FU or doxorubicin, I examined the antitumor activity and adverse
reactions, such as myelotoxicity, immunocompetent organ toxicity, and
gastrointestinal toxicity of combined treatment with chitosan and 5-FU or
doxorubicin in sarcoma 180-bearing mice.
5-FU (12.5 mg/kg twice daily) plus chitosan (150, 375 and 750 mg/kg
twice daily) inhibited the tumor growth as well as 5-FU alone. Chitosan
(150 and 750 mg/kg twice daily) blocked the reduction of blood leukocyte
number caused by 5-FU administration, and it prevented the injury of the
small intestinal mucosa membrane and delayed the onset of diarrhea
induced by 5-FU "Fig. (1), Fig. (2) and Fig. (3)". Furthermore, chitosan
(750 mg/kg twice daily) prevented the reduction of spleen weight induced
by 5-FU in sarcoma 180-bearing mice "Fig. (4)", and the reduction of
lymphocyte, CDS^ and NKl.l.^ T cell numbers induced by 5-FU "Fig.
(5)".
Intraperitoneal doxorubicin (5 mg/kg on days 1 and 8 after inoculation
of tumor cells) significantly inhibited tumor volume and tumor weight,
compared with sarcoma 180-bearing mice. Similarly, doxorubicin plus
chitosan (200 and 800 mg/kg twice daily) also inhibited the tumor growth,
compared with tumor-bearing mice "Fig. (6)". On the other hand, a
remarkable reduction in body weight of mice after 8 days was observed in
the mice receiving intraperitoneal doxorubicin compared with
tumor-bearing mice. Oral administration of chitosan (400 and 800 mg/kg
561
I I sarcoma ISO-bearing mice

I I sarcoma 180-bearing mice
B SFU f^J Chitosan
H ^-^ E 2 I Chitosan
E
o
E
51
5 2<)|- I I ii iii i -a
^- m
' LM
a 0.0"-
5-FU(12.5 mg/kg x2)
Chitosan(ing/kg x2)
-V—1—J—^^^1.
- +
21 <id 0 I-
5-FU(12.5mg/kgx2)
Chitosan(mg/kg x2)
-
-
+
-
+
150
+
375
+
750
Fig. 3. Inhibitory effect of chitosan on gastrointestinal toxicity P»g -*• Inhibitory effect of chitosan on immunocompetent organ
(reduction ofsucrase activity in small intestinal mucosa) toxicity (reduction of spleen weight) induced by 5-FU in
induced by 5-FU in sarcoma 180-bearing mice. sarcoma 180-bearing mice.
Results are expressed as means ± S.E. of 9 mice. KtsnlXs are expressed as means ± S.E. of 9 mice.
I I sarcoma 180-bearing mice I I sarcoma 180-bearing mice
H 5-FU ^ ^ Chitosan H ^'^ [ X J Chitosan

P<0.05
•a
I I
I E
5-FU(12 5mg/kgx2)
Chitosan(ing/kg x2)
mm
Fig. 1. The combined antiitumor activity of 5-FU and chitosan
in sarcoma 180-bearing mice.
750
8
5-FU(12.5 mg/kg x2)

Chitosan(mg/kg x2)
Fig. 2. Inhibitory effect of chitosan on myelotoxicity of 5-FU in

sarcoma 180-bearing mice.
5-FU or 5-FU plus chitosan was administered orally twice "^"y Results are expressed as means ± S.E. of 9 mice.
for 8 days. On day 9, blood and tissues were obtained.
Results are expressed as means ± S.E. of 9 mice.
562
• CDS"^ T Cell H Nkl.l.'^TCell
p<0.05
4.0
3.0
H
+
WD
2.0
o
s
0)
u
+ 1.0
00
Q
U
0.0
5-FU(12.5mg/kgx2) + +
Chitosan(mg/kg x2) 150 750
Fig. 5. Combined effects of S-FU and chitosan on the numbers ofCD8^ andNKLl.^
Tcells in spleen ofC57BL/6 mice.
5-FU or 5-FU plus chitosan was administered orally twice daily for 7 days.
On day 8, the mice were killed by cervical dislocation and their spleens were
quickly removed. CD8"^ and NKl. 1 ."^ cell populations were analyzed by
flow cytometry.
twice daily) prevented the doxorubicin-induced reduction in body weight

after 12 days "Fig. (7)". In addition, the intraperitoneal administration of
doxorubicin to tumor-bearing mice reduced the weight of small intestine
and the sucrase activity of its mucosal membrane. The oral administration
of chitosan (400 and 800 mg/kg twice daily) prevented the
doxorubicin-induced reduction in small intestine weight and sucrase
activity "Fig. (8)". Therefore, it is concluded that the combination of
chitosan and 5-FIJ or doxorubicin might be useful for the prevention of
adverse reactions such as gastrointestinal toxicity, immunotoxicity and
myelotoxicity, caused by 5-FU or doxorubicin without loss of antitumor
activity. Orally administered chitosan was retained for a long period in the
small intestine, suggestive of possible diffusion of chitosan into the
563
intracellular space of villi in the small intestine. Therefore, it seems likely

that the protective mechanism for the 5-FU or doxorubicm-treated
gastrointestinal toxicity by the orally administered chitosan may be due to
the formation of 5-FU- or doxorubicin -chitosan complex m the sntiall
intestinal mucosa through the diffusion of chitosan into the small mtestmal
villi without interfering the anitumor activity of 5-FU or doxorubicin.
I I I I I.. • • I I I I i I
10 12 14
it
10 12 14 Day Day
j DXR(5mg/kg,ip) DXR(5mg/kg,ip)
TDXF
I
it
, DXR(5mg/kg,ip)DXR(5mg/kg,ip)
yoxR
SarcomalSO
Sarcoma 180
Inoculation inoculation
Fig. 6. The combined effects doxorubicin and chitosan Fig. 7. The combined effects ofdoxorubicin and chitosan
on tumor volume in sarcoma 180-bearing mice. on body weight in sarcoma 180-bearing mice.
Results are expressed as means ± S.E. of 10-20 mice •P<0.05. Significantly different from sarcoma 180-bearing mice.
I.
I
I
DXR DXR
(5 mg/kg/wcek, ip) (5 mg/kg/wcek. ip)
(mgfltg X 2/<tay, po) (mg/kg x 2/diy, po)
Fig. 8. The preventive effects of chitosan on doxorubicin-induced gastrointestinal toxicity in sarcoma I

l-bearing mice.
a) small intestine weight; b) sucrase activity in small intestinal mucosa
Results are expressed as means ± S.E. of 10-20 mice.
b) Prevention by Carp Extract of Myelotoxicity and Gastrointestinal

Toxicity Induced by 5-FU without Loss of Antitumor Activity in
564
Tumor-Bearing Mice [3].

Carp (Cyprinus carpi) has been used in Korea, China and Japan as a health
food source. In ancient Chinese medicine, carp was eaten as diuretic, and
as a remedy for eye fatigue. In Japan, carp meat and blood have
traditionally been eaten as a tonic. Although it has recently been thought
that carp extract has antitumor activity, the basis for this hearsay is unclear.
Therefore, to clarify whether carp extract has antitumor effects, the
antitumor effect of carp extract, and the combined effect of 5-FU plus carp
extract on antitumor activity and adverse reactions were investigated in
sarcoma 180-bearing mice. As shown in "Fig. (9)", carp extract had no
effect on survival time in ascites-type sarcoma 180-bearing mice,
indicating that carp extract did not possess direct antitumor activity. Next,
I attempted to investigate the combined effect of 5-FU and carp extract on
antitumor activity in solid-type sarcoma 180-bearing mice. Carp extract
was found to prevent the occurrence of myelotoxicity as determined by the
reduction of leukocyte number, and of gastrointestinal toxicity, as
indicated by the reduction of the weight of the small intestine, induced by
5-FU without loss of the anitumor activity of 5-FU "Fig. (10), Fig. (11)

m S-FU f y j | S-FU + carp extract
P<0.05 pô.o.
Sarcoma ISO-bearing mice

Carp extract (50 mg/mouse x 2/day)
Carp extract (100 mg/mouse x 2/day)
12 16 20
•
Day
0.0
5.FU(12.5mg/kgx2)
Carp extract
(m^mouse x 2)
Mlii
Fig. 9. Effect of carp extract on survival time in asccites-type
sarcoma 180-bearing mice. Fig. 10. Antitumor effect of 5-FU and 5-FU plus carp extract.
5-FU or 5-FU plus carp extract were administered orally twice
Carp extract was administered orally twice daily until death, daily for 8 days. On day 9, blood and tissues were obtained.
starting 12 h after implantation of tumor cells.
and Fig. (12)". These findings indicate that carp extract could be
beneficial as health food source for the prevention of adverse reactions
such as myelotoxicity and gastrointestinal toxicity induced by the cancer
chemotherapy drug 5-FU.
565

I I sarcoma ISO-bearing mice
JIH 5-FU r i / J 5-FU + carp extract
I 5-FU r y j 5-FU + carp extract
5-FU(12.5mg/kgx2) . + + + 5-FU(12.5mg/kgx2) . + + +
Carp extract Carp extract
(mg'mousex2) 50 100 (mg/mousex2) 50 100
Fig. 11. Inhibitory effect of carp extract on myelotoxicity (reduction Fig. 12. Inhibitory effect of carp extract on gastrointestinal toxicity
of leukocyte number) induced by 5-FU. (reduction of the weight of the small intestine) induced by
5-FU.
c) Fish Oils Enhance S-FU-Induced Antitumor Activity without a Loss of

Adverse Reaction of5'FU[4J.
Fish oils that contain high amounts of the n-3 polyunsaturated fatty acids
eiocosapentaenoic acid (EPA, 20:5, n-3) and docosahexaenoic acid (DHA,
22:6, n-3) have been suggested to decrease the risk of development of
cardiovascular disease. Freshwater fish oil carp oil are not rich in n-3
polyunsaturated fatty acids, but tuna oil is rich in n-3 polyunsaturated fatty
acids such as EPA and DHA "Table (1)".
Table 1. Tlie fatty acid components of carp oU and tuna oil.
Fatty acid composition (%) Carp oil Tuna oil

Myristic acid n4:0) 2.0 -
Palmitic acid (16:0) 23.8 17.9
Palmitoleic add (16:1 n-T) 7.8 -
Stearic add (18:0) 3.3 3.8
Oleic add (18:1 n-9) 36.6 17.5
Linoleic add (18:2 n-6) 18.2 3.7
Arachidonic add (20:4 n-6) - 1.9
Eicosapentaenoic add (EPA) 0.9 7.7
(20:5 n-3)
Docosahexaenoic add (DHA) 2.3 26.1
(22:6 n-3)
Others 5.1 21.4
566
First, I examine the antitumor activity of the oral administration of two

fish oils (carp oil and tuna oil) in sarcoma 180-bearing mice. Carp oil and
tuna oil (0.2 and 0.4 ml/mouse) had no effects on tumor growth "Table
(2)".
Table 2. Effects of carp oil and tuna oil on the weights of body and tumor in sarcoma 180-bearing mice.
Mean ± SE
Final body weight (g) Final tumor weight (g) (T/C)
Sarcoma 180-bearing mice 35.5 ± 1.06 3.29 ± 0.78 (100%)
(Control)
Carp oil (0.2 ml/mouse) 36.8 ± 0.89 2.26 ± 0.54 (68.7%)
(0.4 ml/mouse) 37.5 ± 0.55 2.40 ± 0.77 (72.9%)
Tuna oil (0.2 ml/mouse) 38.7 ± 0.68 2.54 ± 0.80 (77.2%)
(0.4 ml/mouse) 36.1 ± 1.21 2.59 ± 0.73_(78.7%)
Results are expressed as mean ± SE of 10 mice in each group.
h)t
Sarcoma 18(M)earing mice (control)
1000 W
5-FU(12.5mg/kg)
1000
Sarcoma 18&4)earmginice(coiiirol)
5-FU(12.5mgAg)
1 5-FU + tuna oiK0.2ml)
5-FU + tuna oiK0.4ml)
800 V
5-FU + carp cMl(0.2inJ/inousc)
S-FU + carp oil(0.4 mlAnousc)
\
' %
!
i^
r'
/
'
1
h-
600 k
400 1
^^
1• 200 1
*Jm
^m* 0'
2 4 6 8 10 12 14 Day 0 2 4 6 8 10 12 14 Day
Fig. 13. Effects of the combination of5-FUplus carp oil (a) and 5-FUplus tuna oil (b) on
tumor growth in sarcomaJSO- bearing mice.
5-FU, 5-FU plus carp oil or 5-FU plus tuna oil was admnistered orally daily for 14 days.
Results are expressed as means ± S.E. of 9-10 mice.
*P<0.05, Significantly different from sarcoma 180-bearing mice.
Next, I examined the combined effects of 5-FU plus two fish oils (carp
oil and tuna oil) on the antitumor activity and adverse reactions compared
to the effects of 5-FU alone (12.5 mg/kg). I found that carp oil (0.4
ml/mouse) or tuna oil (0.2 or 0.4 ml/mouse) enhanced the ability of 5-FU
(12.5 mg/kg) to prevent tumor growth, without increasing adverse
567
400
5-FU (12.5 mg/kg)

300
0 - S-FU + carp oil (0.4 ml/moouse)
5-FU + tuna oil (0.2 ml/mouse)
I 200
20 40 60 100 120
Time (min)
Fig. 14. 5-FU levels in the plasma of mice after oral co-administration of 5-FU plus carp oil or
5-FUplus tuna oil
Results are expressed as means ± S.E. of 5 mice in each group.
•P<0.05, Significantly different from the administration of 5-FU (12.5 mg/kg) alone.
reactions such as myelotoxicity (the reduction of number of leukocytes,

platelet and red cells) (data not shown) and immunocompentent organ
toxicity (the reduction of spleen and thymus weights) "Fig. (13) and Table
(3)".
Table 3. Effects of the combinatioii of 5-FU |dus carp oil or tuna oil on the weights of spleen and thymus
sarcoma 180-bearing mice.
Animal No. Mean:tSE

Spleen (mg) Thymus (mg)
Sarcoma 180-bearing mice
(Control) 10 190.0 ± 30.5 47.95 ± 5.42
5-FU(12.5 mg/kg) 10 149.0 ± 16.8 50.56 ± 3.43
5-FU -H caip oil (0.2 ml/mouse) 10 159.0 ± 23.1 36.69 ± 4.19
+ carp oil (0.4 ml/mouse) 10 158.0 ± 10.3 49,53 ± 3.74
5-FU + tuna oil (0.2 ml/mouse) 10 132.0 ± 4.90 42.23 ± 6.00
+ tuna oil(0.4 ml/mouse) 9 152.2 ± 9.25 50.33 ± 3.61
Results are expressed as mean ± SE of 9 -10 mice in each group.
As shown in "Fig. (14)", the 5-FU levels in the blood of mice were
about 133.8 ± 27.5 and 285.0 ± 19.3 ng/ml, respectively at 5 and 15 min
568
after the oral administration of 5-FU (12.5 mg/kg) and then decreased
rapidly. On the other hand, the blood 5-FU levels after the
co-administration of 5-FU plus carp oil (0.4 ml/mouse) were 99.7 ± 42.4,
48.9 ± 15.6, 39.9 ± 17.2 and 18.6 ± 9.75 ng/ml, respectively, at 5, 15, 30
and 60 min. The blood 5-FU levels after the co-administration of 5-FU
plus tuna oil (0.2 ml/mouse) were 376.4 ± 172.1, 182.6 ± 113.0, 33.9 ±
2.10 and 22.8 ± 5.73 ng/ml, respectively, at 5, 15, 30 and 60 min. The area
under the curve (AUC) (0-120 min) of blood 5-FU levels was reduced by
the oral co-administration of 5-FU with carp oil or tuna oil. Apparent Tmax
was shortened by the oral co-administration of 5-FU with carp oil or tuna
oil. However, AUC (0-4 h) of [6-^H]5-FU incorporation into RNA
fraction of tumor after the co-administration of 5-FU plus carp oil or tuna
oil was similar to that of 5-FU alone "Fig. (15)". These results suggest that
the co-administration of 5-FU plus carp oil or tuna oil enhanced the
5-FU-induced antitumor activity without adverse reactions.
li ^H.5-FU(12.5 mg/kg, 18.5MBq/kg)

- • — ^H-S-FU + carp oil (0.4 ml/mouse)
^H-S-FU + tuna oil (0.2 ml/mouse)
OL
0.0 0.5 1.5 2.0 2.5 3.0 3.5 4.0
Time (h)
Fig. 15. [6'^H] 5'FU incorporation into RNAfractionsof tumor after oral co-administration of
[6'^H]5'FUplus carp oil or [6-3 H]5-FU plus tuna oil

*P<0.05, Significantly differentfromthe administration of 5-FU (12.5 mg/kg, 18.5 MBq/kg) alone.
569
2) Isolation of Antitumor Substance from Basidiomycete Fungus

Agaricus blazei and its Mechanism [5].
The Basidiomycete fungus Agaricus balzei Murill has traditionally
been used as a health food for the prevention of cancer, diabetes,
hyperlipidemia, arteriosclerosis and chronic hepatitis. It has been reported
that A. balzei is used by 300,000 to 500,000 persons for the prevention of
cancer and/or as an adjuvant with cancer chemotherapy drugs after the
removal of a malignant tumor. The hot water extract of A. blazei has
potent antitumor activity in sarcoma 180-bearing mice, and the antitumor
substance was postulated to be the p-(l-6)-glucan fraction. However, the
antitumor effects of lipid fractions have not been well studied. I examined
the antitumor activities of various substances isolated from the lipid
fraction of A. blazei via oral administration to identify the active
substances. Tumor growth was retarded by the oral administration of the
lipid fraction extracted from A. blazei with a chloroform/methanol (1:1,
v/v) mixture at a dose of 800 mg/kg during the 20 days treatment in
sarcoma 180-bearing mice. On the other hand, tumor growth was not
affected by the oral administration of the acetone-insoluble fraction. The
acetone-soluble fraction, which had higher antitumor activity, was divided
into two fractions through treatment with n-hexane. The n-hexane-soluble
and -insoluble fractions (800 mg/kg) also inhibited the tumor growth
"Table (4)".
Table 4. Effects of various lifnd fractions of chloroform : methanol extract (l:l^v/v) (a), acetone- soluble
and -insoluble fractions (b), and n-hexane-soluble and -insoluble fractions (c) on tumor volume at 20 d
and tumor weight at 21 din sarcoma 180-bearing mice^
(a)
\^rious lipid Tumor volume Inhibition Tumor weight Inhibition
fractions mm^ % mg %
Control 4826.9±1150.6 - 4470.0±870.6 -
Chloroform 1087.3± 567.6* 77.5 844.2±425.1* 81.1
methanol extract
(b)
"Various lipid Tumor volume Inhibition Tumor weight Inhibition
Control 928.81250.9 - 827.7±381.2 -
Acetone-soluble 124.1+83.6* 86.6 108.6+83.4* 86.8
fraction
-insoluble 649.1+140.9 30.1 490.5±382.3 40.7
fraction
570
(c)
Various lipid Tumor volume Inhibition Tumor weight Inhibition
Control 766.9±302.9 - 812.0±277.2 -
Hexane-soluble 152.0±74.6* 80.2 163.9±150.7* 79.8
fraction
-insoluble 75.7±24.6* 90.1 54.6±21.4* 93.3
fraction
^Various lipid fractions (800 mg/kg) were orally administered for 20 day in sarcoma 180-bearing mice.
Inhibition ratio (%) was measured as tumor volume or tumor weight of various lipid fraction-treated mice/
tumor volume or tumor weights of control mice. Each value represents the means ± S.E. of 10 mice. *P<0.05,
Significantly different from sarcoma 180-bearing mice.
The substance with the antitumor activity in the n-hexane-insoluble

fraction was isolated through silica gel column chromtography, eluted with
an acetonitrile/methanol (3:2) mixture and identified as ergosterol "Fig.
(16)".
Ergosterol
F i g . 16. Antitum or substance of Agaricus blazei
The oral administration of ergosterol to sarcoma 180-bearing mice

significantly reduced tumor growth at doses of 400 and 800 mg/kg "Fig.
(17)" without adverse reactions, such as the decreases in body, epididynal
adipose tissue thymus, and spleen weights and leukocyte numbers induced
by cancer chemotherapy drugs (data not shown). To clarify the antitumor
activity of ergosterol, I examined the effects of ergosterol on
tumor-induced angiogenesis using two in vivo models. Intraperitoenal
administration of ergosterol at doses of 5, 10 and 20 mgy^g for 5
consecutive days inhibited the neovascularization induced by Lewis lung
carcinoma cell (LLC)-packed chambers "Fig. (18)". Moreover, I examined
that the inhibitory effects of ergosterol on Matrigel-induced
neovascularization. Female C57BL/6 mice were subcutaneously
inoculated with Matrigel containing acidic fibroblast growth factor (aFGF)
571
and heparin with or without ergosterol Ergosterol inhibited the

Matrigel-induced neovascularization "Fig. (19) and Table (5)".
Sarcoma 180-bearingmice (Control)
4000 h + Ergosterol (lOOmg/kg)
+ Ergosterol (200mg/kg)
+ Ergosterol (400 mg/kg)
H- Ergosterol (800 mg/kg)
3000
<D
4 2000 h
1000
Days after inoculation

Fig. 17. Effects of oral administration of ergosterol isolated
fromAgaricus blazei on tumor growth in sarcoma
180'bearing mice.
Solid-type sarcoma 180 was prepared by subcutaneous transplantation into

the right abdomen of mice on day 0. The indicated amounts of ergosterol
were administered orally for 20 consecutive days, starting 12 h after the
implantation of tumor cells.

*P<0.05, Significantly different from sarcoma 180-bearing mice (control).
572
LLC-induced Angiogenesis ^ ,^^ „^ _ ,,^^

(Control) + Ergosterol (5 mg/kg) + Ergosterol (10 mg/kg)
Fig. 18. Effects of ergosterol on neovacidarization in C57BL/6 mice

bearing LLC cell-packed chamber.
Chambers packed with LLC cells were subcutaneously implanted into

a dorsal air-sac of C57BL/6 mice on day 0.
Ergosterol was intraperitoenallyadministered from days 1 to 5.
+ Ergosterol (20 mg/kg)
Table 5. Effects of ergosterol on the weights and hemoglobin contents in the gels 5d after implantation of
Matrigel supfdemented a FGF and heparin^
Treatment Matrigel weight Hemoglobin content

mg mg/Matrigel
Matrigel alone 103.16 ± 10.15* 2.6 ±0.68*
Matrigel + a FGF (1 ng/ml) + heparin 371.60 ± 39.75 21.0 ± 4.00
(64 units/ml) (Control)
Matrigel, a FGF, heparin 185.58 ± 44.40* 6.4 ± 1.86*
+ ergosterol (400 ng/ml)
Matrigel, a FGF, heparin 108.84 ± 9.69* 3.8 ±0.58*
+ ergosterol (800 pig/ml)
^C57BL/6 mice were each injected subcutaneously with 0.5 ml of Matrigel supplemented 1 ng /mL of a FGF
and 64 U/ml of heparin in the presence or absence of ergosterol (400 \k% or 800 (ig/ml). The mice were killed on
d 5 with an overdose of pentobarbital, and the gels were removed, weighed, and then the hemoglobin contents
in the gels were determined. Each value represents the means ± S.E. of 5 mice. *P<0.05, Significantly different
from Matrigel/aFGF/heparin mixture-treated mice.
From these results, it seems likely that the antitumor activity of

ergosterol might be due to direct inhibition of angiogenesis induced by
solid tumors.
573
1 cm
Matrigel atone Matrigel
a FGF{1 ng/ml) Matrigel/a FGF/Heparin
Heparin(64 unrts/ml) + Ergosterol
(400^g/ml) (800^g/ml)
Fig. 19. Photograph of Matrigel 5 daysfater subcutaneous

injection with 0,5 ml of Matrigel alone (a), Matrigel
supplemented with aFGF and heparin in the presence
or absence of ergosterol.
3) Antitumor and Antimetastatic Activities by Natural Products in

Ibmor-Bearing Mice.
a) Isolation of Active Substances from Cassia garretiana Heartwood on

Primary Solid Tumor Growth and Lung Metastasis in Lewis Lung
Carcinoma (LLC)-Bearing Mice and These Mechanisms [6, 7].
574
The heartwood of Cassia garrettiana Craib (Leguminosae) is a Thai

drug called "Sa mae sarn". It has been used as a mild cathartic in folk
medicine. Although it has recently been thought that extracts of C.
garrettiana heartwood have antitumor activity, the basis for this hearsay is
unclear. In addition, the pharmacological effects of this drug have not
been fully investigated and the active substances have not been isolated.
Therefore, to clarify whether the heartwood of C. garrettiana has
antitumor effects, here I first examined the effects of a methanol extract of
C garrettiana heartwood on tumor growth and lung metastasis in
LLC-bearing mice. A methanol extract (500 mg/kg twice daily) of the
heartwood of C. garrettiana inhibited the tumor growth and metastasis to
the lung in LLC-bearing mice "Table (6)".
Table 6. Effects of MeOH extract of Cassia garrettiana heartwood on tumor volume and lung metastasis
in LLC-bearing mice
No. of Tumor volume (mm^ Lung metastasis

Animals Mean ± S.E. (no. of colonies)
Mean ± S.E.
LLC-bearing mice 8 1410.3 ± 764.5 4.75 ± 1.92 (6/8)
(control)
+ MeOH ext. 8 482.3 ±195.1* 2.00 ± 1.25 (3/8)
(500 mg/kg X 2)
Values are expressed as means ± S.E. of 8 mice of each group.
* P<0.05, Significantly different from LLC-bearing mice (control).
PR
OH
OH
Piceatannol: R=H
Piceatannol acetate: R=COCH3
OH
Cassigarcl A
Fig.20. Antitumor and antimetastaic substances of Cassia garrettiana heartwood
Compounds 1 and 2, possessing antitumor and antimetastatic activities,

were isolated from the methanol extract. Compounds 1 and 2 were
identified as cassigarol A and piceatannol (3, 3', 4, 5'-tetrahydroxy
stilbene), respectively, based on the ^H-NMR spectral data and products
575
formed by oxidation with potassium permanganate "Fig. (20)". I examined

the effects of the cassigarol A, piceatannol and piceatannol acetate on
tumor growth in LLC-bearing and on lung metastasis in primary
tumor-removed mice.
2500 r LLC-bearing mice(control)
+ Cassigarol A(50mg/kg x 2/day)
2000
1500
Q 1000
500
0
i i i i i i i i i i i i i i
0 2 4 6 8 10 12 14 Day
Fig. 21. Effects of cassigarol A on tumor growth in LLC-bearing mice.
Solid-type LLC was prepared by subcutaneous transplantation into the right backs of mice
on day 0. Cassigarol A (50 or 100 mg/kg) was administered orallt twice daily for 14 days.
Results are expressed as means ± S.E. of 4-7 mice in each group.
*P<0.05, Significantly different from LLC-bearing mice (control).
As shown in "Fig. (21)", cassigarol A at the dose of 50 mg/kg x 2/day,

significantly inhibited the tumor growth on day 13 whilst at the dose of
100 mg/kg X 2/day, it significantly inhibited the tumor growth on days 11,
13 and 15, as compared to the growth in LLC-bearing mice. On the other
hand, piceatannol did not affect the tumor growth during the experimental
period (through day 14), but piceatannol acetate (100 mg/kg twice daily)
576
significantly inhibited the tumor growth on day 12 "Fig. (22)'
L L C - b e a r i n g m i c e (control)
LLC-bearing mice (control)
piceatannol a c e t a t e ( 5 0 m g / k g x 2 / d a y )
— piceatannol(50mg/kg x 2/day)
piceatannol(100mg/kg x 2/day) piceatannol a c e t a t e ( 1 0 0 m g / k g x 2 / d a y )
0 2 4 6 8 10 1 14 Day 2 4 6 8 10 12 14 Day
2
Fig. 22. Effects of piceatannol (a) and piceatannol acetate (b) on tumor growth in LLC-bearing mice.
Results are expressed as m e a n s ± S.E. o f 4 - 7 m i c e in e a c h group.

* P < 0 . 0 5 , Significantly dififerent fi'om L L C - b e a r i n g m i c e (control).
On day 15, tumor weight reduced by piceatannol acetate (100 mg/kg twice
daily), but piceatannol did not affect "Fig. (23)".
g H LLC-bearing mice
j g a l piceatannol-treated mice
Y^ piceatannol acetate-treated mice
p<0.05
100 . »
I
1
1
i
LLC-removed mice
0
Piceatarmol ^/^ ^r^
(A <A 6 8 10 12 14 16 Day
(mg/kg x 2/day) ' 5U luu -
Piceatannol acetate
Survival time
(mg/kg X 2/day) " 50 100
Fig. 24. Effects of cassigarol A on survival time and survival rate in
carcinectomized mice
Fig. 23. Effects of piceatarmol and piceatannol acetate on tumor weight
On day 15, the solid tumor tissues were removed imder anesthetic
in LLC-bearing mice.
pentobarbital and w e i r e d . Thereafter, cassiagarol A w a s again
Results are expressed as means ± S.E. of 4-7 mice in each group. administered orally twice daily for 17 days, starting 24h after resection
of tumor tissues on day 0. T h e survival time and numl>er of surviving
*P<0.05, Significantly diffaent from LLC-bearing mice (control). tumor-removed mice were determined.
577
Cassigarol A, piceatannol and piceatannol acetate, at the dose of 50 and

100 mg/kg X 2/day, prolonged the survival time and increased the survival
rate as compared to those in tumor-removed mice "Fig. (24) and Fig.
p<0.05
-•••BlfBa- • Q - - - ! l -
30 h
73
40
• carcinectomized mice
• piceatannol (SOmg/kg x2/day)i
• piceattannol(100mg/kgx2/day)
20
piceatannol acetate(50mg/kg x 2/day)
piceatannol acetate(100mg/kg x 2/day)
0»-
Carcinectomized mice
0 2 4 6 8 10 12 14 16 18 20 22 Casigarol A 50 mg/kg x2 100 mg/kg x2
Survival time (day)
Fig. 26. Effeas of cassigarol A on the number of colonies oflung
Fig. 25. Effects of piceatannol and piceatannol acetate on
metastasis in carcinectomized mice.
survival time and survival rate in carcinectomized
Chi day 15, the solid tumor tissues were removed under anesthetic pentobarbital and weighed. Thereafter, piecatannol, piceataimol acetate
"Fig. (25)" or casssigarol A 'Tig. (26)" was again administered orally twice daily for 17 days, starting 24 hours after resection of tumw tissues
on day 0. The survival time and number of surviving tumor-removed mice were determined.
Resuhs are expressed as means ± S.E. of 4-7 mice in each group.
Cassigarol A, piceatannol and piceatannol acetate inhibited the increases

of metastasis to the lung "Fig. (26) and Fig. (27)". Furthermore, to clarify
the antitumor and antimetastatic activities by cassigarol A, piceatannol or
piceatannol acetate, 1 examined the inhibitory effects of the above active
substances on the formation of capillary-like networks (angiogenesis) of
human umbilical vein endothelial cells (HUVECs). Cassigarol A inhibited
the angiogenesis of HUVECs at concentrations of 10 to 100 JAM "Fig.
(28)". Piceatannol also inhibited the angiogenesis of HUVECs at
concentrations of 10 to 100 jiM, but its acetate did not affect "Fig. (29)".
Therefore, it is suggested that the antitumor and/or antimetastatic activities
of cassigarol A, piceatannol might be due to the inhibition of tube
formation (angiogenesis) of vascular endothelial cells.
578
I I Normal mice H Carcinectomized mice

Carcinectomized mice treated with piceatamiol
Carcinectomized mice treated with piceatamiol acetate

15 r
£<a05
p<0,05
10
p<0.05
o
*5 \p<0.05
cd
OL
Normal Carcinectomized mice
mice
piceatamiol
50 100
(mg/kg X 2/day)
piceatamiol acetate 50 100
(mg/kg X 2/day)
Fig. 27. Effects ofpiceatannol andpiceatannol acetate on the number of
metastatic colonies in the lungs of carcinetomized mice.
On day 15, the solid tumor tissues were removed, and then cassigarol A was again
administered orally twice daily for 17 days, starting 24 h after resection of tumor
tissues on day 0. On day 18, the surviving tumor-removed mice were killed and the
metastasis to the lung were observed.
Results are expressed as means A} S.E. of 4-7 mice in each group
579
n CMitrol H i Piceatannol [2 Piceatannol acetate
p<0.05
i! 20
11 « 10
10 50 100
Piceatannol (>xM)
Cassigarol A ( M M ) Piceatannol acetate (M M)
Fig. 28. Effects ofcassigarol A on Matrigel-induced capillary- pig. 29. Effects of piceatannol andpiceatannol acetate on Matrigel-induced
like tube formation by HUVEC capillary-like tube formation by HUVEC.
HUVECs were seeded on the Matrigel in 270 (d of DMEM supplemented with 20% FBS and incubated with the indicated amounts of
cassigarol A, piceattanol or piceatannol acetate for 24 h in a humidified 5% CO2 atmosphere.
Results are expressed as means ± S.E. of 4 experiments.
b) Stilbene Derivatives loslated from the Roots of Polygonum Species

Inhibit Primary Solid-Tumor Growth and Lung Metastasis in LLC-Bearing
Mice and These Mechanism [8, 9].
Stilbenes are naturally occurring phytoalexins found in medicinal plants of
Polygonum species znd Rheum species (Polygonaceae) [10,11]. Among
OH
HO
Resveratrol
CH2OH
23,5,4 '-Te trah ydroxys tilb e ne -

2-0-D-ghicoside
OH
Fig. 30. Stilbenes isolated from the roots of Polygonum species

580
the stilbene derivatives, resveratrol (3,4',5-trihydroxystilbene) and

resveratro-3-O-D-glucoside (piceid) are also found in grapes and red wine.
Previously, I found that piceid and
2,3,5,4'-tetrahydroxystilbene-2-O-D-glucoside reduced the elevation of
lipid levels [12], and that 2,3,5,4'-tetrahydroxystilbene-2-0-D-glucoside
strongly prevented liver damage induced by high lipid peroxidized diets
[13]. Furthermore, I reported that resveratrol strongly inhibited the
formation of 5-lipoxygenase products,
5-hydroxy-6,8,ll,14-eicosatetraenoic acid, leukotrienes B4 and C4, and the
cylcooxygenase product thromboxane B2 from arachidonic acid [14-16].
I found that the inhibitory effect of resveratrol on arachidonic
acid-induced platelet aggregation [14]. Resveratrol and its derivatives
have been further shown to strongly inhibit the degranulation of human
polymorphonuclear leukocytes [16]. Although resveratrol is reported to
contain a cancer chemopreventive agent, the inhibitory action by
resveratrol on distant metastases to other organs from primary
solid-tumors is as yet unproven. In this review, I describe the effects of
three stilbenes "Fig. (30)" isolated from medicinal plants and grapes on
tumor growth and lung metastasis in mice bearing highly metastatic LLC
tumors. 2,3,5,4'-Tetrahydroxystilbene-2-0-D-glucoside and piceid
inhibited tumor growth time-dependently after oral administration of 150
or 300 mg/kg twice daily, respectively "Fig. (31)". These stilbenes also
inhibited lung metastasis in LLC-bearing mice "Table (7)".
Table 7. Effects of 2^,5,4'-tetrahydroxystilbene-2-O-D-glucoside and piceid on

cancer metastasis* lo iung, sfdeen and thymus in LLC-bearing Mice
Metastasis to spleen (mg) Thymus (mg)

Lung(%)
Normal 0/4 (0) 71.72 ± 3.10 56.04 ± 9.21*
LLC-bearing mice
(Control) 3/5 (60) 139.8 ± 42.18 27,33 ± 7.70
+ 2,3^,4'-
Tetrahydroxystilbene-
2-O-D-glucoside 5/5(100) 145.9 ± 53.19 18.64 ± 9.10
(50 mg/kg X 2/day)
(150 mg/kg X 2/day) 1/5 (20) 126.4 ± 35.38 46.31 ± 8.34
+ Piceid
(100 mg/kg X 2/day) 5/5(100) 129.9 ± 45.55 30.79 ± 5.26
(300 mg/kg X 2/day) 1/5 (20) 78.74± 11.58 42.78 ± 7.81
Values are expressed as means ± S.E. from 4 - 5 animals. *P<0.05, significantly different from LLC-bearing
mice (control mice).
581
a) b)
3500 LLC-bearing mice (control)
3500
+ 2,3,5,4'-tetrahydroxystilbene-
2-O-D-glucoside
(50 mg/kg X 2/day)
+ 2,3,5,4'-tetrahydroxystilbene-
Piceid (100 mg/kg x 2/day)
2-0-D-glucoside
2500 X Piceid (300 mg/kg x 2/day)
(150 mg/kg X 2/day) I
E
I 2000 2 2000
1500
>
o 1500
1000 1000
I t i I I
8 12 16 20 24 28 32 Day 8 12 16 20 24 28 32 Day
Fig. 31. Effects of 2,3,5,4'-tetrahydroxystilbene-2-O-D-glucoside (a) and piceid (h) on tumor

growth in LLC-bearing mice.
Solid-type LLC was prepared by subcutaneous transplantation into the right hind paw on
day 0.2,3,5,4'-Tetrahydroxystilbene-2-6>-D-glucoside or piceid was administered orally
twice daily for 32 consecutive days, starting 12 h after tumor implantation.
Results are expressed as means ± S.E. of 4-5 mice in each group.
2,3,5,4'-Tetrahydroxystilbene-2-0-D-glucoside or piceid administered

orally may be converted to an aglycone form of resveratrol by hydrolysis.
Therefore, I examined the effects of resveratrol on tumor growth and lung
metastasis in LLC-bearing mice, DNA synthesis of LLC cells, and
capillary-like tube formation (angiogenesis) of vascular endothelial cells.
Tumor growth and final tumor weight were significantly inhibited by the
intraperitoneally administered resveratrol at doses of 2.5 and 10 mg/kg
"Fig. (32)". Resveratrol (2.5 and 10 mg/kg) significantly reduced the
number of tumor cell colonies that metastasized to the lung compared the
LLC-bearing mice "Fig. (33)". Resveratrol inhibited the DNA synthesis in
LLC cells with a 50% inhibitory concentration (IC50) of 6.8 yM "Fig.
(34)". Treatment with 100 ^M resveratrol for 24 h increased apoptosis to
20.6 ± 1.35 from 12.1 ± 0.36% in LLC cells. In addition, resveratrol
decreased the S phase population at concentrations of 50 and 100 \kM. The
proportion of LLC cells in the G2/M phase of the cell cycle was increased
by the treatment of 50 ^M resveratrol "Table (8)".
HUVEC formed capillary-like networks on Matrigel 24 h after seeding.
Resveratrol dose-dependently inhibited angiogenesis of HUVEC at 5 to
100 fxM "Fig. (35)". These findings suggest that the mechanism of
antitumor and antimetastatic actions of resveratrol might be due to the
582
a)
+ Resveratrol(0.6 mg/kg)
5000 Y
+ Resveratrol(2.5 mg/kg)
+ ResveratroK 10 mg/kg)
3000
1000
0 2 4 6 8 10 12 14 16 18 20 Day
Fig. 32. £;|^c/5 ofresveratrol on tumor volume (a) andfinaltumor weight (b) in LLC-bearing
mice.
Solid-type LLC was prepared by subcutaneous transplantation into the backs on day 0.
Resveratrol was administered intraperitoneally once daily for 21 consecutive days, starting
12 h after implantation of the tumor cells.
inhibition of DNA synthesis in LLC cells and the inhibition of

angiogenesis of vascular endotheUal cells.
Table 8. Effects ofresveratrol on apoptosis Go/Gl, S and G2/M phase of cell cycle in LLC cells^
% of total cells
Concentration Apoptosis Go/Gi S Gz/M
(HM)
None 12.1±0.36 50.0±L97 35.2±L72 14.8±0.29
Resveratrol (5) 9.43±0.51 44.6±0.75 37.9±L25 17.5±0.49
(10) 9.65±0.61 39.9±1,54 43.8±2.12 16.3±0.70
(50) 12.5±0.97 46.8±L15 22.1+1.03* 31.1±0.26*
(100)) 20.6+L35* 52.9±L16 29.2+0.27* 17.8±1.42
^\^lues are expressed as means ± S.E. of 3 experiments. *P<0.05, Significantly different from medium alone.
583
10 r
o 8
o ;7<a05
8 6
cd
0 ^
Resveratrol 0.6 2.5 10
mg/kg
Fig. 33. Effects of resveratrol on the numbers of colonies ofLLC cells
metastasizing to the lung on day 22 in LLC-bearing mice.
*P<0.05, Significantly different from LLC-bearing mice.
584
&
20
3 ,
10 100 1000
luuu - Q 5 JO 5Q 100
Resveratrol ( M M ) Resveratrol ( M M )
Fig. 34. i ^ c / s of resveratrol on ^H-thymidine incorporation into DNA Fig. 35. Effects of resveratrol on Matrigel-induced capillary-
ofLLC cells. like networkformation by HUVEC.
Results are expressed as means ± S.E. of 4 experiments. Results are expressed as means ± S.E. of 4 experiments.
*P<0.05, Significantly diflFerent from medium alone. *P<0.05, Significantly different from medium alone
585
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toxicity and immunocompetent organic toxicity induced by 5-fluorouracil without
loss of antitumor activity in mice. Jpn. 7. Cancer Res, 1999, 90, 765- 774.
[2] Kimura, Y.; Sawai, N.; Okuda, H. Antitumor activity and adverse reactions of
combined treatment with chitosan and doxorubicin intumor-bearing mice. / . Pharm.
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[3] Kimura, Y & Okuda, H. Prevention by carp extract of myelotoxicity
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[5] Takaku, T.; Kimura, Y; Okuda, H. Isolation of an antitumor compound from
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[6] Kimura, Y; Baba, K.; Okuda, H. Inhibitory effects of active substances isolated from
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Lewis lung carcinoma-bearing mice. J. Pharm, Pharmacol, 2000,52, 1287-1295.
[9] Kimura, Y & Okuda, H. Resveratrol isolated from Polygonum
cuspidatum root prevents tumor growth and metastasis to lung and tumor-induced
neovascularization in Lewis lung carcinoma-bearing mice. / . Nutr, 2001, 131,
1844-1849.
[10] Kubo, M.; Kimura, Y; Shin, H.; Haneda, T.; Tani, T.; Namba, K.
Studies on the antifrmgal substance of crude drugs (II). On the roots of Polygonum
cuspidatum Sieb.et Zucc. (Polygonaceae). Shouyakugaku Zasshi, 1981, 35, 58-64.
[11] Kimura, Y; Kozawa, M.; Baba, K.; Hata, K. New constituents of
roots of Polygonum cuspidatum, Planta Med. 1983, 48, 164-169.
[12] Arichi, H.: Kimura, Y; Okuda, H.; Baba, K.; Kozawa, M.;
Arichi, S. Effects of stilbene componets of the roots of Polygonum cuspidatum Sieb.
Et. Zucc. On lipid metabolism. Chem. Pharm, Bull 1982,30, 1766-1770.
[13] Kimura, Y; Ohminami, H.; Okuda, H.; Baba, K.; Kozawa, K.;
Arichi, S. Effects of stilbene components of roots of Polygonum species on liver
injury in peroxizied oil-fed rats. Planta Med 1983,49, 51-54.
[14] Kimura, Y; Okuda, H.; Arichi, S. Effects of stilbenes on
arachidonate metabolism in leukocytes. Biochim Biophys, Acta 1985,834, 275-278.
[15] Kimura, Y; Okuda, H.; Arichi, S. Effects of stilbene derivatives on
archodoudlc metabolism in leukocytes. Biochim, Biophys, Acta 1985,837, 209-212.
[16] Kimura, Y; Okuda, H.; Kubo, M. Effects of stilbenes isolated from
586
medicinal plants on arachidonate metabolism and degranulation in human

polymorphonuclear leukocytes. 7. £mop/iarmaco/. 1995,45, 131-139.
BIOLOGICALLY ACTIVE TRITERPENE

GLYCOSIDES FROM SEA CUCUMBERS
(HOLOTHUROIDEA, ECHINODERMATA)
HUGO D. CHLUDIL, ANA P. MURRAY, ALICIA M. SELDES AND

MARTA S. MAIER*
Departamento de Quimica Orgdnica, Facultadde Ciencias Exactasy

Maturates, Universidad de Buenos Aires, Pabell6n2, Ciudad
Universitaria, (1428) Buenos Aires, Argentina
ABSTRACT: Sea cucumbers are characterized by their content in holothurins,

triterpenoid glycosides that are responsible for the toxicity of these echinoderms. Nearly
100 holothurins isolated in the last twenty years are grouped into three main aglycone
structural types: 3p-hydrox>iiolost-9(ll)-ene, 3p-hydrox5*iolost-7-ene and non-
holostane based aglycones. This communication offers a general view of the structural
characteristics of these saponins and the spectral features in their ^H- and ^^C-NMR and
FAB-MS spectra. Recent advances in the unambiguous spectroscopic characterization of
the triterpenoid skeleton, the substitution patterns and the complete structure of the
oligosaccharide chain are discussed.
INTRODUCTION
The phylum Echinodermata (Greek echinos^ spiny; derma^ skin)

comprises some of the most familiar seashore animals. There are about
7,000 living species widely distributed in all oceans at all depths. The
phylum is divided into five classes: Holothuroidea (sea cucumbers or
holothurians), Asteroidea (starfishes or sea stars), Ophiuroidea (brittle
stars), Crinoidea (sea lilies and feather stars) and Echinoidea (sea
urchins). Triterpenoid and steroid oligoglycosides are predominant and
characteristic secondary metabolites of sea cucumbers and starfishes and
are responsible for their general toxicity [1-6]. Both classes of
echinoderms contain also glycosphingolipids, such as
monohexosylceramides (cerebrosides) and gangliosides [7]. Brittle stars
contain sulfated polyhydroxysteroids [4,8,9] and only two sulfated
steroidal monoglycosides have been reported in the brittle star
588
Ophioderma longicaudum [10]. On the contrary, there is no report of

steroid or triterpenoid glycosides in the classes Echinoidea and Crinoidea.
Several reviews concerning the structures, taxonomic distribution,
evolution and biological activities of sea cucumber triterpenoid
oligoglycosides have been published [11-14]. The purpose of the present
communication is to offer a general view^ of the methods applied in the
structural elucidation of these complex molecules, focusing on recent
examples of cytotoxic, antifungal and virucidal triterpenoid
oligoglycosidesfromour laboratory.
TRITERPENOID GLYCOSIDES
Triterpenoid saponins are typical metabolites of plant origin, but

extensive investigation on marine organisms as sources of new bioactive
metabolites has shown that triterpenoid glycosides are widely distributed
in sea cucumbers. It has been suggested that these saponins have a
defensive role due to their membranotropic action [11]. Penta- and
tetraglycosides containing a norlanostane triterpenoid have been
encountered rarely also in sponges [15].
Most of the triterpenoid glycosides isolated so far from holothurians
present a sugar chain of two to six monosaccharide units linked to the C-3
of the aglycone, which is usually based on a "holostanol" skeleton
[3{3,205-dihydroxy-5a-lanostano-18,20-lactone] (1), Fig.(l) [1].
22 24
o^ ô
R—a
30 31
Fig. (1). Structure of hypothetical holostanol
Only quinovose, glucose, 3-0-methylglucose, xylose and 3-0-

methylxylose are present in the carbohydrate moieties of these glycosides.
The &st monosaccharide unit is always xylose, while 3-0-methylglucose
and 3-0-methylxylose are always terminal. In comparison to steroidal
589
oligoglycosides from starfishes which always contain a sulfate group

attached to C-3 of the aglycone, sixty percent of the triterpenoid
glycosides isolated so far from sea cucumbers have sulfate groups linked
to the monosaccharide units of the oligosaccharide chain. Although most
of them are monosulfated oligoglycosides, several di- and trisulfated
glycosides have been isolated, mainlyfromthe order Dendrochirotida.
Triterpene glycosides are specific for different taxonomic groups of
sea cucumbers and represent good models for studies on biochemical
evolution [16]. They have a wide spectrum of biological effects:
antifungal, cytotoxic, hemolytic, cytostatic and immunomodulatory
activities [12]. These biological activities are a consequence of their
membranotropic action against any cellular membrane containing A^-
sterols. Triterpene glycosides form complexes with these sterols that lead
to the development of single ion channels and larger pores, which cause
significant changes in the physico-chemical properties of membranes
[13]. Sea cucumber cell membranes are resistant to their own toxins due
to the presence of A^- and A^'^ ^-sterols, sulfated A^-sterols and p-xylosides
of sterols instead of thefreeA^-sterols [17].
CHEMICAL STRUCTURES
Nearly 100 different chemical structures of these toxins have been

published in the last 20 years. Most of these triterpenoid oligoglycosides
contain an aglycone based on a "holostanol" skeleton and two main series
can be distinguished: glycosides based on a 3p-hydroxyholost-9(ll)-ene
aglycone and those containing a 3P-hydroxyholost-7-ene skeleton.
Usually aglycones that have a A^'^^ double bond are characteristic of sea
cucumbers belonging to the order Aspidochirota, while those with a A^
unsaturation were generally isolated from animals of the order
Dendrochirotida.
3p-Hydroxyholost-9(ll)-ene aglycones
Bivittoside C, Fig. (2), a hexaglycoside isolated from the sea cucumber

Bohadschia bivittata [18] is the simplest triterpene glycoside with a A^'^^
double bond:
590
R—a
2 Bivittoside C [18] R = [3-0-Me-<Hc<l-^3)-Glo<l-^)H3-0-Me-Glc-(l->3)-Glc-(l->4)-Qui<l-^2)]-Xyl
Fig. (2). Structure of Bivittoside C
A number of glycosides containing aglycones of this series show a

carbonyl group at C-16 (Structure 3, Fig. (3)). With exception of
glycoside 3a, all have an additional A25 double bond in the side chain
3a Ds-Penaustroside D [19] R = [3-O-Me-Xyl<l->3)-Glc<l->4)]-[Qui<l->2)]-Qui-(l->2)^»-0SO5N8hX54

3b Holotoxin Ai [20] R = [3-0-Me-Glc<l-^3)-Glc-(l->4)]-[3-O^Me-Glc-<l->3>XyHl-^)-Qui-(l->2)]-Xyl; A"
3c Holotoxio A [21] R = [3-0-Me-Glc^l->3)-Gic-(l-^)]-[3-0-Me-Glc-(l->3)-Glc-(l->4)-Qui<l->2)]-Xyl; A^
3d Holotoxin Bi [20] R = [Glc<l-^3)-Glc-(l-^)]-[3-0-Me-Glc-(l->3)-X)1-(l-^)-Qai-(l->2)]-Xyl; A^
3e Holotoxin B [21] R = [G^c<l-^3)-Glc-(1^4)]-[3^-Me-G^c-(l->3)-G^c-(l->4>Qui-(1^2)^Xyi; A"
3f Neothynidioside [22] R = 3-0-Me-Glc-(l-y3)-Xyl-<l->4)-Qui-<l->2)^W)S03Na-Xyl; A^'
3g Psolusoside A [23] R = 6-OS03Na-3-^-Me-Glo<l->3)-€-OS03Na-Glc<l->4)-Qui-(l->2)-Xyl; A^'
3h Qadoloside A [24] R = 3-0-Me-Glc-(l-^3)-Xyi-(1^4)-Qui-(l->2)-Xyi; A"
3i Cladoloside B [24] R = [Glc-(l->4)]-[3-0-Me-Glc-<l-^3)-Xyi-<1^4)-Qui-(l->2)]-Xyi; A"
3j Ds-Penaustroside C [19] R = [3-0-Me-Xyi<l-^3)-Gk<l->4)]-[<^<l-->2)]-Qiu-<l->2)-4-OSOjNa-Xyl; A^'
3kHemoiedemoside A [25] R = 3-0-Me-Glc-(1^3)-6-OS03NarGlc-(l-^)-Qiii-(l->2)-4-OS03NarXyl; A"
31 Hemoiedemoside B [25] R = 6-OS03Na-3-0-Me-Glc<l->3)-6-OS03Na.Glc<1^4)-Qiii-{1^2)-4-OS03Na-Xyi;
A^^
3m Caudinoside A [26] R = 3-<9-Me-Glc-(l-^3)-Glo(l-^)-Qui-<l->2)-Xyl; A^
Fig. (3). Structure of 3p-hydro3Qiiolost-9(l l)-en-16-one aglycone based glycosides
Another structural feature is the presence of a 12a-hydroxyl group in

the aglycone, Fig. (4):
591
HO O
4a Bivittoside A [18] R = Qui-(l-^2)-Xyl

4b Bivittoside B [18] R = [3-0-Me-Glc<l- •3)-Gl(K1^4)]-[Qui-(l-^2)].Xyl
4c Bivittoside D [18] R = [3-0-Me-Glc-(l- •3)-Glc-(l->4)]-[3-0-Me-Glc<l->3)-Glc-<l->4)-Qui-(l->2)]-X}d
4d Pervicoside C [27] R = 3-(9-Me-Gl<Kl- •3)-Gld-(l->4)-Qiji<l-^2>4-OS03Na-Xy!
4e Pervicoside B [27] R = 3-0-Me-Gio{\- •3)-Glc-(l->4)-Qui-(l->2)-4-OS03Na-Xyi; A^
Fig. (4). Structure of 3p,12a-dihydro3Qiiolost-9(l l)-ene aglyoone based glycosides
Some glycosides contain two hydroxyl groups at positions 12a and

17a of the holostanol skeketon, Fig. (5):
HO O
5a Echinoside B [28] R = Qui-(1^2)-4-OS03Na.Xyl; R' = H

5b Echinoside A [28] R = 3-0-Me-Glc<l->3>Glo<l->4>Qiii-(l->2>4-OS03Na-Xyl; R* = H
5c 22-Acetoxy-echinoside A [29] R = 3-0-Me-Glc<l->3)-Glc<1^4)-Qui-(l->2)^M3S03Na-Xyl; R* = OAc
5d Holothurin A, [30] R = 3-<9-Me-Glc-(l-^3)-Glc-<l->4)-Qtti-(l->2>4-0S03Na-Xyl; R* = C«
5e 24-Dehydroechinoside B [31] R = Qui-(l->2)^K)SOjNa-XyI; R* = H; A^
5f 24-Dehydroechiiioside A [31] R = 3-0-MeGlc<1^3)-Gd-(l->4)-Qui-<l->2)-4-0S03Na.Xyl; R^ = H; A^
5g 22-Hydroxy-24-dehydroechiiioside A [29] R = 3-0-Me-Glc-(l->3)-Glc-(l->4)-Qui-<l->2)-4-0S03NarX>i; R' =
OH;A^
Fig. (5), Stiucture of 3p,12a, 17a-trihydrox54iolost-9(l l)-«ie aglycone based glycosides
Glycosides 5c, 5d and 5g together with glycosides 6, Fig. (6) and 7,

Fig. (7) are characterized by additional acetoxy or hydroxy groups in the
side chain.
592
OH
R O'
6 24(5)-hydroxy-25-dehydroechinoside A [29] R = 3-0-Me-Glc-(l->3)-Glc-(l->4)-Qui-(l-^2)-4-OS03Na^Xyl
Fig. (6). Structure of a sul&ted tetraglycoside isolatedfixMnthe sea oxccashei Actinopygaflammea
OAc
HO O^ Ô
7a Holothurinosidc B [32] R = [3-0-Me-Glc-(l->3)-CHc-(l->4)-Qui-<l->2)]-[CHc-(l->4)]-Xyl; R ' = OH; A^

7b Pervicoside A (Neothyosidc A) [27] R = 3-C>-Me-Glc-(l->3)-Glc-(l-^)-Qui-(l->2)-4-OS03Na-Xyl; R^ = H
7c Neothyoside B [33] R = Qui-(l->2)-4-OS03Na-Xyl; R ' = H
Fig. (7). Structure of 25-acetoxi-3p,12a-dihydroxjiiolost-9(l l)-€ne aglycone based glycosides
Holothurins A (8a) and B (8b) isolated from the sea cucumber

Holothuria leucospilota [34] as well as Desholothurin A (8d), and
Holothurinosides A (8c), C (8e) and D (8f), Fig. (8)fromHolothuria
forskali [32] are the only examples of glycosides containing the side
chaininafiiranform.
Compounds 3a, 3g-31 and 7c are the only A^'^ ^-glycosides isolated
from sea cucumbers belonging to the order Dendrochirotida. In general,
3|3-hydroxyholost-9(ll)-ene based aglycones were characterized in
holothurins isolated from animals of the order Aspidochirota.
593
8a Holothurm B [34] R = Qui-(1^2)-4-OS03Na-Xyl; R ' = OH

8b Holothurm A [34] R = 3-0-Me-Glc-(l->3)-Glc-(l-^)-Qui-(l-^2)-4-OS03Na-X)4; R^ = OH
8c Holothurinoside A [32] R = [Glc-(1^4)]-[3-0-Me-Glc-(l-^3)-Glc-(l->4)-Qiii-(l->2)]-Xyi; R^ = OH
8d Desholothurm A [32] R = 3-0-Me-CHc-(l->3)-Glc-(l->4)-Qui-(l->2)-Xyl; R ' = OH
8e Holothurinoside C [32] R = 3-0-Me-Glc-(l->3)-Glc-(l-^)-Qui-(l->2)-Xyi; R ' = H
8f Holothurinoside D [32] R = Qui-(1^2)-Xyi; R ' = H
Fig. (8). Structures of glycosides isolatedfromtbe sea cucumbers Holothuria leucospilota and Holothuria
forskalii
3P-HydroxyhoIost-7-ene aglycones
Frondoside B (9a), Cucumariosides A2-4 (9b) and A7-3 (9c), Fig. (9) as
well as several triterpene glycosides isolated from the sea cucumbers
Stichopus chloronotus (lOa-lOh) and Thelenota ananas (lOi, lOj), Fig.
(10) contain the simple 3P-hydroxyholost-7-ene as the aglycone. An
additional acetoxyl group in the side chain is present in compounds 10a-
lOj.
9a Frondoside B [35] R = [3-0-Me-Glc-(l-^3)-6-OS03Na-Glc-(l->4)]-pCyl-<1^2)]-Qui-(l->2)-4-OS03Na.Xyl; A';

A^
9b Cucumarioside K2-A [36] R = [3-0-Me-Glo<l->3)-Glc-(l->4)]-pCyl-(l->2)]-Qui-<l-^2)-Xyl; A^; A^
9c Cucumarioside Ar3 [36] R = [6-OS03Nar3-0-Me-Glc<l->3)-6-OS03Na-Glc-(l-^)]-[Xyl-(l-^2)]-Qui-(l->2)-4-
0S03Na-Xyl;A';A^
Fig. (9). Structures of glycosides isolatedfromthe sea cucumbers Cuctanariafrondosa and Cucumaria
japonica
594
Glycosides lOa-lOj were isolated from Stichopus chloronotus and

Thelenota ananas, two sea cucumbers belonging to the order
Aspidochirota [37].
lOa Stichloroside C, (Stichoposide C) [37] R = [3-0-Me-Glo<l->3)-Glc-(l->4)]-[3-0-Me-ac<l->3)-Xyi-(l-»4)-

Qum-(l-^2)]-Xyi
10b Stichloroside B, (Stichoposide D) [37] R = [3-0-Me-Glc-(l->3)-Glc-(l-^)]-[3-0-Me-Glc-<l-^3)-X)d-(1^4)-
Glc-(l-^2)]-Xyl
10c Stichloroside Aj [37] R = [3-0-Me-<Hc-<l->3)-Glc-(1^4)]-[3-0-Me-Glc-<1^3)-Glc-(1^4)-X5d-(l-^2)l-Xyl
lOd Stichoposide A [37] R = Qui-(l->2)-4-OS03Na-Xyi
lOe Stidioposide B [37] R = (ao<l->2)-Xyl
lOf Stichloroside C2 [37] R = [3-0-Me-Glc-(l->3)-Glc<l->4)]-[3-<9-Me-ac-(1^3)-Xyl-(l->4)-Qui-(l->2)]-X>4; A^'
lOg Stichloroside B2 [37] R = [3-0-Me-Glc-(l->3)-Glc-(l->4)]-[3-0-Me-Glo<l->3)-Xyi-(l-M)-Glc-(l-^2)]-Xyi;
A^^
lOh Stichloroside A2 [37] R = [3-0-Me-Glc-{l->3)-Glc-<1^4)]-[3-0-Me-Glc-(l->3)-CHc-<l-^)-Xyl-(l->2)]-Xyl;
A^
lOi Thelenotoside A [37] R = 3-0-Me-Glc-(l->3)-Xyi-(l-^)-Qui-(1^2)-Xyl
lOj Thelenotoside B [37] R = 3-<9-Me-Glc-(l->3)-X>i-(l->4)-Glc-(l->2)-Xyl
Fig. (10). Structures o f glycosides isolated fixjm the sea cucunibers Stichopus chloronotus and Thelenota
ananas
3p-Hydroxyholost-7-ene aglycones with a carbonyl group at C-16

have been isolated exclusively from the sea cucumber Cucumaria
japonica, Fig. (11).
R—a
l l a Cucumarioside A2-3 [36] R = [3-0-Me-Glc-(l-^3)-Glc-<l-^)]-[Xyi-(l-^2)]-Qui-(l->2)-Xyi

595
l i b Cucumarioside Ar-2 [36] R = [6-OS03Na-3-0-Me-Glc-(l->3)-6-OS03Na-Glc-(1^4)]-[X>d-(l->2)]-Qui-(l->2)-

4-OS03N2hXyl
l i e Cucumarioside Ao-3 [38] R = [3-0-Me-Glc-(l->3)-Xyi-<l-^)]-[Xyl-(l->2)]-Qui-(l-^2)-4-OS03Na.X)d; A"
U d Cucumarioside A,-2 [38] R = [6-OAc^c-(l->3)-Glc-(l->4)]-pCyi-(l->2)]-Qui-(l->2H-OS03Na-Xyl; A^'
l i e Cucumarioside A2-2 [36] R = [3-0-Me-CHc-(l->3)-Glc-(l-^)]-pCyi-(l->2)]-Qui-(1^2)-XyI; A"
l l f Cucumarioside A7-I [36] R = [6-OS03Na-3-0-Me-Glc-(l-^3)-6-OS03Na-Glc-(l->4)]-[X>d-(l->2)]-Qui-(l->2)-
4-OS03Na-Xyl;A^'
l l g Cucumarioside A3 [39] R = [3-(9-Me-ac<l-^3)-6-OS03Na-GlcKl->4)]-[X54-(l->2)]-Qui-(l->2)-4-OS03Na-
Xyi;A"
l l h Cucumarioside A6-2 [39] R = [6-OS03Na-3-0-Me-Gac-(l->3)-Glc-(l->4)]-pCyi-(l->2)]-Qui-(l->2)-4-OS03Na-
Xyl;A"
Hi Cucumarioside A4-2 [36] R = [GIc-(l->3)-ac-<1^4)]-pCyi-(l-^2)]-Qui-(l-^2)-4-OS03Na-Xyl; A^
Fig. (11). Structures of glycosides isolated from the sea cucumber Cucumariajaponica
Another structural feature that has been found only in this series of
aglycones is the presence of an acetoxyl group at C-16. Glycosides with a
p-configuration for this group are shown in Fig. (12).
o^ ô
R—a
12a Frondoside A [40] R = [3-<9-Me-Glc-(l-^3)-Xyl-(l-^)]-PCyl-(l->2)]-Qui-(l->2)-4-OS03Na-Xyl

12b Frondoside Ai [41] R = 3-0-Me-Glc-(l->3)-X>i-(l->4)-Qui-(l->2)-4-OS03Na-Xyl
12c Liouvilloside B [42] R = 6-OS03Na-3-0-Me-Glc-(l->3)-6-OSOjNa-Glc-(l->4)-Qui-(l->2)-4-OS03Na-Xyi
12d Cucumarioside Ao-2 [38] R = [3-0-Me-Glc-(l-^3)-Xyi-(l-^)]-pCyl-(l-^2)]-Qui-(l-^2)-4-OS03Na-Xyl; A^^
12e Neothyonidioside C [43] R = 6-OS03Na-3-6>-Me-Glc-(1^3)-X5d-(l->4)-Qui-(l->2)-4-OS03Na-Xyi; A^
12f Cucumarioside G, [44] R = 3-0-Me-Xyi-(l->3)-GJc-(l->4)-Qui-(l->2)-4-OS03N»-Xy!; A^
12g Liouvilloside A [42] R = 6-OS03Na-3-0-Me-Glc-<l->3)-6-OS03Na-Glc-(1^4)-Qui<l->2)-4-OS03Na-Xyi; A^
12h Cucumarioside C^ [45] R = [3-0-Me-Xyl-(l->3)-Glc<l->4)]-[Xyi-(l->2)]-Qui-(l->2)-Xyi; HE, A^'*
12i Cucumarioside H [46] R = 3-0-Me-Xyl-<l->3)-<31c<l->4)-Qui-(l->2)-4-OS03Na-X>4; 22£'; A^
12k Cucumarioside Cj [45] R = [3-0-Me-X)d-(l->3)-Glc-<l-^)]-pCyl-(l->2)]-Qui-(l->2)-Xyl; 22Z; A^
121 Cucumarioside G3 [47] R = 3-0-Me-ac-(l-^3)-Cac<l->4)-Qui-(l-^2)-4-OS03Na.Xyl; 22Z; A^
Fig. (12). structure of 16p-acetoj^-3p4iydroxjiiolost-7-eoe aglycone based glycosides
Some of the glycosides containing a 16p-acetoxy group also present an

allylic hydroxyl group at C-25, Fig. (13).
596
OH
13a Cucumarioside G4 [47] R = 3-(9-Me-X)4-(l->3)-Glc-{l->4)-Qui-(1^2)Û)S03Na.Xyl

13b Eximisoside A [48] R = 3-0-Me-Glc-(l-^3)-Xyl-(l->4)-Glc-(l->2)-X5d
13c Caldgeroside E [49] R = [6-OS03Na.3-0-Me-Glc<l->3)-a(Kl->4)]-[Glc-(l-^2)]-Qui-{l->2)-4-OS03Na-Xyl
Fig. (13). Structure of 16p-acetoxy-3p,25-dihydrox>iiolosta-7,22-diene aglyccaie based glycosides
Four glycosides isolated from the sea cucumber Cucumaria lefevrei

[50] are the only examples of holothurins with a 16a-acetoxy group in
their aglycones, Fig. (14). Lefevreiosides A2 (14b), B (14c) and C (14d)
show the same monosulfated tetrasaccharide chain and differ in the
degree of unsaturation or the position of the double bond in their side
chains. Lefevreioside Ai (14a) is the desulfated analog of glycoside 14b.
14a Lefevreioside A, [50] R = 3-0-Me-Glc-(l->3)-Glc-(l->4)-Qui-(l-^2)-Xyl

14b Lefevreioside A2 [50] R = 3-0-Me-Glc-(l-^3)-Cac-(l->4)-Qui-<l->2)-4-OS03Na-X>d
14c Lefevreioside B [50] R = 3-0-Me-Glc-(l-^3)-Glc-(l->4)-Qiii-(l->2)-4-OS03Na-Xyi; A^
14d Lefevreioside C [50] R = 3-0-Me-Glc-(l->3)-G!c-(l->4)-Qui-(l->2)-4-OS03Na-Xyl; A^^
Fig. (14). Structures o f glycosides isolatedfixatnthe sea cucumber Cucumaria lefevrei
Several triterpene glycosides isolated from the sea cucumbers

Cucumaria echinata and Pentamera calcigera contain a carbonyl group
at C-23 in the side chain, Fig. (15). This structural feature is absent in 3p-
hydroxyholost-9(ll)-ene aglycones.
597
15a Cucumechinoside C [51] R = 3-(9-Me-Glc-(l->3)-6-OS03Na-ac-(l->4)-Qui-(l->2)-4-OS03Na-Xyl; R^ = H

15b Cucumedimoside F [51] R = 6-OS03Na-3-0-Me-Glc-(l->3)-6-OS03Na^c-(l->4)-Qui-(1^2)-4-OS03Na-Xyl; R' = H
15c Caldgeroside Cj [52] R = [3-0-Me-XyKl->3)-Glc-(l->4)]-[CHc-(l->2)]-Qui-(l->2)-4-OSOjNa-Xyl; R^ = H
15d Caldgeroside D2 [49] R = [3-C>-Me-XyH1^3)-6-OS03Na.Glc-(l->4)]4Glc<l->2)]-Qm-<l-^2)ÔS03Na-X5i; R^ = H
15c Cucumediinoside A [51] R = 3-0-Me-GlcKl->3)-6-OS03Na-Glc-(1^4)-Qui-(l->2)-4-OS03Na-Xyi; R* = O
15f Cucumedimoside B [51] R = 3-O-Me-Glc-(l->3)-2-OSO3N2hXyi-(l->4)-Qui-(l->2)-4-OS03Na-Xyl; R' = O
15g Cucumechinoside D [51] R = 6-OS03Na-3-0-Me-<Hc<l->3)-6-OS03NarGlc<l-->4K^<1^2)-4-OS03Na-Xjd; R^ = O
15h Cucumediinoside E [51] R = 6-OS03Na-3-0-Me-Glc<l->3)-2-OS03Na-X>d<l-^)-C^-(1^2>4-OS03Na-X>d; R^ = O
15i Cucumarioside Ao-1 [38] R= [3-O-Me-Glc-(l->3)-X>i-(l-^)]-[Xyi-<l->2)]-Qui-(l->2)-4-OS03Na-Xyi; R^ = P-OAc
Fig. (15). Structures of glycosides isolated fix>tn the sea cucumbers Cucumaria echinata and Pentamera
calcigera
Recently, we have isolated an antifungal holothurin from the sea

cucumber Psolus patagonicus [53]. Patagonicoside A (16), Fig. (16) is
the &st example of a 3p-hydroxyholost-7-ene aglycone substituted with
12a- and 17a-hydroxy groups.
H%o<^^-
16 Patagonicoside A [53] R = 3-6>-Me-Glc-(l->3)-6-OS03NarGlc-<l->4)-Qui-(l->2)-4-OS03N»-Xyl
Fig. (16). Structure of patagonicoside A, an antifungal oligoglycoside isolated fixxn the sea cucumber Psolus
patagonicus
Non-holostane aglycones
598
Recently, some examples of holothurins having uncommon non-

holostane aglycones have appeared in the literature. These glycosides
have been isolated from seven species of sea cucimibers belonging to the
order Dendrochirota. All are sulfated compounds, the majority
monosulfated at the glucose or xylose units.
Five glycosides contain aglycones with an 18(16)-lactone and a A^-
unsaturation, Fig. (17) and (18).
OAc
17 Psolusoside B [54] R = [6-OSO,Na-Gl(Kl->4)]-[Glc-(l->4)-Glo(l-^2)]-X>i
Fig. (17). Structure of Psolusoside B, isolatedfixmithe sea cucumber Psoltds fabricii
^N^^
•iiiH
18a Cucumarioside G^ [55] R = 3-0-Me-Xyl-(l-^3)-Glc(l->4)-Qui-(l-^2)-4-OS03Na.Xyl

18b Caldgeroside B [52] R= [3-0-Me-X>i-(l->3)-Glc(l->4)]-[Qw-<l->2)]-Qiii-(l-^2)-4-OS03Na-Xyi
18c Caldgeroside C, [52]R = [3-0-Me-X>d<l->3)-CHc(l->4)]-[Glc-(l-^2)]-Qui-(l->2)-4-OS03Na.X>1
18d Caldgeroside D, [49] R = [3-0-Me-Xyl-(l->3)-6-OS03Na-Glc(l-^)]-[CHc<l-^2)]-(>ji-(l->2)-4-OS03Na-X^
Fig. (18). structures of noo4iolostane glycosides isolated fixxn the sea cucumbers Et^ntacta fraudatrix and
Pentamera cahigera
Avilov et al. [56,57] reported three holothurins that are devoid of a

lactonefiinctionand have a shortened side chain. Kurilosides A (19a) and
599
C (19b) contain a 9(ll)-double bond aglycone moiety and 16a-acetoxy

group, Fig. (19).
OAc
R-
19« Kuriloside A [56] R = [3-O.Me-Glc-(1^3)-6-OS03N»<Hc-(l-^)]-[Glc-(l->4)-Qiii-(l->2)]-Xyl

19b Kuriloside C [56] R = [3-(9-Me-Glc-(l->3)-6-OS03Na^c-(l->4)]-[Qui-<l->2)]-Xyl
Fig. (19). Structures of ^yootsides isolatedfixxnthe sea cucumber Duasmodactyla kurilensis
Koreoside A (20) isolated from Cucumaria koraiensis is one of the

two examples of non-holostane glycosides with three sulfete groups in the
oligosaccharide chain, Fig. (20).
COCH,
IIH
20 Koreoside A [57] R = [6-OS03Na-3-C>-Me-<Hc-(l->3)-6-OS03Na-Glc-(l->4)]-PCyl-(l->2)]-Qui-(l->2)-4-

OSOâ-Xyi
Fig. (20). Glycoside isolatedfixxntbe sea cucumber Cucumaria koraiensis
Ds-Penaustrosides A (21a) and B (21b), as well as Frondoside C (21c),

also lack the lactone function and have an additional hydroxyl group at C-
20, Fig. (21).
600
21a Ds-Penaustroside A [19] R = [3-0-Me-Xyi<1^3)-Gic-(l->4)]-[Qui-(l-^2)]-Qui-(1^2)-4-OS03Na-Xyl; R^ = H

21b Ds-Penaustroside B [19] R = [3-0-Me-Xyi-<1^3)4Slc-(1^4)]-[Qui-(l->2)]-Qui-(l->2)-4-OS03Na-X>4; R^ = H;
21c Frondoside C [58] R = [3-0-Me-Xyi<1^3)-ac-(l->4)]-[Qiii-(l->2)]-Qui-<l->2)-4-OS03Na-Xyl; R' = OAc; A^
Fig. (21). Structures of tiOQ4K>lostane glyoosides isolated from the sea cucumbers Pentacta australis and
Cucumariafrondosa
Most of sea cucumber triterpene glycosides are tetra- or

pentaglycosides. The few disaccharides that have been isolated show a
Qui-(1^2)-4-OS03Na-Xyl chain attached to C-3 of the triterpenoid
aglycone [28, 31, 33, 34, 37]. Bivittoside A (4a) and Holothurinoside D
(8f) show no sulfate group while Stichoposide B (lOe) is the only
example of a disaccharide with a glucose unit attached to C-2 of the
xylose unit. Some hexasaccharides have been isolated from sea
cucumbers of the order Aspidochirota: Stichopus japonica [21], Stichopus
chloronotus [37], Parastichopus californius [20] and Bohadschia bivittata
[18]. They are non-sulfated glycosides with a linear 3-0-Me-Glc-(l->3)-
Glc-(l->4)-Xyl chain and a branching of a linear trisaccharide at C-2 of
the xylose unit. The only example with a glucose unit instead of the
terminal 3-0-Me-glucose is Holotoxin Bi (3d).
Most tetrasaccharides show a linear chain with the most common 3-0-
Me-Glc-(l->3)-Glc-(l->4)-Qui-(l->2)-Xyl structure. In some
tetrasaccharides the glucose imit is replaced by a xylose [22, 24, 37, 38,
40, 43, 51] while Cucumariosides Gi (12f) and G4 (13a) show a terminal
3-0-Me-xylose unit. Thelenotoside B (lOj) and Eximioside A (13b) show
a different tetrasaccharide chain: 3-0-Me-Glc-(l-^3)-Xyl-(l->4)-Glc-
(1~>2)-Xyl with no quinovose unit. Non-holostane triterpenoids, such as
Psolusoside B (17), Kuriloside C (19b) and Bivittoside B (4b) are the
only examples of tetrasaccharides with a non-linear chain. Most
tetrasaccharides are sulfated at C-4 of the xylose unit. Additional sulfete
601
groups at C-6 of the 3-0-Me-glucose unit and at C-6 of the glucose unit
have been found in trisulfeted tetraglycosides.
Pentaglycosides isolated from sea cucumbers show a variety of
carbohydrate chains, Fig. (22). Most glycosides contain chains I-IV.
Chain IV is typical for glycosides isolated from the sea cucumber
Pentamera calcigera: Calcigerosides Ci (18c), C2 (15c), Di (18d), D2
(15d) and E (13c). Cucumarioside Ai-2 (lid) is the only example of a
triterpene glycoside containing an acetate group at C-6 of the terminal
glucose unit (chain XII). Pentasaccharide chains with glucose as the
terminal sugar are uncommon and were found in a few glycosides, such
as Cucumarioside A4-2 (Hi) (chain VII), Cladoloside B (3i) (chain X)
and Holothurinoside A (8c) (chain XT).
[3-^-M&<51o<l-^3)-acKl->4)]-pfyHl->2)]<îi<l-^2)]-Xyl-aglyoonea)
[3-a]Vfe<jlc^l-^3>XyKl-^4)]-pfyl-(l-^2)]<îi-(l-^2)]-Xyl^ycoi»
[3-0-Me-XyKl-^3)-Glo<l->4)HGl(Kl->2)]<^-(l->2)]-Xyl-aglyconea^^^
[3^-Me-Xyl-<l->3)-Glc<l->4)HQui-<l->2)]<îi<l->2)]-Xyl-aglyooiie(rV0
[3-O.I^Xyl-(l-^3Kjlo<l-^4)]-pfyK1^2)]<^ji<l->2)]-Xyl-aglycQne(^
[3^-Me-Glc-(l-^3>Glc<l->4)]4Glo<l->4)]<îi-(l-^2)]-Xyl^ycoDe(^
[GlcKl->3Hjlo<l->4)]-pCyKl-^2)]<îi-<l-->2)]-Xyl-aglycone(VII)
[3-aMe-XyHl->3)-C]ao<1^4)HQui<l-^2)]-<^Kl->2)].Xyl-aglycc3oe(Vm^
[3^-Me<jlc^l->3)-Glc<l->4Kîi-(l-^2)HGl<>(l-^4)]- Xyl^ycone (IX)
[Glo<l->4)H3-a.M©<Ho<l->3)-XyKl->4)-Qui-<l->2)]- Xyl-agjycone (X)
[Glo<l->4)]43-^-Me-Gl(Kl-->3)-Glo<l->4)-Qui-<l-^2)]- Xyl-aglycone (XI)
[6-aAc<j!c<l->3)-CHo<l->4)]-pfyl-(1^2)]<îi<l->2)]-Xyl-aglycone(XII)
Fig. (22). Pentaglycoside chains in holotfaurins
Most of the pentasaccharide chains are monosulfated at C-4 of the

xylose unit linked to the aglycone. Only a few disulfeted or trisulfated
pentaglycosides with additional sulfete groups at C-6 of the 3-O-Me-
glucose and glucose units have been isolated [35, 36, 39, 42, 49, 57].
STRUCTURAL ELUCIDATION
Sea cucumber triterpene glycosides are quite fragile molecules. Acidic

hydrolysis of intact holothurins results in the production of artifacts of the
original aglycones due to migration of double bonds and dehydration
reactions [59, 28]. Aqueous acid hydrolysis of glycosides containing a
25(26)-double bond in the aglycone side chain has led to the formation of
artificial 25-hydroxy-genines [21, 60]. To overcome these diflSculties upl-
and ^^C-NMR spectroscopy have been extensively used to determine the
602
Structure of the native aglycones as well as the glycosidic linkages in the

oligosaccharide chain without degradation of the glycosides. Besides, the
development of soft ionization methods, such as fest atom bombardment
(FAB) [61] allowed the mass spectrometric analysis of polar thermally
labile molecules of masses of up to a few thousand Daltons, in particular
for samples which exist as preformed ions in solution. FAB-MS in
positive- and negative-ion modes has been applied to obtain information
on the molecular weight of underivatized glycosides of starfish and sea
cucumbers on the basis of quasi-molecular ions [M+H]^, [M+Na]ând
[M-H]' and [M-Na]", respectively, together with usefiil information on the
saccharide sequence [2].
Nuclear magnetic resonance (NMR) has proved to be a very usefiil
tool for structural elucidation of natural products. Recent progress in the
development of two-dimensional ^H- and ^^C-NMR techniques has
contributed to the unambiguously assignment of proton and carbon
chemical shifts, in particular in complex molecules. The more used
techniques include direct correlations through homonuclear (COSY,
TOCSY, ROESY, NOESY) [62-65] and heteronuclear (HMQC, HMBC)
[66, 67] couplings.
^H-NMR spectra of triterpene glycosides are complicated due to
extensive interproton coupling. The first complete holothurin structures
published in the literature [59, 68] reported only some characteristic
proton signals, such as those due to methyl groups of the triterpenoid
skeleton (I9-CH3, 2I.CH3, 26.CH3, 27-CH3, 3O-CH3, 3I-CH3, 32-CH3),
olefinic protons at C-7, C-11 or those present in the side chain, and
doublets ascribable to the anomeric protons of the oligosaccharide
moiety. Originally, structural elucidation of holothurins was based mainly
on ^^C-NMR data, acid hydrolysis and enzymatic and degradation
reactions. In the last ten years bidimensional NMR experiments have
allowed the assignment of all proton and carbon resonances of the
aglycone and the oligosaccharide chain [32, 35, 39, 40, 48, 52, 57].
NOESY experiments [69] on the intact glycosides have been usefiil in
determining the relative stereochemistry of all chiral centers of the
aglycone. Recently, we have successfiilly assigned all proton and carbon
resonances of a new aglycone in the disulfated tetraglycoside
patagonicoside A (16) by a combination of ^H-^H COSY, COLOC,
HETCOR and NOESY experiments [53]. Fig. (22) shows the NOESY
correlations of the aglycone moiety of Patagonicoside A. Correlations of
603
H-3 with H-1', H-la, H-5a and H-31 confirmed the ^-configuration at C-
3. Of particular interest is the p-configuration of H-9 in 3(3-
hydroxyholost-7-ene based aglycones instead of the characteristic 9a-
configuration in natural steroids and triterpenoids. This proton showed a
characteristic broad doublet at 6 3.02 ppm (in CD3OD) and a strong NOE
correlation with H-19 and H-12p. This last correlation revealed the a-
configuration of the hydroxyl group at C-12. Correlations between H-12
and H-21 evidenced the a-configuration of the hydroxyl group at C-17
and consequently the S configuration at C-20. In this way we were able to
confirm the stereochemistry assigned previously to these carbons by
Kitagawa et al. [27, 28] only on the basis of solvent-induced shifts in the
^H-NMR spectra of the corresponding sapogenols obtained by hydrolysis
of the native saponin.
Fig. (22). NOESY correlations of the agiyoone moiety of Patagooicoide A
Triterpene glycosides of sea cucumbers show characteristic signals due

to the aglycone and the sugar moieties in their ^^C-NMR spectra. The
chemical shifts of aglycone carbons of representative holothurins
containing holostane aglycones are shown in Table 1. Characteristic
resonances for C-3 (5 ca, 88-91 ppm) and C-20 (6 ca. 83-88 ppm) are
observed. Both signals are easily distinguished by DEPT analysis [70].
The presence of a signal at 6 ca. 175-180 ppm is typical for a lactone
carbonyl group ascribable to C-18. The two series of aglycones differing
in the position of the double bond in the triterpenoid skeleton can be
readily distinguished by the chemical shifts of their olefinic carbons.
Holothurins containing a 3p-hydroxyholost-9(ll)-ene based aglycone
such as glycoside 3k show characteristic resonances for a trisubstituted
604
9(ll).double bond at 5 151.0 ppm (s, C-9) and 111.0 ppm (d, C-11). The
presence of an allylic hydroxyl group at C-12a shifts the resonance of C-
11 to 6 ca. 116 ppm (glycosides 4e, 5b, 6 and 8e). Besides, aglycones
with a trisubstituted 7(8)-double bond, as in glycosides 9b, llf, 12a, 13c,
15c and 16, show typical resonances at 6 ca. 120-121 ppm (d, C-7) and
143-148 ppm (s,C-8).
Table 1. ^^C NMR chemical shifts of holostane aglycones
c 3k' 4e^ 5b ^ 6' 8e^ 9b* llf 12a' 13c* 15c* 16^
1 36.1 36.8 36.4 36.1 36.4 36.0 36.8 36.0 35.8 36.0 37.3
2 26.8 27.3 27.0 27.1 27.3 26.9 27.7 26.8 26.6 26.8 27.8
3 88.4 89.1 88.5 88.8 88.8 88.9 90.0 89.2 88.9 88.7 90.8
4 39.5 40.2 39.9 40.1 40.1 39.5 40.4 39.5 39.2 39.4 40.4
5 52.7 53.2 52.7 52.8 52.8 47.9 49.5 48.0 47.7 47.8 50.2
6 20.9 21.4 21.2 21-3 21.2 23.2 24.2 23.3 23.0 23.2 24.0
7 28.3 29.0 28.2 28.4 28.7 119.8 122.7 120.4 120.2 119.8 121.4
8 38.6 40.4 40.8 41.0 40.8 146.6 144.8 145.8 143.2 146.5 148.4
9 151.0 153.6 154.0 154.1 153.5 47.2 48.2 47.2 47.3 47.2 46.1
10 39.7 39.8 39.6 39.8 40.1 35.4 36.7 35.5 35.2 35.4 36.4
11 111.0 116.4 115.6 115.7 116.1 22.8 23.3 22.6 22.3 22.7 35.9
12 31.9 68.5 71.3 71.5 71.5 30.2 30.7 31.4 31.0 30.0 73.6
13 55.7 64.4 58.5 57.8 63.7 58.6 57.9 59.5 59.1 57.5 60.2
14 41.9 46.8 46.3 46.5 46.2 51.2 46.7 47.5 46.9 51.2 52.0
15 51.9 24.3 27.0 36.5 23.6 24.4 53.0 43.6 43.4 34.0 35.7
16 213.4 37.3 35.8 36.8 38.4 34.2 215.3 75.4 74.9 25.5 36.5
17 61.2 47.2 89.1 89.5 47.6 53.0 64.6 54.5 54.2 53.4 90.7
18 176.1 177.5 174.7 174.8 177.5 180.2 180.3 180.6 180.1 179.8 178.5
19 21.9 18.3 20.0 20.1 22.0 23.9 25.0 24.0 23.7 23.9 24.4
20 83.2 84.7 86.9 87.4 83.6 84.1 84.9 85.9 84.9 82.5 88.0
21 26.7 26.4 23.0 23.3 18.7 25.9 27.2 28.4 28.7 27.1 23.0
22 38.3 39.6 36.5 35.2 80.2 39.2 39.2 39.1 41.7 51.8 39.2
23 22.2 23.4 22.2 30.7 28.7 22.9 23.2 22.8 143.2 207.5 23.0
24 37.9 124.5 38.8 75.4 36.7 123.3 38.8 39.6 120.5 52.0 40.7
25 145.4 132.1 28.2 150.0 81.2 131.8 146.5 28.1 70.0 24.3 29.0
26 110.4 25.7 22.6 110.6 28.7 25.5 111.4 22.4 29.6 22.3 23.0
27 22.1 17.7 22.6 18.2 28.0 17.8 23.2 22.9 29.8 22.3 22.9
30 16.4 16.8 16.7 16.8 16.7 17.3 18.4 17.5 17.2 17.3 29.3
31 27.8 28.3 28.0 28.2 27.2 28.6 29.8 28.8 28.1 28.6 17.7
32 20.5 22.1 22.7 22.7 20.1 30.8 32.8 32.4 32.0 30.6 31.2
AcO 171.0 171.0
21.5 21.1
"InPynfi^j-DzO/lnPy-^/s/InCDaOD
605
The position of additional double bonds in the side chains can be

determined from the carbon resonances of the olefinic carbons. A
terminal isopropenyl group in the side chain shows characteristic signals
for the olefinic carbons at 6 ca. 123-124 ppm (C-24) and 132 ppm (C-25)
as well as for the methyl groups attached to C-25 at 6 ca. 25 ppm (C-26)
and 18 ppm (C-27) (glycosides 4e and 9b). The presence of these two
methyl vinyl groups is easily confirmed by the downfield shift of the
methyl singlets in the ^H-NMR spectrum with respect to the
corresponding doublets in a saturated chain. Liouvilloside A (12g), a
virucidal trisulfated triterpene glycoside isolated from the Antarctic sea
cucumber Staurocucumis liouvillei shows two singlets at 6 1.54 ppm (H-
26) and 1.64 ppm (H-27), while Liouvilloside B (12c), the saturated
analog, shows two nearly overlapped doublets (J = 6.6 Hz) at 5 0.83 and
0.84 ppm [42]. Aglycones with a A^^-double bond (3k, 6 and llf) are
characterized by olefinic carbon resonances at 5 ca. 145-150 ppm (C-25,
s) and 110-111 ppm (C-26, t). This disubstituted terminal double bond
shows a diagnostic multiplet for H-26 at 6 4.75 ppm (2H) and a vinyl
methyl signal at 5 1.68 ppm (s, H-27) in the ^H-NMR spectrum [25].
Holothurins that contain a carbonyl group at C-16 (3k and llf) show a
ketone carbonyl signal at 5 ca. 213-215 ppm. Aglycones substituted with
hydroxyl groups at C-12 and C-17 with a-configurations (5b, 6,16) show
a signal at 5 89-90 ppm due to the quaternary C-7. This carbon signal can
be readily distinguished from the C-3 signal by a DEPT experiment.
Holost-7-ene aglycones containing an acetate group at C-16 (12a, 13c)
are characterized by the presence of a singlet at 6 2.0 ppm (CH3CO2) in
their ^H-NMR spectra as well as signals at 6 ca. 171 and 21 ppm for the
carbonyl and methyl groups of the acetate group. Recently, we have
deduced the position of the acetoxyl group at C-16 in Liouvilloside A
(12g) from the chemical shift of the H-16 signal (5 5.63 ppm) and its
correlation with H-17, H-15a and H-15P in the ^H-^H COSY spectrum.
The 16p-configuration was assigned by a NOESY experiment and by
coupling constant analysis for the C-16 proton with the C-17a and C-15
protons. Calculated coupling constant values of 8.9 (Ji5a,i6a), 7.4 (yi5p,i6a)
and 8.9 Hz (Ji6a,\ia) for the most stable conformation of 16(5-
acetoxyholosta-7,24-dien-3P-ol obtained by molecular mechanics were
coincident with experimental and reported values [40] and differed
606
considerably from those calculated for the 16a-isoiner (4.1, 6.9 and 1.2
Hz, respectively) [42].
1 -2
C-NMR data for non-holostane triterpenoid aglycones in intact

glycosides are shown in Table 2.
Table 2. ^^C-NMR chemical shifts of non-holostane aglycones
Carbon 17* 18b** Ds-19a'' 20** 21a*'
1 35.0 35.6 36.5 35.6 36.6

2 26.1 26.6 27.2 27.0 27.3
3 87.8 88.7 88.6 89.1 88.9
4 38.4 39.2 39.5 39.4 39.4
5 46.9 47.2 53.0 48.5 53.2
6 22.4 23.1 21.4 23.4 21.6
7 146.5 122.4 28.6 122.5 28.5
8 121.7 147.3 41.5 147.7 41.7
9 45.2 46.3 149.0 49.3 149.0
10 34.8 35.3 40.0 35.7 40.0
11 20.9 21.6 114.0 22.4 115.3
12 19.3 19.9 35.8 33.6 38.1
13 53.9 56.7 46.4 45.1 45.2
14 45.0 46.0 46.8 53.2 47.6
15 43.7 43.6 42.7 33.6 33.9
16 78.3 81.0 75.9 22.5 22.7
17 59.4 59.1 66.0 62.1 53.3
18 180.2 181.9 16.7 24.9 16.6
19 23.5 23.7 22.4 24.7 23.0
20 82.9 139.9 206.5 211.5 74.2
21 23.0 23.0 30.9 30.7 26.3
22 38.7 113.8 45.7
23 20.7 22.6
24 37.1 40.1
25 144.5 28.3
26 110.1 22.5
27 21.7 22.6
30 16.6 17.1 28.2 17.5 17.0
31 28.1 28.5 17.4 29.0 28.2
32 33.0 33.9 19.6 30.5 19.1
CH3CO 169.0 170.2
CH3CO 21.5 21.0
"InDMSa<4 *InPy^5-D20(4:l) ' Desdfeted analog of 19a
607
Aglycones with a 7(8)-double bond, as structures 17, 18 and 20, show

typical olefinic carbon resonances at 5 146-147 ppm (C-8) and ca. 122
ppm (C-7), while those containing a A^^^^-trisubstituted double bond
(19a, 19b, 21a, 21b and 21c) are characterized by signals at 5 149.0 ppm
(C-9) and 114-115 ppm (C-11). Psolusoside B (17), the &st reported
structure of a non-holostane glycoside with an 18(16)-lactone as well as
holothurins 18a-18d show signals at 6 ca. 180-182 ppm (C-18, s) and 78-
81 ppm (C-16, d). These signals are easily distinguished from the
chemical shifts of C-18 (5 ca. 178-180 ppm) and C-20 (5 ca. 83-88 ppm)
in a A^-holostane aglycone with an 18(20)-lactone.
Characterization of the oligosaccharide chain of hotothurins requires:
a) the identification of each monosaccharide and its anomeric
configuration in the oligosaccharide chain, b) the interglycosidic linkages
and the sequence, c) the position of sulfate groups, and finally the site of
attachment of the oligosaccharide chain to the triterpene aglycone. The
identification of the monosaccharide composition is easily accomplished
by acid hydrolysis of the intact holothurin, derivatization of each
monosaccharide and fiirther analysis by GC and comparison with
standards [42].
FAB-MS is a very usefixl technique for the determination of the
sequence of monosaccharides in the carbohydrate chain of a glycoside.
As shown in Fig. (24) for Liouvilloside A (12g), cleavages can occur on
both sides of the glycosidic linkages with proton transfer. These
cleavages give characteristic fragments that correspond to the sequential
losses of each monosaccharide. In sulfated holothurins, fragment ion
peaks due to the loss of SOsNa are diagnostic for the presence of sulfate
groups in the glycosides. For example, Liouvilloside A (12g) showed
fragment ion peaks at m/z 1355 [M - SOsNa + H + Na]^ 1253 [M -
2S03Na + H + Nalând 1151 [M - 3S03Na + 3H + Na]^ corresponding to
the sequential losses of three sulfate groups.
Although FAB-MS gives information on the sequence of
monosaccharides in the oligosaccharide moiety, it is not possible to
determine the location of the interglycosidic linkages by this method.
NMR has been the method of choice for complete characterization of the
oligosaccharide chain.
608
• 1179(+H+Na) 945(-Hf>fe)
'915(+HfNa) ^534(4Na)
-898(+Na)
-768(+Na)
i-518(4Na)
3-O-Me-Glc- -o- -GlcH-O- -Qd|--o4~Xyl4"0--|-Aglycon

» ' I * I
\ .SOsNa OSOsNa i OSOsNa '
—••lieiC-HfNa) -752(+Na)
Fig. (24). Positive FAB-MSfragpaentationof Liouvilloside A
The ^H-NMR spectra of holothurins show complex and overlapping

signals for hydroxymethine and hydroxymethylene protons of sugar
residues in the downfield region at 8 3.0-5.0 ppm. * Nevertheless, the
anomeric signals usually appear as almost separated doublets at 6 4.7-5.3
ppm in C5D5N:D20 (5:1) with Vi,2 of ca.7.7 Hz and are indicative of the
P-anomers of the pyranose sugars with gluco and galacto configurations
[72]. Another characteristic signal in the ^H-NMR spectra of holothurins
is the methyl doublet resonance of the 6-deoxyhexose quinovose at 6 ca.
1.6 ppm in CsDsNrDaO (5:1). The resonance of the methyl carbon of
quinovose is observed at 5 ca. 18 ppm. Glycosides containing a methoxyl
group attached to C-3 of a glucose or a xylose unit show a typical singlet
at 5 3.6 ppm in their ^H-NMR spectra as well as the corresponding carbon
resonance at 5 ca. 60 ppm.
Comparison of C-NMR data of the oligosaccharide chain of
holothurins with those of reference methyl glycosides has been the
method of choice to determine the interglycosidic linkages [73]. Terminal
sugar residues exhibit remarkable resemblance of their C-NMR data
with those of their respective methyl glycosides. Internal sugar moieties
show deviations in their carbon resonances with respect to the
corresponding methyl glycosides due to glycosidation. Measurement of
the relaxation times (Ti) of the sugar units aided in some cases in the
assignment of carbon resonances of the oligosaccharide chain [28, 60].
Permethylation of the intact non sulfated glycosides or the desulfated
derivatives of sulfeted holothurins followed by GC-MS analysis of the
609
partially methylated alditol acetates [21, 28, 31] has also been used in
order to determine the interglycosidic linkages.
Table 3 shows the ^^C-NMR data for the sugar units of sea cucumber
glycosides containing different pentasaccharide chains.
Table 3. "C-NMR data for the sugar moieties of holotliurins with pentaglycosidic chains
Carbon 12a* Sc** 9a^ Des-lSb**'
r 104.5 103.5 105.7 104.6

2' 81.6 83.3 81.7 83.0
3' 75.3 75.7 75.3 76.9
4' 76.3 77.9 75.8 69.5
5' 64.2 64.1 64.2 65.9
1" 102.2 105.5 102.2 103.0

2" 82.6 75.8 83.9 83.4
3" 75.2 76.3 74.7 75.1
4" 85.3 87.2 87.1 85.5
5" 71.2 71.7 70.6 71.0
6" 18.0 18.0 17.5 17.9
1'" 104.7 104.9 104.3 103.9

2'" 73.6 73.7 73.4 73.5
3'" 86.2 87.8 86.4 86.5
4'" 68.9 69.8 69.7 69.0
5'" 66.0 77.3 74.9 77.1
6'" 62.1 67.6 61.5
1"" 104.5 105.6 104.9 105.1

2"" 74.7 74.9 74.7 74.1
3"" 87.0 87.7 87.5 86.6
4"" 70.6 70.5 78.0 69.6
5"" 77.6 78.3 70.4 66.3
6"" 61.9 62.5 61.8
OMe 60.9 60.7 60.7 60.6
1'"" 105.4 105.3 105.9 105.2

05SJ99
74.9 74.3 75.2 75.8
3'"" 76.6 78.7 76.6 76.2
4»9'" 70.2 71.7 69.7 75.6
C9J»>9
66.6 78.1 64.2 73.4
6"'^^ 62.2 17.9
•lnPy^3-D20(5:l) **InPy^5 ' Py-rfj-DzO (8:2) *toesulfeted analog of 18b "InPy^5-
P20(4:l)
610
One common structural feature is the presence of a xylose unit

attached to C-3 of the aglycone and substituted at C-2' with a quinovose
unit. As shown in Table 2, carbons involved in the interglycosidic
linkages show chemical shifts at 6 ca. 82-88 ppm, shifted downfield from
those expected for the corresponding methyl glycopyranosides.
Glycosides containing a terminal glucose (12a, 8c, 9a) or xylose (Des-
18b) substituted with a methoxyl group at C-3 show an additional signal
at 5 ca. 86-88 ppm due to the substitution at this carbon. Frondosides A
(12a) and B (9a) and the desulfated derivative of Calcigeroside B (Des-
18b) present a branched 2,4-disubstituted quinovose residue with a xylose
unit attached to C-2" in 9a and 12a and a quinovose unit in Des-18b.
This substitution pattern is deduced from the downfield shifts of C-2"
and C-4" of the 2,4-disubstituted quinovose in comparison with those
carbon resonances in a terminal quinovose vmit (Des-18b). On the other
hand, Holothurinoside A (8c) with a 2,4-disubstituted xylose unit attached
at C-3 of the aglycone shows signals at 6 83.3 ppm (C-2') and 77.9 ppm
(C-4') for the carbon atoms involved in the glycosidic bonds.
As shown in Table 3, due to the proximity of carbon resonances of the
different sugar units in the oligosaccharide chain, it is diflBcult to assign
unambiguously each signal on the only basis of comparison with
published data, sometimes performed in different solvents or solvent
mixtures. Recent application of two-dimensional NMR techniques (^H-^H
COSY, relay COSY, HETCOR, COLOC, HMBC and HMQC) to the
structural elucidation of holothurins [32, 35, 39, 40, 48, 52, 57] has
allowed the unambiguous assignment of all ^H and ^^C resonances of the
oligosaccharide chain. The NOESY spectrum of Patagonicoside A clearly
showed the correlations between the protons (Fig. (23)) of the
oligosaccharide chain. These correlations confirmed the iaterglycosidic
linkages deduced previouslyfi-omanalysis of ^H-^H COSY and HETCOR
spectra as well as the site of attachment of the sul&ted xylose unit to the
C-3 of the aglycone [53].
611
Fig. (23). NOESY oorrelatiocis of the oligosaccharide moiety ofpatagonicoide A
Another common structural feature in holothurins is the presence of

one, two or three sulfate units attached to the sugar residues of the
oligosaccharide chain. The location of these groups has been determined
by comparison of ^^C-NMR data of the native glycosides and the
corresponding desulfated derivatives. Desulfetion of the native
holothurins is easily achieved by hydrolysis in a mixture of pyridine and
dioxane at 120X and further purification of the desulfated derivatives by
HPLC [53]. Those holothurins containing an acetoxyl group at C-16, as
Liouvilloside A (12g), are desulfeted by acid hydrolysis in HCl-MeOH in
order to prevent hydrolysis of the acetate group [42].
Table 4 shows the ^H- and ^^C-NMR data for two glycosides,
Patagonicoside A (16) and Hemoiedemoside A (3k), containing the same
disulfeted tetrasaccharide chain and the trisulfated Liouvilloside A (12g),
that differsfiôm16 and 3k in the presence of an additional sulfete group
at C-6 of the terminal 3-0-Me-glucose unit. The three glycosides differ in
their aglycone structures. As observed in Table 4 the esterified carbons
with a sulfete group show downfield shifts of ca, 4-6 ppm with respect to
their desulfeted derivatives, while upfield shifts of ca. 2-3 ppm are
observed for the vicinal carbons. The chemical shifts of these carbons vary
with the solvent used for performing the spectra. For example, the xylose
unit with a sulfate group at C-4', common to all sulfeted holothurins,
shows a characteristic signal for C-4' at 5 77.1 ppm in CD3OD, while the
same carbon resonance is shifted downfield to 5 75.8 and 74.4 ppm in
CsDsN.DaO (5:1) and DMSO-e/6, respectively. Sulfate groups at C'6 of
glucose or a 3-0-Me-glucose residue show typical signals for C-6 at 5
65.6-68.5 ppm in these deuterated solvents.
612
Table 4. *H- and "C-IVMR data for the Sugar Moieties of Patagonicoside A (16),
Hemoiedemoside A (3k) and LiouviUoside A (12g).
16 3k 12g
c Sc*-' SH'(«/mHz) 5c ^'^ 5H"(./inHz) Sc^-^ SH'(>/mHz)
r 105.6 4.46 d (7.9) 104.9 4.69 d (7.1) 104.2 4.32 d (7.3)
T 82.7 3.55 m 82.4 3.69 m 82.0 3.36 m
3' 75.1 (+2.3) 3.78 m 74.8 (+2.6) 4.27 dd (9, 8.7) 14.1 (+2) 3.54 m
4' 77.1 (-6) 4.23 m 75.8 (-5.5) 5,11m 74.4 (-4.8) 3.97 m
5' 63.8 (+2.6) 3.37 m; 63.9 (+2.3) 3.72 m 63.2 (+2.3) 3.19 m
4.17 m 4.75 m
1" 104.8 4.61 d (7.6) 104.6 4.92d/7.7) 103.8 4.49 d (8)

2" 76.3 3.36 m 75.5 3.88 dd 74.9 3.10
3" 75.6 3.55 m 75.6 3.97 m 74.2 3.32 m
4" 873 3.23 m 87.8 3.44 dd (8.7, 86.2 3.03
8.9)
5" 115 3.49 m 71.3 3.66 m 70.4 3.34 m
6" 18.0 1.35 d (6.1) 17.8 1.63 d (6.1) 17.4 1.25 d (5.3)
r" 104.8 4.45 (6.9) 104.6 4.76 d (7.7) 103.1 4.40 d (7.8)
ij'i'tt
74.3 3.41m 74.3 3.95 72.6 3.25 m
3'" 87A 3.60 m 86.5 4.25 85.9 3.49 m
4»»» 10.2 3.42 m 69.9 3.79 68.7 3.20 m
5'" 75.2 (+2.7) 3.69 m 74.7 (+2.7) 4.21 74.2 (+2.1) 3.53 m
6'" 68.5 (-6.1) 4.12 m; 67.5 (-5.7) 4.68 m, 5.14 dd 65.9 (-4.9) 3.78 m, 4.04 dd
(2,10.7) (18,10)
4.38 m
1»»9
105.2 4.57 d (7.9) 104.9 5.29 d (7.8) 103.8 4.47 d (7.9)
2"" 75.4 3.31m 74.5 3.96 m 73.6 3.15 m
3"" 87.6 3.10 m 87.4 3.71m 85.8 3.00
4"" 71.1 3.34 m 70.3 4.02 dd (8.9, 69.3 3.21m
9.3)
«9»»
78.1 3.32 m 113 3.95 m 75.1 (+1.8) 3.34 m
6"" 62.5 3.65 m; 3.85 bd 61.8 4.19 m, 4.43 dd 65.6 (-4.6) 3.83m,4.04dd
(10.5) (2,11.9) (18,10)
OCH3 61.1 3.62 s 60.6 3.85 s 60.1 3.49 s
' Reoofxied at 125 MHz in Me^hanoW4; ^ Italics = interglycx)sidic positions, bold = sul&te positions; (Ac =
6c - ScdesDtfated andog); *" Rfiootded at 500 MHz in Metbanol-</4; ** Recorded at 125 MHz in Vy-dyJhO (5:1); *
RecQided at 500 MHz in Py^5:D20 (5:1); ^ Recorded at 125 MHz in DMSO^;« ReoQided at 500 MHz m
DMSO^
The determination of the position of sulfate groups in holothurins is

important in order to establish structure-activity correlations. Recently,
we have evaluated the antifungal activity of di- and trisulfated glycosides
and their semi-synthetic desulfated analogs against the phytopathogenic
fimgus Cladosporium cucumerinum [25, 53]. We have found that
Hemoiedemosides A (3k) and B (31) and Patagonicoside A (16) wrere
more active than their desulfated analogs. On comparing the antifungal
613
activities of the disulfated glycosides 3k and 16, Hemoiedemoside A

resulted more active. Both glycosides present the same oligosaccharide
chain and differ in the aglycone structure. On the other hand,
Hemoiedemoside B (31) differing from 3k in the presence of a third
sulfate group at C-6 of the terminal 3-0-methylglucose residue is less
active than 31. These results suggest that both the aglycone structure and
the presence and number of sulfete groups at the oligosaccharide chain
play an important role in the antifungal activity of holothurins.
ACKNOWLEDGEMENTS
The authors gratefiilly acknowledge grants from the International

Foundation for Science, Stockhohn, Sweden, the Organization for the
Prohibition of Chemical Weapons, The Hague, The Netherlands,
CONICET, ANPCyT and the Universidad de Buenos Aires. H. D. Chludil
thanks FOMEC-UBA for a fellowship. M. S. M. and A. M. S. are
Research Members of the National Research Council of Argentina
(CONICET).
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SULFUR-CONTAINING NATURAL PRODUCTS

FROM MARINE INVERTEBRATES
MICHELER.PRINSEP
Department of Chemistry, University ofWaikato, Private Bag 3105,

Hamilton, New Zealand.
ABSTRACT: An overview of sulfur-containing natural products isolated from the

marine invertebrate phyla that are commonly studied by natural products chemists, is
provided. The material is arranged by phyla and sulfated compounds are included,
except for the Echinodermata where sulfated saponins and steroids are specifically
excluded. A total of 638 compounds and 530 references are recorded. The review
covers the published literature up until the end of 2001. References to reported
syntheses and comments on biological activities of metabolites are included.
INTRODUCTION
Marine invertebrates such as sponges, bryozoans and tunicates are well

known sources of a wide variety of secondary metabolites or natural
products. Many of these compounds possess novel structures with
unprecedented ring systems or unusual combinations of functional
groups. There is a high incidence of biological activity among the
secondary metabolites of marine invertebrates, which is not surprising,
given the environment that the organisms live in. Marine invertebrates
are either completely sessile, or if able to move, such as is the case for
example, for nudibranchs (sea slugs) and ophistobranchs (sea hares),
they do so only very slowly. Most marine invertebrates lack physical
protection in the form of spines, a sting or a shell, so would seem an
ideal meal for a passing predator. However, these invertebrates flourish
and the fact that they do so, is thought to be due to chemical protection
from predation, overgrowth and infection that their secondary
metabolites convey. For this reason, many research groups worldwide
investigate the natural products of marine invertebrates, primarily
concentrating on their biological activity.
A myriad of structural types are found amongst the secondary
metabolites of marine invertebrates. Halogenation is typical of marine
natural products, which is not unexpected, given that seawater contains
618
very high concentrations of chloride, bromide and iodide ions (559 mM,
0.86 niM and 0.45 |iM respectively) [1]. Sulfur is the fourth most
common element in seawater after chlorine, sodium and magnesium and
the sulfate anion is the second most abundant after chloride [2].
There are many excellent reviews available on marine natural
products in general. The annual reviews by Faulkner [3-20] cover all
secondary metabolites from the major marine phyla. Very few reviews
however, deal solely with sulfur-containing marine metabolites. The
first of these to be published arranged compounds according to structural
types [21]. Another review covers sulfated marine compounds only and
these are arranged by phylum [2]. The most recent review deals with
marine sulfur-containing natural products excluding sulfates and
metabolites are organised according to the sulfur functional groups that
they contain [22]. This review will discuss all sulfur-containing natural
products from the main marine invertebrate phyla studied by natural
product chemists: Bryozoa, Chordata, Cnidaria, Mollusca, Porifera and a
selection of those from the Echinodermata. The vast majority of sulfur-
containing metabolites that have been isolated from echinoderms are
sterol sulfates and saponins and this review will not include coverage of
these but will only deal with other types of sulfur-containing metabolites
from echinoderms. For discussions of the sterol sulfates and saponins of
echinoderms, the reader is referred to other sources [2,3-20]. The
current review concentrates on isolation and biological activity of sulfur-
containing metabolites and covers the literature up to the end of 2001.
As pointed out by Christophersen [21], the distinction between primary
and secondary metabolites is somewhat blurred, therefore, some
compounds that could perhaps be classified most readily as primary
metabolites are included, but macromolecules are specifically excluded.
Every endeavour has been made to be comprehensive, but inevitably
some metabolites may have been overlooked. Metabolites are organised
by phylum and within phyla, compounds are arranged in families, or by
structural type.
Bryozoans
A bryozoan or moss animal is a sedentary colony of minute filter-

feeding individuals called zooids [23]. They are widely distributed
619
throughout the marine environment. There are about 4,000 living

species and over 10,000 as preserved fossils. Apart from a very fev^
freshwâter species, all living bryozoans are marine dwelling [24]. The
zooids that comprise a bryozoan colony each consist of a body wall (the
box or tube) and a polypide. The polypide is made up of a simple
U-shaped gut and an apparatus of tentacles called a lophophore [25].
The inner faces of the tentacles are covered with cilia that beat to
generate a current of water towards the mouth. The supportive outer
body wall is often reinforced by either chitin or calcium carbonate or
both. Zooids can be specialised and take on specific roles within a
colony, such as feeding, brood or funicular (transport or communication)
zooids [25].
Contact with the bryozoan Alcyonidium gelatinosum gives rise to
"Dogger Bank itch", an allergic contact dermatitis. The causative agent
is (2-hydroxyethyl)dimethylsulfoxonium ion (1). Synthesis of 1 was
achieved by base-catalysed condensation of trimethylsulfoxonium
chloride and formaldehyde [26].
OH X"
The sulfur-containing p-carboline alkaloid, l-ethyl-4-methyl-

sulfone-p-carboline (2) was isolated from the New Zealand bryozoan
Cribricellina cribraria, along with several other p-carboline alkaloids.
l-Ethyl-4-methyl-sulfone-P-carboline (2) exhibited only modest
antimicrobial activity, especially compared to some of the other
alkaloids isolated, which were both cytotoxic and antimicrobial [27].
Two sulfur-containing isoquinoline alkaloids, 2-methyl-6,7-

di(methylthio)-2//-isoquinoline-3,5,8-trione (3) and 2-methyl-6-
methylthio-2H-isoquinoline-3,5,8-trione (4) were isolated from a
620
Tasmanian collection of the Australian bryozoan Biflustra perfragilis.

The crystal structure of compound 3 was determined and it exhibited
activity in the brine shrimp assay and against cultures of marine bacteria
but was inactive against human bacterial pathogens [28].
Simultaneously, 7-amino-2-methyl-6-methylthio-"2//-isoquinoline-3,5,8-
trione (5) and compound 3 were reported from a South Australian
collection of the same bryozoan by another research group and named
perfragilins A and B respectively. The bryozoan was referred to as
Membranipora perfragilis but is in fact the same species as B.
perfragilis [28]. Both perfragilins showed activity against the P388
murine leukaemia cell line, with perfragilin B (3) being about one order
of magnitude more active than perfragilin A (5) [29]. Single crystal X-
ray structures of perfragilins A (5) and B (3) were determined [30] and
perfragilin B (3) was later synthesised [31]. These compounds are all
structurally very similar to mimosamycin, which has been isolated from
a terrestrial bacterium and from two marine sponges [28]. This suggests
that the perfragilins are of bacterial origin.
Me-S
The Japanese fouling bryozoan, Dakaira subovoidea was the source

of two 6//-anthra[l,9-Z?c]thiophene derivatives (6) and (7). The structure
of compound 6 was determined by X-ray crystallography [32]. Both
compounds were found to act as antioxidants and were patented for use
as hypolipemics, but no other biological activity was reported [33].
Compound 7 has also been synthesised [34]. Our own studies of the
bryozoan Watersipora subtorquata [35] led to the isolation of the
closely related anthraquinone 5,7-dihydroxy-6-oxo-6//-anthra[l,9-Z?c]
thiophene-l-carboxyhc acid (8), which was identified by nuclear
magnetic resonance (NMR) and mass spectral analysis [36]. Compound
8 exhibited significant cytotoxicity against the African green monkey
kidney cell line, BSC-1 [36].
621
6 R = CH2OH
7 R = C02Me
8 R = CO2H
Two ceramide 1-sulfates (9-10) have been obtained from the

Japanese bryozoan Watersipora cucullata. These compounds are potent
deoxyribonucleic acid (DNA) topoisomerase I inhibitors [37].
HO3SO, C9H19 HO3SO. C7H

7"15
Three new alkaloids, euthyroideones A, B and C (11-13) were

isolated from the New Zealand bryozoan Euthyroides episcopalis [38].
All three compounds contain the unique heterocyclic pyrido(4,3-h)-l,4-
benzothiazine skeleton. The structure of euthyroideone A (11) was
determined by X-ray crystallography, NMR spectroscopy and mass
spectrometry. Euthyroideone B (12) exhibited modest cytotoxicity
against the BSC-1 cell line [38].
11 12 13
Tunicates (Ascidians)
Tunicates or ascidians belong to the phylum Chordata. Ascidiacea is a

class of the subphylum Urochordata (Tunicata) and members of this
622
class are often referred to as tunicates or sea squirts, because their body
is covered with a sack or tunic and many species expel water through a
siphon when disturbed [39]. There are approximately 2,000 living
species of tunicate and of these, ascidians are the most abundant. They
may be solitary or colonial and are sessile, filter-feeding organisms [39].
Biologically active metabolites are quite conmionly found in ascidians
and many of these compounds are derived from amino acids.
Ascidiacyclamide (14), a cytotoxic, cyclic peptide, was isolated from
an unidentified species of ascidian [40]. The absolute configuration was
determined and the structure confirmed by total synthesis [41]. An X-
ray crystal structure was carried out [42] and further X-ray
crystallographic studies determined the conformation of the molecule in
the solid- and solution-states [43].
VN H V
o=< y/^
\ NH HN \
14
Lissoclinum species of tunicates produce a range of cyclic peptides,

which contain thiazole, thiazoline and oxazoline rings. Most belong to
the general families patellamides (octapeptides), lissoclinamides
(heptapeptides) or bistratamides (hexapeptides) [22].
The heptapeptide ulicyclamide (15) and the octapeptide
ulithiacyclamide (16) were the first representatives of a series of cyclic
peptides to be isolated from Lissoclinum patella. Their structures were
elucidated by interpretation of spectral data [44]. A revised structure
was later put forward for ulicyclamide (15) as a result of a detailed
analysis of the fast atom bombardment (FAB) mass spectrum. The same
paper reported the isolation of two more polar cyclic peptides and
another, which was present as a minor component. These heptapeptides
were called lissoclinamides 1-3 (17-19) [45]. An unidentified tunicate
from the Great Barrier Reef contained ulithiacyclamide (16) and
ascidiacyclamide (14) [46]. Two syntheses of ulithiacyclamide (16)
623
have been reported [47,48]. Ulicyclamide (15) was later also

synthesised in high yield by solid phase synthesis [49]. The
conformational properties of ulithiacyclamide (16) were probed using
NMR spectroscopy and molecular mechanics calculations [50].
Ulithiacyclamide B (20) was isolated from L. patella from Pohnpei and
was cytotoxic against the human oral carcinoma cell line, KB [51].
o R
'•\. K'\
O Ph O \ ^ O
15 16 R = CH2CHMe2
20 R = CH2Ph
Y ^
17 18 Rj = Me, R2 = H
19Ri=H,R2 = Me
Three cyclic octapeptides, patellamides A-C (21-23) were isolated

from L. patella and cytotoxicity data for these compounds and for
ulicyclamide (15) and ulithiacyclamide (16) against L1210 murine
leukaemia cells and the human acute lymphoblastic leukaemia (ALL)
cell line CEM were reported [52]. The structures of the patellamides
were later reassigned on the basis of synthetic studies. The proposed
structures of patellamides B (22) and C (23) were synthesised and the
products were shown to differ from the natural products. This led to
new structures being proposed [53,54]. Separate syntheses of
624
patellamide A (21) [55], patellamide B (22) [56] and of patellamides B

and C (22-23) [57] produced compounds that were identical to the
natural products.
oK yr^ o=( y-T
21 22 R = CH2CHMe2
23 R = CH(Me)Et
The spectral data of the patellamides was also reasssigned and the
new assignments used for elucidation of the structures of three new
metabolites, the octapeptide prepatellamide B formate (24) and the
heptapeptides, prelissoclinamide 2 (25), and preulicyclamide (26) [58].
The molecular conformation of patellamide A (21) was determined by
X-ray crystallography [59,60] and the solution conformations of
patellamides B (22) and C (23) were determined by NMR spectroscopy
and molecular dynamics [61]. The octapeptide preulithiacyclamide (27)
is a potent inhibitor of Macrophage Scavenger Receptor and was
isolated from L. patella from Palau along with other known cyclic
peptides [62].
\=M H Û
>=0
o\"r<:/-
25
625
HO-'Z \ HN HN "^ S N .NH

(Qy-NH
H " ' f "^ H " N ' ^ O
PK
26 27
Patellazoles A-C (28-30) are further cytotoxic, thiazole-containing

macrolides isolated from L. patella [63,64]. Structural elucidation of
patellazoles B and C (29-30) relied on interpretation of data that did not
permit stereochemical assignments.
2o R} — H, R.2 — H
29 Ri = H, R2 = OH
30 Ri = OH, R2 = OH
Three cytotoxic peptides, patellamide D (31) and hssoclinamides 4-5

(32-33) were isolated from a Great Barrier Reef specimen of L. patella
and identified by interpretation of spectral data. The peptides were
found within the obligate algal symbiont of the genus Prochloron [65].
Another study of the same Australian L. patella reported lissoclinamide
6 (34), in addition to Hssoclinamides 4-5 (32-33) and patellamide D (31).
The structure of patellamide D (31) was obtained by X-ray
crystallography and its conformation compared with those obtained by
molecular modelling [66]. Patellamide D (31) has been reported to be a
626
resistance-modifying agent in the multidrug resistant CEM/VLBIOO

human leukaemic cell line [67]. The structure of lissoclinamide 5 (33)
was revised as a result of total synthesis [68] and a total synthesis of
lissoclinamide 4 (32) was described [69]. Later, total syntheses of
lissoclinamides 4 (32) and 5 (33) allowed the revision of their
configurations [70].
^6^^.\ ^ a \ ^'^\\
P h / s ^ - / ^P^
31 32 X = thiazoline 34
33 X = thiazole
Lissoclinamides 7 (35) and 8 (36) were isolated from a Great Barrier

Reef specimen of L. patella. Structure-activity studies showed that
lissoclinamide 7 (35) with two thiazoline rings, was the most effective in
vitro cytotoxin of those tested [71]. The stereochemistry of
lissoclinamide 7 (35) was determined by synthesis using Burgess reagent
to form oxazoline and thiazoline rings without scrambling adjacent
chiral centres [72]. Lissoclinamides 9 (37) and 10 (38) were isolated
from an Indonesian L. patella sample [73] while prepatellamide A (39)
was isolated as a minor component of another Indonesian specimen of L.
patella [74].
: o Y
f^NÔ HN O-l"" HN /
^ JL N / VN H "^^Q
ph-^ °
35 X = thiazoline 37
36 X = thiazole
627
38 39
A further cytotoxic, octapeptide, patellamide E (40), was isolated

from L patella from Singapore and the structure was elucidated by
chemical and spectral methods [75]. Patellamide F (41) was isolated
from L patella from north-western Australia and was also cytotoxic.
The structure and absolute stereochemistry of patellamide F (41) were
established by chemical and spectroscopic methods. Patellamide B (22),
ulithiacyclamide (16) and lissoclinamide 3 (19) were also isolated from
the same sample [76]. The octapeptides, patellamide G (42) and
ulithiacyclamides E-G (43-45) were isolated from L. patella from
Pohnpei, along with known series members [77].
I O N ^ OH O ;
•^ NH HN ^ NH HN
f~f H VP fll H Yp
40 Rj = CH(Me)Et, R2 = Me 42
41 Rj = CHMe2, R2 = H
628
- - O
OH f ^ OH O (^ ^ O f ^
"V^N^S "S^N^S ^ ^ ^ N \ ^S
OÎH H J > o^L H JJ oCj fi T
"" /^^ H
HN\ """VN A)
J^ """VN H HN\
S^NYJ>^. ^^Jl^N^^^ S ^ N ^ ^
Ph-^ O OH p^/^ O p ^ ^ O OH
43 44 45
Two cyclic hexapeptides, bistratamides A (46) and B (47) were

isolated from L bistratum. They were found in the obligate algal
symbiont Prochloron [78]. L. bistratum from the Philippines contained
bistratamides C (48) and D (49), with bistratamide D (49) being a central
nervous system depressant [79]. The structure of bistratamide C (48)
was confirmed by total synthesis using enantiomerically pure oxazole
and thiazole amino acids [80], while bistratamide D (49) was
synthesised using a convergent strategy and enantiomerically pure
oxazole, thiazole and oxazoHne segments [81].
r^NH HNÔ ^--f^NH HN"^0
46 X = thiazoline 48 49
47 X = thiazole
L. patella from Fiji contained patellins 1-5 (50-54) [82] and earlier,
solution- and solid-state conformational studies were carried out on
patellin 2 (51), and the structure was determined by X-ray analysis [83].
A Lissoclinum sp. from the Great Barrier Reef yielded patellins 3 (52), 5
(54) and 6 (55) and the heptapeptide trunkamide A (56) [82].
Compounds 50-56 were all identified by interpretation of spectral data
and through use of Marfey's method to determine the absolute
stereochemistry of the constituent amino acids [82]. A total synthesis of
the proposed structure of trunkamide A (56) revealed that the structure
629
of the natural product should be reinvestigated [84] and the structure was
indeed later revised as the result of a total synthesis [85]. Another total
synthesis was later reported [86].
50 51
Solution- and solid-state conformations of tawicyclamides A (57)

and B (58), proline-containing cyclic peptides from a Philippines
specimen of L. patella, were determined by spectroscopic and X-ray
analyses respectively [87]. Patellins 1-6 (50-55), trunkamide A (56) and
the tawicyclamides A-B (57-58) all lack the oxazoline ring present in
most other cyclic peptides isolated from the Lissodinum genus [22].
0 - ^ N - ""
52 R = CH2CHMe2 54
53 R = CHMe2
55 56
630
57 R = CH2Ph
58 R = CH2CHMe2
A number of cyclic peptides have been isolated from the tunicate

Didemnum molle. The structure of mollamide (59), a cytotoxic
heptapeptide isolated from Didemnum molle from the Great Barrier
Reef, was determined by X-ray crystallography and chemical
degradation [88]. Mollamide has also been synthesised [89].
Cyclodidemnamide (60) is a weakly cytotoxic, heptapeptide from D,
molle from the Philippines [90]. Total synthesis of the proposed
structure of cyclodidemnamide gave a product with different spectral
data to those of the natural product, which is thought to be a
stereoisomer [91]. The stereochemistry of one of the two valine residues
in cyclodidemnamide was revised from L-valine to D-valine as a result of
the total synthesis of both isomers [92] and the configuration was also
reassigned as a result of total synthesis [93]. The hexapeptides,
comoramides A (61) and B (62), were isolated from D. molle from
Mayotte lagoon in the Comoros Islands, while the heptapeptides,
mayotamides A (63) and B (64) were isolated from a separate collection
of D. molle from the Comoros Islands [94].
r~^ kÔ
0=( O " N=r
>-NH HN-< ^^
\ P - - O Ph
59 60
631
°y:A: ^"xrx VrT

61 62 63 R = Me
64R = H
A Didemnum sp. from Palau was the source of didemnaketal C (65),

which contains a sulfonic acid group [95]. The in vitro anti-human
immuodeficiency virus (anti-HTV) activity of D. molle from Pohnpei is
associated with the sulfated mannose polysaccharide kakelokelose (66)
[96].
o o o" o
Me02C>,,^s!>sÂA,^^.->^^ -Ôv.^ OSOjNa
HO-^JUQ;^ OSOsNa
NaOsSO-^^"^"^^
66
Lamellarin T-V and Y sulfates (67-70) were isolated from an

unidentified ascidian from the Arabian Sea coast of India [97]. Four
additional lamellarin sulfates, the 20-sulfates of lamellarins B, C and L
and lamellarin G 8-sulfate (71-74) were isolated from Didemnum
chartaceum from the Great Barrier Reef [98]. Unusually long relaxation
times were observed for certain signals in the ^H NMR spectra of these
compounds. Lamellarin a 20-sulfate (75) was isolated from an
unidentified ascidian from India and was an inhibitor of human
immunodeficiency virus type 1 (HTV-l) integrase [99].
632
R,0. .O^Ô
R7O
R.O
OH o R i OMe OR4 OMeORs
67 Ri
Ell = Me, R2 = OMe, R3 = H 71 ^5, R^ = SOsNa, R2 = Me, R3 = H, R4 = Me, R5 = Me, R^ = OMe,
68 Ri = Me, R2 = H, R = H 72 RJ = SOaNa, R2 = Me, R3 = H, R4 = Me, R5 = Me, Re = OMe
69 Ri = Me, R2 = OMe, R3= OH 73 R^ = S03Na, R2 = Me, R3 = Me, R4 = H, R5 = H, R^ = H
70 Ri = H, R2 = H, R3= H 74 Ri = Me, R2 = H, R3 = Me, R4 = H, R5 = S03Na, Re = H
75 Ri = S03Na, R2 = Me, R3 = H, R4 = Me, R5 = Me, Rg = H
Didemnum rodriguesi from New Caledonia contained the unusual

peptidyl alkaloid caledonin (76), that formed a complex with Zn^"^ and
Cu^ ions between thiol and primary amine groups [100]. The
minalemines D-F (77-79) are peptide guanidine derivatives isolated
from a Caribbean collection of D. rodriguesi and contain a sulfamic acid
group [101]. The stereochemistry of cyclodidemniserinol trisulfate (80)
from a Palauan specimen of Didemnum guttatum was partially
determined [102].
f^ N o NH2
. N O "
NH
H H »03? 9 f H U
H2N N^ 'N NH2
H ^
NH O R " O
77 R = C7H15
78R = C8Hn
79R = C9Hi9
OSOaNa
NHS03Na
NaOSOj^
80
633
The structure of the cytotoxic metabolite dendrodoine (81) from the

tunicate Dendrodoa grossularia was determined by X-ray analysis [103]
and it was later synthesised by a convergent route [104].
cxA:^ 81
N
V .Me
Me
The eudistomins are p-carboline alkaloids isolated from Eudistoma

olivaceum. Eudistomins C, E, F, K and L (82-86) all contain a novel
oxathiazepine ring [105]. It was later proposed that the stereochemistry
of eudistomins C, E, F, K and L (82-86) should be revised on the basis
of a nuclear Overhauser enhancement difference spectroscopy (NOEDS)
study of eudistomin K (85) from Ritterella sigillinoides [106].
Eudistomin K sulfoxide (87), an antiviral agent from /?. sigillinoides
[107] was synthesised from eudistomin K (85), and the structure and
absolute configuration of compound 85 were determined by X-ray
analysis [108]. R. sigillinoides also contains debromoeudistomin K (88),
in addition to known eudistomins [109]. A^(10)-Methyleudistomin E
(89) was isolated from E, olivaceum from the Caribbean [110].
82 Ri = H, R2 = OH, R3 = Br, R4 = H 87 89
83 R, = Br, R2 = OH, R3 = H, R4 = H
84 Ri = H, R2 = OH, R3 = Br, R4 = C2H3O2
85 R, = H, R2 = H, R3 = Br, R4 = H
86 Ri = H, R2 = Br, R3 = H, R4 = H
88 Rj = H, R2 = H, R3 = H, R4 = H
Syntheses of both (-)-eudistomin F (84) [111] and of (-)-eudistomin

L (86) [112] have been reported, while (-)-eudistomins C, E, F, K and L
(82-86) were later also synthesised from the corresponding A^-
hydroxytryptamines and D-cysteinal [113].
634
Eudistomidin C (90) was one of three antileukaemic p-carboline

alkaloids isolated from an Okinawan sample of Eudistoma glaucus. It
was identified by spectral methods and synthesis of a derivative [114].
Eudistomidins E (91) and F (92) were isolated from E, glaucus from
Okinawa and identified by spectroscopic techniques [115].
Eudistomidins C (90) and F (92) contain a methyl sulfide group while
eudistomidin E (91) contains a methyl sulfoxide. 14-
Methyleudistomidin C, (93) was isolated from Eudistoma gilboverde
along with known eudistomins [116]. 14-Methyleudistomidin C (93)
exhibited potent cytotoxic activity with IC50 values less than 1 |Lig/mL
against four human tumour cell lines.
SMe
Citorellamine (94) is an indole disulfide dihydrochloride from the

Fijian tunicate Polycitorella mariae. It exhibits potent antimicrobial and
insecticidal activity in addition to cytotoxicity [117]. The structure was
later revised and syntheses of the proposed and true structures carried
out [118].
'NH
k / S f .2HC1
N
H
94
A Didemnum sp. from Rota in the northern Mariana Islands contained

four new p-carboline alkaloids, didemnolines A-D (95-98), together
with known metabolites [119]. The didemnolines are characterised by
substitution at N9 as opposed to CI. A straightforward synthesis of the
didemnolines was reported [120].
635
MeS^N
1 H
Me
95 R = Br 97 R = Br
96R = H 98 R = H
Two antineoplastic 24-membered macrolide sulfates, iejimalides C

(99) and D (100) were isolated from Eudistoma cf. rigida and identified
by interpretation of spectral data [121].
o
Me0s.^x^^x::^>s^^^^0^.Av^'<^^-^jÂ^NHCH0
»X
0S03Na
The structures of two thiazoles (101-102) from Aplidium pliciferum

were elucidated by spectral interpretation and confirmed by synthesis
[122]. A New Zealand species of Aplidium contained a cytotoxic and
antimicrobial 1,2,3-trithiane derivative (103), which on standing at room
temperature for one month in neutral solution, gave 2-vanilloyl
imidazole [123], previously reported as a metabolite of A. pliciferum
[122]. Hysistozoa fasmeriana from New Zealand gave the new (-)-
enantiomer of rran5^-5-hydroxy-4-(4'-hydroxy-3'-methoxyphenyl)-4-(2"-
imidazolyl)-1,2,3-trithiane (103). A second collection of the same
ascidian yielded compound 103 and two dithiane alkaloids,
fasmerianamines A (104) and B (105). The structures were determined
by spectral studies. Both enantiomers of the trithiane 103 exhibited
identical biological activities in a range of assays, including modest
cytotoxic and antimicrobial properties, while the fasmerianamines (104-
105) were inactive [124].
636
S MeO.
J U
OMe
103 104 R = OMe

105 R = OH
Lissoclinotoxin A (106), the antimicrobial constituent of L

perforatum from France, was reported to be a 1,2,3-trithiane derivative
by analysis of spectral data [125]. The structure of lissoclinotoxin A
was later revised to the corresponding pentasulfide, based on
unpublished mass spectral data of a derivative and lissoclinotoxin B
(107), another pentasulfide, was identified by spectral data interpretation
[126]. A Lissoclinum sp. from the Great Barrier Reef, Australia
contained lissoclinotoxin A (106), lissoclinotoxins C (108) and D (109)
and Hssoclins A (110) and B (111) [127]. Lissoclinotoxin C (108) is a
dithiomethyl compound while lissoclinotoxin D (109) is dimeric.
Diplamine (112), a further cytotoxic methyl sulfide-containing
pyridoacridine, was isolated from the tunicate Diplosoma sp. [128] and
later, a total synthesis in 21 steps was reported [129]. Lissoclin
disulfoxide (113) inhibits interleukin-8 receptors and is a dimeric
alkaloid from a South African species of Lissoclinum [130]. The
proposed structure is a symmetrical head-to-head dimer but the spectral
data does not eliminate a symmetrical head-to-tail possibility [17].
NH2
OMe OMe OMe
S-S^ HOY^'SMe HOîyS-S,
S'S
NH2 NH2 NHo

106 107 108 109
637
OMeO OMe
MeS T N ^^ MeS^ y ^S" ^ "SMe
NHR NMe2 NMe2

110 R = COCH2CHMe2
111 R = CO(Me)C=CHMe (E) 113
111 R = COMe
Varacin (114), a cytotoxic compound closely related to

lissoclinotoxin A (106), was isolated from a Fijian sample of L. vareau
and a benzopentathiepin structure was proposed on the basis of spectral
data [131]. Two total syntheses of varacin (114) have been carried out
[132,133] and later, further syntheses were described [134-135]. N,N-
dimethyl-5-(methylthio)varacin (115) and the corresponding trithiane
(116) were obtained from L. japonicum from Palau and 3,4-
desmethylvaracin (117) was isolated from a Eudistoma sp. from Pohnpei
[136]. An inseparable mixture of 5-(methylthio)varacin (118) and the
corresponding trithiane (119) was obtained from a Pohnpeian
Lissoclinum sp. [136]. Three additional antimicrobial polysulfides of the
varacin family (120-122) were isolated from Polycitor sp., collected by
dredging in the Sea of Japan [137].
RoQ
NR4 NR2
114 Ri = Me, R2 = Me, R3 = H, R4 = H2 116 Rj = SMe, R2 = Me2
115 Rj = Me, R2 = Me, R3 = SMe, R4 = Me2 119 Rj = SMe, R2 = Hj
117 Ri = H, R2 = H, R3 = H, R4 = H2 120 R, = H, R2 = H2
118 Ri = Me, R2 = Me, R3 = SMe, R4 = H2
OMe o
's
121
638
The methyl sulfide-containing alkaloids, varamines A (123) and B

(124) were isolated from L. vareau. Their structures were determined by
interpretation of NMR spectral data and by comparison with related
alkaloids. The varamines were cytotoxic towards L1210 murine
leukaemia cells with IC50 values of 0.03 and 0.05 |J.g/mL, respectively
[138]. The varamines (123-124), Hssoclins (110-111) and diplamine
(112) all contain a methyl sulfide group linked to a pyridoacridine ring
system [22].
MeS"
OMe
123 R = Me
124 R = H
The shermilamines are a group of alkaloids containing a thiazinone

ring attached to a pyridoacridine ring system. The structure of
shermilamine A (125), an orange pigment from a species of
Trididemnum from Guam was determined by X-ray analysis [139].
Shermilamine B (126) was later isolated from the same Trididemnum
extract and from a Eudistoma sp. [140]. Shermilamine B (126) was also
isolated from the Red Sea tunicate Eudistoma and the structure was
elucidated on the basis of spectroscopic data [141].
126
Dehydrokuanoniamine B (127) and shermilamine C (128) were

isolated from a Cystodytes sp. from Fiji. Their structures were
determined by analysis of spectroscopic data. These compounds
displayed dose-dependent inhibition of proliferation in human colon
tumour cells in vitro [142]. Shermilamines D (129) and E (130) were
639
isolated from Cystodytes violatinctus from the Comoros Islands, along

with tintamine (131), which has a novel heterocyclic skeleton [143].
Cycloshermilamine D (132) was also isolated from C violatinctus from
the Comoros Islands in extremely low yield and is another
pyridoacridine alkaloid with a novel heterocyclic ring system [144].
129R = NMe2
130R = N(O)Me2
OH 0;5s^N.
NMe2
132
The pyridoacridine alkaloids, kuanoniamines A-D (133-136) were

isolated from an unidentified Micronesian tunicate and its nudibranch
predator, Chelynotus semperL The structures were established by
extensive NMR spectral analysis. Cytotoxicity against KB cells ranged
from IC50 values >10 |Lig/mL for kuanoniamine B (134), 5 [Xg/mL for
kuanoniamine D (136), to 1 |Lig/mL for kuanoniamine A (133) [145].
Kuanoniamine A (133) has also been synthesised [146,147].
133
640
NHR
134 R = COCH2CHMe2
135 R = COEt
136 R = Ac
Polycarpamines A-E (137-141) are unusual sulfur-containing

antifungal agents from Polycarpa auzata from the Philippines. The
structures were elucidated by interpretation of spectral data [148].
Polycarpine (142), a cytotoxic, dimeric, disulfide alkaloid, the
corresponding dihydrochloride (143) and two sulfur-containing related
monomers (144-145) were isolated from Polycarpa clavata from
Western Australia [149]. Polycarpine (142) was also isolated with two
monomers (144,146) from P. aurata from Chuuk [150] and later it was
synthesised in three steps from p-methoxyphenacyl bromide [151].
NMeo NMe2 NMe7

MeO.
Y\S MeO. HO,
MCO^'YTC^
MeSS R OMe MeSS R MeOS OMe
137 R = H 138 R = O 141

140 R = COMe 139 R = S
NH2
OMe
c Me
01U'
MeO Me^^^Y
NH2
jr^'^" ..o^^
VN
142 144 R = OMe 146

143 = .2HC1 145 R = OH
The in vivo antitumour activity of extracts of the tunicate

Ecteinascidia turbinata was noted in the late 1960s [152] but the active
metabolites were only isolated and identified much later by two research
groups. These complex alkaloids were termed the ecteinascidins and are
641
abbreviated as Et with a number representing the value of the highest

mass ion observed in the positive ion FAB mass spectrum. The Harbor
Branch group [153] identified two compounds that were identical to
ecteinascidins 729 (147) and 743 (148), identified at Illinois where
compounds 745 (149), 759A (150), 759B (151) and 770 (152) were also
reported [154]. The stereochemical representations at the 11,13
bridgehead differ between the two groups. Ecteinascidins 759A (150)
and 759B (151) were tentatively assigned as A^-oxides of ecteinascidin
743 (148) [154]. X-ray crystal structures of the N12-formyl derivative
of ecteinascidin 729 and of the natural N12-oxide (153) of ecteinascidin
743 (apparently different from compounds 150 and 151) were
determined [155]. An enantioselective total synthesis of ecteinascidin
743 (148), which entered phase I clinical trials as an anticancer agent,
has been reported [156] and synthesis of 148 from the fermentation
product cyanosafracin B can provide sufficient quantity for clinical trials
[157].
OMe OMe
H0,^Js^Me HO^ Js^^Me
MeO. J ^ -NH
147 Ri = H, R2 = OH 153
148 Rj = Me, R2 = OH
149Ri=Me, R2 = H
150 Ri = Me, R2 = OH, TV-oxide
151 Ri = Me, R2 = OH, N-oxide
152Ri=Me,R2 = CN
Ecteinascidins 597 (154), 583 (155), 594 (158) and 596 (158) are
putative biosynthetic precursors of ecteinascidins and were isolated
from £". turbinata from the Caribbean [158]. A recent review on the
chemistry and pharmacology of the ecteinascidins has been published
[159].
642
OMe OMe OMe
MeO
Four simple sulfates (158-161) were identified as antimicrobial

constituents of Halocynthia roretzi from Japan [160]. Sodium (or
potassium) 2,6-dimethylheptyl sulfate (161) was also found in Polycitor
adriaticus from Croatia [161]. The absolute configuration of 2,6-
dimethylheptyl sulfate (161), which has also been found in other
Mediterranean ascidians, has been determined using Mosher's method
[162].
The Mediterranean ascidian Halocynthia papillosa contained two

cytotoxic sulfates, 6-methylheptyl sulfate (162) and (F)-oct-5-enyl sulfate
(163) [163].
ÔSOjNa ÔSOjNa
162 163
Ascidia mentula from the Mediterranean was the source of two

antiproliferative alkyl sulfates, sodium salts of 3,7,11,15-
tetramethylhexadecane-l,19-diyl disulfate (164) and heneicosane-1,21-
diyl disulfate (165) respectively [164]. Microcosmus vulgaris, also
643
collected in the Mediterranean, was the source of the sodium (or

potassium) salt of (3Z)-4,8-dimethylnon-3-en-l-yl sulfate (166) [165].
ÔSOgNa
OSO.Na
164
NaO^;
165
166
The Mediterranean tunicate Sidnyum turbinatum contained four alkyl

sulfates, 1-heptadecanyl sulfate (167), 1-octadecanyl sulfate (168), sodium
(25)-2,6,10,14-tetramethylpentadeca-l,18-diyl sulfate (169) and 1-hexyl
sulfate (170). The structures were determined by spectroscopic and
chemical methods. All exhibited antiproliferative activity in vitro against
the murine fibrosarcoma cell line, WEHI 164 [166].
NaO.SO NaOiSO
167 168
^0S03Na Na03S0
NaOaSO
169 170
The structure of polyclinal (171), an aromatic sulfate from a

Califomian specimen of Polyclinum planum, was determined by X-ray
crystallography [167].
OH
^CHO
ÔSOsNa
OH
171
644
Uoamines A (172) and B (173) are piperidine alkaloids, isolated from

Aplidium uouo from Maui, Hawaii. They differ only in the geometry of
the 3-thiomethylacrylate ester group [168].
OH
AÂ^ O SMe
H
172 173
Tasmanian collections of Clavelina cylindrica yielded the alkaloids

cylindricines F (174) and G (175), the first thiocyanates isolated from an
ascidian [169]. Cylindricines H-J (176-178) were later isolated from the
same species [170].
,0Ac ,0Ac
SCN
NCS" RH2C
174R = (CH2)4Me 176 R = SCN

175 R = (CH2)2Me 177 R = NCS
A Micronesian ascidian, Nephteis fasicularis, was the source of

fasicularin (179), a tricyclic, thiocyanate-containing alkaloid that was
active in a DNA damaging assay [171]. The structure was confirmed by
total synthesis [172].
C^Hv
NCS.
The virenamides A-C (180-182), thiazole-containing cytotoxic linear

peptides, were isolated from the colonial ascidian Diplosoma virens
collected on the Great Barrier Reef, Australia. Their structures were
645
deduced from NMR spectral data and confirmed using Marfey's procedure
[173]. Virenamides D (183) and E (184) were also obtained from D.
virens from the Great Barrier Reef [174] and virenamide B (181) has been
synthesised [175].
^ . \ ,,
180 181 R = CHMej

182 R = CHjPh
183 184
An enediyne antitumour antibiotic, namenamicin (185) was isolated

from Polysyncraton lithostrotum from Fiji [176].
NHC02Me
HO-^'
wo o^
Y
OH HN.,
185
646
Cnidaria (Coelenterates)
The Cnidaria comprise about 8,000 living species and include jellyfish,
corals, soft corals or gorgonians, sea anemones and hydrozoans. They are
the lowest members of the animal kingdom with cells organised into
specialised organs [177]. Cnidarians have a single internal cavity, which
acts as a stomach and a single opening above, which is encircled by
tentacles and through which food enters and waste escapes [178]. Some
Cnidaria are solitary and consist of a single polyp such as sea anemones
and others are colonial such as corals but all Cnidaria are radially
symmetrical [178]. Many have nematocysts or stinging cells but these
organisms are less likely to contain secondary metabolites for use in
chemical defence, as they are not really required. Terpenoids are very
commonly isolated from this phylum but very few sulfur-containing
compounds have been found in Cnidarians.
The marine hydroid Tridentata marginata contained the aromatic
alkaloids tridentatols A-C (186-188). Tridentatol A (186) inhibited
feeding by the planehead filefish. The structure of tridentatol C (188) was
elucidated by a single crystal X-ray diffraction study [179].
^QXJ SMe ^QXJ NySMe kjk ^

SMe 1 >-SMe
186 187 188 ^
A zoanthid from the Indian Coast, Zoanthus sp., contained the sulfated
sphingolipid hariamide (189) [180].
o
C9H19
Two new ultraviolet (UV) absorbing compounds, palythrine-

threonine-sulfate (190) and palythrine-serine-sulfate (191) were isolated
from the reef-building coral Stylophora pistillata [181].
647
jj OMe
R OH yC
HO ÔSOjH
190R=Me
191 R = H
The sea anemone Anthopleura elegantissima was the source of the

sulfonic acid-containing compound mycosporine-taurine (192) [182],
HOH2CJ X ^ SOH
H
192
Molluscs
The phylum MoUusca comprises approximately 100,000 species, making

it one of the largest animal phyla [177]. The name mollusc means "soft-
bodied". Molluscs are non-segmented, have a head with tentacles and
move by crawling on a foot. For bivalves such as mussels and oysters, the
foot is a digging tool and for cephalopods such as squid and octopuses, it
is formed into tentacles. The outer body covering is termed the mantle
and usually secretes a shell to protect the body [183]. The shell-less
molluscs such as the carnivorous nudibranchs (sea slugs) and herbivorous
ophistobranchs (sea hares), are well known sources of bioactive secondary
metabolites but in many cases the mollusc itself does not produce the
compound but sequesters it from its diet. Similarly, filter-feeding bivalves
have been the sources of large toxic compounds but the actual producers
of these compounds are thought to be microorganisms.
Adenichrome is an Fe (Ill)-containing pigment from bronchial heart of
Octopus vulgaris. It consists of a mixture of closely related peptides
derived from glycine and the isomeric amino acids adenochromines A, B
and C (193-195) [184,185].
648
.CO2H . ro^H
193 Ri= ^-S^ R,= f - S /-< R3 = H
NH NH9
R4N^N R4N^N
R4 = H or Me R4 = H or Me
CO2H .CO2H .CO2H
194Ri = H. R2= ^-S^ R3= ^
^pNH2 =( NH2 NH2
R4N^N
h
.CO2H ,C02H
195 R R2 = H, R3= ^-S
=( NH, NH2
R4Nv^N R4N.^N
The brominated alkaloid neoaplaminone sulfate (196) was isolated

from the sea hare Aplysia kurodai and its structure was determined by
spectral and chemical methods [186]. A. kurodai obtains most, if not all of
its metabolites from the red algae on which it feeds [10].
OMe
Me2N
196
The antineoplastic, cyclic peptide dolastatin 3 (197) was isolated from

the sea hare Dolabella auricularia in small quantities [187]. It bears much
structural resemblance to the cyclic peptides of tunicates. Synthetic
attempts indicated that the original published structure was incorrect
[188]. Three reports of research directed towards synthesis of possible
components of dolastatin 3 (197) failed to help with the correct structure
[189-191]. Reisolation of 197 allowed the determination of the correct
sequence of amino acids in this cyclic pentapeptide and the new structure
was confirmed by synthesis [192]. Synthesis of dolastatin 3 (197) and the
corresponding 12/? diastereoisomer permitted study of the solution
649
conformations by NMR and circular dichroism (CD) spectroscopy and by

molecular forcefield calculations [193,194].
CONH9
The pentapeptide dolastatin 10 (198) was isolated from D. auricularia

and the structure was proposed on the basis of spectral studies [195].
Dolastatin 10 (198) is a very potent antineoplastic agent. The absolute
configuration proposed from spectral analysis was confirmed by synthesis
[196] and dolastatin 10 (198) has subsequently been synthesised by
several other research groups [197-199].
•X"vA
Me, A . , N . ^ ^.j^N-
Me O yK^ Me OMeO M^
198
D. auricularia from Japan contained dolabellin (199), a cytotoxic bis-

thiazole. The structure was elucidated by spectral data examination and
chemical degradation [200]. D. auricularia also contained the cyclic
hexapeptide dolastatin E (200) [201], the stereochemistry of which was
determined by chemical degradation and total synthesis [202]. A further
cytotoxic hexapeptide, dolastatin I (201) was isolated from D. auricularia
from Japan [203] and the stereostructure was confirmed by
enantioselective synthesis [204]. Dolastatin 18 (202) was isolated as a
very minor cytotoxic component of D. auricularia from Papua New
Guinea [205].
650
CI CI
OH
OH
199
H NT H ^ 1
-V- "-^^
yN " N O r^
Ph
'NH HN'^^0
201 202
The ichthyotoxic diacylglycerol umbraculumin C (203) was isolated

from the Mediterranean ophistobranch Umbraculum mediterraneum [206].
The stereochemistry of compound 203 was determined by chemical
interconversion and total synthesis [207].
The nudibranch Glossodoris quadricolor which feeds on the sponge

Latrunculia magnifica, contained the sponge metabolite, latrunculin B
(204) [208], while the nudibranch Chromodoris elisabethina from Guam
and Enewetak in the Marshall Islands contained the icthyotoxic latrunculin
A (205) [209]. Both of these compounds were previously isolated from
the Red Sea sponge, L. magnifica [210,211]. Latrunculin A (205) was
also found in the nudibranch Chromodoris lochi and the sponge, Spongia
mycofijiensis [212].
651
p? -^r?
i^ ^p
X" HN'A X"
204 205
Extracts of the digestive gland of the nudibranch Doris verrucosa

contained the unusual xyloside, 9-(5-deoxy-5-methylthio-P-D-
xylofuranosyl)adenine (206), identified by comparison with synthetic
material [213].
NH2
OH
206
The Australian nudibranch, Cerastoma brevicaudatum contained

(methylthio)furodysinin (207) and dithiofurodysinin disulfide (208) [214],
derivatives of thiofurodysinin acetate (209) that had been isolated earlier
from a sponge [215]. Compounds 207 and 208 are also assumed to be of
sponge origin.
Specimens of the nudibranchs Phyllidia pustulosa and P. varicosa

from the Philippines contained 4a-isothiocyanogorgon-ll-ene (210), in
addition to known compounds [216].
652
SCN
210
Keenamide A (211) is a thiazoline-containing cytotoxic, cyclic

hexapeptide that was isolated from the mollusc Pleurobranchus forskalii,
which is known to feed on ascidians [217].
O
211
Tyrindoxyl sulfate (212) was originally isolated from the gastropod

Murex truncatus and has been used as a source of the dye Tyrian purple
since antiquity. It has also been identified in several other species of
marine molluscs [2].
pSOgNa
^^^SMe
ax ^
212
The kidney of the giant clam Tridacna maxima yielded an arsenic-

containing sugar sulfate (213), the structure of which was determined by
X-ray crystallography [218].
Me
0=As.
HO OH
213
653
Yessotoxin (214) is a polyether from the scallop Patinopecten

yessoensis and has been implicated in diarrhetic shellfish poisoning (DSP).
The structure and partial stereochemistry of yessotoxin were deduced from
spectral data [219]. The relative stereochemistry of yessotoxin and the
structures of two new analogues, 45-hydroxyyessotoxin (215) and
45,46,47-trinoryessotoxin (216) were also established [220]. The absolute
stereochemistry of yessotoxin (214) was determined by NMR
spectroscopy using a chiral anisotropic reagent [221]. The absolute
configuration at C45 in 45-hydroxyyessotoxin (215), isolated from P.
yessoensis, was determined by the use of a modified Mosher's method
[222].
214 Ri = SOgNa, R2 = \
215Ri = S03Na,R2= V
OH
216Ri = S03Na,R2= \'^^^''^tx'^
220Ri = H,R2= V
222Ri = S03Na,R2 =
Two analogues of yessotoxin, homoyessotoxin (217) and 45-

hydroxyhomoyessotoxin (218) were isolated from the digestive glands of
mussels from the Adriatic Sea. Their structures were determined by NMR
spectroscopy and mass spectrometry [223]. Adriatoxin (219), a further
yessotoxin analogue, was isolated from the digestive glands of the mussel
654
Mytilus galloprovincialis from the Adriatic coast of Italy [224]. 1-

Desulfoyessotoxin (220) was isolated from the digestive glands of
Norwegian Af. edulis [225].
HO.SO'
HO3SO'
217 R = H
218 R = OH
OSOaNa
219
Carboxyhomoyessotoxin (221) was isolated from the digestive glands

of mussels from the northern Adriatic Sea [226]. Two further analogues
of yessotoxin, carboxyyessotoxin (222) and 42,43,44,45,46,47,55-
heptanor-41-oxohomoyessotoxin (223) were isolated. Carboxyyessotoxin
(222) was isolated from DSP-infested M. galloprovincialis from the Italian
Coast of the Adriatic Sea [227], while 42,43,44,45,46,47,55-heptanor-41-
oxohomoyessotoxin (223) was also isolated from digestive glands of
mussels from the northern Adriatic. The structure of compound 223 was
determined by NMR spectroscopy and mass spectrometry [228].
655
CO2H
NaOgSO'
111
NaO^SO*
NaO.SO'
223
A chlorosulfolipid (224) has been isolated from the hepatopancreas of

mussels from the northern Adriatic Sea. The structural determination,
including the absolute stereochemistry, was carried out by extensive NMR
spectral analysis and through molecular mechanics and dynamics
calculations [229].
224R = S03H
656
The cockle, Austrovenus stutchburyi from New Zealand contained

brevetoxin Bi (225) [230] and the greenshell mussel, Pema canaliculus
contained brevetoxin B3 (226) [231]. A further brevetoxin analogue,
brevetoxin B2 (227) was isolated from the hepatopancreas of P.
canaliculus [232], while the major toxin in neurological shellfish
poisoning (NSP) associated with P, canaliculus was identified as
brevetoxin B4 (228) [233].
NaOaSO
CO2H
H '-'iV ^ H
226 R = COC13H27, COC15H31
HQ q
)-C02H
227
657
The cytostolic phospholipase A2 inhibitor tauropinnaic acid (229) was

isolated from the Okinawan bivalve Pinna muricata (pen shell). The
structure and stereochemistry at all but one centre were elucidated by
interpretation of spectroscopic data [234].
The genus Conus comprises approximately five hundred species of

predatory cone snails and is therefore, one of the largest, if not the largest,
single genus of marine animals alive. Each species of snail produces a
unique venom with between 50 and 200 components. These sulfur-rich
peptides or conotoxins are neuropharmacologically active and modulate
ion channel function [235]. Any attempt to deal with these toxins within
this review would not be feasible and the reader is referred to other
excellent reviews on the subject [235,236].
Sponges (Porifera)
The phylum Porifera comprises about 5000 living species [177]. Porifera or
"pore-bearers" are filter-feeding organisms, which consist of a main body
cavity perforated by holes. They are considered to be the most primitive of
marine invertebrates, with littie internal organisation [178]. The supporting
skeleton of a sponge consists of spicules composed of calcium carbonate or
silica or of silica spicules and spongin fibres [183]. Sponges in general, and
in particular those without the physical protection of spicules, produce
secondary metabolites that are thought to provide protection against
predation, overgrowth or fouling. Many sponges contain symbiotic
658
microorganisms and thus there is usually some uncertainty as to the true

source of any isolated metabolites. [4].
Isothiocyanates are one of the most common classes of sulfur-
containing marine metabolites and usually occur as terpenes. The
majority of terpene isothiocyanates isolated are from the sponge orders,
Axinellida (Acanthella and Axinella genera), Halichondrida {Cymbastela,
Phakellia, Axinyssa (= Trachyopsis) and Halichondria genera) and
Lithistida {Theonella genus) [22]. Terpene isothiocyanates are often
accompanied by the corresponding isocyanides and formamides.
Biosynthetic studies involving incorporation of ^^C labelled precursors
including 2-isothiocyanopupukeanane (230) into a Hymeniacidon sponge,
revealed that the isocyano group was the precursor of the formamido and
isothiocyano groups, the reverse reactions did not take place and the
sponge did not utilise formate in the isocyano biosynthesis [237].
"K4N^^CS
230
The sponge Axinella cannabina was the source of the sesquiterpenoid,

axisothiocyanate 1 (231) [238]. Later, the structures of axisothiocyanate 2
(232) [239] and axisothiocyanate 3 (233) from A. cannabina were
determined from chemical and spectral data. The latter has a novel
spiro[4,5]decane skeleton [240]. Further study of A. cannabina resulted in
the isolation of axisothiocyanate 4 (234) [241]. The absolute
configurations of axisothiocyanate 1 (231) and axisothiocyanate 4 (234)
were determined from X-ray crystallography and from CD measurements
[242]. Synthesis of racemic axisothiocyanate 4 (234) has been
accomplished [243]. Axisothiocyanate 2 (232) was synthesised in a
stereospecific manner from aromadendrene. The report also stated that
some sesquiterpene isothiocyanates can cause allergic reactions with
headaches, nose itching and rashes [244]. Two additional isothiocyanates
(235-236) were isolated from A. cannabina [245]. A third isothiocyanate
(237) was later isolated from the same extract and axisothiocyanates 1-3
(231-233) were reported to be cytotoxic in vitro [246]. Another
sesquiterpene isothiocyanate (238) was isolated as a minor metabolite of
A. cannabina [241],
659
A Halichondria species of sponge from Oahu, Hawaii contained a

complex mixture of sesqui- and diterpenoids. The sesquiterpenoids
belonged to the a-amorphene (zizanene) series and included an
isothiocyanate (239) [248].
SCN
239
A sesquiterpene isothiocyanate (240) was isolated from the Califomian

nudibranch Cadlina luteomarginata but was presumed to be concentrated
from the Axinella species of sponge which constitutes much of its diet
[249]. Famesyl isothiocyanate (241) was isolated from Pseudaxinyssa
pitys [250], while theonellin isothiocyanate (242) was isolated from the
sponge Theonella cf. swinhoei [251].
NCS
SCN
242
Two further sesquiterpene isothiocyanates were isolated from A.

cannabina as minor metabolites. The first (243) is based on the epi-
eudesmane skeleton while the next (244) is related to alloaromadendrene
[252]. A third isothiocyanate (245) which is a c/^-eudesmane derivative
660
was found in A. cannabina, while the same isothiocyanate was found in

higher yield from Acanthella acuta [253]. Three isothiocyanate
compounds (246-248) were isolated as minor metabolites of A. acuta.
Their structures were elucidated by spectral data interpretation [254].
NCS
NCS
NCS
244 245
A species of Halichondria from the Marshall Islands contained an

isothiocyanate (249) [255] and an isothiocyanate based on the guai-6-ene
skeleton (250) was isolated from an unidentified sponge from Japanese
waters [256]. Acanthella pulcherrima from Australia contained two
isothiocyanates (251-252), in addition to known sesquiterpenes [257].
NCS . NCS
SCN
249
The first sesquiterpene thiocyanate to be isolated from a marine

sponge was (15*,45*,65*,7/?*)-4-thiocyanato-9-cadinene (253) from
Trachyopsis aplysinoides. The structures of this compound and of an
isothiocyanate with a new carbon skeleton (254), were determined by X-
ray analysis and two additional isothiocyanates (255-256) were identified
[258]. Isothiocyanate 254 was synthesised using an oxidative radical
cyclisation reaction as a key step [259].
661
NCS SCN
^ < & 5&-

r SCNJ
00
•Mx; 253 254
/
>
255
i H ""
256
The structure of 5-isothiocyanatopupukeanane (257), a sesquiterpene

isothiocyanate from an Axinyssa species from Guam, was determined by
X-ray analysis [260]. Two isomeric sesquiterpene thiocyanates, 2-
thiocyanatoneopupukeanane (258) and 4-thiocyanatoneopupukeanane
(259) were isolated from an unidentified sponge from Pohnpei and from
Phycopsis terpnis from Okinawa [261]. A sample of Axinyssa (=
Trachyopsis) aplysinoides from Palau yielded a rare thiocyanate, 2-
thiocyanatopupukeanane (260), while two specimens from Pohnpei
yielded 13-isothiocyanatocubebane (261), 1-isothiocyanatoaromadendrane
(262) and 2-thiocyanatoneopupukeanane (258) [262]. This last compound
had previously been assigned different stereochemistry at C2 [261]. (-)-4-
Thiocyanatoneopupukeanane has been synthesised in an enantiospecific
manner (259) [263]. Both enantiomers of 2-thiocyanatoneopupukeanane
(258) have been synthesised from (/?)-carvone [264].
SCN\
NCS , . H '
258 Ri = SCN, R2 = H
^^' 259Ri=H,R2 = SCN ^60 261 262
A sesquiterpene isothiocyanate, halipanicine (263) has been isolated

from an Okinawan specimen of Halichondria panicea [265]. The relative
stereochemistry of halipanicine (263) was established by synthesis [266]
and later, a total synthesis was achieved [267].
SCN
CO
w 263
662
Three new antiparasitic sesquiterpene isothiocyanates, 4-

isothiocyanato-9-amorphene (264), 10-isothiocyanato-4,6-amorphadiene
(265) and 10-isothiocyanato-5-amorphen-4-ol (266) were isolated from a
Fijian specimen of Axinyssa fenestratus. The compounds were identified
by spectral data interpretation [268]. Two isomeric isothiocyanates (267-
268) were isolated from Acanthella klethra from the Great Barrier Reef
and their structures were determined by X-ray crystallography and spectral
data examination [269].
. .Ncs
SCN»
A sesquiterpene thiocyanate, cavemothiocyanate (269) was isolated

from Acanthella cf. cavernosa and the structure was elucidated on the
basis of spectral data. The nudibranch Phyllidia ocellata also contained
cavemothiocyanate [270]. Acanthene B (270) is a sesquiterpene
isothiocyanate isolated from a British Columbian Acanthella sp. [271].
The sesquiterpene thiol, T-cadinthiol (271) was isolated from Cymbastela
hooperi from Kelso Reef on the Great Barrier Reef [272]. A
sesquiterpene isothiocyanate that displayed modest in vitro antimalarial
activity, (-)-9-isothiocyanatopupukeanane (272) was isolated from an
Axinyssa sp. from the Great Barrier Reef [273]. Great Barrier Reef
samples of A. cavernosa contained lO-isothiocyanatocadin-4-ene (273)
[274].
H\^SH ^^^^f^ HTNCS
269 ^^^ 270 271 272 273
Two isothiocyanates, epipolasins A and B and the corresponding (3-

phenylethylamine adducts, epipolasinthioureas A (274) and B (275) were
isolated from the sponge, Epipolasis kushimotoensis. The structures of the
epipolasins were determined by chemical degradation to known
compounds [275]. The structure of epipolasin A is identical to that
663
previously assigned to a metabolite of the nudibranch Cadlina

luteomarginata (240) [249] and the physical data is also similar except for
the sign and magnitude of the optical rotation. The structure of epipolasin
B is identical to that previously assigned to axisothiocyanate 2 (232)
[239]. Synthesis of (-)-(10/?)-10-isothiocyanoaromadendrane indicated
that it was the enantiomer of epipolasin B, previously isolated from E,
kushimotoensis [276].
.-'^"S"'^
CQ
^K!^
w)
^
240 R = NCS 232 R = NCS
274 R = NHC(S)NHCH2CH2Ph 275 R = NHC(S)NHCH2CH2Ph
The structure of a diterpenoid isothiocyanate (276) extracted from a

Halichondria sponge, was determined from chemical and spectral data
[277].
NCS
276
Kalihinols G (277) and H (278) were trace components of a species of

Acanthella from Guam and kalihinol X (279) was isolated from a Fijian
species of Acanthella, All inhibited growth of Bacillus subtilis.
Staphylococcus aureus and Candida albicans [278]. 10-Epi-isokalihinol
H (280) and 15-isothiocyanato-l-epi-kalihinene (281) were isolated from
Acanthella cavernosa from the Seychelles [279]. A Japanese specimen of
A. cavernosa contained a sesquiterpene isothiocyanate (282) and lOP-
formamido-5P-isothiocyanatokalihinol A (283). Structures were assigned
by spectral data interpretation [280]. Phakellia pulcherrima from the
Philippines contained the minor diterpene isothiocyanates kalihinol L
(284), 10-isothiocyanatokalihinol G (285), 10-epi-kalihinol H (286) and
10-isothiocyanatokalihinol C (287) [281]. 10-Epi-kalihinol I (288) and
5,10-bisisothiocyanatokalihinol G (289) were isolated from an Acanthella
sp. from Okinawa [282].
664
HOJ 1 HJ HOj 1 HJ
C N ^ THJ[>
A^^ H CI NCS
277 Ri = NC, R2 = NCS 279 280 281
278 Ri = NCS, R2 = NC
H\.NHCHO NCS
^K - ^
SCN V C SCN ^yJC CN
P V Q
CI NCS
282 283
NCS NCS NCS
HO-
289
A Japanese sponge of the Adociidae family contained 10-

isothiocyanatobiflora-4,15-diene (290), which was identified by spectral
analysis [283]. A. cavernosa from Fiji yielded a diterpene isothiocyanate
(291) [284].
NHCHO
NCS NCS
291
665
Cymbastela hooperi from the Great Barrier Reef contained four

diterpene isothiocyanates (292-295) amongst other diterpenes [285]. An
amphilectene isonitrile (296) was isolated from a Caribbean Cribochalina
sp. [286].
NCS NCS
A series of eighteen long chain, aUphatic a,a)-bis-isothiocyanates

(297-314) and three a-isothiocyano-c?-formyl analogues (315-317) was
isolated from a Fijian species of Pseudaxinyssa [287]. The major
constituents (297), (305) and (315) all have the same length of aliphatic
chain (CI8). Unlike terpenoid isothiocyanates, this series was not
accompanied by the corresponding isocyanides or formamides.
SCNr ^ ( C H 2 ) n / ^ ^ I NCS SCN-(CH2)n NCS SCN (CH2)n-CH0
297n=14 301n = l l 3 0 5 n = 1 6 310n = 13 315 n = 15

298n = 8 302n = 12 306n = 9 311n=14 316 n = 9
299n = 9 303n=13 3 0 7 n = 1 0 312n = 15 317n=16
300n=10 304n=14 308n=ll 313n=17
309n=12 314n=18
Dysidea herbacea contains linear polychlorinated peptides with a

thiazole residue. The metabolites can be divided into the dysidenin, the
isodysidenin and the dysideathiazole series of compounds [22].
Dysidenin (318) was isolated from D. herbacea from Cooktown,
Australia without stereochemical assignments [288]. The structure of
isodysidenin (319), isolated from a sample of D, herbacea from Papua
New Guinea, was determined by X-ray diffraction analysis [289]. It was
proposed that the two compounds differ in stereochemistry at C5 [290].
The absolute configurations were later revised [291].
666
Me Me
^H JL Q H *^ -^H J ^ Q H '^
318 R = Me 319 R = Me, X = CI, Y = CI

325 R = H 322 R = H, X = CI, Y = CI
323R = H,X = C1,Y = H
324R = H,X = H,Y = C1
Two thioacetates, thiofurodysin acetate (320) and thiofurodysinin

acetate (209) were isolated from a Dysidea species from Sydney,
Australia. They were converted by treatment with Raney nickel to a
mixture containing furodysin and furodysinin respectively [214]. These
were the first thiol acetates isolated from natural sources. The absolute
configurations of (-)-(6/?,ll/?)-thiofurodysinin acetate (209), (-)-(6/?,ll/?)-
furodysinin disulfide (208) and (+)-(6/?,ll/?)-methoxythiofurodysinin
acetate lactone (321), isolated from a Fijian specimen of D. herbacea were
determined by chemical interconversion [292].
H OMe
Acs
ô
^""YT^^) ^""^^ O
HA ^
320 209 R = SAc 321
208 R = -S-S- (dimer)
A collection of D. herbacea from near Bowen, Australia yielded 13-

demethylisodysidenin (322), 11 -monodechloro-13-demethylisodysidenin
(323) and 9-monodechloro-l3-demethylisodysidenin (324), all derivatives
of isodysidenin, and a dysidenin derivative, 13-demethyldysidenin (325).
13-Demethylisodysidenin (322) and 13-demethyldysidenin (325) were
epimeric at C5 by direct comparison of the dechlorinated derivatives of
each [293]. Syntheses of both (+)-13-demethyldysidenin (325) and (-)-13-
demethylisodysidenin (322) have been described [294]. The results of this
synthetic study imply that absolute configurations at C2 and C7 in all of
the natural materials are 5, opposite to those assigned by X-ray
crystallography to isodysidenin (319). In an Australian specimen of D,
herbacea, 13-demethylisodysidenin (322) was found to be localised in
cells of the cyanobacterium Oscillatoria spongeliae, while two
sesquiterpenes were associated with the sponge cells [295].
667
Thiofurodysinin (326), a furanosesquiterpene from Dysidea avara

from Australia, was the first report of a sesquiterpene mercaptan from a
sponge [296].
HS^V^r^^x
326
A Palauan species of Dysidea contained 15-acetylthioxyfurodysinin

lactone (327), that binds to human leukotriene B4 (LTB4) receptor. The
structure was determined by spectral data analysis and confirmed by
synthesis involving photo-oxidation of 15-acetylthioxyfurodysinin (328),
which co-occurs with it in the sponge [297,298]. An Australian species of
Euryspongia also contained 15-acetylthioxyfurodysin (329) and 15-
acetylthioxyfurodysinin (328) [299]. A sample of Z). herbacea from the
Great Barrier Reef contained (-)-neodysidenin (330) and the absolute
configuration was determined by capillary electrophoresis of Marfey's
derivatives [300].
H OH
AcS''"V**'"'^f'^^^^>^Q Acs
H/
327
H OH W NH
329 330
The dysideathiazoles A-E (331-335) are a series of polychlorinated

amino acid derivatives from Pacific Island collections of D. herbacea.
The structures were determined by X-ray analyses and the absolute
configurations were determined by X-ray crystallography of a brominated
derivative [301]. Herbamide A (336), a chlorinated amide was isolated
from a Papua New Guinean sample of D. herbacea as a minor component
[302]. D. herbacea from the southern Great Barrier Reef contained a
thiazole (337) amongst other known metabolites [303]. A Dysidea sp.
668
from Okinawa contained the benzothiazole S1319 (338), as a P-

adrenoreceptor agonist [304].
XCI2C. CCI2Y
331 R = H, X = CI, Y = CI 336

332 R = Me, X = CI, Y = CI
333R=:Me,X = Cl,Y = H
334 R = H, X = CI, Y = H
335 R = Me, X = H, Y = H
Me
^ C ^ N ^ , CHCI2
NFP
NHMe
337 y 338
Dysidea avara from the Solomon Islands contained the melemeleones

A (339) and B (340), which were identified by spectroscopic analyses
[305]. They consist of a sesquiterpene linked to a quinone with an
attached taurine residue [22].
SOaH
340
Dysidea sp. from Bararin Island in the Philippines, has yielded the
dysideaprolines A-F (341-346), proline-derived analogues of dysidenin
(318). The barbaleucamides A (347) and B (348), which are structural
analogues of the cyanobacterial metabolite barbamide, were also isolated.
The structures were elucidated by NMR spectroscopic analysis. It is most
probable that all of these compounds are derived from a symbiotic
cyanobacterium found in close association with the Dysidea sp. [306].
669
OMe R
^Â,
X2HC' " ^ ^N Cl^C
.^N,^-^CCl3
R2 O
341 Ri = H, R2 = Me, X = CI, R3 = CHCI2 347 R = H

342 Ri = Me, R2 = Me, X = CI, R3 = CHCI2 348 R = Me
343 Ri = H, R2 = H, X = CI, R3 = CHCI2
344 Ri = H, R2 = Me, X = H, R3 = CHCI2
345 Ri = H, R2 = Me, X = CI, R3 = CH3
346 Ri = H, R2 = Me, X = CI, R3 = CH2CI
The burrowing sponge Siphonodictyon coralliphagum and other

species of the same genus, contain a series of sesquiterpene
hydroquinones. Siphonodictyal D (349), and siphonodictyols G (350) and
H (351) occur as sodium sulfates and the structure of siphonodictyal D
(349) was determined by X-ray crystallography [307]. A deep water
collection of S. coralliphagum contained bis(sulfato)cyclosiphonodictyol
A (352) which inhibits binding of LTB4 to human neutrophils [308].
Na03S0,^^^CH0 Na03S0 OH
349 350
Na03S0^^ Na03S0-f3-0S03Na
"CH2OH
351
670
Agelas nakamurai from Japan produced the sesquiterpene sulfone,

agelasidine A (353), which possessed antispasmodic activity. The
structure was deduced from spectral data [309]. A simple synthesis of
agelasidine A (353) utilised a hetero-Claisen rearrangement [310]. A
biomimetic synthesis of 353 was also reported [311] and another synthesis
of agelasidine A (353) was carried out in three steps from famesol [312].
353
Two diterpene derivatives of hypotaurocyamine, agelasidines B (354)

and C (355) were also isolated from Agelas nakamurai The structures
were determined by interpretation of spectral data. Both are antimicrobial,
inhibit smooth muscle contraction and enzyme activity of Na"*'/K"^-
transporting adenosine triphosphate (ATP)ase [313]. Agelasidine C (355)
has been synthesised [314]. (-)-Agelasidine C (356) and (-)-agelasidine D
(357) were isolated from the Caribbean sea sponge Agelas clathrodes.
The structures were confirmed by interpretation of the spectral data and by
comparison of this data with those of the known antipode (+)-agelasidine
C (355) [315].
NH
H
.N>^NH2
o \ NH
354 355
O'Ô
NH
356 357
Suvanine (358), an acetyl cholinesterase inhibitor was first isolated

from species of Ircinia [316] and then later from a Coscinoderma species
from Fiji and Palau when the structure was revised [317].
671
+
Me, NHo
Me NHo
A Califomian sponge of the Halichondriidae family contained a

sulfated sesterterpene hydroquinone and five sulfated sesterterpenes. The
structures of the halisulfates 1-5 (359-363) were determined by
interpretation of spectral data and a structure was proposed for halisulfate
6 (364). The halisulfates are antimicrobial and antiinflanmiatory [318].
The absolute configuration of halisulfate 3 (361), which was also isolated
from Ircinia sp. from the Philippines, has been determined by application
of the chiral amide method and by chemical degradation techniques [319].
Halisulfate 7 (365) is a sesterterpene sulfate from a Coscinoderma sp.
from Yap, Micronesia [320].
359 360
NaOgSO-
CH20S03Na-0
361 362
672
NaOaSO'
Bioassay directed isolation of serine protease inhibitors from

Coscinoderma mathewsi yielded the 1-methylherbipoline salts (366-367)
of known sesterterpenes halisulfate-1 (359) and suvanine (358) [321].
Coscinosulfate 1 (368), a sesquiterpene sulfate, was isolated from a New
Caledonian collection of C mathewsi. It displayed significant activity as
an inhibitor of the protein phosphatase Cdc25 [322]. A total synthesis
starting from (+)-sclareolide was described [323].
OSOa
Ô Me
367
OS03Na
Sulfircin (369), an antifungal sesterterpene sulfate was isolated as the

N,A^-dimethylguanidinium salt from a deepwater Ircinia species and its
structure was determined by X-ray analysis [324]. Two sesterterpene
sulfates, hipposulfates A (370) and B (371), were isolated from
Hippospongia cf. metachromia from Okinawa and their structures were
elucidated by interpretation of spectroscopic data. Both compounds
possess an enolsulfate functionality [325].
673
NaO^SO^
oso
370R = H
371 R = OH
Akaterpin (372) is an inhibitor of phosphatidylinositol-specific

phospholipase C from a Callyspongia sp. [326]. The relative
stereochemistry of the ring junction in the upper decalin moiety of
akaterpin was shown to be cis by synthesis of model compounds [327].
NaOsSO-f VoSOgNa
Four unstable sulfate esters (373-376) of known furanosesterterpenes

were obtained from Ircinia variabilis and from /. oros from the northern
Adriatic Sea [328]. The 22-(9-sulfates of palinurin (377) and fasiculatin
(378) were isolated from /. variabilis and from /. fasiculata respectively.
Both were toxic to brine shrimp [329]. Ircinianin sulfate (379) was
isolated from /. (Psammocinia) wistarii from the Great Barrier Reef as a
very unstable metabolite [330].
PSO3K
674
379
Adociasulfates 1-6 (380-385) were isolated from a Haliclona (aka

Adocia) sp. from Palau and were all inhibitors of kinesin motor proteins
[331]. Adociasulfate 2 (381) had earlier been shown to inhibit the activity
of the motor protein kinesin by interference with its binding to
microtubules [332]. An Adocia sp. from the Great Barrier Reef contained
adociasulfates 1 (380), 7 (386) and 8 (387), which inhibit vacuolar YC-
ATPase [333]. Adociasulfates 5 (384) and 9 (388) were obtained from
Adocia aculeata from the Great Barrier Reef [334]. The structure of
adociasulfate 1 (380) was confirmed by an enantioselective total synthesis
[335]. Adociasulfate 10 (389) from Haliclona sp. from Palau also inhibits
the kinesin motor proteins [336].
NaOaSO-ZTÔSOsNa
HO^:
380Ri = SO3Na,R2 = SO3Na 381R = S03Na 382
384 Ri = SOsNa, R2 = H 385 R = H
386 Ri = H, R2 = SOsNa
675
H0-/^-0S03Na
OSOaNa
383 387
NaOaSO. COoH
OSOaNa
388 389
Shaagrockols B (390) and C (391) from the Red Sea sponge Toxiclona
toxius, are antifungal hexaprenylhydroquinone disulfates and were
identified by spectral data interpretation [337]. Toxicols A (392), C (393)
and toxiusol (394) are hexaprenoid hydroquinones that were also isolated
from Toxiclona toxius. The structures were determined by spectral data
examination [338].
OSOaNa GSOsNa
0S03Na OSO^Na
390
676
OSOsNa
OSOgNa
392 R = SOgNa 394

393 R = H
A hexaprenyl-hydroquinone sulfate (395) was identified as an H^/K"^-

ATPase inhibitor from a Japanese species of Dysidea [339]. Sarcotragus
spinulosus from deep water contained the Na"^/K"^-ATPase inhibitors
sarcochromenol sulfates A-C (396-398) and sarcohydroquinone sulfates
A-C (399-401) [340]. The structures were determined by spectral data
analysis of the natural products and of derivatives.
NaOaSO'
.rd"*^"
396 n = 5
397 n = 6
398 n = 7
OSOaNa
399 R = H, n = 6
400R = SO3Na,n = 7
401 R = H, n = 8
A heptaprenylhydroquinone derivative (402) was isolated from an

Indian sample of Ircinia fasciculata [341]. Ircinia spinulosa from the
Adriatic contained three sulfated 2-prenylhydroquinones (403-405) that
are toxic to brine shrimp [342]. An Ircinia sp. from New Caledonia
contained a sulfated 2-prenylhydroquinone (406) and a sulfated
furanoterpene (407) [343]. An Australian Sarcotragus sp. contained
octaprenylhydroquinone sulfate (408) and nonaprenylhydroquinone
sulfate (409) as inhibitors of al,3-fucosyltransferase VII [344].
677
OSO^Na
402
0S03Na
ry^^^yiry^ OSO^Na
407
The yellow pigment halenaquinol sulfate (410) has been isolated from
the Okinawan sponge Xestospongia sapra and is a pentacyclic
hydroquinone [345]. The absolute stereochemistry was determined to be
65 by comparing the CD spectrum of a derivative with a theoretically
calculated spectrum [346]. Theoretical calculation of CD spectra of
halenaquinol sulfate (410) isolated from X exigua and X sapra
determined that the absolute stereostructure was 12b5' [347]. The
pentacyclic compound, xestoquinol sulfate (411) has been isolated from an
Okinawan collection of X. sapra and its structure was elucidated on the
basis of spectroscopic data and a chemical conversion [348].
NaOjSO.
OSOaNa
Xestoquinolide B (412) was obtained from Xestospongia cf.

carbonaria and the protein kinase activity of it and related compounds
reported. The structure of this merosesquiterpenoid was elucidated by
spectral data interpretation [349]. A Xestospongia species from the
678
Philippines contained the topoisomerase n inhibitors, secoadociaquinones

A (413) and B (414) [350].
HN"^ H 0 3 S ' ^
0<^^ SO3H 0,s^.x<^^NH
412X = NH,Y = S02 413

orX = S02,Y = NH
Species of Adocia from Truk contained adociaquinone A (415), the

mildly cytotoxic adociaquinone B (416) and 3-ketoadociaquinone (417) as
minor components. All were synthesised [351]. The absolute
configurations of (+)-adociaquinones A (415) and B (416) were
determined by total synthesis [352].
02S^
.If! ° rVô
rxT y^^-^
^0
l yô0
415 R = H2 416
417 R = 0
A number of sulfur-containing cyclic peptides have been isolated from

sponges and many of these are cytotoxic or exhibit other biological
activities. Discodermins A-D (418-421) from Discodermia kiiensis are
antimicrobial, cyclic tetradecapeptides which contain a sulfonic acid group
[353,354]. The structure originally proposed for discodermin A [354] was
later corrected by incorporation of L-proline instead of D-proline [353].
Four additional tetradecapeptides, discodermins E-H (422-425), were
isolated from D. kiiensis from Japan and were cytotoxic and antimicrobial
[355,356]. The amino acid sequence in discodermins A-D (418-421) was
revised, reversing the positions of threonine and asparagine from their
original assignment [355].
679
CONHj
o N>' ^ . ,-^5 ^ .
OH
NH
^N'
"7
Me NH
NH9
1^
H2NOC
N
H
O
418 Rj = H, R2 = H, R3 = Me, R4 = Me, R5 = a

419 Ri = H, R2 = H, R3 = H, R4 = Me, R5 = a
420 Ri = H, R2 = H, R3 = Me, R4 = H, R5 = a
421 Ri = H, R2 = H, R3 = H, R4 = H, R5 = a
422 Rj = H, R2 = H, R3 = Me, R4 = Me, R5 = b
423 R, = H, R2 = H, R3 = Me, R4 = Et, R5 = a
424 Ri = Me, R2 = H, R3 = Me, R4 = Me, R5 = a
425 Ri = H, R2 = OH, R3 = Me, R4 = Me, R5 = a
A deepwater species of Discodermia contained the cytotoxic

depsipeptide, polydiscamide A (426), which was identified by
spectroscopic means, chemical degradation and derivatisation [357].
O HzN^NH
OHC^JÎJ^ t H
HNv
N' > ' "SOjNa
NH
HNO'ITV
^ , ^ C O N H
426
680
Halicylindramides A-C (427-429) are antifungal and cytotoxic

depsipeptides from Halichondria cylindrata from Japan [358]. Two
additional peptides, halicylindramides D (430) and E (431) of which the
former is antifungal and cytotoxic, were also isolated from a Japanese
collection of//, cylindrata [359].
H
***,
V. . O / „ ' O ^ ^ ° / O rf^ H Ph
CONH2
427 Ri = H, R2 = Me
428 Ri = Me, R2 = H
429 Ri = Me, R2 = Me
j ^ O^^NHCHO r S ?ONH2
Ph
O u ^NH
H2N N
" -^ O
H2NOC
430
O. NHCHO
Br-f V-, A-< V-\ CONH2
CONH9
O .-\_Me ^ .pj^
H2N N
^ H
431
Microspinosamide (432), a cyclic peptide incorporating 13 amino acid

residues, was isolated from an Indonesian collection of Sidonops
681
microspinosa. The structure of compound 432 was elucidated by NMR

and mass spectral analyses, and by chemical degradation and
derivatisation studies. This tridecapeptide is the first naturally occurring
peptide to contain a P-hydroxy-p-bromophenylalanine residue.
Microspinosamide (432) inhibited the cytopathic effect of HIV-1 infection
in an in vitro colorimetric assay, with an EC50 value of approximately 0.2
|Lig/mL[360].
f^'^^NH 0w,^^^N H 2
. j^ O O^ H ^ ^ H ^ ''^T ^ f^ H I
OHC.
/=< OH
NH
H9N
O
432
Studies of Theonella sponges from Okinawa resulted in the isolation of

the thiazole-containing cyclic peptides, keramamides F, G, H, J, K, M and
N (433-439) [361-364]. Keramamides F (433) and K (437) were cytotoxic
against KB and LI 210 cell lines and keramamide K (437) contained an
unusual tryptophan residue. A different Theonella sp. from Okinawa
yielded a theonellapeptolide congener (440), which contained a
methylsulfinylacetyl group at the iV-terminus [365]. Synthesis of the
proposed structure of keramamide J (436) indicated that the natural
product structure needs to be re-examined [366].
O O
OHC.
^ OH " Q^ ^ O^XÔ KJ
NH
HN N. J w .OMe
OMe
433
682
OHC
^ ÔMe
435Ri = Br,R2 = OH 437
436Ri = H,R2 = H
438 R = Me
439 R = Et
MeOS
V - NH HN^C
: sV-T-
0 ^cyK^
440
Oriamide (441), a cytotoxic peptide containing the unusual amino

acid, 4-propenoyl-2-tyrosylthiazole, was isolated from a Theonella sp.
from South Africa [367]. Theonella cupola from Indonesia and from
683
Okinawa contained a cytotoxic, cyclic heptapeptide, cupolamide (442)

[368].
NH
X ^ ^
n I
-OSOaNa
' HNy.0 0;:yJ--y'
HO>Â.„, NH O
441 442
Phakellistatin 5 (443), a cytotoxic heptapeptide from Phakellia costata

from Chuuk (Truk) contains a methyl sulfide group [369]. Solution- and
solid-phase syntheses of phakellistatin 5 have been accomplished and the
products were chemically identical but not biologically identical [370].
Hymenamide F (444) from an Okinawan Hymeniacidon sp., is another
cyclic heptapeptide that contains a methyl sulfide group. Structural
elucidation of hymenamide F (444) was carried out through spectral data
analysis of the corresponding sulfoxide [371].
MeS^ H
V^' H ^^-^
H2N-C . . . ^, ^
^ Ph MeS
443 444
The marine red alga Ceratodictyon spongiosum containing the

symbiotic sponge Sigmadocia symbiotica, which was collected from Biaro
Island, Indonesia, yielded two isomers of a bioactive thiazole-containing
cyclic heptapeptide, d^,d5'-ceratospongamide (445) and trans.trans-
ceratospongamide (446). The structures of the ceratospongamides, which
each consist of two L-phenylalanine residues, one (L-isoleucine)-L-
684
methyloxazoline residue, one L-proline residue, and one (L-

proline)thiazole residue, were established through extensive NMR
spectroscopy, as well as chemical degradation and chiral analysis. Cis.cis-
(445) and rran5',rran5-ceratospongamide (446) are stable conformational
isomers of the two proline amide bonds. rran5,rrans-Ceratospongamide
(446) exhibits potent inhibition of secreted phospholipase A2 (SPLA2)
expression in a cell-based model for antiinflammatory activity (ED50 32
nM), whereas the cis.cis isomer (445) is inactive. Trans.trans-
Ceratospongamide (446) was also shown to inhibit the expression of a
human-sPLA2 promoter-based reporter by 90% [372].
' > .
445 446
The cyclic hexapeptide, waiakeamide (447) was isolated from an

Indonesian collection of Ircinia dendroides [373]. Haliclona nigra from
Papua New Guinea contained two cyclic hexapeptides, haligramides A
(448) and B (449). They showed an unusual activity pattern in the
National Cancer Institute (NCI) 60 cell-line panel [374].
Ph"
U l^ "U
V
Me^%
447 448 R = SMe
449 R = S(0)Me
685
The prianosins and the discorhabdins are sulfide-containing

pyrroloiminoquinones. Prianosin A (450) is a potent antileukaemic agent
isolated from the Okinawan sponge Prianos melanos. The structure was
determined by X-ray analysis [375]. Prianosins B-D (451-453) are further
sulfur-containing alkaloids from P. melanos with potent antineoplastic
activity. The structures of the prianosins were elucidated by interpretation
of spectral data [376]. Discorhabdins A (450), B (454) and D (453) are
cytotoxic pigments from a New Zealand species of Latrunculia [377].
Discorhabdin D (453) was also found in an Okinawan species of Prianos
[378]. The structure of discorhabdin A (450) was identical to that
previously reported for prianosin A [375] and the other discorhabdins
were identified through analysis of spectral data. The spectral data of
prianosins C (452) and D (453) were identical to those of 2-
hydroxydiscorhabdin D and discorhabdin D (453) from Latrunculia
brevis, so the structures were therefore revised [379]. Discorhabdin Q
(455) was isolated from L. purpurea and from at least three Zyzzya species
and is cytotoxic [380]. Two Antarctic sponges, Latrunculia sp. and
Negombata sp. contained the antibacterial discorhabdin R (456) [381].
Like the prianosins and the discorhabdins, the batzellines and

isobatzellines are sulfides with a pyrroloquinone skeleton [22]. A
deepwater Batzella sponge contained the alkaloids, batzellines A (457)
and B (458), which possess methyl sulfide groups. The structure of
686
batzelline A (457) was determined by X-ray analysis and that of batzelline

B (458) was proposed on the basis of spectral data analysis and chemical
conversion [382]. Further studies of the deep water Caribbean Batzella
sponge resulted in the isolation of isobatzellines A (459), B (460) and D
(461), which also contain methyl sulfide groups. The structures of the
isobatzellines were proposed on the basis of spectroscopic analyses and
the compounds exhibited cytotoxicity in vitro against P388 cells and
moderate antifungal activity against Candida albicans [383]. BatzelHnes
A (457) and B (458) and isobatzellines A (459) and B (460) have all been
synthesised [384].
SMe SMe
H,N
The Fijian sponge Zyzzya cf. marsailis (now Z fuliginosa) [13]

contained the pyrroloiminoquinone alkaloid makaluvamine F (462).
Discorhabdin A (450) was also found in the sponge [385]. Total synthesis
of makaluvamine F (462) was achieved using hypervalent iodine(in)-
induced reactions [386,387]. Zyzzin (463) is a thiolactam from Zyzzya
{Zyzza) massalis, that during purification, undergoes hydrolysis to the
corresponding lactam [388].
o
X
N NH
N
H
463
Dercitin (464) is a violet acridine alkaloid from a deepwater species of

Dercitus with antitumour, antiviral and immunomodulatory properties in
vitro [389] and with in vivo antitumour activity [390]. The structure was
proposed on the basis of spectral properties. Cyclodercitin (465) was
isolated as a minor alkaloid from a deepwater Dercitus species and three
687
dercitin related compounds, nordercitin (466), dercitamine (467) and

dercitamide (135) were found in a deepwater species of Stelletta. The
structures were determined by spectral data interpretation. All four
compounds inhibited the proliferation of P388 cells in vitro, and all but
cyclodercitin (465) exhibited immunosuppressant activity [391].
466 R = NMe2
467 R = NHMe
135 R = NHCOEt
The structure of the pyridoacridine alkaloid stellettamine (468) from a

deepwater species of Stelletta, was unambiguously determined by X-ray
analysis. This, together with long range ^H-^^C NMR coupling constants
and metal-binding studies on kuanoniamine C (135), a metabolite of the
tunicate Cystodytes [145], recognised to have identical data to that of
dercitamide, led to structural revision of dercitin (464), cyclodercitin
(465), nordercitin (466), dercitamine (467), and dercitamide (135) [392].
The structures of the kuanoniamines and dercitins were confirmed by total
synthesis [393]. Sagitol (469) is a pyridoacridine alkaloid from
Oceanapia sagittaria. It can be obtained from dercitin (464) by singlet
oxygen oxidation, but its CD spectrum suggests that it is not entirely an
artifact [394]. A paper reporting insecticidal activity and cytotoxicity for
kuanoniamines C (135) and D (136) also reported the isolation of an
additional pyridoacridine alkaloid, A^-deacetylkuanoniamine C (470) from
Oceanapia sp. from Truk, Micronesia [395].
469
688
Corallistine (471) was isolated from Corallistes fulvodesmus from

deep water off New Caledonia. The structure was elucidated by X-ray
crystallographic analysis [396]. The unusual alkaloid neamphine (472)
was isolated from Neamphius huxleyi from Papua New Guinea and its
structure determined by X-ray crystallography [397].
MeS^ NH2 O Me
471 472
The antibacterial and cytotoxic bicyclic alkaloid phloeodictine B (473)

was isolated from a New Caledonian species of Phloeodictyon and is
unusual as it contains a cyclic aminoketal functionality. The structure was
proposed on the basis of spectral data [398]. A Phloeodictyon sp. from
New Caledonia contained the cytotoxic and antibiotic alkaloids,
phloeodictines Ci (474) and Ci (475). Their structures were elucidated by
mass spectrometry and NMR spectroscopy [399].
H2N-H ^ \ \ HN-<
NH Ky
474 n = 3
475 n = 2
LatruncuHns A (205) and B (204) are powerful ichthyotoxins isolated

from Latrunculia magnifica. The structure of latrunculin A (205) was
determined by X-ray diffraction analysis to be a 16-membered macrolide
attached to a 2-thiazolidinone moiety [210]. Latrunculin B (204) is a 14-
membered macrolide. The ichthyotoxicity of the latrunculins is probably
due to their haemorrhage causing ability [211]. The structures of two
minor metabolites of L. magnifica, latrunculins C (476) and D (477) were
elucidated by analysis of spectral data [400]. Studies of the action of the
latrunculins on mouse neuroblastoma and fibroblast cells show that they
cause reversible changes in cell morphology [401]. Latrunculin B (204)
has been synthesised by a highly convergent, stereocontroUed route [402].
689
Several reactions of latrunculin B (204) were carried out as part of a study

on structure-activity relationships but the biological activities of the
products were not reported [403]. Two quite different total syntheses of
latrunculin A (205) were briefly communicated [404,405] and full
experimental details were reported later [406,407].
.c^
6,7-Epoxy-latrunculin A (478) and latrunculin M (479) were isolated

from L magnifica. The structures were established by chemical and
spectral methods [408]. Latrunculin A (205) was isolated from
Fasciospongia rimosa collected in Okinawan waters and the absolute
configuration was determined by X-ray analysis [409]. F. rimosa also
contained the cytotoxic latrunculin S (480) and the structure was
elucidated by NMR spectroscopy and chemical correlation with known
congeners [410].
478
Mauritamide A (481), a taurine-containing metabolite, was isolated

from the sponge Agelas mauritiana [411]. The taurine-containing
alkaloids tauroacidins A (482) and B (483) were isolated from a
Hymeniacidon sp. from Okinawa and are tyrosine kinase inhibitors [412].
An imidazole alkaloid, (9£')-clathridine 9-//-(2-sulfoethyl)-imine (484), a
taurine derivative of clathridine, was isolated from the calcareous sponge
690
Leucetta microraphis [413]. Taurospongin A (485) is a sulfated

acetylenic fatty acid derivative from Hippospongia sp. from Okinawa. It
inhibits both DNA polymerase b and HIV reverse transcriptase [414].
B O B ^^
481 Ô^MQ 482 R = Br (95/9/? = 6:4)

483R = H(95/9/?=l:l)
Me
N O ^^ AcO O.
Ct^H
16'^32
H03S
484 485
The pyrrole-imidazole alkaloid taurodispacamide A (486) has been

isolated from the Mediterranean sponge Agelas oroides. The structure
was established from spectroscopic data. Taurodispacamide A (486)
exhibited antihistaminic activity when tested on guinea pig ileum [415].
SO3H
486
The Senegalese sponge Ptilocaulis spiculifer has been shown to

contain dakaramine (487), a tyrosine derivative containing iodine and an
alkyl sulfate (488) as a counterion of dakaramine [416].
I
A ^ O v ^ x - v ^ NMe2
"V'^^^'^^ÔSOsH
487 488
691
Microxine (489), a purine derivative was isolated from the Australian

marine sponge Microxina sp. Microxine was found to weakly inhibit
Cdc2 kinase activity [417].
.N^Ô
NH
489
Three aldose reductase inhibitors (490-492) were isolated from

Dictyodendrilla sp. from Japan and their structures were determined by X-
ray analysis and spectroscopic studies [418].
490 R = Na 492
491 R = H
Hyrtiomanzamine (493), a compound consisting of a 6-hydroxy-P-

carboline associated with a betaine unit, was isolated from Hyrtios erecta
from the Red Sea and the structure elucidated by spectral data
examination. Hyrtiomanzamine exhibited immunosuppressive activity in
the B lymphocytes reaction assay [419].
MeS.
N N.
Me" ^ Me
493
692
The sponge Agelas dispar from the Bahamas contained

pyridinebetaine B (494) [420]. Echinoclathrines B-C (495-496) are
members of a new class of pyridine alkaloids from an Okinawan species
of Echinodathria. They exhibit weak immunosuppressive activity [421].
1
^N-^(CH2)i2SR
+^ 1 H
N
I^SO-
494 495 R = Ac
496 R = H
The sesterterpene pyridinium alkaloid spongidine D (497) was isolated

from a Spongia sp. from Vanuatu as an antiinflammatory agent [422].
S0.H
/olfO'
497
The diketopiperazine cyc/c>-(L-proline-L-thioproline) (498) was

isolated from Tedania ignis but a bacterial origin for the metabolite was
suggested [423]. Cyd(7-(L-proline-L-methionine) (499) was isolated from
Pseudomonas aeruginosa associated with the Antarctic sponge, Isodictya
setifera. The structure was elucidated by spectroscopic methods and
confirmed through synthesis [424].
o o
o
498 499
693
There have been three reports of the same dimeric disulfide. It was
first isolated from an unidentified sponge from Guam and the structure
elucidated by analysis of spectral data. The (E,E) stereochemistry of the
disulfide (500) was defined by comparing the ^^C NMR spectroscopic data
with those of the (E,Z)Asomer (501) that was obtained as an unstable
minor product [425]. Compound 500 was isolated from a species of
Psammaplysilla and was called psammaplin A [426]. It was also isolated
from Thorectopsamma xana, collected from the same location in Guam,
together with a minor dimeric metabolite bisaprasin (502). Both
compounds inhibited growth of Staphylococcus aureus and Bacillus
subtilis [427]. Psammaplin A (bisprasin) (500) was later isolated from a
Dysidea species of sponge and shown to act on Ca^^-induced Ca^"^ release
channels of skeletal muscle [428].
500n= !(£,£)
501 n = 1 (E,Z)
502n = 2
Four minor metabolites, psammaplins B-D (503-505) and

presammaplin A (506) were isolated from Psammaplysilla purpurea, in
addition to psammaplin A (500). Psammaplin B (503) is a thiocyanate
bromotyrosine derivative, while psammaplin C (502) is a sulfanamide.
Psanmiaplin D (505) displayed antimicrobial activity and mild tyrosine
kinase inhibition [429]. The psammaplins Ai (507) and A2 (508) and
aplysinellins A (509) and B (510) were isolated from Aplysinella rhax
from both Pohnpei and Palau. These compounds inhibit famesyl protein
transferase and leucine aminopeptidase [430]. Another sample of A. rhax
from the Great Barrier Reef, Australia contained psammaplin A 11'-
sulfate (511) and bisaprasin 11'-sulfate (512), both of which inhibited
[ H]-l,3-dipropyl-8-cyclopentylxanthine binding to rat brain adenosine Ai
receptors [431].
694
O
H
H T
0
HO'
503 R = SCN 506
504 R = SO2NH2 o
505 R= —S-S^^^^^ A
N OMe
H
507 Ri = H, R2 = SO3', n = 1
508 Ri = SO3-, R2 = 503', n = 2
511 Ri = H, R2 = S03Na, n = 0
^^H OH HO2C
Br Br^
695
34-Sulfatobastadin 13 (513) is an inhibitor of endothelin A receptor

from lanthella sp. from the Great Barrier Reef [432]. Three new bastadin
analogues including 15,34-0-disulfatobastadin 7 (514) and lO-O-
sulfatobastadin 3 (515) were isolated from lanthella basta from Exmouth
Gulf, Western Australia. They showed moderate differential activity as
sarcoplasmic reticulum-Ca^'*"-channel agonists of the skeletal muscle
receptor-protein complex, RylR FKBP12 [433].
NOH
ON,5j^^Na03SO ^>Ss^Br
NOH
NOH
0--V^Br NaOjSO^ OlP

0S03Na H(
v^
Br'
NOH NOH
514 515
lantherans A (516) and B (517) are dimeric tetrabrominated

benzofuran derivatives that were isolated from an Australian lanthella
species. The structures were determined by spectroscopic and chemical
methods. lantheran A (516) includes a (Z,Z)-1,3-butadiene moiety,
whereas iantheran B (517) is the geometric isomer possessing a (Z,£:)-1,3-
butadiene moiety. Both compounds were Na"^/K'^-ATPase inhibitors
[434,435]. lanthesines C (518) and D (519) showed potent NaVK^-
696
ATPase activity and are additional dibromotyrosine derivatives from an

Australian lanthella sp. [436].
516
517 R = S03Na
MeO
N^xvÔ .SOgNa
H
OMe
518
Br, i)H
MeO-
Br
HN^^^^Ôyk Hj^^S03Na
B,A:A^C02H
519
A two Sponge association of a thin crust of Haliclona sp. overlaying an

unidentified choristid (probably not Jaspis) sponge contained two enol
sulfates, presumed to be from the choristid sponge [437]. These enol
sulfates were also found as the sodium salts jaspisin (520) [438] and
isojaspisin (521) [439] and (£)- and (Z)-narains (522-523) [440] from
697
Japanese specimens of Jaspis sp. The jaspisins (520-521) inhibited

hatching of sea urchin embryos and the narains (522-523) induced
metamorphosis in ascidian larvae. Three 3,4-dihydroxystyrene sulfate
dimers (524-526) were also isolated from the same Jaspis species [441].
H0^^^5yx%ÔS03-R H0.,^<^^^^^
520R = Na^ 521R = Na^

522 R = Me2NC(NH2)NH2^ 523 R = Me2NC(NH2)NH2''
"""ij HO^^^XAcHO
"^ 524 "^ ^ SIS
HOsSOvÔ,
"^^^ 526
Aplysillin A (527), with unknown double bond geometries was

isolated from Aplysina fistularis and is a weak thrombin receptor
antagonist [442].
Southern Australian Echinodictyum sp. contained the echinosulfonic

acids A-C (528-530) and echinosulfone (531), which were all antibacterial
[443].
698
528R = Et
529 R = Me
530 R = H
No Sterols with sulfate groups in the sidechain have been isolated from
sponges to date. This contrasts with echinoderms. Many of the sterol
sulfates isolated from echinoderms, especially from ophiuroids (brittle
stars) contain sulfate groups in the sidechain [2].
Halistanol sulfate (532) from Halichondria cf. moorei is a tris-sodium
sulfate salt, which possesses antimicrobial, hemolytic, and ichthyotoxic
activity [444]. It was later isolated from two sponges of the genus
Topsentia as the free sterol and found to inhibit pp60v-src protein tyrosine
kinase activity [445]. The tris(2-aminoimidazolium) salt of halistanol
sulfate (533) was isolated from Topsentia sp. from Japan and is also
antimicrobial [446].
^K^^lX^X)
RO3SO,
I I I R: [ > - !NH2
R= ^ - - NT
ROsSO'
OSO:.Na 6SO3R
532 533
A Sterol disulfate (534) and a sterol trisulfate (535), both closely

related to halistanol, have been found in a species of Halichondria [447]
and in Trachyopsis halichondrioides [448] respectively.
699
H4N"'03SO' .-kAJ
OSOsNa
534 535
A Japanese species of Epipolasis contained five sterol sulfates named

halistanol sulfates A-E (536-540), which differ from the original halistanol
sulfate (532) from Halichondria moorei [449]. Structures were elucidated
by spectroscopic and chemical techniques. Halistanol sulfates F-H (541-
543) are three additional sterol sulfates from Pseudaxinyssa digitata that
inhibit HIV in vitro [450].
Na03S04>s.-4Ct/
3380
H •
0S03Na
b=
536 Ri = a, R2 = H
c=
537 Ri = b, R2 = H
538 Ri = c,R2 = H
539 Ri = d, R2 = H d=
540 Ri = e, R2 = O H
The sterol sulfate, halistanol disulfate B (544) was isolated from a

South African Pachastrella sp. The structure and stereochemistry of
compound 544 were established mainly by interpretation of spectral data.
Halistanol disulfate B (544) was active in the endothelin converting
enzyme (ECE) assay at a micromolar concentration [451]. Three sterol
trisulfates (545-547) have been isolated from the sponges Trachyopsis
halichondrioides and Cymbastela coralliophila [452].
700
Na03Sa
I I J H
NaOjSO'
OSOgNa
544 545 R = ^^.^.^-^^
546 R = ^^Y^
'VV,
547 R = f
The structure of sokotrasterol sulfate (548), isolated from sponges of

the family Halichondriidae was determined by X-ray analysis [453-455].
The steroid, 26-norsokotrasterol sulfate (549), was isolated from the
marine sponge Trachyopsis halichondrioides and was identified by NMR
spectroscopic analysis [456].
HOaSQ
HO3SO'
OSOjNa
548
Ibisterol sulfate (550) is a sulfated sterol from a deepwater Topsentia

sp. that was cytoprotective against HIV-1 in the NCI primary screen [457].
0S03Na
550
701
A Topsentia sp. from Okinawa contained five antimicrobial 14-methyl

sterol sulfates, topsentiasterol sulfates A-E (551-555) [458].
Ophirapstanol trisulfate (556) from deepwater Topsentia ophiraphidites
showed inhibition in the guanosine diphosphate/G-protein RAS exchange
assay [459].
Na03S04
I^T'TI''^
NaOjSO''
HO ^ OSOjNa
551 R =
^ "r"^^
'^ o V-OH
552 R =
^^t^^r=\
-OH
HOÔ
553 R =
o-^ô
554 R =
il \
O
555 R =
OSOaNa
556
702
An unusual 6a-sterol sulfate (557) was isolated from Dysideafragilis,

from the Venetian lagoon and displayed cytotoxicity against two different
tumour cell lines in vitro [460]. Tamosterone sulfates (558-559) are a C14
epimeric pair of polyhydroxylated sterols isolated from a new species of
Oceanapia [461]. The Japanese marine sponge Epipolasis sp. contained
the steroid polasterol B sulfate (560) along with the known compound
halistanol sulfate (532). The structure of compound 560 was determined
on the basis of spectroscopic evidence and a chemical conversion [462].
NaOjSQ
HO Y Y ÔH
GSOsNa OH OH
557 558 R = a-H
559 R = P-H
NaOaSOî 4 y . ^ L i x ^
Na03S0'
OSOjNa
560
An Acanthodendrilla sp. from Japan contained ten steroidal sulfates,

acanthosterol sulfates A-J (561-570). Acanthosterol sulfates I (569) and J
(570) showed antifungal activity against Saccharomyces cervisiae and its
mutants [463]. Clathsterol (571), was isolated from the Red Sea sponge
Clathria sp. The structure was established mainly by interpretation of
spectral data and a chemical transformation. Clathsterol (571) was active
against HIV-1 reverse transcriptase (RT) at a concentration of 10 |LiM
[464]. Toxadocia zumi contains three sterol sulfates (572-574) that are
antimicrobial, cytotoxic, ichthyotoxic and larvicidal [465].
703
RjO.
^ T1 l Tii
R20^
0S03Na OSOsNa OSOaNa
561 562Ri=H,R2 = Ac 564 Ri = H, R2 = Ac
563Ri = H,R2 = H 566 Ri = H, R2 = H
568Ri=Ac,R2 = H 569 Ri = Ac,R2 = H
R,0, NaOjSOi
HI H
v^
OH'
OAc I JL J
«^jdb
NaO^SO' " ^ ^^ NaOaSO^^-^^^
0S03Na
565 Ri = H, R2 = Ac 571 572 R =

567 Rj = H, R2 = H
573 R =
570Ri=:Ac,R2 = H
574R= .
The sterol sulfates haplosamates A (575) and B (576) are inhibitors of

HFV-l integrase from two Philippines Haplosclerid sponges and were
reported to be the first naturally occurring sulfamates [466] but the
structures were revised after re-examination of spectral data [467].
-O
•bP02H0Me
NaOsSO' Y Y ÔR
OH OH
575 R = H
576R = P03H2
704
A sterol sulfate, 3P,4P-dihydroxypregn-5-en-20-one 3-sulfate (577),

was isolated from Stylopus australis from New Zealand and was the first
known sterol sulfate with a 5-pregnene skeleton [468].
HO3SO'
The Pacific deepwater sponge Poecillastra laminaris contained

annasterol sulfate (578), which had glucanase inhibitory activity [469].
NaOaSa 'ÔAc
578
Polymastiamide A (579), an antimicrobial steroid with an unusual side

chain modification involving an amide bond to a non-protein amino acid,
was isolated from the Norwegian marine sponge Polymastia boletiformis.
The structure of polymastiamide A (579) was elucidated by analysis of
spectroscopic data and chemical interconversions [470]. Polymastiamides
B-F (580-584), additional amino acid conjugates of steroids, were later
isolated from the same sponge [471].
O CO2H
'CO2H
NaOsSO'^ > MeO NaOjSa
579 580Ri = H,R2 = OMe

581Ri=Me,R2 = H
705
O CO2H
NaOsSO^^^V""^"--^ 582 Ri = Me, R2 = OMe

583Ri = H,R2 = OMe
584Ri=Me,R2 = H
Echinoclasterol sulfate phenethylammonium salt (585), an antifungal

and cytotoxic steroid, was isolated from the South Australian sponge
Echinoclathria subhispida [472].
^x:!;55^^\^NH3 0380^
585
Three antiviral sterol disulfate orthoesters, orthoesterol disulfates A-C

(586-588) were isolated from Petrosia weinbergi and their structures were
determined by spectral data elucidation [473].
NaOaSa
I I J H
NaOjSO'
586R= / .
587 R=c5^0C
588R:
c^^
706
Weinbersterol disulfates A (589) and B (590) are also antiviral

metabolites from P. weinbergi [474].
589Ri = H,R2 = OH
590Ri = OH,R2 = H
Haliclostanone sulfate (591) is an unusual polyhydroxylated sterol

sulfate from Haliclona sp. from Malaysia [475].
HO' Y ' y ÔH

O l f OH
591
Crellastatin A (592) is the first of a series of cytotoxic bis-steroidal

sulfates isolated from a Crella sp. from Vanuatu [476].
,0S03Na
592 Ri = OH, R2 = OH
593 Ri = H, R2 = OH
594 Rj = OH, R2 = H
595 Ri = H, R2 = H
707
Crellastatins B-M (593-604) are twelve additional cytotoxic, dimeric

4,4'-dimethylsterols from the same Crella sp. [477,478].
,0S03Na
596
.OSOaNa
597 R = OH
598 R = H
.OSOjNa
OSOjNa
600
708
ÔSOjNa
ÔH
r^^.
602
,0S03Na
OSO^Na
ÔH
OSOjNa
Benzylthiocrellidone (605) was isolated from Crella spinulata and the

structure was confirmed by synthesis [479]. It is the first reported
example of a natural product containing a dimedone unit [22].
709
Ph
O S^ OH
00
605
Pateamine (606), a potent cytotoxin containing a dilactone

functionality, was isolated from a New Zealand species of Mycale and
identified by analysis of spectral data [480]. Total synthesis of pateamine
A (606) involved a P-lactam based macrocyclisation [481,482], while
another total synthesis of pateamine employed a concise and convergent
route [483].
A sulfated galactolipid, M-6 (607) was isolated from Phyllospongia

foliascens. M-6 (607) consisted of an inseparable mixture of compounds
with variations occurring in the carboxylic acid portion of the molecule.
Compound 607 has resistant activity against complement fixation in
serological reactions [484].
CH20S03"Na'"
OH hOR
607 Ô"^
R = (a:b=l:2)
a =C0(CH2)6CH=CHC7Hi5
b = C0Ci5H3i
710
The cytotoxic polyether acanthifolicin (608), which is structurally very

similar to okadaic acid, was isolated from Pandaros acanthifolium. It is
unique in having an episulfide group on a long-chain polyether (C38)
backbone. The structure and absolute configuration were determined by
X-ray analysis [485]. Acanthifolicin (608) is thought to be a product of a
microbial or microalgal symbiont of the sponge. Desulfurisation of
acanthifolicin with a Zn/Cu couple yielded okadaic acid [486].
HQH
Mycothiazole (609) is a novel thiazole-containing lipid with

anthelmintic properties isolated from Spongia mycofijiensis. The structure
was established by analysis of spectral data [487]. A total synthesis of (-)-
mycothiazole (609) utilised a convergent strategy. The optical rotation of
the product was the same sign as that of the natural material but
significantly larger [488].
NHC02Me
609
The theonezolides are 37-membered macrocycles, consisting of fatty

acid chains with attached functionalities such as a sulfate ester and a
thiazole [22]. Theonezolide A (610) is a cytotoxic metabolite of Theonella
sp. from Okinawa. The structure was reported without stereochemical
details [489], The structures of theonezolides B (611) and C (612) from a
Japanese Theonella sp. were determined by spectroscopic methods but
without stereochemistry, except at one centre [490].
711
Toxadocials A-C (613-615) and toxadocic acid (616) are sulfated long
chain alcohols isolated from Toxadocia cylindrica that inhibit thrombin.
Their structures were determined by chemical and spectral means
[491,492].
NaOgSO GSOsNa NaOsSO 0S03Na

613 R = CHO
616 R = CO2H
712
NaOsSO OSOgNa CHO NaOaSO OSO^Na
614
OHC NaOaSO 0S03Na
NaOsSO OSOaNa
615
Callyspongins A-B (617-618) are sulfated compounds from a Japanese

sample of Callyspongia truncata. They inhibit fertilisation of starfish
(Asterias amurensis) gametes [493].
"OSO^Na
617 R = SGjNa
618 R = H
Mycale sp. from Japan contained thiomycalolides A (619) and B (620)

as minor metabolites. They are highly cytotoxic glutathione adducts of the
known metabolites mycalolides A and B [494].
OHC
ô
HO2C NH
HO2C o'^-'^s^ I VQ
O OMe
o
619R = 0
620 R = H, 0C0CH(0Me)CH20Me
713
Penares sp. from Japan contained penarolide sulfates Ai (621) and A2

(622), which were a-glucosidase inhibitors [495].
O C4H9 o c.H
•3"7
An Oceanapia sp. collected off the northern Rottnest Shelf, Australia,

has yielded three novel dithiocyanates, thiocyanatins A-C (623-625). The
structures were determined by spectroscopic analysis and confirmed by
total synthesis. The thiocyanatins contain an unprecedented dithiocyanate
functionality and an unusual 1,16-difunctionalised n-hexadecane carbon
skeleton. They possess nematocidal activity [496].
NCS^
SCN ^SCN
623 624
"'SCN
625
Three sulfated ceramides, calyceramides A-C (626-628) were isolated

as inhibitors of neuraminidase from the marine sponge Discodermia calyx.
Their structures were determined by spectroscopic and chemical methods
[497].
NaOjSO.
NaOjSi
714
NaOaSO.
Irciniasulfonic acid (629) was obtained from Ircinia sp. from Japanese
waters. Spectroscopic and chemical analyses revealed it to consist of three
different kinds of acids; common fatty acids, a novel unsaturated branched
CIO fatty acid and an isethionic acid. Irciniasulfonic acid (629) reverses
multidrug resistance in human carcinoma cells caused by overexpression
of membrane glycoprotein [498].
R=
629
Bastaxanthins B, C, D, E, and F (630-634) are novel carotenoid

sulfates from the marine sponge lanthella basta from the Great Barrier
Reef, Australia [499]. The stereostructure of bastaxanthin C (631) was
determined on the basis of infrared (IR), ^H and ^^C NMR, and CD
spectra, and by chemical transformations [500]. Bastaxanthins were also
isolated from /. flabelliformis from the Great Barrier Reef including
bastaxanthin C (631) (major), B (630), D (632), and F (634) and
bastaxanthin G (635) [501]. Bastaxanthin G (635) was not fully
characterised but was the most polar of the carotenoids isolated and was
tentatively described as a disulfate [501],
715
Na03S0' 630 R = CH2OH

631 R = CHO
633 R = CO2H
NaOgSa 632 R = CH2OH

634 R = CO2H
A sulfone (636) is a minor constituent of the Mediterranean sponge

Anchinoe tenacior [502]. Sulfolane (637), a familiar industrial chemical,
was isolated from a mixture of the sponge Batzella sp. and a Lissoclinum
tunicate from Victoria, Australia [503]. It is possibly an absorbed
compound rather than a natural product [12].
(!)
636 637
5-Thio-D-mannose (638), the first example of a naturally occurring 5-

thio-sugar has been isolated from Clathria pyramida [504] and was later
synthesised in 12 steps from D-mannose [505].
HO-
pH HOAOH
638
716
(2-Hydroxyethyl)dimethylsulfoxonium chloride (1), the causative

agent of Dogger Bank Itch which has previously been isolated from the
marine bryozoan Alcyonidium gelatinosum [26], has now been isolated as
a cytotoxic component of the marine sponge Theonella aff. mirabilis
[506].
Echinoderms
The phylum Echinodermata comprises about 7000 living species [177].

Echinoderm means "spiny-skinned" and these organisms are characterised
by the tube feet, which they use to move about. These have suction discs
on the ends, which operate by an internal bulb pumping water in and out
of the foot, causing expansion and contraction. The phylum is sub-divided
into five classes; the asteroids (sea stars), the holothurians (sea
cucumbers), the crinoids (sea lilies), the echinoids (sea urchins) and the
ophiuroids (brittle stars) [178]. As stated in the introduction to this
review, sulfated sterols and saponins, which comprise the majority of
echinoderm metabolites containing sulfur, are not included here.
The histidine derivatives, l-methyl-5-thiolhistidine (639) and its
disulfide (640) were isolated from unfertilised eggs of the sea urchin
Paracentrotus lividus [507] and from those of other echinoderms [508].
Their structures were revised after an unambiguous synthesis [509]. L-
Ovothiol A disulfide (640) was also shown to be the egg release
pheromone of the marine polychaete worm Platynereis dumerilii [510].
Me
CO2H NNf--^NH2
HS /—< S-S
NH2 H2N Y\
N^N-Me H02C^^-^N
639 640 ^^
The ovothiols, a family of mercaptohistidine compounds, have been

isolated from marine invertebrate eggs. Ovothiol B (641) from the scallop
717
Chlamys hastata, ovothiol C from the sea urchin Strongylocentrotus

purpuratus (642) and ovothiol A from the starfish Evasterias troschelli
were isolated from eggs of ovarian tissue [511,512]. The structure of
ovothiol A is identical to that of l-methyl-5-thiolhistidine (639).
CO2H
641 R = H
642 R = Me
The ovaries of the Japanese sea urchin Hemicentrotus pulcherrimus

contained a bitter tasting amino acid, pulcherrimine (643) [513]. A
sulfonoglycolipid isolated from the shell of the sea urchin Anthocidarias
crassispina was a 96:4 mixture of r-0-palmitoyl-3'-0-(6-sulfo-a-D-
quinovopyranosyl)glycerol (644) and the myristoyl counterpart (645)
[514].
OH ^^ OH^
643 644R = Ci5H3i
645R = Ci3H27
Many starfish cause an escape response in usually sessile marine

invertebrates [7]. The starfish Dermasterias imbricata causes the sea
anemone Stomphia coccinea to release its basal disc from the substratum
and swim away on contact. Bioassay-directed fractionation of the starfish
extract led to the isolation of the compound found to elicit this response,
the benzyltetrahydroisoquinoline alkaloid imbricatine (646). The structure
of compound 646 was elucidated by spectral data interpretation. The
amino acid residue in imbricatine is related to the thiol containing amino
acids ovothiols A-C. Imbricatine (646) is active in both L1210 and P388
718
assays [515,516]. The structural elucidation and partial synthesis of

imbricatine (646) were later reported fully [517].
Forbesin (647), a sulfated glycolipid and a disodium salt, eicosane-

1,16-disulfate (648) were isolated from the sea star Asterias forbesi,
Forbesin was also isolated from A. vulgaris [518].
NaOsSO
HO-
MO
647
NaOaSO
OSOgNa
648
The sea star Henricia laeviuscula contained the anthraquinone sodium

isorhodoptilometrin-2'-sulfate (649) [519].
OSOjNa
719
The naphthopyrone, comantherin sulfate (650) was isolated from the

crinoid Comantheria perplexa [520] and three naphthopyrones (651-653)
were isolated from Comanthus parcivirrus timorensis. Both species were
collected off Australia [521].
NaOsSO'
650 651 Ri = OH, R2 = H

652 Ri = OH, R2 = OMe
653Ri = OMe,R2 = OMe
The crinoid Comatula pectinata contained three anthraquinones (654-

656) [522] while two further anthraquinones (657-658) were isolated from
the crinoid Ptilometra sp. l,8-Dihydroxy-3-propyl-9,10-anthraquinone-6-
0-sodium sulfate (657) and l,8-dihydroxy-3-(r-hydroxypropyl)-9,10-
anthraquinone-6-O-sodium sulfate (658) have not previously been isolated
from a natural source. l,8-Dihydroxy-3-(l-hydroxypropyl)-9,10-
anthraquinone-6-O-sodium sulfate (658) was cytotoxic [523].
OH O
NaOgSO. .OMe
NaOsSO'
OR2 0 ORi 0
654 Ri = H, R2 = H 657R = H
655 Rj = Me, R2 = H 658R = OH
656 Ri = Me, R2 = Me
The deepwater stalked crinoid Gymnocrinus richeri contained the

gymnochromes C (659) and D (660) and isogymnochrome D (661). These
compounds have a helical chirality and chiral atoms in the sidechains give
rise to isomers [524].
720
HO 0 OH HO 0 OH HO O OH
Brv
HO"
fWS -%H Brv
HO"JLX
riT if' ^
II ""^
,Br
9SO3H
HO.
TI T11 TL HOs II
rl 1 11^ ^
Br'' Wy^
OH 0 OH
^B,6S03H
\J
Br^
OH 0 OH
JS ^ 'Br
PSO3H
OH O OH
PSO3H
659 660 661
The holothurian Cucumaria frondosa contains 2,6-dimethylnonane-l-

sodium sulfate (662) and 2,4,6-trimethyl-nonane-l-sodium sulfate (663).
The structures were proposed without stereochemical detail [525].
LAjJx.' OSOsNa
662 R = H
663 R = Me
The Japanese sea cucumber Cucumaria echinata contained a

ganglioside CG-1 (664) with neuritogenic activity toward the rat
pheochromocytoma PC-12 cell line [526]. Similar activity was reported
for the ganglioside HPG-8 (665) isolated from the sea cucumber
Holothuria pervicax from Japan [527].
0SO3H
HO^^ÂÔH
HN^3H
..OH
HO' ^ *OHHO^
OH
664
721
OH
loX>^2C0^0.
H03SO'
HO' ^ 'OHHO^
OH
665
A carotenoid sulfate, ophioxanthin (666), was isolated from the

ophiuroid Ophioderma longicaudum from the Mediterranean Sea and
shown to be 5,6,5',6-tetrahydro-P,P-carotene-3,4,3',4-tetraol 4,4'-disulfate
[528]. The carotenoid, dehydroophioxanthin (667) was isolated from the
ophiuroid Ophiocomina nigra off Spain and the structure was determined
by spectral data analysis [529].
(3Z)-4,8-Dimethylnon-3-en-l-yl sodium sulfate (166), which is also

found in the ascidian Microcosmus vulgaris [166], is a sulfated alkene that
was isolated from the ophiuroid Ophiocoma echinata from Colombia
[530].
722
ABBREVIATIONS
ALL = acute lymphoblastic leukaemia

Anti-fflV = anti-human immunodeficiency virus
ATP = adenosine triphosphate
CD = circular dichroism
DNA = deoxyribonucleic acid
DSP = diarrhetic shellfish poisoning
EC50 = effective concentration needed to reduce cell
growth by 50%
ECE = endothelin converting enzyme
ED50 = effective dose needed to reduce cell growth by
50%
FAB = fast atom bombardment
fflV-1 = human immunodeficiency virus type 1
IC50 = inhibitory concentration needed to reduce cell
growth by 50%
IR = infrared
mg = milligram
mL = millilitre
mM = millimolar
[iM = micromolar
nM = nanomolar
NCI = National Cancer Institute
NMR = nuclear magnetic resonance
NOEDS = nuclear Overhauser enhancement difference
spectroscopy
723
NSP = neurotoxic shellfish poisoning

RT = reverse transcriptase
SPLA2 = secreted phospholipase A2
REFERENCES
[I] Faulkner, D.J., Chem, Br., 1995, J7, 680-684.

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[525] Findlay, J.A.; Yayli, N.; Calhoun, L.A.; J. Nat. Prod., 1991, 54, 302-304.
[526] Yamada, K.; Hara, E.; Nagaregawa, Y.; Miyamoto, T.; Higuchi, R.; Isobe, R.;
Honda, S.; Eur. J. Org. Chem., 1998, 371-378.
[527] Yamada, K.; Harada, Y.; Nagaregawa, Y.; Miyamoto, T.; Isobe, R.; Higuchi,
R.; Eur. J. Org. Chem., 1998, 2519-2525.
[528] D'Auria, M.V.; Riccio, R.; Minale, L.; Tetrahedron Lett., 1985,26, 1871-1872.
[529] DAuria, M.V.; Minale, L.; Riccio, R.; Uriarte, E.; J. Nat. Prod., 1991, 54, 606-
608.
[530] Roccagliata, A.J.; Maier, M.S.; Seldes, A.M.; Zea, S.; Duque, C; /. Nat. Prod.,
1997, 60, 285-286.
753
SUBJECT INDEX (Vol. 28) 702

antifimgal activity of 702
Abamectin 434,406 A carapis woodi 387,390
eigmnst Hyalomma spp, 407 in honey bees' tracheal tubes 387
against Phytoseiulus persimilis infestations in Minnesota 390
434 Acaricidal activities 403,406,412,422
against Rhipicephalus spp. 407 against Dermatophagoides
against Tetranichus urticae 434 pteronyssinus 422
Absolute stereostructure 11 against Psoroptes cuniculi 412
of broussonetine C 11 against Rhipicephalus
ofbroussonetineL 12 appendiculatus 406
Aburatubolactam A 139 for killing adult ticks 403
fôm Streptomycessp. 139 of caffeine 422
Aburatubolactam C 139 of Cuminum cyminum 427
fvom Streptomycessp. 139 of essential oils 412,427
(2S)-Abyssinone II 17 of Eucalyptus camaldulensis All
as aromatase inhibitor 17 of eugenol 403
Acacia honey 386 of extract prepared by microwave
aroma of 386 assisted process (MAP) 427
Acanthella 663 of Lavandula angustifolia 412
kalihinol G of 663 of linalool 412
kalihinol H of 663 of Margaritaria discoidea 406
Acanthella cavernosa 663 of Origanum syriacum var. bevanii
10-^/?/-isokalihinolHfrom 663 All
15-isothiocyanato-1 -epi-kalihinene of phenylpropanoid derivatives
from 663 403
Acanthella klethra 662 of Pimenta dioica 403
isothiocyanates from 662 of Pimpinella anisum All
A canthella pulcherhma 660 of Tanacetum vulgare 's extracts
isothiocyanates from 660 427
sesquiterpenes from 660 of P-thujone 427
Acanthifolicin 710 Acaricidal properties 400,406
as cytotoxic agent 710 of benzaldehyde 406
episulfide group of 710 of carvacrol 406
from Pandaros acanthifolium 110 of cedrene 406
similarity to okadaic acid 710 of a-cyclocitral 406
Acanthodendrilla sp. 702 of P-cyclocitral 406
acanthosterol sulfate A of 702 of Euphorbia obovalifolia 's latex
acanthosterol sulfate B of 702 400
acanthosterol sulfate C of 702 of Ficus brachypoda's htex 400
acanthosterol sulfate D of 702 of geraniol 406
acanthosterol sulfate F of 702 of (£)-geranylacetone 406
acanthosterol sulfate G of 702 of a-ionone 406
acanthosterol sulfate H of 702 of linalool 406
acanthosterol sulfate I of 702 of w-cymene 406
acanthosterol sulfate J of 702 of methyl salicylate 406
against Saccharomyces cerevisiae ofnerol 406
754
of nerolidol 406 Adociidae family 664

of nonanal 406 10-isothiocyanatobiflora-4,15-
of P-ocimene 406 diene of 664
of phenylacetaldehyde 406 spectral analysis of 664
of phenylacetonitrile 406 P-Adrenoceptors 183
of a-terpineol 406 with [^H]dihydroalprenolol 183
Acaricide 381,429,435 AflastatinA 127,128
cross-resistance of 429 as aflatoxin inhibitor 128
deguelinas 435 from Streptomyces
effectiveness of 404 griseochromogenes 127
fenazaquinas 429 structure of 128
from Annona squamosa 404 AflastatinB 127
from Azadirachta indica 404 as aflatoxin inhibitor 128
literature about 381 from Streptomyces
Lonchocarpus urucu as 435 griseochromogenes 127
of natural origin 381 Anatoxins 128
pyridabenas 429 from Aspergillus flavus 128
rotenoloneas 435 from Aspergillus nomius 128
rotenoneas 435 from Aspergillus parasiticus 128
tebufenpyrad as 429 from Aspergillus tamarii 128
tephrosinas 435 African tick species 396
A cams siro 382 Amblyomma hebraeum as 396
as mite species 382 Boophilus decoloratus as 396
Acrostalamus fungi 455 Hyalomma sp. as 396
acrostalidic acid from 455 Rhipicephalus appendiculatus as
acrostalic acid from 455 396
isoacrostalidic acid from 455 Rhipicephalus evertsi evertsi as
3-Acyl tetramic acid 111,112,114 396
from Alternaha alternata 114 Agelas dispar 692
from Alternaha longipes 114 from Bahamas 692
from Alternaha tenuis 114 pyridinebetaine B of 692
biosynthetic pathways of 111 Agelas nakamurai 670
from Pyricularia oryzae 114 agelasidine A from 670
tautomeric forms of 112 agelasidine B from 670
Adenichrome 647 antispasmodic activity of 670
Fe(III)-containing pigment as 647 Na^/K^-transporting adenosine
from Octopus vulgaris 647 triphosphate (ATP)ase inhibitor
Adociasp. 674 from 670
adociaquinone A from 678 spectral data of 670
adociasulfate from 674 structure of 670
from Great Barrier Reef 674 synthesis of 670
structure of 674 Agricultural pests 423
Adociasulfates 1-6 674 of forage crops 423
as kinesin motor proteins inhibitors of fruits 423
674 of ornamentals 423
from Haliclona (aka Adocia) sp. of timber 423
674 of vegetables 423
755
Ajoene 432 Ancorinoside A Mg salt 120

acaricidal activity of 432 ficom Ancorinasp. 120
against Tetranyehus urticae 432 Ancorinoside B 120
anticoagulant properties of 432 ficom Ancorinasp. 120
Akaterpin 673 Ancorinoside C 120
as phosphatidylinositol- from Ancorina sp. 120
phospholipase C inhibitor 673 Ancorinoside D 120
from Callyspongia sp. 673 from Ancorinasp. 120
stereochemistry of 673 Annona glabra seeds 430
AlbanolA 17 acetogenins from 430
as aromatase inhibitor 17 against Dermatophagoides
Albanol B 234 pteronyssinus 430
from Morus uralensis 234 against Typhlodromus urticae 430
Alcyonidium gelatinosum sp. 619 asimicinfrom 430
(2-hydroxyethyl) desacetyluvaricin from 430
dimethylsulfoxonium squamocinfrom 430
ion from 619 Annona squamosa 415
Dogger Bank itch by 619 extract of 415
Aldose reductase inhibitor 691 Anthelmintics 331,332
from Dictyodendhlla sp. 691 broad spectrum activity of 332
a-Alkyl-p-hydroxyproline moiety 367 diminished activity of 331
construction of 367 new class of 332
Allelopathic activity 483 Anthocyanidins 275
of natural podolactones 484 cyanidinas 275
of synthetic podolactones 484 delphinidin as 275
Allium sativum (LilidicediQ) 415 malvidinas 275
Alphitolic acid 40 pelargonidin as 275
from Licania heteromorpha var. structure of as 275
heteromorpha 40 Anthocyanins 275,276,277,292
Althiomycin 143 cyanidin-3-glucoside as 275
from Cystobacter fuscus 144 delphinidin-3-glucoside as 275
frovci Myxococcus xanthus 144 for coronary heart disease 292
from Streptomyces althioticus 143 from black grapes 276
from Streptomyces matensis 143 in blackberry 277
Amblyomma 394,397 in blueberry 277
by Beauveria bassiana 397 in cabbage, red 277
by hyperparasitic fimgi 397 in cherry 277
by Metarhizium anisopliae 397 inchokeberry 277
control of 397 in cranberry 277
in goats 394 in currant (black) 277
in sheeps 394 in food plants 276
Amblyomma variegatum 395 in grape (red) 277
repellent properties of 395 in onion 277
[^H]Amine uptake 183 in organe, blood O'uice) 277
of Ginkgo biloba L. 183 in raspberry, red 277
Ancorinoside A 120 in strawberry 277
fvom Ancorinasp. 120 in wines, porto 277
in wines, red 277
756
malvidin-3-glucoside as 275 of gancaonol C 243

pelargonidin-3-glucoside as 275 ofglabrene 241
Anthopleura elegantissima 647 ofglabridin 241
mycosporine-taurine from 647 ofglyasperinD 243
Anti-apoptotic effects 175 ofglycyrin 243
Antiahs toxicaria 203 ofglycyrol 241
antiarone A from 203 ofglycyrrheticacid 241
antiarone B from 203 ofglycyrrhizicacid 241
antiarone E from 204 of isoglycyrol 241
antiarone J from 203 of isolicoflavonol 243
antiarone K from 203 oflicochalcone A 241
ficusins A from 204 oflicochalconeB 241
uses for arrow poison 203 oflicoisoflavoneB 241
Anti-atherosclerotic activity 257,293 of licorice flavonoids 234
of polyphenols 257,293 of licorice-saponin 241
Anti-bacterial activity 140 oflicoricidin 241
of ikarugamycin 140 oflicoricone 243
Anti-carcinogenic activity 257,293 ofliquiritigenin 241
of polyphenols 257,293 ofliquiritin 241
Anti-con vulsant activity 176 of l-methoxyphaseoUidin 243
ofbilobalide 176 of3-(9-methylglycyrol 243
Anti-depressant effects 177 ofvestitol 243
of Ginkgo biloba L. 177 Anti-Human inmiunodeficiency virus
Anti-feedant activity 480 (HIV) activity 225,226
against mammals 480 of antiarone I 226
of l-deoxy-2P,3p-epoxy- of broussoflavonol B 226
nagilactoneA 480 of broussoflavonol C 226
of nagilactone A 480 ofgancaoninR 226
of nagilactone C 480 ofglyasperin A 226
Anti-ftingal activity 66,473,475 ofglycyrol 226
of2-hydroxynagilactoneF 475 ofkazinolB 226
of intrapetacin A 66 of kumatakenin 226
of intrapetacin B 66 ofkuwanonH 225
of LL-Z1271a 473 oflicochalconeB 226
of nagilactone C 475 ofmoracinC 226
of nagilactone E 475 of morusin 225
of oidiodendrolideB 475 of mulberry tree 225
ofoidiolactoneD 475 ofnorartocarpetin 226
Anti-fungal holothurin 597 ofprenylflavones 225
from Psolus patagonicus 597 of wighteone 226
Anti'Helicobacter pylori activities Anti-HIV flavonoids 226
234,241,243 2-arylbenzofiiran as 226
of 6,8-diprenylorobol 243 from Glycyrrhiza species 226
of dihydrolicoisoflavone A 243 from moraceous plants 226
of formononetin 241 Anti-inflammatory activity 200,257,293
of gancaonini 243 of genus Morw^ 200
ofgancaonol B 243 of polyphenols 257,293
757
Anti-metastatic activity 559 of Licania licaniaeflora 63

of natural products 559 of polyphenols 257,293
Anti-microbial activity 62,224,257,293 .of quercetin 3-(9-a-Z-
ofalphitolicacid 62 arabinopyranoside 64
of AMOX 224 of quercetin 3-0-a-Z-
ofbetulinicacid 62 rhamnopyranoside 64,65
of formononetin 224 of quercetin 3-O-P-Z)-
ofgancaonini 224 galactopyranoside 64
ofgancaonolB 224 of quercetin-3 -O-a-L-
of glabrene 224 rhamnopyranoside 64
of glabridin 224 of taxifolin 3-0-a-Z-
ofglyasperinD 224 rhanmopyranoside 64
of glycyrin 224 Anti-pyretic activity 200
of glycyrol 224 of genus M(9rw5 200
of isoglycyrol 224 Anti-radical activity 257,293
of isolicoflavonol 224 of polyphenols 257,293
of Licania heteromorpha var. Anti-stress effects 177
heteromorpha 62 of Ginkgo biloba L. 177
of licochalcone A 224 Anti-tumor activity 517,519,559,560,
of licochalconeB 224 566,211
of licoisoflavoneB 224 of carp oil 566
of licorice flavonoids 224 ofchitosan 560
of licoricidine 224 of Coley's toxin 519
of licoricone 224 offish oils 566
of liquiritigenin 224 of lipids 517
of liquiritin 224 ofmorusin 211
of 3-0-methylglycyrol 224 of natural products 559
of 3|3-0-d5-/?-coumaroyl maslinic of tuna oil 566
acid 62 Anti-tumor substances 569
of 3P-0-cw-/?-coumaroyl alphitolic from Agaricus blazei 569
acid 62 isolation of 569
of 3 ^'O'tranS'P'C0\xmdC[0y\ mechanism of 569
alphitolic acid 62 Anti-tussive activity 200
of 3 p-(9-^r<3f/w-/?-coumaroyl of genus M^rw^ 200
maslinic acid 62 Anti-viral activity 257,293
of polyphenols 257,293 of polyphenols 257,293
of vesititol 224 Anxiolytic activity 177
Anti-neoplasic activity 470 of Ginkgo bilobaL. 177
against P 388 cells 470 Apiculture 390
Anti-oxidant activity 63,179,257,293 acaricides in 390
of Ginkgo bilobaL. 179 Apiguard 391
of 8-hydroxy-naringenin 64 for thymol-based acaricide 391
of kaempferol 3-(9-(2"-P-D- ApilifeVAR 391
xylopyranosyl)-a-Z- as Frakno thymol frame 391
rhamnopyranoside 64,65 a-and P-Apiodionen 117
of kaempferol 3-0-a-I- as topoisomerase inhibitor 117
rhamnopyranoside 64
758
Apis mellifera 387 Artocarpus f[dMono\As 203,216,220

killed by bee larvae 387 against arachidonate 5-lipoxy-
Aplidium pliciferum 635 genase 216
1,2,3-trithiane derivative from 635 against TNF-a release 220
Aplysia kurodai 648 artocarpesin 203
metabolites of 648 artonini 203
neoaplaminone sulfate of 648 morachalcone A 203
AplysillinA 697 Artocarpus alt His 202
as thrombin receptor antagonist Artocarpus communis 202
697 Artocarpus heterophyllus 202
from Aplysinafistularis 697 Artocarpus rigida 202
Apoptosis 175 Artocarpus venenosa 202
ofbilobalide 175 artobiloxanthone from 202
ofginkgolideB 175 artoninE from 202
ofginkgolide J 175 cycloartobiloxanthone from 203
using 3-(4,5-dimethylthiazol-2-yl)- isoprenylated flavonoids from 202
2,5-diphenyl tetrazolium bromide use against inflammation 202
175 use in malarial fever 202
Arachidonate 5-lipoxygenase activity 216 use as folk medicine 202
of artoninE 217 ArtoninE 201,215
of morusin 217 as 5-lipoxygenase inhibitor 215
Arachidonate oxygenase activity 217 from moraceous plants 201
Arachnida 382 Artonini 201
class of 382 from moraceous plants 201
Arjunic acid 28-P-D-glucosyl ester 40 Ascidia mentula 642
from Licania licaniaeflora 40 heneicosane-1,21 -diyl disulfate
Artemisia absinthium 425 from 642
acaricidal activity of 425 3,7,11,15-tetramethylhexadecane-
against aphids 425 1,19-diyl disulfate from 642
as insecticide 425 Ascosalipyrrolidinone A 148
Arthropod-borne diseases 394 against antibacterial activity of 148
of humans 394 against chloroquine-resistant
of livestock 394 Plasmodium falciparum 148
zoonotic as 394 2igmnst Trypanosoma brucei 148
Artobiloxanthone 201 against Trypanosoma cruzi 148
from moraceous plants 201 Aspalathus linearis 272
Artocarpesin 201 AurantosideA 136
from moraceous plants 201 from Theonella swinhoei 136
y4rrocaA77W5 flavones 216 AurantosideB 136
artoninA 216 from Theonella swinhoei 136
artoninB 216 Austrovenus stutchburyi 656
artoninE 216 brevetoxin Bi from 656
cycloartobiloxanthone 216 Autotoxic effects 537
cycloheterophyllin 216 of NO on tumor cells 537
heterophyllin 216 Avermectin derivatives 402
morusin 216 from Boophilus sp. 402
5-lipoxygenase activity of 216 Axinella cannabina 658
axisothiocyanates from 658
759
axisothiocyanate 2 from 658 from marine sponge lanthella basta

axisothiocyanate 3 from 658 714
axisothiocyanate 4 from 658 Bastaxanthin F 714
sesquiterpenoid from 658 as novel carotenoid sulfate 714
Axinyssa fenestratus 662 from lanthellaflabelliformis 714
10-isothiocyanato-4,6- from marine sponge lanthella basta
amorphadiene from 662 1\A
4-isothiocyanato-9-amorphene Batzella spongQ 685
from 662 against Candida albicans 686
10-isothiocyanato-5-amorphen-4- antifimgal activity of 686
ol from 662 batzellines A from 685
Azadirachta indica 401 methyl sulfide groups of 685,686
effects on 5oo/7Mw5 sp. 401 Bee parasites 387
Azadirachta indica dust 389 formic acid in 387
effects on honey production 389 Tropilaelaps clareae as 387
Azadirachta indica oil 388,395 Benzyl benzoate 416
against bee mites 387 digdmstPsoroptes 416
against blood sucking ticks 395 against Sarcoptes mites 416
from azadirachtin 388 against Sarcoptes scabiei 416
effectiveness of 416
BALB/cmice 534 in mange control 416
Meth A sarcoma in 534 Betulinic acid 17,40
Bacillus subtilis ll'i cytotoxic activity of 17
effects of phenols on 223 from Licania heteromorpha var.
Bacterial origin 434 heteromorpha 40
acaricidal agents of 434 from Licania licaniaeflora 40
Barley 260 bom Licania pittieri 40
syringic acid in 260 Bicyclic phloeodictine B 688
vanillic acid in 260 antibacterial activity of 688
Bastaxanthin B 714 cytotoxic activity of 688
as novel carotenoid sulfate 714 cyclic aminoketal from 688
from lanthellaflabelliformis 714 from Phloeodictyon 688
from marine sponge lanthella basta Bicyclo[2.2.2] ring system 363,370
714 construction of 363,370
Bastaxanthin C 714 Biflustra perfragilis sp. 620
as novel carotenoid sulfate 714 2-methyl-6,7-di(methylthio)-2^-
from lanthellaflabelliformis 714 isoquinoline-3,5,8-trione
from marine sponge lanthella basta from 619
714 2-methyl-6-methylthio-2i/-
Bastaxanthin D 714 isoquinoline-3,5,8-trione
as novel carotenoid sulfate 714 from 619
from lanthellaflabelliformis 714 Bioactive compounds 3,4,9,16,22
from marine sponge lanthella basta bom Broussonetia gcmis 3
lU from Broussonetia kazinoki 4,9
Bastaxanthin E 714 from Broussonetia papyrifera
as novel carotenoid sulfate 714 16,22
from lanthellaflabelliformis 714 structure of 9
760
Bioactive monoterpenoids 426 ofgancaoninG 228

carvacrolas 426 ofgancaoninH 228
carvomenthenol as 426 ofgancaonini 228
carvoneas 426 ofgancaoninO 228
citronellol as 426 ofgancaoninP 228
eugenol as 426 ofgancaoninQ 228
geraniol as 426 ofgancaoninR 228
perillyl alcohol as 426 ofgancaoninU 228
4-terpineol as 426 ofgancaoninV 228
thymol as 426 ofgancaoninY 228
Bioavailability 289 of glabranin 228
of catechins 289 of glabranine 228
Biological activity 109,212,228,340 of glabrene 228
ofalbaninD 229 of glabridin 228
ofalbaninF 230 ofglabrocoumarone A 229
ofalbanolA 230 ofglabrol 228
ofalbanolB 229 of glisoflavanone 228
of alvaxanthone 229 ofglyasperin A 228
ofangustoneB 228 ofglyasperinB 228
ofantiaroneB 229 of glyasperin C 228
ofantiaroneF 229 of glyasperin D 228
ofantiaroneG 229 ofglyasperin J 228
ofantiaroneH 229 ofglyasperin K 228
ofantiaronel 229 of glycycoumarin 228
ofantiaroneJ 229 ofglycyrdione A 228
of artobiloxanthone 229 of glycyrin 228
ofartoninE 229 ofglycyrol 228
of bavachalcone 228 ofglyinflaninA 228
ofbroussochalconeB 228 of heterophyllin 230
ofbroussoflavonolB 229 ofhispaglabridin A 228
of broussoflavonol C 229 of3-hydroxyglabrol 228
ofbroussoflavonolE 230 of 14a-hydroxy-MFA 340
ofcudraphenoneB 230 of3-hydroxyparatocharpinC^ 228
ofcudraphenoneD 230 of isoalvaxanthone 230
of cycloartobiloxanthone 230 of isoderrone 228
ofdehydroglyasperinC 228 of isoglycyrol 228
of 3 '-(y,Y-dimethylallyl)-kievitone of isoliquiritigenin 228
228 ofkanzonolB 229
of8-(Y,y-dimethylallyl)-wighteone ofkanzonolG 229
228 ofkanzonolH 229
of echinatin 228 ofkanzonolP 229
of kanzonol R 229
of edudiol 228
ofkanzonolS 229
oferythrininB 229
of kanzonol U 229
of formaononetin 228
of kanzonol V 229
of gancaoinin S 228
of kanzonol W 229
ofgancaoninC 228
of kanzonol X 229
ofgancaoninE 228
of kanzonol Y 229
761
ofkazinolB 230 ofsanggenonB 230

of kazinol E 230 ofsanggenonC 212,230
ofkazinol F 230 ofsanggenonD 212
of kazinol N 230 ofsanggenonM 230
ofKB-1 229 ofsemilicoisoflavoneB 229
ofKB-3 229 of shinpterocarpin 229
ofkumatakenin 229 ofsigmoidin A 229
ofkuwanon 212 ofsigmoidinB 229
ofkuwanonC 230 ofsoroceinF 230
ofkuwanon G 230 oftenuifolinB 229
ofkuwanon H 212 of tetramic acid-containing
ofkuwanon M 212 compounds 109
ofkuwanon R 230 of topozolin 229
oflicochalcone A 229 of wighteone 229
of licochalconeB 229 Biological properties 518,519
of licoflavonol 229 of lipid A 519
of licoisoflavanone 229 Biological studies 35
oflicoisoflavone A 229 on Z/cama genus 35
oflicoisoflavoneB 229 Biosynthesis 15
of licoricidin 229 ofbroussonetine J 15
of licoricone 229 of broussonetine U 15
of licorisoflavan A 229 5/5êco-dehydrocyclopiazonic acid 119
ofmacarangaflavanoneB 229 BivittosideC 589,590
of medicarpin 229 from Bohadschia bivittata 589
of 1-methoxyficifolinol 229 structure of 590
of l-methoxyphaseoUidin 229 Blasticidin 127
ofmoraceinB 230 as antibiotic 127
ofmoracinC 230 from Streptomyces
ofmorusin 212,230 griseochromogenes 127
of morusinol 230 Bombesin receptor antagonists 221
of mulberrin 230 from Morw^ species 221
of mulberrochromene 230 ofkuwanon G 221
ofmulberrofuranB 230 ofkuwanon H 221
of mulberrofliran G 212,230 Boophilus decoloratus 400
of naringenin 229 for Capsicum sp. 400
of norartocarpetin 230 for Euphorbia brachypoda 400
of3-(9-methylgancaoninP 229 for Euphorbia obovalifolia 400
of 4'-0-methylglabridin 229 of Solarium incanum 400
of oxydihydromorusin 230 Boophilus microplus 399,398
of paratocarpin L 229 eugenol of 399
of phaseoluteone 229 isoeugenol of 399
of pinocembrin 229 lethal effect on 398
of 6-prenyleriodictyol 229 synthetic acaricides for 399
of 8-prenyleriodictyol 229 Boophilus ticks 398
of 6-prenylnaringenin 229 in tropical regions 398
ofsanggenolC 230 sub-tropical regions 398
ofsanggenolM 230 BrevetoxinB4 656
ofsanggenonA 212,230 as toxin 656
762
from Perna canaliculus 656 broussonetine N from 5

in shellfish poisoning 656 broussonetine O from 5
Bripiodionen 117,118 broussonetine P from 5
as human cytomegalovirus protease broussonetine Q from 5
inhibitors 118 broussonetinine A from 5
cytotoxicity of 118 broussonetinine B from 5
from Streptomyces sp. 117 broussonol A from 6
BrosimoneA 201 broussonol B from 6
from moraceous plants 201 broussonol C from 6
BrosimoneD 201 broussonol D from 6
from moraceous plants 201 7,4'-dihydroxyflavan from 6
Broussoaurone A 16,17 for increased vision 4
antioxidant activity of 16,17 kazinol A from 6
as cyclooxygenase inhibitor 16,17 kazinolDfrom 5
as neutrophils respiratory burst kazinol E from 6
inhibitor 16 kazinol K from 5
as nitric oxide production kazinol Q from 6
inhibitor 16 kazinol R from 6
as platelet aggregation inhibitor sexual potency of 4
16,17 Broussonetia papyrifera 4,16
Broussoflavan A 16 anti-cancer activity of 4
antioxidant activity of 16 antifungal activity of 4
as platelet aggregation inhibitor 16 antioxidant activity of 4
Broussoflavonol E 17 diaphoretic activity of 4
as platelet aggregation inhibitor 17 in dyspepsia 4
Broussoflavonol F 17 in pregnancy 4
antioxidant activity of 17 laxative activity of 4
antiproliferative activity of 17 Broussonetia zeylanica 4,28
as aromatase inhibitor 17 aromatase inhibitory substance
as cyclooxygenase inhibitor 17 from 4
as platelet aggregation inhibitor 17 broussonetine from 28
Broussoflavonol G 17 cytotoxic flavonoids from 4
antiproliferative activity of 17 3,4'-dihydroxy-2,3 '-bipyridine
antioxidant activity of 17 from 28
Broussonetia kazinoki 4,5 8-hydroxyquinoline-4-
antifimgal activity of 4 carbaldehyde from 28
antiinflammatory activity of 4 8-hydroxyquinoline-4-
antioxidant activity of 4 carbaldehyde oxime from 28
antispasmodic activity of 4 glycosidase inhibitory alkaloids
broussonetine C from 5 from 4
broussonetine D from 5 BroussoninA 16
broussonetine E from 5 antiftmgal activity of 16
broussonetine F from 5 as aromatase inhibitor 16
broussonetine G from 5 BroussoninB 16
broussonetine H from 5 antifungal activity of 16
broussonetine K from 5 Bryozoans 618
broussonetine L from 5 as moss animal 618
broussonetine M from 5 lophophoreof 619
763
sulflir-containing 618 Capsimycin 138

U-shaped gut of 619 antifungal activity of 138
zooids of 619 from Streptomyces strain 13 8
Carboxyhomoyessotoxin 654
C-4-O-acetyl derivative 117 from DSP-infested Mytilus
Cadlina luteomarginata 659 galloprovincialis 654
sesquiterpene isothiocyanate from mussels 654
from 659 structure of 654
Caffeic acid 262 Caribbean collection 632
in apples 262 of Didemnum rodriguesi 632
in blueberries 262 minalemines D from 632
in cider 262 minalemines F from 632
in coffee beverages 262 Carp (Cyprinus carpi) 564
Caffeoylquinic acids 262 antitumor activity of 564
in apples 262 as diuretic 564
in blueberries 262 for eye fatigue 564
in cider 262 Caspase activity 320
in coffee beverages 262 of TNF-a 320
Califomian sponge 671 CassigarolA 575
absolute configuration of 671 effects on tumor growth 575
antiinflammatory activity of 671 Catechins 41,273
antimicrobial activity of 671 (+)-catechin 273
halisulfates 1-5 from 671 (-)-epicatechin 273
in Halichondriidae family 671 epigallocatechin 273
spectral data of 671 epicatechin-gallate 273
Callyspongin A 712 epigallocatechin-gallate 273
as starfish fertilisation inhibitor from Licania densiflora 41
712 from Licania pittieri 41
of Callyspongia truncata 112 Cattle babesiosis 398
Callyspongin B 712 by Babesia bigemina 398
as starfish fertilisation inhibitor by Babesia bovis 398
712 by Ba^î'/a species 398
of Callyspongia truncata 112 Cavemothiocyanate 662
Calpurnea aurea 400 of Acanthella cf cavernosa 662
killing effects on ticks 400 spectral data of 662
Calyceramide A 713 Cell wall conjugates 262
as neuraminidase inhibitors 713 in cereal brans 262
fvom Discodermia QdXyx 713 in spinach 262
Calyceramide B 713 in sugar beet fibre 262
as neuraminidase inhibitors 713 Cerastoma brevicaudatum 651
from Discodermia CdXyx 713 (methylthio)furodysinin from 651
Calyceramide C 713 dithiofiirodysinin disulfide from
as neuraminidase inhibitors 713 651
fxom Discodermia Cd\y\ 713 Ceratodictyon spongiosum 683
Capsicum species 436,400 cw,OT-ceratospongamideof 683
capsaicin from 436 Sigmadocia symbiotica of 683
killing effects of ticks 400 trans, ^/-a/i5-ceratospongamide
of 683
764
Chalcomoracin 210 sinapic (3,5-dimethoxy-4-hydroxy)

frommoraceousplants 210 as 261
Chemical studies 35 Cinnamon bark 259
on Z/cawa genus 35 gallic acid from 259
Chemical reactivity 488 salicylic acid from 259
ofpodolactones 488 syringic acid from 259
Chemotaxonomic study 36 Cisplatin(CDDP) 559
of Parinari gQnns 36 for cancer chemotherapy 559
Chinese crude drug 202 Classification 281
sanggenon A from 202 of ellagitannin oligomers 281
sanggenon C from 202 ofellagitannins 281
sang-Bai-Pi as 202 ofgallotannins 281
Chinese mulberry tree 202 of galloylated proanthocyanidins
isoprenylated flavonoids from 202 281
Morus cathayana as 202 of proanthocyanidins 281
Chlamys hastata 111 ofprodelphinidins 281
from Strongylocentrotus pupuratus of tannins 281
111 Clathsterol 702
Chlorosulfolipid 655 against HIV-1 reverse transcriptase
from hepatopancreas 655 (RT) 702
stereochemistry of 655 from Red Sea sponge Clathria sp.
Chitosan 563 702
effects on doxorubicin-induced Clavelina cylindhca 644
gastrointestinal toxicity 563 cylindricine F from 644
preventive effects of 563 genistic acid 259
Chitosan inhibitory effects 561 Clove buds 259
on gastrointestinal toxicity 561 protocatechuic acid in 259
on immunocompetent organ syringic acid in 259
toxicity 561 Cnidaria 646
on myelotoxicity of 5-FU 561 corals among 646
Cholesterol 373 gorgonians among 646
oxygenation of 373 hydrozoans among 646
Chorioptes mitQS 410 jellyfish among 646
attack on fetlocks 410 living species of 646
Chromodoris elisabethina 650 sea anemones among 646
icthyotoxic latrunculin A from 650 soft corals among 646
Chronic administration 182 Comantherin sulfate 719
of Ginkgo bilobah. 182 from Comantheria perplexa 719
Chrysanthemum cinerahfolium 384 Comatula pectinata 719
pesticide properties of 383 anthraquinones from 719
secondary metabolites from 384 Combined effects 562
Cinnamic acids 261 of 5-FU and chitosan 562
caffeic (3,4-dihydroxycinnamic) Complex I inhibitor 436,437
acid as 261 annonaceous acetogenins as 437
ferulic (3-methoxy-4-hydroxy) capsaicin as 436,437
as 261 deguelinas 436
/?-coumaric (4-hydroxy) acid monotetrahydrofiiranic derivatives
as 261 437
765
piericidin A as 436 effect on central nervous system

rotenoloneas 436 315
rotenone as 436 Crustacea 382
tephrosinas 436 class of 382
cw-Communic acid 456 Cryptocin 123
/m/w-Communic acid 456 against phytopathogenic fimgi 123
ciS' and /rÂ^^-Communic acids 456 Cryptosporiopsis cf. quercina 122
Corallistine 688 from Tryptergyium wilfordii 122
from Corallistes fulvodesmus 688 from Sclerotinia sclerotiorum 123
X-ray crystallography of 688 Cucumaria echinata 720
Coronaridin 234 neuritogenic activity of 720
Coscinoderma mathewsi 672 Cucumariafrondosa 720
as protein phosphatase inhibitor 2,6-dimethylnonane-1 -sodium
672 sulfate from 720
1-methylherbipoline salts from 672 2,4,6-trimethyl-nonane-1 -sodium
serine protease inhibitors from 672 sulfate from 720
sesterterpeneshalisulfate-1 from Cucumariajaponica 595
672 cucumarioside-Ao-3 from 595
suvaninefrom 672 cucumarioside-Ai-2 from 595
total synthesis of 672 cucumarioside-A2-l from 595
Cowdria ruminantium 394 cucumarioside-A2-2 from 595
as rickettsial pathogens 394 cucumarioside-As from 595
Coxiella burnetii 394 cucumarioside-A4-2 from 595
in cattle 394 cucumarioside-A6-2 from 595
insheeps 394 cucumarioside-A7-l from 595
Crella sp. 706,707 cucumarioside-A7-2 from 595
crellastanin A from 706 Cucumaria lefevrei 596
crellastatins B-M from 707 from lefevreioside Ai from 596
Crella spinulata 708 from lefevreioside A2 from 596
benzylthiocrellidone from 708 from lefevreioside B from 596
dimedone unit of 708 from lefevreioside C from 596
Cribricellina cribraria sp. 619 Cucumariosides A7-3 593
P-carboline alkaloid from 619 from Stichopus chloronotus 593
carbolinefrom 619 from Thelenota ananas 593
1 -ethyl-4-methyl-sulfone-P- Cucumariosides A2-4 593
antimicrobial activity of 619 from Stichopus chloronotus 593
Crocetin 314 from Thelenota ananas 593
structure of 314 Cyanogen iodide (ICN) 336
Crocin 315 forcyanation 336
artificial pathway of 314 for cyanation of aromatic
Crocin effects 316,319 compounds 336
on LTP-blocking effects of ofalkenes 336
ethanol 316 Cycloartobiloxanthone 201
on TNF-a-induced cell 319 from moraceous plants 201
Crocus sativus 315 Cyclopiazonic acid 119
ethanol extract of 315 as calcium uptake inhibitor 119
from Penicillium cyclopium 119
766
Cycloshermilamine D 639 of2a-hydroxynagilactoneF 470

from Cystodytes violatinctus 639 of inumakilactone A 468
Cylindramide 139 of inumakilactoneB 468
cytotoxic activity of 139 ofkazinolB 227
from Halichondria cydindrata of I/ca«/a genus 65
139 ofLicania heteromorpha 65
Cymbastela hooperi 665 ofLicania michauxii 65
diterpene isothiocyanates from 665 oflicochalconeB 227
Cymbopogon citratus 398 of licoricidin 227
essential oils from 398 of 15-methoxycarbonyl
Cymbopogon nardus 398 nagilactoneD 468
essential oils from 398 of morusin 227
Cystodytes sp. from Fiji 638 ofnagilactone A 468
dehydrokuanoniamine B from 638 of nagilactone B 468
shermilamine C from 638 of nagilactone C 468
Cytochrome C 321,325 ofnagilactone D 468
from mitochondria 321 of nagilactone E 468
levels in PC-12 cells 325 ofnagilactone F 468
Cytoplasmic proteins 321 ofnagilactone G 468
inapoptosis 321 ofnorartocarpetin 227
Cytotoxic activity 65,217,220,227, ofpodolactoneE 470
468,470
of Antiaris toxicaria (Moraceae) Dakaira subovoidea 620
220 6//-anthra[ 1,9-6c]thiophene
ofantiaronel 227 derivatives 620
ofantiaroneJ 220 2,3-Dehydro-16-hidroxi-nagilactone F
ofantiaroneK 220 464
ofantiaroneL 220 from Podocarpus nagi 464
of artocarpusflavonoids 220 2,3-Dehydro-1-deoxy-nagilactone A 460
ofartoninA 220 from Podocarpus nagi 460
ofartoninB 220 Dehydromoracin C 210
ofartoninE 217,227,220 from moraceous plants 210
ofartoninH 220 2,3-Dehydro-nagilactone A 460
ofbroussoflavonolB 227 from Podocarpus nagi 460
ofbroussoflavonolC 227 Dehydroprenyl flavonoid 203
ofcucurbitacinB 65 from Artocarpus sîQZXQS 203
of cycloheterophyllin 220 from Asian Morus species 203
of 1 -deoxy-2a-hydroxy-nagilactone from Brosimopsis oblongifolia
A 468 203
of 3-deoxy-2a-hydroxynagilactone Demethylmoracin I 18
E 470 as aromatase inhibitor 17
of 2,3 -dihydro-16-hydroxypodolide Dendrodoa grossularia 633
and 16-hydroxypodolide 468 dendrodoine from 633
ofgancaoninR 227 Deoxy-2a-hydroxy-nagilactone A 458
ofglyasperin A 227 from Podocarpus nagi 458
of glycyrol 227 l-Deoxy-2p,3P-epoxynagilactone A 459
of heterophyllin 220 from Podocarpus nagi 459
767
3-Deoxy-2a-hydroxy-nagilactone E 463 Didemnum molle 630

from Ileostylus micranthus 463 comoramide A from 630
from Podocarpus nagi 463 comoramide B from 630
1-Deoxy-nagilactone A 459 cyclodidemnamide from 630
from Podocarpus nagi 459 mayotamide A from 630
3-Deoxy-nagilactone C 459 mayotamide B from 630
from Ileostylus micranthus 459 mollamide from 630
12a-Deoxytetracycline 373 Didemnum rodriguesi 632
hydroxylation of 373 alkaloid caledonin from 632
Dercitinsp. 686 Didemnum sp. 631,634
antitumor activity of 686 anti-human immunodeficiency
antiviral activity of 686 virus (anti-HIV) activity of 631
immunomodulatory properties of didemnaketal C from 631
686 didemnoline A from 634
violet acridine alkaloid from 686 didemnoline D from 634
Dermatophilus congolensis 394 polysaccharide kakelokelose from
skin infection of cattle by 394 631
Desholothurin A 592 2,3-Dihydro-16-hydroxypodolide 462
from Holothuriaforskali 592 from Podocarpus nagi 462
2-Desoxo-15a-methyl-14a-hydroxy-MFA Dihydrochalcone 234,271
347 aspalathin from 272
discovery of 347 from Dracaena loureiri 234
nematocidal activity of 347 in apple juice 271
2-Desoxoparaherquamide A 331 in cider 271
nematocidal activity of 331 in pomace 271
2-Desoxo-PHA 347 nothofagin from 272
discovery of 347 Dihydrodeoxy-nubilactone A 466
Detox 543 from Podocarpus saligna 466
for immunotherapy 543 Dihydromyr 3-rha 41
P-D-glc-nagilactoside B 460 from Licania licaniaeflora 41
from Podocarpus nagi 460 (3',4'-Dihydroxy)benzoylester 40
P-D-glc-nagilactoside D 460 from Licania pyrifolia 40
fxom Podocarpus nagi 460 (2S)-2',4'.Dihydroxy-2"-(l-hydroxy-l-
P-D-glc-nagilactoside E 460 methylethyl)-dihydrofiiro[2,3-
from Podocarpus nagi 460 A]flavanone 17
28-P-D-glucosyl ester 40 as aromatase inhibitor 17
from Licania licaniaeflora 40 2a,27-Dihydroxybetulinic aicd 40
from Licania pyrifolia 40 from Licania pyrifolia 40
Dibenzyltrisulfide 403 3 p,4p-Dihydroxypregn-5-en-20-3-
as oviposition inhibitor 403 sulfate 704
from Petiveria alliacea L. 403 5-pregnene skeleton of 704
Didemnum chartaceum 631 from Stylopus australis 704
lamellarin G 8-sulfate from 631 2a,3a-Dihydroxyurs-12-ene-28-oic 40
lamellarin sulfates from 631 from Licania pyrifolia 40
lamellarin B from 631 Diketopiperazinecvc/(9-(L-proline-L-
lamellarin C from 631 thioproline) 692
lamellarin L from 631
768
bacterial origin of 692 Doxorubicin plus chitosan 560

from ledania ignis 692 tumor growth inhibition by 560
Diketopiperazines 362 Ds-Penaustroside A 599
containing dioxepinoindole ring from sea cucumber 599
362 Ds-Penaustroside B 599
(3Z)-4,8-Dimethyinon-3-en-1-yl sodium from sea cucumber 599
sulfate 721 Dysideaavara 668
as sulfated alkene 721 melemeleone A from 668
from Microcosmus vulgaris 721 spectroscopic analyses of 668
from Ophiocoma echinata 721 Dysidea herbacea 665,666
Dioxepinooxindole ring system 360 dysideathiazole from 665
construction of 360 13-demethylisodysideninfrom 666
Diplosoma sp. 636 13-demethyldysideninfrom 666
diplamine from 636 13-demethylisodysideninfrom 666
Discodermia 138,679 13-demethyldysideninfrom 666
antifungal activity of 139 isodysidenir from 665
cytotoxic activity of 139,679 9-monodechloro-13-demethyl-
deepwater species of 679 isodysidenin from 666
polydiscamide A from 679 1 l-monodechloro-13-demethyl-
Diuretic activity 200 isodysidenin from 666
of genus Morw^ 200 polychlorinated peptides from 665
DNA topoisomerase I inhibitors 621 Dysidea sp. 667,668
ceramide 1-sulfates as 621 15-acetylthioxyfiirodysinin lactone
p-D-nagilactoside C 460 from 667
from Podocarpus nagi 460 P-adrenoreceptor agonist from 668
Docosahexaenoic acid 565 barbaleucamide A from 668
in fish oils 565 benzothiazole S1319 from 668
Dolastatin3 648 dysideaproline A from 668
as antineoplastic agent 648 dysideaproline B from 668
from Dolabella auriculaha 648 dysideaproline C from 668
sequence of 648 dysideaproline D from 668
synthesis of 648 dysideaproline E from 668
Dolastatin 10 145,649 dysideaproline F from 668
antiproliferative activity of 145 from Okinawa 668
as antineoplastic agent 649 proline-derived analogues of
from Dolabella auricularia 649 dysideninfrom 668
Dolastatin 15 145 Dysideathiazole A 667
antiproliferative activity of 145 as polychlorinated amino acid
Doris verrucosa (nudi branch) 651 derivative 667
9-(5-deoxy-5-methylthio-P-D- X-ray analysis of 667
xylofiiranosyOadenine from 651 Dysideathiazole B 667
digestive glands of 651 as polychlorinated amino acid
Doxorubicin 559,560,562 derivative 667
as cancer chemotherapy drug 559 X-ray analysis of 667
gastrointestinal toxicity by 562 Dysideathiazole C 667
immunotoxicity by 562 as polychlorinated amino acid
myelotoxicity by 562 derivative 667
X-ray analysis of 667
769
Dysideathiazole D 667 Eiocosapentaenoic acid 565

as polychlorinated amino acid in fish oils 565
derivative 667 Eliamid 146
X-ray analysis of 667 cytostatic activity of 146
Dysideathiazole E 667 fiingicidal activities of 146
as polychlorinated amino acid nematocidal activity of 146
derivative 667 Ellagic acid 280
X-ray analysis of 667 structure of 280
Dysidin 142 Engorged toxicity 400
antifeedant activity of 142 from Hibiscus rosa-sinensis 400
from Dysidea herbacea 142 from Nicotiana tabacum 400
immunosuppressant activity from Ocimum micranthum 400
of 142 from Quassia simarouba 400
moUuscidal activity of 142 from Ricinus communis 400
from Salvia serotina 400
Echinoclasterol sulfate 705 from Spigelia anthelmia 400
as antifungal 705 from Symphytum officinale 400
as cytotoxic steroid 705 from Stachytarpheta jamaicensis
from Echinoclathria subhispida 400
705 Endotoxins 518
phenethylammonium salt 705 chemistry of 518
Echinoderms 716 Enzymic activity 257
classes of 716 of plant polyphenols 257
histidine derivatives from 716 (-)-Epicatechin 41,289
living species of 716 from chocolate 289
metabolites of 716 from Licania pittieri 41
Echinodictyum sp. 697 3-£/^/-nagilactone C 459
echinosulfonic acid A from 697 from Podocarpus nagi 459
echinosulfonic acid B from 697 3-£/7/-paraherquamide A 354
echinosulfonic acid C from 697 anthelmintic activity of 354
echinosulfone from 697 semi-synthesis of 354
Economic viability 345 Epingaione 402
of 14p-methyl-14-a-hydroxy- growth regulatory activities of 402
MFA 345 Epipolasis sp. 699
of 15a-methyl-14a-hydroxy- halistanol sulfate A from 699
MFA 345 halistanol sulfate B from 699
Ecteinascidia turbinata 640,641 halistanol sulfate C from 699
antitumor activity of 640 halistanol sulfate D from 699
ecteinascidins from 640,641 halistanol sulfate E from 699
EGb 185 Epipolasis kushimotoensis 662
activity against MPTP 184 P-phenylethylamine of 662
effects on neuroendocrine system epipolasins A of 662
185 epipolasins B of 662
effects on phospholipid metabolism epi'StWoWmC 460
185 from Podocarpus nagi 460
protective activity of 184 Epitheaflavic acid 274
formation of 274
770
2p,3P-Epoxypodolide 462 used as insecticides 390

from Podocarpus nagi 462 Estrogen-like activity 227
Equisetin 121 of phenols from moraceous plants
antibiotic activity of 121 227
cytotoxicity of 121 of Glycyrrhiza s^Qcits 227
from Fusarium equiseti 121 (25)-Euchrenone 17
HIV inhibitory activity of 121 as aromatase inhibitor 17
Ergosterol 570,572 Eudistoma cf. rigida 635
as antitumor substance 570 iejimalide C from 635
effect on neovacularization 572 iejimalide D from 635
from Agaricus blazei 570 Eudistoma gilboverde 634
Erythroskyrine 134 methyleudistomidin C 634
from Penicillium islandicum 134 Eudistoma glaucus 634
Essential oil 415,390,408,421,426 eudistomidin C from 634
against sarcoptic mange 415 Eudistoma olivaceum 633
against Varroajacobsoni 390 P-carboline alkaloids from 633
benzyl benzoate in 415 A^(10)-methyleudistomin E
Cedrus deodara (Pinaceae) 415 from 633
from Anonas^p. 392 eudistomin K from Ritterella
from Artemisia absinthium 426 sigillinoides 633
from Artemisia tridentata 408 Eudistoma sip. 637
from Calocedrus decurrens 408 desmethylvaracin from 637
from Chamaecyparis lawsoniana Eupentactafraudatrix 598
(Cupressaceae) 408 from calcigeroside B 598
from Chamaecyparis nootkatensis from calcigeroside Ci 598
408 from calcigeroside D, 598
from Chenopodium spp. 392 from cucumarioside G2 598
from Foeniculum vulgare 408 Euryspongia 667
from Juniperus occidenatalis 408 15-acetylthioxyfiirodysin from 667
from Juniperus occidenatalis 408 Euscaphic acid 40
from Salvia officinalis 392 from Licania pyrifolia 40
from Sequoia sempervirens 408 Euthyroides episcopalis 621
from Tanacetum vulgare 426 euthyroideones from 621
from Thuja plicata 408 Expectorant activity 200
from Thymus vulgaris 392 of genus Morw^ 200
lethality tests of 392
microwave assisted process (MAP) Fabaceae plants 435
427 isoflavonoids from 435
of citronella 421 Fatty acid components 565
of eucalyptus 421 of carp oil 565
of Juniperus viriginiana 408 of tuna oil 565
of Salvia officinalis L. 390 Ferulicacid 262
of spearmint 421 in citrus juices 262
of tea tree 421 in cereal brans 262
of Thymus vulgaris L. 390 in coffee 262
ofwintergreenoils 421 in sugar beet fibre 262
used as bactericides 390
used as fungicides 390
771
Fijian sample 337 5-Fluorouracil (5-FU) 562,559

of Lissoclinum vareau 631 as cancer drug 559
varacin from 637 gastrointestinal toxicity by 562
Fijian tunicate Polycitorella mariae 634 immunotoxicity by 562
citorellamine from 634 levels in mice plasma 567
Flavan-3-ols 273 myelotoxicity by 562
(+)-catechin 273 5-FU plus carp oil 566,568
epicatechin 273 effects on tumor growth 566
Flavanols 272 in sarcoma 180-bearing mice 566
structure of 272 5-FU plus tuna oil 566,568
Flavanones 270 effects on tumor growth 566
eriodictyol 270 in sarcoma 180-bearing mice 566
hesperetin 270 Forbesin 718
hesperidin 270 as sulfated glycolipid 718
in chickpeas 271 eicosane-1,16-disulfate from 718
in citrus fruits 271 from Asterias forbesi 718
in cumin 271 from Asterias vulgaris 718
in hawthorn berry 271 from Henhcia laeviuscula 118
in licorice 271 isorhodopti lometrin-2' -sulfate
in peppermint 271 from 718
in rowanberry 271 Formic acid 384
isosalipurpurin (chalcone) as 271 asmiticide 384
naringenin 270 FrondosideB 593
naringin 270 from Stichopus chloronotus 593
phloretin (dihydrochalcone) from Thelenota ananas 593
as 271 FrondosideC 599
Flavones 266,267,285 from sea cucumber 599
absorption of 285 Fungal metabolites 66,473,475,476
apigenin 267 from Aspergillus flavus IFM
as plant tissue color 267 41934 476
chrysin 267 from Aspergillus fumigatus IFM
diosmetin 267 41243 476
isoorientin 267 from Aspergillus niger H7160B
isovitexin 267 476
lutcolin 267 from Candida albicans 1463D 476
orientin 267 from Candida albicans ATCC
pinocembrin 267 90028 476
vitexin 267 from Candida albicans ATCC
Flavonols 264,265,284 90029 476
from onions 284 from Candida dubliniensis CBS
glycosides of 284 7987 476
inkaempferol 265 from Pichia anomala IFM 47182
in plant foods 265 476
inquercetin 265 from Trichophyton mentagrophytes
structure of 264 KCH1155 476
Flavonoids 208 from Candida guilliermondii IFM
from Morus alba root bark 208 46823 476
772
from Candida kefyr IFM 46921 isorhamnetin-3-O-rutinoside from

476 167
from Candida parapsilosis IFM kaempferol from 167
46863 476 kaempferol-3-0-(2"-0-
from Candida tropicalis IFM glucosyl)rhamnoside from 167
46816 476 kaempferol-3-0-(6"'-0-/7-
from Criptococcus neoformans coumaroyl-2"-0-glucosyl)
ATCC 90112 476 rhamnoside from 167
from Exophiala dermatitidis 476 kaempferol-3.0-[6"'-0-{/7-(7""-0-
glucosyl)coumaroyl} -2"-0-
Gallic acid 280 glucosyl]rhamnoside from 167
structure of 280 kaempferol-3-0-[a-rhamnosyl-
GancaoninR 234 (1 ->2)-a-rhamnosyK 1 ->6)]-p-
from Glycyrrhiza uralensis 234 glucosidefrom 167
Gastrointestinal nematodes 349 kaempferol-3-O-glucoside from
Haemonchus contortus as 349 167
Trichostrongylus colubriformis as kaempferol-3-O-retmoside
349 from 167
Gastrointestinal toxicity 559,560 kaempferol-7-O-glucoside
ofbilobalide 171 from 167
of5-fluorouracil(5-FU) 560 luteolinfrom 167
of chitosan 559 luteolin-3'-(9-glucoside from 167
offish oils 559 4-0-methylpyridoxine from 172
Gel formulation 385 myricetinfrom 167
in Varroajacobsoni 385 myricetin-3-O-rutinoside from 167
8-Geranylapigenin 234 quercetinfrom 167
synthesis of 234 quercetin-3-0-(2"-0-glucosyl)
Ginkgo bilobah, 165,170 rhamnoside from 167
3 '-O-methylmyricetin-3-O- quercetin-3-0-(6"'-0-/7-coumaroyl-
rutinoside from 167 2"-0-glucosyl)rhamnoside
5' -methoxybilobetin from 166 from 167
amentoflavone from 166 quercetin-3-0-[6'"-0-{p-(7""-0-
apigeninfrom 167 glucosyl)coumaroyl} -2"-0-
apigenin-7-O-glucoside from 167 glucosyl]rhamnoside from 167
bilobetinfrom 166 quercetin-3 -0-[6"' -0-/7-coumaroy 1-
biological activities of 165 2"-0-glucosyl)rhamnosyl-7-0-
chemistry of 165 glucosidefrom 167
delphidenon from 167 quercetin-3-0-[a-rhamnosyl-
ginkgetinfrom 166 (1 ->2)-a-rhamnosyl-( 1 ->6)-P-
ginkgolide A from 170 glucosidefrom 167
ginkgolide B from 170 quercetin-3-0-glucoside from 167
ginkgolide C from 170 quercetin-3-O-rhamnoside from
ginkgolide J from 170 167
ginkgolide M from 170 quercetin-3-0-rutinoside (rutin)
isoginkgetin from 166 from 167
isorhamnetin from 167 sciadopitysin from 166
isorhamnetin-3-O-glucoside from
167
773
Glabrene 234 liquiritigenin from 239

from Glycyrrhiza glabra 234 medicarpin from 206
Glossodoris quadricolor 650 1 -methoxyphaseollidin from 206
latruncuiin B from 650 4'-0-methylglabridin from 206
P-Glucuronidase 283 shinpterocarpin from 206
Glucuronides 288 uses as licorice 204
in urine 288 Glycyrrhiza spQCiQS 223
Glycosidase activity 281 bioactive phenolic compounds
of polyphenols 281 from 223
Glycoside naringin 288 Glycyrrhiza uralensis 242
in urine 288 4'-0-methylglabridin from 242
Glycyrol 234 1-methoxyphaseollidin from 242
from Glycyrrhiza uralensis 234 3-(9-methylglycyrol from 242
Glycyrrhiza glabra 239,204,206 6,8-diprenylorobol from 242
8-(y,Y-dimethyallyl)-wighteone dihydroisoflavone A from 242
from 206 gancaonin I from 242
3'-(y,y-dimethylallyl)-kievitone gancaonol C from 242
from 206 gancaonol from 242
edudiol (3,9-dihydroxy-l- glycyrin from 242
methoxy-2-prenylpterocarpan) hispaglabridin A from 242
from 206 isoglycyrol from 242
euchrenone from 206 isolicoflavonol from 242
flavanonefrom 239 licoricone from 242
gancaonin G from 206 shinflavanone from 242
gancaonin H from 206 vestitol from 242
glabrene from 206 Glycyrrhiza glabra var. 205
glabridin from 206 glabrene from 205
glabrone from 206 glabridin from 205
glisoflavanone from 206 glabrol from 205
glyasperin A from 206 3-hydroxyglabrol from 205
glyasperin C from 206 pyranoisoflavan from 205
glyasperin D from 206 Gymnocrinus richeri 719
glycyrrhizic acid from 239 gymnochrome C from 719
glyinflanin G from 206 Gynandropsis gynandra 395
glyinflanin K from 206 repellent properties of 395
hispaglabridin A from 206
3-hydroxyglabrol from 206 Haemonchus contortus 342
3-hydroxyparatocarpin C from 206 Halenaquinol sulfate 677
isoderone semilicoisoflavone B absolute stereochemistry of 677
from 206 as pentacyclic hydroquinone 677
kanzonol U from 206 as yellow pigment 677
kanzonolVfrom 206 from Xestospongia sapra 677
kanzonol W from 206 Halichondria species 659,660
kanzonol X from 206 diterpenoid from 659
kanzonol Y from 206 from Marshall islands 660
kanzonol Z from 206 guai-6-ene skeleton of 660
licorice-saponin G2 from 239 sesqui- and diterpenoids from 659
sesquiterpenoid from 659
774
Halichondria sponge 663 Harzianic acid 135

isothiocyanates from 663 antibiotic activity of 136
Halidona nigra 684 from Trichoderma harzianum 136
from Papua New Guinea 684 Hematologic toxicity 559
haligramides A of 684 with immunosuppression 559
Halidona sp. 706 with leukopenia 559
haliclostanone sulfate from 706 Hepatoprotective activity 257,293
Halicylindramide A 680 of plant polyphenols 257,293
as antifimgal activity 680 Heptaprenylhydroquinone derivative 676
as cytotoxic activity 680 from Irciniafasciculata 676
from Halichondria cylindrata 680 Herbal formulation AV/EPP/14 401
Halicylindramide B 680 Acorus calamus m 401
as antifimgal activity 680 against larvae 402
as cytotoxic activity 680 against nymphs of Boophilus
from Halichondria cylindrata 680 microplus 402
Halicylindramide C 680 Azadirachta indica in 401
as antifimgal activity 680 Cedrus deodara in 402
as cytotoxic activity 680 Eucalyptus globulus in 402
from Halichondria cylindrata 680 Pongamia pinnata in 401
Halipanicine 661 Hexaprenyl-hydroquinone sulfate 676
from Halichondria panicea 661 as H^/K'^-ATPase inhibitor 676
stereochemistry of 661 from Dysidea 676
total synthesis of 661 Himax 416
Halistanol disulfate B 699 acaricidal action of 416
from Pachastrella sp. 699 against demodectic mites 416
in the endothelin converting against psoroptic mites 416
enzyme (ECE) assay 699 against sarcoptic mites 416
stereochemistry of 699 Cedrus deodara of 416
Halistanol sulfate 698 Polyalthia excessa of 416
antimicrobial activity of 698 Polyalthia longifolia of 416
as tris-sodium sulfate salt 698 Hinokiresinol 234
from Halichondria cf. moorei 698 from Chamaecyparis obtusa 234
from Topsentia sp. 698 Histamine release inhibitor 257,293
hemolytic activity of 698 polyphenols as 257,293
ichthyotoxic activity of 698 HMBC and NOB correlations 26
pp60v-src protein tyrosine kinase of 5,7,2' ,4 '-tetrahydroxy-3-
inhibition activity 698 geranylflavone 26
HallactoneA 458 ofisogemichalconeC 25
from Podocarpus hallii 458 Holostanol 588
HallactoneB 462 structure of 588
from Podocarpus hallii 462 Holothuria pervicax 720
from Podocarpus polystachyus ganglioside HPG-8 from 720
462 Holothurinoside A 592
from Podocarpus sellowii 462 from Holothuria forskali 592
Halocynthia papulosa 642 Holothurinoside C 592
6-methylheptyl sulfate from 642 from Holothuria forskali 592
(£)-oct-5-enyl sulfate from 642 Holothurinoside D 592
from Holothuria forskali 592
775
HoIothurinA 592 salicylic acid as 259

Holothuria leucospilota 592 syringic acid as 259
Homoisoflavone 234 vanillic acid as 259
from Dracaena loureiri 234 2a-Hydroxybetulinic acid 40
Honey 385 from Licania pyrifolia 40
formic acid in 385 6(3-Hydroxybetulinic acid 40
Homer-Wadsworth-Emmons from Licania pyrifolia 40
condensation 72 lla-Hydroxybetulinic acid 40
ofstannate 72 from Licania pyrifolia 40
House dust mites 417 Hydroxycinnamic acid derivatives 260
Actinodaphne lancifolia against (2-Hydroxyethyl)dimethylsulfoxonium
419 chloride 716
benzyl benzoate against 418 from Alcyonidium gelatinosum
Cinnamomum camphora against 716
419 ofDogger Bank Itch 716
Cinnamomum japonicum against from Theonella aff mirabilis 716
419 3p-Hydroxyholost-7-ene aglycones 594
Dermatophagoides farinae against Cucumariajaponica from 594
417 14a-Hydroxymarcfortine A 338
Dermatophagoides pteronyssinus synthesis of 338
against 417 15a-Hydroxymarcfortine A 338
Euroglyphus maynei against 417 synthesis of 338
Lindera umbellata against 419 16a-Hydroxymarcfortine A 338
Melaleuca acacoides dLgSLinst 419 synthesis of 338
Melaleuca symphycarpa against 3 '-[y-Hydroxymethyl-(£)-y-
419 methylallyl]-2,4,2',4'-
Neolitsea sericea against 419
tetrahydroxychalcone 11 '-0-
Persia thunbergii digmnst 419
coumarate 16
Taiwania cryptomerioides
as aromatase inhibitor 16
against 418
14p-Hydroxy-MFA 340
tannic acid against 418
synthesis of 340
treatment of 418
14a-Hydroxy-MFA 341
8-Hydroxy eriodictyol 41
from Licania pyrifolia 41 synthesis of 341
8-Hydroxy luteolin 41 3p-Hydroxy-nagilactone A 459
from Licania pyrifolia 41 from Podocarpus nagi 459
8- Hydroxy naringenin 41 15-Hydroxy-nagilactoneD 459
from Licania licaniaeflora 41 from Podocarpus nagi 459
from Licania pyrifolia 41 16-Hydroxy-nagilactoneE 463
2a-Hydroxy ursolic acid 40 from Podocarpus nagi 463
from Licania carii 40 1 P-Hydroxy-nagilactone F 463
from Licania pyrifolia 40 from Podocarpus nagi 463
Hydroxybenzoic acid derivatives 259 3p-Hydroxy-nagilactoneF 463
benzoic acid as 259 from Podocarpus nagi 463
gallic acid as 259 2a-Hydroxy-nagilactone F 464,474
4-hydroxybenzoic acid as 259 against Saccharomyces cerevisiae
isovanillic acid as 259 474
protocatechuic acid as 259 from Ileostylus micranthus 464
776
16-Hydroxypodolide (salignone H) 462 phaechromogenes var

from Podocarpus saligna 462 ikaruganensin 138
Hyperparasites of tick 404 Immunocompetent organic toxicity 560
Beauveria bassiana as 404 of5-fluorouracil(5-FU) 560
Bacillus thuringiensis var. kurstaki /« v//ro activity 470,467
as 404 ofmilanjilactone A 470
Cedecea lapagei as 404 ofmilanjilactoneB 470
Metarhizium anisopliae as 404 ofnagilactoneG 470
Verticillium lecanii as 404 ofnagilactoneF 470
Hypotensive action 200,209,207 of podolactones 467
of isoprenylatedflavonoids 207 Industrial syntheses 71
ofkuwanonG 209 of vitamin A 71
ofkuwanonH 200,209 Inhibitory activity 481
of Morus alba 200 of inumakilactone A 481
of A/orw^ species 207 of inumakilactoneB 481
of mulberrofuran C 209 of inumakilactone C 481
ofmulberrofuranF 209 ofnagilactone A 481
of mulberroftiran G 209 of nagilactone B 481
lO-Hyroxycoronaridine 234 ofnagilactoneG 481
from Tabernaemontana ofpodolactone A 481
penduliflora 234 of podolactone B 481
Hyrtiomanzamine 691 of podolactone C 481
from Hyrtios erecta 691 of podolactone D 481
immunosuppressive activity of 691 ofpodolactone E 481
Hysistozoa fasmeriana 635 Insecticidal activity 477
fasmerianamine A from 635 of 14-epi-ponolactone A 477
fasmerianamine B from 635 ofhallactone A 477
/ra/75'"5 -hydroxy-4-(4' -hydroxy-3' - of hallactone B 477
methoxyphenyl)-4-(2"-imida- ofnagilactone A 477
zolyl)-l,2,3-trithianefrom 635 ofnagilactoneG 477
of nagilactone D 477
lanthella basta 695 ofnagilactone E 477
as Ca^^-channel agonists 695 ofpodolactone A 477
lantheranA 695 ofpodolactone C 477
as NaVK^-ATPase inhibitor 695 of podolide 477
from/a«//ig//a spp. 695 ofsellowinA 477
Ibisterol sulfate 700 Integramycin 147
against HIV-1 700 from Actinoplanessxi. 147
from deepwater Topsentia sp. 700 as HIV-integrase inhibitor 147
Ichthyotoxic diacylglycerol umbraculumin Inumakilactone A 461
C 650 from Podocarpus philippinensis
from Umbraculum mediterraneum 461
650 from Podocarpus macrophyllus
stereochemistry of 650 461
Ikarugamycin 138 Inumakilactone A glucoside 462
antiprotozoal activity of 138 from Podocarpus philippinensis
from Streptomyces 462
777
from Podocarpus macrophylus in soy flour 269

462 in soy sauce 269
Inumakilactone B 461 in soya bean 269
from Podocarpus macrophyllus in soya milk 269
461 in textured soya protein 269
from Podocarpus neriifoiius 461 intofu 269
from Podocarpus polystachyus in tofti yoghurt 269
461 metabolism of 287
Inumakilactone C 466 oestrogenic activity of 268
from Podocarpus macrophyllus Isogemichalcone C 16
466 as aromatase inhibitor 16
Inumakilactone D 466 Isolicoflavonol 17
from Podocarpus macrophyllus as aromatase inhibitor 17
466 Isoprenylated flavonoids 199,203,244
Inumakilactone E 458 chemistry of 244
from Podocarpus macrophyllus biological activity of 199
458 effect on testosterone 5a-reductase
from Podocarpus poly stachyus 244
458 from Cudrania tricuspidata 203
Irciniafasiculata 673 from Cudrania cochinchinenesis
fasiculatin for 673 203
Ircinia spinulosa 676 from Glycyrrhiza sp. 199
brine shrimp toxicity of 676 from medicinal plants 199
2-prenylhydroquinones of 676 kuwanon C as 245
Ircinia variabilis 673 kuwanonEas 245
22-0-sulfates of palinurin from kuwanon G as 245
673 kuwanon Has 245
sulfate esters from 673 kuwanon Las 245
Irciniasulfonic acid 714 morusin as 245
from/rc/wa sp. 714 mulberrofuran A as 245
reverses multidrug resistance 714 mulberrofiiran G as 245
Isobavachalcone 234 oxydihydromorusin as 245
from Morus cathayana 234 Isothiocyanates 658
Isobavachin 234 as marine metabolites 658
from Glycyrrhiza pallidiflora 234 asterpenes 658
Isoflavones 268,269,286,287 from Axinellida order 658
acetyldaidzein 269 from Halichondria genera. 658
acetylgenistin 269 from Halichondrida order 658
aglycones 269 from Lithistida 658
bioavailibity of 286 as precursor of formamido group
daidzein 269 658
genistein 269 5-Isothiocyanatopupukeanane 661
glycosides 269 from Axinyssa species 661
in green split peas 269 Ivermectin 396
inmiso 269 as macrolide antibiotic 396
in plant foods 269 from Streptomyces avermitilis 396
in soy foods 287
in soy cheese 269
778
Janolusimide 146 Ketalized Diels-Alder type adducts 203

bom Janolus cristatus 146 sorocenol B as 203
neurotoxic lipophilic activity of soroceal as 203
146 Koreoside 599
Jaspisin 697 from Cucumaria koraiensis 599
embryos of 697 KuwanonC 210
sea urchin hatching inhibition by from moraceous plants 210
697 KuwanonE 210
from moraceous plants 210
Kae 3-(2"-xyl)rha 41 KuwanonG 201
from Licania pyrifolia 41 from moraceous plants 201
from Licania licaniaeflora 41 KuwanonH 201.222,223
Kae 3-(6"-/?-coum)glc 41 as bombesin receptor antagonists
from Licania densiflora 41 223
Kae3-ara 41 as GRP-induced DNA synthesis
from Licania licaniaeflora 41 inhibitor 222
from Licania pyrifolia 41 from moraceous plants 201
Kae3-rut 41 KuwanonL 210
from Licania apetala var. from moraceous plants 210
apetala 41 KuwanonM 210
Kaempferol 41 from moraceous plants 210
from Licania pyrifolia 41
KazinolA 17 Latrunculia magnifica 689
antioxidant activity of 17 6,7-epoxy-latrunculin 689
as tyrosinase inhibitor 17 latrunculin M from 689 689
as platelet aggregation inhibitor 17 Lissoclinum vareau 638
KazinolB 17 varamine A from 638
as cyclooxygenase 17 Lactacystin 112
as platelet aggregation inhibitor 17 biosynthetic assembly of 112
KazinolC 210 Lactic acid 386
from moraceous plants 210 in honey 386
KazinolE 210 Lancet-shaped follicular mites 410
from moraceous plants 210 as cause of demodectic mange 410
KazinolF 16,210 of Demodex gQUXxs 410
antioxidant activity of 16 Latrunculia 685
as tyrosinase by inhibitor 16 Discorhabdin A from 686
from moraceous plants 210 Latrunculin A 688
KazinolJ 210 as ichthyotoxins 688
from moraceous plants 210 from Latrunculia magnifica 688
KazinolM 210 2-thiazolidinone moiety of 688
from moraceous plants 210 Lavandula angustifolia Miliar 411
KazinolN 210 essential oil of 411
from moraceous plants 210 constituents of 411
KeenamideA 652 LC/ESI-MS analyses 290
as cyclic hexapeptide 652 of sulfated tea catechins 290
as cytotoxic agent 652 of urinary glucuronid 290
from Pleurobranchus forskalii 642 Lepidium sativum 400
in tick toxicity 400
779
Licania apetala var. apetala (E. May) myricetin 3 ',5 '-dimethylether-3-0-

Fritsch 58 P-D-glucopyranoside from 49
kaempferol 3-(9-rutinoside from myricetin 3'-methylether-3-0-P-D-
58 galactopyranoside from 49
myricetin 4'-(9-a-L- myricetin 3-(9-(2"-0-a-L-
rhamnopyranoside from 58 rhamnopyranosyl)-a-L-
phytochemical studies of 38 rhamnopyranoside from 49
quercetin 3-0-rutinoside from 58 myricetin 4'-methylether-3-0-a-L-
quercetin 3-0-a-L- rhamnopyranoside from 49
arabinopyranoside from 58 naringenin 8-hydroxy-4'-methyl
quercetin 3-0-a-L- ether from 49
rhamnopyranoside from 58 phytochemical studies of 38
quercetin 3-0-P-D- Licania heteromorpha var. heteromorpha
galactopyranoside from 58 Bentham 38,53
taxifolin 3-(9-a-L- alphitolic acid from 53
rhaninopyranoside from 58 betuiinic acid from 53
Licania carii Cardozo 38,42 myricetin 3,4'-di-(9-a-L-
betuiinic acid from 42 rhamnopyranoside from 53
2a-hydroxyursolic acid from 42 myricetin 3-0-a-L-
maslinic acid from 42 rhamnopyranoside from 53
myricetin 3'-methyl-3-0-rutinoside myricetin 3-0-P-D-
from 42 galactopyranoside from 53
myricetin 3-0-(2"-0-P-D- myricetin 4'-methylether-3-0-a-L-
xylopyranosyl)-a-L-rhamno- rhanmopyranoside from 53
pyranoside from 42 myricetin 4'-methylether-3-0-P-D-
myricetin 3-0-rutinoside from 42 galactopyranoside from 53
myricetin 3-0-P-D- myricetin 4'-methylether-3-0-P-D-
galactopyranoside from 42 glucopyranoside from 53
myricetin 3-0-p-D- myricetin 7-methylether 3,4'-di-0-
glucopyranoside from 42 a-L-rhamnopyranoside from 53
phytochemical studies of 38 3P-0-cw-/?-coumaroyl alphitolic
quercetin 3-0-(2"-0-P-D- acid from 53
xylopyranosyl)-a-Z- 3P-0-c/5'-/7-coumaroyl maslinic
rhamnopyranoside from 42 acid from 53
quercetin 3-O-rutinoside from 42 3 P-(9-cw-/7-coumaroy l-2a-
quercetin 3-(9-P-D- hydroxy-urs-12-en-28-oic acid
galactopyranoside from 42 from 53
quercetin 3-0-p-D- 3P-0-/ra/75-/7-coumaroyl alphitolic
glucopyranoside from 42 acid from 53
P-sitosterol 3-(9-P-D- 3P-(9-rra/M'-/7-coumaroyl maslinic
glucupyranoside from 42 acid from 53
ursolic acid from 42 3 P-0-rra«5-/7-coumaroyl-2a-
Licania densiflora YAtxrAioonXQ 38,49 hydroxy-urs-12-en-28-oic acid
3 ',4'-dimethylmyricetin-3-0-p-D- from 53
glucopyranoside from 49 phytochemical studies of 38
myricetin 3' ,5' -dimethylether-3 -O- Licania intrapetiolaris Spruce
a-L-rhamnoside from 49 (ex. Hook) 38
780
cucurbitacin B from 59 2a-hydroxyursolic acid from 45

intrapetacin A from 59 lupeol from 42
intrapetacin B from 59 maslinic acid from 45
phytochemical studies of 38 oleanolic acid from 44
Licania licaniaeflora (Sagot) Blake 38,56 phytochemical studies of 38
arjunic acid 28-P-D-glucosyl ester p-sitosterol 3-0-P-D-
from 57 glucopyranoside from 45
betuiinic acid from 57 P-sitosterol from 42
maslinic acid from 57 tormentic acid 28-P-D-
olean-12-ene-2a,3P-diolfrom 57 glucopyranosyl ester from 45
oleanolic acid 3-O-a-L- tormentic acid from 45
arabinopyranoside from 57 2,3,27-trihydroxylup-12-en-28-oic
oleanolic acid from 57 acid 3-(3'-4'-dihydroxybenzoyl
pomolic acid from 57 ester) from 45
phytochemical studies on 38 ursolic acid 3-0-a-L-
tormentic acid 28-P-D-glucosyl arabinopyranoside from 45
ester from 57 ursolic acid from 44
ursolic acid from 57 uvaolfrom 44
Licania pittieri FrmiCQ 38,42 Licorice 204,205
catechinfrom 42 anti-hepatitic principles from 205
epicatechin from 42 antitussives from 205
phytochemical studies of 38 anti-ulcer compounds from 205
quercetinfrom 42 dihydrophenanthrene from 205
quercetin 3-0-a-L- dihydrostilbenes from 205
arabinopyranoside from 42 flavor from 205
quercetin 3-0-a-L- Glycyrrhiza species from 205
rhamnopyranoside from 42 isoliquiritigenin from 205
quercetin 3-O-P-D- isoliquiritin from 205
galactopyranoside from 42 liquiritigenin glycosides from
quercetin 3-0-P-D- 205
glucopyranoside from 42 liquiritin apioside from 205
ursolic acid from 42 liquiritin from 205
Licania pyrifolia GnsobdLch 38,42 sweetening agents from 205
a-amyrin from 42 Linalool 413
betulin from 42 analysis of 413
betuiinic acid from 44 efficacy of 413
2,3-dihydroxylup-12-en-28-oic Liouvilloside A 608
acid 3-(3',4'-dihydroxybenzoyl massfragmentationof 608
ester) from 44,45 Lipid A 518,519,523,525,526,529,534,
2a,3a-dihydroxyurs-12-ene-28-oic 537,538
acid from 45 acute effects of 523
euscaphic acid 28-p-D- antitumor activities of 518
glucopyranosyl ester from 45 association with
immunomodulators 534
euscaphic acid from 45
effect on acquired immunity 529
1 la-hydroxybetulinic acid
effect on cytokine production 526
from 44
effect on innate immunity 525
6P-hydroxybetulinic acid from 44
781
from Escherichia coli 519 Liquiritigenin 234

from Salmonella typhimurium 519 from Glycyrrhiza species 234
in cancer vaccination 537 Lissoclinum bistratum 628
to treat tumor-bearing animals 538 bistratamide A from 628
treatment with 534 bistratamide B from 628
Lipid A DT 5461 534 bistratamide C from 628
for C38 colon carcinoma 535 bistratamide D from 628
for Lewis lung carcinoma 535 Lissoclinumjaponicum 637
for MH134 hepatoma 535 dimethyl-5-(methylthio) varaein
for MM46 mammary carcinoma from 637
535 Lissoclinum patella 622,623,625,626,
for murine Meth A fibrosarcoma 627,629
535 ascidiacyclamide from 622
treatment with 534 heptapeptide ulicyclamide from
Lipid A OM-174 536 622
antitumor activity of 536 patellamide A from 623
treatment with 536 patellamide B from 623
Lipid A ONO-4007 535 patellamide C from 623
treatment with 535 patellamide E from 627
Lipid A tolerance 521 patellamide F from 627
by anti-LPS antibodies 522 patellamide G from 627
a-Lipomycin 135 patellazole A from 625
from Staphylococcus aureofaciens patellazole B from 625
135 patellazole C from 625
as antibacterial agent 135 patellins 1-6 from 629
p-Lipomycin 135 prepatellamide A from 626
from Staphylococcus aureofaciens tawicyclamide A from 629
135 tawicyclamide B from 629
as antibacterial agent 135 trunkamide A from 629
Lipopolysaccharides (LPS) 518,520,521, ulithiacyclamide B from 623
523,528,533,539 ulithiacyclamide from 622
acute effects of 523 Lissoclinum perforatum 636
antigenic properties of 518 1,2,3-trithiane derivative from 636
biological properties of 518 Lissoclinum sp. 637
by PROb colon cancer cells 533 trithiane from 637
chemical properties of 518 Lissoclinum sp. from Great Barrier Reef
detoxificaiton of 520 636
effects on angiogenesis 528 lissoclinotoxin A from 636
effects on blood flow 528 lissoclinotoxin C from 636
from Pantoea agglomerans 539 lissoclinotoxin D from 636
from Salmonella abortus 539 LL-Z1271a 465
in human macrophages 521 from Acrostalagmus 465
in peritoneal carcinomatosis 533 from Oidiodendron griseum 465
phase I trials with 539 LL-Z1271Y 465
receptor for 520 from Acrostalagmus 465
Rhodobacter sphaeroides lipid A from Oidiodendron griseum 465
as 521
782
Luteolin 286 Marcfortine 331

absorption of 286 synthesis of 331
Luteolin-7-(9-p-glucoside 286 Marcfortine A (MFA) 331,332,336,372
absorption of 286 antiparasitic activity of 332
Lycopersicon hirsutum f. glabratum asnematocide 331
(Solanaceae) 433 conversion to paraherquamides
2-undecanone from 433 372
methyl ketones: 2-tridecanone from from Penicillium roqueforti
433 331,332
Lydicamycin 126 microbial hydroxylation of 346
as antibacterial agent 126 reaction of 336
from Streptomyces lydicus 126 spiroalkyl analogs of 355
Lyngbya majuscula 143 structure of 332
pukeleimide C from 143 Marcfortine A's Reaction 336
Lyrophagus longior 419 with cyanogen iodide (CNI) 336
against 1,8-cineole 420 Margaritaria discoidea 395
against camphor 420 acaricidal properties of 395
against Eucalyptus globulus against Amblyomma variegatum
(Myrtaceae) 419 395
against fenchone 420 Marmesin 18
against Lavandula stoechas 419 antifungal activity of 18
against Lavandula angustifolia Maslinicacid 40
419 from Licania carii 40
against Imalool 420 from Licania licaniaeflora 40
against linalyl acetate 420 from Licania pyrifolia 40
against Mentha x piperita Matrigel 573
(Lamiaceae) 419 photograph of 573
against menthol 420 MauritamideA 689
against menthone 420 as taurine-containing metabolite
689
Malic conjugates 262 from Agelas mauritiana 689
in grapes 262 Melaleuca species (Myrtaceae) activity
in lettuce 262 418
in spinach 262 against Dermatophagoides
in wines 262 pteronyssinus 418
Malonomicin 118 against Melaleuca argentea 418
antiprotozoal activity of 118 against Melaleuca dealbata 418
from Streptomyces rimosus var. against Melaleuca saligna 418
paromomycinus 118 Melinis minutiflora (Poaceae) 398
Maltophilin 140 a- and p-pinene from 398
as antiftingal agent 140 MelophlinB 117
from Stenotrophomonas Membranotropic action 588
maltophilia 140 of triterpenoid glycosides 588
MalyngamideA 142 Menthol 391
structure of 142 effects on honey bees 391
Mange mites 409 Metabolism 283
transmission of 409 of dietary flavonoids 283
treatment of 409
783
Meth A fibrosarcoma 534 Mitomycin 559

antitumoral effects of 534 as anticancer drug 559
15-Methoxycarbonyl-nagilactone D 458 Molluscicidal activity 59
from Podocarpus nagi 458 of Licania carii 60
14P-Methyl-14a-hydroxy-MFA 340 of Licania densiflora 60
synthesis of 340 of Licania heteromorpha var.
15a-Methyl-14a-hydroxy-MFA 342 heteromorpha 60
synthesis of 342 of Licania licaniaejlora 60
15P-Methyl-14a-hydroxy-MFA 342 of Licania pittieri 60
synthesis of 342 of Licania pyrifolia 60
1 -Methyl-4-phenyl-1,2,3,6-tetrahydro- Molluscs 647
pyridine (MPTP) 184 outer body of 647
dopaminergic neurotoxicity of 184 Monoamine oxidase activity 181
Microspinsodamide 680 of Ginkgo bilobaL. 181
as cyclic peptide 680 Monophosphoryl lipid A 537
from Sidonops microspinosa 681 from Salmonella minnesota 537
p-hydroxy-/7-bromophenylalanine Morachalcone A 201
residue of 681 from moraceous plants 201
in HIV-1 infection 681 MoracinN 18
structure of 681 as aromatase inhibitor 18
Microxine 691 Morusin 201,202
as Cdc2 kinase inhibitor 691 from moraceous plants 201
from Microxina sp. 691 from Morus alba L. root bark of
Milanjilactone A 462 202
from Podocarpus milanjianus 462 Morusin hydroperoxide 210
Milanjilactone B 464 from moraceous plants 210
from Podocarpus milanjianus 464 Morwi" flavonoids 215
Mirabimide E 144,145 effects on arachidonate metabolism
effects on solid tumours 145 215
from Scytonema mirabile 144 Mulberroftiran C 210
Mites 423 from moraceous plants 210
as Oligonychus sp. (Acari: tetrany- Mulberroftiran F 210
chidae) 423 from moraceous plants 210
as Phyllocoptruta oleivora 423 Mulberroftiran G 210
as Tegolophus australis (Acari: from moraceous plants 210
Eriophyidae) 423 Mycalesp. 712
as Tetranychus sp. 423 as cytotoxic glutathione adducts
Miticidal properties 390,414 712
as Azadirachta indica 414 thiomycalolide A of 712
ofestragole 414 Mycothiazole 710
ofeugenol 414 as novel lipid 710
of linalyl acetate 414 anthelmintic properties of 710
ofmenthol 390 from Spongia mycofijiensis 110
ofterpenes 414 Myelotoxicity 559
Mitogenicity 530 ofchitosan 559
of lipid A 530 offish oils 559
to B lymphocytes 530 Myr3-(2"-rha)rha 41
from Licania densiflora 41
784
Myr 3-(2"-xyl)rha 41 Myr4'-rha 41

from Licania carii 41 from Licania apetala var. apetala
from Licania pyhfolia 41 41
Myr 3,4'-dirha 41 Myr 7-OMe-3,4'-dirha 41
from Licania heteromorpha var. from Licania heteromorpha var.
heteromorpha 41 heteromorpha 41
Myr3'4-diOMe-3-glc 41 Myricetin (Myr) 41
from Licania densiflora 41 fvom Licania densiflora 41
Myr3'4-diOMe-3-rha 41 from Licania pyrifolia 41
fvom Licania densiflora 41 Mytilus galloprovincialis 654
Myr3'OMe-3-gal 41 from Adriatic coast of Italy 654
from Licania densiflora 41 from Mytilus edulis 654
Myr3'OMe-3-glc 41 in fish oils 565
fvom Licania densiflora 41 n-3 polyunsturated fatty acid from
Myr3'OMe-3-rut 41 565
from Licania carii 41
Myr3-ara 41 NagilactoneA 458
from Licania licaniaeflora 41 from Podocarpus macrophyllus
Myr 3-gal 41 458
from Licania carii 41 fvom Podocarpus nagi 458
from Licania densiflora 41 from Podocarpus philippinensis
from Licania heteromorpha var. 458
heteromorpha 41 from Podocarpus polystachyus
from Licania licaniaeflora 41 458
Myr3-glc 41 NagilactoneB 458
from Licania carii 41 from Podocarpus nagi 458
from Licania densiflora 41 NagilactoneC 458
Myr3-rha 41 from lleostylus micranthus 458
from Licania densiflora 41 from Podocarpus halli 458
from Licania heteromorpha var. from Podocarpus nagi 458
heteromorpha 41 from Podocarpus nivalis 458
from Licania pyrifolia 41 from Podocarpus macrophyllus
Myr3-rut 41 458
from Licania carii 41 fvom Podocarpus purdeanus 458
Myr3-xyl 41 NagilactoneD 458
from Licania densiflora 41 from Podocarpus nagi 458
Myr 4'OMe-3-gal 41 NagilactoneE 461
from Licania heteromorpha var. fvom Podocarpus nagi 461
heteromorpha 41 NagilactoneF 464
Myr4'OMe-3-glc 41 from Podocarpus macrophilus 464
from Licania heteromorpha var. from Podocarpus milanjianus 464
heteromorpha 41 from Podocarpus nagi 464
Myr4'OMe-3-rha 41 from Podocarpus sellowii 464
fvom Licania densiflora 41 Nagilactone G 462
from Licania heteromorpha var. from Podocarpus milanjianus 462
heteromorpha 41 from Podocarpus sellowii 462
785
Nagilactonel 464 Neolitsea sericea 422

from Podocarpus nagi 464 24Z-ethylidenelanost-8-en-3-
NagilactoneJ 466 one from 422
from Podocarpus nagi 466 24-methylenelanost-8-en-3-
Nagilactoside A 459 one from 422
from Podocarpus nagi 459 lanostanes from 422
Nagilactoside F 460 Nephteis fasicularis 644
from Podocarpus nagi 460 source of fasicularin 644
Nagilactoside G 460 Neuronal cell 315
from Podocarpus nagi 460 crocin's effect on 315
Namenamicin 645 Neuroprotective properties 173,177
from Polysyncratn lithostrotum of Ginkgo biloba L. 173,177
645 Neurotrophic activity 174,177
Naphthoquinones 408,429 in neural injury 174
action of 429 of Ginkgo biloba L. 177
from Calceolaria andina 408 New lupane derivatives 46
mitochondrial respiration inhibition from Licania pyrifolia 46
by 429 Nicotiana tabacum, Solanaceae 415
structure of 408 A^-Methyl-D-aspartate (NMDA)
Narains 697 receptors 184
from Jaspis sipQCiQS 697 EGb as antagonist of 184
in ascidian larvae 697 radioligand binding of 184
metamorphosis induction by 697 NMDA-induced currents 319
(25)-Naringenin 17 in hippocampal neurons 319
aromatase inhibition by 17 Non-holostane triterpenoids 600
Naringenin 288 bivittosideB 600
Naringenin chalcone 271 kurilosideC 600
in juice 271 psolusosideB 600
in ketchup 271 Nor- or bisnorditerpQnoids 454
in tomato skin 271 nagilactone C 454
Natural podolactones 457 nagilactone E 454
7,9(ll)-dienolideas 457 oidilactone C 454
7a,8a-epoxy-9(ll)-enolideas 457 NubilactoneA 463
a-pirone [8(14),9(11)-dienolide] as from Podocarpus nubigena 463
457 (3/?)-Nyasol 234
Natural products 573,617 from Anemarrhena asphodeloides
antimetastatic activity of 573 234
antitumor activity of 573 (35)-Nyasol 234
from marine invertebrates 617 from Anemarrhena asphodeloides
in tumor-bearing mice 573 234
sulfiir-containing 617 Nyasol (cw-hinokiresinol) 234
Neamphius huxleyi 688 from Anemarrhena asphodeloides
neamphine from 688 234
Neoflavone dimer 1 234
from Pistacia chinensis 234 Oceanapiasp. 713
Neoflavone dimer 2 234 dithiocyanates from 713
from Pistacia chinensis 234 nematocidal activity of 713
thiocyanatin A from 713
786
thiocyanatin B from 713 OncoVax-P 545

thiocyanatin C from 713 against prostate cancer 545
total synthesis of metabolites of monophosphoryl lipid A in 545
713 Ophioxanthin 721
3P-0-c/.y-/7-coumaroyl alphitolic acid 40 5,6,5' -6' -tetrahydro-P, P-carotene-
from Licania heteromorpha var. 3,4,3',4'-tetraol 4,4'-disulfate as
heteromorpha 40 721
3p-0-c/6:-/7-coumaroyl maslinic acid 40 2is carotenoid sulfate 721
from Licania heteromorpha var. from Ophioderma longicaudum
heteromorpha 40 721
3 P-0-c/5'-/7-coumaroyl-2a-hydroxy ursolic Order aspidochirota 594
acid 40 stichloroside A from 594
from Licania heteromorpha var. stichloroside Ai from 594
heteromorpha 40 stichloroside A2 from 594
Oestrogenic activity 257,293,302 stichloroside B from 594
of plant compounds 257,302 stichloroside Bi from 594
of polyphenols 293 stichloroside C\ from 594
Oidiodendrolide B 465 stichloroside C2 from 594
from Oidiodendron truncatum 465 Stichopus chloronotus 594
Oidiodendrum griseum All Oriamide 682
terpenoid dilactones from 472 as cytotoxic peptide 682
Oidiolactone C 465 Oteromycin 147
from Oidiodendron griseum 465 from unidentified fiingus 147
from Oidiodendron truncatum 465 3p-0-rra«5-p-coumaroyl alphitolic acid
Oidiolactone D 465 40
from Oidiodendron griseum 465 from Licania heteromorpha var.
from Oidiodendron truncatum 465 heteromorpha 40
Oidiolactones 475 3P-0-/ra«5-/7-coumaroyl maslinic acid 40
antiftingal activities of 475 from Licania heteromorpha var.
from Oidiodendrum truncata 475 heteromorpha 40
01ean-12-ene-2a,3p-diol 40 3 P-0-/ra/75-/7-coumaroyl-2a-hydroxy
from Licania licaniaeflora 40 ursolic acid 40
Oleanolic acid 40 from Licania heteromorpha var.
from Licania pittieri 40 heteromorpha 40
from Licania licaniaeflora 40 Ovothiols 716
Oleficin 135 from marine invertebrate eggs 716
antibiotic activity of 135 ofmercaptohistidines 716
from Streptomyces parvulus 135 Oxalic acid 386
Oligoglycosides 589 in honey 386
from starfishes 589 toxicity of 386
Oligosaccharide moiety 611 4-Oxo-flavonoids 263
of patagonicoide A 611 structure of 263
NOESY correlations of 611 16-OxoparaherquamideB 372
8-OMe apigenin 41 conversion of 372
from Licania densiflora 41 3-(9-a-L-arabinoside 40
from Licania licaniaeflora 40
from Licania pyrifolia 40
787
from 705
Paraherquamides 331,332 orthoesterol disulfate B
fromMFA 332 from 705
synthesis of 332 orthoesterol disulfate C
Paraherquamide A 331,367 from 705
antiparasitic activity of 332 weinbersterol disulfate A
asymmetric synthesis of 367 from 706
conversion of PHB to 335 Phakellistatin 5 683
from Penicillium paraherquei 331 as cytotoxic heptapeptide 683
hydroxylationofMFA 335 from Phakellia costata 683
structures of 332 methyl sulfide group of 683
total synthesis of 367 solid-phase synthesis of 683
(+)-Paraherquamide B 359 Phase I trial 540
conversion of MFA to 333 with monophosphoryl lipid A 540
stereocontrolled synthesis of 358 Phenolic compounds 200
Patagonicoide A 603 from Morus alba 200
NOESY correlations of 603 from Morus bombycis 200
Pateamine 709 from Morus Ihou 200
as cytotoxin 709 kuwanon G as 200
dilactone functionality of 709 Pheromones 428
from New Zealand species of against Tetranychus mites 428
Mycale 709 famesol as 428
total synthesis of 709 naphthoquinones as 428
Patellamides 624 nerolidolas 428
as macrophage scavenger receptor structure of 428
inhibitor 624 Phomasetin 122
from Lissoclinum patella 624 as HIV inhibitor 122
X-ray crystallography of 624 from P/zowa sp. 122
p-Coumaric acid 262 PhylUdia pustulosa 651
in cereals brans 262 4a-isothiocyanogorgon-l 1-ene
in spinach 262 from 651
in sugar beet fibre 262 Phyllospongia foliascens 709
p-Coumaroylquinic acids 262 in serological reactions 709
in sweet cherries 262 sulfated galactolipid M-6 from
Penares sp. 713 709
as a-glucosidase inhibitor 713 Phylum echinodermata 587
penarolide sulfate A from 713 asteroideain 587
Pentaglycosides 601 crinoideain 587
from sea cucumbers 601 echinoideain 587
Pentamera calcigera 598 holothuroidea in 587
from calcigeroside B 598 ophiuroidea in 587
from calcigeroside Ci 598 secondary metabolites from 587
from calcigeroside Di 598 Physarorubinic acid 133
from cucumarioside G2 598 from Physarum polycephalum 13 3
Petrosia weinbergi 705,706 Phytolacca dodecandra 400
antiviral activity of 705 tick toxicity of 400
antiviral metabolites from 706 Piericidinas 435
orthoesterol disulfate A as inhibitor of 435
788
Pimenta dioica 398 PodolactoneB 461

against Boophilus microplus 398 from Podocarpus neriifolius 461
essential oils from 398 PodolactoneC 461
Pimpinella specks 431 from Podocarpus neriifolius 461
(1 £)-propeny 1-4-hydroxybenzene from Podocarpus milanjianus 461
from 431 PodolactoneD 461
against Typhlodromus telahus 431 from Podocarpus neriifolius 461
epoxy-anoltiglate from 431 Podolactone E
epoxy-pseudoisoeugenolisobutyrate from Ileostylus micranthus 463
from 431 from Podocarpus neriifolius 463
epoxy-pseudoisoeugenoltiglate Podolactones 453,454,455,495
from 431 antifeedant activity of 454
isoeugenolisobutyrate from 431 anti-inflammatory activity of 453,
phenylpropanoids from 431 454
pseudoisoeugenolisobutyrate anti-tumor activity of 453,454
from 431 biogenetic pathway for 455
Piperazinedione 359 from fimgi 465
Piquerol A 404,405 from Ileostylus micranthus 454
acaricidal activity of 404 from Podocarpus spQQxts 453
from Piqueria trinervia 404 fiingicidal activity of 453,454
toxicity to gravid female ticks 404 growth regulatory activity of
Piquerol B 404 454,453
acaricidal activity of 404 insecticidal activity of 453,454
from Piqueria trinervia 404 synthesis of 495
toxicity to gravid female ticks 404 Podolide 462
Plant polyphenol 257,281,300 from Podocarpus gracilor 462
anthocyanins as 257 Pohnpei 661
bioactivity of 257 13-isothiocyanatocubebane
epidemiological studies on 300 from 661
flavanolsas 257 1 -isothiocyanatoaromadendrane
flavanones as 257 from 661
flavonesas 257 2-thiocyanatoneopupukeanane
in humans 281 from 661
isoflavones as 257 4-thiocyanatoneopupukeanane
occurrence of 257 from 661
proanthocyanidins as 257 thiocyanates from 661
structure of 257 Polonovski-Potier reaction 337
Plant regulatory activity 4809 Polycarpamine A 640
ofpodolactoneE 480 as disulfide alkaloid 640
Platelet-activating factor (PAF) 188 as sulfiir-containing antifiingal
as potent phospholipid agent 640
inflammatory mediator 188 from Polycarpa aurata 640
PNU-141962 353 from Polycarpa auzata 640
isotopic labeling of 353 from Polycarpa clavata 640
Podocarpus totara 454 polycarpine as 640
podolactones from 454 synthesis of 640
PodolactoneA 461 Polycarpamine B 640
from Podocarpus neriifolius 461 as disulfide alkaloid 640
789
as sulfur-containing antifungal in syringic acid 259

agent 640 in vanillic acid 259
from Polycarpa aurata 640 PR 1388 465
from Polycarpa auzata 640 from Oidiodendron griseum 465
from Polycarpa clavata 640 from Oidiodendron truncatum 465
polycarpine as 640 Pramanicin 149,150
synthesis of 640 as antifiingal agent 149
Polycarpamine C 640 biosynthesis of 150
as sulfur-containing antifungal ftom g^^nus Stagonospora 149
agent 640 Predator mites 434
from Polycarpa aurata 640 as pathogen 434
from Polycarpa auzata 640 Neozygites adjarica as 434
from Polycarpa clavata 640 Neoseiulus fallacis as 434
synthesis of 640 Phytoseiulus persimilis as 434
Polycarpamine D 640 Proanthocyanidins 278,292
as sulfur-containing antifungal in apples 278
agent 640 degradation of 292
from Polycarpa aurata 640 in grapes 278
from Polycarpa auzata 640 in pears 278
from Polycarpa clavata 640 in strawberrys 278
synthesis of 640 structure of 278
Polycarpamine E 640 4-Propenoyl-2-tyrosylthiazole 682
as sulfiir-containing antifungal from Theonella sp. 682
agent 640 Propionibacterium acnes 534
from Polycarpa aurata 640 Proton-translocating NADH:Q
from Polycarpa auzata 640 oxidoreductase 435
from Polycarpa clavata 640 high-affinity inhibitors of 435
synthesis of 640 Psammaplin A (bisprasin) 693
Polycephalin B 134 effects on Bacillus subtilis 693
structure of 134 from Dysidea species 693
Polycephalin C 134 from Psammaplysilla 693
structure of 134 Psammaplysilla purpurea 693
Polyclinum planum 643 antimicrobial activity of 693
polyclinal from 643 as tyrosine kinase inhibitor 693
Polyenoyltetramic acids 132 presammaplin A from 693
Polymastiamide A 704 psammaplin B from 693
as antimicrobial steroid 704 psammaplin C from 693
from Polymastia boletiformis 704 psammaplin D from 693
Polyphenol glycoside 281 Pseudaxinyssa 665
hydrolysis of 281 a,a)-bis-isothiocyanates of 665
PonalactoneA 463 Fijian species of 65
from Podocarpus nakaii 463 a-isothiocyano-(9-formyl analogues
Ponalactone A glucoside 463 of 665
from Podocarpus nakaii 463 Pseudaxinyssa pitys 659
Potato tubers 259 famesyl isothioycyanate from 659
in gallic acid 259 PsolusosideB 598
in protocatechuic acid 259 from Psolus fabricii 598
in salicyclic acid 259
790
Psoroptes mites 410 Que3-rha 41

in bull fattening management 410 from Licania apetala var.
spread of 410 apetala 41
Ptilocaulis spiculifer 690 from Licania densiflora 41
alkyl sulfate from 690 from Licania licaniaeflora 41
diS Senegalese sîongQ 690 from Licania pittieri 41
dakaramine from 690 from Licania pyhfolia 41
Ptilometrasp. 719 Que 3-rut 41
anthraquinones from 719 from Licania apetala var.
Pulcherrimine 717 apetala 41
from Hemicentrotus pulcherrimus from Licania carii 41
1\1 from Licania densiflora 41
Pyrethrins 405 Quercetin 41,283
against Hyalomma anatolicum 405 from Licania densiflora 41
against Rhipicephalus haemaphy- from Licania pittieri 41
saloides 405 from Licania pyrifolia 41
Pyridoacridine alkaloids 639 in plasma 283
cytotoxic effects on KB cells 639 Quercetin glucuronides 284
from Chelynotus semperi 639 as plasma metabolites 284
kuanoniamine A as 639 Quercetin-3-glucoside 285
kuanoniamine B as 639 bioavailability of 285
kuanoniamine C as 639 Quercetin-3-rutinoside 285
kuanoniamine D as 639 in foods 285
NMR spectral analysis of 639 Quercetin-4'-glucoside 285
Pyrrole-imidazole alkaloid in foods 285
taurodispacamide A 690 Quercetin-glycosides 285
antihistaminic activity of 690 bioavailability of 285
from Agelas oroides 690 from onions 285
Que 3-(2"-xyl)rha 41 Rec-assay 225

from Licania carii 41 of 6-prenyleriodictyol 225
from Licania pyrifi)lia 41 of 8-prenyleriodictyol 225
Que3'OMe-3-glc 41 ofgancaoninC 225
from Licania apetala var. apetala of isoliquiritigenin 225
41 of licoisoflavanone 225
ôm Licania densiflora 41 of licoisoflavoneB 225
from Licania licaniaeflora 41 of semilicoisoflavone B 225
Que3-ara 41 Red wine 274
from Licania pittieri 41 (+)-catechin in 274
from Licania pyrifolia 41 (-)-epicatechin in 274
Que3-gal 41 Resveratrol 582
from Licania apetala var. apetala effects in LLC-bearing mice 582
41 effects on tumor volume 582
from Licania carii 41 effects on tumor weight 582
from Licania densiflora 41 Retinoid chemistry 69
from Licania licaniaeflora 41 recent progress in 69
from Licania pittieri 41 Retinol (vitamin A) 69
in mammalian food 69
791
Reutericyclin 127 Sanggenon C 201

as antibiotic 127 from moraceous plants 201
Rosmarinic acid 262 Sanggenon D 210
in culinary herbs 262 from moraceous plants 210
in mixed herbs 262 Sanggenon M 234
in stuffing 262 from Morus cathayana (root bark)
Rotenoneas 435 234
as inhibitor of 435 Sanggenon O 201
RT-PCR analyses 323 from moraceous plants 201
of tumor necrosis factor TNF-a Saponin 204
323 Glycyrrhiza aspera from 204
Glycyrrhiza eurycarpa from 204
Sa mae sam 574 Glycyrrhiza glabra from 204
antitumor activity of 574 Glycyrrhiza inflata from 204
as mild cathartic 574 Glycyrrhiza korshinskyi from 204
Cassia garrettiana Craib 574 Glycyrrhiza uralensis from 204
from cassigarol A 574 Sarcoptes mitts 410
from piceatannol 574 cause of human scabies 410
from piceatannol acetate 574 symptoms of 410
Saffron {Crocus sativus L.) 313,314,315 Sarcoptes scabiei 409
anti-tumor activity of 314,315 cause of mange mites 409
as coloring agent 313 cause of scabies 409
for flavoring 313 Sea cucumbers 597
uses in medicine 313 calcigeroside Cj from 597
SalignoneA 466 calcigeroside D2 from 597
from Podocarpus saligna 466 cucumarioside Ao-1 from 597
Salignone B 466 cucumechinoside A from 597
from Podocarpus saligna 466 cucumechinoside B from 597
Salignone! 462 cucumechinoside C from 597
from Podocarpus saligna 462 cucumechinoside D from 597
Salignone J 466 cucumechinoside E from 597
from Podocarpus saligna 466 cucumechinoside F from 597
Salignone K 466 triterpene glucosides from 601
from Podocarpus saligna 466 Secondary metabolites 3,432,617
Salignone L 466 acaricidal activity of 432
from Podocarpus saligna 466 against Tetranychus urticae 432
Salignone M 463 biological activity of 617
from Podocarpus saligna 463 epitaondiol as 432
Salvia officinalis 391 epitaondiol diacetate as 432
essential oils from 391 epitaondiol monoacetate as 432
Sang-Bai-Pi 209 from Broussonetia kazinoki 3
as herbal medicine 209 from Broussonetia papyrifera 3
sanggenon C from 209 from Broussonetia zeylanica 3
sanggenon D from 209 from Stypopodium flabelliforme
Sanggenon A 201 432
from moraceous plants 201 halogenation of 617
Sanggenon B 210 ofbryozoans 617
from moraceous plants 210 of marine invertebrates 617
792
ofnudibranchs (sea slugs) 617 Sokotrasterol sulfate 700

of ophistobranchs (sea hares) 617 from Halichondriidae family 700
ofsponges 617 Sophoraflavanone 234
oftunicates 617 from Anaxagorea luzonensis 234
stypetriol triacetate as 432 Soroceal 201
SellowinA 461 from moraceous plants 201
from Podocarpus sellowii 461 Soybean isoflavones 286
SellowinB 461 in humans 286
from Podocarpus sellowii 461 Spasmolytic activity 257,293
Sellowin C 458 of plant polyphenols 257,293
from Podocarpus sellowii 458 SpinosynA 405
Sesquiterpene cadina-4,10( 15)-dien-3-one SpinosynB 405
402 from Gynandropsis gynandra
from Hyptis verticillata 402 (L.) 405
Sesquiterpenes 419,659 structure of 405
from Neolitsea sericea 419 Spinosyns 404
from Taiwania cryptomeriodes from Saccharopolyspora
419 spinosa 404
ShaagrockolB 675 Spiro oxmAoXt 371
anifimgal activity of 675 formation of 371
from red sea sponge Toxiclona Spiroalkyl analogs 357
toxius 675 biological activity of 357
spectral data of 675 ofMFA 357
Sidnyum turbinatum 643 Sponges (Porifera) 657
1-heptadecanyl sulfate from 643 filter-feeding organisms of 657
1-hexyl sulfate from 643 living species of 657
1-octadecanyl sulfate from 643 Stellettamine 687
sodium (25)-2,6,10,14-tetra- A^-deacetylkuanoniamine C from
methylpentadeca-1,18-diyl sulfate 687
from 643 from Stelletta sp. 687
SigmoidinB 234 total synthesis of 687
from Glycyrrhiza uralensis 234 Stereoselective synthesis 74
Sinapic acid 262 of i^ retinal 74
in broccoli 262 Steroidal monoglycosides 587
in citrus juices 262 from Ophioderma longicaudum
in kale 262 588
in leafy brassicas 262 6a-Sterol sulfate 702
Siphonodictyon coralliphagum 669 cytotoxicity against 702
bis(sulfato)cyclosiphonodictyol from Dysideafragilis 702
A 669 from Venetian lagoon 702
siphonodictyal D from 669 Sterol sulfates haplosamate A 703
siphonodictyols G from 669 as HIV-1 integrase inhibitors 703
Sodium (or potassium) 2,6-dimethylheptyl Stilbene derivatives 579
configuration of 642 2,3,5,4'-tetrahydroxystilbene-2-0-
from Halocynthia roretzi 642 D-glucoside as 579
from Polycitor adriaticus 642 from Polygonum species 579
sulfate 642 in LLC-bearing mice 579
piceidas 579
793
resveratrol as 579 as antileukaemic agents 685

tumor growth inhibition by 579 from Prianos melanos 685
Stomphia coccinea 1\1 prianosin A as 685
benzyltetrahydroisoquinoline structure of 685
imbricatine from 717 Sulfircin 672
by starfish Dermasterias imbricata as antifimgal sesquiterpene
causes 717 sulfate 672
Storage mites 423 as A^, A^-dimethylguanidinium
Acarus siro as 423 salt 672
Lepidoglyphus destructor as 432 hipposulfates A from 672
Streptolygidin 130 Sulfolane 715
antibiotic activity of 130 from Batzella sp. 715
as bacterial RNA polymerase from Lissoclinum tunicate 115
inhibitor 130 Sulfone 715
as terminal DNA transferase Anchinoe tenacior 715
inhibitor 130 as constituent of Miditerranean
from Streptomyces lydicus 130 sponge 715
structure elucidation of 130 Sulfiir-containing cyclic peptides 678
Structural elucidation 601 biological activities of 678
of sea cucumber triterpene cytotoxicity of 678
glycosides 601 Discodermin A as 678
Structure/activity relationship 404 Discodermin B as 678
of eugenol 404 Discodermin C as 678
Stylophora pistillata 646 Discodermin D as 678
palythrine-threonine-sulfate from sponges 678
from 646 Suvanine 670
Stylosanthes sp. 400 as acetyl cholinesterase inhibitor
against Boophilus microplus 400 670
against Haemaphysalis intermedia from Coscinoderma species 670
400 from Ircinia species 670
against Rhipicephalus sanguineus Suzuki cross-coupling reaction 73
400 Swem oxidation 340
Stylosanthes hamata 399 Synapic potential mediation 318
against Boophilus microplus 399 by non-NMDA receptors 318
Stylosanthes humilis 399 Synthesis 503,508
against Boophilus microplus 399 of (±)-3p-hidroxinagilactone F
Stylosanthes scabra 400 503
against Boophilus microplus 400 of3p-hydroxy-13,14,15,16-
against Haemaphysalis intermedia tetranorlabda-7,9(l l)-dien-
400 (19,6p),(12,17)-diolide 508
against Rhipicephalus sanguineus ofLL-Z1271a 495
400 of nagilactone F 499
34-Sulfatobastadin 13 695 ofOidiolactoneC 505
as endothelin A receptor inhibitor
695 Tannic acids 280
from Janthella sp. 695 structure of 280
Sulfide-containingpyrroloiminoquinones
685
794
Tartaric conjugates 262 Tetramicacid 109,110

in coffee 262 biological activity of 110
Tauropinnaic acid 657 biosynthesis of 110
as phospholipase A2 inhibitor 657 ^^C-NMRof 113
from Pinna muricata 657 derivatives of 109
stereochemistry of 657 1,5-dihydro-4-hydroxy-2H-pyrrol-
Taurospongin A 690 2-oneas 109
as DNA polymerase inhibitor 690 from Chaetomium globosum 125
as HIV reverse transcriptase from lactic acid bacteria 126
inhibitor 690 from Melophlus sarassinorum 116
as sulfated acetylenic fatty acid IR values of 113
derivative 690 2,4-pyrrolidinedione as 109
Hippospongia sp. 690 spectral properties of 111
Taxifolin 3-rha 41 structure of 110,111
from Licania apetala var. synthesis of 110
apetala 41 UV spectra of 113
from Licania licaniaeflora 41 Tetramic acid magnesium salt 115
Taxisol 559 from Pseudomonas magnesiorubra
as anticancer drug 559 115
Tea tree {Melaleuca alternifolia, Tetranichus milQS 433
Myrtaceae) 415 neem extracts against 433
activity against Psoroptes cuniculi Tetranorditerpenoid dilactones 454
415 from Acrostalagmus genus 454
oil of 415 from Aspergillus wentii 454
Tenuazonic acid 114 from Oidiodendron griseum 454
analogues of 115 from Oidiodendron truncatum 454
antibacterial activity of 115 Tetranychus urticae AIAÂIS
antiviral activity of 115 adults of 424
as growth inhibitor 115 effects of Amaryllidaceae family
biosynthesis of 114 on 425
for peptide inhibition 115 effects of Combretum glutinosum
from Alternaria alternata 114 on 424
from Alternaria longipes 114 effects of Combretum micranthum
from Alternaria tenuis 114 on 424
from Phoma sorghina 114 effects of Glossostemon bruguieri
from Pyricularia oryzae 114 on 425
Tertiary amines 373 effects of Juglans regia 424
oxidation of 373 effects of Melia azedarach extracts
Tetrahydroglabrene 234 on 424
synthesis of 234 effects of Prosopis chilensis on
2,4,2',4'-Tetrahydroxy-3'-prenylchalcone 424
16 effects of Sambucus nigra on 424
aromatase inhibition by 16 effects of Taraxacum officinale
5,7,2' ,4' -Tetrahydroxy-3 -gerany Iflavone (Asteraceae) on 424
17 QffQCts of trigyna on 424
aromatase inhibition by 17 leaf-dipping method against 424
(2S)-5,7,2',4'-Tetrahydroxyflavanone 17 reared on Brassica rapa 425
aromatase inhibition by 17 reared on Canna indica 425
795
reared on Conyza dioscoridis 425 Ticks toxicity 403

reared on Trigonellafoenum' byeugenol 403
graecum 425 byisoeugenol 403
Theaflavin 274 by methyleugenol 403
formation of 274 bysafrole 403
Theaflavingallates 274 Tirandamycin A 131
formation of 274 biological activity of 131
Thearubigins 274 from Streptomyces tirandis 131
formation of 274 Topsentiasp. 701
Theonella sponges 681 in guanosine diphosphate/G-protein
keramamides F from 681 RAS exchange assay 701
theonellapeptolide congener from sulfates of 701
681 topsentiasterol sulfate A from 701
thiazole-containing cyclic peptides topsentiasterol sulfate B from 701
from 681 topsentiasterol sulfate C from 701
Theonezolides 710 topsentiasterol sulfate D from 701
as cytotoxic agents 710 topsentiasterol sulfate E from 701
from Theonella sp. 710 Tormentic acid 40
Therapeutic vaccines 541 from Licania licaniaeflora 40
against cancer 541 from Licania pyrifolia 40
Thioacetates 666 Total synthesis 502
absolute configuration of 666 of nagilactone F 502
conversion to fiirodysin 666 ToxadocialA 711
from Z>v^Wea species 666 as sulfated long chain alcohols 711
(I5*,45*,65*,7/?*)-4-Thiocyanato-9- from Toxadocia cylindrica 711
cadinene 660 thrombin inhibition by 711
from Trachyopsis aplysinoides ToxadocialB 711
660 as sulfated long chain alcohols 711
X-ray analysis of 660 from Toxadocia cylindrica 711
5-Thio-D-mannose 715 thrombin inhibition by 711
as naturally occurring 5-thio- ToxadocialC 711
sugar 715 from Toxadocia cylindrica 111
from Clathria pyramida 115 thrombin inhibition by 711
Thiofiirodysinin 667 Toxic essential oils 393
asfiiranosesquiterpene667 from Apis mellifera 393
from Dysidea avara 667 from Varroajacobsoni 393
Thymol 391 Toxiclona toxius 675
employed as Frakno thymol frame toxicol A from 675
391 toxiusol from 675
Thymovar 391 Trachyopsis halichondrioides 700
employed as Frakno thymol frame 26-norsokotrasterol sulfate from
391 700
Thymus vulgaris 391,415 Tracheal mites 389
against Knemidocoptes pilae 415 effects ofvegetable oils on 389
essential oils from 391,415 Triandamycin B 131
Tick-borne diseases 394 from Streptomyces flaveolus 131
in livestock 394 Trichostrongylus colubriformis 342
796
Tridacna maxima 652 Tyrophagus putrescentiae 421

arsenic-containing sugar sulfate against 1,8-cineole 421
from 652 against fenchone 421
Tridentata marginata 646 against isomers of caryo-
tridentatol A from 646 phyllene 421
tridentatol B from 646 against linalool 421
tridentatol C from 646 against linalyl 421
Trididemnum sp. 638 against menthone 421
from Guam 638 against myrtanol 421
shermilamine A from 638 against Pinus halepensis 420
P-Triketone 117 digdxnsX Pinus nigra 420
from Apiosordaria effusa 117 against Pinus pinaster 420
Triterpene glycoside 596,587,589 against Pinus pinea 420
antifiingal activity of 589 against pinene 421
cytotoxic activity of 589 against pulegone 421
cytostatic activity of 589 against a-terpinene 421
from Cucumaria echinata 596 against y-terpinene 421
from Pentamera calcigera 596 against terpineol 421
from sea cucumbers 587 against valencene 421
hemolytic activity of 589
immunomodulatory activity of 589 UoamineA 644
Tropical ixodid ticks 404 from Aplidium uouo 644
Hyalomma genera as 404 UoamineB 644
Rhipicephalus genera as 404 of Aplidium uouo 644
Tryptophan derivative 369 3-thiomethylacrylate ester
construction of 369 group of 644
Tumor 532 Urbalactone 459
effectof lipid A on 532 from Podocarpus urbanii 459
in host response to LPS 531 Ursolic acid 18,40
Tumor growth in LLC-bearing mice 581 from Licania carii 40
effects of 2,3,5,4'-tetrahydroxy- from Licania licaniaeflora 40
stilbene-2-O-D-glucoside on 581 from Licania pittieri 40
effects of piceid on 581 from Licania pyrifolia 40
Tumor necrosis factor-a (TNF-a) 219 inhibition of HI V-1 protease
as tumor promoter 219 dimerization by 18
by okadaic acid 219 Uvaria pauciovulata ATI
Tunicates (Ascidians) 621 effects on Dermatophagoides
active metabolites of 622 pteronyssinus All
ascidiacyclamide from 622 squamocin from 422
bistratamides from 622 structure of 422
from Phylum chordata 621
lissoclinamides from 622 Vancoresmycin 150
patellamides from 622 activity against gram-positive
Tyrindoxyl sulfate 652 bacteria 150
as Tyrian purple dye 652 from Amycolatopsin sp. 150
from Murex truncatus 652
797
Varroajacobsoni 387 Wheat flour 259

honey bees tolerant to 387 vanillic acid in 259
toxicity of 387 syringic acid in 259
VarroaxmiQS 383,384,392
as honey bee parasites 384 Xanthobaccin A 140
biological activity of 392 asfiingitoxicmetabolite 140
Vasoprotective effects 302 Xanthobaccin B 140
of green tea 302 asftmgitoxicmetabolite 140
Vermisporin 125 Xanthobaccin C 140
antimicrobial activity of 125 asfiingitoxicmetabolite 140
from Ophiobolus vermisporis 125 Xestoquinolide B 677
Vernonia amygdalina 400 from Xestospongia cf.
tick toxicity of 400 carbonaria 611
Veterinary medicine 383 protein kinase activity of 677
for ectoparasites control 383 spectral data of 677
Vetiver grass 399
for controlling ticks 399 Yessotoxin 653
Phetchabunas 399 analogues of 653
'Si Sa Kef as 399 from Patinopecten yessoensis 653
'Uthai Thani' as 399 in diarrhetic shellfish poisoning
VirenamideA 644 (DSP) 653
as cytotoxic linear peptides 644
from Diplosoma virens 644 Zoanthus sp. 646
Virenamide B 644 sphingolipid hariamide from 646
as cytotoxic linear peptides 644 zoanthid A from 646
from Diplosoma virens 644 Zyzzya cf marsailis 686
Virenamide C 644 discorhabdin A from 686
as cytotoxic linear peptides 644 makaluvamine F from 686
from Diplosoma virens 644,645 total synthesis of 686
Vitamin A synthesis 71
by Baadische Anilin by 71
by Hoffrnann-LaRoche 71
by Rhone-Poulenc 71
SodaFabrik 71
Waiakeamide 684
as cyclic hexapeptide 684
from Ircinia dendroides 684
Watersipora subtorquata 620
5,7-dihydroxy-6-oxo-6//-
anthra[ 1,9-6c]thiophene-1 -
carboxylic acid from 620
Wentilactone A 465
from Aspergillus wentii 465
Wentilactone B 465
from Aspergillus wentii 465

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studies in

Natural Products Chemistry

Vol. 1 Stereoselective Synthesis (Part A)

© 2003 Elsevier Science B.V. All rights reserved.

Electronic Storage or Usage

First edition 2003

Library of Congress Cataloging in Publication Data

British Library Cataloguing in Publication Data

In various segments of the scientific community, natural products research comes in

Obviously, for progress to be realized on a widespread basis, general dissemination of

Bioactive compounds from the genus Broussonetia

Chemical and biological studies on Licania genus

Recent progress in retinoid chemistry

Bioactive tetramic acid metabolites

Chemistry and biological activities oi Ginkgo biloba

Chemistry and biological activities of isoprenylated flavonoids from

Plant polyphenols: Structure, occurrence and bioactivity

Promising pharmacological actions of crocin in Crocus sativus on the central

Synthesis and modification of marcfortine and paraherquamide class

Acaricides of natural origin, personal experiences and review of

Podolactones: A group of biologically active norditerpenoids

Prevention of cancer chemotherapy drug-induced adverse reaction, antitumor

Biologically active triterpene glycosidesfromsea cucumbers (holothuroidea,

Sulfur-containing natural products from marine invertebrates

Subject Index 753

Kazuo Abe Graduate School of Pharmaceutical Sciences, The

Alejandro F. Barrero Department of Organic Chemistry, Institute of

Anna Rita Bilia Dipartimento di Scienze Farmaceutiche, Universita di

Alessandra Braca Dipartimento di Chimica Bioorganica e Biofarmacia,

Lorenzo Bramati ITB-CNR, Via F. Ui Cervi, 93 - 20090 (MI), Italy

Dominique L. Cartier FRE 2125 CMIS, 6 rue de TUniversite 29000 Quinq)er,

Hugo D. Chludil Departamento de Quimica Organica, Facultad de Ciencias

Guido Flamini Dipartimento di Chimica Bioorganica e Biofarmacia, Via

Toshio Fukai School of Pharmaceutical Sciences, Toho University, 2-2-

Nolwenn Gauthier Cancer Immunotherapy Research Laboratory, Ecole

Emilio L. Ghisalberti Department of Chemistry, University of Western

M. Haga Department of Hygienic Chemistry, Faculty of

Yoshio Hano School of Pharmaceutical Sciences, Toho University, 2-2-

Jean-Francois Jeannin Cancer Immunotherapy Research Laboratory, Ecole

Yoshiyuki Kimura Second Department of Medical Biochemistry, School of

A. Douglas Kinghom Program for Collaborative Research in the Pharmaceutical

Roger Labia FRE 2125 CNRS, 6 rue de I'Universite 29000 Quimper,

Byung H. Lee Preclinical Development, Pharmacia Animal Health, 7000

Dongho Lee Chemistry and Life Sciences, Research Triangle Institute,

Departamento de Quimica Organica, Facultad de Ciencias

Grupo de Productos Naturales, Centro de Quimica

Markus Minoggio ITB-CNR, Via F. Hi Cervi, 93 - 20090 (MI), Italy

Jose F. Quilez Del Moral Department of Organic Chemistry, Institute of

Ivano Morelli Dipartimento di Chimica Bioorganica e Biofarmacia,

Satoshi Morimoto Graduate School of Pharmaceutical Sciences, Kyushu

Taro Nomura School of Pharmaceutical Sciences, Toho University, 2-2-

Takashi Ochiai Faculty of Pharmaceutical Sciences, Fukuoka University,

Alena Pance Cancer Immunotherapy Research Laboratory, Ecole

Piergiorgio Pietta ITB-CNR, Via F. Ui Cervi, 93 - 20090 (MI), Italy

Cosimo Pizza Dipartimento di Scienze Farmaceutiche, Universita di

Michele R. Prinsep Department of Chemistry, University of Waikato, Private

Daniele Reisser Cancer Immunotherapy Research Laboratory, Ecole

Hiroshi Saito Graduate School of Pharmaceutical Sciences, The

K. Sasaki Department of Hygienic Chemistry, Faculty of

Alicia M. Seldes Departamento de Quimica Organica, Facultad de Ciencias

Hiroshi Shimeno Faculty of Pharmaceutical Sciences, Fukuoka University,

Yukihiro Shoyama Graduate School of Pharmaceutical Sciences, Kyushu

Shinji Soeda Faculty of Pharmaceutical Sciences, Fukuoka University,