New Strategies for Identifying Natural Products of Ecological Significance from Corals

Luiz Fernando Cappa de Oliveira

Manzamine A (1) known as a cytotoxin exhibited potent antimalarial activity with 40% recovery 60 days after single intraperitoneal (i.p.) injection against mice infected with the malaria parasite Plasmodium berghei. Pachastrisamine (6), a simple derivative of a sphingosine, isolated from a sponge, Pachastrissa sp. showed a high level of cytotoxicity. New sesquiterpene carbonimidic dichlorides and related aldehydes (812) have been isolated as cytotoxic constituents of the sponge Stylotella aurantium and shown to have moderate cytotoxicity. Cyclic undecapeptides, barangamides AD (1316), have been isolated from the sponge Theonella swinhoei. Their structures were established by spectroscopic analysis and chemical reaction.

Chemical and biological investigation of the cultured marine soft coral Xenia elongata led to the isolation of two new diterpenes (2, 3). Their structures were elucidated using a combination of NMR and mass spectrometry. Biological evaluations and assessments were determined using the specific apoptosis induction assay based on genetically engineered mammalian cell line D3 deficient in Bak and Bax and derived from a mouse epithelial cell. The diterpenes induce apoptosis in low micromolar concentrations. The results indicate that the previously isolated compound (1) affects cell in a manner similar to that of HSP90 and HDAC inhibitors and in a manner opposite of PI3 kinase/mTOR inhibitors. Compound (3) inhibits selectively HDAC6 in high micromolar concentrations. Keywords: structural and functional characterization of marine drug; marine biotechnology; bioactive compounds and bioproducts; drug discovery and development

Studies in Natural Products Chemistry Volume 43 This page intentionally left blank Studies in Natural Products Chemistry Volume 43 Edited by Atta-ur-Rahman, FRS International Center for Chemical and Biological Sciences H.E.J. Research Institute of Chemistry University of Karachi Karachi, Pakistan Amsterdam • Boston • Heidelberg • London • New York • Oxford Paris • San Diego • San Francisco • Singapore • Sydney • Tokyo Elsevier The Boulevard, Langford Lane, Kidlington, Oxford, OX5 1GB, UK Radarweg 29, PO Box 211, 1000 AE Amsterdam, The Netherlands First edition 2014 Copyright © 2014 Elsevier B.V. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher. 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Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN: 978-0-444-63430-6 ISSN: 1572-5995 For information on all Elsevier publications visit our web site at store.elsevier.com Printed and bound in Great Britain 14 15 16 17 18 11 10 9 8 7 6 5 4 3 2 1 This book has been manufactured using Print On Demand technology. Each copy is produced to order and is limited to black ink. The online version of this book will show color figures where appropriate Contents Contributors Preface 1. Total Synthesis of Diterpenoid Pyrones, Nalanthalide, Sesquicillin, Candelalides A–C, and Subglutinols A, B xiii xvii 1 Tadashi Katoh Introduction Total Synthesis of ()-Sesquicillin [Zhang and Danishefsky, 2002] Synthetic Strategy Total Synthesis Total Synthesis of ( )-Nalanthalide and (+)-Sesqucillin [Katoh et al., 2006, 2010] Total Synthesis of ( )-Nalanthalide Total Synthesis of (+)-Sesquicillin Total Synthesis of ( )-Candelalide A–C [Katoh et al., 2005, 2009] Total Synthesis of ( )-Candelalide A Total Synthesis of ( )-Candelalide B Total Synthesis of ( )-Candelalide C Total Synthesis of ( )-Subglutinols A, B Total Synthesis of ( )-Subglutinol A [Hong et al., 2009, 2010] Total Synthesis of ( )-Subglutinol A [Katoh et al., 2011] Total Synthesis of ( )-Subglutinol B [Hong et al., 2009, 2010] Total Synthesis of ( )-Subglutinol B [Katoh et al., 2011] Conclusion Acknowledgements References 2. Chemical Diversity of Vibsane-Type Diterpenoids and Neurotrophic Activity and Synthesis of Neovibsanin 2 4 4 6 7 7 12 14 14 19 21 25 25 29 32 34 35 37 38 41 Miwa Kubo, Tomoyuki Esumi, Hiroshi Imagawa, and Yoshiyasu Fukuyama Introduction Vibsane-Type Diterpenoids The Stereochemistry of Vibsanins B (1) and C (2) The Absolute Configuration of Vibsanin F (3) 11-Membered Ring Vibsane-Type Diterpenoids 7-Membered Ring Vibsane-Type Diterpenoids Rearranged Vibsane-Type Diterpenoids (Neovibsanins) 41 42 43 45 49 50 56 v vi Contents Biological Activities of Vibsane-Type Diterpenoids Neurotrophic Activity of Neovibsanins Synthesis of Vibsane-Type Diterpenoids A Minimal Structural Core of Neovibsanin Required for Neurotrophic Activity Conclusion Acknowledgments References 3. Natural and Synthetic Alkamides: Applications in Pain Therapy 61 62 64 72 75 75 75 79 Marı́a Yolanda Rios and Horacio F. Olivo Introduction Biosynthesis Capsaicinoids Capsaicin Capsaicin’s Mechanism of Action Capsaicin SARs Pharmaceutical Formulations Based on Capsaicinoids Affinin (Spilanthol) Sanshools Piperine and Piperovatin Conclusions Acknowledgments References 4. Alkaloids as Inhibitors of Monoamine Oxidases and Their Role in the Central Nervous System 79 85 89 91 92 95 100 100 103 106 110 111 113 123 Carolina Dos Santos Passos, Claudia Simoes-Pires, Amelia Henriques, Muriel Cuendet, Pierre-Alain Carrupt, and Philippe Christen Introduction Therapeutic Potential of Monoamine Oxidase Inhibition in Neurological Disorders Depression Parkinson’s Disease Other Neurodegenerative Diseases Smoke and Alcohol Cessation Alkaloids as Monoamine Oxidase Inhibitors Indole Alkaloids Isoquinoline Alkaloids Piperidine Alkaloids Desoxypeganine Other Alkaloids Conclusion References 123 124 125 126 126 127 127 128 134 137 138 139 141 142 Contents vii 5. Furanocoumarins: Biomolecules of Therapeutic Interest 145 José Antonio Del Rı́o, Licinio Dı́az, David Garcı́a-Bernal, Miguel Blanquer, Ana Ortuño, Enrique Correal, and José Marı́a Moraleda Introduction Furanocoumarins Furanocoumarin Biosynthesis Furanocoumarins in Nature: Distribution and Sources Furanocoumarin Analytic Methods Extraction from Plant Material Sample Purification Purification by Column Chromatography Purification by Thin-Layer Chromatography High-Performance Liquid Chromatography Supercritical Fluid Chromatography and CE Gas Chromatography Activity of Furanocoumarins Therapeutical Use of Furanocoumarins Mechanisms of Action of Furanocoumarins Skin Disorders Noncutaneous Autoimmune Diseases Solid Organ Transplant Rejection Graft Versus Host Disease Cutaneous T-Cell Lymphoma Cancer Microorganism Infections Other Diseases or Clinical Complications Conclusions Acknowledgments References 6. Interactions Between Natural Health Products and Antiretroviral Drugs 146 147 147 149 161 161 162 163 163 164 166 166 167 168 169 170 174 176 178 180 181 184 185 185 186 187 197 Marı́a José Abad Martı́nez, Luis Miguel Bedoya del Olmo, and Paulina Bermejo Benito Introduction Interactions Between Natural Health Products and Antiretroviral Drugs The Replication Cycle of HIV Existing Antiretroviral Drug Classes Nucleoside Reverse Transcriptase Inhibitors Non-nucleoside Reverse Transcriptase Inhibitors Protease Inhibitors Entry Inhibitors Integrase Inhibitors Guidelines on the Use of Antiretroviral Therapy for HIV Infection 197 199 200 202 202 203 204 205 205 206 viii Contents Examples of Clinical Interactions Between NHPs and Antiretroviral Drugs St. John’s Wort Garlic Grapefruit Milk Thistle Ginkgo Ginseng Concluding Remarks Acknowledgments References 7. Lichens: Chemistry and Biological Activities 207 207 209 211 211 212 213 214 215 216 223 Sammer Yousuf, M. Iqbal Choudhary, and Atta-ur-Rahman Introduction Lichen Chemistry: A Brief History Chemical Structure and Diversity Biosynthesis of Lichen Substances Shikimic Acid Pathway Polymalonate Pathway Mevalonic Acid Pathway Lichen-Derived Secondary Metabolites and Their Functions Biological Activities of Secondary Metabolites of Lichens Antibacterial, Antibiotic, and Antifungal Activities Antiprotozoal Activity Antiviral Activity Cytotoxicity and Antitumor Activities Antioxidant Properties Antidiabetic Properties Enzyme Inhibition Properties Antipyretic and Analgesic Properties Conclusion References 8. Chemistry and Bioactivities of Royal Jelly 223 224 226 227 227 227 229 229 232 233 240 240 245 249 251 253 255 255 256 261 Eleni Melliou and Ioanna Chinou Introduction Chemical Constituents Identified in RJ Fatty Acids Biological Properties Antimicrobial Activities Antioxidative Activity Estrogenic Activity Activities in Reproductive System in Male Rats Tonic/Biostimulating Properties 261 262 262 270 270 272 273 274 275 Contents Immunomodulating Properties Neuronal Function Properties Antidepressant Activities Antihypertensive Activity Insulin-Like Activities Wound Healing and Skin Improving Properties Properties Against Rheumatoid Arthritis Cytotoxic Activities Protective Activities Properties in Dentistry Allergic Reactions and Hypersensitivity Concluding Remarks References 9. Synthetic Cannabinoids: Synthesis and Biological Activities ix 277 278 279 279 280 281 282 283 283 283 283 284 286 291 Joel Schlatter Introduction Distribution of Synthetic Cannabinoids Legality and Regulation Synthesis of Synthetic Cannabinoids Biological Activities of Synthetic Cannabinoids Epidemiological Data Current Medicinal Purposes Discussion and Conclusion References 10. New Strategies for Identifying Natural Products of Ecological Significance from Corals: Nondestructive Raman Spectroscopy Analysis 291 292 293 294 295 305 305 307 309 313 Lenize Fernandes Maia, Beatriz Grosso Fleury, Bruno Gualberto Lages, Joel Christopher Creed, and Luiz Fernando Cappa de Oliveira Introduction Natural Products from Cnidaria A New Method for Identifying Natural Products from Cnidaria Raman Spectroscopy: Basic Principles Instrumentation Application of Raman Scattering to Marine Natural Products: An Overview Characterization of Metabolites from Marine Organisms Raman Spectroscopy Applied to Biologically Relevant Natural Products Concluding Remarks Acknowledgments References 313 314 327 328 334 335 335 341 343 344 344 x Contents 11. Insulin Resistance as a Target of Some Plant-Derived Phytocompounds 351 Mohamed Eddouks, Amina Bidi, Bachir EL Bouhali, and Naoufel Ali Zeggwagh Introduction Insulin Resistance Phytocompounds Targeting of Insulin Resistance Amorfrutins Bassic Acid Caffeic Acid Christinin-A Cinnamaldehyde Cryptoleptine Diosgenin Epicatechin Galactomannan 4-Hydroxybenzaldehyde Lagerstroemin and Flosin B Mangiferin Marsupin and Pterostilbene Myricetin Oleanolic Acid Oleuropeoside Paeoniflorin Stevioside Ursolic Acid Vanillin Conclusion Acknowledgment References 12. Saponins Produced by Gypsophila Species Enhance the Toxicity of Type I Ribosome-Inactivating Proteins 351 352 355 355 355 356 357 357 358 359 359 360 361 361 362 363 364 364 365 366 366 367 368 368 370 370 375 Idris Arslan Saponins Gypsophila Saponins Ribosome-Inactivating Proteins Saponins as Cytotoxic Agents Acknowledgment References 375 376 377 378 379 380 Contents 13. Biologically Active Compounds from the Genus Uncaria (Rubiaceae) xi 381 Anjaneya Swamy Ravipati, Narsimha Reddy, and Sundar Rao Koyyalamudi Introduction Ethnobotany of Uncaria spp. Herbal Formulations Containing Uncaria spp. Chotoko Gou-teng Kampo Gud Kuiyangling Phytochemistry of Uncaria spp. Alkaloids Terpenoids Flavonoids Extraction, Isolation, Purification, and Identification of Novel Compounds Bioactivities Cytotoxicity Antiinflammatory Activities Antibacterial Activity Antiviral Activity Antimutagenic Activity Activity Against Vascular Diseases Immunostimulation Activity Hypotensive Effects CNS-Related Activity and Effects on Locomotion Response Activity Against Vascular Dementia and Ischemia Structural Diversity of Compounds from Uncaria spp. Structure and Activity Relationship of Bioactive Compounds from Uncaria spp. Alkaloid Biosynthesis in U. tomentosa Concluding Remarks References 382 384 384 385 385 385 385 385 386 386 389 389 390 392 392 397 397 398 398 398 399 400 400 401 401 402 403 404 404 14. Asymmetric Phase-Transfer Catalysis as a Powerful Tool in the Synthesis of Biologically Active Chiral Complex Natural Products 409 Guddeangadi N. Gururaja and Mario Waser Introduction PTCs Derived from Cinchona Alkaloids 409 414 xii Contents Michael Addition Reactions a-Alkylation Reactions Epoxidations Alkylative Dearomatization/Annulation PTCs Derived from Binaphthol a-Alkylation Reactions Mannich Reactions Michael Reactions PTCs Derived from Tartaric Acid Mannich Reactions Michael Reactions a-Alkylation Reactions Synopsis References 15. Challenges of Biopesticides Under the European Regulation (EC) No. 1107/2009: An Overview of New Trends in Residue Analysis 414 415 422 423 424 425 427 428 429 429 430 430 431 433 437 Juan José Villaverde, Beatriz Sevilla-Morán, Pilar Sandı́nEspaña, Carmen López-Goti, and José Luis Alonso-Prados Introduction European Legislation for Sustainable Use of Pesticides and Challenges that Biopesticides Have to Face Evolution of Regulation at the European Level Regulation (EC) No. 1107/2009: Data Requirements and Uniform Principles Directive 2009/128/EC: IPM Practices and Current Barriers for Biopesticide Registration Advantages and Disadvantages of “Biopesticides” and the Controversial Use of this Term Requirements in Analytic Methods to Fulfill the Actual European Legislative Framework Analytic Techniques Applied to the Analysis of Biopesticide Residues Sample Preparation Techniques Chemical Analysis: Instrumental Techniques and Future Trends Analysis of Biopesticide Residues in Environmental Matrices Conclusions References Index 438 440 440 443 445 448 449 451 452 467 473 475 478 483 Contributors Numbers in Parentheses indicate the pages on which the author’s contributions begin. Marı́a José Abad Martı́nez (197), Department of Pharmacology, Faculty of Pharmacy, University Complutense, Ciudad Universitaria s/n, 28040 Madrid, Spain José Luis Alonso-Prados (437), Plant Protection Products Unit, DTEVPF, INIA, Madrid, Spain Idris Arslan (375), Biomedical Engineering, Faculty of Technology, Pamukkale University, Denizli, Turkey Paulina Bermejo Benito (197), Department of Pharmacology, Faculty of Pharmacy, University Complutense, Ciudad Universitaria s/n, 28040 Madrid, Spain Amina Bidi (351), Moulay Ismail University, Errachidia, Morocco Miguel Blanquer (145), Cell Therapy Unit, Hospital Universitario Virgen de la Arrixaca, Faculty of Medicine, University of Murcia, Murcia, Spain Bachir EL Bouhali (351), Moulay Ismail University, Errachidia, Morocco Pierre-Alain Carrupt (123), School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Geneva, Switzerland Ioanna Chinou (261), Department of Pharmacognosy and Chemistry of Natural Products, Faculty of Pharmacy, University of Athens, Panepistimiopolis Zografou, Athens, Greece M. Iqbal Choudhary (223), H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, Pakistan, and Department of Biochemistry, Faculty of Sciences, King Abdulaziz University, Jeddah, Saudi Arabia Philippe Christen (123), School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Geneva, Switzerland Enrique Correal (145), Instituto Murciano de Investigación y Desarrollo Agrario y Alimentario (IMIDA), La Alberca, Murcia, Spain Joel Christopher Creed (313), Departamento de Ecologia, IBRAG, Rua São Francisco Xavier, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, RJ, Brazil Muriel Cuendet (123), School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Geneva, Switzerland Licinio Dı́az (145), Plant Biology Department, Faculty of Biology, University of Murcia, Murcia, Spain Luiz Fernando Cappa de Oliveira (313), Núcleo de Espectroscopia e Estrutura Molecular, Departamento de Quı́mica, Instituto de Ciências Exatas, Universidade Federal de Juiz de Fora, Juiz de Fora, Minas Gerais, Brazil xiii xiv Contributors Luis Miguel Bedoya del Olmo (197), Department of Pharmacology, Faculty of Pharmacy, University Complutense, Ciudad Universitaria s/n, 28040 Madrid, Spain Mohamed Eddouks (351), Moulay Ismail University, Errachidia, Morocco Tomoyuki Esumi (41), Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima, Japan Beatriz Grosso Fleury (313), Departamento de Ecologia, IBRAG, Rua São Francisco Xavier, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, RJ, Brazil Yoshiyasu Fukuyama (41), Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima, Japan David Garcı́a-Bernal (145), Cell Therapy Unit, Hospital Universitario Virgen de la Arrixaca, Faculty of Medicine, University of Murcia, Murcia, Spain Guddeangadi N. Gururaja (409), Institute of Organic Chemistry, Johannes Kepler University Linz, Altenbergerstr. 69, 4040 Linz, Austria Amelia Henriques (123), Laboratory of Pharmacognosy, Faculty of Pharmacy, Universidade Federal do Rio Grande do Sul, UFRGS, Porto Alegre, Rio Grande do Sul, Brazil Hiroshi Imagawa (41), Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima, Japan Tadashi Katoh (1), Laboratory of Medicinal and Synthetic Chemistry, Department of Pharmaceutical Sciences, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai Japan Sundar Rao Koyyalamudi (381), School of Science and Health, University of Western Sydney, Locked Bag 1797, Penrith South DC NSW 1797, Australia, and Departments of Biochemistry, The Children’s Hospital at Westmead, Sydney, NSW 2145, Australia Miwa Kubo (41), Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima, Japan Bruno Gualberto Lages (313), Programa de Pós-Graduação em Ecologia e Evolução, Instituto de Biologia Roberto Alcântara Gomes, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, RJ, Brazil Carmen López-Goti (437), Plant Protection Products Unit, DTEVPF, INIA, Madrid, Spain Lenize Fernandes Maia (313), Núcleo de Espectroscopia e Estrutura Molecular, Departamento de Quı́mica, Instituto de Ciências Exatas, Universidade Federal de Juiz de Fora, Juiz de Fora, Minas Gerais, Brazil Eleni Melliou (261), Department of Pharmacognosy and Chemistry of Natural Products, Faculty of Pharmacy, University of Athens, Panepistimiopolis Zografou, Athens, Greece José Marı́a Moraleda (145), Cell Therapy Unit, Hospital Universitario Virgen de la Arrixaca, Faculty of Medicine, University of Murcia, Murcia, Spain Horacio F. Olivo (79), Medicinal and Natural Products Chemistry, The University of Iowa, Iowa City, Iowa, USA Contributors xv Ana Ortuño (145), Plant Biology Department, Faculty of Biology, University of Murcia, Murcia, Spain Carolina Dos Santos Passos (123), Laboratory of Pharmacognosy, Faculty of Pharmacy, Universidade Federal do Rio Grande do Sul, UFRGS, Porto Alegre, Rio Grande do Sul, Brazil Atta-ur-Rahman (223), H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, Pakistan Anjaneya Swamy Ravipati (381), School of Science and Health, University of Western Sydney, Locked Bag 1797, Penrith South DC NSW 1797, Australia Narsimha Reddy (381), School of Science and Health, University of Western Sydney, Locked Bag 1797, Penrith South DC NSW 1797, Australia Marı́a Yolanda Rios (79), Centro de Investigaciones Quı́micas, Universidad Autónoma del Estado de Morelos, Morelos, Mexico José Antonio Del Rı́o (145), Plant Biology Department, Faculty of Biology, University of Murcia, Murcia, Spain Pilar Sandı́n-España (437), Plant Protection Products Unit, DTEVPF, INIA, Madrid, Spain Joel Schlatter (291), Laboratory of Forensic Toxicology, Department of Biology, University Hospital of Jean Verdier—APHP, Bondy, France Beatriz Sevilla-Morán (437), Plant Protection Products Unit, DTEVPF, INIA, Madrid, Spain Claudia Simoes-Pires (123), School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Geneva, Switzerland Juan José Villaverde (437), Plant Protection Products Unit, DTEVPF, INIA, Madrid, Spain Mario Waser (409), Institute of Organic Chemistry, Johannes Kepler University Linz, Altenbergerstr. 69, 4040 Linz, Austria Sammer Yousuf (223), H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, Pakistan Naoufel Ali Zeggwagh (351), Moulay Ismail University, Errachidia, Morocco This page intentionally left blank Preface This represents the 43rd volume of this long-standing series, which have been published regularly under my editorship over the last quarter of a century. In Chapter 1, Tadashi Katoh, the total synthesis of biologically active diterpenoid pyrones—nalanthalide, sesquicillin, candelalides A–C, and subglutinols A, B—is reviewed with a particular focus on the synthetic methodology and strategy. In Chapter 2, Fukuyama et al. discuss the structural diversity, neurotrophic activity, and synthesis of vibsane-type diterpenoids. Rios and Olive, in Chapter 3, describe the molecular basis of the immunomodulatory and analgesic activities of alkamides. They also discuss the chemical interactions of these natural products with their biological receptors. In Chapter 4, Christen et al. illustrate the role of certain alkaloids, particularly those of the indole and quinolone type, for the inhibition of monoamine oxidases with potential applications in the treatment of central nervous system disorders. Antonio Del RÍo et al. review the structures of furanocoumarins, their occurrence in plants, analytical methods, pharmacological properties, and therapeutic uses in Chapter 5. As the human immunodeficiency virus (HIV)-infected patients are treated with the highly active antiretroviral therapy (HAART), the interactions of the drugs being used have become a major concern. Keeping this in view, Martinez et al., in Chapter 6, present an overview of the effects of herbal medicines on antiretroviral drug-metabolizing and transporting enzymes. They particularly focus on potential herb–antiretroviral drug interactions, as well as interactions at the pharmacodynamic level. Chapter 7 contributed by Yousuf et al. summarizes the structural classes of different polyphenolic secondary metabolites produced by the lichens as well as their diverse biological and pharmacological activities. In Chapter 8, Melliou and Chinou present an interesting review on the chemistry and bioactivities of Royal Jelly, which is secreted by Apis mellifera, and is known for its biological significance. In Chapter 9, various biological roles of synthetic cannabinoids have been discussed by Schlatter. Corals are known to produce ecologically significant secondary metabolites. Fleury et al., in Chapter 10, review the use of Raman spectroscopy for the analysis of corals for several ecological effects. Chapter 11 by Eddouks et al. provides a review of 26 selected phytocompounds with beneficial roles for the treatment and prevention of insulin resistance associated with diabetes mellitus. Furthermore, Arslan in Chapter 12 demonstrates the increased toxicity of ribosome-inactivating proteins in the presence of saponins, produced by Gypsophila. In Chapter 13, Koyyalamudi et al. review the compounds isolated from the genus Uncaria, having biological significance. xvii xviii Preface Gururaja and Waser describe the use of asymmetric phase-transfer catalysis for the synthesis of biologically active complex natural products in Chapter 14. In Chapter 15, Villaverde et al. present an overview of the new trends in residue analysis, and the definition of biopesticide with reference to the European Regulation (EC) No. 1107/2009. I would like to thank Ms. Taqdees Malik, Ms. Darshna Kumari, and Ms. Humaira Hashmi for their assistance in the preparation of this volume. I am also grateful to Mr. Mahmood Alam for the editorial assistance. Atta-ur-Rahman, FRS International Center for Chemical and Biological Sciences, (H.E.J. Research Institute of Chemistry), University of Karachi, Karachi, Pakistan Chapter 1 Total Synthesis of Diterpenoid Pyrones, Nalanthalide, Sesquicillin, Candelalides A–C, and Subglutinols A, B Tadashi Katoh Laboratory of Medicinal and Synthetic Chemistry, Department of Pharmaceutical Sciences, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai Japan Chapter Outline Introduction 2 Total Synthesis of ()-Sesquicillin [Zhang and Danishefsky, 2002] 4 Synthetic Strategy 4 Total Synthesis 6 Total Synthesis of ( )-Nalanthalide and (+)-Sesqucillin [Katoh et al., 2006, 2010] 7 Total Synthesis of ( )-Nalanthalide 7 Total Synthesis of (+)-Sesquicillin 12 Total Synthesis of ( )-Candelalide A–C [Katoh et al., 2005, 2009] 14 Total Synthesis of ( )-Candelalide A 14 Total Synthesis of ( )-Candelalide B 19 Total Synthesis of ( )-Candelalide C Total Synthesis of ( )-Subglutinols A, B Total Synthesis of ( )-Subglutinol A [Hong et al., 2009, 2010] Total Synthesis of ( )-Subglutinol A [Katoh et al., 2011] Total Synthesis of ( )-Subglutinol B [Hong et al., 2009, 2010] Total Synthesis of ( )-Subglutinol B [Katoh et al., 2011] Conclusion Acknowledgements References Studies in Natural Products Chemistry, Vol. 43. http://dx.doi.org/10.1016/B978-0-444-63430-6.00001-1 © 2014 Elsevier B.V. All rights reserved. 21 25 25 29 32 34 35 37 38 1 2 Studies in Natural Products Chemistry INTRODUCTION In recent years, a number of diterpenoid pyrones and related compounds have been isolated from microorganisms, particularly from fungal strains [1–6]. Several of these natural products have been reported to exhibit a wide variety of biological properties such as insecticidal [1,3c], antihypertensive [3d], bronchospasmolytic [3d], anti-inflammatory [3d], laxative [3d], anticancer [3e], and immunosuppressive activities [3–6]. In most cases, however, further biological studies including structure–activity relationships (SARs) are severely restricted probably because of the limited structural diversity of microorganisms. Consequently, the development of efficient and flexible synthetic methods for this class of natural products and related compounds is quite desirable and worthwhile from the viewpoint of medicinal chemistry and pharmaceuticals. In 2001, the Merck research group reported the isolation and structure elucidation of nalanthalide (1, Fig. 1) from the culture broth of Nalanthamala sp. [2]. This natural product was found to be a novel blocker of the voltage-gated potassium channel Kv1.3 (IC50 ¼ 3.9 mM) [7]. A closely related diterpenoid pyrone, sesquicillin (2), wherein the g-pyrone ring of 1 is replaced by an a-pyrone ring, was isolated from the culture broth of Acremonium sp. by Erkel et al. [3a]. This natural product was first classified as an inhibitor of glucocorticoid-mediated signal transduction [3a]. Sesquicillin has been reported to strongly induce G1 phase arrest in human breast cancer cell lines [3b]. Recently, four additional and new sesquicillin analogues (named sesquicillins B–E) were isolated from the culture broth of Albophoma sp. in addition to 2 (renamed sesquicillin A) [3c]. These substances were reported to exhibit insecticidal and cytotoxic activities [3c]. The gross structure and stereochemistry of 1 and 2 have been determined by extensive NMR spectroscopic studies [2,3a], but their absolute configurations have not been assigned. These natural products consist of a trans-decalin skeleton connected with a fully substituted g- or a-pyrone ring via a methylene linkage involving five asymmetric carbon centers [2,3a,c]. Subsequent to the discovery of nalanthalide, the structurally similar diterpenoid pyrones—candelalides A (3), B (4), and C (5)—were successively isolated from a culture broth of Sesquicillium candelabrum by the Merck research group in 2001 [5]. These natural products were also found to be novel blockers of the voltage-gated potassium channel Kv1.3 (IC50 ¼ 3.7 mM for 3, 1.2 mM for 4, 2.5 mM for 5) [5]. Kv1.3 channels plays pivotal roles in the control of membrane potential in human T cells, wherein it sets the resting potential. The blocking of Kv1.3 causes the membrane depolarization of human T cells, and this prevents Ca2+ entry required for T cell activation [7]. These processes lead to the diminution of lymphokine release and synthesis from the calciumdependent pathway, thus suppressing the activation and proliferation of human T cells [7]. Consequently, nalanthalide and candelalides A–C are expected to Chapter 1 Total Synthesis of Diterpenoid Pyrones 3 FIGURE 1 Structures of nalanthalide (1), sesquicillin (2), candelalides A–C (3–5), and subglutinols A (6), B (7). be promising new agents for the treatment of T cell-mediated autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, and insulin-dependent diabetes [2,5,7]. The gross structure and stereochemistry of 3–5 have been determined by extensive spectroscopic studies including 2D NMR experiments, whereas their absolute configurations have not been confirmed [5]. Candelalides A–C possess a novel tricyclic decahydro- or dodecahydro-1H-benzo[f] chromene skeleton (ABC ring system) connected to a fully substituted g-pyrone ring via a methylene linkage involving five to seven asymmetric carbons [5]. Interestingly, the candelalide with the most complex structure, 4, exhibits the most potent Kv1.3 blocking activity [5]. 4 Studies in Natural Products Chemistry In 1995, Strobel and Clardy et al. reported the isolation and structural elucidation of two novel immunosuppressive diterpenoid pyrones, subglutinols A (6) and B (7) from the endophytic fungus Fusarium subglutinans [6]. The immunosuppressive activities of 6 and 7 are comparable; they were found to be equipotent in the mixed lymphocyte reaction (MLR) assay (IC50 ¼ 0.1 mM) and the thymocyte proliferation (TP) assay (IC50 ¼ 0.1 mM) [6]. However, their mechanism of action remains unclear. In comparison, cyclosporine A showed similar potency in the MLR assay and was 104 more potent in the TP assay [6]. Hence, subglutinols are also anticipated to be promising candidates or new leads for novel classes of immunosuppressive agents [6]. The relative stereochemistry of 6 and 7 was determined by extensive NMR spectroscopic and X-ray diffraction analysis, but their absolute configurations were not established [6]. These diastereomeric natural products (6 and 7) possess a novel tricyclic dodecahydronaphtho[2,1-b]furan skeleton (ABC ring system) in which transfused AB rings are particularly characteristic features [6]. Owing to the unique structural features as well as the attractive biological properties, considerable attention has been focused on the total synthesis of the diterpenoid pyrones (1–7). In 2003, we embarked on a project directed at the total synthesis of this class of natural products with the aim of determining the unknown absolute configuration as well as disclosing the SARs. Our earnest endeavors culminated in completing the enantioselective total synthesis of 1 in 2006 [8,9], 2 in 2010 [9], 3 in 2005 [10,11], 4 and 5 in 2009 [11], and 6 and 7 in 2011 [12]. In these synthetic studies, the absolute configurations of 1–5 were established. The total synthesis of racemic sesquicillin [()-2] was accomplished by Zhang and Danishefsky [13]. Recently, Hong et al. reported the first total synthesis of 6 and 7 in an enantioselective manner, which led to the determination of their absolute configurations [14]. They also reported that 6 exhibits significant potential as an immunosuppressant with dose-dependent osteogenic activity [14]. In this chapter, the total syntheses of 1–7 are reviewed with a particular focus on synthetic strategy. TOTAL SYNTHESIS OF ()-SESQUICILLIN [ZHANG AND DANISHEFSKY, 2002 [13]] Synthetic Strategy Zhang and Danishefsky reported the first total synthesis of ()-2 [13]. Their retrosynthetic plan for ()-2 is illustrated in Scheme 1. The first crucial step in this contemplated scheme is envisaged to start with the stereoselective Eschenmoser– Claisen rearrangement [15] of allyl alcohol 13 to construct the requisite transdecalin portion 11 via intermediate 12. Rearrangement precursor 13 is accessible starting from ()-5-methyl-Wieland–Miescher ketone (15) via trans-decalone 14. The second critical step is envisioned to involve the aldol-type coupling reaction of methyl ester 10 with the known aldehyde 9 [16] to assemble the requisite Chapter 1 Total Synthesis of Diterpenoid Pyrones 5 P S SCHEME 1 Retrosynthetic plan for ()-sesquicillin [()-2] according to Zhang and Danishefsky. TBS, tert-butyldimethylsilyl. 6 Studies in Natural Products Chemistry carbon framework for a pyrone ring formation. The coupling product 8 would then be transformed to the target ()-2 by a-pyrone ring formation under basic conditions. Intermediate 10 would then be derived from the rearrangement product 11 by one-carbon homologation. Total Synthesis As shown in Scheme 2, the synthesis of intermediate 13, a precursor of the crucial Eschenmoser–Claisen rearrangement, was carried out starting from 14, E E SCHEME 2 Synthesis of intermediate 13. Tf, trifluoromethanesulfonate; PCC, pyridinium chlorochromate; DMSO, dimethyl sulfoxide; PPTS, pyridinium 4-toluenesulfonate; EE, ethoxyethyl. Chapter 1 Total Synthesis of Diterpenoid Pyrones 7 which was prepared from 15 in two steps [17]. Thus, compound 14 was converted to aldehyde 16 in 56% overall yield via a four-step operation including the stereoselective reduction of the C3 carbonyl group in 14, the protection of the resulting b-alcohol as its O-tert-butyldimethylsilyl (TBS) ether, hydroboration followed by oxidative treatment, and the pyridinium chlorochromate (PCC) oxidation of the resulting primary alcohol. The subsequent Wittig reaction of 16 using Ph3P+CHMe2I afforded olefin 17 (85%), whose ethylene acetal moiety was removed by acid treatment to furnish ketone 18 in 96% yield. Formylation at the C8 position of 18 followed by the protection of the hydroxy group in the resulting enol provided ethoxyethyl (EE) enol ether 19. The reduction of the C9 carbonyl group in 19 with NaBH4 and subsequent acid treatment formed aldehyde 20 in 80% overall yield from 18. Finally, the reduction of the formyl group in 20 with NaBH4 afforded requisite intermediate 13 in quantitative yield. After obtaining intermediate 13, the crucial Eschenmoser–Claisen rearrangement was investigated (Scheme 3). Thus, the treatment of 13 with N,N-dimethylacetamide dimethyl acetal in m-xylene at reflux temperature resulted in the formation of the expected rearrangement product 11 with an 87% yield and high stereoselectivity (>20:1) with respect to the C9 position. The subsequent superhydride reduction of the N,N-dimethylamide moiety in 11 followed by the mesylation of the resulting alcohol and cyanide displacement afforded nitrile 21 in 99% yield in three steps. Compound 21 was then converted to methyl ester 10 in 50% overall yield via a three-step sequence of reactions including the diisobutylaluminum hydride (DIBAL) reduction of the nitrile function, oxidation, and methyl esterification. The second crucial reaction was achieved by treating 10 with lithium diisopropylamide (LDA) followed by the addition of aldehyde 9 [16], forming the desired coupling product 22 in 62% yield. Compound 22 was further transformed to b,d-diketo ester 8 in 81% overall yield via a four-step sequence including Dess–Martin oxidation, the deprotection of the TBS group, the acetylation of the liberated hydroxy group, and the removal of the ethylene acetal moiety. Finally, compound 8 was subjected to 1,8-diazabicyclo[5.4.0] undec-7-ene (DBU)-induced enolization/lactonization, leading to the formation of the target ()-2 in 61% yield. This total synthesis was accomplished in 3.8% overall yield in 26 steps from ()-15. TOTAL SYNTHESIS OF ( )-NALANTHALIDE AND (+)-SESQUCILLIN [KATOH ET AL., 2006 [8], 2010 [9]] Total Synthesis of ( )-Nalanthalide Synthetic Strategy We reported the first total synthesis of naturally occurring 1 in 2006 [8,9]. Our retrosynthetic plan is outlined in Scheme 4. We envisioned that target molecule 8 Studies in Natural Products Chemistry S SCHEME 3 Synthesis of ()-sesquicillin [()-2]. Ms, methanesulfonyl; DIBAL, diisobutylaluminum hydride; TMS, trimethylsilyl; LDA, lithium diisopropylamide, DMAP, 4-dimethylaminopyridine; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene. Chapter 1 Total Synthesis of Diterpenoid Pyrones 9 C N Ref. [17] SCHEME 4 Retrosynthetic plan for ( )-nalanthalide (1) according to Katoh et al. 1 would be formed by a coupling reaction between the appropriately substituted trans-decalin aldehyde 25 (accessible from intermediate 26) and the fully substituted 3-lithio-g-pyrone 23 (available from the corresponding bromide 24). This coupling reaction is synthetically challenging at the synthetic chemistry level, because the C9 formyl group in decalin 25 lies in a sterically congested axial orientation. The advanced key intermediate 26, possessing both a hydroxymethyl group at C9 and an exo-methylene moiety at C8, would be formed by the strategic [2,3]-Wittig rearrangement [18] of stannylmethyl ether 28 (accessible from allyl alcohol 13), where we believed that the stereocenter at C9 and exomethylene function at C8 would be simultaneously formed via intermediate carbanion 27. Intermediate 13 should, in turn, be derived from 14 [17], which is readily prepared from enantiomerically pure 15 (>99% ee). 10 Studies in Natural Products Chemistry Total Synthesis At first, as shown in Scheme 5, we pursued the synthesis of intermediate 28, a substrate for the key [2,3]-Wittig rearrangement, starting from known enantiomerically pure 14 [17]. The route to allyl alcohol 13 from 14 has been established (30% overall yield in 11 steps) in Danishefsky’s total synthesis of ()-2 (cf. Section “Total Synthesis of ()-Sesquicillin [Zhang and Danishefsky]”) [13]. Therefore, we decided to follow the Danishefsky’s route with some improvements of the reaction steps and conditions (cf. 29 ! 30 ! 18, 19 ! [31] ! 20), which allowed an increase in the total SCHEME 5 Synthesis of the intermediate 28. Chapter 1 Total Synthesis of Diterpenoid Pyrones 11 yield of 13 (49% overall yield in 11 steps). Thus, the reduction of the C3 carbonyl group in 14 with LiAlH4 afforded the desired b-alcohol 29 in 98% yield as a single stereoisomer. The subsequent hydroboration of 29 followed by oxidative treatment and simultaneous protection of the two hydroxy groups in the resulting diol provided bis-TBS ether 30 in 85% yield in the two steps. Compound 30 was then converted to ketone 18 in 77% overall yield via a threestep operation including the selective deprotection of the TBS group on the C4 side chain and the ethylene acetal moiety at C9 by acid treatment, Dess– Martin oxidation, and Wittig olefination using Ph3P+CHMe2I . To introduce a formyl group at the C8 position, compound 18 was treated with HCO2Et in the presence of NaH to afford the corresponding enol, whose hydroxy group was protected as its EE ether to produce enol ether 19 in 92% yield in two steps. The subsequent NaBH4 reduction of the C9 carbonyl group in 19 followed by reaction with methanesulfonyl chloride (MsCl) provided the desired a,b-unsaturated aldehyde 20 (86% yield in two steps) via intermediate mesylate 31. Finally, compound 20 was converted to the requisite stannylmethyl ether 28 in 98% overall yield through a two-step sequence involving the NaBH4 reduction of the formyl group followed by the stannylmethylation [19] of the resulting alcohol 13 with n-Bu3SnCH2I in the presence of KH and 18-crown-6. With the key intermediate 28 synthesized, we next investigated the critical stereocontrolled [2,3]-Wittig rearrangement of 28 to construct the requisite decalin system 26, which has both a hydroxymethyl group at C9 with correct stereochemistry and an exo-methylene function at C8 (Scheme 6). After SCHEME 6 [2,3]-Wittig rearrangement of the stannylmethyl ether 28. 12 Studies in Natural Products Chemistry screening several reaction conditions, we found that the designed [2,3]-Wittig rearrangement proceeded smoothly and cleanly in a completely stereoselective manner by treatment with n-BuLi in hexane from 50 to 0 C for 12 h. The expected rearrangement product 26 was produced in 92% yield as a single stereoisomer with respect to the C9 position. The structure and stereochemistry of 26 were confirmed by extensive spectroscopic analysis including 600 MHz 1H NMR NOESY experiments. The stereochemical outcome observed for this [2,3]-Wittig rearrangement can be rationalized by considering that the attack of intermediate carbanion 27, generated in situ by the tin/lithium exchange of stannane 28, on the C9 olefinic carbon preferentially occurs from the less hindered a-face of the molecule under the influence of the b-oriented axial methyl group at the decalin junction, leading to 26 as the single product. Having obtained the requisite trans-decalin portion 26, we further investigated the synthesis of the first target 1 by assembling the trans-decalin and g-pyrone segments (Scheme 7). After the Dess–Martin oxidation of 26 (quantitative yield), the crucial coupling reaction of the resulting aldehyde 25 with 3-lithio-g-pyrone 23 (cf. Scheme 4) was successfully achieved by an initial bromine/lithium exchange of 3-bromo-2-methoxy-5,6-dimethyl-4Hpyran-4-one (24) and subsequent reaction with 25 from 78 to 55 C for 1 h. The desired coupling product 32 was obtained in 87% yield as a mixture of epimeric alcohols (ca. 1:1), which was very difficult to separate. It is noteworthy that the regiochemical integrity of the sensitive exo-methylene moiety at C8 was maintained during the coupling reaction. The removal of the sterically hindered hydroxy group from 32 was achieved by applying the Barton– McCombie procedure [20], forming the desired deoxygenated product 33 in 82% yield in two steps. Finally, the deprotection of the sterically congested TBS group in 33 by treatment with BF3Et2O followed by conventional acetylation provided 1 in 81% overall yield. The comparison of the optical rotations of the synthetic material and natural 1 led to the assignment of the absolute configuration of natural 1. This total synthesis was completed in 20.6% overall yield in 21 steps from 15. Total Synthesis of (+)-Sesquicillin Synthetic Strategy In 2010, we achieved the first total synthesis of naturally occurring 2 [9]. Our synthetic plan is outlined in Scheme 8. The g-pyrone moiety present in 1 is considered to be an equivalent to vinylogous methyl ester; therefore, the hydrolysis of this moiety followed by spontaneous tautomerization to a-pyrone would form 2 via the plausible intermediates 34 and 35. To the best of our knowledge, the method for the conversion of 1 to 2 was hitherto unknown; hence, this approach posed a considerable challenge from the synthetic viewpoint. Chapter 1 Total Synthesis of Diterpenoid Pyrones 13 N SCHEME 7 Synthesis of ( )-nalanthalide (1). AIBN, 2,20 -azobisisobutyronitrile. Synthesis Initial attempts to realize the direct conversion of 1 to 2 under conventional basic conditions (1 M NaOH, MeOH, rt ! reflux) were unsuccessful (Scheme 9). The expected hydrolysis of the g-pyrone moiety in 1 followed by the tautomerization of g-pyrone to a-pyrone proceeded smoothly and cleanly at reflux temperature; however, the unfavorable deprotection of the acetyl group occurred during the reaction, producing de-O-acetylsesquicillin (37) in good yield (83%). Therefore, we decided to pursue the synthesis of 2 in a step-by-step manner from de-O-acetylnalanthalide (36), which is the most advanced intermediate of the nalanthalide synthesis (cf. Scheme 7, but 14 Studies in Natural Products Chemistry N T S SCHEME 8 Synthetic plan for (+)-sesquicillin (2) according to Katoh et al. the structural formula is not shown). Thus, the hydrolysis of the g-pyrone moiety in 36 under basic conditions (1 M NaOH, MeOH, reflux) provided the desired product 37 (83% yield), having an a-pyrone ring, whose two hydroxy groups were simultaneously acetylated to form the corresponding diacetate 38 (88% yield). Finally, the chemoselective deprotection of the acetyl group on the pyrone ring in 38 under mild basic conditions resulted in the formation of 2 in 88% yield. This synthesis determined the absolute configuration of natural 2. This total synthesis was achieved in 14.5% overall yield in 23 steps from 15. TOTAL SYNTHESIS OF ( )-CANDELALIDE A–C [KATOH ET AL., 2005 [10], 2009 [11]] Total Synthesis of ( )-Candelalide A Synthetic Strategy In 2005, we accomplished the first total synthesis of 3 [10,11]. Our retrosynthetic plan is shown in Scheme 10. We envisioned that the target molecule 3 would be derived from hydroxy aldehyde 39 (accessible from disilyl ether 40) via intramolecular hemiacetal formation followed by dehydration. Chapter 1 15 Total Synthesis of Diterpenoid Pyrones N S SCHEME 9 Synthesis of (+)-sesquicillin (2). Intermediate 40 would be produced through a coupling reaction between the appropriately functionalized decalin aldehyde 41 (available from alcohol 42) and 3-lithio-g-pyrone 23. Intermediate 42 would be formed through the strategic [2,3]-Wittig rearrangement of stannylmethyl ether 43. On the basis of the results accumulated from the nalanthalide synthesis (cf. Section “Total Synthesis of ( )-Nalanthalide” and Scheme 6), we expected that the C9 stereogenic center and the C8 exo-methylene function in the product 42 would be simultaneously formed. Intermediate 43, in turn, would be derived from 14 [17]. Total Synthesis ( )-Candelalide A was synthesized starting from trans-decalone 14 as shown in Scheme 11. Thus, the stereoselective reduction of the C3 carbonyl group in 14 with L-selectride produced the desired a-alcohol 44 in 91% yield as a single stereoisomer. After the hydroboration of 44 followed by the deprotection 16 Studies in Natural Products Chemistry C C C Ref. [17] SCHEME 10 Retrosynthetic plan for ( )-candelalide A (3) according to Katoh et al. TES, triethysilyl. of the ethylene acetal moiety, the two hydroxy groups in the resulting ketone were differentially protected as TBS and triethylsilyl (TES) ethers, providing the corresponding disilyl ether 45 in 67% overall yield from 44. Formylation at the C8 position in 45 followed by the protection of the resulting enol as an EE ether furnished enol ether 46 in 92% yield in two steps. The subsequent NaBH4 reduction of 46 and the mesylation of the resulting alcohol produced the desired a,b-unsaturated aldehyde 47 in 88% overall yield. Compound 47 was further converted to the requisite stannylmethyl ether 43 in 84% overall yield via a two-step sequence involving the NaBH4 reduction of the formyl group and the stannylmethylation of the resulting alcohol. The crucial [2,3]-Wittig rearrangement of 43 via intermediate carbanion 48 proceeded smoothly and cleanly in a stereoselective manner, which gave Chapter 1 Total Synthesis of Diterpenoid Pyrones SCHEME 11—CONT’D 17 18 Studies in Natural Products Chemistry C SCHEME 11 Synthesis of ( )-candelalide A (3). TBAF, terea-n-butylammonium fluoride. the desired product 42 (78% yield) with a small amount of its C9 stereoisomer (not shown, 9% yield). The structure and stereochemistry of these rearrangement products were unambiguously confirmed by 1H NMR NOESY spectra. By the Dess–Martin oxidation of 42 (98% yield), the critical coupling reaction between the resulting aldehyde 41 and 3-lithio-g-pyrone 23 was successfully achieved. The expected coupling product 49 was obtained in 95% yield as a mixture of epimeric alcohols (ca. 8:1). The removal of the hydroxy group from 49 was carried out by applying the Barton–McCombie procedure [20], leading to the desired deoxygenated product 40 in 72% overall yield. Compound 40 was further transformed to aldehyde 50, a precursor of the key cyclization reaction, in 81% overall yield by the selective deprotection of the TBS group and the Dess–Martin oxidation of the liberated hydroxy group. The subsequent deprotection of the TES group in 50 with tetrabutylammonium fluoride (TBAF) resulted in the formation of the expected cyclized hemiacetal 51 in 99% yield via intermediate hydroxy aldehyde 39 (not isolated). Finally, the dehydration of 51 was achieved by reaction with MsCl and Et3N, leading to the target 3 in 87% yield. This synthesis established the absolute configuration of natural 3. The total synthesis of 3 was completed in 11.8% overall yield in 22 steps from 15. Chapter 1 Total Synthesis of Diterpenoid Pyrones 19 Total Synthesis of ( )-Candelalide B Synthetic Strategy In 2009, we presented the first total synthesis of 4, which represents the most complex structure among candelalides A–C [11]. Our synthetic plan was designed as shown in Scheme 12. We envisaged that target molecule 4 would be produced through the 6-exo cyclization of epoxy alcohol 52 followed by the inversion of configuration at the C12 hydroxy group. The advanced key intermediate 52 would be derived from aldehyde 53, which should be accessible from diene 54 by functional group manipulation and deprotection or vice versa. Intermediate 54 would be formed through the coupling reaction of decalin aldehyde 55 (accessible from alcohol 56) and 3-lithio-g-pyrone 23. Intermediate 56 would be prepared through the [2,3]-Wittig rearrangement of stannylmethyl ether 57 in the same manner asdescribed above. Intermediate 57 would be available from the common intermediate 44 (cf. Scheme 11). SCHEME 12 Retrosynthetic plan for ( )-candelalide B (4) according to Katoh et al. 20 Studies in Natural Products Chemistry Total Synthesis First, as shown in Scheme 13, we investigated the synthesis of the key intermediate 64, possessing the requisite carbon framework and functional groups. The synthesis was performed by a reaction sequence similar to that described in the section on the synthesis of 3 (cf. see Section “Total Synthesis” under Section “Total Synthesis of ( )-Candelalide A” and Scheme 11). Thus, the common intermediate 44 was converted to decalone 58 in 97% overall yield via a two-step operation involving the acid hydrolysis of the ethylene acetal moiety and the TES protection of the hydroxy group. Subsequent formylation at C8 in 58 and the EE protection of the resulting enol gave enol ether 59 in 88% yield in two steps. After the NaBH4 reduction of the C9 carbonyl group in 59, the resulting alcohol was subjected to dehydration, providing a,bunsaturated aldehyde 60 in 94% overall yield from 59. Compound 60 was then successfully converted to stannylmethyl ether 57 in 81% overall yield via a two-step sequence involving NaBH4 reduction and stannylmethylation. The critical [2,3]-Wittig rearrangement of 57 afforded the desired product 56 in 76% yield. After the Dess–Martin oxidation of 56 (98% yield), the coupling reaction of the resulting aldehyde 55 with 3-lithio-g-pyrone 23 was achieved under the same conditions described above. The desired coupling product 61 was obtained in 86% yield as an inseparable mixture of epimeric alcohols. The removal of the hydroxy group from 61 led to the desired deoxygenated product 54 in 72% overall yield. After the Lemieux–Johnson oxidation of 54 (85% yield), the resulting aldehyde 53 was allowed to react with a Grignard reagent (Me2C]CHMgBr) to furnish the desired products 62 (42% yield) and 63 (40% yield) as a mixture of epimeric alcohols, which can be separated by silica-gel column chromatography. The subsequent hydroxydirected epoxidation of 62 and 63 delivered the corresponding epoxides 64 (84% yield) and 65 (80% yield) as single diastereomers. We next investigated the final route that led to the completion of the total synthesis of 4 as shown in Scheme 14. Critical to the sequence was the construction of the highly substituted tetrahydropyran ring (A ring) present in 4. To this end, the TES protecting group in 64, which has favorable stereochemistry at the epoxide ring for the subsequent ether cyclization, was removed by treatment with TBAF at room temperature, which provided the liberated epoxy alcohol 52 in quantitative yield. The exposure of 52 to PPTS resulted in the formation of the requisite cyclization product 66 (79% yield). Finally, the inversion of the configuration at the C12 hydroxy group in 66 was achieved by oxidation with tetra-n-propyl ammonium perruthenate (TPAP) followed by the NaBH4 reduction of the resulting ketone with complete stereoselectivity, resulting in the production of the target 4 in 72% yield in two steps. The absolute configuration of natural 4 was determined by this total synthesis. The total synthesis of 4 was completed in 3.6% overall yield in 24 steps from 15. Chapter 1 Total Synthesis of Diterpenoid Pyrones 21 Total Synthesis of ( )-Candelalide C Synthetic Strategy In 2009, we also reported the first total synthesis of 5 [11]. Our retrosynthetic plan is outlined in Scheme 15. We envisioned that target molecule 5 would be produced by the construction of the tetrahydropyran ring (A ring) through the 6-exo cyclization of hydroxy epoxide 67, where the requisite stereogenic SCHEME 13—CONT’D 22 Studies in Natural Products Chemistry SCHEME 13 Synthesis of intermediate 64. acac, acetylacetonate; TBHP, tert-butyl hydroperoxide. center at C13 in 5 is established. Intermediate 67 would be derived from the common intermediate 50 (cf. Scheme 11) by sequential functional group manipulation and deprotection. Total Synthesis As shown in Scheme 16, we examined the synthesis of 5 starting from the common intermediate 50. The sequence involved the stereocontrolled formation of the isopropanol-substituted tetrahydropyran ring (A ring) present in 5 as the crucial step. Thus, to set up the requisite homoprenyl side chain at the C4 position, compound 50 was initially subjected to Wittig olefination using Ph3P+CHMe2I , which provided the desired product 68 (82% yield). The subsequent chemoselective epoxidation of the C13–C14 olefinic double bond in 68 was efficiently achieved by reaction with 1 equi. of 3-chloroperoxybenzoic acid (mCPBA) in the presence of NaHCO3, producing C SCHEME 14 Synthesis of ( )-candelalide B (4). TPAP, tetra-n-propyl ammonium perruthenate; NMO, 4-methylmorpholine N-oxide. E C SCHEME 15 Retrosynthetic plan for ( )-candelalide C (5) according to Katoh et al. 24 Studies in Natural Products Chemistry C E SCHEME 16 Synthesis of ( )-candelalide C (5). mCPBA, 3-chloroperoxybenzoic acid. the desired epoxide 69 in 98% yield as a mixture of diastereomers (a-/bepoxide 1:1). It is noteworthy that the C8 sensitive exo-olefin moiety remained intact during epoxidation. Finally, the removal of the TES protecting group from 69 by exposure to TBAF triggered the expected 6-exo cyclization of the liberated alcohol 67 to produce the target 5 as the sole product in 43% yield. Interestingly, another possible product 71 (13-epi-candelalide C) via the diastereomeric epoxide 70 was not obtained in this cyclization reaction. The completion of the total synthesis of 5 led to the absolute configuration of natural 5. This total synthesis was achieved in 4.7% overall yield in 22 steps starting from 15. Chapter 1 Total Synthesis of Diterpenoid Pyrones 25 TOTAL SYNTHESIS OF ( )-SUBGLUTINOLS A, B Total Synthesis of ( )-Subglutinol A [Hong et al., 2009 [14a], 2010 [14b]] Synthetic Strategy In 2009, Hong et al. reported the first total synthesis of naturally occurring ( )-subglutinol A (6), which led to the determination of the absolute configuration of natural 6 [14]. The retrosynthetic plan for 6 is shown in Scheme 17. The first crucial step in this scheme is envisaged to be the Cu(I)-mediated intermolecular SN20 alkylation of phosphate 77 (accessible from intermediate 78) with Grignard reagent 76 to construct intermediate 75, possessing both a 2-(1,3-dioxolan-2-yl)ethyl group at C9 with correct stereochemistry and an exo-methylene moiety at C8. Target molecule 6 would be derived from intermediate 75 via the olefin cross-metathesis of 75 followed by the aldol-type coupling reaction of methyl ester 74 with aldehyde 73, and the final formation of an a-pyrone ring on 72. The second critical step is envisaged to be the reductive deoxygenation of g-hydroxy ketone 79 to construct the characteristic tetrahydrofuran ring (A ring) with correct stereochemistry at the C12 position. Cyclization precursor 79 would be synthesized from the common intermediate 29 accessible from 15 (cf. Schemes 2 and 5). A similar strategy that included the aldol-type coupling reaction (74 + 73 ! 72) and the a-pyrone ring formation (72 ! 6) has been explored by Danishefsky and Zhang (cf. Section “Total Synthesis of ()-Sesquicillin [Zhang and Danishefsky]”), while the Cu(I)mediated SN20 reaction (77 ! 75) and the construction of the A ring (79 ! 78) are quite unique and challenging from a synthetic viewpoint. Total Synthesis The key intermediate 78 was prepared starting from 29 as shown in Scheme 18. Thus, ozonolysis of 29 provided lactol 80 in quantitative yield. The addition of (triisopropylsilyl)ethynyllithium (TIPSdC^CdLi) to lactol 80 followed by MnO2 oxidation led to g-hydroxy ketone 79, a key substrate for the subsequent cyclization reaction, in 85% yield in two steps. The crucial reductive cyclization of 79 was efficiently achieved by treatment with BF3 OEt2 in the presence of a reducing agent such as Et3SiH. The desired cyclized product 82 was obtained in 91% yield as a single diastereomer. In the reduction step, a hydride would be added to the oxocarbenium ion intermediate 81 from a direction opposite to the C17 methyl group. The deprotection of the TIPS group in 82 followed by the partial reduction of the alkyne function gave alkene 78 in 96% overall yield. The completion of the total synthesis of 6 is outlined in Scheme 19. Compound 78 was converted to allyl alcohol 86 by following the procedures established by Danishefsky et al. [13] and Katoh et al. [8–11]. Thus, the 26 Studies in Natural Products Chemistry P A S O R Ref. [17] SCHEME 17 Retrosynthetic plan for ( )-subglutinol A (6) according to Hong et al. TIPS, triisopropylsilyl. Chapter 1 Total Synthesis of Diterpenoid Pyrones 27 SCHEME 18 Synthesis of intermediate 78. acid-catalyzed acetal deprotection of 78 afforded ketone 83 in 91% yield. The formylation of 83 followed by EE-protection provided EE enol ether 84 in 67% overall yield. The subsequent reduction of 84 and dehydration gave aldehyde 85 (94% yield) in two steps. The further reduction of 85 afforded 86 in 95% yield. After the conversion of 86 to the corresponding phosphate 77 (92% yield), the crucial Cu(I)-mediated intermolecular SN20 addition of Grignard reagent 76 to 77 in the presence of CuI2LiCl provided intermediate 75 in 64% yield as a single diastereomer in good regioselectivity (SN20 /SN2 5:1). The simultaneous deprotection and oxidation of 75 using Jones reagent followed by ester formation, furnished methyl ester 87 in 44% yield in two steps. The olefin cross-metathesis of 87 with 2-methylpropene smoothly proceeded to give intermediate 74 in 91% yield. The subsequent aldol reaction of 74 with aldehyde 73 followed by Dess–Martin oxidation provided b-keto ester 72 in 53% yield in two steps. Finally, the deprotection of the 1,3-dithiane moiety in 72 followed by DBU-mediated cyclization resulted in the target 6 (53% yield in two steps), which led to the determination of the absolute configuration of natural 6. This total synthesis was accomplished in 2.1% overall yield in 24 steps from 15. 28 Studies in Natural Products Chemistry E A M S SCHEME 19 Synthesis of ( )-subglutinol A (6). Chapter 1 Total Synthesis of Diterpenoid Pyrones 29 Total Synthesis of ( )-Subglutinol A [Katoh et al., 2011 [12]] Synthetic Strategy In 2011, we reported the second total synthesis of 6 [12]. Our retrosynthetic plan is outlined in Scheme 20. We envisioned that target molecule 6 could be synthesized by the formation of the characteristic tetrahydrofuran ring (A ring) through an internal SN2-type cyclization of tosylate 88 followed by the conversion of the g-pyrone moiety into a-pyrone. In the cyclization step, we expected that the requisite stereogenic center at C12 in 6 would be stereospecifically established. Key cyclization precursor 88 would be prepared from decalin aldehyde 89 (accessible from alkene 90) by carbon chain extension at the C4 side chain. Relying on our previous studies on the synthesis of candelalides S Ref. [17] SCHEME 20 Retrosynthetic plan for ( )-subglutinol A (6) according to Katoh et al. Ts, 4-toluenesulfonyl. 30 Studies in Natural Products Chemistry A–C, intermediate 90 was expected to be synthesized by a coupling reaction of the appropriately substituted decalin aldehyde 91 with 3-lithio-g-pyrone 23. Intermediate 91 would be prepared from the common intermediate 29 available from 15 (cf. Scheme 5). Total Synthesis As shown in Scheme 21, we first pursued the synthesis of the advanced key intermediate 98 possessing the requisite hydroxyhomoprenyl side chain at C4 with the correct stereochemistry at C12. The route to aldehyde 89 from the starting material 29 is similar to that described in Section “Total Synthesis of ( )-Candelalide B” (from 44 to 53 in Scheme 13). Thus, the acid hydrolysis of the ethylene acetal moiety in 29 followed by the protection of the hydroxy group gave TES ether 92 in 93% yield in two steps. The subsequent formylation of 92 and EE-protection afforded EE enol ether 93 in 79% yield in two steps. After the NaBH4 reduction of 93, the resulting alcohol was dehydrated to give a,b-unsaturated aldehyde 94 in 83% overall yield from 93. Compound 94 was then converted to stannylmethyl ether 95 in 93% overall yield. The [2,3]-Wittig rearrangement of 95 resulted in the formation of alcohol 96 (75% yield), which was then oxidized to the corresponding aldehyde 91 (92% yield). The subsequent coupling reaction of 91 with 3-lithio-g-pyrone 23 afforded the expected product 97 in 93% yield as an inseparable mixture of epimeric alcohols (ca. 1:1). The removal of the hydroxy group from 97 provided the deoxygenated product 90 (72% yield in two steps), which was then subjected to site-selective Lemieux–Johnson oxidation to give the requisite aldehyde 89 in 77% yield. It is noteworthy that the sensitive exo-methylene moiety at C8 remained intact during Lemieux– Johnson oxidation event. To set up the requisite hydroxyhomoprenyl side chain at C4, compound 89 was allowed to react with a Grignard reagent (Me2C]CHMgBr), which provided the desired products 98 (12R, 71% yield) and its C12 epimer 99 (12S, 18% yield). The stereoselectivity of this nucleophilic addition can be explained, as depicted in 89A, by assuming that the attack of the nucleophile on the formyl group preferentially occurs from the less hindered Si-face under the influence of the bulky O-TES group at C3, giving the desired stereoisomer 98 as the major product. After obtaining the requisite intermediate 98, we then directed our attention to the synthesis of target 6 as shown in Scheme 22. The sequence involved the stereocontrolled formation of the tetrahydrofuran ring and subsequent conversion of the g-pyrone moiety into a-pyrone as the crucial steps. To this end, the removal of the TES protecting group from 98 followed by treatment with TsCl resulted in the formation of the desired cyclized product 99 in 80% overall yield as a single stereoisomer. We believe that the cyclized product 99 was formed from intermediate tosylate 88 (not isolated) Chapter 1 Total Synthesis of Diterpenoid Pyrones 31 through an internal SN2-type cyclization reaction. Finally, the hydrolysis of the g-pyrone moiety in 99 under basic conditions followed by the spontaneous tautomerization of g-pyrone to a-pyrone resulted in the formation of the target 6 in 90% yield. This total synthesis was achieved in 7.9% overall yield in 21 steps from 15. SCHEME 21—CONT’D 32 Studies in Natural Products Chemistry SCHEME 21 Synthesis of intermediate 98. S SCHEME 22 Synthesis of ( )-subglutinol A (6). Total Synthesis of ( )-Subglutinol B [Hong et al., 2009, 2010] Synthetic Strategy The first total synthesis of ( )-subglutinol B (7) was also achieved by Hong et al. in 2009 [14]. The absolute stereochemistry of this natural product was Chapter 1 Total Synthesis of Diterpenoid Pyrones 33 verified by this synthesis [14]. The retrosynthetic plan for 7 is outlined in Scheme 23. The most crucial step in this scheme is envisaged to be the tandem olefin cross-metathesis (CM)/SN20 reaction of the common intermediate 29 (cf. Scheme 5) to form the characteristic tetrahydrofuran ring (A ring) with correct stereochemistry at the C12 position (29 ! 101). Intermediate 101 would be transformed to target molecule 7 through the aldol-type coupling reaction of methyl ester 100 with aldehyde 73 in a manner similar to that described above (cf. Section “Total Synthesis of ( )-Subglutinol A [Hong et al., 2009, 2010]” and Scheme 19). Total Synthesis As shown in Scheme 24, the synthesis of the key intermediate 101 to ( )subglutinol B (7) began with the olefin cross-metathesis reaction of 29. Thus, the reaction of 29 with allyl chloride in the presence of the Grubbs secondgeneration catalyst and subsequent intramolecular SN20 reaction of the resulting hydroxy allyl chloride 102 (tandem CM/SN20 reaction) led to the desired cyclized product 101 in 53% yield as a single stereoisomer. Compound 101 was successfully converted to the target 7 via the aldol-type coupling reaction of methyl ester 100 with aldehyde 73 by employing a reaction sequence similar to that described in Section “Total Synthesis of ( )-Subglutinol A [Hong et al., 2009, 2010]” (cf. Scheme 19). This total synthesis was accomplished in 4.4% overall yield in 19 steps starting from 15. S Ref.[17] T SCHEME 23 Retrosynthetic plan for ( )-subglutinol B (7) according to Hong et al. 34 Studies in Natural Products Chemistry A S SCHEME 24 Synthesis of ( )-subglutinol B (7). Total Synthesis of ( )-Subglutinol B [Katoh et al., 2011] Synthetic Strategy We also accomplished the total synthesis of ( )-subglutinol B (7) in 2011 [12]. Our retrosynthetic plan for 7 is outlined in Scheme 25. The target molecule 7 would be produced through an internal SN2-type cyclization of tosylate 103, which should be available from the common intermediate 98 (cf. Scheme 22) by the inversion of configuration at the C12 hydroxy group. Total Synthesis The synthetic route to 7 starting from the common intermediate 98 is shown in Scheme 26. The crucial inversion of configuration at the C12 hydroxy group was successfully achieved via an oxidation/reduction sequence. Thus, the oxidation of 98 with TPAP provided ketone 104 in 88% yield. The expected stereoselective reduction was realized by treating 104 with DIBAL, affording the desired product 99 in 71% yield along with the undesired, but recyclable, 98 in 28% yield. In this reaction step, the possible transition state 104A is involved, for which hydride attack on the carbonyl group preferentially occurs from the less hindered Re-face. After the deprotection of the TES group in 99 (86% yield), the resulting diol 105 was subjected to the crucial SN2-type cyclization under the same conditions described previously (cf. 98 ! [88] ! 99 in Scheme 22). The desired product 106 was obtained in 83% yield as a single stereoisomer via the proposed intermediate tosylate Chapter 1 Total Synthesis of Diterpenoid Pyrones 35 S SCHEME 25 Retrosynthetic plan for ( )-subglutinol B (7) according to Katoh et al. 103 (not isolated). Finally, the alkaline hydrolysis of 106 gave the target 7 in 82% yield. This total synthesis was achieved in 3.5% overall yield in 24 steps from 15. CONCLUSION In this chapter, the total synthesis of biologically attractive diterpenoid pyrones—nalanthalide (1), sesquicillin (2), candelalides A–C (3–5), and subglutinols A (6), B (7)—has been summarized with particular focus on their synthetic strategies. It is of interest to look at each respective method for approaching the target molecules. Our method explored various features: (i) strategic [2,3]-Wittig rearrangement of stannylmethyl ethers (28 ! [27] ! 26 in Scheme 6, 43 ! [48] ! 42 in Scheme 11, 57 ! 56 in Scheme 13, 95 ! 96 in Scheme 21), (ii) coupling reaction of decalin portions with a common g-pyrone ring [25 + 23 (prepared from 24) ! 32 in Scheme 7, 41 + 23 ! 49 in Scheme 11, 55 + 23 ! 61 in Scheme 13, 91 + 23 ! 97 in Scheme 21], (iii) formation of the characteristic di- or tetrahydropyran rings and tetrahydrofuran rings (50 ! [39] ! 51 ! 3 in Scheme 11, 52 ! 66 in Scheme 14, 69 ! [67] ! 5 in Scheme 16, 98 ! [88] ! 99 in 36 Studies in Natural Products Chemistry S SCHEME 26 Synthesis of ( )-subglutinol B (7). Chapter 1 Total Synthesis of Diterpenoid Pyrones 37 Scheme 22, 105 ! [103] ! 106 in Scheme 26), and (iv) conversion of the g-pyrone moiety into the corresponding a-pyrones (36 ! 37 in Scheme 9, 99 ! 6 in Scheme 22, 106 ! 7 in Scheme 26). On the basis of the present study, we are currently synthesizing additional analogues of 1 and 3–7 with the aim of exploring their SARs. In addition, further investigations to identify the mechanism of action of 6 and 7 using the synthetic samples are in progress in our laboratories. ACKNOWLEDGEMENTS Our study was financially supported in part by a Grant-in-Aid Scientific Research on Priority Area (No. 17035073 and No. 18032065), a Grant-in-Aid Scientific Research (C) (No. 18590013 and No. 21590018), a Grant-in-Aid for High Technology Research Program at Private Universities (2005–2009), and a Grant-in-Aid for the Strategic Research Foundation Program at Private Universities (2010–2014) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (MEXT). ABBREVIATIONS acac AIBN DBU DIBAL DMAP DMSO EE IC50 LDA mCPBA Ms NMO NMR NOESY PCC PPTS TBAF TBHP TBS TES TIPS Tf TMS TPAP Ts acetylacetonate 2,20 -azobisisobutyronitrile 1,8-diazabicyclo[5.4.0]undec-7-ene diisobutylaluminum hydride 4-dimethyl- aminopyridine dimethyl sulfoxide ethoxyethyl half maximal (50%) inhibitory concentration lithium diisopropylamide 3-chloroperoxybenzoic acid methanesulfonyl 4-methylmorpholine N-oxide nuclear magnetic resonance nuclear overhauser effect spectroscopy pyridinium chlorochromate pyridinium 4-toluenesulfonate terea-n-butylammonium fluoride tert-butyl hydroperoxide tert-butyldimethylsilyl triethysilyl triisopropylsilyl trifluoromethanesulfonate trimethylsilyl tetra-n-propyl ammonium perruthenate 4-toluenesulfonyl 38 Studies in Natural Products Chemistry REFERENCES [1] Viridoxins A and B, see: S. Gupta, S.B. Krasnoff, J.A.A. Renwick, D.W. Roberts, J.R. Steiner, J. Clardy, J. Org. Chem. 58 (1993) 1062–1067. [2] Nalanthalide, see: M.A. Goetz, D.L. 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Slaughter, M.A. Goetz, Org. Lett. 3 (2001) 247–250. [6] Subglutinols A and B, see: J.C. Lee, E. Lobkovsky, N.B. Pliam, G. Strobel, J. Clardy, J. Org. Chem. 60 (1995) 7076–7077. [7] (a) For recent reviews on the voltage-gated potassium chanel Kv1.3 as a novel therapeutic target for autoimmune disorders, see: H. Wulff, Expert Opin. Ther. Targets 20 (2010) 1759–1765. (b) W. Nguyen, B.L. Howard, D.S. Neale, P.E. Thompson, P.J. White, H. Wulff, D.T. Manallack, Curr. Med. Chem. 17 (2010) 2882–2896. (c) H. Peng, D.J. Huss, J. Neurosci. 30 (2010) 10609–10611. (d) S. Rangaraju, V. Chi, M.W. Pennington, K.G. Chandy, Expert Opin. Ther. Targets 13 (2009) 909–924. (e) H. Wulff, M. Pennington, Curr. Opin. Drug Discov. Dev. 10 (2007) 438–445. [8] T. Abe, K. Iwasaki, M. Inoue, T. Suzuki, K. Watanabe, T. Katoh, Tetrahedron Lett. 80 (2006) 3251–3255. [9] T. Oguchi, K. Watanabe, H. Abe, T. Katoh, Heterocycles 80 (2010) 229–250. [10] K. Watanabe, K. Iwasaki, T. Abe, M. Inoue, K. Ohkubo, T. Suzuki, T. 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This page intentionally left blank Chapter 2 Chemical Diversity of Vibsane-Type Diterpenoids and Neurotrophic Activity and Synthesis of Neovibsanin Miwa Kubo, Tomoyuki Esumi, Hiroshi Imagawa and Yoshiyasu Fukuyama Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima, Japan Chapter Outline Introduction Vibsane-Type Diterpenoids The Stereochemistry of Vibsanins B (1) and C (2) The Absolute Configuration of Vibsanin F (3) 11-Membered Ring VibsaneType Diterpenoids 7-Membered Ring VibsaneType Diterpenoids Rearranged Vibsane-Type Diterpenoids (Neovibsanins) 41 42 43 45 49 50 Biological Activities of VibsaneType Diterpenoids Neurotrophic Activity of Neovibsanins Synthesis of Vibsane-Type Diterpenoids A Minimal Structural Core of Neovibsanin Required for Neurotrophic Activity Conclusion Acknowledgments References 61 62 64 72 75 75 75 56 INTRODUCTION The genus Viburnum consists of about 150 species of shrubs or small trees that were previously included in the family Caprifoliaceae. However, recent classifications based on molecular phylogeny have put them in the family Adoxaceae [1]. They are distributed in the temperate Northen Hemisphere, with a few species extending into tropical regions in South America and Studies in Natural Products Chemistry, Vol. 43. http://dx.doi.org/10.1016/B978-0-444-63430-6.00002-3 © 2014 Elsevier B.V. All rights reserved. 41 42 Studies in Natural Products Chemistry Southeast Asia, and about 15 species are distributed in Japan [2]. There is a long history of the folk medicinal use of Viburnum species. For example, the dried bark of V. opulus L., which is known as “Cramp Bark,” is used to alleviate painful menstrual cramps as well as a sedative [3]. Native American women took black haw (V. prunifolium L.) to treat the menopause and menstrual cramps [4]. The genus Viburnum has been documented to contain a variety of compounds such as iridoids, terpenoids, and aromatic compounds [5,6]. Among the chemical contents of Viburnum species, vibsane-type diterpenoids are considered to be characteristic of the Viburnum species because they have not been found in other higher plants. In this review [7], we focus on the structural diversity, biological activities, and synthesis of vibsane-type diterpenoids, in particular, synthetic studies and neurotrophic activity of neovibsanin A and B are emphasized. VIBSANE-TYPE DITERPENOIDS In 1980, Kawazu reported the isolation of vibsane-type diterpenoids from the leaves of Viburnum odoratissimum var awabuki (K. Kouh) Zabel ex Rumpler, and they were shown to consist of a unique fumulane skeleton with an additional C-5 unit, Fig. 1. However, there had been little interest in their chemical structure and biological activity since their discovery. Since 1996, we have continued to investigate the vibsane-type diterpenoids that are specific to Viburnum species as well as their biological activities, resulting in the discovery of about 60 new diterpenoids. They possess unique structures, some of which have unexpected chemical reactivity and interesting biological activity. The first vibsanetype diterpenoids that were reported by Kawazu were vibsanins A (17), B (1), and F (3), which possess an 11-membered ring, and vibsanins C (2)–E (16), which have a 7-membered ring, Fig. 2 [8]. Their stereochemistry has remained unexplored except for that of vibsanin E (16) [9]. Since these diterpenoids consist of a new carbon skeleton, we have proposed the new term of “Vibsane” for diterpenoids possessing a fumulane carbon framework with an additional isoprene unit [10]. 19 5 4 6 7 8 10 11 9 20 12 18 3 2 1 13 14 Fumulane FIGURE 1 Carbon skeletons of fumulane and vibsane. Vibsane 16 15 17 Chapter 2 Structural Diversity, Neurotrophic Activity, and Synthesis Vibsanin B (1) Vibsanin C (2) Vibsanin F (3) Vibsanin A (17) Vibsanin D Vibsanin E (16) 43 FIGURE 2 Vibsanins A–F, which were isolated from Viburnum awabuki by prof. Kawazu. The Stereochemistry of Vibsanins B (1) and C (2) It was necessary to determine the absolute stereochemistry of vibsanins B (1) and C (2) before discussing the structures of the newly isolated vibsane-type diterpenoids. The 1H NMR of 1 showed two kinds of broad signals at room temperature, but a pair of the sharp signals was observed at 0 C. This phenomenon indicated that vibsanin B (1) is present in solution as two conformational isomers [11]. Two conformers, 1a and 1b, were elucidated on the basis of NOESY data and J values, Fig. 3. The main conformer 1a demonstrated the NOE correlations shown in Fig. 3 and had a large J8,9 (9.3 Hz) value. These NMR data suggested that 1a adopts a chair-like conformation for the sequential bonds from C-5 to C-10 with a dihedral angle of 180 between C8-H and C9-H and takes a transoid geometry for the a,b-unsaturated ketone at C-4–C-6, whereas 1b consists of a boat conformation and has a cisoid form according 44 Studies in Natural Products Chemistry FIGURE 3 The two conformers, 1a and 1b, elucidated by NOESY, and the lowest energy conformers CT and BC obtained by MM2 calculations. The arrows show the NOE. to NOE analysis and its small J8,9 (2.2 Hz). Additionally, 1a and 1b were consistent with the two most stable conformers, CT and BC, found by the MM2 calculations [12]. In the course of the VT experiments for 1 in DMSO-d6, we found that 1 induced an irreversible change at 110 C. To elucidate this thermal transformation in detail, a solution of 1 in toluene was refluxed for 1 h to give rise to four products, which eventually were found to correspond to 7-membered ring vibsanin C (2) (85.9%) and its stereoisomers 2a (11.2%), 2b (1.6%), and 2c (0.2%), Scheme 1. Vibsanin C (2) and 5-epi-vibsanin C (2b) are natural products, but 2a and 2c have been not found in nature. The formation of the four 7-membered ring vibsanins can be ascribed to the oxy-Cope rearrangement of 1. The major product, vibsanin C (2) and its 5-epimer 2b, which contain a D8,9 E-olefin, are considered to rearrange through the CT and BC conformers, respectively; whereas, 2a and 2c, which contain a D8,9 Z olefin, are presumably transformed through the CC and BT conformers, which were found to be within 6 kcal/mol of the global minimum energy by MM2 calculations, Fig. 4. Thus, the absolute configuration of the 11-membered ring vibsanin B (1) turns out to be completely correlated with that of the 7-membered ring vibsanin C (2) via an oxy-Cope rearrangement. Next, the enol ester group of 2 was saponified under basic conditions, which was followed by an intramolecular aldol condensation reaction to give rise to the aldehyde 2d, which was converted to the bromophenyl carbamate 2e. X-ray crystallographic analysis of 2e unambiguously established the absolute 5S, 10S, and 11S configurations of vibsanin C (2), Scheme 2. This result means that vibsanin B (1) has chiral centers of 7R, 8R, and 11S. Thus, the absolute structures of 1 and 2, which were previously unsolved, have been established as shown in Fig. 5 [13]. Chapter 2 45 Structural Diversity, Neurotrophic Activity, and Synthesis O O OH O O Vibsanin C (2) (85.9%) O OH O O HO O OH O 110 °C O 2a (11.2%) Toluene O O Vibsanin B (1) O OH O O 2b (1.6%) O OH O O O 2c (0.2%) SCHEME 1 Oxy-Cope rearrangement of vibsanin B (1). The Absolute Configuration of Vibsanin F (3) Although vibsanin F (3), which was isolated from the leaves of V. odoratissimum var. awabuki in 1980, belongs to the simplest 11-membered ring structure of the vibsane-type diterpenoids, its stereochemistry has never been solved [8]. We have decided to unambiguously determine the absolute stereochemistry of 3 via its asymmetric synthesis [14]. Vibsanin F (3) has three chiral centers, among which C-7 and C-11 are anticipated to be 7S and 11S, respectively, based on those of vibsanin B (1) as shown in Fig. 6. However, the chirality of C-6 may be 3a (6S) or 3b (6R). 46 Studies in Natural Products Chemistry O O R1O HO R1O OH HO HO R2 R2 CC (491.1 KJ mol–1) BT (483.4 KJ mol–1) FIGURE 4 CC and BT conformers leading to 2a and 2c, respectively. OH O 5 O O 10 O 11 O (1) 2M NaOH MeOH (2) BrPhNCO DABCO toluene 1 OR s OHC s s 2d R = H 2e R = CONHPhBr Vibsanin C (2) 5 10 11 SCHEME 2 Conversion of 2 to the p-bromophenyl carbamate derivative 2e and an ORTEP diagram of the molecular structure of 2e. First, we selected 3a as the first synthetic target. The synthetic procedures used are outlined in Schemes 3 and 4. Asymmetric epoxidation of the allyl alcohol 4 by the Sharpless protocol [15] provided 5, which had 6S and 7S chiral centers that corresponded to those of 3a. Regioselective epoxidation of 5 with m-chloroperbenzoic acid exclusively gave the diepoxide 6 as a diastereomeric mixture. The primary hydroxyl group of 6 was converted to its triflate, with Chapter 2 47 Structural Diversity, Neurotrophic Activity, and Synthesis O OH O OH 7R O 10R O 11S O O HO 5S 8R 11S O Vibsanin C (2) Vibsanin B (1) FIGURE 5 Absolute configurations of vibsanins B (1) and C (2). OH O 7S OH O 7S 6S 6R 11S 11S 3b 3a FIGURE 6 Possible structures of 3a and 3b for vibsanin F (3). (a) HO HO O (b) HO 5 4 O O 6 O O O O O (d) (c) (e) O O OH O MeO2C CO2Me 7 MeO2C 8 9 SCHEME 3 Reagents and conditions: (a) Ti(OiPr)4, L-(+)-DET, TBHP, MS4A, CH2Cl2, 35 C, 99% (92% ee); (b) MCPBA, CH2Cl2, 0 C, 91%; (c) Tf2O, Et3N, THF, 78 C, then 5 equiv the dianion of methyl acetoacetate generated with NaH and n-BuLi, THF, 0 C, 94%; (d) NaH, 15-crown-5, DMSO, then 5-iodo-2-methylpent-2-ene, 77%; and (e) 10 mol% Pd(PPh3)4, DMSO, 90 C, 60%. which a large excess of the dianion of methyl acetoacetate was reacted at 0 C to give rise to 7 in high yield. The introduction of a 4-methyl-3-pentenyl unit using sodium hydride and 15-crown-5 in DMSO gave rise to the precursor 8 in good yield, which was required for the subsequent palladium-catalyzed macrocyclization. Subjecting 8 to the Tsuji–Trost reaction using 10 mol% Pd(PPh3)4 in DMSO afforded the sole product 9 in a moderate yield [16]. 48 Studies in Natural Products Chemistry O 9 O OH (a), (b) OTBDMS (c), (d) HO OTBDMS MsO 11 10 O O O OTBDMS (f), (g) CHO 12 OH (i) 14 OH OH O LiAlH 4 OH LiAlH 4 THF, reflux 45% 3a CHO (h) 13 O (e) THF, reflux 45% 15 Vibsanin F (3) SCHEME 4 Reagents and conditions: (a) TBDMSCl, Et3N, 4-DMAP, CH2Cl2, 74%; (b) LiAlH4, THF, 0 C; (c) MsCl, Et3N, 4-DMAP, CH2Cl2, 0 C; (d) DBU, toluene, 120 C, 60% over three steps; (e) NaBH4, DMPU, 55 C; (f ) TBAF, THF, 100%; (g) Dess–Martin periodinane, Et3N, CH2Cl2, 100%; (h) PhSH, AIBN, benzene, 90 C, 48%; and (i) NaBH4, CeCl3, MeOH, 0 C, 56%. The stereoselective formation of 9 can be explained by assuming that transition states A and B, as shown in Fig. 7, are involved in this cyclization. If the nucleophilic displacement of the p-allylpalladium intermediate with the anion of the b-ketoester moiety proceeds through a product-like transition state, transition state A, which should lead to the more stable product 9 with a pseudoequatorial C-6 unit and a pseudoaxial methyl group, probably favors over transition state B, producing a less stable product with the opposite stereochemistry at the quaternary center. Although the 11-membered ring was diastereoselectively constructed, the Z-geometry of the trisubstituted olefin in 9 has to be converted to E-geometry. First, the alcohol 9 was protected with TBDMSCl, before the reduction of both carbonyl groups with LiAlH4 to give rise to the diol 10, which was then mesylated, Scheme 4. The resultant dimesylate was subjected to elimination of the secondary mesylate under basic conditions, giving rise to the monomesylate 11 in a moderate yield over three steps. Subsequent reductive demesylation Chapter 2 49 Structural Diversity, Neurotrophic Activity, and Synthesis A B Pseudoaxial Pseudoaxial MeO O O O O 9 L Pd L OH MeO Pseudoequatorial O L O 9 Pd L HO Pseudoequatorial FIGURE 7 Possible transition states, A and B, for palladium-catalyzed cyclization of 8. of 11 was successfully achieved using a NaBH4–DMPU system, resulting in the formation of 12 in good yield. The aldehyde 13, which was derived from 12, was treated with AIBN and thiophenol to produce the desired E-olefin 14. Finally, the conjugate aldehyde in 14 was reduced by the Luche protocol to afford 3a. The 1H NMR of 3a, however, was not identical to that of natural vibsanin F (3). Thus, each epoxide ring of the synthetic product 3a and vibsanin F was reduced with LiAlH4, resulting in the preparation of the same diol 15. All the spectroscopic data for both diols were identical to each other, and therefore, the absolute configuration of vibsanin F has been established to be 3b with 6R, 7S, and 11S forms. 11-Membered Ring Vibsane-Type Diterpenoids Vibsane-type diterpenoids consist of three subtypes, 11-membered ring, 7-membered ring, and the rearranged types. These diterpenoids occur exclusively in V. odoratissimum var awabuki, V. odratissimum Ker Gawl, V. suspensum Lindl, and V. sieboldi Miq. The 11-membered ring and 7-membered ring vibsane-type diterpenoids are common to these plants. Most 11-membered ring vibsanins consist of a fumulane-like skeleton containing a b,b-dimethylacrylate group at the C-8 position except for vibsanin F (3). Vibsanin A (17) [8] and vibsanins P (18)–T (22) [17], which contain oxidatively modified C-6 units at the C-11 position, were isolated from both Japanese and Taiwanese V. odoratissimum as shown in Fig. 8. Since the conformations of the 11-membered ring vibsanins A and P–T, which bear a 6,7-epoxide ring, are fixed, analysis of their NMR can be performed normally. On the other hand, vibsanin B (1) and vibsanols A (23) and B (24) [18], which contain a cross-conjugated diene, show complex NMR signals due to the presence of several kinds of conformational isomers. Therefore, careful structure elucidation should be performed by a combination of VT NMR experiments and MM2 calculations [13]. 50 Studies in Natural Products Chemistry O OH R1 O OH O OH O O O O R2 R1 R2 Vibsanin B (1) Vibsanin A (17) CH2OH OH Vibsanin P (18) CH2OH O OH O Vibsanin Q (19) CH2OH OMe OH Vibsanol A (23) Vibsanin R (20) CH2OH O OH OH OH O Vibsanin S (21) CH2OH O Vibsanin T (22) CHO OH HO Vibsanol B (24) FIGURE 8 11-Membered ring vibsanins. 7-Membered Ring Vibsane-Type Diterpenoids The 7-membered ring vibsanins are made up of a diverse range of compounds, which possess two ketones and an isoprene unit attached to the 7-membered ring. There are two basic types, the (5S,10R) and (5R,10R) stereoisomers. Vibsanins C (2), G (25), H (27), and 18-O-methylvibsanin G (26), which belong to the (5S,10R) type, occur commonly in Viburnum species [19,20]. Vibsanins I (30), J (31), K (32), and 18-O-methylvibsanin K (33) [21] as well as 14,15-epoxyvibsanin C (34) were isolated from V. odoratissimum var. awabuki in Tokushima [19,21] and V. odoratissimum in Taiwan, respectively. On the other hand, 5-epi-vibsanins C (35), H (36), I (38), K (39), and their 18-Omethyl and/or 15-O-methyl congeners 37 and 40 [22] have been found in all Viburnum species except for V. suspensum, Fig. 9A. Vibsanin M (41), which possesses a D4,5 double bond [20] and a bicyclic vibsanin N (46) were isolated from V. odoratissimum and V. odoratissimum var awabuki in Taiwan [23]. Furthermore, aldovibsanins A (42)–C (45) as depicted in Fig. 9B were isolated from V. odoratissimum [20,24]. Chapter 2 51 Structural Diversity, Neurotrophic Activity, and Synthesis Additionally, interesting tricyclic 7-membered vibsanins are shown in Fig. 10. Vibsanin E (16) and 16-hydroxyvibsanin E (47) [25], which were isolated from V. odoratissimum var awabuki, have an ether bond between C-15 and C-18; whereas, cyclovibsanins A (48) and B (49), 15-Omethylcyclovibsanin B (50), and 3-hydroxy-15-O-methylcyclovibsanin A (51), which are composed of a tricyclo[6.3.2.00,0]tridecane skeleton, contain CdC bonds between C-18 and C-16 or C-17. Cyclovibsanins have no oxygen atom between C-18 and C-15 [26]. A OR1 O 18 5 O O OR1 O 5 O O 10 O O R2 R2 R1 R2 R1 R2 OH Vibsanin G (25) H OH 18-O-methylvibsanin G (26) Me Vibsanin H (27) H 5-Epi-vibsanin C (35) H 5-Epi-vibsanin H (36) H OH 5-Epi-15-O-methylvibsanin H (37) Me 5-Epi-vibsanin I (38) H OMe OH 15-O-methylvibsanin H (28) 15,18-O-dimethylvibsanin H (29) H 15 OMe Me OMe OOH Vibsanin I (30) OOH 5-Epi-vibsanin K (39) H OOH 5-Epi-18-O-methylvibsanin K (40) H Me OOH O Vibsanin J (31) H Vibsanin K (32) H O O OH O OOH 18-O-methyl- Me vibsanin K (33) 14,15-epoxyvibsanin C (34) OOH O Vibsanin M (41) H O FIGURE 9 (A) 7-Membered ring vibsanins. (Continued) 52 Studies in Natural Products Chemistry B H R2 OH O O H OH R1 OHC H OHC H Aldovibsanin B (44) Aldovibsanin A (42) R1 = Me, R2 = OH R1 = 7-Epi-alodvibsanin A (43) H OH, R2 = Me OH O O CO2H O O O OHC H H OH OH Vibsanin N (46) Aldovibsanin C (45) FIGURE 9—CONT’D (B) Another 7-membered ring vibsanins. O O O 3 Vibsanin E (16) 18 O O O 3-Hydroxyvibsanin E (47) O R R=H R = OH R3 O O Cyclovibsanin A (48) 15-O-methylcyclovibsanin A (49) 15-O-methylcyclovibsanin B (50) 3-Hydroxy-15-O-methylcyclovibsanin A (51) R1 15 R2 R1 = Me, R2 = OH, R3 = H R1 = Me, R2 = OMe, R3 = H R1 = OMe, R2 = Me, R3 = H R1 = Me, R2 = OMe, R3 = OH FIGURE 10 7-Membered ring tricyclic vibsanin. Vibsanin E (16) can be readily converted from vibsanin C (2) via a cationic process (a) by treating it with BF3OEt2 as shown in Scheme 5 [25]. On the other hand, another plausible biosynthetic pathway for cyclovibsanins is (b) as indictaed in Scheme 5, in which a proton is eliminated from one of two methyl groups in 52 to give rise to 52a. Dehydration produces the exomethylene ketone 52b, the C-4 carbonyl group of which is then protonated to trigger cyclization through the cationic intermediate 52b, resulting in the formation of a tricyclic framework such as that seen in the cyclovibsanins. Chapter 2 F3B 53 Structural Diversity, Neurotrophic Activity, and Synthesis O O– OH BF3OEt2 O RO OH O CH2Cl2, –78 °C a RO b H 2 b R = COCH=CMe2 52 50% a O– OH –H 2 O O H RO O OH O O RO 16 52a 52b O RO O O Cyclovibsanins RO 52c SCHEME 5 Conversion of vibsanin C (2) to vibsanin E (16) and a plausible biosynthetic pathway for cyclovibsanins. Furanovibsanins, a diverse range of 7-membered ring vibsanins, which are presumed to be produced by two ketones at the C-4 and C-7 positions, have also been found. The examples shown in Fig. 11 are furanovibsanin A (53) and its 3-O-methyl congener 54, which are presumably derived from 3-hydroxyvibsanin E. The additional examples are furanovibsanin B (55) and its 7-epimer 56, and furanovibsanins C (57)–G (61). These diterpenoids were isolated from V. odoratissimum var awabuki, collected in Tokushima [27]. We have already proposed such a plausible biogenetic pathway for three subtypes of vibsane-type diterpenes as vibsanin B (1) could be transformed into vibsanin C (2) by a Cope-type reaction, based on the results of thermal reactions of 1 [13,19]. Additional isolation of these furanovibsanins compel us to elaborate their biosynthetic process after 2 is produced. Thus, our proposed biosynthetic sequences leading to the furanovibsanins from 2 are outlined in Schemes 6 and 7. Biogenetic conversion of compounds 53–56 and 60 from vibsanin C (2) can be rationalized by a cationic process like (a) in Scheme 6 followed by an acetal formation between C-4 and C-7 ketones and intramolecular addition of oxygen nucleophiles. This is based on the fact that this type of tricyclic formation can be readily realized by 54 Studies in Natural Products Chemistry O R1 OR O R2 O CHO O O O O OMe H Furanovibsanin A (53) R = H Furanovibsanin B (55) R1 = Me, R2 = OMe 3-O-methylFuranovibsanin A (54) R = Me 7-Epi-furanovibsanin B (56) R1 = OMe, R2 = Me OMe OMe O O CHO O CHO O O O H H Furanovibsanin D (58) Furanovibsanin C (57) OMe MeO O O OMe O O O OMe O O Furanovibsanin E (59) Furanovibsanin F (60) O OMe CH2OH O OMe O Furanovibsanin G (61) FIGURE 11 7-Membered ring furanovibsanins. BF3OEt2-mediated conversion of 2 to a tricyclic vibsanin E (16) [25]. On the other hand, compounds 57 and 58 are not likely to follow a cationic cyclization (a) since there is no proof where an isopropyl group on the C-14 position originates. One possible way is that a protonation onto the D14 double bond in 2 may produce less stable secondary cation on C-14, which can trigger a cyclization to result in the formation of cyclohexane ring having a isopropyl Chapter 2 55 Structural Diversity, Neurotrophic Activity, and Synthesis H+ OH O 7 4 5 O Vibsaninin C (2) O O RO 15 14 OH OH (a) 2 RO OH O 18 R = COCH RO CMe2 A (b) 57 and 58 O O OH CHO H O 53–56, and 60 H OH O + O RO O RO H RO H B C SCHEME 6 Plausible biosynthetic pathway of furanovibsanins 53–58 and 60. O O OH O 7 4 O OH O 18 5 O2 O OH 2 RO 15 14 RO RO 2 D E c) d) OH O OH2 OH OH H+ OR A O OR d) (c) OH2 c) -H2O OR (d) RO -H2O F 59 61 G SCHEME 7 Plausible biosynthetic pathway of furanovibsanins 59 and 61. group on the C-14 position, but the polarization of the D2 double bond which must participate in this cyclization is reverse and thus this type of cationic process is not likely to occur. As vibsanin C is produced from vibsanin B by the Cope-type reaction, compounds 57 and 58 are also postulated to be formed via a nonionic process. Namely, intramolecular ene reaction might involve in the formation of a cyclohexane ring between C-2 and C-14 as well as of an aldehyde function at C-18. Following pathway (b) as outlined in Scheme 6, it is postulated that the prenyl side chain of vibsanin C could be cyclized onto C-2 by an intramolecular ene reaction, resulting in the formation of a bicyclic 56 Studies in Natural Products Chemistry aldehyde B, and then its tautomer C could make an acetal giving rise to 57 and 58. To be precise, compound 57 should be derived through process (b) in Scheme 6 from 5-epi-vibsanin C (35) [22]. This nonionic process can rationalize not only where an isopropyl group originates but also why bicyclic 7-membered ring vibsanins bearing an isopropyl group such as 57 and 58 accompany an aldehyde group on the C-18 position. We postulate that compounds 59 and 61 may be formed from vibsanin C (2) by oxidation process. Since vibsane-type diterpenes bearing a hydroperoxide group were frequently isolated from V. odoratissimum var awabuki, [22,28], we envisaged biogenetic pathway involving an oxidation step as outlined in Scheme 7. Namely, intramolecular acetal formation between C-4 and C-7 ketones leads to furan D, and then autoxidation of furan ring occurs to give endo-peroxide E, followed by a series of ring opening reaction, dehydration and addition of alkoxyl groups, giving rise to 59 and 61 via pathways (c) and (d), respectively. However, we have no evidence to support this biosynthetic route leading to 59 and 61. Rearranged Vibsane-Type Diterpenoids (Neovibsanins) The rearranged vibsane-type diterpenoids (neovibsanins), which contain a b,b-dimethylacrylate ester and an isoprene unit-substituted cyclohexene ring core fused to a tetrahydrofuran ring, are rarely occurring natural products as shown in Fig. 12. In 1996, the first rearranged vibsane-type diterpenoids, neovibsanins A (62) and B (63), were isolated from V. odoratissimum var awabuki [11]. Recently, we have ascertained the presence of neovibsanin in the fresh leaves of V. odoratissimum var awabuki and also found that neovibsanin (86) is changed to neovibsanins A (62) and B (63) when kept in methanol at room temperature, indicating that neovibsanins A and B are artifacts derived from neovibsanin [21]. Since neovibsanins A (62) and B (63) were reported, a number of rearranged vibsane-type diterpenoids have been found in V. odoratissimum var awabuki, V. suspensum, and V. sieboldi, and have become known as a characteristic compound group of the Viburnum species as summarized in Fig. 12. Among them, neovibsanin C (64) is the first example of a natural product with a macrocyclic structure formed through an endo-peroxide group [29]. The unusual structure of 64 was established by converting into it from neovibsanin B (63) as outlined in Scheme 8. The reduction of 64 with Zn in EtOH-AcOH resulted in the formation of the diol 83, which was treated with methanol under acidic conditions to give rise to the methyl acetal 84. The acetal 84 was also derived from neovibsanin B (63) by photosensitized oxidation, followed by reduction of the formed peroxy group [7-epi-neovibsanin D (66)]. In addition, when 66 was treated with pTsOH in anhydrous benzene, 64 was generated in good yield, presumably by an acetal exchange reaction. Thus, the structure of neovibsanin C (64) including its absolute configuration was established, Scheme 8. H O H R1 O HO O H O R2 O O O O O O O O O O Neovibsanin (86) Neovibsanin C (64) Neovibsanin A (62) R1 = OMe, R2 = Me Neovibsanin B (63) R1 = Me, R2 = OMe H R1 O O R2 O O O O O O O O O O OR OR OOH Neovibsanin D (65) R1 = OMe, R2 = Me 7-Epi-neovibsanin D (66) R1 = Me, R2 = OMe O O Neovibsanin H (67) R = H Neovibsanin I (69) R = H 2-O-methylneovibsanin H (68) R = Me 2-O-methylneovibsanin I (70) R = Me O O O O O H 14-Epi-neovibsanin F (73) R = H OH 14-Epi-18-oxoneovibsanin F (75) 14-Epi-15-O-methylneovibsanin F (74) R = Me O O O O O O O O O O H O OR 15-O-methylneovibsanin F (72) R = Me O O O OR Neovibsanin F (71) R = H O O O O O OMe H 15-O-methyl-18-oxoneovibsanin F (76) Neovibsanin G (77) 14-Epi-neovibsanin G (78) 6 MeO O O 7 O MeO O O O O O H O H Neovibsanin J (79) O Neovibsanin K (80) Neovibsanin P (81) O O O O O OMe Spirovibsanin A (82) FIGURE 12 Rearranged vibsane-type diterpenoids (neovibsanins). O 58 Studies in Natural Products Chemistry H O O O EtOH-AcOH O O O Zn O O H HO O O OH Neovibsanin C (64) 83 p-TsOH benzene p-TsOH MeOH H O MeO H O O O O Ph3P O Benzene MeO O O OOH 7-Epi-neovibsanin D (66) OH 84 O2, Rose Bengal hν H O O MeO O O Neovibsanin B (63) SCHEME 8 Chemical correlation of neovibsanin B to neovibsanin C. Neovibsanins H (67) and I (69) [30], and their 2-O-methyl congeners 68 and 70 are neovibsanins that do not possess an acetal group. Similar compounds, neovibsanins F (71) and its 14-epimer 73, and 14-epi-18oxoneovibsanin F (74) which contain an additional 6-membered ring were isolated from V. suspensum [30]. Spirovibsanin A (82) is the first example of a nor-vibsane-type diterpenoid [28]. Recently, neovibsanins J (79), K (80), and P (81) as depicted in Fig. 12, in which two hydroxy groups at the C-4 and C-18 positions are involved in acetal formation on the C-7 carbonyl, were found [31]. It has been shown that a 7-membered ring vibsanin C (2) can be derived from vibsanin B (1), an 11-membered ring vibsane-type diterpenoid, by Chapter 2 59 Structural Diversity, Neurotrophic Activity, and Synthesis thermal oxy-Cope rearrangement. However, no formation of neovibsanins was observed even upon heating 1 under acidic or basic conditions. It should be noted that neovibsanins A (62) and B (63) can be produced from vibsanin B (1) by photochemical reaction. Surprisingly, irradiation of vibsanin B (1) in benzene with a high pressure Hg lamp afforded 87, which possesses a neovibsanin-framework, in 4% yield, along with 85 (18%) and 2c (27%), Scheme 9 [11]. These compounds have been never found in natural sources. OH O O OH O O O RO H OH RO RO 85 (18%) 87 (4%) 2c (27%) hν benzene 1h O OH OH RO R: COCH=C(CH3)2 1 hν MeOH 1h R1 H O OH OH O R2 OHC O O MeO RO MeO Neovibsanin A (62) (12%) Neovibsanin B (63) (20%) 88 (9%) SCHEME 9 Photochemical reaction of vibsanin B (1). 89 (8%) 60 Studies in Natural Products Chemistry When the photochemical reaction of 1 was carried out in MeOH, neovibsanins A (62) and B (63) were produced in 12% and 20% yields, respectively, in addition to 88 (9%) and 89 (8%) as over-reacted products [32]. The generation of 88 and 89 was presumably due to a series of fragmentation and cationic cyclizations triggered by the methanolysis of a b,b-dimethylacryl ester group [32]. Furthermore, irradiation of 1 in 50% aqueous MeOH for 1 h directly yielded neovibsanin (86). This photochemical reaction of 1 forces the E/Z isomerization of the double bond at C-5 to generate (5Z)-vibsanin B (85) as shown in Scheme 9. The MM2 calculations for 85 and 1, in which the C-12–C-17 side chain was replaced with a t-butyl group, were performed using MacroModel® [12] and provided the most stable conformers, 1-(5Z) and 1-(5E), for each molecule as shown in Fig. 13. In the case of 1-(5Z), the distance between the C-4 carbonyl and the C-7 OH is 1.65 Å, whereas 1-(5E) has a distance of 4.98 Å as depicted in Fig. 13. This means that a 1,7-hydrogen shift from the OH group at C-7 to the carbonyl at C-4 readily occurs in 1-(5Z), but not in 1-(5E). This hydrogen shift not only causes a breakage of the C-7/C-8 bond as well as cyclization between C-10 and C-4, leading to the neovibsanin-framework, but also undergoes another oxy-Cope rearrangement via 85 to give rise to a 7-membered ring (8Z)-10-epi-vibsanin C (2c), which is not formed by the thermal oxyCope rearrangement of vibsanin B (1). Taking the aforementioned results into consideration, we wish to propose plausible biosynthetic mechanisms for neovibsanins, as outlined in Scheme 10. The biosynthesis of all neovibsanins would start from the key 5 7 4 1.65 Å 8 9 5 4 7 10 8 1-(5Z) (450.7 kJ mol–1) 9 10 1-(5E) (461.8 kJ mol–1) FIGURE 13 The most stable conformers, 1-(5Z) and 1-(5E), for (5Z)-vibsanin B (85) and vibsanin B (1), respectively, obtained by MM2 calculation. Chapter 2 O H+ O H O 61 Structural Diversity, Neurotrophic Activity, and Synthesis a OH H OH H+ HO RO O HO O a b RO RO 1 b B A b R = COCH=CMe2 H H O HO O d O a O O O RO RO OHR1 RO c d E D C c Neovibsanins A – D Neovibsanins F and G Neovibsanins H and I SCHEME 10 Plausible biosynthetic pathways for the formation of various neovibsanins from vibsanin B (1). intermediate A, which is derived from vibsanin B (1). The C-18 hydroxy group undergoes a 1,4-addition to yield B. In the case of route a, a hemiacetal E is generated, leading to neovibsanins A–D. Likewise, route b gives the intermediate allyl cation C prior to dehydration, which is trapped by some nucleophiles such as water to afford neovibsanins H and I (route c) and causes cationic cyclization to give D, resulting in the formation of neovibsanins F and G (route d). Additionally, neovibsanins J and K are presumably converted from the key intermediate A via the production of a bicyclic acetal of the C-7 carbonyl containing C-18 and C-4 hydroxy groups [31]. However, a question still remains about how the key intermediate A is generated from vibsanin B (1) by enzyme-catalyzed reaction. BIOLOGICAL ACTIVITIES OF VIBSANE-TYPE DITERPENOIDS The leaves of V. odoratissimum var awabuki had been used as a fish poison for catching fishes in Okinawa and Southeast Asia for a long time. In 1980, Kawazu reported the first isolation of a piscicidal compound, vibsanin A, 62 Studies in Natural Products Chemistry and a plant growth inhibitor, vibsanin B (1) [8]. Later, many vibsane-type diterpenoids were reported, among which some diterpenoids such as vibsanin C (2), 5-epi-vibsanin C (35) [22], vibsanol A (23) [18], and vibsanins K (32) and P (18) [20,33] exhibited significant and/or moderate cytotoxicity against tumor cells. Additionally, vibsanin B (1) and neovibsanin F (71) showed moderate toxic activity in a brine shrimp lethality assay [30]. Neurotrophic Activity of Neovibsanins A remarkable pathological symptom of Alzheimer’s disease (AD) is the loss of neuronal cells in the brain. Correspondingly, the overall strategy for treatment of AD is to prevent neuronal death or to produce new neuronal cells in the degenerative regions. Neurotrophins, such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), and glial cell line-derived neurotrophic factor (GDNF), are recognized as important regulatory substances in the nervous system [34]. Thus, neurotrophins are expected to have therapeutic efficacy for the treatment of AD. However, they cannot cross brain–blood barrier because of the property of their high molecular polypeptide and are easily metabolized by peptidases under physiological conditions [35]. To address this issue, considerable efforts have been made to find small molecules that have neurotrophic properties or are capable of enhancing the action of NGF in appropriate cell populations [36]. Rat pheochromocytoma (PC12) cells have been used as a good in vitro model of neuronal differentiation. After stimulation with NGF, PC12 cells differentiate to extend neurites and develop the characteristics of sympathetic neurons [37]. For example, two iridoids, picrosides I and II [35] and some clerodanetype diterpenoids, 6a,7a-dihydroxyannonene, and 7a, 20-dihydroxyannonene [38] have been demonstrated to show neurite outgrowth-promoting activity in NGF-mediated PC12 cells. Also some synthetic compounds, N-benzyloxycarbonyl-Leu-Leu-leucinal (ZLLLal) [39], AIT-082,39 SR577 [37,40] and Aroclor 1254 [41], have been reported to accelerate the action of NGF in PC12 cells. Recently, we have found that neovibsanin (86), and neovibsanins A (62) and B (63) have neurotrophic properties. Namely, they promote neurite outgrowth of NGF-mediated PC12 cells at concentrations ranging from 10 to 40 mM [42,43]. Evaluation was carried out for PC12 cells neurite outgrowth according to a previously reported experiment procedure [37,44]. As shown in Fig. 14, neovibsanin (B), neovibsanin A (C), and neovibsanin B (D) significantly promoted neurite outgrowth from NGF (10 ng/mL)-treated PC12 cells at 40 mM. Among three compounds, neovibsanin A seems less potent than neovibsanin and neovibsanin B. However, all of them had no effect on morphology of PC12 cells in the absence of NGF. Additionally, other vibsane-type Chapter 2 Structural Diversity, Neurotrophic Activity, and Synthesis A B C D 63 FIGURE 14 Morphology of PC12 cells under treatments of neovibsanin (86), neovibsanin A (62), and neovibsanin B (63) PC12 cells were cultured in DMEM/2% HS + 1% FBS and treated by (A) NGF 10 ng/mL, (B) neovibsanin 40 mM + NGF 10 ng/mL, (C) neovibsanin A 40 mM + NGF 10 ng/mL, and (D) neovibsanin B 40 mM + NGF 10 ng/mL. diterpenoids such as 7-membered and 11-membered ring subtypes have not been so far found to show neurotrophic activity in PC12 cells. In Fig. 15, quantitative analysis of the percentage of cells with neurites and the neurite length extending from the cell bodies indicated that neovibsanin (86) and neovibsainin B (63) significantly increased the percentage of PC12 cells bearing neurites and the neurite length compared with those of NGFmediated PC12 cells at concentrations ranging from 5 to 40 mM, and, in the degree of activity, neovibsainin B (62) was likely to be less that neovibsanin (86). On the other hand, neovibsanin B (63) promoted efficiently the neurite outgrowth from of NGF-mediated PC12 cells in a dose-dependent manner at concentrations from 10 to 40 mM. In comparison of the percentage of cells with neurites and the average neurite lengths as summarized in Fig. 16, neovibsanin B (63) seemed to be the most potent NGF-potentiator among three compounds. This result consists with morphological evaluation. It is assumed from these results that a stereochemistry on the acetal carbon may be related with affecting neurite outgrowth activity. 64 Studies in Natural Products Chemistry FIGURE 15 Neurite outgrowth-promoting activities of neovibsanin, neovibsanin A, and neovibsanin B in PC12 cells were cultured in DMEM/2% HS + 1% FBS with or without 10 ng/mL NGF and different concentrations of neovibsanin, neovibsanin A, and neovibsanin B for 48 h. PC12 cells were fixed and quantified for the percentage of cells bearing neurites and the primary neurite length. Over 40 fields were randomly selected under microscope for analysis of the percent of cells with neurites. At least 200 cells were selected for calculating the neurite length. Data were expressed as means SE. ***P < 0.001 compared with NGF only by one-way ANOVA followed by Bonferroni post hoc means comparison. ###P < 0.001 versus control by Student’s t-test. SYNTHESIS OF VIBSANE-TYPE DITERPENOIDS These unique molecular architectures and significant biological activities have strongly motivated organic chemists to devote their efforts to the syntheses of the vibsane-type diterpenoids. So far, the vibsane-type diterpenoids whose total syntheses have been achieved are only four molecules, that is ()-2-Omethylneovibsanin H (68) [45], ()-neovibsanin B (63) [46], ()-vibsanin Chapter 2 65 Structural Diversity, Neurotrophic Activity, and Synthesis H O HO O O Neovibsanin (86) H R1 O O R2 O O O Neovibsanin A (62): R1 = OMe, R2 = Me Neovibsanin B (63): R1 = Me, R2 = OMe FIGURE 16 Comparison of neurite outgrowth-promoting activities of neovibsanin, neovibsanin A, and neovibsanin B in PC12 cells. The method was the same as that in Fig. 15. Data were expressed as means SE. Difference between groups was tested with Student’s t-test. ***P < 0.001 compared with NGF only. E (16) [47,48], and ( )-neovibsanin G (77) [49]. From a synthetic point of view, the stereocontrol in the successive stereogenic centers involved in these diterpenoids has been challenging. Williams’ pioneering synthetic studies have addressed the issue of diastereoselective creation of the stereocenters involved in vibsane natural products. In fact, through many approaches toward the total syntheses of these molecules, the synthetic efforts have resulted in formation of diastereomers of the natual products as shown in Fig. 17, that is, (6S)-vibsanin F (3a) [14], ()-5,10-bis-epi-vibsanin E (16a) [50], ()-5,14-bis-epi-spirovibsanin A (82a) [51,52], and ()-4,5bis-epi-neovibsanins A (62a) and B (63a) [53]. Recently, Williams reported the comprehensive reviews highlight his group’s accomplishments in the total synthesis of vibsane-type diterpenpids [54,55]. Thus, This review focuses on our independent synthesis of neovibsanin B (63), which is not only a significant neurotrophic mimic but also the most challenging molecule among vibsane-type diterpenoids. 66 Studies in Natural Products Chemistry O6 O OH O 5 O O O O O 10 O O 3a 14 16a O R2 O O 4 O OMe H O 5 H 82a H R1 O 5 O MeO O O Neovibsanin B (63) 62a R1 = OMe, R2 = Me 63a R1 = Me, R2 = OMe FIGURE 17 Synthesized diastereomers of vibsane family and neovibsanin B (63). O O O O O O (a), (b) O O (c) N + OH 90 91 HO DMPMO OH (d) 92a 92b DMPMO O O (g), (h) (e), (f) 93a, b N DMI 94 OTBS 95 SCHEME 11 Reagents and conditions: (a) (COCl)2, benzene, reflux; (b) TBSOCH]CHCH] CH2, MeLi, CH2Cl2, DME, 20 C, 79%; (c) DMI, 200 C, 58%; (d) DIBAL, THF, 70 C to rt, 89%; (e) Bu2SnO, toluene, reflux, and then 2,4-DMPMCl, TBAI, toluene, reflux; (f ) Dess– Martin reagent, CH2Cl2, 0 C, 70%; (g) Bu3P, HCHO, aq. MeOH/CHCl3, rt, 84%; and (h) TBSCl, imidazole, CH2Cl2, 0 C, 99%. In 2009, Imagawa and Nishizawa reported the first synthesis of ()neovibsanin B (63) [46]. They employed an intramolecular Diels–Alder reaction of 91 at 200 C in dimethyl imidazolidinone (DMI) as a solvent for constructing the core cyclohexenone ring of neovibsanin, leading to a mixture of 92a and 92b (9:1) in 58% yield. DMI plays a crucial role in accelerating this reaction rate and thus makes it suitable for scale-up. Further manipulation of Diels–Alder adducts 92a and 92b involves introduction of a hydroxymethyl group by Baylis–Hillman reaction with formaldehyde to provide the key intermediate 95, Scheme 11. It should be noted that 1,2-addition of nucleophiles to the C-4 ketone was dominated by addition of the less hindered undesired face to afford Chapter 2 Structural Diversity, Neurotrophic Activity, and Synthesis 67 exclusively products having the undesired stereochemical disposition at C-4 [45,53,56]. Imagawa and Nishizawa overcame this steric drawback by devising tactic of using the oxygen of 2,4-dimethoxybenzyl (2,4-DMPM) group at C-10 to coordinate with the organolithium reagent and deliver the propargyl group from the same face. Reaction of excess lithio ethylpropiolate 96 with a toluene solution of 95 at 78 C successfully proceeded to give rise to the adduct 97 with the correct stereochemistry in 87% yield as a single diastereomer. 1,2Addition of 96 to the C-4 ketone in 95 was completely controlled by the coordination of the two oxygen atoms of 2,4-DMPM group with 96, which is shown in Fig. 18, to give 97 having the desired b-configuration at C-4. The triple bond of 97 was reduced with Red-Al to the a,b-unsaturated ester 98, which was in turn treated with TBAF for the deprotection of the TBS group, thereby triggering the subsequent Michael addition and lactonization to give rise to a tricyclic lactone 99 having the desired stereochemistry in good yield. The Deprotection of 2,4-DMPM in 99 by DDQ oxidation was troublesome due to the acidity of in situ formed DDQH. To avoid acidic conditions, this oxidation was carried out in a two phase system of CH2Cl2 and NaCl saturated-phosphate buffer to give a desired alcohol, which was protected again as the TBS group, giving rise to 100 in good yield. The resulting 100 was reacted with Tebbe reagent, and then treated with PPTS in methanol afforded a mixture of 102a and 102b (1:4.5), which were readily separated by HPLC. The major 102b was converted to the aldehyde 103, which was treated with KHMDS to generate a potassium enolate. This was in situ trapped with 3,3-dimethylacryloyl chloride completing the first total synthesis of ()-neovibsanin B (63), Scheme 12. Recently, we reported efficient construction of the chiral all-carbon quaternary center with a vinyl moiety that would permit postfunctional group manipulation by the conjugate addition of lithium divinyl cuprate to (4S,20 E)3-(60 -TBDPS-30 -methylhex-20 -enoyl)-4-phenyloxazolidin-2-one (104), and demonstrate that this method provides a versatile chiralquaternary carbon source 106 for the synthesis of natural products by its use to the synthesis of (+)-bakuchiol, Scheme 13 [57]. We have decided to apply this methodology to create the C-11 chiral quaternary carbon involved in vibsane natural products. As the first target, we selected the Imagawa–Nishizawa intermediate 95 that is the core framework of neovibsanin natural products. The asymmetric FIGURE 18 Chelated control of the 2,4-DMPM group with 96. DMPMO CO2Et O 10 4 Li OTBS DMPMO HO HO OTBS 4 96 CO2Et DMPMO CO2Et OTBS (a) Toluene –78 °C 87% 95 (b) DMPMO O O (c), (d) O TBSO TBSO (e) O O TBSO O 101 H H H O R2 (f) O 100 99 R1 H H H O 98 97 O 102a: R1 = OMe, R2 = Me 102b: R1 = Me, R2 = OMe (g), (h) MeO O O O 103 (I) O O MeO O O Neovibsanin B (63) SCHEME 12 Reagents and conditions: (a) Red-Al, THF, 78 C, 87%; (b) TBAF, THF, rt, 87%; (c) DDQ, CH2Cl2/phosphate buffer, NaCl, 0 C, 83%; (d) TBSCl, imidazole, 0 C to rt, 99%; (e) Tebbe reagent, pyridine, THF-toluene; (f ) PPTS, MeOH, 0 C, 91%; (g) TBAF, THF, rt, 99%; (h) SO3 pyridine, Et3N, DMSO, rt, 89%; and (i) KHMDS, THF, then 3,3-dimethylacryloly chloride, 78 C, 60%. O O O O (CH2=CH)4Sn PhLi, CuCN N Ph –78 to –50 °C O OH N Ph R OTBDPS OTBDPS OTBDPS 104 105 OH 106 DMPMO O OTBS 11 (+)-Bakuchiol Imagawa–Nishizawa intermediate 95 SCHEME 13 Enantioselective construction of the chiral all-carbon quaternary center at C-11. O O O N R Ph –78 to –50 °C O O (CH2=CH)4Sn PhLi, CuCN, Et2O Ph OTBDPS 87% O 85% OTBDPS OTBDPS 109 108 107 OH (a), (b), (c) N 11S (11S:11R = 95:5) (d), (e) Br Br I (f), (g), (h), (i) 89% OTBS 83% OTBDPS 110 OTBDPS 111 SCHEME 14 Reagents and conditions: (a) 30% H2O2, LiOH, THF–H2O, 0 C; (b) EtOH, EDC, DMAP, CH2Cl2, rt; (c) LiAlH4, THF, 0 C; (d) PCC, celite, CH2Cl2, 0 C; (e) CBr4, PPh3, CH2Cl2, 0 C; (f ) n-BuLi, THF, 78 C, then (CH2O)n; (g) Bu3SnH, AIBN, THF, reflux; (h) I2, CH2Cl2, 0 C; and (i) TBSCl, DMAP, Et3N, CH2Cl2, rt. 1,4-additition reaction of (CH2]CH)2Cu(CN)Li2 to 107 bearing (R)-4phenyl-2-oxazolidinone was employed to give 108 as a diastereomeric mixture of 95 (11S): 5 (11R) in good yield. Each diastereomer was readily separated by silica-gel chromatography. The optically pure (11S)-108 was converted to (2Z,11S)-111 according to the procedures outlined in Scheme 14. 70 Studies in Natural Products Chemistry Previously, we reported that the modified Negishi palladium(0)-catalyzed carbonylative cyclization of ()-111 provided the cyclohexene-1-one derivative ()-114, which corresponds to the cyclohexene ring of neovibsanin, Scheme 15 [58]. We examined this reaction in detail (Table 1). First, the reaction was performed using 5 mol% PdCl2(PPh3)2 and Et3N (1.5 equiv) in MeO2C O MeO2C 10 O OTBS OTBS 11 I OTBS OTBDPS 5 mol % PdCl2(PPh3)2 36 h (10R, 11S)-112 OTBDPS OTBDPS (10S, 11S)-112 O MeO2C OTBS OTBS 111 OTBDPS OTBDPS 113 114 SCHEME 15 Negishi’s Pd(0)-catalyzed cyclic carbopalladation-carbonylative tandem reaction of (2Z,11S)-111. TABLE 1 Pd(0)-Catalyzed Carbopalladation-Carbonylative Reaction of (2Z,11S)-111 under CO atmosphere (4 MPa) Base Entry (1.5 equiv) Solvent 1 Et3N 2 Et3N 3 Et3N 4 Et3N 5 Et3N 6 7 Et3N K3PO4 8 i-Pr2NEt 9 DABCO a MeCN/PhH (1:1) MeCN/PhH (1:1) MeCN/PhH (1:1) MeCN/PhH (1:1) 1,4Dioxane MeOH MeCN/PhH (1:1) MeCN/PhH (1:1) MeCN/PhH (1:1) MeOH (equiv) Temp. ( C) 112 (10R:10S)a 114 111 1113 (%) 4 100 11 (1.1:1) 0 6 52 48 100 54 (2.4:1) 10 2 0 48 60 49 (2.7:1) 13 0 21 24 60 69 (2.6:1) 14 0 0 4 100 0 0 0 90 – 4 100 100 6 (1.4:1) 16 (1.4:1) 13 18 4 0 52 3 4 100 0 0 0 85 4 100 24 (1.6:1) 0 9 0 Ratio was determined by 1H NMR spectroscopy in CDCl3 (300 MHz). Chapter 2 71 Structural Diversity, Neurotrophic Activity, and Synthesis MeCN/PhH (1:1) containing 4 equiv of MeOH at 100 C in an autoclave, which gave rise to the desired diastereomeric mixture of 112 in 11% yield along with ca. 50% of 114 containing a small amount of 113 (6%) (Table 1, entry 1). On the other hand, the addition of an excess amount (48 equiv) of MeOH to this reaction system dramatically increased the yield of 112 to 54%, contaminated with 2% of the noncyclic ester 114 (entry 2). The use of high pressure (8 MPa) was found to be ineffective at suppressing the generation of 114 (entry 3), but low temperature (60 C) was able to decrease the generation of 113 (entry 3). After several trials, we found that the following reaction conditions; 24 equiv of MeOH, 4 MPa CO, temperature of 60 C, led to the formation of 112 alone in ca. 70% yield as a diastereomeric mixture (10R:10S ¼ 2.6:1) (entry 4). Each diastereomer of 111 was readily separated by silica-gel column chromatography. It should be noted that 114 was exclusively generated in high yield when 1,2-dioxane was used as solvent (entry 5). With (10R,11S)-112 in hand, we focused on the last few steps for the synthesis of Imagawa–Nishizawa’s intermediate 95, Scheme 16. Treatment of (10R,11S)-112 with n-Bu4NF containing acetic acid gave 115, and the MeO2C MeO2C O OTBS O MeO2C OTBS (a) O OTBS (b), (c) 70% 69% OH OTBDPS 115 (10R, 11S)-112 HO HO O 116 OH OTBS DMPMO OTBS + OH (f) OTBS 45% 117 (29%) (g) 95% 118 (26%) (e) DMPMO (d) 119 43% H O OTBS (+)-95 O O MeO O O (+)-Neovibsanin B (63) SCHEME 16 Reagents and conditions: (a) n-Bu4NF, AcOH, THF, rt; (b) (COCl)2, DMSO, 78 to 10 C, then Et3N; (c) Me2CHP+Ph3I , n-BuLi, THF, 0 C; (d) DIBAL-H, CH2Cl2, 78 C; (e) NaBH4, EtOH, rt; (f ) 2,4-DMPM-trichloroacetoimidate, 10 mol% CSA, CH2Cl2, 20 C; and (g) Dess–Martin periodinane, CH2Cl2, rt. 72 Studies in Natural Products Chemistry resultant hydroxy group was oxidized by Swern oxidation to its aldehyde, which was subjected to Wittig olefination to give the dimethyl olefin 116 in 69% yield over two steps. Reduction of 116 with DIBAL-H provided the cyclic hemiacetal 117 and diol 118 in 29% and 26% yields, respectively. The cyclic hemiacetal 117 was reduced with NaBH4 to give 118. The selective protection of the primary alcohol in 118 using the reaction conditions, 2,4DMPM-trichloroacetoimidate in the presence of 10 mol% CSA gave rise to the desired 2,4-DMPM-ether 119 in 45% yield. Finally Dess–Martin oxidation of 119 afforded the Imagawa–Nishizawa’s intermediate (+)-95 as an optically active form ([a]D ¼ + 20.1 (c 1.05, MeOH)) in 95% yield. Thus, the first enantiocontrolled formal synthesis of (+)-neovibsanin B was accomplished [59]. A Minimal Structural Core of Neovibsanin Required for Neurotrophic Activity Neovibsanins have been shown to display neurite outgrowth activity in PC12 cells, suggesting that neovibsanin-type compounds could be a candidate for the development of novel therapeutic agents to treat neurodegenerative diseases such as AD [43]. While neovibsanins consist of tricyclic unusual diterpenoid structure, it is important to clarify the structural factors of the neovibsanin skeleton that are essential for exerting its biological activity for further studies on drug development. In 2009, Williams et al. reported that the stereochemistry at the 4 and 5 positions on the neovibsanin skeleton has very little effect on neurotrophic activity via the synthesis of unnatural 4,5-bi-epi-neovibsanins [53]. In the same year, we completed the first total synthesis of ()-neovibsanin B, which shows identical activity as that of natural (+)-63 [46]. Having structure–activity information in hand, we designed and synthesized a number of structurally simplified derivatives 120–123, which either lack side chains from the neovibsanin skeleton or a part of the skeleton to clarify the structure required for an onset of neurite outgrowth activity as shown in Figs. 19 and 20. The neurite outgrowth activity of these synthetic derivatives were accessed by using PC12 cells (JCRB0733). As results, 120a-a and 120a-b significantly promote NGF (20 ng/mL)-mediated neurite outgrowth at the same level as that of natural 63 as depicted in Fig. 21. These findings imply that the side chains of neovibsanin are unimportant in terms of the onset of neurite outgrowth activity. Quantitative-activity evaluation for derivatives 120a–d revealed that increasing the bulkiness of the acetal moiety leads to decreased activity of the corresponding derivatives, Fig. 22. Moreover, the activity of the corresponding a-isomer was slightly greater than that of the b-isomer, Fig. 22. Compound 120h, which bears an allyl group, showed very weak activity, but 120e, 120f, 120g had no activity any more. Interestingly, hemiacetal 120i shows significant cytotoxicity as well as weak neurotrophic activity. The bicyclic derivative 121, intermediate Chapter 2 73 Structural Diversity, Neurotrophic Activity, and Synthesis H H MeO O O RO O Bistetrahydofutanyl moiety Remove side chains R1 121 O 5 R2 H 120 O 18 O 4 O 2 Side chains The necessity of double bond The necessity of acetal H Neovibsanin A (62): R1 = MeO, R2 = Me O O H MeO Neovibsanin B (63): R1 = Me, R2 = OMe O O O H 122 124 FIGURE 19 Structurally simplified derivatives designed for structure–activity related studies. H MeO H EtO O O H n-PrO O O a-MeO : 120a-a b-MeO : 120a-b O O O a-EtO : 120b-a b-EtO : 120b-b a-PrO : 120c-a b-PrO : 120c-b a-BuO : 120d-α b-BuO : 120d-b a/b 1 : 3 a/b 1 : 2 a/b 1 : 1.6 α/β 1 : 2 H O H n-BuO O H O O O No activity H HO O O O O No activity 120e a/b 1 : 2.7 120f H O 120i a/b 1 : 3.2 O O No activity H O O O Low activity 120g a/b 1 : 3.3 FIGURE 20 The structures of acetal derivatives. 120h a/b 1 : 3 74 A Studies in Natural Products Chemistry B C FIGURE 21 The neurite outgrowth promoting activities of 120a-a and 120a-b. (A) PC12 cells in the presence of 20 ng/mL of NGF as control. No remarkable morphological change was observed. (B) A notable neurite growth was observed in the presence of 10 mM of 120a-b and 20 ng/mL of NGF. (C) A significant neurite growth was observed in the presence of 10 mM 120a-a and 20 ng/mL NGF. FIGURE 22 Quantitative assays for neurite outgrowth activities of acetal derivatives PC12 cells were cultured in two 24-well plates in DMEM + 10% HS and 5% FBS for 24 h at a density of 2 103 cells/cm2. The medium was then changed to DMEM + 2% HS and 1% FBS supplemented with NGF (10 ng/mL) in the absence or presence of test compound. After 4 days the length of the longest neurites were quantified. Data are expressed as the mean SE (n ¼ 149). **P < 0.01 versus control; Dunnett’s/test. lactone 122, and saturated compound 123 were found to be inactive. These results indicate that a tricyclic acetal structure with double bond, which is represented as structure 120a, should be the essential structural core for neovibsanins to retain neurite outgrowth-promoting activity. Thus, the simple structure core of neovibsanins required for maintaining the neurite outgrowth-promoting activities has been derived from structure–activity relationships [48]. We are currently attempting to synthesize a large amount of 120a for doing further mechanistic studies as well as for conducting AD model animal experiments. Chapter 2 Structural Diversity, Neurotrophic Activity, and Synthesis 75 CONCLUSION Since Prof. Kawazu’s first report in 1980, over 80 vibsane-type diterpenoids have been found and the new term of “Vibsane” has now become accepted. These diterpenoids not only show a rich structural diversity but also occur specifically in a few Viburnum species. Thus, they are very interesting natural products from chemical and taxonomic points of view, and moreover, their chemical diversity makes them a valuable chemical library. As neovibsanin A and B have been found to exhibit interesting neurotrophic activity, it is expected that new biological activities will be discovered among the vibsane-type diterpenoids. Recently, vibsane-type diterpenoids have attracted much attention from organic chemists due to their unique structures and important biological activities [54,56,60–64]. We hope that this review has stimulated much attention in synthetic studies of these diterpenoids as well as in biological studies of neovibsnains. ACKNOWLEDGMENTS We thank our colleagues for their dedication toward these projects of vibsane-type diterpenoids and whose names are listed in the literature cited in references. These works were supported by Grant-in-Aids for Scientific Research from the Ministry of Education, Culture, Sports, and Technology of Japan and the Open Research and MEXTSenryaku grants from the Promotion and Mutual Aid Corporation for Private School of Japan. 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Olivo{ *Centro de Investigaciones Quı´micas, Universidad Autónoma del Estado de Morelos, Morelos, Mexico { Medicinal and Natural Products Chemistry, The University of Iowa, Iowa City, Iowa, USA Chapter Outline Introduction Biosynthesis Capsaicinoids Capsaicin Capsaicin’s Mechanism of Action Capsaicin SARs 79 85 89 91 92 95 Pharmaceutical Formulations Based on Capsaicinoids Affinin (Spilanthol) Sanshools Piperine and Piperovatin Conclusions Acknowledgments References 100 100 103 106 110 111 113 INTRODUCTION Alkamides (N-alkylamides, alkenamides, or alkenylamides) are bioactive natural amides possessing an aromatic or aliphatic unsaturated fatty acid residue [R1CO] linked to an aliphatic or aromatic amine residue [R2] (Fig. 1). These alkamides belong to a promising group of natural alkaloids with demonstrated insecticidal [1,2], antimalarial [3–5], antibacterial [6,7], or plant protection activities [8]. In mammals, alkamides have shown immunostimulant [9–13], analgesic, anti-inflammatory or antioxidant properties [14], yet to be developed and applied to therapies related to these properties [15]. Alkamides have a wide chemical structural diversity and exert an important variety of biological–pharmacological effects via multiple mechanisms of action and targets. These compounds have high ethnopharmacological importance and Studies in Natural Products Chemistry, Vol. 43. http://dx.doi.org/10.1016/B978-0-444-63430-6.00003-5 © 2014 Elsevier B.V. All rights reserved. 79 80 Studies in Natural Products Chemistry O R1 R2 N FIGURE 1 General structure of alkamides. for this reason there have been attempts to systematize the information available regarding their chemistry, occurrence, and functionality [16]. More than 300 alkamides have been isolated as natural products. The most common sources of these natural alkaloids are plants belonging to Asteraceae, Solanaceae, Rutaceae, and Piperaceae families, although their presence has been demonstrated at least in 35 other plant families [16,17]. According to their molecular architecture, these alkaloids could be considered chemotaxonomic markers. For example, alkamides isolated from Asteraceae plants showed a characteristic linear C8–C18 fatty acid chain, being C10–C12 alkyl acid the most frequently isolated. The alkyl acid chain commonly includes two double bonds, although those with one, three, or four double bonds have also been isolated. In many cases, these alkamides also possess one or two carbon–carbon triple bonds, characteristic only present in Asteraceae. The most characteristic amine residue in Asteraceae alkamides is the N-isobutyl group, but N-2-methylbutyl, N-3-methylbutyl, N-phenethyl, piperidinyl (or piperidide), 2,3-dehydro-piperidinyl (or piperideide), pyrrolidinyl, and pyrrolidyl groups were also found. Some representative examples of alkamides from Echinaceae (Asteraceae) are shown in Fig. 2. Capsaicinoids are characteristic alkamides from Solanaceae family and are isolated solely from plants belonging to Capsicum genus. They are the metabolites responsible for the hot and pungent sensation that occurs when one bites Capsicum fruits known as “chili pepper” [17–19]. All capsaicinoids possess an N-vanillylamide residue (N-4-hydroxy-3-methoxybenzyl amine group) attached to a linear or branched fatty acid core, Fig. 3. These natural alkaloids differ only in the length of the fatty acid chain, which varies in the presence of a single unsaturation and in chain length from C7 up to C11, C14, C16, or C18. Medium-length branched-chain fatty acids are unusual metabolites in plants but common in the Solanaceae family. The distinctive feature from alkamides isolated from Piperaceae family is the presence of an aromatic ring, commonly a 3,4-methylenedioxyphenyl group (although p-methoxyphenyl and 3,4,5-trimethoxyphenyl have also been found) linked to the end of an unsaturated C3–C16 fatty acid chain [20]. The amine group commonly found is an N-isobutyl, pyrrolidinyl, or piperidinyl group, Fig. 4 [21–23]. Four genuses from the Rutaceae family produce alkamides: Evodia, Pleiospermium, Zanthoxylum, and Glycosmis, the latter two being the most productive. More than 50 aliphatic [24,25] and aromatic [5,26] alkamides have been isolated from Zanthoxylum genus, which are responsible for its R2 R5 R3 N H R1 C8–C18 linear saturated or unsaturated chain R3 R6 N H R4 N-isobutyl R3=R5=R6=H; R4=CH3 N-2-methylbutyl R3=CH3; R4=R5=R6=H N N-phenethyl R3=H N-tyramidyl R3=OH N-(O-methyl-tyramidyl) R3=OCH3 N Piperidide Pyrrolidinyl ∆2,3 = piperideide ∆2,3;4,5 = pyrrolidyl N H N H O O Undeca-2E,4Z-diene-8,10-diynoic acid isobutylamide Dodeca-2E,4Z-diene-8,10-diynoic acid 2-methylbutylamide O O Undeca-2Z,4E-diene-8,10-diynoic acid isobutylamide Dodeca-2Z,4E-diene-8,10-diynoic acid isobutylamide O O Dodeca-2E,4Z-diene-8,10-diynoic acid isobutylamide Undeca-2Z-en-8,10-diynoic acid isobutylamide O Trideca-2E,7Z-diene-10,12-diynoic acid isobutylamide O Dodeca-2Z,4E-diene-8,10-diynoic acid isobutylamide O Dodeca-2E,4E,10E-triene-8-ynoic acid isobutylamide FIGURE 2 Selected examples of alkamides from Echinaceae (Asteraceae). 82 Studies in Natural Products Chemistry OCH3 N H OH O Trinordihydrocapsaicin 2,4,6-Nonatrienamide O 6-en-Homocapsaicin I O Homocapsaicin I O Homocapsaicin II O Homodihydrocapsaicin I O Homodihydrocapsaicin II O Myrvanil O Dinordihydrocapsaicin Caprilyc acid vanillylamide (octylamide) O O Nonivamide O O Norcapsaicin 4-en-Norcapsaicin 4-en-6-methylNorcapsaicin Nordihydrocapsaicin O Decylic acid vanillylamide Capsaicin 4-en-Capsaicin Dihydrocapsaicin 5-en-7-methyl-Capsaicin O O O Palvanil O O O Stevanil O Olvanil O O O O Livanil O Linvanil FIGURE 3 Capsaicinoids from Capsicum genus (Solanaceae). pungent taste and their use in toothache, urinary and venereal ailments, antioxidant, anthelmintic, and relaxing activities, being used also for rheumatism and lumbago. Some examples of these alkamides are illustrated in Fig. 5. Sulfur-containing alkamides represent the typical chemical profile of plants from Glycosmis genus. These alkamides accumulate particularly in the leaves [27–29]. Some plants of this genus have been used in Thailand, Sri Lanka, and Malasia as traditional medicines for treatments of abscess, scabies, and snakebite [30]. Alkamides from Glycosmis show antifungal [31,32] Chapter 3 83 Natural and Synthetic Alkamides R2 R1 N H C2 -C14 Unsaturated chain 3 R R4 N R6 N-isobutyl R6 = H N-3-acetoxy-isobutyl R6 = OAc Pyrrolidinyl R5 R7 3 4 N 5 R –R = OCH2O; R = H R3,R5 = H; R4 = OCH3 R3,R4 ,R5 = OCH3 Piperidinyl R7 = H,H (piperidide) 5,6-dihydro-2(1H)pyridinone ∆3,4,R7 = O R7 N N N H O O O R7 = H,H 4 O H3CO O H3CO O n = 11 Trachyone n = 6 Isopiperolein B Piplartine O O 4,5-Dihydropiperlongumine O O n O 3 O R7 = H,H O O n OCH3 O Pellitorine Tric hosttachine Piperine O R7 = O O O O O Pipernonaline n=2 n=5 n=7 n=8 Laetispicine Guineensine Brachystamide B Pergumidiene FIGURE 4 Representative alkamides from Piper (Piperaceae). and antiherpes simplex virus activities [33,34]. With the exception of simple methylamides, the amine substituent of these alkamides are characterized by the presence of phenethyl or styryl groups that may be linked to different prenyloxy or geranyloxy groups in the para position (Fig. 6). A series of these alkamides isolated from the leaf extracts of different species of Glycosmis were synthesized because of their strong antifungal and insecticidal properties [35–37]. 84 Studies in Natural Products Chemistry H N NH NH OH O H3CO Alatamide O OH CH3 α-Sanshool (hidroxy-α-sanshool) N O Lanyuamide I O Ailanthamide H3CO O O O Lanyuamide II CH3 O N Lanyuamide III (hidroxy-lanyuamide III) O O H3CO γ-Sanshool (hidroxy-γ-sanshool) N-(4-methoxyphenetyl)-N-methylbenzamide FIGURE 5 Some alkamides from Zanthoxylum (Rutaceae). O CH3 N H N-methylthiocarbamate CH3 S 5 CH3 O 3 O R2 1 N R1 2,3 2⬘,3⬘ (E); R1 = CH3; R2 = H Dehydrothalebanin A Δ , Δ Dehydrothalebanin B Δ2,3, Δ2⬘,3⬘(Z); R1 = CH3; R2 = H 2,3 2⬘,3⬘ (Z); R1 = CH3; R2 = OCH3 Glycrophylamide Δ , Δ Thalebinin Δ2⬘,3⬘(Z); R1 = CH3; R2 = H O O 2,3 O Puginamide Δ ; R1 = H; R2 = 4 3 2 R1 = OH; R2 = CH3 Sakambullin R1 = OCH3; R2 = CH3 O-Methylsakambullin O Glypatvin A O R1 = H; R2 = R4 CH3 S O O Gerambullin R7 O O 2,3 1 2 3 Penimide A Δ (E); R = CH3; R = O; R = H 2,3 Penimide B Δ (Z); R1 = CH3; R2 = O; R3 = H 2,3 1 2 Methoxypenimide B Δ (Z); R = CH3; R = O; R3 = OCH3 2,3 Krabin Δ (E); R1 = CHO; R2 = H.H; R3 = H,H 2,3 Isokrabin Δ (Z); R1 = CHO; R2 = H.H; R3 = H,H 5 R1 = H; R2 = CH3 Dambullin R3 R2 1 N 2 R1 S 4 R1 N H S O O R1 1⬘ 3⬘ CH3 N 2⬘ S CH3 Ritigalin R1 = O Niranin R1 = H,H 2⬘,3⬘ (E) Dehydroniranin A R1 = H; Δ 2⬘,3⬘ (Z) Dehydroniranin B R1 = H; Δ O R2 O O CH3 N R1 R3 R5 R6 R2 R1 = R2 = R3 = R4 = R5 = H; R6 = R7 = CH3 Gerambullol R1 = R2 = R3 = R4 = R5 = H; R6 = CH3; R7 = CH2OH β-Hydroxygerambullin R1 = R3 = R4 = R5 = H; R2 = OH; R6 = R7 = CH3 β-Hydroxygerambullol R1 = R3 = R4 = R5 = H; R2 = OH; R6 = CH3; R7 = CH2OH β-Hydroxygerambullal R1 = R3 = R4 = R5 = H; R2 = OH; R6 = CH3; R7 = CHO Sakerine R1 = R2 = R4 = R5 = H; R3 = OH; R6 = R7 = CH3 Sakerinol A R1 = R2 = R4 = R5 = H; R3 = OH; R6 = CH3; R7 = CH2OH O-Methylsakerinol A R1 = R2 = R4 = R5 = H; R3 = OCH3; R6 = CH3; R7 = CH2OH Sakerinol B R1 = R2 = R4 = R5 = H; R3 = OH; R6 = CH2OH; R7 = CH3 Sakerone R1 = R2 = R4 = H; R3 = OH; R5 = O; R6 = CH3; R7 = CH3 Sakerol R1 = R2 = R4 = H; R3 = OH; R5 = OH; R6 = CH3; R7 = CH3 Isosakerol R1 = R2 = R5 = H; R3 = OH; R4 = OH; R6 = CH3; R7 = CH3 Methylgerambullal R1 = CH3; R2 = R3 = R4 = R5 = H; R6 = CH3; R7 = CHO FIGURE 6 Representative alkamides from Glycosmis (Rutaceae). Finally, alkamides with both acid and amine aromatic residues have been isolated from plants of several other families, like Euphorbiaceae, Annonaceae, Rutaceae, Convolvulaceae, Menispermaceae, Brassicaceae, Aristolochiaceae, etc. These alkamides possess a phenethyl group on the amine residue, with cinnamoyl or benzoyl groups as the acid group, Fig. 7 [17]. Chapter 3 85 Natural and Synthetic Alkamides Benzylphenethylamides Cinnamoylphenethylamides R R O 1 N H 4 HO R3 2 R R1 OCH3 OCH3 H OCH3 R1 O N H R2 HO R2 R3 R4 OH H OH Feruloyltyramine OH OH OH Feruloyldopamine OH OH OH Coumaroyldopamine OH H OH N-cis-feruloyloctopamine R 1 OH R2 OH N-[2-(3,4dihydroxyphenyl)ethyl]3,4-dihydroxybenzamide OCH3 H dihydroalatamide FIGURE 7 Aromatic alkamides from diverse family of plants. BIOSYNTHESIS The structural variability of the alkamides is the result of genetic characteristics of each plant family. Even subtle structural differences indicate the origin of each compound in a particular group within the same family. For example, within Asteraceae family, Anthemideae and Heliantheae are the only two tribes that make alkamides, suggesting that one or more element of alkamide biosynthesis is absent in the remainder tribes [38]. Until today, many aspects from the alkamide biosynthesis remain unknown. In Asteraceae, alkamides result from the combinatorial ligation of alkylamines apparently derived after decarboxylation from amino acids with short- and medium-chain fatty acids derived from the polyketide route. Feeding experiments of labeled precursors showed that alkamides derived from linear C18-fatty acids through the sequence oleic acid-linoleic acid-crepenynic acid (crepenynate [10-14C])-enediynic acid (methyl enediynoate [16-14C]) (Fig. 8) [39]. It has been suggested that the highly unsaturated alkamides may be regarded as biosynthetically advanced, whereas the corresponding alkamides with only one or two double bonds appear to be more primitive [40]. In Heliantheae, C14 alkamides arise from a C18 acetylenic precursor which lose four carbons by a double b-oxidation [39]. Oxidative degradation at the terminal methyl group of C14 alkamides leads to the loss of two or three carbons (C12 and C11 alkamides, respectively), typical from Spilanthes and Echinaceae plants. Feeding experiments with [15N]valine, L-[2H8]valine, and L-[2H8]phenylalanine showed enrichment at the amine residue of these natural alkamides showing the amino acid origin of this region of the molecule [38,41]. The biosynthetic pathway for capsaicinoids has been established and several genes involved in important steps of transformation of key precursors have been identified. In such manner, it is known that the genes Pal, Ca4h, and Comt are involved in the phenylpropanoid pathway; Kas, Acl, and Fat encoding for the enzymes involved in fatty acid metabolism; and the spicy 86 Studies in Natural Products Chemistry 6 Fatty acid biosynthesis 6 COOH Oleic acid 3 6 COOH Linoleic acid 3 O -H2 Dehydrocrepenynic acid 6 COOH 6 OH O 6 β-Oxidation -CH3 CO2 β-Oxidation -CH3 CO2 C14 Alkamides - O O Leucine COOH COOH 6 -CO2 Amine C16 Alkamides [O] 3 3 NH2 COOH COOH 4 C18 Alkamides - O Valine [O] 6 -CH3CO 3 Amine O Enediynic acid COOH Amine NH2 6 COOH -H2 3 - O Phenylalanine 6 COOH OOH 3 NH 2 6 COOH Crepenynic acid O2 [O] -CH 3CO COOH 2 HOOC -CO2 H 6 COOH Amine COOH C15 Alkamides C12 Alkamides [O] -CO2 C11 Alkamides FIGURE 8 Biosynthetic aspects from the alkamide production in Asteraceae plants. and pungent properties are associated with Pun1 locus presence [42–46]. However, a detailed understanding of their biosynthetic pathway gene regulation and the identification of the involved enzymatic machinery remains elusive [47]. Some models for capsaicinoid biosynthesis have been developed based on literature and metabolic databases that incorporate work in Capsicum, related genera, and model organisms [48]. In light of the current understanding, capsaicinoids are biosynthesized by the capsaicin synthase catalyzed condensation reaction, Fig. 9. Capsaicin synthase condenses branched fatty acid residues with vanillylamine, which comes from two different convergent pathways. The phenylpropanoid pathway provides vanillylamine and its precursors from phenylalanine. The anabolism of branched amino acids leucine or valine, which are deaminated and subsequently elongated by addition of two-carbon sequences derived from acetyl-CoA via the fatty acid synthase, provides branched fatty acid residues [49–52]. Elongation cycles continue Fatty acid synthesis NH3 O O O - BKDH O - BCAT O O - HSCoA O -CO2 α-KG Glu α-Ketoisovalerate Valine Isobutyrate O 3 X MalonylCoA SCoA O SCoA KAS, ACL FAT, ACS Isobutyryl CoA C10 branched fatty acid residue O 2 X MalonylCoA NH3 O O O O α-KG Glu O -CO2 α-Ketoisocaproic acid Leucine Isovalerate SCoA O HSCoA O- O- - BCAT, branched chain amino acid transferase α-KG, α-ketoglutarate Glu, BKDH, 3-methyl-2-oxobutanoate dehydrogenase KAS, β-ketoacyl ACP synthase ACL, acyl carrier protein FAT, acyl-ACP thioesterase ACS, acyl-CoA synthase C9 branched fatty acid residue SCoA Capsaicin synthase Isovaleryl CoA O N n H OH O 3 X MalonylCoA SCoA n =3–5 C11 branched fatty acid residue O O OH CoAS OCH3 COMT CoAS OH OH SAM Caffeoyl CoA CPR OCH3 pAMT HCHL O OH Acetyl-CoA Feruloyl CoA Vanillin AA + H 3N OCH 3 OH KA Vanillylamine -H2O CA3H O2 O 2e- O OH Coumaroyl CoA O CA4H HSCoA -O CoAS 4CL OH -H2O PAL OOC + NH -O O2 p-Coumarate Cinnamate -NH4 CPR 2e- FIGURE 9 Fatty acid and phenylpropanoid pathways for biosynthesis of capsaicinoids. 3 Phenylalanine OCH 3 PAL, phenylalanine ammonia lyase CA4H, cinnamic acid 4-hydroxylase CPR, Cytochrome P450 reductase 4CL, 4-coumaroyl-CoA ligase CA3H, coumaric acid 3-hydroxylase COMT, caffeic acid O-methyltransferase SAM, S-adenosyl-methionine HCHL, hydroxycinnamoyl-CoA pAMT, putative aminotransferase AA, amino acid KA, Keto acid 88 Studies in Natural Products Chemistry O COOH H3CO COSCoA + NH2 H2N H N 2 Phenylalanine COOH N H3CO O OCH3 Lysine MalonylCoA Piperlongumine O COSCoA O + N-piperoyl transferase HN O O O N Piperine FIGURE 10 Biosynthetic pathway of Piper alkamides. Phenylpropanoid pathway O Cysteine O H3C S Methionine Mevalonate pathway 8-Hydroxygeranyl N CH3 FIGURE 11 Biosyntetic pathway of sulfur-containing alkamides from Glycosmis genus. until the fatty acid is enzymatically released by the thioesterase (FAT), which participates in the regulation of chain length, affording capsaicinoids with branched even-number of carbons in the acyl moieties [53]. The desaturation to the unique trans configuration occurs before the thioesterase FAT liberates the free fatty acids characteristic of the capsaicinoids [54]. Many aspects of the biosynthesis of Piperaceae alkamides remain unknown. Some steps from piperlongumine biosynthesis were investigated [55]. Feeding experiments with L-[U-14C]phenylalanine, L-[U-14C] lysine, [2-14C]sodium acetate and DL-[2-14C]tyrosine, demonstrated incorporation of L-[U-14C]phenylalanine and L-[U-14C]lysine into the alkamides, whereas [2-14C]sodium acetate and DL-[2-14C]tyrosine showed no incorporation. It is probable that phenylalanine renders piperoylCoA via elongation of the corresponding cinnamic acid with malonylCoA, Fig. 10. It has been demonstrated that N-piperoyltransferase catalyzes the amide formation by joining piperoylCoA to a piperidine, pyrrolidine, or isobutylamine, to deliver the Piper alkamides series like capsaicin [56]. It is reasonable to assume that all structurally related amides isolated from Piper species are biosynthesized by the same mechanism. It has been assumed that Glycosmis alkamides are derived from the amino acid cysteine or methylthiopropenic acid [57]. These acids are combined with amines mostly represented by phenethyl or phenethenyl (styryl) groups arising from the phenylpropanoid pathway, Fig. 11. The para position of the aromatic moiety amines can be further linked to various prenyloxy and geranyloxy groups, Chapter 3 Natural and Synthetic Alkamides 89 derived from mevalonate [27]. The sulfide group of these alkamides can also be oxidized to sulfones [34] and sulfoxides [58] or shortened by oxidation [16]. All theories of the natural origin of these alkamide series should be studied in more detail. There is a lack of information on the chemical, enzymatic, and genetic aspects on the biosynthesis that should be studied. Plants containing alkamides are widely used as condiments in the preparation of meals in many parts of the world. India, China, Mexico, Malaysia, and Thailand are only a few countries with records of high consumption and long tradition in their use, not only as part of their food, but also as traditional medicines used for centuries. Independently of the genus or family to which the plants producing alkamides belong, they all have some common features. They produce an unpleasant spicy sensation, a pungent, and acid taste. They cause intense ptyalism (salivation) and tingling when in contact with the mouth, tongue, gums, teeth, cheeks, palate, and lips. This initial feeling is commonly followed by numbness, paresthesia, and anesthesia, which may take from a few minutes to a long and variable period of time. Alkamides, at concentrations in the ppm range, are responsible for these properties and for many other pharmacological activities largely documented in the phytochemistry literature for these plants, known in many places of the world as “the toothache medicine.” A small piece of the fresh part of these plants (leaves, roots, flowers, or bark, depending on the species) is chewed to alleviate toothache. For this health benefit, the use of whole extracts and infusions, whose chemical composition is not well characterized, administered topically is a frequent practice. Alkamides are absorbed by buccal mucosa, avoiding all disadvantages of the oral administration such as instability to stomach acid conditions and poor and slow absorption [59]. Other important pharmacological activities reported for plants producing alkamides are insecticidal, antioxidant, antimalarial, and antiviral. Alkamides are largely used as immunostimulants, immunomodulators, and antiinflammatory agents. Also, they are employed in myofascial pain, osteoand rheumatoid arthritis, and systemic lupus. Some formulations from these plants represent multimillionaire sales in the United States and some European countries, where they are used for their economic potential. This review is a compilation on the literature available that aims to describe the state of the art of the most important alkamides and the plants producing them, including aspects on the chemical structures contributing to their analgesic activity and their mechanism of action. CAPSAICINOIDS Peppers, commonly known as “chili,” are used in many countries worldwide. “Chili” or products derived from them are consumed in varied amounts depending on each culture and each country, with Mexico, India, Brazil, Thailand, China, and Malasia as the highest consumers. Currently, there are 90 Studies in Natural Products Chemistry about 20 wild and five cultivated species of Capsicum “chili” known. Cultivated species are Capsicum annuum var. annuum, Capsicum frutescens, Capsicum baccatum var. pendulum, Capsicum chinense, and Capsicum pubescens which include hot and sweet peppers of high economic value. The pericarp of all Capsicum fruits includes vesicles filled with “oleoresins” composed mainly from alkaloids known as capsaicinoids or N-vanillyl-acylamides. These are responsible for the aroma, color, pungency, acidity, and hot sensation of the fruits of Capsicum species. Its intake ranges from pleasant to painful sensations responsible of its popular use in the cuisines from several cultures around the world. These properties make capsaicinoids highly appreciated and useful as pungent additives of commercial importance to the food and pharmaceutical industries in the world market. Pungency, burn, and itchy sensations are caused in humans because capsaicinoids interact and stimulate TRP receptors (nociceptors) in the mouth, skin, and mucoses responsible for the sensations of pain, heat, and acidity. These nocicepors are also responsible for analgesic effects, pain relief, and anesthesia, due to their ability to desensitize sensory neurons [60]. More than 20 different natural capsaicinoids have been found in Capsicum fruits [61]. However, capsaicin and dihydrocapsaicin are the most predominant alkamides in nature [17]. Other natural capsaicinoids included in Capsicum species are norcapsaicin, nordihydrocapsaicin, homocapsaicin, and homodihydrocapsaicin (Fig. 3) [60,62]. Pharmacological properties of capsaicinoids include cancer prevention [63], cardiovascular and gastrointestinal benefits [64], antiarthritic pain control, anti-inflammatory and antioxidant activities [65], and weight loss properties. Despite the mechanism of action for which capsaicinoids produce weight loss is not fully understood, clinically, they play a beneficial role as part of an antiobesity management program [66]. Because of these multiple biological properties, capsaicinoids are currently important targets for synthesis to study pharmacological behavior [67]. Studies on the structure–activity relationship (SAR) between the acyl chain length and the pungency of capsaicinoids reveal that an optimum chain of nine carbons (such as capsaicin and dihydrocapsaicin) causes the strongest sensation of pungency in humans. The loss or gain of a single carbon atom leads to loss of approximately half the pungency as in nordihydrocapsaicin, homocapsaicin, and homodihydrocapsaicin [68]. Capsaicinoids with a longer or shorter acyl chain than C9 have less pungency and capsaicinoids with a C18 chain or longer do not show this property [69]. On the other hand, chemical profile from sweet pepper shows capsinoids as their major constituent. Capsinoids are capsaicinoid analogs, bearing similar structures except for the central linkage; while capsaicinoids possess an amide moiety, capsinoids include an ester moiety (Fig. 12). Comparing with capsaicinoids, capsinoids are nonpungent and do not produce the unpleasant discomfort showed for capsaicinoids [70], probably due to their higher lipophilicity or hydrolysis before they reach the nerve endings that desensitize the sensory neurons [71–73]. Capsinoids have very light Chapter 3 91 Natural and Synthetic Alkamides OCH3 O OH O Nordihydrocapsiate O Capsiate O Dihydrocapsiate O Homocapsiate O Homodihydrocapsiate FIGURE 12 Representative capsinoids from sweet peppers. R1 N H OCH3 O OCH3 H OH Capsaicinoid OH Vanillin FIGURE 13 Structural similarities between capsaicinoids and vanillin. analgesic properties. These studies demonstrate the importance of chain length and the presence of the nitrogen atom to ensure the pungent activity shown for the capsaicinoids. Examination of the capsaicinoid structures reveals a common feature, namely an aromatic ring possessing a methoxy and hydroxyl substituents. Because of the similarity of these structures to the natural substance vanillin (Fig. 13), these pungent substances are collectively known as “vanilloids” [74]. The high predominance of capsaicin in Capsicum fruits makes this alkamide the most studied to evaluate the pharmacological activity of capsaicinoids. Capsaicin is also the capsaicinoid most widely used as pharmaceutical. CAPSAICIN Capsaicin [8-methyl-N-vanillyl-6-nonenamide; (E)-N-(4-hydroxy-3-methoxybenzyl)-8-methyl-6-nonenamide] includes three important chemical regions: trans-alkenyl fatty acid residue, amide, and vanillyl group (Fig. 14). The exact combination of these regions is responsible for capsaicin’s pharmacological activities. 92 Studies in Natural Products Chemistry Amide O N H OCH3 OH Alkenyl fatty acid Vanillyl FIGURE 14 The three regions of capsaicin. This natural alkamide is considered a safe and effective topical analgesic that also possesses antioxidant and anticancer properties. Initially, its strong pungency and nociceptive activity limited its use. However, although its topical application initially produces an uncomfortable burning sensation, it eventually leads to analgesia [75]. The biological effects of capsaicin were not well understood until it was discovered that it exerts two distinct actions on sensory neurons, an immediate but temporary excitation followed by a long-lasting desensitization. This interesting molecule has been studied in vitro and in animal models using topical and parenteral routes of administration. In this manner, capsaicin is frequently used in topical formulations to treat various sources of pain including arthritis, twists, inflammation, chronic pain states, bone cancer pain, muscle pain, and in treating toothache [76]. It is also used in the management of pain from posttherapeutic neuralgia and diabetic neuropathy, osteoarthritis, and rheumatoid arthritis [75]. It is widely known that capsaicin is the principal ingredient of several medical preparations and self-defense sprays [76]. Pretreatment with capsaicin, either by infiltration or by proximal perineural application inhibited both, heat-hyperalgesia and guarding-pain behaviors caused by plantar incision in mice through common mechanisms [77]. Capsaicin is currently being studied by several research groups in both pharmaceutical industry and universities worldwide because of this interesting behavior against painful processes. CAPSAICIN’S MECHANISM OF ACTION The pharmacological mechanism of action of capsaicin has been extensively reviewed [78]. In 1997, Julius and coworkers isolated and cloned the molecular target of capsaicin, a cellular transmembrane protein and a nonselective cation-permeable pore region (channel) named transient receptor potential vanilloid subtype 1 (TRPV1, also known as vanilloid receptor for its interaction with capsaicinoids) [79]. The TRPV1 receptor is found in primary afferent polymodal C-fibers and Ad-fibers. C-Fibers are responsible for the burning sensations, while Ad-fibers are responsible for the temperature (cold and hot), pressure (mechano), and pain sensations. Both fibers are located in Chapter 3 Natural and Synthetic Alkamides 93 small sensory neurons from central and peripheral nociceptor endings present on nerves. These are referred to as “capsaicin-sensitive” neurons, which transmit information about noxious stimuli to the central nervous system [80–84]. It is believed that not all small sensory nerve fibers are nociceptors, a subgroup of them is also implicated in mediating itch sensation [85,86]. Thus, it is highly likely that some of them are involved in mediating sensation while others mediate nociception. The burning sensation and several of the phenotypic effects caused by capsaicin occurs via its direct interaction with the TRPV1 receptor. TRPV1 is sensitive to activation by a wide variety of exogenous and endogenous physical and chemical stimuli. This receptor has been implicated in multiple disease states such as nociception, neurogenic inflammation, thermal inflammatory hyperalgesia, neuropathic and visceral pain, urinary incontinence, chronic cough, and irritable bowel syndrome [87]. Because of these multiple disease states, the TRPV1 receptor presents a validated target for the discovery of lead chemical probes and the development of novel pharmacologic interventions. TRPV1 antagonists are of value in suppressing gastrointestinal hyperalgesia related to inflammation and other circumstances [88]. The TRPV1 channel is also capsaicin sensitive and is responsible for the sensations induced by various spices and food additives [88,89]. The importance of TRPV1 as a pain sensor was validated by both knockout of the TRPV1 gene in mice and knock-down of TRPV1 by RNA interference [90]. Vanilloid receptor TRPV1 is involved in sensing and integrating a multitude of noxious stimuli (pain transduction) and plays a central role in inflammatory pain. This receptor shows preference for calcium, being activated by capsaicin, vanilloids, piperine, temperatures over 43 C (thermosensation), pH below 5.5 (acidosis), endogenous and exogenous agonists (endovanilloids and anandamide) and indirectly proinflammatory agents (bradykinin and prostaglandins) [89,91]. Furthermore, TRPV1 is a well recognized and validated target for the discovery of new analgesic drugs for neuropathic pain treatment [92]. Administration of capsaicin and dihydrocapsaicin excites the activity of medial thalamic neurons, which play an important role in pain perception and detection of thermal and mechanical nociception [69,93]. Probably, other natural alkamides also exert their effects on the pain-conducting fibers in a similar manner. These nerve fibers can transmit both thermal and mechanic pain sensations as well as itch. Capsaicin is an agonist of the TRPV1 receptor. When capsaicin binds to the TRPV1 receptor it causes an opening of the channel to allow Ca2+ and Na+ entrance into the sensory neurons. Intercellular Ca2+ accumulation desensitizes nerves and depolarization occurs. The next step in this process is peptidergic, known as exocytosis, consisting of the release of sensory neuropeptides, among them substance-P, whose vascular actions lead to neuroinflammation [94]. Other interactions with the TRPV1 receptor by Calcitonin gene related peptide somatostatin, neuronin A, and 94 Studies in Natural Products Chemistry kassinin will produce an acute moderate to intense burning and stinging sensation, which can be perceived as painful or itchy. Through this mechanism of action capsaicin acts as a potent respiratory irritant, inducing sneezing, cough, profuse mucus secretion [95], initial hypersensitization or hyperalgesia and pain. A strong and beneficial analgesic action follows this initial step, explained by the acute cellular influx of predominantly Ca2+, and potassium cations. This high intracellular cation level (with a calcium: sodium permeability ratio starting at about 8:1 increasing to about 25:1 during prolonged capsaicin exposures) [96] is sufficient to activate the calcineurin pathway [91,97]. The TRPV1 channel activity is also modulated by its phosphorylation status, dependent of protein kinases (PK) such as PKA, PKC, and calcium/calmodulin dependent kinase II (CaΜΚΙΙa), which sensitizes its functions. TRPV1 dephosphorylation is achieved by protein phosphatase 2B (also known as calcineurin) and b-arrestin-2. This dephosphorylation desensitize its activity [98] and subsequently desensitize the nociceptor fibers, which causes a lose of function, causing degeneration of the pain signaling pathway and the sensory nerve endings regeneration. Thus, highly efficacious TRPV1 agonists such as capsaicin, can dephosphorylate the channel in a calcium dependent fashion via the calcineurin pathway leading to its desensitization. This mechanism of action constitutes the basis of therapeutic use of capsaicin in reducing the painful states caused, for example by rheumatoid arthritis [68], postherpetic neuralgia, postmastectomy pain syndrome, and diabetic neuropathy. TRPV1 is present in the upper and lower respiratory tracts, increasing nasal and cough sensitivity, promoting fluid secretion, airway narrowing, and bronchoconstriction. In animal models, it plays an essential role in induced cough in a variety of conditions associated with airway hyperresponsiveness, including acid reflux, asthma, interstitial lung disease, and chronic obstructive pulmonary disease [99]. Intraperitoneal injection of capsaicin prevented respiratory depression and diminished severe airway responses, abating bronchoconstriction [99]. These and other mechanisms may explain the analgesic effect of TRPV1 agonists, including the pain relief afforded by topical application of capsaicin in patients with neuropathic pain [100]. Several reviews on the TRPV1 pharmacology related to the capsaicinoid activities are available in the literature [87,101–106]. Factors that promote the open state of TRPV1 receptor facilitate pain, being nociceptive. Conversely, factors that favor its closed state might act as analgesic agents [107]. It is believed that a slow activation of the TRPV1 receptor also produces a slow depolarization and increases intracellular Ca2+ into Ad- and C-fibers. This slow rate of activation results in significant sodium channel inactivation and an analgesic effect, but less activation of the fibers (less burning and stinging sensation). The depolarization produced by capsaicin, however, is fast, showing sensory afferents always spike before their sodium channels become inactivated (before analgesia) [108]. Thus, the quantification of Chapter 3 Natural and Synthetic Alkamides 95 neurotransmitters by presynaptic cells or the changes in intracellular Ca2+ concentration in in vitro assays for agonism (Ca2+ influx into dorsal root ganglia neurones) are good data to report neuronal synaptic activity and predictive of analgesic activity. On the other hand, agonist activity in vitro is predictive of antinociceptive activity and analgesia in vivo [109]. The efficacy of capsaicin application in the treatment of C-fiber disorders is dose dependent. Significant relief from burning pain could be achieved and could persist for up to several months after a single capsaicin exposure [110]. In this manner, capsaicin binds to the same group of nociceptors which lead to the sensation of pain, heat, and acid. Then lead for a reduction in pain and inflammation by depleting the neurotransmitter pain signaling [111]. This effect has been observed for example when an intraarticular injection of capsaicin is administrated to reduce the mechanical hyperalgesia induced in osteoarthritis [112]. Additionally, capsaicin appears to be effective in protecting bone from osteoarthritic damage, supporting the hypothesis that capsaicinsensitive sensory neurons contribute to bone lesions. Therefore capsaicin may be useful for the development of new therapeutic approaches to pain control and prevention of osteoarthritis-dependent bone loss [113]. CAPSAICIN SARs Initial burning pain caused by capsaicin was considered a potential limitation to its use and development as active principle in formulations for the treatment of pain and itch [68]. Since its discovery, several studies have been focused on the design and generation of more potent and effective capsaicinoids with less or without pungent action and less toxic effects than capsaicin to be used as analgesics. Studies on TRPV1 agonists or antagonists have focused on separating the excitatory effects of capsaicin analogs from the antinociceptive properties of these molecules, with the purpose to develop the ideal vanilloid, which might provide the perfect analgesic effects without the side effects caused by vanilloid receptor agonists [114]. Several chemical synthesis of capsaicin and analogs have been reported, mainly by amidation of vanillylamine with fatty acid derivatives [115–130]. Lipase-catalyzed transacylation using Novozyme 435 (lipase B from Candida antarctica), lipase AK, and lipase PS have also proved to be effective to obtain these compounds [131–134]. Using these synthetic methods, several agonists and antagonists of TRPV1 capsaicin receptors have been obtained and evaluated in pain treatment and sensory hyperreactivity and its SAR has been defined. Each molecular region of capsaicin has been analyzed by means of systematic structural studies of the pungent and antinociceptive SARs. These studies revealed that capsaicin, dihydrocapsaicin, and N-vanillyloctylamide (Fig. 3) are approximately equipotent, suggesting that either the overall size or the hydrophobicity (or both) are more important than the double bond and the branched side chain. Variation on fatty acid length, from C7 to C11, 96 Studies in Natural Products Chemistry have the greatest efficacy, constituting compounds with highly potent analgesic activity in mice, being nonivamide (C9 fatty acid) virtually identical to natural capsaicin. Nonivamide, however, retains the high pungency characteristic of capsaicin. Longer fatty acid residues (C12–C17) eliminate the analgesic activity. However, the presence of unsaturations restores and potentiates activity. Compounds possessing C18 aliphatic monounsaturated side chains turn active again, like olvanil (a capsaicin agonist also known as NE-19550, N-9-Z-octadecenoyl-vanillamide, and N-vanillyloleamide), which is 10-fold more potent than capsaicin in TRPV1 activation in the in vitro and in vivo assays [89,129]. The importance of fatty acid size and hydrophobicity is also emphasized by the lack of activity shown by compounds with short side chains, with polar functional groups attached at the end of hydrophobic chains and with longer than C18 hydrophobic chains. On the other hand, ciscapsaicinoids like olvanil and oleylhomovanillamide (NE-28345) are very efficacious, demonstrating that the stereochemistry, which is trans in natural capsaicinoids, and the position of the double bond, which is at C5, C6, or C7 in natural capsaicinods, is not critical [130]. A further increase in potency was obtained by the introduction of a phenylacetyl substituent on the acyl moiety of olvanil, as discovered for phenylacetylrinvanil, the most potent capsaicinoid reported to date (aprox. 500-fold more potent than capsaicin) [89]. Capsaicinol [117], olvanil, rinvanil and phenylacetylrinvanil (IDN5890) [121] are less pungent than capsaicin and are capsaicinods useful in studies of pain and human-TRPV1 (hTRPV1) mode of action (Fig. 15). Therefore, capsaicin’s affinity for TRPV1 results from the length of its hydrocarbon chain, which allows it to bind very strongly to the receptor. Nordihydrocapsaicin, homocapsaicin I, and homodihydrocapsaicin I have slight structural variations (Fig. 3) in the hydrocarbon chain, altering the affinity OCH3 N H OH OH O Capsaicinol O Oleylhomovanillamide O Rinvanil Phenylacetylrinvanil OH COCH2Ph FIGURE 15 Fatty acid region modifications for capsaicin. O Chapter 3 97 Natural and Synthetic Alkamides of the fatty acid for the receptor, as well as binding sites [135]. This is the reason why these compounds are less burning, although they also show less analgesic activity [108]. Capsaicinoids and endovanilloids in general are highly lipophilic compounds and must cross the cell membrane to act on their intracellular binding site(s) on TRPV1. In fact, charged capsaicin analogs cannot cross the cell membrane and are only effective when they are applied intracellularly [89]. Only one carbon between the nitrogen and the aromatic ring, as found in capsaicin, appears to be the optimum length required in the amine region of the capsaicinoid molecule to show maximum effect, Fig. 16. Reverse amides are equipotent. N-Methylation in all cases leads to reduction or loss of activity. Direct attachment of the aromatic ring to the nitrogen atom causes the compounds to lose their activity. Therefore, the benzylic carbon appears to be a requisite for activity. A thiourea replacing the amide group confers the highest potency [128]. An exhaustive study of the SARs on the aromatic ring led to several conclusions, Fig. 17. Compounds with 3-methoxy-4-hydroxybenzyl substitution pattern (vanillyl) are the most potent, being crucial for high analgesic activity. Substitution at 2, 5, and 6 positions, either singly or in any combination on the aromatic ring, leads to poorly active or inactive compounds. Blocking or alkylating the 4-phenol reduces or removes activity. Removal of the 4-phenol leads to loss of agonistic activity. Variation of the 4-substituent removes or decreases activity. Removal of the 3-methoxy group reduces activity. Interchanging the phenol and methoxy substituents at positions 3 and 4 decreases activity and replacing the substituent at the 3-position to an alkyl group leads to reduction or abatement of activity [127]. On the other hand, removal of one of the two oxygens in the aromatic ring in capsaicin conduces to the loss of the activity probing that both atoms are essential to the analgesic activity [124]. Capsaicin becomes inactive when its aromatic structure includes a methylenedioxy group [127]. Inspired in the discovery of the powerful vanilloid antagonism of 50 -iodoresiniferatoxin and of the remarkable effect of aromatic iodination on the improving of the analgesic activity, a systematic investigation on the aromatic halogenation of capsaicinoids was performed to assess if reversal of Thiourea group Reverse amide S N H N H Highest potency O OCH3 OH OCH3 OH Optimum length required Necesary for the activity O OCH3 N H OH N H Weakly active or inactive FIGURE 16 Amide region modifications for capsaicin. H N OCH3 O Equipotent OH N-methylation O N CH3 Reduction or loss of activity OCH3 OH 98 Studies in Natural Products Chemistry Decrease activity Reduction or abatement of the activity OH N H OCH3 OR N H OH Esentials to a high analgesic activity Inactive O N H N H O * * OCH3 OH * *Sustitution lead to poorly active or inactive compounds Remotion reduce activity Remotion lead to loss of agonist activity Blockade or alkylation reduces or removes activity Weak activity N H OCH3 X OH X = I > Br > Cl N H Moderated analgesic activity OCH3 NH2 FIGURE 17 Aromatic ring SARs for capsaicin. vanilloid activity was also possible in the vanillamide residue. The TRPV1 activity of halovanillamides depends of the site of halogenation, the effect being maximal at C-60 and on the nature of the halogen, with iodine being more efficient than bromine or chlorine in reversing the agonistic activity [120]. A series of 4-amino capsaicin analogs were prepared to investigate the bioisosteric replacement of 4-phenol group, and all of these compounds exhibited moderate or weak potency in their analgesic test. However, all these compounds retained the high pungency and side effects characteristic of capsaicin: sedation, vasodilation, ptosis, and decrease of respiration [123]. Models arising from these studies suggest that the side chain of capsaicin (lipophilic moiety) may bind linearly in an extended form through the lipid interface of the transmembrane channel, while the vanilloid moiety might interact with residues in the cytosolic region linking these domains [136]. This idea is reinforced exploring the agonistic activity of the two enantiomers of (S)- and (R)-a-fluorocapsaicin by quantification of increases in intracellular Ca2+ in dorsal root ganglia neurons. Both synthetic fluorinated isomers evoked increased intracellular Ca2+ concentration in similar agonistic response than capsaicin. Inhibition of the activity of both enantiomers by an antagonist to TRPV1 receptor indicates that both enantiomers have the same capsaicin binding site without enantiomerical differentiation, indicating that Chapter 3 99 Natural and Synthetic Alkamides H N R1 C8H17 O OR2 OCH3 R1 = Br R1 =H R2 = R1 = I O R2 = O OCH3 OCH3 R1 = H NO2 R2 = R1 = Br NO2 R2 = H3CO H3CO OCH3 OCH3 O O R1 = H R2 = NO2 O O R1 = Br R2 = NO2 H3CO H3CO OCH3 OCH3 R1 = Br NO2 R2 = H3CO OCH3 FIGURE 18 Some caged-nonivamide analogs. the side chain binds in an extended conformation from the amide bond directly along a molecular axis [116]. A rational to decrease the irritating properties of capsaicin and increase the ease and safety of its use is introducing a photolabile protecting group (process known as caging), that initially produces a biologically inactive capsaicin derivative. A photolysis process releases the biologically active molecule once introduced into the system under study. This caging process allows a temporal and spatial control over when and where the compounds are released, controlling activation or inactivation of the receptor [137–147]. Some caged TRPV1 agonists and antagonists based on the structure of nonivamide alkamide have been developed (Fig. 18). These compounds showed to be inactive in the absence of UV irradiation. However, they showed fast activation of TRPV1 receptors under UV irradiation [123]. Saturated fatty acids increase the expression of the inflammatory genes interleukin 1b, macrophage inflammatory protein 1 (MIP-1), interleukin 6 and 8 (IL-6 and IL-8) in adipose tissue and liver. They have been used as 100 Studies in Natural Products Chemistry models to elucidate the mechanism by which capsaicin improves free fatty acid-induced inflammation. THP-1 Cells were treated with palmitate in the presence and absence of capsaicin and measured gene expression of MIP-1 and IL-8 in human acute monocytic leukemia cell (THP-1) macrophages. Capsaicin increased the gene expression of carnitine palmitoyltransferase 1 and the b-oxidation of palmitate, reduced the gene expression of macrophage inflammatory MIP-1 and IL-8 and significantly reduced palmitate-stimulated activation of c-Jun N-terminal kinase, c-Jun, and p38 [65]. An exhaustive analysis of the structural implications of capsaicinoids on their metabolism by P450 enzymes and how they contribute to both detoxification and bioactivation processes in humans has been proposed. Cyclization, dehydrogenation to afford the terminal diene and imides, oxidation at the end of the carbon chain to render primary and tertiary alcohols, and demethylation and aromatic hydroxylation, are typical reactions in these processes, yielding metabolites with limited pharmacological and toxicological effects via reduction in their affinity for TRPV1 [148]. PHARMACEUTICAL FORMULATIONS BASED ON CAPSAICINOIDS Application of capsaicin and some derivatives to the management of diverse chronic pain syndromes have been demonstrated using different application forms, such as creams, patches, lotions, nasal sprays, and injectables [90,92,149–153]. Salonpas® Hot patch and Allergy Buster® homeopathic nasal spray are based on Capsicum extracts. Qutenza® patches [95,154,155], Zostrix and Capzasin-HP creams and Adlea (ALRGX-4975) injectable solution are formulated using capsaicin as the active principle [156,157]. Finally, Civamide including both a cream and a nasal solution is based on capsaicin Z-isomer (zucapsaicin) [68]. Some of these pharmaceutical forms are under development or commercially available for the treatment of diverse pain conditions including minor aches, pain relief, arthritic pain, postherpetic neuralgia, HIV associated neuropathy, peripheral neuropathic pain in nondiabetic adults, neuropathic pain associated with postherpetic neuralgia, nerve pain, and damage associated with shingles and noncontrolled diabetes, osteoarthritis, and cluster headache, among others. AFFININ (SPILANTHOL) The roots of H. longipes (Asteraceae), just like the fruits of Capsicum species, are used traditionally in Mexico as spice, flavoring, insecticide, antimicrobial, antiparasitic, buccal analgesic, and anesthetic in traditional medicine since the Náhuatl civilization [158–160]. Chewing a piece of H. longipes root creates an intense nonpungent numbness and tingling sensation in the mouth, stimulating salivation [161]. It has also been reported that it produces analgesia Chapter 3 101 Natural and Synthetic Alkamides Amide O N H Alkenyl fatty acid Isobutyl FIGURE 19 Affinin molecular regions. and antiinflammation in dental and oral pathologies in humans [162], suggesting the presence of compounds with analgesic and/or anti-inflammatory properties. Affinin (spilanthol, N-isobutyl-2(E),6(Z),8(E)-decatrienamide) induces saliva flow and is the main bioactive component of the roots of H. longipes. Its chemical structure contains the three regions of capsaicin (Fig. 19) [163–165]. It was shown through a bioassay-directed study that affinin evokes the release of GABA [163] because of the use of H. longipes as analgesic in the treatment of oral ulcers and pain toothache [166]. The analgesic effect of this plant was associated with the presence of affinin [163]. A low combined dose of ethanolic extract of H. longipes diclofenac showed antihyperalgesic effect in the Hargreaves model of thermal hyperalgesia induced by carrageenan. This dose combination interacts synergically and may represent a therapeutic advantage for the clinical treatment of inflammatory pain [162]. In the same context, H. longipes ethanolic extract, affinin, and isobutyl-decanamide displayed a marked anti-inflammatory effect on mouse ear edema test [167]. These results are in accordance with the fact that affinin permeates the skin [59] and buccal mucosa [15]. H. longipes extract and affinin have antinociceptive effects in the acetic acid-induced writhing and capsaicin tests in mice [168]. It has been suggested that this effect may be due to a reduction of the release of neuropeptides in both central and periferal afferents, producing antinociception, suppressing neurogenic as well as inflammatory nociception. The precise mechanisms and sites by which these agents induce antinociception are currently under investigation. Interactions with opioidergic, GABAergic, and serotoninergic systems as well as the cyclic GMP-K+ channel pathway have an important modulatory role in this antinociceptive action. The relation between analgesic effect and in vivo DNA damaging potential of H. longipes ethanolic extract was examined. Its antinociceptive, mutagenic, and cytotoxic effects were measured and histopathological studies from liver, heart, kidneys, spleen, lung, and brain provided evidence that the ethanolic extract exerts analgesic effects with no genotoxic effects [169]. H. longipes acetone extract has sedative effects, but affinin is not involved in this effect. Additionally, both H. longipes acetone extract and affinin are not mutagenic [170,171]. One 28-day study in rats maintained on a diet containing 0, 130, 1300, or 13000 ppm of H. longipes extract equivalent to 5.5, 57, and 572 mg/kg body 102 Studies in Natural Products Chemistry weight/day of affinin for males and 6.5, 64, and 629 mg/kg for females, respectively, revealed no deaths or clinical signs of toxicity in any test group. In accordance with the European Food Safety Authority, the use of affinin (spilanthol) as flavoring in the European industry corresponds to an intake of 24 mg/capita/day on the basis of this Maximized Survey-derived Daily Intake approach [172]. From the nine species included in Heliopsis genus, H. longipes is the only species chemically analyzed, being an excellent source of affinin, which could be isolated in 45% yield from dried extract. H. longipes is also source from other promising related natural alkamides, which are yet to be evaluated as anti-inflammatory and analgesic agents (Fig. 20) [163,173]. Twenty six affinin analogs were synthesized by amidation from unsaturated acids with simple aliphatic amines and evaluated for their sensory properties, being affinin the most active compound [174]. Affinin is also the main component in at least five other species of Asteraceae family: Wedelia parviceps [175], Acmella ciliata, A. oleracea, A. oppositifolia [176] and Spilanthes acmella [177,178]. They all have been used as a spice and also in folk medicine since ancient times to cure severe toothache, infections of throat and gums, stomatitis, paralysis of tongue, antiseptic, analgesic, and immunemodulators. S. acmella extract and affinin (spilanthol) are formulated as Buccaldol® and Indolphar® commercial gels, indicated for local painful mouth issues and minor mouth ulcers [15]. Affinin inhibits the major human P450 enzymes involved in drug metabolism [179] and the NO production in a murine macrophage cell line, efficiently downregulates the production of inflammatory mediators IL-1b, IL-6, and TNF-a, and attenuates the expression of COX-2 and iNOS. These N H O O OH OH OH O O O O FIGURE 20 Affinin-related alkamides isolated from Heliopsis longipes. O N H Chapter 3 103 Natural and Synthetic Alkamides findings are suggestive that affinin can be a useful inhibitor of inflammatory mediators and is potentially applicable for COX-2 selective nonsteroidal anti-inflammatory drugs [180]. Currently, biosynthesis and production of spilanthol from in vitro cells and tissue cultures of S. acmella are promising tools useful for the purpose of scale-up processes [177,181,182]. Another specie used as anesthetic is Acmella decumbens and its content of alkamides has been established [183]. On the other hand, the anesthetic alkamide (2E,4E,8Z,10E)-N-isobutyl-2,4,8,10-dodecatetraenamide was isolated from the toothache plant Acmella oleracea and at least from nine other plants (Fig. 21) [184]. SANSHOOLS The roots, stem-barks, fruits, and seeds of several Zanthoxylum species (Rutaceae) have been used as food additives [185,186] and to treat toothache [187,188]. Zanthoxylum genus is represented by 250 species in the world. These species evoke an irritant, tingling, pungent, cooling, and anesthetic sensation on the tongue that is distinct from the sensation evoked by capsaicin because it lacks the painful sensation [189,190]. Although Zanthoxylum species are popular analgesic drugs and it is known that these properties are due to their high alkamide production, few efforts have been made to determine which metabolites and their contribution to the analgesic properties. For example, 50 years ago, the local anesthetic pellitorine was identified from the roots of Zanthoxylum zanthoxyloides [191]. However, since this pioneering finding, few efforts have been made to investigate the analgesic properties of this natural substance and its synthetic derivatives [192,193]. Pellitorine does not induce saliva flow. It only produces a numbing sensation on the tongue at 10 ppm concentration. However, its isomer, cis-pellitorine shows pungent and warming sensations at the same low concentration (Fig. 22) [192]. Similarly to pellitorine, many other structurally related pungent alkamides are commonly isolated from Zanthoxylum species and all of them are still needed to be studied biologically [194]. O N H FIGURE 21 N-isobutyl-2E,4E,8Z,10E-dodecatetraenamide. O O N H Pellitorine FIGURE 22 Pellitorine geometrical isomers. N H cis-Pellitorine 104 Studies in Natural Products Chemistry O α-Sanshool N O H R1 β-Sanshool N O H R1 ε-Sanshool N O H R1 γ-Sanshool N OH R1 δ-Sanshool N H R1 FIGURE 23 Sanshools (R1 ¼ H) and hydroxy-sanshools (R1 ¼ OH) families. Sanshools are linear polyunsaturated fatty acid amides and major contributors to the characteristic taste of Zanthoxylum plants [195]. They are among the few Zanthoxylum alkamides studied for their saliva flow and analgesic properties. The series of lipophilic trans- and cis-isomers known as (a-, b-, d-, g-, and e-) sanshools and (a, b-, d-, g-, and e-) hydroxy-sanshools are shown in Fig. 23 [190,196]. a-Hydroxy-sanshool has been found to excite the lingual branch of the trigeminal nerve fibers that conduct tactile, temperature, and pain sensations in the mouth, activating low and high threshold cool receptors as well as low threshold mechanoreceptors in neurons mediating innocuous sensations, distinct from previously described capsaicin. This compound produces tingling sensation on the tongue, being useful as a model for studies of paresthesia. a-, b-, and e-Hydroxyl-sanshools were evaluated using taste testing with humans. b-Hydroxy-sanshool was inactive while a- and e-isomers were active, showing that the cis C6–C7 double bond configuration is necessary for biological activity, but not the C10–C11 double bond configuration [197]. a-Hydroxy-sanshool stimulates sensory neurons innervating the mouth by targeting two chemosensitive members of the transient receptor potential (TRP) channel, TRPV1 and TRPA1 as its molecular targets in these sensory neurons [25,194,196–199]. As previously discussed, TRPV1 receptors are involved in sensing a multitude of noxious stimuli. However, TRPA1 receptors respond specifically to cold and pungent compounds. a-Hydroxysanshool causes depolarization in sensory neurons with concomitant firing of action potentials and evokes robust inward currents, causing Ca2+ influx in cells [25]. This natural alkamide excites neurons through a unique mechanism involving inhibition of pH- and anesthetic-sensitive two-pore potassium channels KCNK3, KCNK9, and KCNK18 [200]. b-Hydroxy-sanshool does not excite sensory neurons. However, g-sanshool is also a potent agonist of TRPV1 with an EC50 value of 5.3 mM, activity that explains its pungent and tingling sensation and its use as a natural anesthetic for toothache. Sanshools show only marginal vanilloid activity, both in TRPV1 activation and in Chapter 3 Natural and Synthetic Alkamides 105 sensory assays for pungency [201]. b-Sanshool and g-sanshool inhibited human ACAT-1 and -2 activities being promising therapeutic agents for the treatment of hypercholesterolemia and atherosclerosis [202]. A lipophilic extract from the fruit husks of Z. bungeanum maximum rich in a- and b-hydroxy-sanshools has been validated as a skin soothing agent and anti-itching cosmetic ingredient (commercial name Zanthalene®), because it inhibits synaptic transmission potently, showing a short “paralytic pungency” and an immediate “lifting” although a modest long-term antiwrinkle effects. This extract relaxes subcutaneous muscles and acts as a topical lifting agent for wrinkles [201]. A series of eleven new synthetic alkylamides was designed to analyze the effect of length, unsaturation position, and configuration of the fatty acid chain on the TRPV1 and TRPA1 channel activation by means of intracellular calcium [Ca2+] increment measurements (Fig. 24). Modification of the unsaturations on the alkyl region seems to have little influence on the TRPV1 activation, but Z-olefins are crucial in the activation of TRPA1 receptors. The modification of the amide part at (5Z)N-(2-hydroxy-2-methylpropyl)-dodec-5-enamide by serine [(S,Z)-2-dodec-5enamido-3-hydroxypropanoic acid] or glutamic acid [(S,Z)-2-dodec-5enamidopentanedioic acid] moieties resulted in a significant decrease of TRPA1 activation, probably due to steric hindrance or electronic interactions (Fig. 24). (Z)-2-Dodec-5-enamidoacetic acid was a more potent agonist on TRPA1 channels. Alkylamide (S,Z)-2-dodec-5-enamidopropanoic acid exhibited specificity toward TRPA1. Finally, modification of fatty acid chain using analogs of (S,Z)-2-dodec-5-enamidopropanoic [(S,E,Z)-2-[dodeca-2,6-dienamido]propanoic acid, (Z,Z,Z)-2-[octadeca-9,12,15-trienamido]propanoic acid, and (Z)-2-[hexadeca-9-enamido]propanoic acid] conduced to loss of activity, FIGURE 24 Synthetic alkamide analogs of sanshool. 106 Studies in Natural Products Chemistry underlying the fact that both the alkyl chain tail and the polar amide head of the alkylamides play a role in TRPA1 activation [198]. PIPERINE AND PIPEROVATIN Piper genus (Piperaceae) includes over 1000 species widely distributed throughout the tropical and subtropical regions of the world, some of which have commercial, medicinal, and economical importance [203]. Piper species have been used in China, India, Latin America, and West Indies in traditional medicine as sedatives, to alleviate pain, for the treatment of rheumatism, arthritic conditions, toothaches, and snake bites [20,21,204]. P. nigrum and P. longum (long pepper) are the most popular and used species of this genus. They are perennial climbers native of India, whose seeds are used in cuisine in many countries for their spicy properties and pungency. P. nigrum fruits are highly appreciated because they produce a spice seed used in seasoning in the entire world. When its fruits are collected when red, they produce black pepper, but when they are collected ripen, they produce white pepper [205]. P. nigrum and P. longum are conventionally used as immune enhancers in Indian traditional medicine [206]. P. chaba, a third species of Piper genus, is traditionally used in rheumatic pain. Its methanol extract showed a significant dose dependent analgesic activity in both, acetic acid-induced writhing and tail flick test in mice [207]. Decades of investigations in the natural products chemistry of the Piper genus yielded an important amount of monomeric and dimeric alkamides of diverse molecular architectures contributing to their biological activities [20–23,203,208–219]. Specifically, [4 + 2] dimers were isolated in P. chaba while [2 + 2] dimers were isolated in P. nigrum and P. longum, in both cases together with monomeric amides. The n-hexane extracts from different parts of nineteen Piper species (whole plant, stipites, or fruit) were tested for their anti-inflammatory activity against cyclooxygenase-1 (COX-1) and 5-lipoxygenase (5-LOX), showing evidence that extracts of several of these species act as in vitro inhibitors of both enzymes [213]. Piper alkamides have been reported to possess various activities, like ACAT inhibition (hyperlipidemia and atherosclerosis treatment) [220], cytotoxic [203], antimicobacterial and antibacterial [209], antifungal [210,211], insecticidal [221,222], antiprotozoan [22], anxiolytic, antidepressant and anti-inflammatory [23], and analgesic [20,21]. These Piper alkamides all include a characteristic methylendioxy group, which makes them inhibitors of cytochrome P450 metabolism. Piperine is the major constituent found within the fruits of P. nigrum, P. longum, and many other Piper species. This natural alkamide is responsible for the taste and smell of pepper. It is estimated that each person in the United States consumes a daily intake of 21 mg of piperine [223]. Biologically, this Chapter 3 107 Natural and Synthetic Alkamides O O N O FIGURE 25 Piperine structure. compound displays several pharmacological properties, including pain relief, anticonvulsant, antidepressant, antimetastatic, chemopreventive, anxiolytic, sedative, antioxidant, anti-inflammatory, and immunomodulatory properties [206]. Its anxiolytic and antidepressant activity have been explained by its capability to inhibit monoamine oxidase activity and increase the levels of noradrenaline and serotonin in some regions of the mouse brain [224]. Piperine shares structural similarity with capsaicin, and also produces a sharp, peppery, and burning taste when in contact with the mouth, behaving like capsaicin, being a strong natural agonist (activator) of the vanilloid receptor TRPV1 channel, acting essentially by a similar mechanism (Fig. 25) [225]. In fact, piperine is a less potent, but more effective activator of the hTRPV1 receptor than capsaicin. hTRPV1 quickly desensitizes in response to piperine but not to capsaicin. This desensitization occurs because piperine displays a greater degree of cooperativity than capsaicin [205]. It is not clear why piperine exhibits an improved desensitization–excitation ratio compared to capsaicin. However, this desensitization could be interpreted as an indication that piperine-mediated activation of hTRPV1 involves more hTRPV1subunits having a greater number of interacting sites (additional binding sites) on the hTRPV1 receptor than those required for capsaicin [226]. For these reasons, piperine and related compounds are attractive targets for the design and synthesis of improved TRPV1 agonists that can be developed into clinically useful drugs [204]. Surprisingly, very little is known about piperine’s SARs. In this context, fourteen piperine analogs were evaluated at 100 mM concentration in HEK293 cells expressing the hTRPV1, with the purpose of measuring its TRPV1 activities (Fig. 26) [204]. Tetrahydropiperine, D2-dihydropiperine, piperine, and 5-(30 ,40 methylendioxy phenyl)-2E,4E-pentadienoic acid morpholine amide displayed high Ca2+ responses indicating that methylenedioxy aromatic group and a four-carbon atom chain (without importance of the double bond presence in the middle chain) between the aromatic ring and the amide are important for the activity. 5-(30 ,40 -Methylendioxy phenyl)-2E,4E-pentadienoic acid 4-chlorophenyl amide, 5-(30 ,40 -methylenedioxy phenyl)-2E,4E-pentadienoic acid isobutyl amide, 3-(30 ,40 -methylenedioxyphenyl)-2E-propenoic acid piperidine amide, and 3-(30 ,40 -methylendioxyphenyl)-2E-propenoic acid isobutyl amide were inactive, probably because of the volume of the isobutylamine and chlorobenzene groups, the charge density of the nitrogen atom or the chain length. 3-(40 -Hydroxy-30 -methoxyphenyl)-2E-propenoic acid piperidine amide, 3-(40 -hydroxy-30 -methoxyphenyl)-2E-propenoic acid isobutyl amide, Active Inactive O O O Natural and synthetic piperine analogs N Tetrahydropiperine O O N Half of activity ∆2-Dihydropiperine O O O O O N Piperine H3CO N HO O N O O 5-(3¢,4¢-methylendioxy phenyl)-2E,4E pentadienoic acid morpholine amide 5-(4¢-hydroxy-3¢-methoxyphenyl)2E,4E-pentadien-piperidine amide O H3CO N H HO 5-(4¢-hydroxy-3¢-methoxyphenyl)2E,4E-pentadien isobutyl amide FIGURE 26 SAR for piperine analogs. N H O 5-(3¢,4¢-methylendioxy phenyl)-2E,4E pentadienoic acid 4-chlorophenyl amide O O O Cl O O N H O 5-(3¢,4¢-methylenedioxy phenyl)-2E,4Epentadienoic acid isobutyl amide O O N O 3-(3¢,4¢-methylenedioxyphenyl)-2E propenoic acid piperidine amide O O N H O 3-(3¢,4¢-methylendioxyphenyl)-2E propenoic acid isobutyl amide O H3CO N HO 3-(4¢-hydroxy-3¢-methoxyphenyl)-2E propenoic acid piperidine amide O H3CO N H HO 3-(4¢-hydroxy-3¢-methoxyphenyl)-2E propenoic acid isobutyl amide O H3CO N O HO 3-(4¢-hydroxy-3¢-methoxyphenyl)-2E propenoic acid morpholine amide O H3CO N S HO 3-(4¢-hydroxy-3¢-methoxyphenyl)-2E propenoic acid thiomorpholine amide Chapter 3 109 Natural and Synthetic Alkamides 3-(40 -hydroxy-30 -methoxyphenyl)-2E-propenoic acid morpholine amide, and 3-(40 -hydroxy-30 -methoxyphenyl)-2E-propenoic acid thiomorpholine amide also lacked activity, reinforcing the importance of both, an intact aromatic methylenedioxy group and a four-carbon chain for the activity. Half of the activity was recovered when the chain length was back to four carbons, such in 5-(40 -hydroxy-30 -methoxyphenyl)-2E,4E-pentadien-piperidine amide and 5-(40 -hydroxy-30 -methoxyphenyl)-2E,4E-pentadiene isobutyl amide. The presence of the four-carbon chain makes an important contribution to the activity. The amino acid region of the TRPV1 ion channel protein required for activation by piperine and some of its analogs remains to be identified. In contrast to capsaicin which includes a vanilloid aromatic residue, piperine possesses a methylenedioxy group, which is essential for its activity. Opening of the methylenedioxy group creates a vanilloid residue, but it reduces or abates the activity. These differences in the pharmacological profile between piperine and vanilloid amides suggest that piperine and its analogs bind to TRPV1 at a site different from the vanilloid binding site [204]. At doses of 100 mg/Kg, piperine has antirheumatic effect in animal models and anti-inflammatory effects on IL1b-stimulated rheumatoid arthritis fibroblast-like synoviocytes, acting by inhibition of the production of two important proinflammatory mediators, IL6 and PGE2 [227]. Piperine also exerts antinociceptive effects in a model of visceral inflammation pain in mice [223] and shows a dose dependent synergistic effect on antinociception induced by nimesulide in the acetic acid-induced writhing test in mice. In the formalin test, nimesulide alone did not modify nociceptor mediated pain, while a combination of nimesulide with piperine significantly decreased it. In inflammatory pain, duration of formalin induced behavior indicated a synergistic activity of piperine with nimesulide. These findings suggest that piperine could be used as a biological enhancer when is coadministered with nimesulide [228]. Two additional Piper alkamides with demonstrated antinociceptive activity are laetispicine and piperovatine (Fig. 27), isolated from P. laetispicum [208], and P. piscatorum and P. ovatum [229], respectively. Similar to established local anesthetics, piperovatine produces tongue-numbing because it is a potent stimulator of neuronal intracellular calcium increase, similar in duration and character to other voltage-gated sodium channel agonists. The exact nature of piperovatine’s interaction with the sodium channel remains unknown. The predominant mode of local anesthetic action is the blockage O O H3CO O N H N H O Laetispicine Piperovatine FIGURE 27 Natural piperine analogs, laetispicine, and piperovatine. 110 Studies in Natural Products Chemistry of voltage-gated sodium channel which increases sodium conductance across the neuronal membrane [229]. Piperovatine has been isolated also from P. callosum, P. hancei, P. alatabaccum, Ottonia anisum, O. corcovadensis, O. ovata, and O. vahlii [230]. Alkamides that are used as spice and to produce anesthetic effects have also been isolated from other species. These alkamides have been isolated from Ottonia propinqua [231,232], O. frutescens [233], and Matricaria pubescens [234]. CONCLUSIONS Alkamides are alkaloids of restricted distribution in plants. Although they have been isolated in more than 35 family plants, their presence is usually limited to obtain the individual alkamides in only a few milligrams. However, they are the most abundant components in Asteraceae, Solanaceae, Rutaceae, and Piperaceae families. Even within these four families, their presence is restricted to a few genera: included within Anthemideae and Heliantheae tribes in Asteraceae, Capsicum in Solanaceae, Zanthoxylum and Glycosmis in Rutaceae, and Piper in Piperaceae. Structurally, alkamides possess an acidic and an amino moieties. Both acidic and amino moieties are characteristic depending on the plant family. Today, more than 300 alkamides have been isolated as natural products and more than 100 of their synthetic analogs have been prepared. A large variety of biological activities have been described for these compounds. However, three activities are predominantly important: analgesic attributed to the alkenylamides isolated from some Asteraceae, Capsicum and Piper species; immunomodulatory associated to the characteristic N-alkynylamides found in some of the Asteraceae plants; and antifungal and insecticidal shown for the sulfurated alkamides from Glycosmis. The distribution of alkamides has been described, but important aspects on their chemistry and pharmacology still remains unknown, for example, a complete understanding on biosynthesis, SARs, mechanism of action, and pharmacokinetic parameters are lacking, especially for Asteraceae, Piperaceae, and Rutaceae alkamides. Biologically, the more studied alkamides are the capsaicinoids, especially because of capsaicin, the most abundant capsaicinoid in Capsicum. Research on pungent and analgesic properties of capsaicin led to the discovery of its therapeutic target, the TRPV1 receptor, a key molecular target responsible for its analgesic properties. Several studies using capsaicin and its TRPV1 receptor led to the understanding of its analgesic and anesthetic properties, its mechanism of action and its structure–analgesic activity relationship, knowledge that has been applied to the analysis of the mode of action of agonist and antagonist analogs. The structure–analgesic activity studies suggest that all alkamides include the key amide fragment as responsible for the Chapter 3 Natural and Synthetic Alkamides 111 analgesic activity. However, variation of the alkyl residues in the fatty acid and/or amine groups, confers distinctive characteristics to each alkamide that may promote their use. For example, the presence of the vanilloid residue in capsaicin ensures its pungent properties, an adverse characteristic for its medicinal use. However, many other natural alkamides that lack this structural fragment do not show this unpleasant drawback. This suggests that the adverse pharmacological aspect of vanilloids can be dissociated from its analgesic activity, making the other more than 400 natural and synthetic alkamides known up to date, different to capsaicinoids because of the lack of the vanilloid fragment, potential, and promising candidates to be assayed as analgesic and/or anesthetic agents. Despite its adverse effects, capsaicin is currently being used as the active principle in several pharmaceutical formulations to treat diverse pain conditions, establishing the bases to the eventual use of related alkamides with the same purpose. A drug with analgesic activity, long-lasting effect, and no pungency promises to be of great clinical value. To find that analgesic agent, it is necessary to generate rational data to establish the scientific basis for its use. To demonstrate its use in pain treatment, it is necessary to have a reliable and suitable evaluation model of its effect. The development of models to evaluate the effect and mechanism of action of analgesic drugs is very important. There is a lack of models for pain. This lack of models has been the main restriction in the discovery of therapeutic agents, for example, to treat dental pain. Several Aristolochiaceae, Solanaceae, Asteraceae, and Rutaceae plant species produce an anesthetic sensation when in contact with the mouth, tongue, and lips. They are popularly known as “anesthesia” due to these effects. The whole plants, some parts of them, or their extracts and preparations are being used as local anesthetic and toothache remedies in traditional medicine. A common characteristic of these plants is their alkamide content, being likely that their use is based on the presence of these natural alkaloids. Affinin, sanchools, hydroxysachools and piperine and analogs has shown their analgesic activities. Synthetic analogs of these alkamides have been prepared to study their analgesic effects. These findings suggest that alkamides are excellent candidates for continuing a search for the ideal alkamide analgesic. In the last years, important advances have been made on the molecular structure and the nociceptive and analgesic properties of the alkamides. However, with exception of capsaicin, their SARs remain to be studied. ACKNOWLEDGMENTS This work was financially supported by CONACyT (Grant number 79584-Q). M. Y. R. thanks CONACyT for a sabbatical fellowship (Grant Number 178520). We are grateful to Enrique Salazar-Leyva for technical assistance. 112 Studies in Natural Products Chemistry ABBREVIATIONS 4CL 5-LOX AA ACAT-1 ACAT-2 Acl (ACL) ACP ACS ALRGX-4975 BCAT BKDH CA3H Ca4h (CA4H) CaΜΚΙΙα c-Jun CoA Comt (COMT) CONACyT COX-1 COX-2 CPR DNA EC50 Fat (FAT) GABA GMP-K+ HCHL HEK293 HIV hTRPV1 IDN5890 IL-1b IL-6 IL-8 iNOS KA Kas (KAS) KCNK18 KCNK3 KCNK9 lipase AK lipase PS 4-coumaroyl-CoA ligase 5-lipoxygenase amino acid acetyl coenzyme A acetyltransferase 1 acetyl coenzyme A acetyltransferase 2 acyl carrier protein acyl carrier protein acyl-CoA synthase adlea branched chain amino acid transferase 3-methyl-2-oxobutanoate dehydrogenase coumaric acid 3-hydroxylase cinnamic acid 4-hydroxylase calcium/calmodulin dependent kinase II protein encoding by humans JUN gene coenzime A caffeic acid O-methyltransferase consejo nacional de ciencia y tecnologı́a cyclooxigenase 1 cyclooxigenase 2 cytochrome P450 reductase deoxyribonucleic acid effective concentration acyl-ACP thioesterase g-Aminobutyric acid potassium guanosine monophosphate hydroxycinnamoyl-CoA human embryonic kidney 293 cells human immunodeficiency virus human-TRPV1 phenylacetylrinvanil interleukin 1b interleukin 6 interleukin 8 induced nitric oxide synthase keto acid β-ketoacyl ACP synthase two-pore potassium channel subfamily K member 18 two-pore potassium channel subfamily K member 3 two-pore potassium channel subfamily K member 9 lipase from Pseudomonas fluorescens lipase from Pseudomonas cepacia Chapter 3 Natural and Synthetic Alkamides MIP-1 NE-19550 NE-28345 NO Novozyme 435 p38 P450 pal (PAL) pAMT PGE2 PK PKA PKC Pun1 RNA SAM SAR THP-1 TNF-a TRP TRPA1 TRPV1 UV a-KG 113 macrophage inflammatory protein 1 olvanil oleylhomovanillamide nitric oxide lipase B from Candida antarctica mitogen-activated protein kinase cytochrome P450 phenylalanine ammonia lyase putative aminotransferase prostaglandin E2 protein kinase protein kinase A protein kinase C gene for pungency 1 ribonucleic acid S-adenosyl-methionine structure activity relationship human acute monocytic leukemia cell tumor necrosis factor alpha transient receptor potential transient receptor potential cation channel subfamily A, member 1 transient receptor potential vanilloid 1 ultraviolet a-ketoglutarate REFERENCES [1] B. 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This page intentionally left blank Chapter 4 Alkaloids as Inhibitors of Monoamine Oxidases and Their Role in the Central Nervous System Carolina Dos Santos Passos*, Claudia Simoes-Pires{, Amelia Henriques*, Muriel Cuendet{, Pierre-Alain Carrupt{ and Philippe Christen{ *Laboratory of Pharmacognosy, Faculty of Pharmacy, Universidade Federal do Rio Grande do Sul, UFRGS, Porto Alegre, Rio Grande do Sul, Brazil { School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Geneva, Switzerland Chapter Outline Introduction 123 Therapeutic Potential of Monoamine Oxidase Inhibition in Neurological Disorders 124 Depression 125 Parkinson’s Disease 126 Other Neurodegenerative Diseases 126 Smoke and Alcohol Cessation 127 Alkaloids as Monoamine Oxidase Inhibitors 127 Indole Alkaloids 128 Isoquinoline Alkaloids 134 Piperidine Alkaloids 137 Desoxypeganine 138 Other Alkaloids 139 Conclusion 141 References 142 INTRODUCTION Monoamine oxidases (MAOs) are mitochondrial outer membrane-bound flavoenzymes that catalyze the degradation of biogenic amines, more specifically the oxidative deamination of several important neurotransmitters, including 5-hydroxytryptamine (5-HT) (or serotonin), histamine, and the catecholamines dopamine, noradrenaline, and adrenaline. There are two isoforms Studies in Natural Products Chemistry, Vol. 43. http://dx.doi.org/10.1016/B978-0-444-63430-6.00004-7 © 2014 Elsevier B.V. All rights reserved. 123 124 Studies in Natural Products Chemistry of MAO, MAO-A and -B, which differ with respect to amino acid sequence, distribution in the body tissues, and substrate/inhibitor specificity. MAOs play an important role in several neurodegenerative diseases such as Huntington’s disease, Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis, as well as in depression. Furthermore, they are suspected to be inhibited in cigarette smokers and in alcoholic subjects. Several classes of natural products can modulate MAO activity. One of these classes is the alkaloids, which are nitrogenous secondary metabolites essentially from plant origin. They are one of the largest groups of chemicals found in nature. Most alkaloids are quite toxic and produced by the plants as a defense against herbivores. This class of compounds is not only characterized by a great structural diversity but also by a great diversity of pharmacological effects. Many varieties of alkaloids have remarkable structural similarities with neurotransmitters in the central nervous system (CNS), including dopamine, serotonin, and acetylcholine. They can either mimic (agonists) or block (antagonists) the activity of neurotransmitters leading to numerous physiological and psychological effects. Among the alkaloids with activity on the CNS are those able to inhibit MAO-A and -B with various potencies, such as indole, isoquinoline, piperidine, quinazoline, tetrahydroisoquinoline, tropane, and tryptamine derivatives. The aim of this chapter is to summarize the MAO inhibitory profiles of several of these alkaloids and highlight their importance in the search for novel MAO inhibitors to treat neurodegenerative diseases and neuropsychiatric disorders. They might be particularly promising lead compounds for discovering and developing novel clinical drugs. THERAPEUTIC POTENTIAL OF MONOAMINE OXIDASE INHIBITION IN NEUROLOGICAL DISORDERS Monoamine oxidases (MAOs, EC 1.4.3.4) are a family of flavin-dependent metabolic enzymes that catalyze the oxidative deamination of biogenic and xenobiotic amines. They play an important role in motor and mood control, as well as in the regulation of motivation and other brain functions. Two isoenzymes, MAOA and -B, are distinguishable on the basis of their in vitro substrate specificity and inhibitor sensitivity [1]. MAO-A has a higher affinity for 5-HT, and to lesser extent, for noradrenaline and dopamine. It is inhibited by low concentrations of clorgyline, whereas MAO-B is more specific toward benzylamine, 2-phenylethylamine, and is inhibited by selegiline (Deprenyl) [2,3]. The reaction catalyzed by MAO generates hydrogen peroxide (H2O2), the corresponding aldehyde, and ammonia (from primary amines) as shown in Fig. 1 or a substituted amine (from secondary amines). Both MAO-A and -B are tightly associated with the outer membrane of the mitochondria, and only a small part of both enzymes is found within the microsomal fraction. While MAOs located in peripheral tissues and in the blood–brain barrier seem to exert a protective role through the oxidation of Chapter 4 125 Alkaloids as Inhibitors of Monoamine Oxidases R H2 C O MAO NH2 + H2O 2H2O + FAD R C H + NH3 H2O2 + FADH2 FIGURE 1 Oxidative deamination of a primary amine catalyzed by mononoamine oxidase. amines in the blood (metabolic barrier), MAO isoenzymes in the CNS have more specific functions [2]. As low levels of MAO-A were detected in serotonergic neurons, selective MAO-A inhibitors were shown to increase brain 5-HT and to exert an antidepressant effect. MAO-B is also present in serotonergic neurons and, by degrading other amines, it may contribute to the purity of 5-HT delivered to the synaptic cleft. Both MAO-A and -B are found in noradrenergic neurons. Within synaptosomes of these neurons, MAO-A plays an important role in the deamination of noradrenaline and dopamine in the hypothalamus and striatum, respectively. On the other hand, MAO-B exerts a major role in the extraneuronal dopamine metabolism when dopamine uptake is impaired [2]. MAO inhibitors can be used for the treatment of neurodegenerative diseases, depression, and stroke, as well as tissue damage associated with oxidative stress, nicotine (smoking), and alcohol addiction [2]. Selectivity and reversibility are the main factors to consider while determining the usefulness of MAO inhibitors to treat neurological diseases. For instance, nonselective inhibitors have been avoided in the treatment of extraneuronal MAO-Bdependent pathologies (e.g., Parkinson’s disease) because of the “cheese reaction.” This reaction consists of a marked hypertensive response due to the increase of unchanged tyramine levels in the blood following the ingestion of food rich in tyramine, such as cheese. This is a consequence of the inhibition of MAO-A, the predominant isoform in the stomach and intestine responsible for the metabolization of dietary pressor amines [4]. However, the common trend to avoid the prescription of MAO-A inhibitors is now undergoing some changes since the discovery that these inhibitors can also improve the motor function in patients suffering from Parkinson’s disease [5,6]. MAO-B levels are up to fivefold higher in the brain of the aging population, corroborating the use of MAO-B inhibitors in age-related neurodegenerative diseases. An increase in MAO-B levels results in dopamine depletion and increases in toxic and reactive catalytic by-products, such as dopanal and H2O2 [1]. Depression Selective MAO-A inhibition in the CNS is responsible for the antidepressant effect of MAO inhibitors clinically used, leading to increased levels of dopamine, 5-HT, and noradrenaline [1]. Reversible MAO-A inhibitors have 126 Studies in Natural Products Chemistry demonstrated particular efficacy in the treatment of depression in elderly patients [7]. Despite the efficacy of MAO-A inhibitors as antidepressants, these drugs are usually reserved for patients who failed to respond to the first-line therapy (tricyclic antidepressants), due to the risk of hypertensive crisis (the cheese reaction) and the interaction with serotonergic drugs (serotonin syndrome) [6]. MAO-B inhibitors do not have antidepressant activity and are devoid of the cheese reaction side effect, unless concentrations are high enough to also inhibit MAO-A [2]. Parkinson’s Disease MAO-B inhibitors are currently used as monotherapy in the treatment of Parkinson’s disease, mainly at the early stages following the diagnosis. This increases the endogenous dopamine levels in the affected regions of the brain and postpones the beginning of levodopa treatment (which carries the risk of motor complications). Along with disease progression, the use or association of other drugs, such as dopaminergic agonists, levodopa, and COMT inhibitors, is considered [8–10]. While the MAO-B selective inhibitors are still recommended for the clinical management of Parkinson’s disease, their use has been recently revisited by several authors. Studies showed that the selective inhibition of MAO-A or -B did not alter the steady-state dopamine levels in brain. However, increases in dopamine activity and the subsequent behavioral changes are observed when both isoforms are almost completely inhibited [11]. Acute and chronic treatments with MAO-A and MAO-B inhibitors have similar effects on enzyme activity. The chronic treatment with the selective MAO-B inhibitor L-deprenyl further increases dopamine release due to the action of one of its metabolites, L-amphetamine [11–13]. Other Neurodegenerative Diseases Several neurodegenerative diseases such as Huntington’s disease, Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis have pathological characteristics in common. These include misfolding of proteins that tend to aggregate, impairment of the ubiquitin–proteasome system responsible for the elimination of highly ubiquitinated toxic protein aggregates, oxidative stress, excitotoxicity, and inflammatory processes. Few studies have shown that MAO inhibitors could countervail some of these processes through various neuroprotective mechanisms, such as the interaction with the mitochondrial outer membrane, as well as the upregulation of antiapoptotic proteins and neurotrophic factors [2,6]. Human MAO-A and -B were shown to be upregulated in the basal ganglia of the brain of Huntington’s disease patients. The increase of MAO activity correlated with the severity of the pathology [14]. A mouse model of Huntington’s Chapter 4 Alkaloids as Inhibitors of Monoamine Oxidases 127 disease was also used to demonstrate that specific MAO-A knockout and the intrastriatal administration of the specific inhibitor clorgyline were both able to reduce striatal damage and oxidative stress. One possible explanation was that dopamine contributed to striatal damage caused by impaired mitochondrial function via its metabolization by MAO, followed by the production of toxic oxygen-based free radicals [15,16]. Smoke and Alcohol Cessation MAO-A and -B were shown to be inhibited in cigarette smokers. This is one of the reasons why MAO inhibitors were suggested to be useful in smoking cessation and continued abstinence [17,18]. This effect might be related to the maintenance of a level of MAO inhibition to which smokers were exposed [2]. As a matter of fact, an increase in dopamine levels was detected in the limbic system of smokers, contributing to the reinforcement of nicotine and other addictive drugs [19]. Clinical trials with MAO-B specific inhibitors (selegiline and EVT302) failed to improve smoking abstinence rates compared to placebo [20,21]. On the other hand, the study of the polymorphism in the promoter region of the MAOA gene in alcohol-dependent, heavily smoking men showed that there was evidence for a MAOA gene-associated effect on the quantity of cigarettes smoked. Longer alleles in the promoter region are associated with increased MAO-A activity. Individuals with the 4-repeat alleles MAOA genotype consume more cigarettes per day than those with the 3-repeat allele genotype (p < 0.05) [22]. Moreover, a recent study demonstrated an increase in MAO-A binding in the prefrontal and anterior cingulate cortex during acute withdrawal from heavy cigarette smoking. These findings revive the interest in the use of selective MAO-A inhibitors for smoking cessation, which needs further investigation [23]. Regarding alcoholism, some studies showed low MAO activity levels in alcoholic subjects, but these results could not be confirmed by subsequent studies. Several other factors such as gender, metabolic profile, and concomitant smoking may alter MAO activity [19]. Interestingly, a study showed that the levels of MAO-B activity were significantly increased in dependent subjects submitted to alcohol withdrawal, even after bias correction for smoking and gender [24]. Further studies are needed to better understand the role of alcohol abuse on MAOs inhibition and their implications on alcohol withdrawal. ALKALOIDS AS MONOAMINE OXIDASE INHIBITORS Alkaloids have been extensively investigated for their effects on MAO-A and -B. Several classes of these secondary metabolites are known as potent MAO inhibitors. Among those able to inhibit MAO-A and -B with various potencies are the indoles, isoquinolines, piperidines, quinazolines, tetrahydroisoquinolines, 128 Studies in Natural Products Chemistry tropanes, and tryptamines. Several projects on the optimization of original scaffolds of natural origin by chemical synthesis have already been started and have produced interesting and very potent mono- and multifunctional inhibitors [25–27]. However, the pharmacomodulation of these natural scaffolds generally based on local (quantitative) structure–activity relationship ((Q)SAR) and molecular modeling approaches are beyond the scope of this chapter centered on natural alkaloids. Indole Alkaloids b-Carboline Alkaloids The b-carboline alkaloids are a large group of natural and synthetic indole alkaloids that possess a common tricyclic pyrido[3,4-b]indole ring structure. These molecules can be categorized according to the saturation of their N-containing six-membered ring and, consequently, they present marked differences in the basicity of this nitrogen atom. Unsaturated members are named as fully aromatic b-carbolines (bCs; Fig. 2A), whereas the partially or completely saturated ones are known as dihydro-b-carbolines (DHbCs; Fig. 2B) and tetrahydro-b-carbolines (THbCs; Fig. 2C), respectively [28]. Regarding their biological effects, the b-carboline alkaloids may interact selectively with specific enzymatic targets leading to a variety of pharmacological activities [29]. The inhibitory effects of bCs on MAO-A and -B have been evaluated in enzymes obtained from various organisms revealing details related to their potency, selectivity, and modes of action. The harmala alkaloids were able to inhibit both MAO isoenzymes, possessing various potency and selectivity according to the saturation of the six-membered ring containing one nitrogen atom and substituents of the b-carboline system [30–32]. In nature, these compounds are reported to occur in a number of plants, including Banisteriopsis caapi and Peganum harmala, the extracts of which are traditionally used for their medicinal and psychotropic properties [33]. The main harmala alkaloids (1–12) and their IC50 values for MAO-A and -B inhibition are summarized in Table 1. The mode of inhibition of both MAO-A and -B by bC alkaloids is reversible and competitive, according to kinetic studies [30,34] using human (hMAOs) and rat (rMAOs) enzymes [32,34]. FIGURE 2 Building blocks ring systems of b-carboline alkaloids. Chapter 4 129 Alkaloids as Inhibitors of Monoamine Oxidases TABLE 1 MAO Inhibition By Harmala Alkaloids Subclass Alkaloid MAO IC50 (mM) MAO-A IC50 (mM) MAO-B IC50 (mM) bC Harmine (1) 0.013a 0.002b, 0.005c 20b, >1c 6-MeO-harmane (2) 0.71a Harmol (3) 0.50a 0.018b Harmane (4) 0.45a 0.34d >25d Norharmane (5) 3.55a 6.47d 4.68d Harmaline (6) 0.016a 0.003b 25b 6-MeO-harmalan (7) 1.20a Tetrahydro-harmine (8) 1.77a 0.074b >100b Tryptoline (9) 6.20e >25e 1-Methyl-tryptoline (10) 16e >25e Banisteroside A (11) 4.90b >100b Banisteroside B (12) 22b >100b DHbC THbC a Cytosolic fraction from rat liver containing both MAO-A and -B. The substrate was labeled 5-hydroxy[side-chain-2-14C]tryptamine creatinine sulfate ([14C]5-HT) at a final concentration corresponding to 0.1 mM [30]. b hMAO-A and -B (BD Gentest Supersomes) and kynuramine as substrate in concentrations corresponding to its Km for MAO-A and -B [31]. c rMAOs from liver homogenate and the substrates [14C]5-HT (for MAO-A) and [14C] phenylethylamine (for MAO-B) [32]. d hMAO-A and -B (BD Gentest Supersomes) and kynuramine as substrate [34]. e hMAO-A and -B (BD Gentest Supersomes) and kynuramine as substrate [35]. Regarding SAR, the experimental data of MAO-A and -B inhibition by b-carboline alkaloids indicate that the fully aromatic bCs (harmine, 1), and the DHbCs (harmaline, 6) are more potent inhibitors than the THbCs (tetrahydro-harmine, 8) [30,31]. Compounds 1 and 6 displayed about the same potency for inhibiting the oxidative deamination of 5-HT by rat liver MAOs, with IC50 values of 0.013 and 0.016 mM, respectively (see Table 1) [30]. These data are corroborated by studies using hMAO-A, in which 1 and 6 inhibited this enzyme with IC50 of 0.002 and 0.003 mM, respectively (see Table 1) [31]. The diminution of the inhibitory activity of THbCs may be a consequence of variations in the 3D structure of the N-containing sixmembered ring: a flat geometry seems to be more adequate to interact with the different parts of the MAOs active sites [36]. Additionally, it was demonstrated that 8 was about 30-fold less potent in inhibiting hMAO-A (IC50 of 1.77 mM) than 1 and 6 (see Table 1) [31]. The substituent at C-7 also seems to be important for MAO-A inhibition. Compound 1, 130 Studies in Natural Products Chemistry which possesses a methoxy group at C-7, was a more potent MAO-A inhibitor than harmol (3) and harmane (4), which contain a hydroxyl group and a hydrogen at this position, respectively (see Table 1) [30,31]. Moreover, the 7-substituted bCs (1 and 6) presented higher affinity for MAO-A than the corresponding 6-substitued analogues (6-MeO-harmane, 2 and 6-MeO-harmalan, 7) (see Table 1) [30]. The lack of the methyl substituent at C-1, such as in norharmane (5), seemed to reduce about 10-fold the inhibitory activity on MAO-A. Although 5 was a weaker MAO-A inhibitor than 4, it inhibited MAO-B with IC50 values close to those able to inhibit MAO-A, opposite to other harmala alkaloids, which seem to be selective for inhibition of the A isoform [34]. Finally, the presence of a seven-membered ring at C-1 and N-2, such as in banisteroside A (11) and B (12) [31], seems to be another feature causing the lack of MAO-A activity. The subtle variations in activity reported here could suggest that the selectivity of substrates and inhibitors for MAO-A or -B can be determined at two levels. The first one is the access to the substrate binding site through the hydrophobic entrance cavity (MAO-B) and the putative channel (MAO-A). The second possibility is by differential interactions (hydrophobic stacking and/or H-bonds) with specific residues once the molecule has entered the substrate binding site [36]. R3 R3 R2 N H N R2 R1 N H N R1 1 R1 = CH3; R2 = OCH3; R3 = H 6 R1 = CH3; R2 = OCH3; R3 = H 2 R1 = CH3; R2 = H; R3 = OCH3 7 R1 = CH3; R2 = H; R3 = OCH3 3 R1 = CH3; R2 = OH; R3 = H 4 R1 = CH3; R2 = R3 = H 5 R 1 = R 2 = R3 = H R3 R2 R3 N H N R4 R1 8 R1 = CH3; R2 = OCH3; R3 = H; R4 = H R2 N N H H R1 O R4 HO OH 9 R1 = R2 = R3 = R4 = H 11 R1 = (α)OGlc; R2 = OCH3; R3 = H; R4 = (β)OH 10 R1 = CH3; R2 = R3 = R4 = H 12 R1 = (β)OGlc; R2 = OCH3; R3 = H; R4 = (α)OH Chapter 4 Alkaloids as Inhibitors of Monoamine Oxidases 131 Deeper structural studies by crystallography, molecular modeling, and SAR were performed until recently to modulate the potency and the selectivity of synthetic harmine (1) derivatives (see e.g., [37]). These studies showed that lipophilic substituents, replacing the methyl group of the methoxy moiety at C-7, increased the potency for MAO-A inhibition in comparison with 1. Additionally, it was found that synthetic compounds containing a cyclohexyl group as substituent at C-7 were more potent MAO-B inhibitors than 1. Docking simulations demonstrated that this cyclohexyl chain points to a lipophilic pocket in the “entrance cavity” of the human MAO-B active site. THbC are also described as constituents of spider venom. The major THbC isolated from Parawixia bistriata venom was identified as 6-hydroxytrypargine (13), being also named PwTX-I. This compound occurs at low abundance in spider venom, and this is the reason why Saidemberg et al. [38] proposed a Pictet–Spengler synthesis of 13 for its complete functional characterization. The two synthetic enantiomers resulting from this synthesis, (+)-PwTX-I and ( )-PwTX-I, were analyzed for MAO-A and -B inhibition displaying IC50 values ranging from 8 to 39 mM. The results demonstrated no significant differences in the inhibitory effects of these two enantiomers on MAO-A and -B activity. Moreover, these THbCs seemed to be slightly selective for MAO-B inhibition. The kinetic studies showed that (+)-PwTX-I and ( )-PwTX-I were noncompetitive inhibitors on both MAO-A and -B, differing from the competitive inhibition described for the harmala alkaloids [38]. Monoterpene Indole Alkaloids Some monoterpene indole alkaloids (MIAs) have been evaluated regarding their inhibitory activity on MAOs. Yohimbine (14) displayed a weak rMAO inhibition obtained with a partially purified liver mitochondrial preparation. These effects were observed with concentrations of 100 mM (10–20% inhibition) and 1000 mM (30–40% inhibition) [39]. In another study, isomers of 14, tested at 1000 mM, showed weak inhibition (5–15%) of rMAOs obtained with a hypothalamic tissue homogenate [40]. The antitumour agents vinblastine 132 Studies in Natural Products Chemistry (15) and vincristine (16), tested at 200 mM, inhibited the oxidation of benzylamine by rMAOs from brain mitochondria, possessing about the same qualitative potency [41]. Subsequent kinetic experiments indicated that 15 acted as a reversible and competitive MAO-B inhibitor with an estimated Ki of 0.77 mM [41]. Echitovenidine (17) is an indole from Alstonia venenata fruits possessing a vincadiformine skeleton. This compound was able to inhibit 47% of tyramine oxidation by rMAOs from brain mitochondria at 300 mM and 24% at 30 mM [42]. N N N H H H3CO H O N H H H3CO O O O OH 14 17 N OH H N N H H3CO O H3CO H N H R H3CO O OH O O 15 R = CH3 16 R = CHO MIAs occurring in Psychotria species from the neotropics have also been evaluated on MAO assays. In experiments using brain mitochondrial fractions as a source of rMAO-A and -B, strictosidinic acid (18), lyaloside (19), and strictosamide (20) showed weak to very weak inhibition of MAO-A and -B with IC50 values ranging from 117 to 475 mM for MAO-A inhibition, and 645 to >1000 mM for MAO-B inhibition (see Table 2) [43,44]. Alkaloids 18, 19, and 20, together with other MIAs also isolated from neotropical Psychotria (21–27), were tested on hMAO-A and -B, showing inhibition levels Chapter 4 133 Alkaloids as Inhibitors of Monoamine Oxidases TABLE 2 Rat and Human MAO-A and -B Inhibition By Monoterpene Indole Alkaloids from Psychotria Species MIA rMAO-A IC50 (mM)a rMAO-B IC50 (mM)a hMAO-A IC50 (mM)b Strictosidinic acid (18) 475 >1000 >100 Lyaloside (19) 118 724 182 >100 Strictosamide (20) 133 646 141 >100 Pauridianthoside (21) Angustine (22) E-vallesiachotamine (23) Z-vallesiachotamine (24) Vallesiachotamine lactone (25) Prunifoleine (26) 14-Oxoprunifoleine (27) 19 hMAO-B IC50 (mM)b 316 c 138 d 120 d 126 d 134 d 41 d 81 1.10 2.14 0.85 0.87 7.41 6.92 a rMAO-A and -B from brain mitochondrial fractions; substrate kynuramine in concentrations close to its Km for MAO-A and -B inhibition; deprenyl and clorgyline were used as selective inhibitors to differentiate MAO-A from -B [43,44]. b hMAO-A and -B (BD Gentest Supersomes) [45]. c Reversible and competitive inhibition [45]. d Irreversible inhibition [45]. similar to those verified on the rat enzymes (see Table 2) [45]. Among the MIAs evaluated on hMAOs, angustine (22) inhibited hMAO-A in a reversible and competitive way, while compounds 23–27 behaved as irreversible hMAO-A inhibitors. The IC50 values calculated for hMAO-A inhibition by Psychotria MIAs varied from 0.85 to 182 mM, while the IC50 values for hMAO-B inhibition ranged from 40 to 316 mM. As observed for most of the harmala alkaloids, MIAs seemed to possess selectivity for the A isoenzyme [45]. 134 Studies in Natural Products Chemistry Isoquinoline Alkaloids The protoberberine alkaloids jatrorrhizine (28) and berberine (29) were shown to inhibit rMAOs from brain mitochondria [46]. Compound 28 displayed MAO-A and -B inhibition with IC50 values of 4 and 62 mM, respectively. In this same study, berberine (29) inhibited only MAO-A with an IC50 of 126 mM [46]. The difference in potencies between 28 and 29 could be explained by the phenol hydroxyl group present at C-2 in jatrorrhizine (28) and absent in berberine (29), which possesses a methylenedioxy moiety between C-2 and C-3 [46]. Compound 29 was also evaluated together with palmatine (30) in assays using brain homogenates from mouse (mMAOs) as an enzyme source. In these experiments, it was demonstrated that both 29 and 30 inhibited MAO activity with IC50 values of 98 and 91 mM, respectively [47,48]. Furthermore, 29 and 30 behaved as noncompetitive inhibitors with Ki values of 44 and 59 mM, respectively, in assays using kynuramine as substrate [49]. Another protoberberine isoquinoline alkaloid, coptisine (31), displayed inhibitory effects on mMAO-A from brain, without affecting MAO-B activity. Compound 31 inhibited MAO-A activity in reactions using kynuramine as substrate in a reversible and competitive way, with an IC50 of 1.8 mM [50]. The benzophenanthridine alkaloid sanguinarine (32) inhibited Chapter 4 135 Alkaloids as Inhibitors of Monoamine Oxidases mMAO activity in brain homogenates with an IC50 of 25 mM. The kinetic analysis by Lineweaver–Burk reciprocal plots indicated that 32 behaved as a noncompetitive MAO inhibitor with respect to the substrate kynuramine. The estimated Ki was 22.1 mM [51]. Higienamine (33), a simple tetrahydroisoquinoline (TIQ), inhibited 42% MAO-A activity at 150 mM. The kinetic studies indicated that this inhibition was noncompetitive to kynuramine, with a Ki of 188 mM [47]. Some TIQ, benzyltetrahydroisoquinoline, and tetrahydroxyberberine alkaloids known for their inhibitory activity on MAO can be formed within the human body. An example of such a TIQ is salsolinol (34), a weak MAO inhibitor formed by the direct condensation of acetaldehyde and dopamine. Following the metabolization of alcohol (oxidized to acetaldehyde) and dopamine, 34 can be found in rat brain [52], where it acts as a MAO inhibitor competitive to 5-HT, suggesting selectivity to MAO-A. The estimated Ki values for MAO inhibition by 34 were between 30 and 285 mM (see Table 3) [52–56]. In addition, the potency of 34 for MAO-A inhibition TABLE 3 MAO-A and -B Inhibition of TIQs Alkaloids MAO-A Ki (mM) Mode of Inhibition MAO-B Ki (mM) Mode of Inhibition 110a, 140b, 31c Ca, Cb, Cc 52,000a, NIc NCa 38d Cd (S) salsolinol (34S) 284c, 150d Cc, Cd Tetrahydropapaveroline (35) 820a, 200b Ca, Cb 5000a NCa 2,3,9,11-Tetrahydroxyberberine (36) 50a Ca 3800a Ca Norsalsolinol (37) NId, 47e, 65f Me, Mf 699d NCd N-methyl-norsalsolinol (38) 61d, 81e, 71f Cd, Me, Cf 289d NCd N-methyl-(R)salsolinol (39R) 36d Cd N-methyl-(R)salsolinol (39S) 81d Cd TIQ Alkaloids (R) salsolinol (34R) C, Competitive inhibition; NC, noncompetitive inhibition; M, mixed type inhibition; NI, no inhibition. a rMAOs from brain homogenate. MAO-A activity was evaluated using serotonin as substrate, and MAO-B activity was evaluated with the substrate benzylamine. Experiments performed with racemic mixtures [52]. b rMAOs from brain homogenate. MAO-A activity was evaluated using serotonin as substrate. Experiments performed with racemic mixtures [53]. c hMAO-A isolated from placenta; hMAO-B isolated from liver; substrate kynuramine in concentrations close to its Km for MAO-A and -B [54]. d hMAO-A and -B from human brain synaptosomal mitochondria; substrate kynuramine; deprenyl and clorgyline were used as selective inhibitors to differentiate MAO-A from -B [55]. e hMAO-A from placenta [55]. f hMAO-A from liver [55]. 136 Studies in Natural Products Chemistry varied according to the enantiomeric form: the R enantiomer of 34 was about twofold more potent for MAO-A inhibition than the S enantiomer [54,55]. O O + + R N O N O O O O 28 R = OH 29 30 R = CH3 O O + N O O O + N O O O 31 32 HO NH HO OH 33 Meyerson et al. [52] and Minami et al. [55] also evaluated the effects of other TIQ alkaloids (35–39) on the enzymatic oxidation of different substrates by MAOs (see Table 3) [52,55,56]. The in vitro data for MAO inhibition indicated that most of these alkaloids were MAO-A selective, except for compound 35, which seemed to be a nonspecific inhibitor of rMAOs from brain homogenates [52]. The SAR of isoquinoline derivatives was reported in details by Bembenek et al. [54] and Thull et al. [57]. These studies reinforced that isoquinoline compounds are often selective toward MAO-A and helped to clarify the relative importance of steric, lipophilic, and polar interactions in modulating MAO-A inhibitory activity [49]. Chapter 4 137 Alkaloids as Inhibitors of Monoamine Oxidases HO HO N R1 R2 R3 34R R1 = H; R2 = CH3; R3 = H 34S R1 = CH3; R2 = H; R3 = H 37 R1 = R2 = R3 = H 38 R1 = R2 = H; R3 = CH3 39R R1 = H; R2 = R3 = CH3 39S R1 = CH3; R2 = H; R3 = CH3 HO HO NH HO N HO OH OH OH 35 36 OH Piperidine Alkaloids Kong et al. [58] demonstrated that the alkaloid piperine (40) could inhibit MAOA and -B from rat brain mitochondria in a dose-dependent manner, with IC50 values of 49 and 91 mM, respectively. The kinetic experiments with 40 on MAO-A indicated inhibition of mixed type, with Ki and KI data of 36 and 26 mM, respectively. On the other hand, the inhibition of MAO-B by 40 was shown to be competitive, with a Ki of 79 mM. Other studies also evaluated the effects of piperine (40) on MAO-A and -B activity. Lee et al. [59] determined the potential of 40, isolated from Piper longum extracts, in inhibiting mMAOA and -B in brain mitochondrial fractions. In these experiments, 40 competitively inhibited both MAO-A and -B with IC50 values of 21 mM (Ki ¼ 19 mM) and 7 mM (Ki ¼ 3.19 mM), respectively. In addition, the MAO-A and -B inhibition by 40 seemed to be reversible, as demonstrated by the recovery of percentages higher than 95% of MAO-A and -B activity after dialysis experiments [59]. Taken together, the data of Kong et al. [56] and Lee et al. [57] indicate that 40 seemed to be more selective for rMAO-A, and mMAO-B. In a recent study using brain mitochondria as a source of rMAO-A and -B, Mu et al. [60] demonstrated that 40 inhibited both MAO-A and -B with IC50 values of 0.40 and 0.26 mM, respectively. Finally, inhibition studies using human enzymes revealed that 40 was about 100-fold more selective for hMAO-B inhibition: hMAO-A IC50 ¼ 59 mM and hMAO-B IC50 ¼ 0.48 mM [61]. 138 Studies in Natural Products Chemistry An in vivo study aiming to investigate the antidepressant effect of 40 and of antiepilepsirine (41) showed that both compounds, at doses of 10–20 mg/kg, possessed minor inhibitory activity on MAO-A and -B, when compared with the in vitro enzymatic experiments [62]. Other piperidine alkaloids from Piper longum were also evaluated for MAO inhibition [63]. Guineensine (42) showed significant MAO inhibitory activities with IC50 values of 139.2 mM. The inhibition assays were performed using brain mitochondrial fractions as source of mMAOs, kynuramine as substrate, and clorgyline and deprenyl as selective MAO-A and -B inhibitors, respectively. Desoxypeganine Desoxypeganine (43) is a quinazoline alkaloid isolated from Peganum harmala and is able to inhibit the enzymes MAO-A (IC50 ¼ 2.0 mM) [64] and acetylcholinesterase (IC50 ¼ 17 mM) [65]. This compound showed some activity for the pharmacological treatment of alcohol abuse to reduce craving and depression [66,67]. Inhibitor 43 was subjected to clinical trials aiming at the assessment of its oral bioavailability, pharmacokinetics profile, and tolerability in healthy volunteers submitted to a single-dose (50, 100, 150, or 200 mg) [67] and multiple-dose regimens (50 and 100 mg; 3 days) [66]. These preliminary studies indicated that 43 had a linear and dose-proportional pharmacokinetics, oral bioavailability, plasma half-life, renal excretion, and an adequate safety profile. These features led to further clinical investigations with 43. Chapter 4 Alkaloids as Inhibitors of Monoamine Oxidases 139 Other Alkaloids Quinine (44), cinchonicinol (45), and cinchonaminone (46), isolated from Cinchonae cortex (Cinchona succiruba, Rubiaceae), inhibited MAO from bovine plasma in assays using benzylamine as the substrate [68]. The IC50 values calculated for MAO inhibition were 16 mM (44), 12 mM (45), and 32 mM (46), and kinetic experiments indicated that quinine (44) acted as a competitive MAO inhibitor. The tropane alkaloids atropine (47) and hyoscine (48) slightly inhibited MAO when evaluated at 100 mg/mL (inhibition corresponding to 9% for 47, and 15% for 48) and 200 mg/mL (inhibition corresponding to 14% for 47, and 20% for 48) [69]. Seven indoloquinazoline alkaloids isolated from Evodia rutaecarpa inhibited rMAO-A and -B from brain mitochondria: 1-methyl-2-undecyl-4(1H)-quinolone (49), 1-methyl-2-nonyl-4(1H)-quinolone (50), 1-methyl-2-[(Z)-6-undecenyl]4(1H)-quinolone (51), evocarpine (52), 1-methyl-2-[(6Z,9Z)-6,9-pentadecadienyl]-4(1H)-quinolone (53), dihydroevocarpine (54), and echinopsine (55). The IC50 values for MAO-A inhibition ranged from 240 to >400 mM, whereas the IC50 values for MAO-B inhibition ranged from 2.3 to >400 mM. Among these compounds, the most potent inhibitor for both MAOs was 50, which displayed a 100-fold selectivity for MAO-B. Alkaloids 49, 50, and 54, which differ from one another by C2H4 in the length of the C-2 saturated hydrocarbon chain, showed MAO-B inhibitory activities with IC50 values of 19, 2.3, and 215 mM, respectively, indicating that the longer the aliphatic side chain is, the weaker the inhibitory effect on MAO-B activity is. Additionally, the SAR comparison of the IC50 values among the compounds with same length of the side chain suggested that the presence of double bonds could improve the potency of MAO-B inhibitory activity. Finally, kinetic studies determined that 53 acted as a MAO-B competitive inhibitor, with Ki of 3.8 mM [70]. 140 Studies in Natural Products Chemistry O N 49 R = 50 R = 51 R = 52 R = 53 R = 54 R = 55 R = H R Chapter 4 Alkaloids as Inhibitors of Monoamine Oxidases 141 CONCLUSION In the important search for new drugs to treat age-related diseases, research on MAOs inhibition plays an important role. In this respect, some classes of alkaloids demonstrated multiple biological activities on CNS. The indole and isoquinoline alkaloids showed particularly interesting activities as competitive and noncompetitive inhibitors. The selectivity between MAO-A and -B inhibition is an important factor to decide on the use of a compound for a specific application. Because several alkaloids with small chemical differences were isolated, it was often possible to retrieve SAR information. Even if the range of activity, selectivity, and mode of inhibition varies greatly between classes of alkaloids and also within those classes, a general trend shows a more important selectivity for MAO-A. Few leads were identified, but recent advances have demonstrated that the most promising MAOs inhibitors have to be multifunctional [71]. Without doubt, nature is an important source of novel scaffolds. Considering the lack of a global in silico strategy to predict MAOs inhibition potency, it is mandatory to perform systematic in vitro assays for the inhibition of MAOs from any new natural compound isolated. As soon as the most promising hits are experimentally identified, the modulation of multifunctional activities becomes easier [72,73]. Indeed, the hit selection and lead optimization techniques are now sufficiently mature to rapidly drive via adequate focused virtual libraries the pharmacomodulation of natural scaffolds toward more potent multifunctional MAOs inhibitors [74–80]. ABBREVIATIONS 5-HT βCs CNS COMT DHβCs FAD H2O2 hMAO hMAO-A hMAO-B MAO-A MAO-B MAOs MIAs mMAO mMAO-A mMAO-B 5-hydroxytryptamine β-carbolines central nervous system catechol-O-methyl transferase dihydro-β-carbolines flavin adenine dinucleotide hydrogen peroxide human monoamine oxidase human monoamine oxidase A human monoamine oxidase B monoamine oxidase A monoamine oxidase B monoamine oxidases monoterpene indole alkaloids mouse monoamine oxidase mouse monoamine oxidase A mouse monoamine oxidase B 142 PwTX-I (Q)SAR rMAO rMAO-A rMAO-B THβCs TIQ Studies in Natural Products Chemistry 6-Hydroxytrypargine (quantitative) structure-activity relationship rat monoamine oxidase rat monoamine oxidase A rat monoamine oxidase B tetrahydro-β-carbolines tetrahydroisoquinoline REFERENCES [1] D.E. Edmondson, C. Binda, J. Wang, A.K. Upadhyay, A. Mattevi, Biochemistry 48 (2009) 4220. [2] M.B.H. Youdim, D. Edmondson, K.F. Tipton, Nat. Rev. Neurosci. 7 (2006) 295. [3] J.C. Shih, K. Chen, M.J. Ridd, Annu. 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Chapter 5 Furanocoumarins: Biomolecules of Therapeutic Interest José Antonio Del Rı́o*, Licinio Dı́az*, David Garcı́a-Bernal{, Miguel Blanquer{, Ana Ortuño*, Enrique Correal{ and José Marı́a Moraleda{ *Plant Biology Department, Faculty of Biology, University of Murcia, Murcia, Spain { Cell Therapy Unit, Hospital Universitario Virgen de la Arrixaca, Faculty of Medicine, University of Murcia, Murcia, Spain { Instituto Murciano de Investigación y Desarrollo Agrario y Alimentario (IMIDA), La Alberca, Murcia, Spain Chapter Outline Introduction Furanocoumarins Furanocoumarin Biosynthesis Furanocoumarins in Nature: Distribution and Sources Furanocoumarin Analytic Methods Extraction from Plant Material Sample Purification Purification by Column Chromatography Purification by Thin-Layer Chromatography High-Performance Liquid Chromatography Supercritical Fluid Chromatography and CE Gas Chromatography Activity of Furanocoumarins 146 147 147 149 161 161 162 163 163 164 166 166 167 Therapeutical Use of Furanocoumarins 168 Mechanisms of Action of Furanocoumarins 169 Skin Disorders 170 Noncutaneous Autoimmune Diseases 174 Solid Organ Transplant Rejection 176 Graft Versus Host Disease 178 Cutaneous T-Cell Lymphoma 180 Cancer 181 Microorganism Infections 184 Other Diseases or Clinical Complications 185 Conclusions 185 Acknowledgments 186 References 187 Studies in Natural Products Chemistry, Vol. 43. http://dx.doi.org/10.1016/B978-0-444-63430-6.00005-9 © 2014 Elsevier B.V. All rights reserved. 145 146 Studies in Natural Products Chemistry INTRODUCTION Coumarins are phenolic compounds widely distributed in the plant kingdom. The isolation of coumarin was first reported by Vogel in 1820 [1,2]. The name coumarin originates from a Caribbean word coumarou for the Tonka tree (Dipteryx odorata Willd, Leguminosae), which shares the characteristic smell of these compounds and was known botanically at one time as Coumarouna odorata Aubl. Naturally occurring coumarins, which are classified by their benzopyran2-one nucleus, have been isolated from numerous plants, particularly members of the Apiaceae, Rutaceae, and Fucaceae, as well as from some genera of Leguminosae. A comprehensive collection of structures has been described for them [3–6]. Plant coumarins originate from the shikimate and general phenylpropanoid pathways, yielding cinnamic acid as the immediate product [7,8], which is diverted in various ways. The pattern of coumarins has been proposed as a parameter of taxonomic identification [9–12]. Coumarins are lactones with the basic structure of 1,2-benzopyrone (1) (Fig. 1): most are oxygenated at C-7 position and have isoprenoid chains, attached to a carbon or oxygen or both [7]. According to their structure, coumarins are classified as (I) simple coumarins, (II) furanocoumarins, (III) pyranocoumarins, or (IV) pyron-ring substituted coumarins and its hydroxylated, alkoxylated, and alkylated derivatives, along with their glycosides [7]. Furanocoumarins are subdivided into linear type, generically known as psoralens, where the furan ring is attached at carbons 6 and 7, such as psoralen 5 6 7 8 10 9 4 3 O 2 1 1 O O O O 2 O O O 3 O O OH O O O O O O 4 O 5 O FIGURE 1 Structures of main furanocoumarins in vegetal kingdom. (1) Umbelliferone. (2) Psoralen. (3) Angelicin. (4) Nodakenetin. (5) Archangelicin. Chapter 5 Biomolecules of Therapeutic Interest 147 (2) (Fig. 1), and angular type, generically known as angelicins, where the ring is attached to carbons 7 and 8 of the coumarin structure, for example, angelicin (3) (Fig. 1). Generally, dihydrofuranocoumarins such nodakenetin (4) (linear type) [13] and archangelicin (5) (angular type) [14] have a reduced furan ring. 7-Hydroxycoumarin, commonly known as umbelliferone (1), is often regarded as the parent, both structurally and biogenetically, of the more complex coumarins [15]. Another commonality among coumarins is the presence of isoprenoid chains, frequently of one unit, but often of two or three units, attached to a carbon or oxygen or both. The prenyl group may be found as the simple 3-methylbut-2-enyl unit but is often encountered as the corresponding epoxide or vicinal glycol or in a variety of oxidized and skeletally rearranged forms [8]. Biogenetically, an additional heterocyclic ring can be formed when the prenyl group interacts with an o-phenolic group. The structural variations of this type encountered in natural coumarins mostly include dihydrofuran, hydroxydihydropyran, and their derivatives, furan and dihydropyran. FURANOCOUMARINS Furanocoumarins have been used in folk medicine for a long time. The Indian sacred book Atharva Veda describes the treatment of leukoderma (vitiligo) using a poultice from a plant now known as Psoralea corylifolia, and the ancient Egyptians used Ammi majus for the same disorder. The first furanocoumarin, 5-methoxypsoralen, was isolated in 1838 by Kalbrunner from bergamot oil. Furanocoumarins are a therapeutically important subtype and have various clinical applications. They are found in roots but are more concentrated in fruits and leaves, where they are usually stored in resins as components of the essential oil. The most outstanding property of furanocoumarins is their great ability to sensitize cells to visible light, sunlight, and, especially, nearultraviolet light. This results in strong toxicity, mutagenicity, and possibly carcinogenicity. The mechanism of action is well known. After intercalation into the double helix of the DNA and molecular complexing, the lightactivated furanocoumarins react with the pyrimidine bases, especially with thymine. Since furanocoumarins are strong phototoxic compounds, their presence in a plant has been demonstrated to be a protective mechanism against phytopathogenic microorganisms and herbivores. FURANOCOUMARIN BIOSYNTHESIS Umbelliferone is considered the precursor of furanocoumarins [16]. It is first prenylated in the 6- (for linear furanocoumarins) or 8-position (for angular furanocoumarins) to yield demethylsuberosin and osthenol, respectively. Dimethylallyl diphosphate is required for the 6-prenylation [17]. Demethylsuberosin is 148 Studies in Natural Products Chemistry transformed into marmesin by marmesin synthase (cytochrome P450), which catalyzes the instant cyclization without releasing an intermediate, presumably due to delocalization of the double-bond electrons by the 7-hydroxy group forming the dihydrofuran ring from the ortho-prenylated phenol. Marmesin is transformed into psoralen by psoralen synthase (cytochrome P450), which catalyzes the oxidative carbon–carbon chain (Fig. 2) [18,19]. Psoralen synthase was found to operate by eliminating acetone and one hydrogen from position 30 [17]. Psoralen synthase is very specific for (+)-marmesin and does not accept the ( )stereoisomer (nodakenetin) as a substrate. Moreover, an analogous reaction sequence converts osthenol to (+)-columbianetin catalyzed by columbianetin synthase, and (+)-columbianetin is transformed in angelicin catalyzed by angelicin synthases (Fig. 3) [20,21]. The psoralen synthase gene from A. majus was recently cloned and expressed in yeast cells [22]. The gene was classified as CYP71AJ1 and represents the first cloned monooxygenase sequence committed to coumarin biosynthesis. The homologous enzyme for the angular furanocoumarins has not been isolated to date. The hydroxylation of psoralen at the 5- and/or 8-position is probably necessary for the formation of bergaptol (5-hydroxypsoralen), xanthotoxol (8hydroxypsoralen), and 5,8-dihydroxypsoralen. Only psoralen 5-monooxygenase catalyzes the subsequent hydroxylation of psoralen to bergaptol, in the presence of molecular oxygen, and NADPH has been characterized as a cytochrome P450 enzyme from A. majus [20]. 8′-Prenyltransferase Umbellliferone HO O O Angular furanocoumarins 6′-Prenyltransferase Demethylsuberosin HO O O Marmesin synthase O (+) Marmesin H O O O Psoralen synthase Psoralen O O O Psoralen 5-monooxigenase O O O Bergapten O Psoralen 8-monooxigenase OH Bergaptol OCH3 o-methyltranferase Xanthotoxol Omethyltranferase O O O Bergaptol O OH Xanthotoxol OCH3 O O O O OCH3 Xanthotoxin O O OCH3 Isopimpinellin FIGURE 2 Biosynthesis pathway of lineal furanocoumarins. O Chapter 5 149 Biomolecules of Therapeutic Interest 6⬘-Prenyltransferase Umbellliferone HO O O Linear furanocoumarins 8⬘-Prenyltransferase Osthenol HO O O Columbianetin synthase O O (+) Columbianetin O H OH Angelicin synthase Angelicin Monooxygenase O-Methyl-tranferase O O Monooxygenase O O-Methyl-tranferase MeO MeO MeO Sphondin Pimpinellin O O O O O O FIGURE 3 Biosynthesis pathway of angular furanocoumarins. The action of at least two distinct O-methyltransferases (OMTs), a methyl group, is transferred to bergaptol or xanthotoxol to obtain bergapten and xanthotoxin [7,23–29]. The isopimpinellin formation (5,8-dimethoxypsoralen) pathway is uncertain. Precursor feeding studies with Ruta graveolens plants, however, revealed that bergapten or xanthotoxin may be converted further to isopimpinellin with a slight bias towards xanthotoxin, posing the question as to the existence of another set of OMTs as well as bergapten 8-hydroxylase and xanthotoxin 5-hydroxylase. FURANOCOUMARINS IN NATURE: DISTRIBUTION AND SOURCES Coumarins are widely distributed in the plant kingdom, especially in the families Apiaceae, Rutaceae, Leguminosae, and Compositae [30,31]. Generally, the highest level of coumarins is found in the fruits, followed by roots, stems, and leaves. In Table 1 are shown the main linear furanocoumarins present in the plant kingdom and vegetable sources. On the other hand, in Table 2 are shown the main angular furanocoumarins and their presence in different plants. TABLE 1 Structures of Plant Linear Furanocoumarins and Their Presence in Plant Kingdom Linear Furanocoumarins Name Synonym Psoralen 7-H-Furo(3,2-g)(1)benzopyran-7-one Molecular Formula O Bergapten Plant Source/References O O 4-Methoxyfuro(3,2-g)benzopyrane-7-one OCH3 O O O Apiaceae Pastinaca sativa [26] Apium graveolens [265] Petroselinum sativum [24] Angelica sp. [266,267] Coriandrum sativum [268] Glehnia littoralis [269] Leguminosae Psoralea corylifolia [266] Rutaceae Ruta graveolens [270] Citrus aurantifolia [271] Moraceae Ficus carica [272] Apiaceae Heracleum laciniatum [273] Pimpinella major [274] Pastinaca sativa [26] Heracleum sphondylium [275] Ammi majus [276] Angelica sp. [266,267] Levisticum officinale [277] Apium graveolens [265] Petroselinum sativum [24] Peucedanum tauricum [51] Rutaceae Citrus limon [270] Citrus paradisi [278] Xanthotoxin 9-Methoxyfuro[3,2-g][1]benzopyran-7-one O O O OCH3 Isopimpinellin 4,9-Dimethoxy-7H-furo(3,2-g)(1)benzopyran-7one OCH3 O O O OCH3 Bergaptol OH Xanthotoxol Apiaceae Pastinaca sativa [26] Apium graveolens [265] Petroselinum sativum [24] Heracleum sphondylium [279] Rutaceae Citrus limon [270] Apiaceae Peucedanum tauricum [51] 4-Hydroxy-7H-furo(3,2-g)(1)benzopyran-7-one O Apiaceae Pastinaca sativa [26] Apium graveolens [265] Petroselinum sativum [24] Ammi majus [276] Angelica sp. [266,267] Rutaceae Ruta graveolens [270] Citrus aurantifolia [279] O O Apiaceae Cnidium monnieri [280] 9-Hydroxy-7H-furo(3,2-g)(1)benzopyran-7-one O O O OH Continued TABLE 1 Structures of Plant Linear Furanocoumarins and Their Presence in Plant Kingdom—Cont’d Linear Furanocoumarins Name Synonym 5,8-Dihydroxypsoialen 4,9-Dihydroxy-7H-furo[3,2-g][1]benzopyran-7one Molecular Formula Plant Source/References Apiaceae Cnidium monnieri [280] OH O O O OH Isoimperatorin Apiaceae Glehnia littoralis [269] Angelica lucida [281] Rutaceae Ruta graveolens [270] 4-((3-Methyl-2-butenyl)oxy)-7H-furo(3,2-g)(1) benzopyran-7-one O O Imperatonin O O 9((3-Methyl-2-butenyl)oxy)-7H-furo(3,2-g)(1) benzopyran-7-one O O O O Apiaceae Angelica dahurica [282] Pastinaca sativa [26] Ammi majus [276] Rutaceae Clausena anisata [283] 8-Hidroxybergapten 9-Hydroxy-4-methoxy-7H-furo[3,2-g][1] benzopyran-7-one Apiaceae Angelica dahurica [284] OCH3 O O O OH 5-Hidroxyxanthotoxin 4-Hydroxy-9-methoxy-7H-furo[3,2-g][1] benzopyran-7-one Apiaceae Peucedanum zenkeri [285] OH O O O OCH3 Oxypeucedanin 4-((3,3-Dimethyloxiranyl)methoxy)-7H-furo(3,2g)(1)benzopyran-7-one Apiaceae Peucedanum ostruthium [286] Rutaceae Clausena anisata [287] O O O Heraclenin O O Apiaceae Heracleum candicans [288] 9-(2,3-Epoxy-3-methylbutoxy)-7H-furo(3,2-g)(1) benzopyran-7-one O O O O O Continued TABLE 1 Structures of Plant Linear Furanocoumarins and Their Presence in Plant Kingdom—Cont’d Linear Furanocoumarins Name Synonym Phellopterin 4-Methoxy-9-[(3-methyl-2-butenyl)oxy]-7Hfuro(3,2-g)[1]benzopyran-7-one Molecular Formula Plant Source/References Apiaceae Angelica dahurica [267] Seseli elatum [289] OCH3 O O O O Cnidilin Apiaceae Cnidium dubium [290] Sphenosciadium capitellatum [69] Angelica dahurica [267] 9-Methoxy-4-[(3-methyl-2-buten-1-yl)oxy]-7Hfuro[3,2-g][1]benzopyran-7-one O O O O OCH3 Oxypeucedaninhydrate 5-Benzofuranacrylic acid, 4-(2,3dihydroxy-3-methylbutoxy)-6-hydroxy-, d-lactone Apiaceae Peucedanum ostruthium [291] O O O O O Heraclenol Apiaceae Heracleum candicans [288] 9-(2,3-Dihydroxy-3-methylbutoxy)-7H-furo[3,2g][1]benzopyran-7-one O O O O OH HO Byakangelicin 9-{[(2R)-2,3-Dihydroxy-3-methylbutyl]oxy}-4nnethoxy-7H-furo[3,2-g]chromen-7-one Apiaceae Angelica dahurica [267] OCH3 O O O O O Cnidicin Apiaceae Angelica dahurica [267] 4,9-Bis[(3-methyl-2-butenyl)oxy]-7H-furo[3,2-g] [1]benzopyran-7-one O O O O O Continued TABLE 1 Structures of Plant Linear Furanocoumarins and Their Presence in Plant Kingdom—Cont’d Linear Furanocoumarins Name Synonym Bergamottin 4-((3,7-Dimethyl-2,6-octadienyl)oxy)-, (E)-7Hfuro(3,2-g)(1)benzopyran-7-one Molecular Formula Plant Source/References Rutaceae Citrus paradisi [278] O O 8-Geranoxypsoralen O O Xanthotoxol geranyl ether O O O O Apiaceae Heracleum canescens [292] Heracleum pinnatum [292] Byakangelicol 9-[[(2R)-3,3-Dimethyl-2-oxiranyl] methoxy]-4-methoxy-7H-furo[3,2-g][1] benzopyran-7-one Apiaceae Angelica dahurica [267] OCH3 O O O O O Epoxybergamottin 4-[[(2E)-5-(3,3-Dimethyloxiranyl)-3methyl-2-pentenyl]oxy]-(9CI)7H-furo[3,2-g][1] benzopyran-7-one Rutaceae Tetradium daniellii [293] O O O 6,7-Dihydroxybergamottin 4-[(6,7-Dihydroxy-3,7-dimethyl-2-octenyl)oxy]-, (E)-H-furo[3,2-g][1]benzopyran-7-one O O Rutaceae Citrus paradisi [278] OH OH O O O O TABLE 2 Structures of Plant Angular Furanocoumarins and Their Presence in Plant Kingdom Angular Furanocoumarins Name Synonym Angelicin 2H-Furo[2,3-h]chromen-2-one Molecular Formula O O Isobergapten Apiaceae Angelica sp. [294,295] Pastinaca sativa [296] Ammi majus [296] Apium graveolens [287] Heracleum laciniatum [273] Leguminosae Bituminaria bituminosa [297] Moraceae Ficus carica [272] O Apiaceae Pimpinella magna [294] Heracleum lanatum [295] Pastinaca sativa [296] 5-Methoxy-2H-furo(2,3-h)-1-benzopyran-2-one OCH3 Sphondin O O O 6-Methoxy-2H-furo(2,3-h)-1-benzopyran-2-one H3CO O Plant Source O O Apiaceae Heracleum thomsoni [273] Pastinaca sativa [298] Heracleum lanatum [299] Pimpinellin 5,6-Dimethoxy-2H-furo[2,3-h]-1-benzopyran-2-one OCH3 H3CO Isobergaptol O O O Apiaceae Heracleum thomsoni [302] 5-Hydroxy-2H-furo[2,3-h]-1-benzopyran-2-one OH O O 5,6-Dihidroxyangelicin 5-Benzofuranacrylic acid,4,6,7-trihydroxy-,glactone O Apiaceae Angelica glabra [303] Ligusticum acutilobum [304] OH HO O O Lanatin Apiaceae Ammi majus [24] Angelica archangelica [300] Heracleum laciniatum [273] Pimpinella saxifraga [301] O Apiaceae Heracleum thomsoni [305] 5-[(3-Methyl-2-butenyl)oxy]-(9CI)2H-furo[2,3h]-1-benzopyran-2-one O O O O Continued TABLE 2 Structures of Plant Angular Furanocoumarins and Their Presence in Plant Kingdom—Cont’d Angular Furanocoumarins Name Synonym Heratomin 6-Isopetenyloxyangelicin Molecular Formula Plant Source Apiaceae Heracleum thomsoni [306] O O O O Chapter 5 Biomolecules of Therapeutic Interest 161 Apiaceae plants, which are very rich in coumarins, compose a large family with 240–300 genera and over 3000 species. The presence of furanocoumarins has been described in more than 410 species. The angular furanocoumarins are mainly present in genera Heracleum, Pastinaca, and Pimpinella and are almost absent from Angelica, Peucedanum, Prangos, and Seseli. On the other hand, the linear furanocoumarins are present in Prangos, Angelica, and Peucedanum. Most of these plants contain four linear furanocoumarins, which are psoralen, bergapten or 5-methoxypsoralen (5-MOP), xanthotoxin or 8-methoxypsoralen (8-MOP), isopimpinellin or 5,8-dimethoxypsoralen (5,8-MOP). Angelicin is present in Angelica archangelica, which gave its name to the molecule. Rutaceae is a family that consists of 150 genera and around 1600 species. Coumarins have been characterized in 178 rutaceous species from 50 genera [32]. The four main linear furanocoumarins, psoralen, 5-MOP, 8-MOP, and 5,8-MOP, are present in R. graveolens and R. pinnata, along with other minor linear furanocoumarins, such as oxypeucedanin. Citrus bergamia plants usually contain 5-MOP and other linear furanocoumarins (bergamottin and bergaptol). Different Moraceae species contain bergapten and psoralen. Xanthotoxin has only been described in F. carica. The Leguminosae consist of over 600 genera and 12,000–17,000 species, but the occurrence of coumarins is known in only 155 species of the genera Psoralea and Coronilla. These plants contain the most simple linear and angular furanocoumarins, which are psoralen and angelicin, respectively. These two furanocoumarins have been found in most of the Psoralea species that have been analyzed. In contrast, some Coronilla species, such as C. juncea and C. scorpioides, lack angelicin. FURANOCOUMARIN ANALYTIC METHODS Extraction from Plant Material To date, at least 1300 coumarins have been identified, and many new coumarin structures are being reported each year [33,34]. A variety of methods have been developed for the extraction and identification of known and novel coumarins. Plants are complex matrices, producing a range of secondary metabolites with different functional groups and polarities. Furanocoumarins occur in plants as aglycones or glycosides. Because furanocoumarins have differentiated chemical structures and various hydrophilic and hydrophobic properties, many solvents with increasing polarity and elution strength are used in classical extraction methods (Soxhlet extraction, maceration, percolation, and ultrasonic-aided extraction) [35,36]. Glowniak et al. [37] demonstrated that the application of mixed binary or ternary solvents of dichloromethane, chloroform, trichloroethylene, ether, petrol, and tetrachloromethane leads to a significant increase in the extraction efficiency. Härmälä et al. [38] investigated 162 Studies in Natural Products Chemistry 20 organic solvents to apply them in the extraction of coumarins from the root of A. archangelica. Physicochemical parameters, such as density, viscosity, surface tension, and number of carbon atoms in a molecule, were compared in relation to concentrations of polar and no polar substances obtained in extraction processes. Chloroform was chosen as the most efficient solvent for the extraction of coumarins from plant material. In general, aglycones are soluble in petrol, benzene, oil ether, chloroform, diethyl ether, and alcohols (methanol and ethanol), but they are not soluble or only weakly soluble in water. Glycosides are soluble in water and alcohols. Another method for the isolation of furanocoumarins from plant materials is based on lactone type of coumarin structure. Alcohol or water–alcohol solutions of KOH break the lactone ring in coumarins (in a boiling water bath), giving rise to coumaric acids. Then, after acidification, these acids cyclize to coumarins again, and these coumarins can be extracted using ether. There are many disadvantages in the described method because coumarins are labile substances and are susceptible to acids and bases, which can destroy their epoxide structure and ester bonds in side chains. Supercritical fluid extraction (SFE) has been used for the extraction and separation of furanocoumarins from various subspecies of A. archangelica [39,40]. The fluid is characterized by a high diffusion coefficient, low viscosity and density, and lack of surface tension. The fluid has very good penetration and the ability to dissolve the substances in this matrix [38,41]. Other methods for extracting from plant material include ultrasonification, microwave-assisted solvent extraction in open and in pressurized modes, and accelerated solvent extraction, also called pressurized liquid extraction with exhaustive extraction in a Soxhlet apparatus [42,43]. In Pastinaca sativa and Archangelica officinalis fruits, higher yields of hydrophobic furanocoumarins (bergapten, imperatorin, and phellopterin) were extracted with pressurized liquid extraction in Soxhlet apparatus [41–50]. Sample Purification The next step in sample preparation is to purify the crude extract. Plant extracts contain much ballast material, both nonpolar (chlorophylls and waxes) and polar, such as tannins or sugars. Most often liquid–liquid extraction is used, which takes advantage of solubility differences of hydrophobic substances, which have affinity for nonpolar solvents, and hydrophilic substances, which have an affinity for aqueous solutions. Purification can also be achieved by solid-phase extraction (SPE). This method has been developed for the purification of furanocoumarins from Peucedanum tautaricum Bieb [51,52]. Sidwa-Gorycka et al. [53] used SPE to purify the furanocoumaric fractions obtained from A. majus L. and R. graveolens L. using octadecyl-SPE microcolumns. Chapter 5 Biomolecules of Therapeutic Interest 163 Purification by Column Chromatography Furanocoumarins can be fractionated on an aluminum oxide column eluted with a mixture of different solvents, such as petroleum ether, chloroform, and ethanol, or on silica gel column eluted sequentially with a mixture of hexane, chloroform, and ethanol [54]. Furanocoumarins were extracted from Heracleum sibiricum L. (Apiaceae) fruits by gravitation column chromatography using silica gel and a mixture of benzene, ethyl acetate, and chloroform [55]. Vacuum liquid chromatography on silica gel was developed for the isolation of oxypeucedanin from the leaves of Prangos uloptera [56]. Another useful adsorbent for column chromatography is Florisil, which was used to fractionate furanocoumarins obtained from fruits of Peucedanum alsaticum L. and P. cervaria (L.) Lap [57]. Purification by Thin-Layer Chromatography The physicochemical properties of furanocoumarins depend upon their chemical structure, specifically the presence and position of functional hydroxy or methoxy groups, and alkyl chains. Several analytic methods for the quality control of furanocoumarins in plant materials, such as thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), high-performance liquid chromatography–mass spectrometry (HPLC–MS), high-speed countercurrent chromatography (HSCCC), gas chromatography (GC), gas chromatography–mass spectrometry (GC–MS), capillary electrophoresis (CE), and pressurized capillary electrochromatography (pCEC), have been reported [58,59]. Several adsorbents have been applied for the chromatographic analysis of furanocoumarins, for example, silica gel, alumina, polyamide, and Florisil [60], using a mixture of benzene, acetone, toluene, ethyl acetate, ethylic ether, methanol, and hexane as eluent [61]. The furanocoumarins present in fruits of H. sibiricum L. have been analyzed by two-dimensional TLC on silica gel. The chromatograms were analyzed using UV light and daylight, after spraying with a solution of iodine or Dragendorff’s reagent [55]. Waksmundzka-Hajnos et al. [62] described the use of diol and cyanopropyl–silica for the separation of 10 furanocoumarin standards. In the separation of furanocoumarins, the best results were obtained using CN–silica, firstly chromatographed with the use of normal-phase system and then in a reversed-phase system. The use of graft TLC of coumarins was also reported [63–65]. The coumarins on thin-layer chromatograms are usually revealed by UV light at 365 nm, before or after treatment with an ethanol solution of potassium hydroxide or with ammonia vapor. Sometimes, the fluorescent color is associated to determine the type of functional group [54,66,67]. 164 Studies in Natural Products Chemistry After the separation of furanocoumarin, spectroscopic methods can be used to identify and quantify these phenolic compounds. Coumarin shows absorption bands at 274 and 311 nm, which have been attributed to the benzene and pyrone rings, respectively. Methyl substitution at C-5, C-7, or C-8 leads to a bathochromic shift of the 274 nm maximum but leaves the 311 nm maximum practically unchanged [68]. 7-Oxygenated coumarins show strong absorption bands at about 217 and 315–330 nm with weak peaks or shoulders at 240–250 nm. Linear furanocoumarins show four zones of absorption at 205–225, 240–255, 260–270, and 298–316 nm. Angular furanocoumarins can readily be distinguished from linear forms since the maxima at 242–245 and 260–270 are absent [69]. Different chemical tests have been described to identify furanocoumarins. When solutions of psoralen are illuminated with UV light, a blue fluorescence is issued. The addition of a little sodium hydroxide solution leads to yellow fluorescence in UV light. Methoxsalen, in the presence of sulphuric acid, gives an orange-yellow color that changes to light green. Xanthotoxin gives an instant precipitate with Wagner’s reagent (I2 and KI) and yellow coloration with dilute HNO3. Imperatorin gives an intense deep orange coloration with a few drops of sulfuric acid and finally changes to brown; with Marqui’s reagent, one rapidly changes to brown; Tollen’s reagent (ammoniacal AgNO3) produces a silver mirror; Fehling’s solution produces a brick-red precipitate of cupric oxide, and nitric acid imparts a distinct yellow color [70]. High-Performance Liquid Chromatography Furanocoumarins can also be examined by means of HPLC. This technique has been shown to be a very efficient system for the separation of this group of compounds. HPLC methods have been reported for the determination of psoralens in callus cultures, in vitro culture, serum, dermis, plants, citrus essential oils, and phytomedicines [71–74] (see Table 3). Linear furanocoumarins, such as psoralen, bergapten, xanthotoxin, and isopimpinellin isolated from three varieties of Apium graveolens, were examined by normal-phase HPLC equipped with a variable wavelength detector set at 250 nm [75]. Reversed-phase HPLC is the most widely to quantify furanocoumarins. The quantitative analysis of some furanocoumarins from P. sativa fruits was performed by RP-HPLC in system C18 eluted with methanol and water in gradient elution [42,43,76]. The quantitative analysis of furocoumarins present in P. alsaticum and P. cervia was performed by UPLC (ultra-performance liquid chromatography) using a column packed with 1.7 mm C18 [57]. Liquid chromatography coupled with mass spectrometry (LC–MS) technique is becoming increasingly popular. In particular, the introduction of atmospheric pressure chemical ionization (APCI) has dramatically influenced the possibilities for analyzing poorly ionizable compounds. Coumarins can be Chapter 5 165 Biomolecules of Therapeutic Interest TABLE 3 Chromatographic Methods in the Analysis of Furanocoumarins Plant Material Columns Eluent Detector References Apium graveolens (Apiaceae) Normal phase Ethyl acetate (0.1%), formic acid (0.1%) in chloroform 250 nm [75] Pastinaca sativa (Apiaceae) C18 Methanol and H2O gradient Diode array [42,43] Citrus bergamia (Rutaceae) C18 Methanol and 5% acetic acid gradient Diode array [76] Angelica dahurica (Apiaceae) C18 66:34 (v/v) methanol water UV [59] Brosimum gaudichaudii (Moraceae) CLC-ODS Acetonitrile–water 55:45 (v/v) UV (223 nm) [74,77] Peucedanum tautaricum (Apiaceae) Hypersil ODS C18 Methanol–water gradient Acetonitrile–water gradient UV [51] Melilotus officinalis (Leguminosae) RP-18 Solvent A: water and orthophosphoric acid (1:10,000), Solvent B: methanol, Solvent C: acetonitrile UV [78] Heracleum candicans (Apiaceae) Ovens Solvent A: water– acetonitrile–thf, Solvent B: ACN– methanol–thf UV (310 nm) [79] Dorstenia multiformes (Moraceae) detected in both positive and negative ion modes. Whereas the positive ion mode often generates higher yields, the noise level is lower in the negative ion mode, improving the quality of the signals. So, preliminary investigations regarding the polarity used are very important. A sensitive, specific, and rapid LC–MS method has been developed and validated for the simultaneous determination of xanthotoxin, psoralen, isoimpinellin, and bergapten [80]. A paper by Zheng et al. [81] reported the quantitation of eleven coumarins including furocoumarins in Angelica dahurica, using HPLC–ESI-MS/MS. Yang et al. [82] developed a method for the characterization of linear furanocoumarins in Radix glehniae by LC–MS by the 166 Studies in Natural Products Chemistry combination of two scan modes: multiple ion monitoring-informationdependent acquisition-enhanced product ion mode (MIM-IDAEPI) and precursor scan information-dependent acquisition-enhanced product ion mode (PREC-IDA-EPI) on a hybrid triple quadrupole-linear ion trap mass spectrometer. This method permits the characterization of trace furanocoumarins. High-performance liquid chromatography–diode array detection–electrospray ionization tandem mass spectrometry (HPLC/DAD/ESI-MSN) has been used for the chromatographic fingerprint analysis and characterization of furocoumarins in the roots of A. dahurica [83] and other herbal medicines [59,84]. HSCCC equipped with an HPLC system for separation and purification of furanocoumarins from crude extracts of plant materials has also been described. Psoralen and isopsoralen were isolated and purified from P. corylifolia using HSCCC [85]. This technique has been used for the isolation and purification of furanocoumarins from A. dahurica [86] and from Cnidium monnieri (L.) Cusson [87]. Supercritical Fluid Chromatography and CE Supercritical fluid chromatography (SFE) has been used for the separation from furocoumarins of essential oils. The best separation was obtained on a pentafluorophenyl phase with CO2–EtOH as mobile phase [88]. In few cases, CE was chosen to quantify furanocoumarins. Psoralens present in roots and aerial parts of Chrysanthemum segetum L. were analyzed with electrophoresis apparatus with UV detection at 280 nm [89]. The best overall separation was obtained on an uncoated silica capillary using a buffer solution of boric acid and borax in water. In another assay, micellar electrokinetic capillary chromatography was used in the separation of coumarins contained in Angelicae tuhou [90], with sodium dodecyl sulfate, sodium borate, and dihydrogenphosphate as electrolyte. In Fructus Cnidii extracts, pCEC was used for the separation and determination of coumarins [58]. Gas Chromatography GC has predominantly been used for the identification and quantitative analysis of furocoumarins in preparations and raw plant materials. The GC data can be used to determine the structure and estimate the retention time of analogous coumarins [54]. A number of methods have been described for the analysis of furanocoumarins using capillary GC [24,91]. A gas chromatographic method was used to determine osthole content in Fructus Cnidii extract, with a DBTM-5 column a flame ionization detector FID [92]. The analysis of psoralen, bergapten, pimpinellin, and isopimpinellin has been carried out by GC-FID with a capillary fused silica [71]. Chapter 5 Biomolecules of Therapeutic Interest 167 ACTIVITY OF FURANOCOUMARINS Phenolic compounds exhibit a wide range of biological activities, and they can act as analogues of cellular signal compounds or substrates. These molecules usually have several phenolic hydroxyl groups in common, which can dissociate into negatively charged phenolate ions. The mechanisms of action of coumarins are complex, but they can generally be divided into two lightdependent and light-independent processes. Photoactive or photosensitizer compounds when irradiated can cause toxic reactions in living cells [93]. This can be especially important when the photoactive compounds react with arthropods, bacteria, or fungi, affecting human, animal, or crop health. Furanocoumarins are the main class of plant photosensitizers [94,95]. Absorption of light generates highly reactive excited electronic states, which can interact either with biomolecules or with oxygen, generating the highly toxic reactive oxygen singlet excited state. UV radiation elicits the synthesis of endogenous photosensitizers by the plant defense mechanisms [94,96]. In relation to uncoupling activity and the effects of furanocoumarins on respiration, imperatorin, a furanocoumarin from Imperatoria ostruthium L. and A. majus L., inhibited respiration and phosphorylation of isolated mitochondria in the presence of succinate. Several coumarins have been reported to inhibit glycolysis and oxidative phosphorylation. Coumarin and xanthotoxin can lower the oxygen uptake by meristematic cells of Apium cepa root tips [97]. They also induce morphological changes in the mitochondrial matrix to make it dense. The inhibition of mitosis may be caused by a lack of intracellular energy or changes in mitochondrial structure [98]. Another molecular mode of action of furanocoumarins is related with protein modification. Proteins have multiple functions: enzymes, transporters, ion channels, receptors, microtubules, structural proteins, etc. Conformational changes alter their properties and can prevent effective cross talk between proteins themselves and between proteins and other targets. Polyphenols can interact with proteins by forming hydrogen bonds and ionic bonds with electronegative atoms of the peptide bond or the positively charged side chains of basic amino acids, respectively. Xanthotoxin is a phototoxic furanocoumarin that acts as a P450 monooxygenase inhibitor. Different P450 monooxygenases are involved in the biosynthesis of T-2 toxin in Fusarium sporotrichioides. The addition of xanthotoxin to liquid cultures of F. sporotrichioides blocks T-2 toxin production and induces accumulation of trichodiene, the hydrocarbon precursor of trichothecenes. Quantitative reverse-transcriptase PCR indicated that several of the genes in the toxin biosynthetic pathway were upregulated by xanthotoxin, with Tri4 showing the highest increase in expression. These results indicate that, while xanthotoxin inhibits specific P450 monooxygenase activity, it also has an effect on gene expression [99]. 168 Studies in Natural Products Chemistry The enzymes involved with DNA replication, DNA repair, DNA topoisomerase, and transcription are important targets in all organisms. Inhibitors of these systems are often active against bacteria, fungi, and animal cells. The DNA itself can be modified by compounds with reactive groups, such as epoxides. Interaction between furanocoumarin and DNA occurs in two steps: (a) intercalation of coumarin between two base pairs of the DNA and (b) covalent photoconjugation through 3,4 and 20 ,30 double bonds of bifunctional furanocoumarins [100]. Furanocoumarins such as angelicin, which only form monoadducts (involving the 3,4 double bond), induce mutation at a frequency comparable to ultraviolet light of 254 nm [101]. Interference with DNA, protein biosynthesis, and related enzymes can induce complex chain reactions in cells. Furanocoumarins can photoreact with unsaturated fatty acids. For example, angelicin can form an adduct with linolenic acid. The unsaturated fatty acids may have important role in the phosphatidylinositol system, and furanocoumarin adducts may change the regulatory function of this system. Also, these adducts may inhibit phospholipase and thus prevent the activation of protein kinase C. The greatest extent of photobinding of xanthotoxin and 4,6,30 -trimethoxy was found in the lipid fraction followed by the protein and nucleic acid fractions [98]. THERAPEUTICAL USE OF FURANOCOUMARINS Although furanocoumarins have been used by Indian and Egyptian civilizations for more than 3000 years to treat several skin disorders, it was only in the middle of the twentieth century when the photosensitizing and pigmentstimulating agents in these plants were identified. Indians and Egyptians recognized that the ingestion of boiled extract of leaves, seeds, or roots of several plants, such as A. majus or P. corylifolia, and subsequent sunlight exposure were therapeutic for vitiligo. Since this discovery, extensive research on different aspects of therapeutical interest of furanocoumarins and other related biomolecules has been realized. The modality of treatment that uses these natural compounds has been termed photochemotherapy. Different types of phototherapy include broadband UVB (290–320 nm), narrowband UVB (311–313 nm), UVA1 (340–400 nm), and psoralen and UVA light (PUVA) photochemotherapy (320–400 nm) [102,103]. PUVA therapy consists of the patient receiving total body irradiation with ultraviolet A (UVA) light several times a week after taking a psoralen. Most commonly available psoralens for photochemotherapy include 8-methoxypsoralen or 8-MOP (also called methoxsalen or xanthotoxin), 5-methoxypsoralen or 5-MOP (or bergapten), angelicin, 4,50 -dimethylangelicin, and 4,50 ,8-trimethoxypsoralen or 4,50 ,8-trimethoxypsoralen (TMP) [104]. Psoralen is taken 2 h before irradiation and can be administered orally (5-MOP or 8-MOP) or topically, either painted onto the skin surface or, more frequently, using a bath delivery system Chapter 5 Biomolecules of Therapeutic Interest 169 in which the patient soaks for 15 min in a weak psoralen solution, followed immediately by UVA exposure. This last modality of administration of the psoralen is called bath PUVA [103,104]. Administration of psoralen (usually 8-MOP) in a dilute bath water solution seems to be an effective alternative to the more widely used systemic application, avoiding side effects such as nausea, vomiting, elevation of liver transaminases, or even photodamage to the eyes. Furthermore, it reduces cumulative UVA doses [105]. Moreover, bath PUVA results in a uniform cutaneous absorption and a homogeneous skin distribution of the psoralen. Furthermore, psoralens can be employed using a technique called extracorporeal photopheresis (ECP). This modality is a cell-based immunomodulatory therapy involving the separation of leukocyte-rich plasma from patient followed by ex vivo administration of a psoralen and UVA radiation before reinfusion [106]. ECP was first reported in 1987 by Edelson et al., who used it in a clinical trial for the treatment of cutaneous T-cell lymphoma [107], and actually is being increasingly considered as a safe and promising immunomodulatory therapy with multiple and diverse clinical applications. At the present time, many therapeutical uses have been given to furanocoumarins together with UV light, including the treatment of many skin diseases such as vitiligo, psoriasis, systemic lupus erythematosus, mycosis fungoides, Sézary syndrome, and pemphigus vulgaris; the treatment of other types of noncutaneous disorders, including autoimmune diseases such as Crohn’s disease, type 1 diabetes mellitus, multiple sclerosis, and rheumatoid arthritis; and also the treatment of solid organ transplant rejection and graft versus host disease (GVHD). Mechanisms of Action of Furanocoumarins Although the exact mechanism of action of PUVA therapy is not yet fully understood, this modality of treatment is beneficial for more than 20 different cutaneous diseases. It has been previously reported that PUVA treatment induces apoptosis of pathogenically relevant cells (including T cells, mast cells, and keratinocytes), which is of key importance in the treatment of psoriasis, mycosis fungoides, and atopic dermatitis. Also, PUVA induce the enhancement of melanocyte proliferation for vitiligo, the decreased release of histamine from both basophils and mast cells for histaminic disorders such as urticaria pigmentosa, the inhibition of adhesion molecules and proinflammatory cytokines by keratinocytes in inflammatory diseases, and the inhibition of antigen-presenting function of Langerhans cells. On the other hand, ECP is in general used for the treatment of systemic or multifocal diseases. ECP is divided in three steps: leukapheresis, photoactivation with 8-MOP and UVA light (1–2 J/cm2), and reinfusion of buffy coat to the patient to close the cycle. During UVA irradiation phase, 8-MOP binds covalently to leukocytes’ DNA leading them to cell cycle arrest and induction 170 Studies in Natural Products Chemistry of apoptosis within 48–72 h. These preapoptotic leukocytes are reintroduced into the peripheral circulation where they are phagocytosed by specialized antigen-presenting cells such as immature dendritic cells. In the absence of a proinflammatory milieu, the internalization of self-antigens embodied in these apoptotic cells by dendritic cells inhibits their immunostimulatory properties and renders them tolerogenic, producing augmented levels of antiinflammatory cytokines such as interleukin (IL)-10 and transforming growth factor-beta (TGF-b), decreased levels of proinflammatory cytokines such as IFN-a, IL-12, and tumor necrosis factor-alpha (TNF-a); and increased CCR7-mediated migration of dendritic cells to secondary lymphoid organs [108]. Importantly, other studies have shown that intravenous infusion of apoptotic cells after ECP induces T regulatory cell differentiation from naive CD4+ T-cell population in response to TGF-b and IL-10 produced by tolerogenic dendritic cells, together with the suppression of antigen-specific responses mediated by effector T cells. This increase in the levels of circulating and functional T regulatory cells has been closely related with the clinical effectiveness of ECP [108–111]. Taken together, these modulations in the immune responses induced by ECP lead to the maintaining of the peripheral tolerance, which is very important in the treatment of many diseases such as GVHD, solid organ transplant rejection, or autoimmune diseases. In the treatment of other diseases such as cutaneous T-cell lymphoma, ECP has shown an antitumor effect. The processing of the apoptotic lymphocytes by antigen-presenting cells induces a clonal cytotoxic response, which targets the malignant T-cell population, process mediated by increased levels of TNF-a and interferon-gamma (IFN-g) produced by monocytes and lymphocytes after ECP. Skin Disorders Vitiligo is an acquired idiopathic pigmentary disorder of the skin and hair characterized by the loss of functional melanocytes from the epidermis, with the apparition of well-circumscribed asymptomatic white macules. This disease affects up to 1–4% of population. The cause of vitiligo is unknown, but it has been reported that it may arise from autoimmune, genetic, oxidative stress, neural, or viral causes, which finally contribute to melanocyte destruction resulting in the characteristic depigmented lesions [112]. Recent papers have emphasized the involvement of CD8+ cytotoxic T cells in melanocyte destruction [113–115]. Phototherapy, including narrowband ultraviolet B and PUVA, is an alternative treatment to the use of topical and systemic steroids, topical calcineurin inhibitors, topical vitamin D analogues, or surgical treatment. Only patients with extensive vitiligo are considered suitable for this type of treatment [116,117]. Using oral PUVA with 8-MOP, 5-MOP, and TMP, the irradiation is usually administered twice a week with at least 1 or 2 days between treatments. Chapter 5 Biomolecules of Therapeutic Interest 171 For all oral PUVA protocols, the initial dose is based on the skin type and can oscillate until asymptomatic mild erythema is observed in the vitiligo lesions [118–120]. The most common PUVA short-term side effects include cutaneous or ocular phototoxicity, nausea, and other manifestations such as insomnia, headache, and lightheadedness after ingestion of psoralens [117,118,121]. Patients usually need months of treatment (100–200 treatments), with about 70% of patient responding after 1–2 years. The expected response is related to the development of multiple perifollicular macules of repigmentation and contraction in size. Nevertheless, if after 4–6 months or 30–50 treatments, the patient does not respond, PUVA should be discontinued. Stable disease, for at least a year, is usually easier to treat. Completely repigmented areas can be stable for a decade or more without relapse, but if a lesion is not fully repigmented, reversal of acquired pigmentation may occur when treatment is discontinued [104,122]. KUVA and topical PUVA are alternative treatments to oral PUVA in vitiligo patients. KUVA uses an organic biomolecule derivate of 1,4-benzopyrone and furan called khellin as photosensitizer, and this is not phototoxic. Topical PUVA may be suitable for small lesions, while UVA alone is of limited benefit [122]. Cytotoxic CD8+ cutaneous lymphocyte antigen (CLA)+ T cells may have a crucial role in the pathogenesis of vitiligo, and the presence of melanocytespecific CD8+ CLA+ T cells seems to be closely related to the activity of the disease as the presence of this type of cells is associated with melanocyte destruction [123]. There are some studies reporting a good correlation between the clinical recovery and the modulation of CD8+ CLA+ T-cell number in peripheral blood after PUVA (25% reduction of CD8+ CLA+ T cells in comparison with pretreatment levels). This finding reinforces the therapeutic potential of PUVA treatment [124]. In summary, PUVA therapy in vitiligo patients increases the number and activity of epidermal melanocytes in all areas and decreases the degenerative changes in both melanocytes and keratinocytes. The reversal of degeneration in both leukodermic and apparently normal skins after PUVA points towards the role of this modality in both repigmentation and protection against further depigmentation [125]. Psoriasis is another chronic and autoimmune skin disorder characterized by raised, scaly, and reddened patches (or plaques), which result from hyperproliferation of the epidermis and inflammation of both epidermal and dermal layers [126]. This disease is also characterized by periods of remission and relapse. Psoriasis is mediated by activated T cells [127] and activated dendritic cells located in psoriatic plaques. These cells release proinflammatory cytokines, including both TNF-a and IFN-g, that increase blood vessel synthesis, vasodilatation, and keratinocyte proliferation [128]. Historically, the long-term management of psoriasis has been complicated by a variety of treatment-related factors, including inconsistent efficacy over time, the risk of significant cumulative toxicities, and patient dissatisfaction 172 Studies in Natural Products Chemistry and noncompliance. In 1974, a group of Harvard scientists started to use oral PUVA for the treatment of psoriasis. Repeated exposures (18–20 treatments) to UVA after oral ingestion of 8-MOP led to the disappearance of the psoriatic lesions [129]. Importantly, therapeutic efficacy of PUVA was drastically enhanced combining PUVA and oral administration of aromatic retinoid derivates such as etretinate 10 days before to starting PUVA therapy. This modality, called RePUVA, was able to reduce both the time and number of treatments necessary for complete clearance of psoriatic lesions (30%) and the UVA cumulative dose required to clear psoriasis (56%) [130,131]. Balneotherapy using 8-MOP and UVA radiation represents an alternative modality for the treatment of this skin disease [132]. 5-Methoxypsoralen, another linear furanocoumarin, has been also employed for the treatment of psoriasis. In comparative clinical trials of parallel design, psoriasis clearance rates of >90% or >97% were observed in similar numbers of patients (60–77%) receiving oral PUVA 5-methoxypsoralen or oral PUVA 8-methoxypsoralen treatments [133]. Generally, 5-methoxypsoralen patients required a greater total UVA exposure than 8-methoxypsoralen recipients to achieve clinical response. However, the incidence and severity of adverse events were generally lower in PUVA using 5-methoxypsoralen than in PUVA with 8-methoxypsoralen. These adverse events include short-term cutaneous and gastrointestinal unfavorable effects. The long-term tolerability of 5-methoxypsoralen has yet to be fully established. Decreased epidermal proliferation is considered to be the main mechanism of action of PUVA in the treatment of psoriasis. Once excited by UVA, psoralens can react with molecular oxygen, producing reactive oxygen species that cause mitochondrial dysfunction and lead to apoptosis of skin Langerhans cells, keratinocytes, and lymphocytes [134]. PUVA further decreases epidermal cell proliferation by noncompetitively binding to epidermal growth factor receptors and directly altering the cell surface membrane. Pemphigus vulgaris is a rare, potentially fatal, autoimmune mucocutaneous blistering disease. Both genders are equally affected with the mean age of onset in the sixth and seventh decade of life. The patients present skin lesions, typically flaccid blisters, which can be recurrent and relapsing and are located on the entire body surface and on the mucous membranes of the mouth. Pathology of pemphigus vulgaris is characterized by the in vivo deposition of autoantibodies on the keratinocyte cell surface. These antibodies, which are also present in the circulation, are typically directed against a 130 kDa protein called desmoglein-3. Additional autoantibodies against desmoglein-1 have been also detected [135]. ECP using 8-methoxypsoralen has been applied to patients with persistent pemphigus vulgaris who had previously needed large doses of immunosuppressive drugs to control their disabling disease, showing long-term remission and absence of several side effects [136–138]. On the other hand, ECP using psoralens is effective for the treatment of severe, therapy-resistant atopic Chapter 5 Biomolecules of Therapeutic Interest 173 dermatitis and in clearing or reducing erosive lesions in patients with lichen planus [139–142]. Atopic dermatitis is an immune-mediated, relapsing skin disorder for which treatment options include a variety of emollients, corticosteroids, immunomodulators, and phototherapy [143]. Atopic dermatitis patients who failed to respond to standard first- and/or second-line therapies showed substantial skin responses to ECP treatment within 6–10 cycles, indicating that photopheresis may have beneficial therapeutic effect in patients who are refractory to conventional therapy [144]. In addition, lichen planus is a chronic mucocutaneous disease that can develop on the skin, mouth, scalp, genitals, or nails. Oral and topical PUVA are therapeutic options for extensive lichen planus. However, this disease is usually more resistant than psoriasis, requiring more treatment sessions and higher cumulative UVA doses, with earlier relapses. Alternatively, combined PUVA–retinoid therapy may accelerate clearing of lichen planus manifestations [104]. Systemic lupus erythematosus is an autoimmune disease that causes affectation not only of the skin but also of other organs, resulting in photosensitive skin eruptions, arthritis, serositis, nephritis, and hematologic abnormalities. It is produced by circulating autoantibodies, immune complexes, and complement deposition that leads to cell and tissue injury [145]. In several open clinical trials, patients with systemic lupus erythematosus have been treated with ECP using 8-MOP, showing a significant response to the treatment, with no or minor side effects [146]. Systemic sclerosis or scleroderma is a chronic multisystem disorder of unknown etiology clinically characterized by a thickening of the skin produced by an accumulation of connective tissue, which affects other internal organs such as the gastrointestinal tract, lungs, heart, liver, and kidneys and microvasculature abnormalities. Although systemic sclerosis is not curable by now, especially in patients with aggressive disease, new treatment modalities include the use of minocycline, lung transplantation, etanercept, thalidomide, and PUVA [147,148]. Extracorporeal photochemotherapy using 8-MOP has been used in patients with diffuse systemic sclerosis, in a schedule of 2 successive days monthly for at least 50 months. Using this treatment, an improvement or stabilization was noted in most patients in skin thickening, joint mobility, and pulmonary function and in symptoms including Raynaud’s phenomenon, dyspnea, fatigue, dysphagia, arthralgias, and cutaneous ulcers [149]. It has been also reported that ECP contributes to the restoration of disproportional autoimmune responses and attenuates fibrotic processes, thus decelerating the disease progression [150]. Altogether, ECP is well tolerated in the management of early-onset diffuse systemic sclerosis and may provide an increasingly beneficial clinical outcome. Other skin manifestations treated or prevented by the use of psoralen by means of PUVA and/or ECP procedures are summarized in Table 4. Taken together, PUVA and ECP seem to be a good choice for the treatment or prevention of many skin diseases of different etiologies, although 174 Studies in Natural Products Chemistry TABLE 4 Skin Diseases Treated or Prevented Using PUVA or Extracorporeal Photopheresis (ECP) PUVA/ECP in therapy of disease Pemphigoid Epidermolysis bullosa acquisita Atopic eczema Discoid lupus erythematosus Subacute lupus erythematosus Dermatomyositis Solar urticaria Nephrogenic systemic fibrosis Alopecia areata Urticaria pigmentosa Pansclerotic morphea Eosinophilic fasciitis Eczema Eosinophilic pustular folliculitis Granuloma annulare Parapsoriasis Pityriasis lichenoides Pityriasis rosea Polycythemia vera Transient acantholytic dermatoses Palmoplantar pustulosis Lymphomatoid papulosis Prurigo nodularis Pityriasis rubra pilaris Scleromyxedema PUVA/ECP in prevention of disease Polymorphous light eruption Hydroa vacciniforme Solar urticaria Persistent light reaction Chronic actinic dermatitis Actinic reticuloid Erythropoietic protoporphyria Sun-induced nonmelanoma skin cancer the mechanism of action by which these procedures are effective is poorly described to date. Noncutaneous Autoimmune Diseases Aside from their utility for the treatment of multiple skin manifestations, psoralens and UVA radiation could be used as a therapeutic alternative for several immune-mediated disorders as Crohn’s disease and ulcerative colitis. Both are chronic inflammatory diseases of the gastrointestinal tract and are collectively known as inflammatory bowel disease. This disorder is produced by a dysfunction of the immune system that leads to the accumulation of abundant lymphocytes and monocytes in the mucosa of the bowel, together with the secretion of cytokines and proinflammatory mediators. There are several genetic, environmental, and physiological factors that contribute to the pathogenesis of inflammatory bowel disease [151]. Alternatively to the use of steroids, other types of therapies are needed due to the apparition of steroid resistance and the side effects related with its short- and long-term use. ECP with 8-MOP and UVA has been recently reported as a potential therapy for refractory Crohn’s disease [152]. After at least 24 weeks of stable concomitant ECP therapy, patients with moderate Chapter 5 Biomolecules of Therapeutic Interest 175 disease achieved a good clinical response, although patients with severe disease showed limited improvement [153,154]. Type 1 diabetes mellitus is another autoimmune disease produced by the lymphocytic infiltration of the pancreas and the subsequent destruction of insulin-producing beta cells that leads to increased blood and urine glucose [155]. Since the autoimmune background of the disease was discovered, several types of immune therapies have been used, including the use of psoralens by ECP. Several clinical trials using ECP with 8-MOP and UVA radiation have been carried out. In one of them, ECP-treated patients secreted significantly more C peptide in urine than control patients. C peptide is a marker that measures endogenous insulin production. Furthermore, the insulin dose/kg body weight needed to achieve satisfactory glycosylated hemoglobin (HbA1c) values (which is related to levels of blood glucose) was always lower in the photopheresis group [156]. Recent studies suggested that ECP is effective in the inhibition of autoimmune processes against beta cells from pancreatic islets by maintaining T regulatory and Th2 cells-associated immunoregulation, which possess a protective role in the development of this disease [157,158]. Together, these results suggest that psoralen-mediated therapies could be beneficial in the treatment of autoimmune diseases such as type 1 diabetes mellitus. Multiple sclerosis is a relapsing and finally progressive disorder of the central nervous system causing white matter demyelination of variable prognosis. Clinical symptoms include sensory disturbances, unilateral optic neuritis, diplopia, limb weakness, gait ataxia, neurogenic bladder, and bowel symptoms. Although it is believed to be an autoimmune disorder, with involvement of both the humoral and the cellular components, there are also genetic and environmental factors involved in its pathogenesis [159]. In patients who fail initial treatment with high dose of steroids, other therapies may be of benefit, including interferon-1b, glatiramer acetate, immunoglobulin, anti-TNF-a, mitoxantrone hydrochloride, therapeutic plasma exchange, and ECP [160,161]. A preliminary double-blind, placebo-controlled trial of ECP using 8-MOP found this therapy to be a safe and tolerable method, but it did not affect significantly the course of chronic progressive multiple sclerosis and in secondary forms could only transiently improve the clinical picture [162]. However, in a more recent pilot study with patients with relapsing–remitting multiple sclerosis, ECP was also a safe and tolerable technique suggesting that this treatment might be useful as a therapeutic alternative in patients not responsive to or not eligible for traditional immunomodulating or immunosuppressive treatments [163]. Rheumatoid arthritis is a systemic autoimmune disorder produced by an inflammatory response of the synovium of the joints and is accompanied by hyperplasia of synovial cells, excess of synovial fluid, and apparition of fibrous tissue in the synovium, leading together to the destruction of the articular cartilage and ankylosis or fusion of the joints. In a pilot study published 176 Studies in Natural Products Chemistry by Malawista et al., ECP using 8-MOP was applied to rheumatoid arthritis patients, showing a reduction in number and degree of joint affectation after 12–16 weeks of therapy. This preliminary study suggests that ECP may be effective at least in the short term in certain patients with rheumatoid arthritis [164]. Solid Organ Transplant Rejection Treatment with psoralen-derived therapies has become relevant to other autoreactive disorders that may also benefit from an enhanced T regulatory cell response including solid organ transplants rejection. In this condition, the immune system of the organ recipient recognizes proteins on the surface of the cells of the transplanted organ (i.e., major histocompatibility antigens) as foreign and responds by attacking the donor organ. The immune response can be limited by the use of donor organs with similar major histocompatibility antigens and by the use of immunosuppressive drugs that reduce the immune response to the transplanted organ. The standard treatment for solid organ rejection is immunosuppression. However, several complications and risks such as severe infections from immunosuppressive therapies remain a significant problem, and alternative treatments are needed. The use of ECP to prevent or treat rejection of transplanted solid organs dates back to 1985, when the first transplantation of UVA-radiated rat kidney after administration of 8-MOP was reported [165]. Four years later, the first ECP use in heart xenotransplantation and allotransplantation in primates (from cynomolgus monkey to baboon or from baboon to baboon, respectively) was described, showing an absence of hyperacute rejection and an increased graft survival in the treated group [166,167]. These studies demonstrated that ECP can suppress recipient immune responses to donor xeno- or alloantigens improving graft survival, while maintaining the ability to respond to other unrelated antigens. After these initial approaches in animal models, ECP has been used in humans for the prevention and/or treatment of several solid organ transplant rejections, including kidney, heart, lung, pancreas, and liver. The year of introduction of ECP for treating rejection of each type of transplant is indicated in Table 5. Importantly, ECP is effective for patients resistant to conventional treatment, particularly if started early. Besides reversal of allograft rejection, a reduction in immunosuppressive therapy has also been frequently achieved [173,174]. The first clinical application of psoralen and UVA radiation in transplantation was the treatment of kidney allograft acute rejection [168]. This first clinical attempt was performed using PUVA therapy on the donor kidneys previously to its transplantation into the patients. Importantly, fewer rejection episodes were reported in patient receiving treated organs compared with untreated counterparts after 3 months of the transplant. Furthermore, this Chapter 5 177 Biomolecules of Therapeutic Interest TABLE 5 Use of Extracorporeal Photopheresis for Prevention or Treatment of Acute Rejection Episodes after Solid Organ Transplantations Solid Organ Transplant Year of Introduction References Kidney 1987 [168] Heart 1992 [169] Lung 1995 [170] Pancreas 1995 [171] Liver 2000 [172] group of patients also showed a lower rate of infectious complications. After this initial clinical study, ECP using 8-MOP and UVA radiation has been employed to treat recurrent and/or steroid-refractory acute rejection episodes in kidney transplantation [174–177]. The first report of the successful use of ECP in human cardiac transplant recipients was 20 years ago and described the efficiency of the ECP as a complementary treatment for acute rejection in a heart allotransplantation [169]. Using the combination of 8-MOP and UVA radiation, eight of nine episodes of heart rejection were successfully reversed by photopheresis, also achieving a reduction in the inflammatory graft infiltrate. Recurrent acute rejection of heart allotransplant is another indication for ECP [178], and due to the reduction of the B lymphocyte and cytotoxic antibody levels after the photopheresis procedure, it could be indicated in hypersensitized patients [179]. Moreover, the benefits of ECP have been demonstrated on patient survival and allograft function during multiple and/or refractory rejection [180,181]. Several reports showed a beneficial effect of ECP in the prevention of coronary allograft vasculopathy, a leading cause of death after heart transplantation [182–184]. Lung transplantation is the standard therapy for end-stage lung deficiency resulting from chronic obstructive pulmonary disease and an increasing number of pulmonary fibrosis cases. Acute lung allograft rejection occurs in 50–70% of lung transplant recipients and typically occurs in the first 6–12 months after lung transplantation. Chronic rejection of the lung allograft remains the most common cause of death in lung transplant recipients after the first year of transplant. Despite potent immunosuppressive maintenance treatments incorporating calcineurin inhibitors, mycophenolate mofetil, and corticosteroids, the development of chronic allograft rejection continues to decrease the long-term survival of lung transplant recipients [185]. ECP has been also employed from 1995 as a therapy for the treatment of lung transplant rejection when conventional therapies do not induce an adequate 178 Studies in Natural Products Chemistry response [186,187]. Importantly, ECP is not associated with a higher risk of infections, a common complication when using immunosuppressant therapy. In some reports, lung transplant patients who did not respond to standard immunosuppressive drugs, with deterioration of graft function due to refractory bronchiolitis obliterans syndrome or persistent acute rejection, after ECP they experienced stabilization of lung function and/or symptoms. Furthermore, ECP seems to be effective to treat patient with life-threatening combined heart and lung graft rejection [188–191]. In the case of pancreas transplantation, only one case has been reported corresponding to a patient that received kidney and pancreas transplants and after treated by ECP for acute rejection [171]. Unfortunately, in this study, the function of the pancreas allograft before or after photopheresis was not reported. The first experience using ECP in liver transplant was published by Lehrer et al. In this report, authors treated a patient with hepatic allograft rejection refractory to high-dose corticosteroid and lymphocytolytic therapy with several sessions of ECP showing a complete reversal of acute cell-mediated rejection and absence of opportunistic infections or other adverse events [172]. Also, hepatic rejections occurring with hepatitis C virus recurrence, steroid-resistant acute rejection, and acute rejection in a major ABO mismatched liver graft were particularly considered as elective indications for ECP [173]. Importantly, ECP has been extensively investigated for prophylaxis of hepatic allograft rejection [192]. For this purpose, ECP has been used to delay the introduction of calcineurin inhibitors among high-risk liver transplant recipient to avoid this drug toxicity and for prevention of acute cellular rejection among ABO-incompatible liver transplant recipients. Also, ECP was efficient when it was applied in hepatitis C virus-positive patients with the aim of reducing the immunosuppressive burden and improving sustainability and efficacy of preemptive antiviral treatment. Altogether, ECP could be considered as a safe and efficient procedure for the treatment and prophylaxis of hepatic allograft rejection. Graft Versus Host Disease Hematopoietic stem cell transplantation is an intensive therapy used for the treatment of high-risk hematologic malignant disorders and other lifethreatening hematologic and genetic diseases. GVHD remains the most frequent and serious complication following allogeneic hematopoietic stem cell transplant and limits the extensive application of this important procedure. GVHD is an immunologic disorder that affects several organs, including the gastrointestinal tract, liver, skin, and lungs. It occurs when transplanted donor T cells respond to foreign histocompatibility antigens presented by host antigen-presenting cells, resulting in significant morbidity and mortality due to immune- and cytokine-mediated tissue injury [193,194]. The disease can be acute or chronic and may present with minimal to severe, life-threatening Chapter 5 Biomolecules of Therapeutic Interest 179 symptoms. Acute GVHD was defined as arising before day 100 posttransplant, whereas chronic disease happened after that time [195]. Despite improvements in posttransplant immunosuppression, up to 30% of HLA-identical marrow graft recipients and up to 90% of patients receiving marrow from unrelated donors still develop significant acute GVHD [196]. Recent advances in the understanding of the pathogenesis of GVHD have led to new approaches to its management, including using those that preserve the graft versus leukemia effect following allogeneic transplant. Prevention of GVHD has been limited to either the depletion of donor T cells before transplantation or the use of broadly immunosuppressive drugs that cause profound immunodeficiency and toxicities such as hypertension, diabetes, renal failure, and aseptic bone necrosis [197,198]. Alternatively, ECP appears to be an effective strategy for both acute and chronic GVHD, even in patients who are refractory to conventional immunosuppressive therapy [197,199]. The therapeutic effect of ECP for GVHD appears to involve induction of apoptosis in psoralen plus UVA-exposed lymphocytes, modulation of monocyte-derived dendritic cell differentiation, increased production of antiinflammatory cytokines, decreased dendritic cell antigen-presenting function, and induction of regulatory T cells that establish immune tolerance [200,201]. Due to the lack of efficacy of the existing treatments, there has been an emerging interest in investigating the utility and benefits of ECP with 8-MOP and UVA light in the treatment of GVHD. Small pilot and early-phase trials suggest that ECP is an effective treatment for both acute and chronic GVHD. Szcepiorkowski et al. showed that for steroid-refractory acute GVHD, overall response rates varied from 52% to 100% depending on site of involvement, ranging from 40% to 83% for skin, 63% to 100% for gastrointestinal tract, and 27% to 71% for liver involvement with complete responses outnumbering partial responses [186]. A summary containing the percentages of partial or complete responses achieved after ECP in acute GVHD patients is shown in Table 6. In chronic GVHD response rates, steroid-resistant patients ranged from 35% to 75% with liver or gastrointestinal complications and 60–80% with skin symptom improvement [208]. Improved survival rates of 38–73% were noted for patients receiving ECP [209,210]. There was an absence of long-term side effects compared with the observed using steroid treatment such as bone necrosis, growth retardation, and cataracts [186,211]. Taken together, ECP has shown particular promise in the skin manifestation of acute GVHD and to be a steroid-sparing modality for chronic GVHD. Although the mechanisms of action are still poorly understood and the optimal therapeutic regimens, including the schedule, duration, and approach to tapering and discontinuation, have not been well delineated, ECP offers hope for this treatment-resistant complication of hematopoietic stem cell transplantation. ECP warrants further evaluation and development in order to define the optimal therapeutic approach and the most appropriate patient population. 180 Studies in Natural Products Chemistry TABLE 6 Extracorporeal Photopheresis Studies Performed in Patients with Acute Graft Versus Host Disease % Complete or Partial Response No. of Patients Skin Involvement Liver Involvement Gut Involvement % Overall Survival Sniecinski et al. [202] 11 50 9 60 64 Greinix et al. [203] 6 100 100 – 100 Miller et al. [204] 4 100 50 100 50 Greinix et al. [205] 21 81 67 0 57 Salvaneschi et al. [206] 9 89 20 60 67 Dall’Amico et al. [199] 14 79 57 70 57 Messina et al. [207] 33 82 60 75 R: 69; NR: 12 Greinix et al. [200] 59 93 65 74 R: 59; NR: 11 Author R, responders; NR, nonresponders. Cutaneous T-Cell Lymphoma Cutaneous T-cell lymphoma describes a heterogeneous group of rare lymphoproliferative disorders that are characterized by the accumulation of malignant T-cell clones that home to the skin. Their etiology and pathogenesis are currently unknown. The most common form of the disease is called mycosis fungoides, which accounts for approximately 60% of new cases. The hematologic manifestation of cutaneous T-cell lymphoma is called Sézary syndrome and accounts for only 5% of cases. It has worse prognosis than mycosis fungoides, and the median survival of patients is less than 3 years from diagnosis [212]. Treatment of cutaneous T-cell lymphoma can be topical skin-directed or systemic. Topical options are used in the early skin-localized stage of the disease and include emollients, steroids, UVB radiation, retinoids, and PUVA. PUVA can produce relatively long-lived remissions. However, it is also associated with short-term side effects of oral psoralen intake, including nausea, vomiting, inconsistent gastrointestinal absorption, and long-term complications such as photosensitivity and the potential for development of skin cancer Chapter 5 Biomolecules of Therapeutic Interest 181 [213]. On the other hand, systemic treatment is applied to patients with widespread disease and may consist of single-agent or combination chemotherapy, achieving response rates of up to 80% with complete responses of 30%. Additional systemic treatments include interferon-a, IL-12, monoclonal antibodies directed against the malignant lymphocytes, and ECP [214]. ECP was approved for the treatment of mycosis fungoides in 1987 based on data that demonstrated an overall combined response rate for advanced disease of 58%, including 15% with complete remission of lesions [107]. After that, many publications have shown the clinical benefit of ECP in this disease. The standard schedule for ECP in the treatment of cutaneous T-cell lymphoma usually involves treatment on 2 successive days at 2- to 4-week intervals, which is generally continued for up to 6 months. In patients who show a good response, treatment intervals may be gradually lengthened to maintain this efficacy with fewer treatment cycles [215]. A meta-analysis of 19 studies in patients at all stages of cutaneous T-cell lymphoma showed an overall response rate higher than 55% using ECP. ECP was also effective in the treatment of Sézary syndrome, showing an overall response rate of 43% [216]. Although ECP is highly effective in some patients with cutaneous T-cell lymphoma, combination with other therapies can enhance its efficacy in later disease stages or in patients with insufficient response to ECP monotherapy. Some studies demonstrated that the combination of ECP and interferon-a was more effective in patients with cutaneous T-cell lymphoma than ECP alone [217,218]. Also, combination therapy of ECP and bexarotene, a novel oral retinoid, or together with granulocyte–macrophage colony-stimulating factor (sargramostim) seems to be a promising alternative treatment to this disease, obtaining good overall response rates [219,220]. Therefore, ECP, used as monotherapy or in combination with others, could be recommended as first-line treatment for all stages of advanced or refractory cutaneous T-cell lymphoma. Cancer Different compounds of the family of the coumarins have been described as agents with potent antitumor activity. There are some publications showing that the coumarins 1,2-benzopyrone and 4-hydroxycoumarin or warfarin can be used to prevent or delay the recurrence of malignant melanoma [221]. In a multicenter double-blind trial, these coumarins were administered daily for 2 years after surgery in patients with high risk of melanoma, showing a reduction of the recurrence of this disease in the coumarin-treated group (31%) compared with the placebo-treated group (71%). Moreover, there were no toxic effects related with the use of these coumarins. Psoralen and 5-methoxypsoralen, two of the major active furanocoumarins present in isolated extract from leaves and other aerial components of the fig tree Ficus 182 Studies in Natural Products Chemistry carica, have shown antiproliferative effects on the human melanoma cell line C32, providing a new perspective in developing new formulations potentially useful in PUVA therapy for the treatment of malignant melanoma and other cutaneous diseases [222]. 4-Hydroxycoumarin has been also described as an effective drug for the treatment of small cell lung cancer, a tumor cell type characterized by a coagulation-associated pathway [223,224]. It has been reported that anticoagulation agents such as heparin and 4-hydroxycoumarin prevent tumor formation by limiting the ability of tumor cells to adhere to the pulmonary microvasculature, a process that occurs during tumor metastasis [225]. 4-Hydroxycoumarin in combination with a histamine H2-receptor antagonist called cimetidine has also shown an intensification of the inhibition of the adhesion together with other antimetastatic effects on breast cancer cells [226]. Other authors have also demonstrated that the coumarin 1,2-benzopyrone in combination with cimetidine can produce important antitumor responses in some patients with advanced renal cell carcinoma [227]. There are also some reports showing the potential effect of the coumarin 1,2-benzopyrone for the treatment of prostatic carcinoma [228,229]. Fortyeight patients with metastatic hormone naive or hormone refractory prostatic carcinoma were orally treated with 1,2-benzopyrone daily and evaluated monthly for toxicity and response in a multicenter phase II clinical trial. Toxicity was limited to nausea and vomiting in some patients. The results showed no complete responses, but partial responses were achieved in 8% of treated patients with low tumor loads. The remaining patients progressed after 1–12 months. These studies demonstrated that 1,2-benzopyrone is a relatively nontoxic coumarin, but further trials are needed in order to improve the efficacy of this compound for the treatment of prostatic carcinoma. Also, Myers et al. demonstrated the inhibition of growth of the human malignant prostatic cell lines DU145 and LNCaP after incubation with this coumarin [230]. 7-Methoxy-8-(3-methyl-2-butenyl) coumarin, also called osthole, is a coumarin derivate extracted from many plants such as C. monnieri that has been used in traditional Chinese medicine as therapy for eczema, cutaneous pruritus, Trichomonas vaginalis infection, and sexual dysfunction due to its antiallergic, anti-inflammatory, and antibacterial effects. Recently, osthole has been described as an inhibitor of tumor cell growth and a potent inductor of apoptosis for leukemia and lung cancer cell lines [231–233]. Furthermore, it has been also reported that osthole is able to inhibit the migration and invasion of breast cancer cells and thereby it could represent a promising therapy to prevent the metastasis of this type of tumor [234]. Importantly, some osthole derivates bearing aryl substituents at 3-position of coumarin have shown 100-fold improved inhibition of growth of the human breast cancer cell lines MCF-7 and MDA-MB231 [235]. In other reports, osthole was found to Chapter 5 Biomolecules of Therapeutic Interest 183 inhibit proliferation through cell cycle arrest and induced apoptosis of hepatocellular carcinoma [236]. Altogether, osthole could be an interesting compound for the development of new anticancer agents for several types of tumors. In addition, auraptene, a prenyloxycoumarin isolated from members of Citrus sp., has been shown to be effective in inhibiting the development of liver, skin, tongue, and esophageal tumors and colitis-related colon cancers in some animal models [237–239]. Regarding leukemia, the derivate 8-nitro-7-hydroxycoumarin can induce apoptosis of the human leukemic cell lines HL-60 and K562 through the alteration of cell cycle and inhibition of DNA synthesis [240]. Another derivate of coumarin called esculetin or 6,7-dihydroxycoumarin was also able to arrest the cell cycle of the HL-60 cells [241]. Thus, these coumarin derivates could be elected as potential candidates to use them as drugs with antiproliferative effects in leukemia patients. Psoralens have shown effect in the regulation of human cervical carcinoma cell proliferation using the antisense technology [242,243]. This technique has been used to regulate gene expression in a sequence-specific manner, which enables suppression of the proliferation of cancer cells and exploration of the function of targeted genes. It has been improved using the photo-crosslinking compound TMP as a carrier that allows to the antisense oligo to reach the target gene into DNA and inhibiting the subsequent specific protein biosynthesis. Psoralen also confers to antisense oligos a higher resistance to be degraded by cellular nucleases. Thus, psoralen-conjugated oligo complementary to the initiation codon region of human papillomavirus HPV18-E6*-mRNA after UVA irradiation inhibited drastically the human cervical carcinoma cells proliferation. The psoralen-conjugated antisense DNA has significant potential to regulate gene expression and could be a promising concept for the cancer therapy. In order to improve the actual therapies against different types of tumors, nanomedicine has developed new strategies directed to increase the efficacy of drugs targeting tumors and uptake by tumors. It has been proposed the use of nanomaterials to locally activate therapeutic agents and limit the side effects of systemic administration of the drug that may negatively impact quality of life of patients [244,245]. Thus, a combined treatment of targeted drug delivery and localized X-ray activation would allow to induce the death of only those cells that both took up the nanodrug. The negative side effects associated with nonspecific uptake of chemotherapeutic compounds and radiation doses could be importantly reduced. Anticancer drugs have been recently developed in which scintillating nanoparticles (or nanoscintillators) can be used to activate psoralen in deep tissues. One of the most effective scintillating nanoparticle configurations is the combination of a psoralen such as 8-MOP and a fragment of the HIV-1 TAT cell-penetrating/nucleartargeting peptide anchored to UVA radiation-emitting Y2O3 (yttrium oxide) nanoscintillators. Using this combination, Scaffidi et al. showed the first 184 Studies in Natural Products Chemistry evidence of drug-associated, X-ray-activated reduction in the PC-3 human prostate cancer cell growth for psoralen tethered to scintillating nanomaterials [246]. Further analyses of this and other formulations as well as their in vivo animal models efficacy will be evaluated in the future. Microorganism Infections Certain antimicrobial properties have been attributed to furanocoumarins against some types of microorganism such as bacteria, virus, fungi, protozoa, and yeasts. Mycobacterium tuberculosis is one of the most deadly human pathogens, causing an estimated two million deaths and eight million of new cases each year worldwide. The cure for this bacterial disease is sometimes complicated by the appearance of resistance to antibiotics used for the treatment [247]. Treatment failure of first-line drug regimens requires the use of second-line drugs. However, some circulating M. tuberculosis strains are also resistant to these second-line drugs [248]. Several years ago, it was demonstrated that the psoralen 8-MOP has significant in vitro activity against M. tuberculosis strain H37Rv and Mycobacterium avium intracellulare [249,250]. More recently, it has been reported that the psoralen 8-MOP has antimycobacterial activity and in vitro synergistic activity in combination with the first-line antituberculosis drugs isoniazid, rifampicin, and ethambutol [251]. Yoshimura et al. demonstrated that 8-MOP in combination with UVA radiation (PUVA treatment) importantly inhibited the growth of both methicillin-susceptible and methicillin-resistant Staphylococcus aureus in a dose-dependent manner in vitro [252]. In chronic brucellosis, Brucella abortus infects macrophages, thus eluding the immune response [253]. When immunostimulatory drugs such as coumarin were administered, the symptoms of chronic brucellosis disappeared. These results have encouraged the use of coumarin in other chronic infections such as mononucleosis (Epstein–Barr virus), mycoplasmosis (Mycoplasma pneumoniae), toxoplasmosis (Toxoplasma gondii), and Q fever (Coxiella burnetii). Also, another antiplasmodial coumarin derivate called 5,7dimethoxy-8-(30 -hydroxy-30 methyl-10 -butene)-coumarin has been isolated from the roots of the plant Toddalia asiatica. This finding supports the traditional use of this plant for the treatment of malaria [254]. Furthermore, angelicin, libanorin, psoralen, and aurapten isolated from extracts of the plant Diplotaenia damavandica have shown antifungal activity against Candida albicans, Cryptococcus neoformans, Cladosporium cucumerinum, Saccharomyces cerevisiae, and Aspergillus niger [255,256]. Transmission of viral diseases through blood products is a problem in transfusion medicine. It has been demonstrated that the combination of 8-MOP and UVA radiation, 4-aminomethyl-TMP, and TMP is able to inactivate a wide range of animal viruses in serum or plasma without any Chapter 5 Biomolecules of Therapeutic Interest 185 adverse effects on the biological and biochemical properties and function of platelets, thus reducing the risk of viral transmission from platelet transfusion. The mechanism of action of the psoralen and UVA radiation in the viral inactivation is attributed to an oxygen-dependent disruption of membranes [257,258]. Other Diseases or Clinical Complications Besides the many therapeutic approaches of the furanocoumarins and other related molecules commented before, it has been described some punctual uses of them for the treatment of other diseases or clinical complications. In this regard, a recent study investigated the efficacy of the combination of coumarin and troxerutine therapy for the protection of salivary glands and mucosa in patients undergoing head and neck radiotherapy. Both compounds had a beneficial effect in the treatment of radiogenic sialadenitis and mucositis [259]. Moreover, ECP using 8-MOP and UVA radiation has been evaluated during a clinical trial to analyze its efficacy for the treatment of clinical restenosis after percutaneous transluminal coronary angioplasty [260]. After this study, ECP has been shown to be effective in reducing restenosis in patients undergoing balloon percutaneous transluminal coronary angioplasty with and without stent deployment. However, the use of this procedure in this complication needs further investigations. In a double-blind randomized trial with patients with chronic filiaritic lymphoedema and elephantiasis, coumarin treatment showed a very significant reduction of all grades of this tropical disease [261]. Another trial carried out using coumarin demonstrated that in lymphoedema of the arms and legs, this compound reduced the swelling in the absence of side effects [262]. In addition, the combination treatment of coumarin and the flavonoid troxerutin (Venalot®) has been effective for the treatment of several inflammatory and edematous states and to strengthen the microvasculature [263]. The compound 6,7-dimethoxycoumarin or scoparone isolated from the herb Artemisia scoparia possesses vasodilator and hypolipidemic effects and has been tested in rabbits with hyperlipidemia and diabetogenic condition. Administration of this coumarin for a week was able to reduce total cholesterol and triglycerides to normal values. Furthermore, scoparone treatment retarded the characteristic pathomorphological changes associated with hyperlipidemia such as intimal thickening or accumulation of fatty streaks and foam cells [264]. CONCLUSIONS The peculiar biochemical behavior of furanocoumarins and related biomolecules when irradiated with UVA light has made possible their clinical use in 186 Studies in Natural Products Chemistry several diseases, offering scarce side effects in comparison with the other treatment options for these illnesses. However, the exact mechanism by which these molecules exert their effect remains to be described in detail, and important questions regarding their use in clinical settings, such as length of treatment or design of specific protocols, concomitant use of immunosuppressive therapy, long-term side effects, assessment of therapy efficacy, and cost-effectiveness, continue to remain unanswered. Thus, additional in vitro assays and more animal models are needed in the future to elucidate the full mechanisms of action of these molecules. Also, further clinical studies using the actual available or the future discovered compounds are required and convert these natural products in a promising field of extensive research in order to find new therapies or improve the existing ones. ACKNOWLEDGMENTS This study was partly funded by Grant BFU2010-19599 provided by Spanish Ministry of Science and Innovation and the Spanish Cell Therapy Network (TerCel) of Spanish National Institute of Health Carlos III. ABBREVIATIONS 5,8-MOP 5-MOP 8-MOP CD CE CLA ECP GC–MS GVHD HbA1c HSCCC IFN-g IL kDa OMT pCEC PUVA SFE SPE TGF-b 5,8-dimethoxypsoralen or isopimpinellin 5-methoxypsoralen or bergapten 8-methoxypsoralen or xanthotoxin cluster differentiation capillary electrophoresis cutaneous lymphocyte antigen extracorporeal photopheresis gas chromatography–mass spectrometry graft versus host disease glycosylated hemoglobin high-speed countercurrent chromatography interferon-gamma interleukin kiloDalton O-methyl transferase pressurized capillary electrochromatography psoralen and UVA light supercritical fluid extraction solid-phase extraction transforming growth factor-beta Chapter TLC TMP TNF-a UVA 5 Biomolecules of Therapeutic Interest 187 thin-layer chromatography 4,50 ,8-trimethoxypsoralen tumor necrosis factor-alpha ultraviolet A REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] Bruneton, J., Intercept Ltd. 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This page intentionally left blank Chapter 6 Interactions Between Natural Health Products and Antiretroviral Drugs Marı́a José Abad Martı́nez, Luis Miguel Bedoya del Olmo and Paulina Bermejo Benito Department of Pharmacology, Faculty of Pharmacy, University Complutense, Ciudad Universitaria s/n, 28040 Madrid, Spain Chapter Outline Introduction Interactions Between Natural Health Products and Antiretroviral Drugs The Replication Cycle of HIV Existing Antiretroviral Drug Classes Nucleoside Reverse Transcriptase Inhibitors Non-nucleoside Reverse Transcriptase Inhibitors Protease Inhibitors Entry Inhibitors Integrase Inhibitors 197 199 200 202 202 203 204 205 205 Guidelines on the Use of Antiretroviral Therapy for HIV Infection 206 Examples of Clinical Interactions Between NHPs and Antiretroviral Drugs 207 St. John’s Wort 207 Garlic 209 Grapefruit 211 Milk Thistle 211 Ginkgo 212 Ginseng 213 Concluding Remarks 214 Acknowledgments 215 References 216 INTRODUCTION After its identification in 1981 as a novel distinct immunodeficiency syndrome (“acquired” rather than “primary”) characterized by a depletion of CD4 T cells and an expansion of activated CD8 T cells, in 1983 acquired immunodeficiency syndrome (AIDS) was finally associated with human immunodeficiency virus (HIV) in a causative way [1,2]. Following the Studies in Natural Products Chemistry, Vol. 43. http://dx.doi.org/10.1016/B978-0-444-63430-6.00006-0 © 2014 Elsevier B.V. All rights reserved. 197 198 Studies in Natural Products Chemistry development of a diagnostic tool, a huge mass of information on the epidemiology of the disease was rapidly collected. The overwhelming impact HIV has on the world is undeniable; by the end of 2009, there were 33.3 million people in the world living with HIV, with 1.8 million deaths in that year alone [3–5]. Furthermore, the high rate of death can be directly attributed to the lack of available medications; only 36% of the infected population received adequate antiretroviral therapy (ART). The natural course of HIV infection is characterized by a progressive loss of CD4 T cells leading to severe immunodeficiency. A decrease in CD4 T cells below 200 cells/mm3 is the threshold at which the risk of opportunistic infections dramatically increases. The advances in the knowledge of HIV biology, pathogenesis, and therapy, and their dramatic positive consequences on HIV-related morbidity and mortality, are quite unique in the history of medicine [6–10]. Today, highly active antiretroviral therapy (HAART), a treatment paradigm using three or more antiretroviral drugs in combination, is potent, convenient, capable of reducing HIV blood concentrations to undetectable values within a few weeks of the initiation of treatment, and of inducing a robust and sustained CD4 T cell gain. For adherent patients with undetectable viral loads, HIV has become a chronic manageable disease in an aging and genetically diverse population. Although the need for primary or secondary prophylaxis of opportunistic infections has declined due to potent HAART, many patients require treatments for other concomitant conditions such as cardiovascular disease, hyperlipidemia, hypertension, diabetes, gastrointestinal conditions, osteoporosis, or renal disease, which may be manifestations of long-term drug toxicity, increasing age, or the virus itself [11–19]. Furthermore, treatment may be required for other indications, including hepatitis coinfection, psychiatric illness, substance abuse, oncology diagnoses, or solid-organ transplantation [20–25]. Current treatment, when available, is not without limitations such as high pill burden, occurrence of adverse events, and particularly, the development of resistance and crossresistance between drug classes [26–28]. Despite intensive research efforts, a cure for HIV infection remains elusive. However, as a result of improved antiretroviral treatment, the disease has become a chronic manageable illness in Western countries with infected patients generally living longer. Patients therefore require medications not only for HIV infection but also for related or unrelated comorbidities, providing further challenges for healthcare providers and patients alike. This has made the management of HIV-infected patients increasingly complex, not only because of expanding therapeutic choices but also due to the emergence of resistance and the potential long-term toxicity of antiretroviral agents. As million of patients with HIV are put on treatment with HAART, drug interactions have become a major concern for healthcare providers [29–34]. Drug–drug interactions (DDIs) are common as a result of interacting metabolic pathways, and it is of great importance that DDIs of newly available Chapter 6 Interactions Between NHPs and Antiretroviral Drugs 199 therapies should be investigated to determine optimal treatment conditions and provide maximum therapeutic effect. INTERACTIONS BETWEEN NATURAL HEALTH PRODUCTS AND ANTIRETROVIRAL DRUGS The concomitant use of natural health products (NHPs), dietary supplements, and HAART is a common reality, with up to 60% of HIV-infected individuals reporting the use of NHPs in a recent survey [35–37]. There are numerous concerns associated with broad and unreported use of NHPs, including the risk of potential drug interactions or safety. One aspect of safety is the risk of adverse events due to pharmacological interactions between NHPs and conventional therapies [38–50]. These are often underestimated for two main reasons: consumers generally consider herbal medicinal products “safe” because of their natural origin; and they are often taken without consulting a physician as they are self-care products [51]. A review of research studies into patients’ communication of the use of complementary and alternative medicines (CAMs) to their medical practitioner found nondisclosure rates of 23–72%, and three main reasons for nondisclosure [52,53]: concern about a negative response from the medical practitioner, the fact that the medical practitioner does not ask, and the perception that because medical practitioner work within a biomedical framework they have no knowledge of CAMs. Individuals living with HIV/AIDS may take a variety of NHPs or other forms of CAMs due to the belief that complementary therapies and NHPs are synergistic with antiretrovirals: to help boost the immune system and aid in repairing the damage caused by the virus; to help prevent the occurrence of opportunistic coinfections; to help alleviate HIV-related conditions such as dementia, depression, and wasting; to increase their amount of systemic antioxidants; to improve their sense of general well-being; to reduce the side effects of various HIV drugs; or due to a desire for increased control over the disease process [54]. Although most NHPs are generally considered safe, these products contain pharmacologically active compounds that could potentially affect biological processes that regulate common drug disposition pathways [55]. In the case of NHPs, recognizing interactions may be complicated because of the number of new variables involved. Many products are available on the market, and plant extracts contain numerous different types of chemical compounds with various pharmacological properties. Furthermore, the composition of an extract may vary depending on its geographic origin, the stage of growth of the plant at harvest, postharvest treatments, standardization criteria, and stability. In some cases, herbal medicinal products may also be subject to contamination and errors in identification and concentrations [56]. DDIs occur when one therapeutic agent alters either the concentration (pharmacokinetic interaction) or the biological effect of another agent (pharmacodynamic interaction). They are caused by four main mechanisms: altered 200 Studies in Natural Products Chemistry drug absorption, altered renal elimination, additive effects or toxicities, and altered metabolism of drugs [57–59]. Some NHPs have been shown to induce or inhibit important metabolic pathways that are involved in the metabolism of certain HAART [60–70]. The plasma concentrations of these drugs may be reduced and therefore their efficacy diminished or, on the contrary, increased, which would exacerbate their toxicity. As a result, there have been reports of cases of antiretroviral toxicity or viral failure and development of drug resistance secondary to NHPs–antiretroviral interactions [71]. The most important pathway for drug metabolism is the family of liver enzymes known as cytochrome P450 (CYP450), particularly CYP3A4, which is responsible for the metabolism of 50% of currently available drugs, including HAART [72]. This family of liver enzymes also include CYP1A2, CYP2C9, CYP2C19, and CYP2D6. The second most important drug interaction in HIV patients on HAART is altered efflux mechanisms, such as P-glycoprotein (P-gp), which is responsible for transporting a range of compounds out of the intestinal epithelial cells and back into the intestinal lumen [73,74]. P-gp and other transporters from the ATP-binding cassette (ABC) transporter superfamily (e.g., ABCG2) are not only involved in drug transport in the gut but also in other processes related to drug disposition such as tubular secretion. Several reports have indicated that P-gp plays an important role in oral drug absorption [73]. There is therefore a high potential for drug interactions in this population, as some antiretrovirals such as protease inhibitors (PTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs) are both substrates and inhibitors or inducers of CYP450 hepatic enzymes and drug transporters [32]. Negative consequences of drug interactions include viral breakthrough and development of resistance, suboptimal disease/symptom management, or drug toxicity and possible nonadherence. In this review, we have aimed to provide an overview of the effects of herbal medicines on antiretroviral drug-metabolizing enzymes, focusing on potential herb–antiretroviral drug interactions, as well as interactions at the pharmacodynamic level. Despite the heightened awareness of herb–HIV drug interactions in the media and scientific journals, the investigations are limited in scope and methodological quality. Clinicians and the public do not have high-quality information to guide their decision to use NHPs. Additional research is warranted into both pharmacokinetic methodology and NHPs–HIV drug interactions. THE REPLICATION CYCLE OF HIV A working knowledge of the HIV replication cycle is essential for understanding the mechanism of action of antiretrovirals [10,75]. From the very early hours of its penetration into the body, HIV provokes a cascade of events that will set up the viral clock for the patient’s entire life. Several factors are predictors of more rapid HIV disease progression, including the severity of Chapter 6 Interactions Between NHPs and Antiretroviral Drugs 201 clinical symptoms, the intensity of viral replication at the time of primary infection, and the CD4 cell count. Anti-HIV drugs are classified into groups according to their activity on the replicative cycle of HIV, which can be roughly divided into seven steps: virus–cell adsorption, virus–cell fusion, uncoating, reverse transcription, integration, budding, and maturation [76–78]. HIV is an enveloped virus that contains two copies of viral genomic ribonucleic acid (RNA) in its core. In addition to the copies of RNA, the viral core also contains enzymes required for HIV replication: reverse transcriptase (RT), integrase (IN), and protease (PT). The first step in the HIV replication cycle is the interaction between the envelope proteins of the virus (gp120) and specific host cell surface receptors (e.g., CD4 receptor) of the host cell. In the second step, the virus binds to the chemokine receptors CXC-chemokine receptor 4 (CXCR4) and CC-chemokine receptor 5 (CCR5), resulting in conformational changes in the envelope proteins. This ultimately results in the fusion of the viral envelope and the host cytoplasmic membrane. Fusion creates a pore through which the viral capsid enters the cell. Once HIV has entered the cell, it must disarm and highjack the intracellular machinery for its own benefit. Following entry into the cell, the viral RT enzyme catalyzes the conversion of viral RNA into deoxyribonucleic acid (DNA). This viral DNA enters the nucleus and becomes inserted into the chromosomal DNA of the host cell (integration). This process is facilitated by the viral enzyme IN. Expression of the viral genes leads to the production of precursor viral proteins. These proteins and viral RNA are assembled at the cell surface into new viral particles, and leave the host cell by a process called budding. During the process of budding, they acquire the outer layer and envelope. At this stage, the PT enzyme cleaves the precursor viral proteins into their mature products. If this final phase of the replication cycle does not take place, the released viral particles are noninfectious and noncompetent to initiate the replication cycle in other susceptible cells. In the last few years, it has been demonstrated that in the early phases of infection HIV preferentially targets CCR5+CD4+memory T lymphocytes in the gastrointestinal tract. This results in a rapid, massive, and possibly permanent destruction of CD4 cells, rupture of the intestinal mucosa, and penetration of microbial translocation products in the systemic circulation [79]. At the same time, all body compartments, including the central nervous system, become infected. Although it is often symptomatic, primary HIV infection is seldom recognized, because symptoms are nonspecific, consisting of fever, malaise, generalized lymphadenopathy, pharyngitis, diarrhea, and rash, sometimes associated with abnormal laboratory results. After primary HIV infection, a chronic asymptomatic phase of variable duration ensues with symptomatic disease usually developing when CD4 cell count falls to below 350 cell/mm3, and characterized by the occurrence of several AIDS- or non-AIDS-associated events (mainly infections or tumors). In the absence of treatment, death is unavoidable. However, there is a minority of patients, the so-called elite 202 Studies in Natural Products Chemistry controllers, who are able to spontaneously control the infection and maintain low viremia and high CD4 cell count in the absence of therapy. The very high level of HIV replication during primary HIV infection, and the high activation of the immune system represent unfortunate optimal conditions to ensure HIV transmission from one individual to another. Plasma HIV RNA levels are usually high, with an elevated risk of transmitting the infection; formulating an early diagnosis is therefore very important not only for the infected individuals, but also for the whole community [80]. Several factors are involved in driving the magnitude of the HIV reservoir size such as CD4 T cell nadir and viral replication which determines the size of the viral reservoir. The fact that HIV reservoirs remain relatively stable after prolonged therapy suggests that the harm caused by HIV to the immune system during untreated infection creates the immunological conditions that favor the survival and persistence of virus-infected cells [81]. Two major forms of viral latency coexist in vivo: preintegration latency refers to unintegrated HIV DNA that is unstable and will either degrade or integrate into the host cell genome, usually following cell activation. This form of latency is established after partial or complete block of the viral life cycle at steps prior to the integration of viral DNA. Postintegration latency refers to the presence of integrated HIV DNA in cells that are not actively producing viral particles. This latent state is extremely stable, and is limited only by the lifespan of the infected cell and its progeny. Cytokines may modulate the mechanisms responsible for the establishment of these two forms of viral latency [82]. While CD4 cell decline is the most specific feature of HIV infections, its mechanism has not been totally clarified. Current opinion is that several factors contribute, the most important being a direct effect of HIV on CD4 cells and a generalized state of inflammation and activation, perhaps due to the chronic translocation of microbial products from the infected gut lumen into the systemic circulation. Successful long-term ART is able to reduce, but not eliminate, the burden of inflammation, which is likely to be causatively associated with some troubling complications of HIV infection [83]. EXISTING ANTIRETROVIRAL DRUG CLASSES Currently, there are six classes of antiretroviral agents, with 25 approved for single drug treatment, and 12 as fixed dose combination by the U.S. Food and Drug Administration (US-FDA) [7,77,78,84–91]. They are divided into six classes according to their mechanism of action. Nucleoside Reverse Transcriptase Inhibitors The group of nucleoside reverse transcriptase inhibitors (NRTIs) is composed of various nucleoside analogs: zidovudine, stavudine, lamivudine, didanosine, Chapter 6 Interactions Between NHPs and Antiretroviral Drugs 203 zalcitabine, abacavir, tenofovir, and emtricitabine. Inside the host cell, these products need to be metabolically activated by phosphorylation. They inhibit the activity of RT by termination of chain elongation. NRTIs were the first to be approved and form the backbone of HIV treatment. They are preferred as first-line drugs due to their favorable pharmacokinetic profile and low risk of DDIs. These agents are not substrates, or inducers or inhibitors of any of the major CYP450 enzymes; for this reason, clinically significant DDIs are unlikely to occur with other drugs that undergo hepatic metabolism. However, several studies in vitro revealed a direct interaction of these drugs (tenofovir, emtricitabine) with members of the ABC family [92]. These studies also provide evidence for different pathways for transcriptional modulation of expression between efflux transporters. However, NRTIs have low genetic barrier for drug resistance, and continued treatment is reported to accumulate mutations that cause resistance and cross-resistance to agents within the class [93,94]. Moreover, the current drugs in this class are associated with bone marrow suppression and high mitochondrial toxicity. All NRTIs can, to a much lesser extent, inhibit the activity of normal cellular DNA polymerases, most notably the mitochondrial DNA polymerase g. This NRTI-associated inhibition of mitochondrial function may account for certain drug-specific adverse effects, for example, elevated serum lactate and resulting lactic acidosis, as well as disorders of the liver, muscles, adipose tissue, and peripheral nerves. Co-formulations of the NRTIs tenofovir–emtricitabine and abacavir–lamivudine have emerged as the most frequently prescribed backbones [95,96]. However, there is always a balance between efficacy and toxicity/adverse effects and a good combination therapy may require monitoring of the patient’s drug therapy. Non-nucleoside Reverse Transcriptase Inhibitors This group include efavirenz, etravirine, nevirapine, delviradine, and rilpivirine. These drugs do not need metabolic activation and have half-lives. They inhibit RT in a noncompetitive manner by binding to the enzyme in a pocket far from the active site. Because drugs in this class are extensively metabolized via the CYP450 enzyme system, drug interactions are problematic and higher among NNRTIs than NRTIs. Nevirapine and efavirenz are inducers of the hepatic CYP3A4. Delviradine, on the other hand, inhibits CYP3A4. Efavirenz also inhibits CYP2C9 and CYP2C19, albeit to a lesser extent. Both nevirapine and efavirenz are metabolized by CYP2B6 as well as CYP3A4. Through this complex interaction with the CYP450 enzyme system, NNRTIs may change the metabolism of and thus lower (nevirapine, efavirenz), or increase (delviradine), the plasma levels of coadministered drugs that are metabolized by the CYP450 system. Similarly, drugs that induce or inhibit CYP450 activity may have an effect on the plasma concentrations of NNRTIs. 204 Studies in Natural Products Chemistry Efavirenz also interact directly with members of the ABC family, resulting in a decrease in ABC functionality [92]. The barrier to HIV resistance is relatively low for available NNRTIs and for the second generation of NNRTIs, efavirenz, nevirapine, delviradine, and rilpivirine [93,97,98]. Single-point mutations in RT can inactivate all members of this class, with the exception of etravirine. Given this lowresistance barrier, NNRTIs are often used early in therapy when the probability of HIV resistance to these agents is lowest, and the combined protective effect of three fully active drugs is optimal. NNRTIs are an integral part of initial treatment regimen along with one or two NRTIs and a PTI. In developing countries, nevirapine or efavirenz are commonly included in the initial regimen due to their efficacy, low cost, and convenient dose schedule. Nevirapine is also safe in pregnancy and has been extensively used to prevent vertical transmission. However, nevirapine-based regimen has been reported to cause fatal cutaneous hypersensitivity and hepatotoxicity. Protease Inhibitors This group include ritonavir, indinavir, saquinavir, nelfinavir, amprenavir, fosamprenavir, atazanavir, tipranavir, and darunavir. HIV PT is a complex enzyme composed of two identical halves with an active site located at the base of the cleft. It is responsible for the cleavage of the large viral precursor polypeptide chains into smaller, functional proteins, thus allowing maturation of the HIV virion. This process takes place in the final stages of the HIV life cycle. Inhibition of the PT enzyme results in the release of structurally disorganized and noninfectious viral particles. PTI therapy is often complicated by potential drug interactions due to the PTIs’ potent inhibition of CYP3A4 and P-gp [99]. PTIs are substrates for the CYP450 system (primarily CYP3A4) and are themselves, to varying degrees, inhibitors of this system, with ritonavir being the most potent inhibitor. The potential for drug interactions with ritonavir is further increased by its inhibition of CYP2D6 (although to a lesser extent compared with CYP3A4) and its induction of CYP1A2. Most PTIs are administered in combination with ritonavir, which boosts the effect of other PTIs by inhibiting the CYP3A4 isoenzyme. As CYP3A4 metabolizes PTIs, coadministration of ritonavir allows a reduced dosage of these PTIs. PTIs have high genetic barrier for drug resistance, and the use of low ritonavir as a boosting agent is now considered as a first-line option for patients who do not respond to or tolerate an initial treatment regimen. However, the mutations that arise from selection pressure from any PTI can grant crossresistance to other drugs in the same class. Most PTIs, except nelfinavir, are available with ritonavir boosting and have been approved and are the preferred option for an initial ART [100,101]. The limitations of PTIs include insulin resistance, dyslipidemia, hypertriglyceridemia, high risk of coronary artery disease and clinically significant interactions with other drugs Chapter 6 Interactions Between NHPs and Antiretroviral Drugs 205 (antimicrobials, anticonvulsants, psychotropics, etc.), including NHPs. Although NHPs and PTIs can influence the same drug disposition pathways, the reason for the incidence of adverse drug interactions occurring with HIV/AIDS patients is multifactorial. Many of these factors are strictly pharmacologic interactions or expected toxicity inherent in drug classes, whereas some are related to altered immunity. Entry Inhibitors This group comprises CCR5 and fusion inhibitor maraviroc and enfuvirtide. HIV entry into the host cell is a multistep process, involving the attachment of virus to CD4 and chemokine receptors (CCR5 and CXCR4). A complex interaction between host cell receptors and viral glycoprotein brings both viral and host cell membranes in close proximity, resulting in fusion. This knowledge has helped to identify novel drug classes as fusion inhibitors and chemokine receptor antagonists [102–104]. Pharmacologically, sequential inhibition of the successive steps of viral entry pathway by the combination of a fusion inhibitor and a chemokine receptor blocker would result in synergism. However, due to the ability of HIV to use multiple coreceptors for entry and to the diversity of the HIV env gene, it is difficult to identify patients who will respond effectively to the treatment, and the optimal use of these drugs will require a high degree of clinical acumen [105]. Inhibitors of HIV entry have targeted the conformational rearrangement of gp41 (enfurvitide) and the gp120–CCR5 interaction (maraviroc). The need for twice-daily injections with enfurvitide, along with the local adverse effects that accompany these injections, has limited its clinical use. Common adverse effects also include neutropenia and an increased risk of bacterial pneumonia. Resistance to enfurvitide is conferred by amino acid substitutions in the heptad repeat 1 region of gp41. Maraviroc, the first approved CCR5 antagonist, has seen limited clinical use to date, in part because it is active only against CCR5-using viruses, and thus requires an expensive blood coreceptor usage test prior to use. According to the product labeling, enfurvitide is not metabolized by CYP450, and its use is unlikely to result in significant drug interactions. However, the entry inhibitor maraviroc has the most interactions because it is metabolized through the CYP450 system. The daily dosage must be adapted in the case of association with a CYP450 inducer or an inhibitor. The efficacy of maraviroc has been demonstrated in patients failing other antiretrovirals classes, but its potential for first-line or switch therapy must be proved in future trials. Integrase Inhibitors HIV IN catalyzes both viral-cDNA processing and integration into the cellular genome by strand transfer. IN strand transfer inhibitors prevent viral DNA 206 Studies in Natural Products Chemistry from integrating into host DNA by inhibiting the IN enzyme involved in strand transfer [106–108]. The first agent of this class is raltegravir, which was approved in October 2007 by the US-FDA for both treatmentexperienced and naı̈ve patients [109]. The rational for prescribing this drug in treatment-naı̈ve patients is to preserve NNRTIs and PTIs for future regimens [110]. Raltegravir demonstrated significant antiviral activity against HIV isolates resistant to a variety of antiretroviral drugs, such as PTIs, NRTIs, and NNRTIs, and may be used as a component of both first-line and salvage antiretroviral regimens. However, resistance to raltegravir develop easily, which may limit its long-term effectiveness [111,112]. HIV resistance to raltegravir is conferred by amino acid substitutions that occur in proximity to IN catalytic residues, although the genetic barrier to resistance to integrase inhibitors (INIs) is considered to be low. Raltegravir is primarily metabolized by glucuronidation. There are at present insufficient data reported to determine whether promoter polymorphism in glucuronidation enzymes has any clinically relevant effect on the safety or activity profile of this drug. These qualities have prompted the search for agents with once-daily dosing, a more robust barrier to resistance, and a resistance profile with a limited overlap with that of raltegravir [113]. Elvitegravir, recently approved by the US-FDA, has the benefit of being part of a one-pill, once-daily regimen, but suffers from extensive cross-resistance with raltegravir. Dolutegravir is the most advanced second generation of INIs, and has good tolerability, oncedaily dosing with no need for a pharmacological enhancer, and relatively good cross-resistance with raltegravir. GUIDELINES ON THE USE OF ANTIRETROVIRAL THERAPY FOR HIV INFECTION There is a general consensus for treating patients with symptoms ascribed to HIV infection. Current Department of Health and Human Services guidelines recommend ART for all patients with a history of an AIDS-defining illness or severe symptoms of HIV infection, regardless of CD4 T cell count. Opinions on when to start ART in asymptomatic patients have varied widely, ranging from the “treat early, treat hard” approach of the early HAART years—with an emphasis on HIV RNA values—to the current, more conservative guidelines, delaying therapy until a certain threshold of CD4 cell counts has been reached. Current recommendations of when to start treatment of HIV are based on estimates of the risks of developing AIDS or death. Current anti-HIV protocols are fairly individualized, based on the patient’s medical history, CD4 T cell count, viral load, and resistance assays. During HAART, a combination of three or more drugs is administered. This combinational therapy reduces the emergence of resistant virus particles, which represents a major problem in anti-HIV therapy. The basic configuration of antiretroviral regimens is unchanged. The most common initial regimen Chapter 6 Interactions Between NHPs and Antiretroviral Drugs 207 consists of a NNRTI or PTI, with two NRTIs [85,86,90,114,115]. Each class of antiretroviral drugs has the potential to cause toxicities, many of which are shared by drugs likely to be used concomitantly in HIV-positive patients. This complicates the treatment, causes difficulty in causability assessment, and may require treatment withdrawal in serious life-threatening reactions. Long-term use of HAART has been reported to produce morphological and metabolic abnormality syndrome, especially hypertriglyceridemia; this in turn has increased the risk of cardiovascular and cerebrovascular diseases in patients receiving ART [116]. EXAMPLES OF CLINICAL INTERACTIONS BETWEEN NHPs AND ANTIRETROVIRAL DRUGS Clinical significant DDIs, frequently seen in patients on HAART, can adversely affect patient care and complicate ART. An understanding of drug interaction classification and mechanisms is essential for predicting their occurrence. Drug interactions can be classified into two broad categories: pharmacokinetic and pharmacodynamic interactions. Although both types occur with HAART, pharmacokinetic drug interactions are much more common, and mainly involve drug metabolism, due to the fact that NNRTIs and PTIs are extensively metabolized via the CYP450 enzyme system. In general, pharmacokinetic interactions are considered clinically significant when there is at least a 30% change in maximum drug concentration (Cmax), minimum (trough) concentration, or the area under the concentration time curve (AUC). PTIs and NNRTIs have a relatively narrow therapeutic range, and antiviral activity closely correlates with their plasma concentrations. Statistically, NHPs may be more likely than prescription drugs to inhibit and/or induce the enzymatic pathways involved in the metabolism of HAART agents [45]. Inducing the metabolic enzymes that clear HAART may decrease the concentration of these drugs, thereby decreasing their efficacy and possibly leading to treatment failure. These induction effects are more critical in treatment-experienced patients, and in patients who may already have low HAART drug plasma concentrations due to poor adherence to therapy, malabsorption, or inadequate dosing. Conversely, inhibiting these metabolic enzymes can increase HAART drug concentrations and exacerbate their toxicity. Selected DDIs associated with NHPs and HAART are detailed below (Table 1). St. John’s Wort St. John’s wort, Hypericum perforatum L. (Guttiferae), is one of the most widely used herbal antidepressants. Several systematic review report St. John’s wort to be more effective than placebo and equally effective as synthetic antidepressant drugs in the short-term treatment of depressive disorders, TABLE 1 Some Examples of Interactions Between Herbal Products and ART Medicinal Plant Major Active Ingredients Indications Hypericum perforatum L. Hyperforin Depression Allium sativum L. Sulfur compounds, flavonoids Hypercholesterolemia and vascular disease Citrus paradisi Macfad Furanocoumarins, bergamottine, flavonoids Inflammation, infections and cardiovascular disease Sylibum marianum L. Gaert. Ginkgo biloba L. Flavanolignans Terpene lactones, bilabolides, flavonoids, lignans Panax ginseng C.A. Meyer Ginsenosides, kaempferol Mechanism of Action Drug Candidates for Potential Interactions References Indinavir, nevirapine, lamivudine Saquinavir, darunavir, ritonavir [42,44,46,48,60,63,66–68,117–124] Indinavir, saquinavir, amprenavir [44,66,67,129] Hepatitis and liver cirrhosis Induction or inhibition of CYP450 and P-gp Induction or inhibition of CYP450 and/or P-gp Inhibition of intestinal CYP3A4 and P-gp Inhibition of CYP450 indinavir [47,50,130–134] Neurodegenerative disorders and peripheral vascular disease Enhancer performance, promoter vitality Inhibition or induction of CYP450 and/or P-gp Inhibition and/or induction of CYP450 or P-gp Ritonavir, lopinavir, raltegravir, efavirenz Ritonavir [44,135–140] [44,46,47,60,66–68,117,119,125–128] [47,57,59,65,67,141–147] Chapter 6 Interactions Between NHPs and Antiretroviral Drugs 209 including major depression [148]. Experimentally, St. John’s wort and its active ingredient hyperforin have been shown to inhibit the reuptake of several neurotransmitters such as serotonin, noradrenaline, dopamine, glutamate, and g-aminobutyric acid. Because depression is commonly observed in HIVinfected patients, there is a significant potential for the combined use of this herb and HAART drugs, and therefore, for interactions between these drugs. As a monotherapy, St. John’s wort has an encouraging safety profile. However, a number of clinical reports indicate the possibility of significant interactions—mainly pharmacokinetic—with prescribed drugs. It is a potent inducer of CYP3A4 and, depending on the dose, duration, and route of administration, it may induce or inhibit other CYP450 isoenzymes and P-gp. Clinical evidence confirms St. John’s wort as a CYP3A4 inducer capable of decreasing plasma concentrations of NNRTIs and PTIs such as indinavir and nevirapine, leading to drug failure [42,44,46,48,60,63,66–68,117–120]. Two clinical drug interaction studies, a single-sequence drug interaction study with indinavir, and a population pharmacokinetic study with nevirapine, revealed a reduction in antiretroviral drug concentrations after coadministration of St. John’s wort [121–124]. With indinavir, the AUC decrease by 57%, while with nevirapine the apparent oral clearance increase by 35%. Other CYP450 and P-gp substrates whose pharmacokinetic profile has been reportedly altered by H. perforatum include other antiretroviral drugs such as NRTIs (lamivudine). The Word Health Organization’s Collaborating Center for International Drug Monitoring has reported one case of reduction of drug activity in an indinavir–lamivudine–stavudine regimen coadministered with St. John’s wort, associated with an increase in HIV plasma RNA viral load [123]. St. John’s wort is therefore contraindicated in patients taking antiretroviral drugs that are metabolized by the CYP3A4 pathway. Hyperforin may be the most active inducer of CYP3A4 found in St. John’s wort; the use of a low hyperforin preparation of the plant may reduce the risk of DDIs. However, because the antidepressant activity of St. John’s wort has been linked to its hyperforin content, a low hyperforin preparation may be less efficacious; moreover, other St. John’s wort components may be involved in CYP3A4 induction. Given the availability of alternative, effective antidepressants, and the potential for deleterious drug interactions, the coadministration of St. John’s wort and HAART should be avoided. For this reason, in 2000 the US-FDA published a health advisory concerning the risk of drug–herb interactions. Garlic Garlic, Allium sativum L. (Amarillidaceae), is the most widely sold botanical dietary supplement in the United States, where it is used therapeutically to prevent hypercholesterolemia and subsequent vascular disease [149]. Its major 210 Studies in Natural Products Chemistry bioactive phytochemicals are the sulfur compounds aliin, allicin, diallyl disulfide, diallyl sulfide, 5-allyl-L-cysteine; garlic also contains numerous flavonoids/isoflavonoids (nobiletin, quercetin, rutoside, and tangeretin), polysaccharides, saponins, and terpenes (citral, geraniol, linalool, and a- and b-phellandrene). Garlic extracts are very commonly used by HIV-infected patients to treat several opportunistic infections, and are alleged to have antihyperlipidemic, antioxidant, and antimicrobial activities. However, clinical investigations highlighted a small number of potential, pharmacokinetic interactions between garlic and drugs, including HAART [44,46,47,60,66–68,117,119]. Piscitelli et al. [125] analyzed garlic’s interactions with the PTI saquinavir (a P-gp and CYP3A4 substrate), and found two different pharmacokinetic responses, possibly due to saquinavir metabolites. In six volunteers, saquinavir exposure decreased after garlic intake and returned to near-baseline values after 10 garlic-free days. It is worth noting that a reduction in these concentrations occurred in only six of the nine subjects studied. In another three, there were no changes in saquinavir pharmacokinetic after garlic supplementation, but a large reduction after the 10-day washout period. Because there were similar reductions in the magnitude of all concentration parameters, it is likely that garlic decreased saquinavir bioavailability, perhaps secondary to induction of intestinal CYP3A4 and/or P-gp. These parameters remained 30–40% below baseline after a 10-day washout period, suggesting that there may be a long-lived systemic metabolite of garlic, or a production of saquinavir metabolites that autoinduce metabolism. Unfortunately, this study lacked a control group, and the reduction in saquinavir concentrations in the washout period could be a result of a time-dependent autoinduction effect rather than a drug interaction. Saquinavir induces P-gp expression in vitro and could reduce its own concentration over time. More recently, Berginc et al. [126] investigated the mechanisms of interactions between the PTIs, saquinavir and darunavir, and garlic supplements. Garlic extracts significantly inhibited CYP3A4 metabolism of both drugs, and modulated hepatic distribution of the corresponding saquinavir and darunavir metabolites. The competition between saquinavir and garlic constituents for the same binding site on the efflux transporter and the positive cooperative effect between darunavir and garlic phytochemicals—which bind to separate binding places on transporter—are the most likely mechanisms explaining the plasma profile changes which could occur in vivo during concomitant consumption of antiretrovirals and garlic supplements. The phytochemicals inducing changes in the distribution of saquinavir and darunavir were most probably flavonoids and lipophilic organosulfur compounds, respectively. Another trial showed that acute dosing of garlic over 4 days did not significantly alter the single-dose pharmacokinetics of the PTI ritonavir (a CYP3A4 and P-gp substrate), although the reason for the discrepancy is presently unclear [127]; this suggested that a longer duration of garlic therapy may be required to observe a significant decrease in ritonavir plasma concentration. One case Chapter 6 Interactions Between NHPs and Antiretroviral Drugs 211 report described two HIV-infected patients who were taking garlic supplements and developed severe gastrointestinal toxicity after starting ritonavir therapy [128]. Symptoms recurred after rechallenge with low-dose ritonavir, suggesting that elevated ritonavir concentrations were not the cause. Ritonavir may have inhibited the metabolism of garlic, thereby leading to a pharmacokinetic interaction, or it may have enhanced the toxic effect of garlic on the intestinal tract, thereby leading to a pharmacodynamic interaction. Until further information is available, patients taking antiretrovirals that are P-gp and CYP3A4 substrates should be monitored where there is concomitant use of garlic. Grapefruit Citrus paradisi Macfad (Rutaceae) is the botanical name of grapefruit, whose juice is widely known to affect drug bioavailability in humans [44,129]. Grapefruit juice contains flavonoids, a large class of plant polyphenolic secondary metabolites that have various pigmental and antimicrobial functions, and which are also found in many other plants and vegetables. They are studied for their antioxidant, anti-inflammatory, and antimicrobial activity, and ability to prevent cardiovascular diseases. The fact that grapefruit raises the bioavailability of many drugs has been attributed to the irreversible inhibition of intestinal CYP3A4 and P-gp by flavonoids. However, the main inhibitory effect on intestinal CYP3A4 and P-gp is attributed to some furanocoumarins and bergamottine, which together could lead to various clinically important pharmacokinetic alterations. A number of studies have confirmed the high risk of DDIs between HAART and grapefruit juice. Based on clinical studies, grapefruit may increase the bioavailability and adverse effects of PTIs such as indinavir, saquinavir, and amprenavir [66,67]. In spite of the known health benefits of grapefruit juice, its consumption in combination with drugs requires caution. Milk Thistle Milk thistle, Silybum marianum L. Gaert. (Asteraceae), is one of the most widely sold herbal medicinal products for treating hepatitis and liver cirrhosis [150]. It is also used after chemotherapy as a liver protectant to improve longterm treatments. The seeds are the active part of the plant, and the main phytochemical is silymarin, a mixture of four flavanolignans: silybin, isosilibilin, silichristin, and silidianin. Silybin is the major active constituent of the mixture and it is often used pure for clinical investigations. Silymarin protects cells from radical-induced damage by boosting endogenous antioxidant enzymes; it also been reported to inhibit the activation of proinflammatory cascades, and increase the expression of anti-inflammatory cytokines. Patients use milk thistle in a variety of clinical settings, but it is most frequently used in gastrointestinal clinics to help treat hepatitis and cirrhosis. S. marianum is 212 Studies in Natural Products Chemistry commonly taken by patients who are coinfected with HIV and hepatitis, as it is presumed to promote liver health. However, interactions between milk thistle and/or silymarin and HAART drugs can occur, and may lead to clinical consequences [47,50,130]. Two uncontrolled clinical studies revealed trends toward reduction in concentrations of the PTI indinavir after coadministration of milk thistle. The indinavir AUC was not significantly reduced over the 8-h dosing interval, although the final Cmax determined 8 h after the last indinavir dose was significantly decreased (by 25%) [131]. The authors concluded that their results indicated a very low risk of clinically relevant interactions with indinavir therapy. The same results were obtained by Di Cenzo et al. [132], using a different study design in 10 healthy volunteers. It was found that silymarin failed to influence the pharmacokinetic of indinavir in healthy subjects, but it cannot be concluded that silymarin would not have influenced the pharmacokinetic of indinavir at higher dosages. These results suggest that silymarin and milk thistle extract did not inhibit CYP1A2, CYP2D6, CYP2E1, and CYP3A4 in in vivo human studies. Moreover, milk thistle intake does not affect systemic metabolism of UDPglucuronosyl transferase (UGT) substrates because plasma concentrations of flavanolignans in vivo are not sufficient to inhibit the drug glucuronidation. However, in vitro investigations implicate milk thistle extract and/or silymarin as inhibitors of human CYP3A4, CYP2C9, CYP2D6, and CYP2E1 [133,134]. Therefore, concentrations of orally administered silymarin may be sufficient to compete for CYP450 binding sites in the liver and gut wall, and this apparent lack of in vitro–in vivo correlations may be due to the following factors: poor bioavailability, large interindividual variations in silymarin absorption, lower CYP450 binding affinities of silymarin conjugates, interproduct variability in silymarin content, or poor dissolution characteristics of milk thistle dosage forms. Moreover, silymarin inhibited in vitro recombinant UGT1A1, UGT1A6, UGT1A4, UGT2B7, and UGT2B15, and in vivo UGT systems. Despite its popularity, limited information is available on the safety, interactions with other drugs, and the mechanisms of interaction of silymarin and milk thistle. Patients who want to use this plant should be advised about the potential for interactions in order to reduce therapeutic failure or increased toxicity of conventional drug therapies. Ginkgo Herbal medicinal products containing Ginkgo biloba L. (Ginkgoaceae) are used to prevent and treat neurodegenerative disorders, tinnitus, some types of glaucoma, and peripheral vascular disease. Due to its perceived pharmacological benefits, ginkgo products are being sold throughout the world as dietary supplements or over-the-counter drugs. It has also been reported that ginkgo products were ranked as one of the top-selling dietary supplements Chapter 6 Interactions Between NHPs and Antiretroviral Drugs 213 in the United States and Europe [151]. The main pharmacologically active phytochemicals are the terpene lactones ginkgolides A, B, and C, and bilabolides. The standardized special G. biloba extract (EGb761) has several pharmacological activities against the cerebral dysfunctions associated with brain aging and degenerative dementia. One of the proposed mechanisms for the neuroprotective functions of EGb761 is that it protects neurons from low-density lipoprotein receptor-related protein (LRP) ligands, as in the case of b-amyloid peptide-induced neurotoxicity. Recent studies suggest that the interaction of HIV Tat protein with LRP, with the resulting disruption of the normal metabolic balance of LRP ligands, may contribute to AIDSassociated neuropathologies, including dementia. These findings raise the possibility of using EGb761 as an alternative strategy to treat HIV-induced neurological disorders [152]. For this reason, EGb761 is a popular herbal product among HIV-infected patients due to its positive effects on cognitive function. Although the safety of G. biloba is promising, accumulated data show evidence of significant interactions with medications, including HAART, which can place individual patients at great risk [44,135,136]. However, the findings in humans are contradictory, and further studies are needed to elucidate the role of G. biloba in altered drug absorption due to CYP450 and P-gp alterations. Ginkgolides and bilabolide were found to inhibit CYP1A2, CYP3A, and CYP2C9 weakly or negligibly in human liver microsomes, while the flavonoids kaempferol, quercetin, apigenin, myricetin, and tamarixetin inhibited CYP1A2 and CYP3A [137]. Quercetin, amentoflavone (biflavonoid), and the lignan sesamin were inhibitors of CYP2C9. Robertson et al. [138] conducted a single-sequence longitudinal study in which 13 healthy volunteers received 120 mg of EGb761 twice daily for 28 days, and on day 27, a single dose of ritonavir-associated lopinavir (a CYP3A4 substrate). The results showed no significant effects on the pharmacokinetic of lopinavir with ritonavir. According to the authors, the pharmacokinetic of lopinavir was not affected by the induction effect of ginkgo on CYP3A4 because of simultaneous CYP3A4 inhibition due to ritonavir. Additionally, clinical data were lacking on the potential inhibitory or inductive effect of G. biloba on the pharmacokinetic of raltegravir, an INI, although concomitant use was not recommended [139]. Investigations demonstrated that G. biloba did not reduce raltegravir exposure. The potential increase in Cmax of raltegravir is probably of minor importance, given the large intersubject variability of the pharmacokinetic of raltegravir and its reported safety profile. Another antiretroviral drug whose pharmacokinetic profile has been reportedly altered by G. biloba is the NNRTI efavirenz [140]. Ginseng In the Orient, Panax ginseng C.A. Meyer has been used as a drug for more than 2000 years [153]. At present, ginseng is one of 12 medicinal herbs most 214 Studies in Natural Products Chemistry commonly used in America and is the best-known and most highly valued herb in Korea, China, and Japan. In particular, Korean and Siberian ginseng is traded for high prices on the international market. Since the late 1960s, a number of studies have been performed to identify the active ingredients of ginseng and their functions. Ginseng is considered to be an adaptogenic agent that enhances physical performance, promotes vitality and increases resistance to stress and aging, and possesses immunomodulatory activity [154–156]. Its major active constituents include ginsenosides (panaxosides, more than 150 isolated), sterols, flavonoids, peptides, vitamins, polyacetylenes, minerals, b-elemine, and choline [157]. Ginsenosides are considered to be major pharmacologically active constituents, and approximately 12 types of ginsenosides have been isolated and structurally identified. However, interactions can occur between ginseng and/or ginsenosides and HAART drugs, and may lead to clinical consequences [47,57,59,65,67]. In human microsomal studies, P. ginseng extract and individual ginsenosides showed various inhibitory and/or inducing effects on CYP450. The individual ginsenosides (Rb1, Rb2, Rc, Rd, Re, Rf, and Rg1) show the inhibitory effect on CYP1A1 only at a high concentrations (over 100 mg/ml) [141]. Ginsenoside Rd had only a weak inhibitory activity against CYP3A4, CYP2D6, CYP2C19, and CYP2C9, whereas ginsenoside Re and ginsenoside Rf substantially increased the activity of CYP2C19 and CYP3A4 [142]. However, there are only a few case reports in human studies regarding ginsenoside–drug interactions via CYP450 and/or P-gp systems. No reports of ginsenoside glucuronidation were found in the literature. In a pharmacokinetic study in which ginsenoside Rd was administered intravenously to volunteers, no glucuronidated metabolites were detected in the rat S9 liver fraction and in clinical pharmacokinetic studies [143,144]. Another component of ginseng, the flavonoid kaempferol, exhibited a marked inhibition of P-gp-mediated efflux of the HIV PTI ritonavir by increasing its cellular uptake in Caco-2 cells [99]. In human clinical studies, P. ginseng extract is unlikely to alter the disposition of coadministered drugs that are primarily dependent on the CYP2D6 or CYP3A4 pathway [145,146]. Other studies show that, independently, Korean ginseng intake has beneficial effects on the slow decrease of CD4 T cells and on serum-soluble CD8 levels in HIV-1-infected patients, although the human leukocyte antigen factor was also significantly associated with the rate of CD4 T cell depletion in the Korean population [147]. CONCLUDING REMARKS Patients undergoing HAART regularly use medicinal plants and this often occurs without the knowledge of the doctor or pharmacist. There is evidence that herbal preparations can cause pharmacokinetic and pharmacodynamic interactions that represent a potential risk in patients under HAART. NHPs are not inert substances. Although we found examples of clinically Chapter 6 Interactions Between NHPs and Antiretroviral Drugs 215 nonsignificant interactions with antiretroviral agents, there are others who can have serious consequences on the treatment’s efficacy or toxicity. The clinical consequences of herb–drug interactions vary from being well-tolerated to moderate-to-serious adverse reactions, or possibly life-threatening events. Undoubtedly, the early and timely identification of herb–drug interactions is imperative for preventing potentially dangerous clinical outcomes [158,159]. The evidence available in the literature indicates various mechanisms through which this can occur. By interacting with conventional medication, herbal remedies may precipitate manifestations of toxicity, or at the other extreme, lead to therapeutic failure. There is often a lack of scientific rigor in studying these interactions. Inadequate reporting makes it very difficult to determine whether a particular herb–drug interaction has occurred. Proper documentation is necessary, providing all the relevant information and a clear description of adverse event. For the most part, NHPs are not standardized. Data from published studies may sometimes be suboptimal, contradictory, and outdated, and new studies are urgently needed. NHPs should be studied using the same rigorous scientific criteria as conventional drugs whenever possible. In the meantime, caution should be exercised and clinicians should always be vigilant to the possibility of interactions between NHPs and antiretroviral drugs in their patients. Although one or two reports may not warrant an outright contraindication to combinations of herbal remedies and HAART, precautions do need to be exercised by obtaining this information from the medical history divulged by patients during counseling sessions. This review provides information on commonly used herbs and their potentials DDIs with HAART within the levels of evidence currently available. ACKNOWLEDGMENTS The technical assistance of Ms. Brooke-Turner is gratefully acknowledged. 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[158] X.W. Chen, E.S. Serag, K.B. Sneed, J. Liang, H. Chew, S.Y. Pan, S.F. Zhou, Curr. Med. Chem. 18 (2011) 4836–4850. [159] E.F. Fang, T.B. Ng, in: E.F. Fang, T.B. Ng (Eds.), Antitumor Potential and Other Emerging Medicinal Properties of Natural Compounds, Part VIII, Springer, Heidelberg, Germany, 2013, pp. 407–418. This page intentionally left blank Chapter 7 Lichens: Chemistry and Biological Activities Sammer Yousuf*, M. Iqbal Choudhary*,{ and Atta-ur-Rahman* *H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, Pakistan { Department of Biochemistry, Faculty of Sciences, King Abdulaziz University, Jeddah, Saudi Arabia Chapter Outline Introduction 223 Lichen Chemistry: A Brief History 224 Chemical Structure and Diversity 226 Biosynthesis of Lichen Substances 227 Shikimic Acid Pathway 227 Polymalonate Pathway 227 Mevalonic Acid Pathway 229 Lichen-Derived Secondary Metabolites and Their Functions 229 Biological Activities of Secondary Metabolites of Lichens 232 Antibacterial, Antibiotic, and Antifungal Activities Antiprotozoal Activity Antiviral Activity Cytotoxicity and Antitumor Activities Antioxidant Properties Antidiabetic Properties Enzyme Inhibition Properties Antipyretic and Analgesic Properties Conclusion References 233 240 240 245 249 251 253 255 255 256 INTRODUCTION Lichens are unique, stable, and self-supporting symbiotic associations of fungi with microalgae and/or cyanobacteria [1]. They are among the slowest growing organisms with remarkable tolerance to adverse atmospheric conditions (extreme temperature change, salinity increase, drought, poor nutrition, etc.) and may potentially reach an age of thousands of years [2,3]. According to Studies in Natural Products Chemistry, Vol. 43. http://dx.doi.org/10.1016/B978-0-444-63430-6.00007-2 © 2014 Elsevier B.V. All rights reserved. 223 224 Studies in Natural Products Chemistry an estimate, lichens comprise more than 25,000 species with 98% Ascomycota fungal partners [4,5]. Lichens are found in a wide range of habitats, ranging from plains to the highest mountains of tropical to arctic regions under xeric to aquatic conditions [4–6]. They occur on or within rocks on soil, trees, shrubs, trunks, and animal carapaces and on man-made undisturbed surfaces like bricks, leather, wood, etc. [6–8]. Since ancient times, lichens have been used for diverse purposes. They are widely known as sources of color dyes. The purple pigment from Roccella species was used for dyeing “togas” by Romans. Ancient Romans also extracted a brown pigment, named “crottal,” from Evernia, Ochrolechia, and Parmelia species. The pH paper impregnated with litmus, a mixture of water-soluble dyes that are extracted from lichens of the genus Roccella, is still used by chemists [2]. In additions to dyeing, lichens have been used for cosmetic purposes [3]. Lichens are also widely used as biodetectors due to their high sensitivity to air pollutants, such as sulfur, different heavy metals, and nitrogen [9–11]. In the Ayurvedic system of medicine, lichens are used for the treatment of bronchitis, asthma, leprosy, burning sensation, spleen enlargement, and heart diseases. Some are also used as “blood purifier.” In the Unani medicinal system, lichens are used for the treatment of various stomach disorders, liver pain, inflammation, vomiting, etc. [1,12,13]. Lichens Parmelia sulcata Taylor and Peltigera apthosa (L.) Wild. are used for the treatment of rabies and “Thrush” in children, respectively [13]. This review mainly focuses on biological activities of the various lichen species and their different chemical compounds, responsible for diverse biological properties, reported in the literature until June 2012. LICHEN CHEMISTRY: A BRIEF HISTORY The first report of chemicals from lichens was contributed by William Nylander, a Finnish botanist and entomologist in the mid-ninetieth century [2,14]. However, the first systemic study of lichen chemistry was carried out by Friedrich Wilhelm Zopf, a German scientist [2,15]. Characterization of many chemical compounds from lichens was successfully achieved by Japanese researchers. Especially, Asahina and Shibata et al. have contributed immensely in exploring the chemical diversity of lichens. They studied the biosynthesis of lichen substances, and introduced the process of microcrystallization to the study of chemistry of lichen substances [2,16,17]. This procedure was based on the fact that different lichen substances crystallize in a characteristic manner in the presence of certain reagents and can thus be identified microscopically. This rapid technique allows the identification of major constituents produced by hundreds of lichen species. However, the technique is not suitable for minor constituents. In 1952, Wachtmeiste introduced paper chromatography to isolate and identify secondary metabolites of lichens [18]. Chapter 7 Lichens: Chemistry and Biological Activities 225 The relationship between the paper chromatographic Rf values and chemical structures of lichen secondary metabolites was described by Mitsuno in 1952 [19]. Due to limitations of paper chromatography for separation of structurally diverse secondary metabolites of lichens, Ramaut introduced the use of thin layer chromatography (TLC) in 1963 for the separation of lichen compounds, belonging to depsides and depsidones classes. Pastuska’s solvent phase (benzene–dioxane–glacial acetic acid) [20,21] was used in this process. After these reports, TLC was extensively used by different researchers to study specific groups of compounds which are produced by lichens. However, the use of several solvent systems and diverse conditions by different authors, made the results impossible to compare and the purification and identification of lichen compounds fairly complex. The problem was partially solved by Culberson and coworkers in 1970. They developed a method, based on the TLC Rf values, for the identification of different classes of lichen compounds [22–24]. Culberson reported the first use of high-performance liquid chromatography (HPLC) on crude extracts of lichens in 1972. They used normal silica gel columns with isocratic solvent mixtures (hexane, isopropyl alcohol, and acetic acid). Gas chromatography was not found to be suitable due to low viability and thermal unstability of lichen compounds. In 1978, the same group reported for the first time the use of reverse phase HPLC for the purification of lichen-based secondary metabolites. They used RP-18 columns and water– methanol–acetic acid mixture as eluent to purify different compounds, belonging to depsides and depsidones classes [25–27]. After these reports, the isocratic system was extensively utilized for the purification of such types of compounds. In 1979, Strack et al. reported the use of gradient solvent systems and purified 13 phenolic compounds from lichens [28]. A standard HPLC method for the identification and quantification of aromatic compounds in lichen extracts was developed by Huovinen et al. in 1987 [29]. They used reverse phase (RP-8 and RP-18) columns as stationary phases and eluted the extracts with a mixture of methanol and phosphoric acid. Benzoic acid and bis-(2-ethyl-hexyl)-phthalate were used as internal standards in these separations [29]. This method was improved by Feige et al. in 1993 by replacing bis-(2-ethyl-hexyl)-phthalate with soloromic acid as internal standard [30]. The improved method was found to be more suitable for the identification of chloroxanthones and long-chain depsides in lichen extracts. Later, an HPTLC method was developed to identify the lichen substances [6]. The first data of more than 400 substances, purified from lichens, were compiled and published by Culberson in three books [2,31]. Till June 2012, the scientific literature contains reports of more than 1050 lichen substances, purified by modern chromatographic methods especially HPLC [32,33] and identified by spectroscopic techniques including mass, 1D, and 2D 1H- and 13 C-NMR spectroscopy, and X-ray diffraction analyses [6]. 226 Studies in Natural Products Chemistry CHEMICAL STRUCTURE AND DIVERSITY Lichens are well-known sources of phenolic secondary metabolites of different classes, including mononuclear phenols (e.g., orcinol (1) and b-orcinol (2)), quinones (e.g., parietin (3)), dibenzofurans (e.g., pannaric acid (4)), depsidones (e.g., salazinic acid (5)), depsones (e.g., picrolichenic acid (6)), depsides (e.g., homosekikaic acid (7)), g-lactones (e.g., protolichesterinic acid (8)), pulvinic acid derivatives (e.g., vulpinic acid (9)), and xanthones (e.g., lichexanthone (10)). In addition to these commonly found compounds, several unique constituents have also been reported from lichens. A cyclic depsipeptide, known as arthogalin (11), was reported from an endemic species of lichen of Galapagos Islands [34]. Unique phenylalanine-derived scabrosin esters (12–16) were reported from a lichen species, Xanthoparmelia scabrosa (Vain.) Hale [35]. The lichens, native to central Asia, are unique in producing halogenated secondary metabolites, such as brominated depsidones (17 and 18) [36] and brominated acetylenic fatty acids (19–26) [37]. Rezanka and coworkers [38–41] reported the isolation of unique monotetrahydrofuranic acetogenins (27–30), g-lactone containing long-chain fatty acids (31–33), and a macrolactone glycoside (34) from the lichens which are native to central Asia. Umbilicaxanthosides are mono- and di-prenylated xanthone glucosides (35 and 36), reported only from Umbilicaria proboscidea (L.) Schrader of the Ural mountains [41]. Similarly, lichenized ascomycete Collema cristatum var. marginale is known to produce photoprotecting glycosylated mycosporine collemin A (37) [42]. R R5 R1 R4 R2 R3 Orcinol (1), R = CH3, R1 = R3 = R5 = H, R2 = R4 = OH b-Orcinol (2), R = R3 = CH3, R1 = R5 = H, R2 = R4 = OH Methyl orsellinate (57), R = COOCH3, R1 = R4 = CH3, R2 = H, R3 = R5 = OH Phenyl orsellinate (58), R = COOPh, R1 = R4 = CH3, R2 = H, R3 = R5 = OH Orsellinic acid (79), R = COOH, R1 = R4 = CH3, R2 = H, R3 = R5 = OH Rhizonyl alcohol (83), R = R3 = CH3, R1 = CH2OH, R2 = OH, R4 = OCH3, R5 = H Rhizonyl aldehyde (84), R = R3 = CH3, R1 = CHO, R2 = OH, R4 = OCH3, R5 = H Ethyl orsellinate (90), R = COOC2H5, R1 = R4 = CH3, R2 = H, R3 = R5 = OH Ethyl heamatomate (92), R = R4 = OH, R1 = COOC2H5, R2 = CH3, R3=H, R5 = CHO Methyl-b-orcinolcarboxylate (93), R = R3 = CH3, R1 = COOCH3, R2 = R4 = OH, R5 = H Atraric acid (99), R = R4 = OH, R1 = COOCH3, R2 = R5 = CH3, R3 = H, R4 = OH Chapter 7 Lichens: Chemistry and Biological Activities OH O 227 OH R1 R O Parietin (3), R = CH3, R1 = OCH3 Emodine (39), R = OH, R1 = CH3 R R7 R7 R6 R2 R5 O R3 R4 Pannaric acid (4), R = R5 = OH, R1 = R4 = H, R2 = R7 = CH3, R3 = R6 = COOH Umbilicaxanthoside A (35), R = R2 = R5 = H, R1 = O-β-D-Glc., R3 = R7 = OH, R4 = OCH3, R6 = Umbilicaxanthoside B (36), R = R2 = R5 = H, R1 = O-β-D-Glc-(1-4)-β-D-Glc., R3 = R7 = OH, R4 = OCH3, R6 = BIOSYNTHESIS OF LICHEN SUBSTANCES Diverse biosynthetic pathways are involved in the production of a wide range of secondary metabolites produced by lichens. Shikimic acid, polymalonate, and mevalonic acid pathways are known to be mainly responsible for the biosynthesis of secondary metabolites in lichens [43]. Shikimic Acid Pathway Pulvinic acid (38) and its derivatives (e.g., vulpinic acid (9)) and other cyclic phenolic derivatives are mainly synthesized through shikimic acid pathway (Scheme 1) in lichens. These classes of compounds are widely distributed in lichens of the family Stictaceae and are usually obtained by the fusion of two phenylpyruvate units. Polymalonate Pathway Lichen metabolites belong to terphenylquinone derivatives (e.g., emodine (39)). Structurally unique depside class of compounds (e.g., hyperhomosekikaic acid (40)), and depsidones (e.g., sublobaric acid (41)) are synthesized via polymalonate pathway (Scheme 2). These compounds are synthesized by the fungus only when it is in symbiotic association with alga (lichenized). The enzymes polyketide synthases (PKSs) are responsible for regulating the assembly of carbon back bone in many secondary metabolites, in cluster with other genes involved in the synthetic pathway. Armaleo and coworkers have 228 Studies in Natural Products Chemistry COOH PO COOH P H HO O Aldol-type reaction O HO COOH NAD HO D-Erythrose-4-P Aldol-type reaction HOPH4 H OH Phosphoenol pyruvate (PEP) PO O OH O OH D-Arabino-heptulosonic acid-7-phosphate (DAHP) OH OH 3-Dehydroquinic acid H2O HO COOH COOH HO NADPH O OH Dehydration and enolization OH 3-Dehydroshikimic acid Dehydration and enolization H2O H2O Phenolic compounds COOH HO OH O OH Shikimic acid OH OH Gallic acid and other phenolic compounds SCHEME 1 Shikimic acid pathway for the biosynthesis of polyphenols. 4 HSCoA 4 CO2 O CoA S C Acetyl Co-A + O O CH3 CoA S NADPH NADP O H2O S CoA O O O O O O O 4 CoA-SCo-CH2-COOH 3 Malonyl CoA Malonyl Co-A 3 Malonyl CoA 3 HSCoA, 3 CO2 3 HSCoA, 3 CO2 O O O O S 3H2O, HSCoA, CO2 OH O O OH O O CoA O 3H2O, HSCoA, CO2 OH CH3 O OH Chrysophanolanthrone CH3 HO Emidianthrone OH R R = CH3 R = CH2OH R = COOH SCHEME 2 Polymalonate pathway for the biosynthesis of polyphenols. O OH CH3 Emodin Hydroxyemodin Emodic acid Chapter 7 229 Lichens: Chemistry and Biological Activities reported for the first time the role of PKS enzyme cluster in the synthesis of depsides and depsidones at genetic level [44]. Mevalonic Acid Pathway This pathway is mainly involved in the biosynthesis of different types of terpenes (e.g., 16b-aceteoxyhopane-6a-22-diol (42) and zeorin (43)). However, only a few di- and triterpenes are reported from different species of lichens [1]. The pathway is described in Schemes 3 and 4. Lichen-Derived Secondary Metabolites and Their Functions Many lichen secondary metabolites are reported to have various biological functions and they are produced in response to various environmental factors O CH3 H SCoA + SCoA O O Nucleophile Acetyl CoA Biological Claisen-type condensation CH3 SCoA O O Electrophile Biological Aldoltype condesation Acetyl CoA (NADPH + H+) HO + H2O - HSCoA CoASH +H - HSCoA CH3 O HOOC OH O PP = P OH O O P 3R-Mevalonic acid OH (ATP) OH CH3 O HO HOOC OPP Mevalonic acid diphosphate - CO2, - H2O isomerase OPP Gamma,gamma-dimethyl allyl-pyrophosphate OPP Isopentenylpyrophosphate (activated isoprene) OPP Geranyl pyrophosphate (precursor of monoterpene) SCHEME 3 Mevalonate pathway for the biosynthesis of monoterpenes. 230 Studies in Natural Products Chemistry OPP Geranyl pyrophosphate (precursor of monoterpene) - HOPP + OPP OPP Farnesyl pyrophosphate (precursor of sesquiterpene) - HOPP + OPP OPP Geranylgeranylpyrophosphate GGPP (Precursor of diterpene) 2GGPP, - HOPP, tail to tail linkage 2FPP, - HOPP, tail to tail linkage Squalene (Precursor of many cyclic triterpenes) SCHEME 4 Mevalonate pathway for the biosynthesis of di- and triterpenes. [2,3]. Some of them work as light filters to protect against intense radiations [3] or as preventive agents against damages caused by grazing herbivorous animals and the lethal effects of pathogenic microorganisms [2]. Lichens of Antarctica or alpine zones of Chile, the regions with depleted ozone layer, are reported to have an increased production and accumulation of rhizocarpic acid (44), protective against high UV-B radiations [45]. Many other metabolites, derived from shikimic acid pathway, are known to have characteristic UV-absorbing functional groups, such as conjugated lactone in depsidones (45–49) and conjugated carbonyls in usnic acids (50, 51). Their presence further supports the hypothesis that lichens produce them for protection against intense exposure to UV light [3,46,47]. The UV-B absorbing and photoprotecting properties of collemin A (37), produced by C. cristatum (Weber) Weber ex F.H. Wigg. var. marginale have been studied by Torres et al. They discovered that collemin A can protect UV-B-induced cell destruction in a dose-dependent manner. It can also partially prevent pyrimidine dimer formation in cultured human keratinocytes [3,42]. Unusual macrolactone glycosides (52–53) and acetylinic lipids (19–28), obtained from Cetraria islandica (L.) Ach., are suspected to maintain the membrane integrity and liquid water balance in lichens occurring in regions with low temperatures [3,48,49]. Polysaccharides and polyols account for up Chapter 7 231 Lichens: Chemistry and Biological Activities to 57% of the extractable lichen compounds and are known to have frostprotecting (antifreeze) properties [50,51]. These lichen-derived proteins, due to their antifreezing properties, are also used as food preservatives [3]. There are literature reports that some lichen-based secondary metabolites are involved in maintaining equilibrium in symbiotic association of fungi with microalgae and/or cyanobacteria [2,52]. The role of lichen-derived substances in facilitating their attachment to the substrate (rocks, trees, shrubs, trunks, etc.) has also been reported by Rundel in 1978 [53]. CH3 R2 O O RO OH O O R1 O HO Salazinic acid (5), R = OH, R1 = CHO, R2 = CH2OH Isidiophorin (82), R = OCH3, R1 = CH = CHCOCH3, R2 = CH3 H 3C O H3CO HOOC HO O CH3 O Picrolichenic acid (6) R5 R R4 R6 O R7 O R1 R2 R3 R8 Homosakikaic acid (7), R = C3H7, R1 = R7 = H, R2 = R8 = OCH3, R3 = R4 = OH, R5 = COOH, R6 = C5H11 Hyperhomosakikaic acid (40), R = R6 = C5H11, R1 = R7 = H, R2 = R8 = OCH3, R3 = R4 = OH, R5 = COOH Atranorine (78), R = R2 = R6 = OH, R1 = CHO, R3 = CH3, R4 = H, R5 = R8 = CH3, R6 = COOCH3, R7 = H Lecanoric acid acid (80), R = CH3, R1 = R4 = R8 = H, R2 = R3 = OH, R5 = CH3, R6 = COOH, R7 = OH Diffractaic acid (86), R = R2 = OCH3, R1 = CHO, R3 = CH3, R4 = H, R5 = R8 = CH3, R6 = COOH, R7 = H Sakikaic acid (88), R = R6 = C3H7,R1 = R7 = H, R2 = R8 = OCH3, R3 = R4 = OH, R5 = COOH 232 Studies in Natural Products Chemistry HOOC CH2 O O C13H27 Protolichesterinic acid (8) O O O RO R1 R2 Vulpinic acid (9), R = CH3, R1 = OH, R2 = H Pulvinic acid (38), R = R1 = R2 = H Pinastric acid (55), R = CH3, R1 = OH, R2 = OCH3 CH3 H3CO O OH O OCH3 Lichexanthone (10) BIOLOGICAL ACTIVITIES OF SECONDARY METABOLITES OF LICHENS Along with other applications, the most important use of lichen-derived substances is in traditional medicines for the treatment of human and animal diseases. Various lichen species of the genus Usnea are widely used as analgesic (for pain relief ) in different countries of Asia, Europe, and Africa [5,54]. Usnea densirostra Taylor has been used for the treatment of many health disorders in Argentinian folk medicines [55]. Lichen species of the genus Parmelia and Umbilicaria are used for the treatment of many health disorders such as infections, diarrhea, skin diseases, epilepsy, convulsions, cranial remedies, and as purgative in the Chilean medicinal system [56]. The lichen Ramalina thrausta (Ach.) (Nyl.) is used in Finland to cure wounds in athlete’s foot, various skin diseases, sore throat, and toothache [5]. Cetraria islandica (L.) Ach. is known as a cough remedy since ancient times [7]. Moreover, this alpine lichen is still commercially available as a cold remedy named Broncholind® by MCM Klosterfrau (Köln, Germany). In addition to this, many lichen species are used in traditional medicinal systems for the treatment of bleeding piles, diabetes, bronchitis, heart Chapter 7 233 Lichens: Chemistry and Biological Activities and blood diseases, pulmonary bronchitis, and dyspepsia [57]. There are also reports in the literature that many lichen-derived secondary metabolites possess a wide range of biological activities, including antioxidant, antiinflammatory, antiproliferative, antibacterial, antifungal, enzyme inhibition, antitumor, cytotoxicity, etc. These scientific studies further support the use of lichens in traditional medicine in various regions of the world. Antibacterial, Antibiotic, and Antifungal Activities The antibacterial activities of lichens have been known for decades. Burkholder and coworkers in 1944 reported for the first time the antibacterial properties of lichen extracts [58]. Extensive studies by Vartia on different species of lichens indicated that their antibacterial potential is related to their types, solvent used for extraction, and the bacterial strains to be targeted [5,59]. H H O HN O O O O H NH O H Arthogalin (11) O O H R S O H O R1 S N O N O O O Dimethyl scabrosin ester (12), R = R1 = CH3 Dipropyl scabrosin ester (13), R = R1 = C3H7 Methyl propyl scabrosin ester (14), R = CH3, R1 = C3H7 Methyl pentyl scabrosin ester (15) R = CH3, R1 = C5H11 Dipentyl scabrosin ester (16) R = C3H7, R1 = C5H11 Ranković and coworkers have evaluated the acetone, aqueous, and methanolic extracts of the Serbian lichens Cladonia furcata (Huds.) Schrad., Parmelia 234 Studies in Natural Products Chemistry pertusa Schaer, Parmelia caperata (L.) Hale., P. sulcata, Hypogymnia physodes (Nyl.) Nyl., Lasallia pustulata L., Umbilicaria polyphylla (L). Baumg, Usnea pustulata C. W. Dodge, and U. cylindrica against various species of bacteria and fungi. The strongest antimicrobial activities were observed with methanol extracts of P. pertusa and P. sulcata, whereas extracts of P. caperata and U. cylindrica showed only weak activity. They further concluded that Bacillus mycoides and Candida albicans were the most sensitive bacterial and fungal strains, respectively, against the lichen extracts tested [60,61]. CH3 O R O H3CO OR2 Br O R1 Acarogobien A (17), R = R2 = H, R1 = CH3 Acarogobien B (18), R = Br, R1 = CHO, R2 = 7 COOMe Br 18-Bromo-(5E,17E)-octadeca-5,17-diene-15-yonoic acid methyl ester (19) COOMe 5 Br 8-Bromooctadeca-5,7,17-triynoic acid methyl ester (20) COOMe Br 5 Br 16,18-Dibromo-(5E,17E)-octadeca-5,17-diene-5,7-diyonoic acid methyl ester (21) COOMe Br Br 7 18,18-Dibromo-17-octadecene-5,7-dynoic acid methyl ester (22) Chapter 7 235 Lichens: Chemistry and Biological Activities The antibacterial activity of 69 lichen species from New Zealand was studied by two research groups. They reported the inhibitory effects of lichen extracts against Bacillus subtilis, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, and Streptococcus pneumoniae [62,63]. Antibacterial effect of the acetone, methanol, and petroleum extracts of Usnea ghattensis G. Awasthi against Bacillus megaterium, B. licheniformis, B. subtilis, and S. aureus was studied by Karagoz et al. [64]. The antibacterial potential of ethanolic and aqueous extracts of 11 Turkish lichen species was evaluated. A potent antibacterial activity was reported for the ethanol extract of Ramalina farinaceae (L.) Ach. and aqueous extract of Peltigera polydactyla (Neck.) Hofmm. [64]. Phytochemical studies on the anti-Gram-positive extracts of Usnea hirta (L.) have revealed that the activity was due to the presence of hirtusneanoside (54), pinastric (55), and evernic acids (56) [65,66]. The antibacterial activity of methanolic extracts of Evernia prunastri (L.) Ach., H. physodes (L.) (Nyl.), Flavoparmelia caperata (L.) Hale, and Cladonia foliacea (Huds.) Willd. was reported by Mitrović et al. against 15 strains of bacteria. They discovered that the methanolic extracts of H. physodes and C. foliacea have the strongest antibacterial activity against gram positive bacteria [67]. The Antarctic lichens are rich sources of antibacterial compounds. Gram-positive S. aureus and B. subtilis were found to be sensitive against the methanolic extracts of four lichens, collected in Antarctica [68]. Three new depsidones were isolated from lichen Neuropogon and were found to be moderate growth inhibitors of Mycobacterium vaccae [69]. Various lichen-based aromatic phenols, such as methyl- (57) and phenyl-orsellinate (58), due to their antimicrobial properties, are considered to be equally useful preservatives as commercial preservatives [70]. ()-Usnic acids (50 and 51) are the most common compounds in lichen species. They interact with the gut digestive flora of ruminants [71]. Usnic acid is also used as a clinical antibiotic (Binan® and Usno®), as well as topical antiseptic in many products, such as Camillen 60 Fudes spray, and Gessato® shaving treatment from Italy [3]. Usnic acid is also reported as an inhibitor of bacterial biofilm formation on polymeric material and it is therefore used in coatings on medical devices [72]. It is also active against resistant pathogenic strains of S. aureus, Mycobacterium aurum [73], and Listeria monocytogenes [74]. Br 7 COOMe 18-Bromo-(5E,17Z)-octadeca-5,7-diene-15-ynoic acid methyl ester (23) 236 Studies in Natural Products Chemistry COOMe Br 18-Bromo-(5E,15Z)-octadeca-5,5-diene-11,13,17-triynoic acid methyl ester(24) Br COOMe 5 O OH 18-Bromo-9-hydroxy-12,13-trans-epoxy-(10E,15Z)-octadeca-10,155-diene-17ynoic acid methyl ester (25) COOMe O Br CH3 CH3 18-Bromo-5,6-trans-endomethylene-7,11,15-trimethyl-(8E,10Z)-octadeca-8,10diene-17-ynoic acid methyl ester (26) R H 3C HOH 2C O O Tornabeatin A (27), R = 7 5 Tornabeatin B (28), R = 7 7 Tornabeatin C (29), R = 11 Tornabeatin D (30), R = 14 Chapter 7 237 Lichens: Chemistry and Biological Activities R CH2 R1 O O OH Murolic acid (31), R = βCOOH, R1 = 11 OH Protoconstipatic acid (32), R = αCOOH, R1 = 11 OH Allo-murolic acid (33), R = βCOOH, R1 = 11 Weak antibacterial activity of various anthraquinones against B. subtilis was reported by Ivanova and coworkers [3]. The traditional use of lichen C. islandica as antiulcer remedy was supported by the activity of protolichesterinic acid (8) against Helicobacter pylori [75]. Antimicrobial activity of stictic (59), protocetraric (60), and fumarprotocetraric acids (61) against bacteria and fungi was evaluated by Ranković and Misić. They observed that bacteria were more sensitive to these compounds, as compared to fungi. Lobaric (62), salazinic (5), and protolichesterinic acids (8) were also tested against nonpathogenic M. aurum (similar sensitivity profile to Mycobacterium tuberculosis), with a high MIC (125–250 mg/mL), except for usnic acids (50 and 51) that showed an MIC of 50 mg/mL [73]. In 2007, Gupta and coworkers reported the activity of nine lichen species against M. tuberculosis strains (H37Rv and H37Ra) for the first time. They found that the ethanol extracts of F. caperata (L.) Hale and Heterodermia leucomela (L.) Poelt. exhibit MIC ¼ 250 mg/mL against M. tuberculosis (H37Rv and H37Ra strains), whereas the ethanol extracts of lichen species Everniastrum cirrhatum (Fr.) Hale ex Sipman, Rimelia reticulata (Taylor) Hale & Fletcher, and Stereocaulon foliolosum (Nyl.) were found to be active against H37Rv strain at a concentration of 500 mg/mL [76]. OH O OH HO HO HO HO O OH O OH O OH HO O N H O OH O O O OH H3C O O O O OH HO 12 NH2 Gobienine A (34) Collemin A (37) 238 Studies in Natural Products Chemistry O OCH3 O O O NH OH Rhizocarpic acid (44) Ivanova et al. in 2002 reported a moderate antimicrobial activity of neuropogonines A, B, and C (46–48) which were obtained from Neuropogon, an Antarctic lichen species [69]. Salazinic acid (5) and its di-O-propyl, -butyl, -pentyl, and -hexyl ester derivatives were evaluated for their antibacterial activity against E. coli and S. aureus and found to be active against both organisms. They observed that the elongation of chain has no significant effect on their antibacterial potential. Salazinic acid (5) showed growth inhibition of E. coli only [77]. In 2012, Bucukoglu and coworkers reported the antimicrobial potential of gyrophoric acid (63) against Klebsiella pneumoniae and Morganella morganii [78]. O R O R3 R4 O R1 O H R6 R5 Panarin (45), R = Cl, R1 = OH, R3 = R6 = CH3, R4 = OCH3, R5 = H Neuropogenin A (46), R = OH, R1 = H, R3 = R5 = CH2OH, R4 = H, R6 = CH3 Neuropogenin B (47), R = H, R1 = OH, R3 = CH2OH, R4 = OCH3, R5 = COOH, R6 = CH3 Neuropogenin C (48), R = H, R1 = OH, R3 = CH2OH, R4 = H, R5 = COOH, R6 = CH3 Neuropogenin D (49), R = H, R1 = OH, R3 = CH2OH, R4 = OH, R5 = COOH, R6 = CH3 Protocetraric acid (60), R = R6 = H, R1 = OH, R3 = CH2OH, R4 = OH, R5 = COOH Virensic acid (94), R = H, R1 = R4 = OH, R3 = R6 = CH3, R5 = COOH Methylvirenate (95), R = H, R1 = R4 = OH, R3 = R6 = CH3, R5 = COOCH3 The antimicrobial activity of the crude extract of Parmelia perlata (Huds.) Ach. and its two constituents 64 and 65 (4-amino-3-hydroxy-6-methoxy-2methylcyclohexa-1,3-diene-1-carbaldehyde and 5-amino-2-ethoxy-4-methylcyclohexa-1,3-diene-1-carboxylic acid, respectively) was reported by Thippeswamy and coworkers. Compound 64 was found to be significantly more active against Corynebacterium michiganensis, whereas compound 65 was active against Pseudomonas solanacearum [79]. Chapter 7 239 Lichens: Chemistry and Biological Activities Crude extract of Usnea steineri Zahlbr. and its major phenolic compounds usnic acids (50 and 51) was investigated by Lucarini and coworkers against Mycobacterium kansassi (ATCC 12478) and M. avaium (ATCC 15769). The acetone extract and purified usnic acid showed promising growth inhibition of both the strains with MIC in the range of 16–32 and 8–16 mg/mL, respectively [80]. OH O O R H3C HO O OH H O (+)Usnic acid (50), R = (–)Usnic acid (51), R = CH3 CH3 OR O HOOC R1O 3 O HO OR2 4 18 11 O (52), R = R1 = R2 = H, 3S, 4R, 18R (53), R = R1 = R2 = H, 3S, 4R, 18S OH COOCH3 O OH OH O O OH OH O OH O O HO HO OH Hirtusneanoside (54) O 240 Studies in Natural Products Chemistry Antifungal activity of an anthraquinone parietin (3), obtained from Caloplaca cerina (Ehrh. ex Hedw.) Th. Fr., was reported by Manojlovic and coworkers [81]. The antifungal activities of extracts of Usnea florida (Ach.) Motyka and Protousnea poeppigii (Nees & Flot) Krog. were also evaluated in 2008, with no significant activity against Aspergillus and Candida strains. Interestingly, a secondary metabolite from P. poeppigii, divaricatinic acid, showed promising activity against pathogenic strains of Microsporum and Trichophyton with MIC values between 50 and 100 mg/mL [82]. The antifungal activity of extracts of E. prunastri (L.) Ach. and H. physodes (L.) (Nyl.) was studied by Mitrović and coworkers. They were found to be active against yeast and filamentous fungi, respectively [67] (Table 1). Antiprotozoal Activity Antileishmanial activity of pannarine (66), 10 -chloropannarine (67), and (+)-usnic acid (50) against promastigote forms of Leishmania species was reported by Fournet and coworkers [83]. Weak trypanocidal activity of triterpenes 68–70 and depsides 71 and 72, isolated from Pseudocyphellaria corrifolia (Müll. Arg.) Malme, has been reported by Fritis and coworkers against Trypanosoma cruzi [84]. Trypanosomiasis is a common vector-borne disease in tropical regions of the world. Successful remedies of the disease are much sought for (Table 2). Antiviral Activity Some secondary metabolites of lichens were also reported to have antiviral effects. Perry and coworkers in 1999 reported the antiviral activity of ()-usnic acids (50 and 51) against the Polio and Herpes simplex type 1 viruses [63]. The viricidal activity of parietin (3), a secondary metabolite of Teloschistes chrysophthalmus (L.) Th. Fr., was reported against Junin and Tacaribe arena viruses [85]. Tobacco mosaic virus inhibition potential of lichenan (73), a metabolite of many lichens, was also reported in the literature [86]. Usnic acid (50) was also reported to exhibit activity against same viruses as tested for parietin (3) [6] (Table 3). R3 COOR4 R O O R1 R5 R2 Evernic acid (56), R = R3 = OH, R1 = OCH3, R2 = R5 = CH3, R4 = COOH Methyl evernate (70), R = R5 = OH, R1 = OCH3, R2 = R3 = R4 = CH3 Sphaerophorin (74), R = CH3, R1 = OCH3, R2 = R3 = OH, R4 = COOH, R5 = C7H15 Lecanorin (91), R = R1 = R3 = OH, R2 = R5 = CH3, R4 = H TABLE 1 Antibacterial and Antifungal Constituents from Different Lichen Species Compound Activity Lichen Source References Hirtusneanoside (54) Pinastric acid (55) Evernic acid (56) Antibacterial Usnea hirta, Ramalina farinaceae (L.) Ach., Peltigera polydactyla (Neck.) Hofmm. [65,66] Methyl orsellinate (57) Phenyl orsellinate (58) Moderate inhibitors of Mycobacterium vaccae Neuropogon spp. [70] (+)-Usnic acid (50) ( )-Usnic acid (51) Interact with the gut digestive flora of ruminants, clinical antibiotic, topical antiseptic, active against resistant pathogenic strains of Staphylococcus aureus, Mycobacterium, and Listeria monocytogens Cladonia arbuscula (Waltr.), C. laptoclata, Heterodea mulleri (Hampe) (Nyl.), Pseudocyphellaria glabra (Hook. f. & Taylor) C. W. Dodge, P. homoeophylla (Nyl.) Dodge & Sticta, Nephroma arcticum (L.) Torss., Alectoria ochroleuca (Hofmm.), Ramalina farinacea (L.) Ach., Usnea campestris R. Sant., U. longissima Ach., U. misaminensis (Vain.) Motyka, U. venosa [1,3,71–74,80] Protolichesterinic acid (8) Active against Helicobacter pylori Cetraria islandica (L.) Ach. [75] Stictic acid (59) Protocetraric acid (60) Fumaprotocetraric acid (61) Lobaric acid (62) Salazinic acid (5) Protolichesterinic acid (8) Antimycobacterium Parmotrema dilatatum (Vain.) Hale, Parmotrema tinctorum (Nyl.) Hale, Pseudoparmelia sphaerospora (Nyl.) Hale, and Usnea subcavata (Motyka) [60,73] Continued TABLE 1 Antibacterial and Antifungal Constituents from Different Lichen Species—Cont’d Compound Activity Lichen Source References Salazinic acid (5) Inhibition of Escherichia coli and Staphylococcus aureus Cetraria islandica (L.) Ach., Parmotrema dilatatum (Vain.) Hale, Parmotrema tinctorum (Nyl.) Hale, Pseudoparmelia sphaerospora (Nyl.) Hale, and Usnea subcavata (Motyka) [77] Gyrophoric acid (63) Active against Klebsiella pneumoniae and Morganella morganii Cetraria islandica (L.) Ach. [78] 4-Amino-3-hydroxy-6-methoxy-2methylcyclohexa-1,3-dienecarbaldehyde (64) 5-Amino-2-ethoxy-4hydroxycyclohexa-2,4diene-carboxylic acid (65) Active against Corynebacterium michiganensis and Pseudomonas solanacearum Parmelia perlata (Huds.) Ach. [79] Parietin (3) Antifungal Caloplaca cerina (Ehrh. ex Hedw.) Th. Fr. [81] Chapter 7 243 Lichens: Chemistry and Biological Activities TABLE 2 Antiprotozoal Constituents from Different Lichen Species Compound Activity Lichen Source References (+)-Usnic acid (50) 10 -Chloropannarine (67) Pannarine (68) Active against Promastigotes of Leishmania Erioderma leylandi (Taylor) Mull. Arg., Psoroma palladium (Nyl.), Protousnea malacea (Stirt) Krong. [83] 2-((3S,5aR,5bR,11aS,11bR,13bS)5a,5b,8,8,11a,13bHexamethylicosahydro-1Hcyclopenta[a]chrysen-3-yl) propan-2-ol (66) Hopan-22-ol (68) Hopan-16b-22-diol (69) Hopan-6a,7b-22-triol (70) Methyl evernate (71) Tenuiorin (72) Active against Trypanosoma cruzi Pseudocyphellaria corrifolia (Müll. Arg.) [84] TABLE 3 Antiviral Constituents from Different Lichen Species Compound Activity Source References Parietin (3) Against Junin and Tacaribe arena viruses Teloschistes chrysophthalmus (L.) Th. Fr. [85] (+)-Usnic acid (50) ( )-Usnic acid (51) Against Polio, Herpes simplex type 1, Junin, and Tacaribe arena viruses Cladonia arbuscula (Waltr.), C. laptoclata, Heterodea mulleri (Hampe) (Nyl.), Pseudocyphellaria glabra (Hook. f. & Taylor) C. W. Dodge, P. homoeophylla (Nyl.) Dodge & Sticta, Naphroma arcticum (L.) Torss., Alectoria ochroleuca (Hoffm.), Ramalina farinacea (L.) Ach., Usnea campestris R. Sant., U. longissima Ach., U. misaminensis (Vain.) Motyka, U. venosa [6] Lichenan (73) Against Tobacco mosaic virus Cetraria islandica (L.) Ach., Evernia prunastri (L.) Ach. [86] 244 Studies in Natural Products Chemistry O CH3 O R OR1 O H O O HO O Stictic acid (59), R = OCH3, R1 = H Vesuvianic acid (81), R = OCH3, R1 = C2H5 O O O H O O OH OH O H O O O HO Fumaprotocetraric acid (61) R O R1 O R2 R4 R5 O R3 R7 R6 Sublobaric acid (41), R = C2H5CO, R1 = R3 = R4 = H, R2 = OCH3, R5 = OH, R6 = COOH, R7 = C5H11 Lobaric acid (62), R = COC4H9, R1 = R3 = R4 = H, R2 = OCH3, R5 = OH, R6 = COOH, R7 = C5H11 Pannarine (66), R = R3 = R5 = CH3, R1 = Cl, R2 = OCH3, R4 = H, R5 = OH, R6 = CHO, R7 = OH 1'-Chloropannarine (67), R = R4 = R7 = CH3, R1 = R6 = Cl, R2 = OH, R3 = CHO, R5 = OCH3 a-Alectoronic acid (76), R = R7 = CH2COC5H11, R1 = R4 = H, R2 = R5 = OH, R6 = COOH Diploicin (77), R = R7 = CH3, R1 = R3 = R4 = R6 = Cl, R2 = OH, R5 = OCH3 Chapter 7 Lichens: Chemistry and Biological Activities 245 COOR OH O O HO O O OR1 OR2 Gyrophoric acid (63), R = R1 = R2 = H Umbilicaric acid (89), R = R2 = H, R1 = CH3 Tenuiorin (72), R = R2 = CH3, R1 = H Cytotoxicity and Antitumor Activities Cytotoxicity of n-hexane, diethyl ether, and methanolic extracts of eight lichen species Cladonia convoluta (Lam.) Cout., C. rangiformis Hoffm., E. prunastri (L.) Ach., F. caperata (L.) Hale, Parmotrema perlatum (Huds) M. Cholsey, Plastismatia glauca (L.) W.L. Culb. & C.F. Culb, Ramalina cuspidata (Ach.) (Nyl.), and Usnea rubicunda Strit. were evaluated against two murine and four human cancer cell lines, by Bézivin and coworkers. The n-hexane extracts of these lichens were found to be more active than the methanolic extracts [6,87]. ( )-Usnic acid (51) was reported to be cytotoxic against various cancer cell lines, including prostate carcinoma, Lewis lung carcinoma, human brain metathesis, breast adenocarcinoma, human chronic myelogenous leukemia, and human glioblastoma cell lines [33,88]. ()-Usnic acids (50 and 51) decreased proliferation of the human breast and lung cancer cell lines without causing DNA damage [89]. The antiproliferative effect of ()-usnic acids (50 and 51) against K-562 (human leukemia cells) and HEC-50 (endometrial carcinoma cells) was reported by numerous groups [5,90,91]. The depsidones pannarin (66) and sphaerophorin (74), isolated from Sphaerophorus globosus (Huds.), were reported to inhibit the cell growth and induce apoptosis in human prostate carcinoma (DU-145) and melanoma M14 cells [92,93]. 246 Studies in Natural Products Chemistry H H CH3 CH3 CH3 CH3 R3 CH3 H H3C H CH3 R CH3 R2 R1 16 - Actoxyhopane-6 -22-diol (42), R = OCOCH3, R1 = H, R2 = R3 = OH Zeorin (43), R = OH, R1 = R2 = H, R3 = OH Hopan-22-ol (68), R = R1 = R2 = H, R3 = OH Hopan-16β-22-diol (69), R = R1 = H, R2 = R3 = OH Hopan-6α-7β-22-triol (70), R = R1 = H, R2 = R3 = OH Tenuiorin (72), R1 = R2 = R3 = OH, R = H R NH2 R1 OH R2 4- Amino-3-hydroxy-6-methoxy-2- methylcyclohexa-1,3-dienecarbaldehyde (64), R = OCH3, R1 = CHO, R2 = CH3 5- Amino-2-ethoxy-4-hydroxycyclo hexa-2,4-dienecarboxylic acid (65), R = COOH, R1 = OC2H5, R2 = H The antitumor activity of protolichesterinic acid (8) (C. islandica (L.) Ach.) against breast- and mitogen-stimulated lymphocyte cancer cell lines was reported by Russo and coworkers [94]. Müller in 2005 studied the possible mechanism of action of protolichesterinic acid (8). The inhibition of 5-lipoxygenase enzyme and its nonspecific binding with DNA polymerase-b and ligase-1 was found to be the main mechanisms of its action [7]. Correche and coworkers evaluated a series of nine depsidones, four depsides, and a tridepside gyrophoric acid (63) for their cytotoxicity in lymphocyte cell culture and reported that depsidones were more cytotoxic as compared to depsides [95]. Lobaric acid (62) was also studied for its antitumor activity against three malignant cell lines of erythro-leukemia. It was found to exert a significant reduction in DNA synthesis [96] (Table 4). Another group of researchers, Ogmundsdottir et al., have reported the antiproliferative potential of lobaric acid (62) against 10 human cancer lines named, capan-1 and -2, PANC-1 (pancrease), NCI-H1417 (lung cell), PC-3 (prostate), T47-D (breast), AGS (stomach) NTH:OVCAR-3 (ovaries), WiDr (colorectal), HL-60, K-562, and JURKAT (acute promyelocytic, erythro cell, TABLE 4 Cytotoxic and Antitumor Constituents from Different Lichen Species Compound Activity Source References (+)-Usnic acid (50) ( )-Usnic acid (51) Cytotoxic against various cancer cell lines, including prostate carcinoma, Lewis lung carcinoma, human brain metathesis, breast adenocarcinoma, human chronic myelogenous leukemia and human glioblastoma Antiproliferative against K-562 (human leukemia cells) and HEC-50 (endometrial carcinoma cells) Active against Mycobacterium kansassi and Mycobacterium avaium Cladonia arbuscula (Waltr.), C. laptoclata, Heterodea mulleri (Hampe) (Nyl.), Pseudocyphellaria glabra (Hook. f. & Taylor) C. W. Dodge, P. homoeophylla (Nyl.) Dodge & Sticta, Nephroma arcticum (L.) Torss., Alectoria ochroleuca (Hofmm.), Ramalina farinacea (L.) Ach., Usnea campestris R. Sant., U. longissima Ach., U. misaminensis (Vain.) Motyka, U. venosa, U. steineri Zahlbr. [5,33,80,88–91] Pannarin (66) Sphaerophorin (74) Inhibits cell growth and induce apoptosis in human prostate (DU-145) and human melanoma M14 cell lines Sphaerophorus globosus (Huds.) [92,93] Protolichesterinic acid (8) Antitumor activity against breast and mitogenstimulated lymphocytes cancer cell lines Cetraria islandica (L.) Ach. [7,94] Salazinic acid (5) Gyrophoric acid (63) Stictic acid (59) Cytotoxic against lymphocyte cell culture Parmelia nepalensis Taylor., Parmelia tinctorum (Nyl.) [95] Lobaric acid (62) Antitumor activity against malignant cell lines of erythro-leukemia Antiproliferative against capan-1 and -2, PANC-1 (pancrease), NCI-H1417 (lung cell), PC-3 (prostate), T47-D (breast), AGS (stomach) NTH:OVCAR-3 (ovaries), and JURKAT (acute promyelocytic, T-Cell, and erythro cell leukemia cell lines) Parmotrema dilatatum (Vain.) Hale, Parmotrema tinctorum (Nyl.) Hale, Pseudoparmelia sphaerospora (Nyl.) Hale, and Usnea subcavata (Motyka) [95,96] Continued TABLE 4 Cytotoxic and Antitumor Constituents from Different Lichen Species—Cont’d Compound Activity Source References Variolaric acid (75) a-Alectoranic acid (76) Cytotoxic against B16 murine melanoma cells Ochrolechia parella (L.) A. Massal [97] Salazinic acid (5) Antitumor activity against human HCT-8, MDA-345, SF-295, and MB-435 cell lines Cetraria islandica (L.) Ach., Parmotrema dilatatum (Vain.) Hale, Parmotrema tinctorum (Nyl.) Hale, Pseudoparmelia sphaerospora (Nyl.) Hale, and Usnea subcavata (Motyka) [98] Diploicin (77) Cytotoxic against B16 (murine melanoma) and HaCaT (human keratinocyte) cell lines Buellia canescens (Dicks.) De Not. [99] Pannarine (66) Inhibitor of DU-145 prostate carcinoma and M14 (human melanoma) cell lines Sphaerophorus globosus (Huds.) [96,100] Protolichesterinic acid (8) HeLa cell lines Parmotrema dilatatum (Vain.) Hale, Parmotrema tinctorum (Nyl.) Hale, Pseudoparmelia sphaerospora (Nyl.) Hale and Usnea subcavata (Motyka), Ramalina farinacea (L.) Ach., Cladonia furcata (Huds.) Schrad., Ochrolechia androgyna (Hoffm.) Arn., Parmelia caperata (L.) Ach., and Parmelia conspresa (Ach.) Ach. [101] Chapter 7 249 Lichens: Chemistry and Biological Activities and T-Cell leukemia cell lines) [95,97]. Millot and coworkers isolated variolaric (75) and a-alectoronic acids (76) from Ochrolechia parella (L.) A. Massal and evaluated their cytotoxic potential against B16 murine melanoma cells [98]. Salazinic acid (5) and its di-O-alkyl (propyl, butyl, pentyl, and hexyl) derivatives were tested by Micheletti and coworkers in 2009 against HCT-8, MDA-345, SF-295, and MB-435 human tumor cell lines. They concluded that cytotoxic activity of salazinic acid (5) increases with the elongation of the alkyl chain [99]. OH OH O HO O O O HO OH OH HO O OH O O OH OH O HO O OH n Lichenan (73) The cytotoxicity of diploicin (77) was evaluated by Millot and coworkers in 2009 against B16 (murine melanoma) and HaCaT (human keratinocyte) cell lines [100]. Pannarin (66), a secondary metabolite from lichen Diploicia canescens, was tested by two different research groups for its inhibitory potential against DU-145 prostate carcinoma [100] and M14 (human melanoma) cell lines with positive results [96]. Recently, the cytotoxicity of protolichesterinic acid (8) against HeLa cell lines has been evaluated by Brisdelli and coworkers. Its activity is based on its ability to induce cell death through activation of caspases-3, -8, and -9 [101]. Antioxidant Properties Impressive antioxidant properties of crude extracts of lichens have been reported, but ironically the secondary metabolites obtained from lichens have not shown any promising antioxidant property. Due to the presence of large quantities of polyphenolic compounds, lichens are expected to exhibit antioxidant activities [3,102]. Cuculloquinone from Flavocetraria cucullata (Bellardi) Kärnefelt & A. Thell. exhibited up to 80% inhibition of 2,2diphenyl-1-picrylhydrazil (DPPH)-free radical where BHT was used as a standard. The activity was found to be twofold higher than that of standard [103]. Bhattarai and coworkers have reported the antioxidant potential of five lichen species, collected in Antarctica, containing 30–35% phenolic contents. Extracts of all species were found to be antioxidant in nature. They also evaluated the antioxidant properties of methyl orsellinate (57), atranorine (78), 250 Studies in Natural Products Chemistry orsellinic acid (79), and lecanoric acid (80) and concluded that the total phenolic contents and antioxidant activities have a direct correlation [104]. The lipid peroxidation inhibition and DPPH-scavenging potential of phenolic compounds, vesuvianic acid (81), stictic acid (59), usnic acid (50), isidiophorin (82), rhizonyl alcohol (83), rhizonaldehyde (84), pulmonarianin (85), and diffractaic acid (86), as well as a steroid ergosterol peroxide (87), isolated from Lobaria pulmonaria (L.) Hoffm. and Usnea longissima Ach., were reported by Atalay and coworkers in 2011. All compounds showed a good DPPH radical scavenging potential, except usnic and diffractaic acids, which were found to be inactive in the assay [105]. O OH O HO O O O Virolaric acid (75) O CH3 O H3CO OH O O H O O H Pulmonarianin (85) Antioxidant activity of many lichen compounds, isolated from Parmotrema grayana Hue, Heterodermia obscurata (Nyl.) Trevisan, Cladonia sp., and Roccella montagnei Bel. were evaluated by Thadhani and Choudhary et al. by using superoxide radical (SOR), DPPH, and nitric oxide (NO%) radical scavenging assays [106,107]. They reported promising antioxidant activity of sekikaic acid (88, IC50 ¼ 82.0 0.3 mM), lobaric acid (62, IC50 ¼ 97.9 1.6 mM), and lecanoric acid (80, IC50 ¼ 91.5 2.1 mM), as compared to the standard propyl gallate (IC50 ¼ 106.0 1.7 mM). Methyl-b-orscellinate Chapter 7 251 Lichens: Chemistry and Biological Activities (57, IC50 ¼ 84.7 0.1 mM) was found to be a potent nitric oxide scavenger, as compared to the standard rutin (IC50 ¼ 86.8 1.9 mM). In 2012, Bucukoglu and coworkers evaluated the antioxidant potential of extracts of six Umbilicaria species (U. aprina (Nyl.), U. cylindrica, U. decussata (Vill.) Zahlbr., U. leiocarpa DC., U. nylanderiana Zahlbr., and U. virginis (Schaer.) Schol.). The methanol extracts exhibited a moderate DPPH radical scavenging property. These researchers also evaluated the antioxidant activity of different acids, isolated from lichens. Umbilicaric acid (89) exhibited the highest activity (68.14%) in DPPH radical scavenging assay [78]. Radical scavenging activity is a reliable indicator of the antioxidant potential of compounds against oxidative stress and associated health disorders (Table 5). CH3 CH3 H3C CH3 H3C CH3 O O H HO Ergosterol peroxide (87) Antidiabetic Properties Choudhary and coworkers in 2011 have evaluated for the first time the protein antiglycation potential of orsellinic acid (79), ethyl orsellinate (90), lecanoric acid (80), gyrophoric acid (63), licanorin (91) and ethyl heamatomate (92), isolated from the lichen Parmotrema cooperi (J. Steiner & Zahlbr.) Sérus. Among all, compound 92 (IC50 ¼ 220.55 1.16 mM) was found to be the most potent in the series, more active than rutin (IC50 ¼ 294.50 1.5 mM) which was used as a standard in this assay [108]. Inhibition of glycation of protein in hyperglycemia (diabetes) is an approach for delaying the on-set of late diabetic complications such as diabetes neuropathy, nephropathy, retinopathy, cardiovascular disorders, and strokes. The potent a-glucosidase inhibitory activity of methyl orscinollinate (57), methyl b-orscinolcarboxylate (93), and zeorin (43) was also patented by the same group in 2011 [109]. a-Glucosidase inhibitors are extensively used for treatment of postprandial hyperglycemia, obesity, and viral infections (Table 6). TABLE 5 Antioxidant Constituents Isolated from Different Lichen Species Compound Activity Source References Methyl orsellinate (57) Atranorine (78) Orsellinic acid (79) Lecanoric acid (80) Antioxidant Stereocaulon alpinum Laurer, Ramalina terebrata Hook., and Taylor, Caloplaca regalis (Vain.) Zahlbr [104] (+)-Usnic acid (50) Stictic acid (59) Vesuvianic acid (81) Isidiophorin (82) Rhizonyl alcohol (83) Rhizonyl aldehyde (84) Pulmonarianin (85) Diffractaic acid (86) Ergosterol peroxide (87) Lipid peroxidation inhibition and DPPH radical scavenger Lobaria pulmonaria (L.) Hoffm., U. longissima Ach. [105] Methyl orsellinate (57) Lobaric acid (62) Lecanoric acid (80) Sakikaic acid (88) Active in superoxide radical (SOR), DPPH, and nitric oxide-scavenging assays Parmotrema grayana Hue, Heterodermia obscurata (Nyl.) Trevisan, Rocella montagnei Bel. [106] Umbicaric acid (89) DPPH radical scavenger Umbilicaria aprina (Nyl.), Umbilicaria cylindrical, Umbilicaria decussata (Vill.) Zahlbr., Umbilicaria leiocarpa DC., Umbilicaria nylanderiana Zahlbr., Umbilicaria virginis (Schaerer.) Schol. [78] Chapter 7 253 Lichens: Chemistry and Biological Activities TABLE 6 Antidiabetic Constituents Isolated from Different Lichen Species Compound Activity Source References Homosckikaic acid (7) Gyrophoric acid (65) Orsellinic acid (79) Ethyl orsellinate (90) Lecanorin (91) Antigylcation Parmotrema cooperi (J. Steiner & Zahlbr.) Sérus [108] Methyl orsellinate (57) Methyl-borcinolcarboxylate (93) Zeorin (43) a-Glucosidase inhibition Parmotrema grayana, Rocella montagni, Psudocyphellaria crocata (L.) Vain, Stereocaulon alpinum Laur. Different species of lichens, for example, Anaptychia, Lecanora, Parmelia, Naephroma, and Plaodium [109] O O O R H3CO HO OH OCH3 (96) R = H (97) R = COOH Enzyme Inhibition Properties Lichen constituents have also shown inhibitory activities against several clinically important enzymes. Neamati et al. reported the enzyme inhibitory potential of 17 lichen acids, including depsidones, depsides, and their synthetic derivatives, against HIV-1 integrase. Virensic acid (94), stictic acid (59), and methyl ester of virensic acid (95) showed enzyme inhibition with IC50 ¼ 3 mM [90,110]. Lobaric acid (62) was found to be a potent inhibitor of the contractile activity of smooth muscles of guinea pig taenia coli, from guinea pig induced by ionophore A23187 at a dose of 5.8 mM. Compound 254 Studies in Natural Products Chemistry 62 also found to inhibit the ionophore A23187-induced generation of cysteinyl-leukotrienes at the dose of 5.5 mM [111]. It also inhibited arachidonate 5-lipoxygenase enzyme, obtained from porcin leucocytes (IC50 ¼ 7.3 mM), with fourfold greater activity than its activity against cyclooxygenase [112]. Seo and coworkers have reported potent protein tyrosine phosphatase 1B (PTP1B) inhibitory activity of lobaric acid (62) and two other pseudo-depsidones (96 and 97) with IC50 values of 0.87, 6.86, and 2.48 mM, respectively [113] (Table 7). Protein tyrosinase phosphatase 1B (PTP1B) enzyme inhibitory activity of four diterpene furanoids, isolated from Antarctic lichen Huea species, was reported by Cui and coworkers in 2012. Among them, huaefuranode A (98) was found to be a noncompetitive inhibitor of the target protein PTP1B with IC50 ¼ 13.9 mM [114]. Tyrosinase inhibitors are used to control the level of TABLE 7 Enzyme Inhibiting Constituents Isolated from Different Lichen Species Compound Activity Source References Stictic acid (59) Virensic acid (94) Methyl virensate (95) Inhibitors of HIV-1 integrase Sulcaria virens (Taylor) Bystrek ex Brodo & D. Hawksw [89,110] Lobaric acid (62) Inhibitors of the contractile activity of guinea pig taenia coli smooth muscles Inhibitor of ionophore A23187induced generation of cysteinylleukotrienes, porcin leukocyte arachidonate 5-lipoxygenase Parmotrema dilatatum (Vain.) Hale, Parmotrema tinctorum (Nyl.) Hale, Pseudoparmelia sphaerospora (Nyl.) Hale, and Usnea subcavata (Motyka) [111,112] Lobaric acid (62) Pseudo-depsidones (96) and (97) Inhibitors of tyrosine phosphatase 1B (PTP1B) Stereocaulon alpinum Laurer ex Funck [113] Hueafuranoid A (98) Inhibitor of tyrosine phosphatase 1B (PTP1B) Antarctic lichen Huea species [114] Lecanorin (91) Atraric acid (99) Inhibitors of urease enzyme Parmotrema cooperi (J. Steiner & Zahlbr.) Sérus [108] Chapter 7 255 Lichens: Chemistry and Biological Activities L-DOPA, which is in turn associated with skin disorders, Parkinson’s disease, schizophrenia, etc. Choudhary and coworkers have evaluated different phenolic constituents of P. cooperi (J. Steiner & Zahlbr.) Sérus against urease and found licanorin (91) and atraric acid (99) as significant inhibitors. Atraric acid (99) was found to be more active than the thiourea (IC50 ¼ 21 0.001 mM) [108]. Overexpression of urease enzyme by infectious bacteria is responsible for various pathologies, including peptic ulcers urolithiasis, formation of infectious kidney stones, polynephritis, hepatic coma, and urinary catheritis. Soil urease is also known to cause economic loss by rapid urea degradation. Inhibition of urease enzyme by small molecules is an effective strategy to counteract these problems. OH O HO CH3 CH3 CH3 O Hueafuranoid A (98) Antipyretic and Analgesic Properties Lichen substances are also known to have activities against pain, fever, and inflammation. Vijayakumar in 2000 reported a dose-depend antiinflammatory effect of (+)-usnic acid (50) isolated from R. montagnei Bel. in rats. For this purpose chronic and acute models were employed [115]. (+)-Usnic acid (50) was also found to possess analgesic property in mice and antipyretic effect in lipopolysaccharide-induced fever in vivo [54]. CONCLUSION Lichens are fascinating organisms with equally fascinating chemical compositions. Their ability to survive in harsh and constrained conditions is largely dependent upon their chemical diversity. This brief review illustrates lichens to be powerful sources of highly oxygenated aromatic compounds with diverse biological activities. Till June 2012, more than 1000 secondary 256 Studies in Natural Products Chemistry metabolites have been obtained from different lichen species. However, there is still a need of extensive studies to discover unexplored chemical diversity of lichens as well as to evaluate their pharmacological potential. Study of the role of chemicals in symbiotic or mutualistic relationship of lichen partners (fungi and alga or cyanobacteria) and their role in their survival and longevity can provide useful leads for possible human use. 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This page intentionally left blank Chapter 8 Chemistry and Bioactivities of Royal Jelly Eleni Melliou and Ioanna Chinou Department of Pharmacognosy and Chemistry of Natural Products, Faculty of Pharmacy, University of Athens, Panepistimiopolis Zografou, Athens, Greece Chapter Outline Introduction Chemical Constituents Identified in RJ Fatty Acids Biological Properties Antimicrobial Activities Antioxidative Activity Estrogenic Activity Activities in Reproductive System in Male Rats Tonic/Biostimulating Properties Immunomodulating Properties Neuronal Function Properties 261 262 262 270 270 272 273 274 275 277 Antidepressant Activities Antihypertensive Activity Insulin-Like Activities Wound Healing and Skin Improving Properties Properties Against Rheumatoid Arthritis Cytotoxic Activities Protective Activities Properties in Dentistry Allergic Reactions and Hypersensitivity Concluding Remarks References 279 279 280 281 282 283 283 283 283 284 286 278 INTRODUCTION Royal jelly (RJ) is a milky-white to yellowish creamy and acidic material with a slightly pungent odor and taste [1,2], which is a secretion of the mandibular and hypopharyngeal glands of young worker nurse honeybees (Apis mellifera Hymenoptera, Apidae). It is an essential food for feeding all young larvae and temporarily (up to and no more than 3 days) to the brood of workers and drones, but it is a sole food of the queen bee for both her larval and adult life [3]. The significant role of RJ is to provide both nutrition and protection for Studies in Natural Products Chemistry, Vol. 43. http://dx.doi.org/10.1016/B978-0-444-63430-6.00008-4 © 2014 Elsevier B.V. All rights reserved. 261 262 Studies in Natural Products Chemistry fast-developing honeybee larvae [4,5] and is also the key driving force in honeybee caste determination. A fertile egg develops into either a sexually perfect future queen bee with fully mature ovaries for reproduction and a longer life span or a sexually immature worker depending strictly on the time and amount of RJ intake during their larval development [6]. Moreover, RJ possesses high nutritional values due to the abundant amounts of proteins (amounting up to 50% of its dry weight) [7,8], free amino acids, lipids, vitamins, and sugars. In the framework of our scientific studies for RJ [9–15], we report in this chapter a detailed bibliographic investigation on the chemistry and biological properties of RJ, throughout the international literature. Chemical Constituents Identified in RJ Chemically, fresh RJ comprises water (50–70%), proteins (9–18%), carbohydrates (7–18%), fatty acids and lipids (3–8%), mineral salts (ca. 1.5%), and small amounts of polyphenols and vitamins. The lyophilized product contains <5% of water, 27–41% of proteins, 22–31% of carbohydrates, and 15–30% of fats [16,17]. Fatty Acids The fatty acid components constitute a unique and, from several aspects, very interesting characteristic of RJ. About 80–90% (on dry weight) of the fatty fraction of RJ is constituted by free fatty acids with exceptionally rare and unusual structures (Fig. 1). Contrary to carboxylic acids with 14–20 carbon atoms that are usually found in animal or plant materials, RJ contains mainly small chain (8 up to 12 carbon atoms) hydroxy fatty acids (Table 1), dicarboxylic acids (Fig. 2), monohydroxyacids, and derivatives (Fig. 3) and dihydroxyacids (Fig. 4). The main fatty acid of RJ is trans-10-hydroxy-2-decenoic acid (1) and is followed by the saturated derivative 10-hydroxydecanoic acid (2) (Fig. 1). It is noteworthy that fatty acid 1 has never been detected in any other natural raw material or even in other bee products [9,10,16–24]. It should be noted that many fatty acids have been identified only with GC or GC–MS and without NMR data (except those reported in [10]). More specifically for many dihydroxyacids, their identification is rather tentative OH HO O Trans 10-hydroxy-2-decenoic acid (1 1) (Weaver et al., [18], Lercker et al., [19]) FIGURE 1 The main fatty acids of RJ. OH HO O 10-Hydroxydecanoic acid (2 2) (Weaver et al., [18], Lercker et al., [19]) TABLE 1 Fatty Acids that Have Been Identified in RJ Name Refs Name Refs Butyric acid, octanoic acid, oleic acid, succinic acid, octadecanoic acid, eicosanoic acid, tetracosanoic acid, hexadecanoic acid, 7-oxooctanoic acid, 3-methyl-3-hydroxyglutaric acid, 9,10dihydroxy-2-decenoic acid, 9,10-dihydroxydodecanoic acid, 9-oxo-2-decenoic acid (9-ODA), 11,12dihydroxy-2-dodecenoic acid, 13-hydroxy-2-tetradecenoic acid, 2-hydroxyoctanoic acid, 3-methyl-3-hydroxyglutaric acid, 14-hydroxytetradecanoic acid [16] 5,10-Dihydroxydecanoic acid, 8,9-dihydroxydecanoic acid, 3-hydroxydecanedioic acid, 1,12-dodecanedioic acid, 7-hydroxy-2-octenoic acid, 3,10dihydroxydodecanoic acid, 8-hydroxy-2-octenoic acid, 2-octene-1,8-dioic acid, 8-hydroxy-2-decenoic acid, (Z)-9-hydroxy-2-decenoic acid, (Z)-9-HDA, 3,9dihydroxydecanoic acid, 8,9-dihydroxydecanoic acid, 10-hydroxy-2-dodecenoic acid, 11-hydroxy-2-dodecenoic acid, 3-hydroxydecanedioic acid, 1,12-dodecanedioic acid, 10,12dihydroxydodecanoic acid [16,17] 9-Hydroxynonanoic acid, 3-hydroxyundecanoic acid, 5,10-dihydroxydecanoic acid [18,19] 12-Hydroxydodecanoic acid, 12-hydroxy-2-dodecenoic acid, 3-hydroxyoctanoic acid, 13-hydroxytetradecanoic acid, 2-dodecene-1,12-dioic (traumatic) acid, 10-hydroxydodecanoic acid, 12-hydroxydodecanoic acid, 12-hydroxy-2-dodecenoic acid, 11-hydroxyundecanoic acid [16,17,20] Hexanedioic acid, nonanedioic acid, eptanedioic acid [18] 6-Hydroxydecanoic acid [19] Suberic acid (octanedioic acid) [16,18] 7-Hydroxyoctanoic acid [16–19] (E)-9-Hydroxy-2-decenoic acid, (E)-9-HDA, 9-hydroxydecanoic acid [16,17,19] 10,11-Dihydroxydodecanoic acid [10,16,17,20] Continued TABLE 1 Fatty Acids that Have Been Identified in RJ—Cont’d Name Refs Name Refs 10-Hydroxydecanoic acid, 10-HDAA, (E)-10-hydroxy-2-decenoic acid, 10-HDA [10,16–20] 8-Hydroxyoctanoic acid, 3,12-dihydroxydodecanoic acid, 3,13-dihydroxytetradecenoic acid [10,16–18,20] Sebacic acid (decanedioic acid), 3,10-dihydroxydecanoic acid [10,16–19] 3,11-Dihydroxydodecanoic acid, 3-hydroxydecanoic acid, 3-HDA, 11,12-dihydroxydodecanoic acid, 3-hydroxydodecanedioic acid, 11-hydroxydodecanoic acid [10,16,17] 2-Decene-1,10-dioic acid [16–18,20] 10-Acetoxy-2-decenoic acid, 10-acetoxydecanoic acid, 3-hydroxydecanoic acid methyl ester, 11-oxododecanoic acid [10] Chapter 8 OH O 265 Chemistry and Bioactivities of Royal Jelly O O O OH OH Hexanedioic acid (3 3) OH Heptanedioic acid (4 4) (Weaver et al., [18]) OH O O O O OH OH Octanedioic acid (5 5) (Weaver et al., [18]) OH Nonanedioic acid (6 6) (Weaver et al., [18]) OH OH O O O O OH OH Sebacic acid (7) (Weaver et al., [18], Lercker et al., [19]) 8) Trans 2-decenedioic acid (8 (Weaver et al., [18]) OH O O OH OH 3-Hydroxydodecanedioic acid (Melliou and Chinou [10]) FIGURE 2 Dicarboxylic acids. and in some cases doubtful. Other aliphatic compounds mainly reported by Isidorov et al. [17] are the following: Aliphatic alcohols: 2,3-butanediol, 2,3-butanediol, meso-glycerol, 1-hexa decanol, and 1-tetracosano. Aliphatic aldehydes: 3-methylbutanal, 2-methylbutanal, 2-methyl-2-butenal, and hexanal Aliphatic ketones: 2-pentanone, 2-heptanone, 2-nonanone, and 9-hydroxy2-nonanone [10] Lactones: d-decalactone and d-octalactone [10] Hydrocarbons: n-pentacosane, n-heptacosane, n-nonacosane, 9-hentriacontene, 7-hentriacontene, n-hentriacontane, and 9-tritriacontene Many aromatic components (Fig. 5) have also been identified in RJ. The following are the other aromatic components that were mainly reported as traces by Isidorov [16,17]: pyrocatechol, hydroquinone, 2-methoxy-p-cresol, methyl salicylate, methyl benzoate, benzaldehyde, phenol, 2-methoxyphenol, toluene, benzoic acid, 4-hydroxyhydrocinnamic acid, 266 Studies in Natural Products Chemistry O HO OH OH OH O 8-Hydroxyoctanoic acid (9 9) (Weaver et al., [18], Lercker et al., [19] HO 7-Hydroxyoctanoic acid (10) OH O O OH OH 9-Hydroxynonanoic acid (1 11) 6-Hydroxydecanoic acid (11) O OH OH OH OH O 9-Hydroxydecanoic acid (12) (Lercker et al., [19] 9-Hydroxy-2-decenoic acid (14) (Lercker et al., [19], Brown and Felauer, [21]) O OH HO O OH OH 3-Hydroxyundecanoic acid (13) (Weaver et al., [18], Lercker et al., [19] 11-Hydroxyundecanoic acid (15) O O OH O OCH3 OH 11-oxododecanoic acid (Melliou and Chinou [10]) 3-Hydroxydecanoic acid methyl ester (Melliou and Chinou [10]) O O OH CH3 OH 11(S)-Hydroxydodecanoic acid (Melliou and Chinou [10]) O 10-Acetoxy-2-decenoic acid (Melliou and Chinou [10]) O OH O CH3 O 10-Acetoxy-decanoic acid O OH (Melliou and Chinou [10]) FIGURE 3 Monohydroxyacids and derivatives. 4-hydroxy-3-methoxyphenylethanol, p-coumaric acid, caffeic acid, and nicotinic acid. It is also noteworthy that methylparaben that is widely used as a synthetic food preservative has been found as a natural constituent of RJ [10]. Sterols 24-Methylenecholesterol constitutes the most important sterol of RJ with the percentage 49–58% of total sterols. This is followed by b-sitosterol Chapter 8 267 Chemistry and Bioactivities of Royal Jelly Dihydroxyacids (Weaver et al., [18], Lercker et al., [19]) O HO OH O HO OH OH OH 5,10-Dihydroxydecanoic acid (17) 3,10-Dihydroxydecanoic acid (16) O HO OH OH 3,12-Dihydroxydodecanoic acid (18) O OH OH OH 3,13-Dihydroxytetradecanoic acid (19) OH O OH OH 10(R),11(R)-dihydroxydodecanoic acid (Melliou and Chinou [10]) O HO OH OH 11(R),12-dihydroxydodecanoic acid (Melliou and Chinou [10]) O OH OH OH 3,11-Dihydroxydodecanoic acid (Melliou and Chinou [10]) FIGURE 4 Dihydroxyacids. (19–24% of total sterols), isofucosterol (9–16% of total sterols), campesterol (6–7% of total sterols), and desmosterol (0.5–4.5% of total sterols) [25]. Remarkable is the existence of testosterone in RJ even in very small concentrations in the order of 11–12 ng/g [26]. Vitamins The content of RJ in vitamins has been the subject of several studies, since the first study [27] showed that it is exceptionally rich in vitamins. In the Table 2, the results appear with regard to the content of RJ in water-soluble vitamins [28]. In other reports [29], the content in vitamins is near the minimal values of Table 2. Also, traces of vitamin C have been found, while it should be noted that vitamins A, D, and K are completely absent. Pterins 1 g fresh RJ contains an average of 25 mg biopterin and 3 mg of neopterin [30]. 268 Studies in Natural Products Chemistry OH OH O OH OH p-Hydroxybenzoic acid (20) (Brown et al., [21], Barbier et al., [22]; Isidorov et al., [17]) OH O p-Hydroxyphenylacetic acid (21) (Weaver et al., [18]) OH OCH3 COOCH3 COCH3 Methyl paraben (Melliou and Chinou [10]; Isidorov et al., [17]) Acetovanillone (Melliou and Chinou [10]) OH OH OCH3 CH2CH2COOH COCH3 p-Hydroxyacetophenone (Melliou and Chinou [10]) 3-(4-Hydroxy-3-methoxyphenyl)propionic acid (Melliou and Chinou [10]) FIGURE 5 Aromatic components [21–24]. TABLE 2 Vitamin Content Vitamins mg/gr of RJ Thiamin 1.44–6.70 Riboflavin 5.0–25 Pyridoxine 1.0–48.0 Niacin 48–88 Pantothenic acid 159–265 Viotin 1.1–19.8 Folic acid 0.130–0.530 Inositol 80–350 Chapter 8 269 Chemistry and Bioactivities of Royal Jelly Sugars The main sugars that are contained in RJ are mainly fructose and glucose in relatively constant proportion similar with that of honey, with fructose as the prevailing sugar. In many cases, fructose and glucose calculated together constitute 90% of total sugars. The sucrose content varies considerably from sample to sample. Other sugars that are detected in much smaller quantities are maltose, trehalose, melibiose, ribose, and erlose [24,31,32]. A more detailed analysis of the contained sugars has been performed by Isidorov [17,20] and the following sugars or related derivatives have been identified: arabinofuranose, arabinitol, ribitol, methyl a-fructopyranoside, a-sorbofuranose, methyl b-fructofuranoside, pinitol, a- and b-fructofuranose, inositol, b-fructopyranose, b-glucofuranose, gluconic acid, g-lactone, a- and b-glucopyranose, a- and b-galactopyranose, mannitol, sorbitol, glucitol, chiro-inositol, myo-inositol, gluconic acid, 2-(acetamido)-2-deoxygalactopyranose, 2-(acetamido)-2-deoxyglucopyranose, a-lactulose, sucrose, a- and b-maltose, a-cellobiose, a- and b-maltulose, turanose, trehalose, b-palatinose, leucrose, a-cellobiose, a-isomaltose, gentibiose, b-isomaltose, raffinose, 1-kestose, erlose, melezitose, and maltotriose. Although there are several studies about the free amino acids in RJ (Table 3) [9], very recently, it was reported by Isidorov [17] that only proline could be detected and that the rest of the amino acids reported previously in literature are coming from larvae and not from RJ per se. Miscellaneous [17] Lactic acid, glycolic acid, glyceric acid, malic acid, erythritol, isothreonic acid, threonic acid, arabinoic acid, g-lactone, ribonic acid, g-lactone, quinic acid, urea, adenosine (adenine riboside), and guanosine. TABLE 3 Amino Acids Amino Acids mg/kg Amino Acids mg/Kg Alanine 0.08 Lysine 3.72 Arginine 0.59 Phenylalanine 0.06 Aspartic acid 0.26 Proline 8.00 Glutamic acid 1.00 Serine 0.13 Glycine 0.10 Threonine 0.04 Histidine 0.23 Tyrosine 0.04 Isoleucine 0.05 Valine 0.06 Leucine 0.05 Lysine 3.72 270 Studies in Natural Products Chemistry Metals The total content in ash in fresh RJ is roughly 1% and around 2–3% in the lyophilized RJ. The main metals that have been detected are K, Ca, Na, Zn, Fe, Cu, and Mn [9] with K having the highest concentration. Proteins The proteins constitute an important part of RJ components. It is calculated that 50% on dry weight of RJ corresponds in proteins [33,34]. The majority of proteins (MPJPs) of the RJ belong in a wider family, of which nine members with molecular weights from 49 to 87 kDa have been identified up to today. The proteins MRJP1, MRJP2, MRJP3 (that exist in five isomers), MRJP4, and MRJP5 represent about 82% of total content of proteins that is contained in the RJ [8,35–37]. The MRJP1 has molecular weight 57 kDa as monomer or 350 kDa as hexamer. The MRJP3 is a glycoprotein (70 kDa) that has been isolated from RJ. With the exception of the yellow protein of Drosophila melanogaster, which is related with the polymerization of precursors of forms of melanin, the proteins MRJPs have not been reported in non-insect species [37]. Also, it is generally acceptable that the particular proteins that exist in the royal jelly constitute source of nitrogen and essential amino acids for the larvae bees. Up to today, however, there have not been reported data in the bibliography that would prove the implication of these proteins in the process of growth of larva. Also, Fujiwara et al. in 1990 [38] found a protein in the RJ which had molecular weight around 55 KDa, which they named “royalisin.” BIOLOGICAL PROPERTIES RJ has been demonstrated to possess several pharmacological activities in vitro, in vivo in experimental animals, and through clinical studies, including vasodilative and hypotensive activities, increase of growth rate, and antitumor, antimicrobial, antihypercholesterolemic, and anti-inflammatory activities. Moreover, recently, immunomodulatory activities and estrogen-like effects have been published [9,10], while its potential therapeutic value in inhibiting joint destruction in rheumatoid arthritis has been also proved. Earlier biological investigations that have been assured by recent studies have shown that RJ has an insulin-like activity. Therefore, RJ has been widely used in commercial medical products, health foods, and cosmetics in many countries for more than 35 years [39]. Antimicrobial Activities A peptide fraction was isolated from RJ, where the N-terminal amino acid sequence of the major peptide within the fraction was V–T–C–D–L–L–S–F–K–G. Chapter 8 Chemistry and Bioactivities of Royal Jelly 271 This sequence corresponded to the honeybee defensin royalisin of MW 5523 Da that has been shown to exert antibacterial activity against some Gram-positive bacteria. Diffusion tests on agar plates showed that the peptide fraction had an inhibitory effect against the honeybee pathogen Paenibacillus larvae subsp. larvae, the primary pathogen of American foulbrood disease, as well as against other Gram-positive bacteria such as Bacillus subtilis and Sarcina lutea. Moreover, the peptide fraction was shown also to possess antifungal effect against the model fungus Botrytis cinerea [40]. Peptides isolated from natural fonts have been the object of several studies aimed at finding new molecules possessing antibacterial activity. In a recent study on peptides, originally isolated from RJ, the jelleins, and some analogs having a UV reporter at the N- or C-terminus, it was find out that they are mainly active against Gram-positive bacteria; interestingly, they act in synergy with peptides belonging to the family of temporins such as temporin A and temporin B against Staphylococcus aureus A170 and Listeria monocytogenes [41]. The antimicrobial activity of RJ’s peptide defensin 1 has been also published in another study where it was determined that it is able to inhibit in vitro growth of the pathogen causing American foulbrood (AFB) (P. larvae subsp. larvae) [42]. Greek RJ extracts of different polarities and their isolated compounds were studied for their antimicrobial activity against six Gram-negative and Grampositive bacterial strains, two oral pathogens (S. mutans and S. viridans), and three human-pathogen fungi (C. albicans, C. tropicalis, and C. glabrata). The results of these tests showed interesting and promising antimicrobial activity as some of the isolated compounds such as 3-hydroxydodecanedioic acid exhibited very strong antimicrobial activity against all assayed microorganisms (MIC 0.17–0.36 mg/mL). Methylparaben and 9-hydroxy-2-nonanone showed specific activity against S. aureus and S. epidermidis, while the higher activity against all assayed fungi was exhibited by sebacic acid (MIC 0.15–0.20 mg/mL). It was noteworthy that both methanolic and dichloromethane extracts showed distinguished antimicrobial spectrum of activity [9,10]. RJ has been also studied for its in vitro and in vivo antimycotic activity against dermatophytes. The results of the in vivo studies showed an antimycotic activity against Epidermophyton floccosum, Microsporum canis, and Microsporum gypseum; while all tested strains of Trichophyton sp. appeared resistant even with the maximum assayed concentration [43]. Several honeybee products including RJ have been evaluated for their ability to inhibit the growth of 40 yeast strains of Candida albicans, Candida glabrata, Candida krusei, and Trichosporon spp. using the broth microdilution method. MIC values for RJ and fluconazole (used as standard) were 0.06–1 and 0.02–96 mg/mL, respectively, demonstrating that RJ could potentially control some fluconazole-resistant fungal strains [44]. 10-hydroxy-2-decenoic acid (10-HDA), main component of RJ, is well known for its antimicrobial 272 Studies in Natural Products Chemistry properties [9,10]. Streptococcus mutans associated with pathogenesis of oral cavity, gingivoperiodontal diseases, and bacteremia following dental manipulations, while the genes that encode glucosyltransferases (gtfs) especially gtfB and gtfC are important in S. mutans colonization and pathogenesis. In a recent study, it was proved that 10-HDA prevents gtfB and gtfC expression efficiently in the bactericide subconcentrations and it could effectively reduce S. mutans adherence to the cell surfaces [45]. Antioxidative Activity The in vitro antioxidative effects of some RJs have been evaluated using a lipid peroxidation model, showing that they were not so strong [9] and somehow independent by heat treatment contrary to the activities of honey [46]. In another experiment, RJ was collected from larvae of different ages that were transferred in artificial queen bee cells for 24, 48, and 72 h. RJ harvested from the 1-day-old larvae 24 h after the graft displayed predominant antioxidant properties, including scavenging activity of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and inhibition of linoleic acid peroxidation. Regardless of the initial larval age, lower antioxidant activities were observed in the RJ harvested later than 24 h. In addition, higher contents of polyphenolic compounds were determined in the RJ harvested 24 h than that harvested 48 or 72 h after the graft, implying that the polyphenolic compounds may be the major component for giving the antioxidant activities in RJ, showing that the time of harvest and the initial larval age did affect strongly the antioxidant potencies in RJ [1]. In a recent study, the in vivo antioxidative activities of RJ were determined against radiation-induced oxidative stress in male albino rats, after the animals were exposed to a fractionated dose of gamma radiation. RJ was administrated (g/Kg/day) 14 days before exposure to the first radiation fraction and the treatment was continued for 15 days till the end of the study. Gamma-irradiation induced hematologic, biochemical, and histological effects in the animals while, in the RJ-treated irradiated group, significantly ameliorated the changes induced in serum lipid profile, as well as in all hematologic parameters. It was concluded that all previous amelioration observed in RJ treated rats might be due to the antioxidant capacity of RJ’s active constituents [47]. In a comparable in vivo experiment, the possible protective role of RJ on radiation-induced brain damage in male rats was studied, which were also irradiated with gamma radiation, and RJ was administered before and after irradiation per day for 7 days. It was resulted that an increase in brain tissue malondialdehyde (MDA) concentrations was detected, while significant decreases in catalase (CAT) and superoxide dismutase (SOD) activities in irradiation alone group were detected when compared to control group. Increases in MDA were relatively well prevented by RJ, while the administration of RJ increased activity of SOD and CAT enzymes and decreased MDA level in the brain tissue. Moreover, on histopathologic examination, RJ reduced edema, necrosis, Chapter 8 Chemistry and Bioactivities of Royal Jelly 273 vasodilation, and neuronal degeneration. The obtained results demonstrated once more that RJ-treated animals were protected from the damage in the brain during irradiation, relating again this beneficial effect of RJ to protection from oxidative injury [48]. Moreover, the in vivo protective effect of RJ on chronic lambda-cyhalothrin (LCT) (a synthetic pyrethroid insecticide) toxicity in mice was studied. The oral treatment of the animals with RJ significantly ameliorated the indices of hepatotoxicity, nephrotoxicity, lipid peroxidation, and genotoxicity induced by LCT in both doses of RJ (100 or 250 mg/kg of body weight) used that provided significant protection, while its strongest effect was observed at the dose of 250 mg/kg of body weight, suggesting that RJ is a potent antioxidant against LCT-induced toxicity, and its protective effect is dose-dependent [49]. The in vivo protective effect of RJ against cisplatin-induced renal oxidative stress in rats has been studied, showing that the administration of cisplatin (CP) to rats induced a marked renal failure, characterized with a significant increase in serum BUN and uric acid concentrations, and they had higher kidney MDA and lower GSH-Px, SOD, and CAT activities. In the groups that were administered RJ in association with CP, improvement was observed in some oxidative stress parameters and certain other biochemical parameters [50]. In another comparable in vivo study to 24 rats, the effects of RJ against oxidative stress caused by CP injury of the kidneys and liver were detected, by measuring tissue biochemical and antioxidant parameters and investigating apoptosis immunohistochemically. RJ elicited a significant protective effect towards the liver and kidney by decreasing the level of lipid peroxidation (MDA), concluding that RJ may be used in combination with cisplatin in chemotherapy to improve cisplatin-induced oxidative stress parameters and apoptotic activity [51]. The effect of RJ on CD3+, CD5+, CD45+ T-cell, and CD68+ cell distribution in the colon of rats with acetic acid-induced colitis was studied. This study has shown that RJ has anti-inflammatory, cell regeneration effect in the colon of rats with acetic acid-induced colitis, confirming its antioxidant potential [52]. The in vivo protective effect of RJ against the hematopoiesis dysfunction in X-irradiated mice has been also investigated. When RJ (1.0 g/kg, p.o., or 0.5 g/kg, i.p.) was administered every day beginning 2 weeks before X-irradiation (10 Gy), a significant increase in the number of leukocytes and erythrocytes was observed in mice treated with RJ, as compared with X-irradiated control. These results suggested that the protective effect of RJ against hematopoietic dysfunction could be expressed through an increase in the number of hematopoietic stem cells by the induction of hematopoietic factor such as GM-CSF and IL-3 [53]. Estrogenic Activity An experiment has been conducted to determine whether natural RJ paste administered orally or intramuscularly (i.m.) in conjunction with exogenous 274 Studies in Natural Products Chemistry progesterone is associated with improved reproductive responses in 30 ewes. The results demonstrated that RJ treatments in conjunction with exogenous progesterone were capable of improving estrus response and pregnancy rate [54]. Potential estrogenic activities of RJ were investigated using various approaches. RJ competed for binding of 17b-estradiol to the human estrogen receptor a and b, but its affinities were weak compared with diethylstilbestrol and phytoestrogens. The reporter gene expression assays suggested that 0.1–1 mg/ml RJ activated estrogen receptors, leading to enhanced transcription of a reporter gene through an estrogen-responsive element. These findings provide evidence that RJ possesses estrogenic activities through interaction with estrogen receptors followed by endogenous gene expressions [55]. In another study, four pure compounds isolated from RJ (10hydroxy-2-decenoic acid, 10-hydroxydecanoic acid, trans-2-decenoic acid, and 24-methylenecholesterol) are evaluated for their estrogenic activities by a ligand-binding assay for the estrogen receptor (ER) b. All of them inhibited binding of 17b-estradiol to ER-b, although more weakly than diethylstilbestrol or phytoestrogens. Expression assays suggested that these compounds activated ER, as evidenced by enhanced transcription of a reporter gene containing an estrogen-responsive element. Treatment of MCF-7 cells with these compounds enhanced their proliferation, but concomitant treatment with tamoxifen blocked this effect. Exposure of immature rats to these compounds by subcutaneous injection induced mild hypertrophy of the luminal epithelium of the uterus, but was not associated with an increase in uterine weight. These findings provide evidence that these compounds contribute to the estrogenic effect of RJ [56]. Furthermore, the possible effects of three RJ fatty acids (FAs) (10hydroxy-2-decenoic, 3,10-dihydroxydecanoic, and sebacic acids) on estrogen signaling were investigated in various cellular systems. The incoming data proposed a possible molecular mechanism for the estrogenic activities of RJ’s components that, although structurally entirely different from E2, mediate estrogen signaling, at least in part, by modulating the recruitment of ER-a, ER-b, and coactivators to target genes [13]. Moreover, the effectiveness of a herbal formula containing RJ (together with other natural components such as evening primrose oil, damiana, and ginseng) was evaluated in 120 women with menopausal symptoms. The outcome was measured by the Menopause Rating Scale II (MRS-II) and there was a statistically significant improvement in the MRS-II score after 2 and 4 weeks of treatment, concluding that RJ in this product may be beneficial and a safe natural promoter of health and well-being in women during the menopausal transition [57]. Activities in Reproductive System in Male Rats It is already referred that RJ contains naturally testosterone [26] and empirically that it improves male reproductive functions. That was the main reason that RJ was tested in vivo for its potential influence to the reproductive Chapter 8 Chemistry and Bioactivities of Royal Jelly 275 functions in male rats treated with nicotine, which has caused different degrees of testicular abnormalities. Testicular function was assessed in rats chronically treated with RJ (1 mg/g body wt/day) and/or nicotine at a concentration of 0.005% that simulate the intake of nicotine by heavy smokers. Nicotine administration significantly lowered serum acid phosphatase and testosterone values and caused chronic inflammation of testicular interstitium. The results showed a potential improvement for the treated with RJ animals, while further investigations are needed to clarify the hazards of different doses of RJ versus its benefits particularly when taken by heavy smokers [58]. The in vivo control of adverse effects of RJ on the reproductive system of puberty male rats was investigated, after daily administration of RJ to rats at doses 200, 400, and 800 mg/kg for 4 weeks. The dietary exposure to RJ did not affect body weight, but the organ coefficients for the pituitary and testis in the high-dose group were decreased significantly compared with the control group, and significant changes in the microstructure of the testis were also observed. No significant differences in sperm count were observed; however, the sperm deformity rate in the high-dose group increased significantly. In total, high-dose RJ oral administration adversely affected the reproductive system of the assayed rats, but the unfavorable effects were alleviated by cessation of administration [59]. Properties Against Osteoporosis As RJ contains testosterone [24] and possesses steroid hormone-type activities [54–57], it was hypothesized that it may have beneficial effects on osteoporosis. In a recent study, both an ovariectomized rat model and a tissue culture model were used. The results of the study indicated that RJ was almost as effective as 17b-estradiol in preventing the development of bone loss induced by ovariectomy in rats. In tissue culture models, RJ increased calcium contents in femoraldiaphyseal and femoral-metaphyseal tissue cultures obtained from normal male rats; however, in a mouse marrow culture model, it neither inhibited the parathyroid hormone (PTH)-induced calcium loss nor affected the formation of osteoclast-like cells induced by PTH. Therefore, these results suggested that RJ may prevent osteoporosis by enhancing intestinal calcium absorption, but not by directly antagonizing the action of PTH [60]. In a comparable study, it was investigated whether RJ and bee pollen reduce the bone loss due to osteoporosis in oophorectomized female rats model. It was concluded that RJ and bee pollen after a 12-week treatment decrease the bone loss due to osteoporosis, proposing that these results may contribute to the clinical practice [61]. Tonic/Biostimulating Properties RJ is widely used as a food additive as well as for its biostimulating effects. In order to prove this kind of bioactivities, a series of experiments have been 276 Studies in Natural Products Chemistry published. A stimulation of cell growth in the U-937 human myeloid cell line by RJ protein (DIII protein with molecular weight of 58 kDa) was obtained in an experiment, while the growth-stimulating activity of the DIII protein was shown to be relatively heat and pH stable [62]. In another study, it was represented that the larvae of the insect cerambycid beetle (Cerambyx cerdo) reared on an artificial diet containing RJ caused several metabolic changes accelerating its development process, increasing the number of molts, and decreasing the protease activity [63]. Moreover, the life span-extending effects of RJ and its related substances on the nematode Caenorhabditis elegans were determined. The results demonstrated that truly RJ and its substances extended life span in C. elegans, suggesting that RJ may contain longevity-promoting factors, while further analysis and characterization of the life span-extending agents in RJ are needed that may lead to the development of nutraceutical interventions in the aging process [64]. The in vivo antifatigue effect of fresh RJ in mice was recently investigated. The mice were accustomed to swimming in an adjustable-current swimming pool; they were subjected to forced swimming 5 during 2 weeks, and the total swimming period until exhaustion was measured. The swimming endurance of the RJ group significantly increased compared with the other groups. The mice in the RJ group showed significantly decreased accumulation of serum lactate and serum ammonia and decreased depletion of muscle glycogen after swimming compared with the other groups. These findings suggested that RJ could ameliorate the physical fatigue after exercise, and this antifatigue effect of RJ seemed to be associated with the freshness of RJ, possibly due to its content of 57 kDa protein [65,66]. In a clinical study, it was aimed to investigate the effects of different levels of RJ supplementation on biochemical parameters in male swimmers. All participants were trained by swimming totally 20 km in 2 h on 5 days a week for 4 weeks. A supplementation of 500 mg, 1 g/day, and 2 g/day of RJ throughout the 30-day exercise program was not significantly effective in the swimmers [67]. Moreover, it was recently reported of TRPA1 activation by fatty acids from RJ, proving that activation of TRPA1 and TRPV1 induces thermogenesis and energy expenditure enhancement. The activation of human TRPA1 and TRPV1 by RJ extracts was measured and found that the hexane extract contains TRPA1 agonists. The main functional compounds in the hexane extract were trans-10-hydroxy-2-decenoic acid (10-HDA) and 10-hydroxydecanoic acid (10-HDAA), most characteristic fatty acids of RJ, concluding that the main function of RJ is TRPA1 activation by 10-HDA and 10-HDAA [68]. The protective effect of RJ on immune dysfunction in aged mice was investigated after oral administration of RJ (1 g/kg) to mice daily for 1 month. The results showed that through the normalization of T-cell differentiation in the thymus by RJ, the ability to provide T cells to the periphery and the ability of T cells to produce cytokines and to induce systemic immune responses, mainly Th1 responsiveness, were recovered in aged mice [69]. Chapter 8 Chemistry and Bioactivities of Royal Jelly 277 Immunomodulating Properties RJ and especially its protein components have been shown to possess immunomodulatory activity. In order to study a possible in vivo immunomodulatory effect of RJ, an established rodent model in mice and rats fed with RJ before and after immunization with sheep red blood cells (SRBC) was used. The results indicated that RJ exhibited immunomodulatory properties by stimulating antibody production and immune-competent cell proliferation in mice or depressing humoral immune functions in rats. Both phenomena, though species-related in this model, could probably be reversed by changing the dose or the route of RJ application [70]. 10-hydroxy-2-decenoic acid (10-HDA), among the major fatty acids of RJ, was tested for its effects on T-cell functions in mice. The results showed that 10-HDA (1, 5, and 25 mg/kg, i.p. 5 days) significantly antagonized the inhibition of delayed type hypersensitivity (DTH) induced by cyclophosphamide, but it had no effect on DTH in normal mice. The mitogen concanavalin a that induced proliferation of T cell from the mouse spleen was increased by 50–100 mg/L 10-HDA in vitro, while its effect on the T subpopulation was studied by monoclonal antibody indirect immunofluorescence method. The results showed that 10-HDA enhanced the number of L3T4+ (T(H)) lymphocytes while in doses 25, 50, and 100 mg/L, with concanavalin a, it increased the IL-2 secretion from mouse splenocytes in a concentration-dependent manner, proving clearly that 10-HDA had an immunomodulatory effect on T-cell functions [71]. The in vivo immunomodulatory effects of RJ were further studied, investigating the suppression of allergic reactions by RJ in immunized mice after oral administration of RJ (1 g/kg). The results suggested that RJ suppressed antigen-specific IgE production and histamine release from mast cells in association with the restoration of macrophage function and improvement of Th1/Th2 cell responses in mice [72]. It has been studied previously using in vitro and in vivo experimental systems that Perilla frutescens leaf extract (PFE) exhibits antiallergic functions through inhibiting IgE production by way of downregulating the production of the Th2 cytokines IL-4, IL-5, and IL-10, which play important roles in allergic responses. Based on those tests, it was further examined whether RJ exhibits antiallergic functions similar to those of PFE using an immediate hypersensitivity model, in which mice were immunized with ovalbumin (OVA)–alum. The results of the study suggested that RJ exhibits antiallergic functions through a different mechanism from that of PFE [73]. In some recent studies, it was investigated the influence of RJ fatty acids on the immune system and especially the effect of 10-hydroxy-2-decenoic acid (10-HDA) and 3,10-dihydroxy-decanoic acid (3,10-DDA), isolated from RJ, on the immune response using a model of rat dendritic cells (DCs)-T-cell cocultures. Both fatty acids, at higher concentrations, inhibited the proliferation of allogeneic T cells, where the effect of 10-HDA was stronger and 278 Studies in Natural Products Chemistry was followed by a decrease in interleukin-2 (IL-2) production and downregulation of IL-2 receptor expression. The results confirmed the immunomodulatory activity of RJ fatty acids and suggested that DCs are a significant target of their action [11]. In another publication, the effect of an RJ extract and pure isolated compounds was studied using an in vitro rat T-cell proliferation assay. It was resulted the complexity of biological activity of RJ and suggested that its water extract possessed the most potent immunomodulatory activity in vitro [12]. Moreover, the effect of 3,10-DDA was tested on maturation and functions of human monocyte-derived dendritic cells (MoDCs). It was showed that 3,10-DDA stimulated maturation and Th1 polarizing capability of human MoDCs in vitro, which could be beneficial for antitumor and antiviral immune responses [14]. The efficacy of RJ as a potent in vivo immunomodulator in the restoration of alcoholic liver injury in mouse model was also determined. RJ modulated important immune phenomena on alcoholic liver injury. These findings provide evidence that RJ might have the capacity to restore the function of the immune system in individuals with alcoholic liver diseases [74]. The effect of 10-hydroxy-2-decenoic acid (10-HDA) from RJ on LPS-induced cytokine production was investigated in murine macrophage cell line RAW264 cells, where 10-HDA inhibited LPS-induced IL-6 production dose-dependently, but did not inhibit TNF-a production. 10-HDA inhibited LPS-induced NF-kB activation in a dose-dependent fashion. The results suggested that 10-HDA showed anti-inflammatory effects and could be a therapeutic drug candidate for inflammatory and autoimmune diseases associated with IkB-Χ and IL-6 production [75]. The in vivo influence of apitherapy diet on erythrocytes in wistar rats with experimentally CCL4-induced hepatotoxicity was determined, showing that an apitherapy diet and RJ in laboratory animals improved the hematologic parameter values to normal levels (number of erythrocytes, RBC; hemoglobin, HGB; hematocrit, HCT; mean corpuscular volume, MCV, VEM; mean corpuscular hemoglobin, MCH, HEM; mean corpuscular hemoglobin concentration, MCHC, CHEM; red cell distribution width, RDW) [76]. Neuronal Function Properties Trimethyltin (TMT) is a toxic organotin compound that induces acute neuronal death selectively in the hippocampal dentate gyrus (DG) followed by cognition impairment; however, the TMT-injured hippocampal DG itself is reported to regenerate the neuronal cell layer through rapid enhancement of neurogenesis. Neural stem/progenitor cells (NS/NPCs) are present in the adult hippocampal DG and generate neurons that can function for the cognition ability. It was investigated whether RJ stimulates the regenerating processes of the TMT-injured hippocampal DG and found that orally administered RJ significantly increased the number of DG granule cells and simultaneously improved the cognitive impairment. Furthermore, it was shown that RJ Chapter 8 Chemistry and Bioactivities of Royal Jelly 279 facilitated neurogenesis of cultured NS/NPCs. These results, taken together with previous observations, suggested that the orally administered RJ may be a promising avenue for ameliorating neuronal function by regenerating hippocampal granule cells that function in the cognition process [77]. The in vivo neuroprotective effect activity of RJ in male rabbits, on traumatic spinal cord injury (SCI), was studied. Significant improvement was observed in RJ treated group, 24 h after SCI, with respect to control, while RJ treatment mostly prevented lipid peroxidation and also augmented endogenous enzymatic or nonenzymatic antioxidative defense systems and RJ treatment significantly decreased the apoptotic cell number induced by SCI [78]. The pharmacological actions of RJ on the nervous system were examined showing that RJ may activate effectively neuronal functions [79]. Antidepressant Activities In vivo antidepressant-like activity of 10-hydroxy-2-decenoic acid (10-HDA) in stress-inducible depression-like mouse model was conducted. The animals were evaluated by the tail-suspension test, elevated plus-maze test, and openfield test at 1 day after the end of stress exposure. The results demonstrated that 10-HDA and RJ were effective in ameliorating the stress-inducible symptoms of depression and anxiety, intraperitoneally administered, while RJ given by the oral route was less effective [80]. Antihypertensive Activity In order to clarify the potential physiological function of RJ, the gastrointestinal enzyme production of antihypertensive peptides from RJ was studied, showing that protease N-treated RJ (ProRJ) and peptides from ProRJ (IleTyr (IY), Val-Tyr (VY), and Ile-Val-Tyr (IVY)) inhibited angiotensin I-converting enzyme (ACE) activity, and they have an antihypertensive effect in repeated oral administration for 28 days on spontaneously hypertensive rats (SHR). These results suggested that peptides contributed to the antihypertensive effect of ProRJ, which was computed to be about 38%. Therefore, it is considered that intake of RJ (and its peptides), as a functional food, would be beneficial for improving blood pressure in people with hypertension [81–84]. Effects of RJ on Serum Lipoprotein Metabolism In Vivo and in Humans RJ was studied in order to assess the size and consistency of its potential effect on serum lipids in experimental animals and humans. The data from animal studies were pooled, where possible, and statistically evaluated by Student’s t-test, while meta-analysis was used for the evaluation of human trials. It was found that RJ significantly decreased serum and liver total lipids 280 Studies in Natural Products Chemistry and cholesterol levels in rats and rabbits and also retarded the formation of atheromas in the aorta of rabbits fed a hyperlipidemic diet. Meta-analysis of the controlled human trials of RJ to reduce hyperlipidemia showed a significant reduction in total serum lipids and cholesterol levels and normalization of HDL and LDL as determined from decrease in b/a lipoproteins. The best available evidence suggested that RJ at approximately 50–100 mg/day decreased total serum cholesterol levels by about 14% and total serum lipids by about 10% in the group of patients studied [85]. In another study, the effect of RJ on the nicotine-induced atherogenic lipoprotein profile was evaluated. The changes in lipid and lipoprotein cholesterol, RJ (1 mg/g body weight/ day), and/or nicotine (0.05 mg/ml in drinking water) concentrations were tested in adult male rats, during 8 weeks. In total, the data demonstrated that RJ was a potential antiatherosclerotic agent capable of improving the nicotine-induced atherogenic lipoprotein profile [86]. Rat models with experimental hyperlipidemia were also fed with lyophilized RJ 700 mg/kg daily for 6 weeks. Results showed that lyophilized RJ could reduce cholesterol level and increase high-density lipoprotein cholesterol level. It could also increase the red cell deformability (RCD) and decrease plasma fibrinogen level. It could be suggested that RJ could be used in preventing and treating hyperlipidemia and improving highly coagulant status of blood [87]. The same results appeared when the pharmacological effect of pure 10-hydroxy-2-decenoic acid (10-HDA) from RJ was studied in the same in vivo animal model [88]. Very recently, the effects of RJ supplementation on serum lipoprotein metabolism in humans were examined, in 15 volunteers, which were divided into an RJ intake group (n ¼ 7) and a control group (n ¼ 8). The RJ group took 6 g/day for 4 weeks. Their serum total cholesterol (TC) and serum low-density lipoprotein (LDL) decreased significantly compared with those of the control group (p < 0.05). There were no significant differences in serum high-density lipoprotein (HDL) or triglyceride concentrations. The results suggested that dietary RJ decreases TC and LDL by lowering small VLDL levels [89]. Insulin-Like Activities An aqueous extract of RJ produced hypoglycemia when injected into larvae of Manduca sexta. The application of specific radioimmunoassay to the partially purified extract showed that RJ contains several insulin-like peptides, the major immunoreactive component of which had an apparent mol. wt similar to that of bovine insulin. These results suggested the existence of a peptide in the honeybee having both biological and structural similarities to vertebrate insulin [90]. Partially purified extracts from honeybees (Apis mellifera), and extracts from their separated heads, cross-reacted in a porcine insulin radioimmunoassay. The active extracts displaced porcine insulin from rat liver insulin receptors and showed insulin-like activity with rat adipocytes that could be abolished with bovine insulin antiserum. The presence of an insulin Chapter 8 Chemistry and Bioactivities of Royal Jelly 281 like-structure of close similarity to mammalian in RJ was demonstrated, as is also present in the head of the bees, indicating high evolutionary conservation of the molecule [91]. Contrary to the results of the previous studies, chronically diabetic rats prepared by a single i.v. injection of streptozotocin were used to study whether RJ possesses a hypoglycemic reaction, where orally RJ administration of 10, 100, and 1000 mg/kg/day did not show any insulinlike activity [92]. Due to the controversy of the conclusions of earlier biological investigations that have shown that RJ has insulin-like activity, however, there have so far been no clinical trials to support these findings. The potential effect of RJ ingestion on the glucose metabolism of 12 healthy humans was studied. They underwent the standardized oral glucose tolerance test (OGTT) and afterward a second OGTT after ingestion of 20 g of RJ. Serum glucose levels after 2 h and the area under the curve for glucose were significantly lower (P ¼ 0.041) after RJ administration. This insulin-like activity of RJ that seems to act even after passage through the human stomach could lead to the development of new interesting concepts in diabetology after further clinical appropriate trials [93]. Wound Healing and Skin Improving Properties Oral RJ administration of 10, 100, and 1000 mg/kg/day in chronically diabetic rats prepared by a single i.v. injection of streptozotocin was studied whether it could augment wound healing. RJ showed some anti-inflammatory activity by decreasing exudation and collagen formation in granulation tissue formation in the cotton pellet method. RJ also shortened the healing period of desquamated skin lesions. Thus, RJ possesses an anti-inflammatory action and is able to augment wound healing [92]. The effect of a cream containing propolis (1%) and RJ (0.5%) on 13 patients with sensitive skin of different etiologies was examined. After the application in the period of 2–4 weeks after usual therapy procedure, it was concluded that the effect was positive regardless the etiology of the skin sensitiveness [94]. In another in vivo study, it was shown that RJ increases collagen production by normal hamster fibroblasts in the presence of ascorbic acid (AA) or ascorbic acid 2-O-alpha-glucoside (AA-2G). The effects of a combination of RJ with AA-2G on collagen synthesis were much greater than those of the combination of RJ with AA. RJ showed significant inducing activity on fibroblasts in the presence of AA-2G. These results suggested a possible mechanism for TGF-b production induced by RJ and AA-2G, occurring in conjunction with collagen synthesis in normal hamster fibroblasts [95].The influence of 10-hydroxy-2-decenoic acid (10-HDA)from in RJ in correcting skin barrier dysfunction was also evaluated. The activity of Hydroxydecine®, its synthetic counterpart, was evaluated in vitro on the regulation of epidermal differentiation markers, ex vivo on the inflammatory response and restoration of skin barrier function, and in vivo on UV-induced xerosis in healthy human volunteers. Hydroxydecine® 282 Studies in Natural Products Chemistry thus proved its efficacy in activating keratinocyte differentiation processes in vitro, restoring skin barrier function and reducing inflammation ex vivo, and hydrating dry skin in vivo [96]. Pure apisin a 350 kDa, among the main glycoprotein of RJ, was isolated and investigated together with RJ, if they promote cell proliferation and the production of collagen in normal human neonatal skin fibroblasts, NB1 RGB cells. Moreover, it was investigated whether apisin promotes the differentiation of MC3T3-E1, a mouse osteoblastic cell line, with calcium and hydroxyapatite production. RJ and apisin induced proliferation and collagen production in NB1RGB cells and promoted the differentiation of MC3T3E1into osteoblasts. These findings suggested that RJ has proliferation- and differentiation-inducing functions partly due to apisin [97]. In another recent study, the hypopigmentary mechanism of RJ in a mouse melanocyte cell line, B16F1, was studied. Treatment of B16F1 cells with RJ markedly inhibited melanin biosynthesis in a dose-dependent manner. Since the key enzyme in the melanin synthetic pathway is tyrosinase, many depigmenting agents in the treatment of hyperpigmentation act as tyrosinase inhibitors. The results of the experiment suggested that RJ reduces melanin synthesis by downregulation of tyrosinase mRNA transcription and serves as a new candidate in the design of new skin-whitening or therapeutic agents [98]. Moreover, RJ studied in vitro and showed high protection against ultraviolet B-induced photoaging in human skin fibroblasts via enhancing collagen production. The effects of RJ and 10-HDA on UVB-induced photoaging were tested by measuring procollagen type I, transforming growth factor (TGF)-b1, and matrix metalloproteinase (MMP)-1 after UVB irradiation. The UVB-irradiated human skin fibroblasts treated with RJ and 10-HDA had increased procollagen type I and TGF-b1 productions. The protective effects of RJ ovariectomy-induced skin aging were examined in vivo by determining the same as previously factors (type I procollagen and (MMP)-1) in female rats. The level of procollagen type I protein was increased in the dorsal skin of ovariectomized rats fed with a dietary supplement containing 1% RJ extract, but the level of MMP-1 was not altered. In particular, the amount of collagen recovered was close to the normal level showing that RJ may protect against skin aging by enhancing collagen production in rats with ovariectomy-induced estrogen deficiency [99]. Properties Against Rheumatoid Arthritis Rheumatoid arthritis synovial fibroblasts (RASFs) are known to produce matrix metalloproteinases (MMPs) and cause joint destruction. 10-HDA tested how to inhibit the activities of MMPs: with RASFs isolated from rheumatoid tissues by enzymatic digestion, cultures in monolayers were treated with 10-HDA (0.5, 1, and 2 mM). The molecular investigation revealed that Chapter 8 Chemistry and Bioactivities of Royal Jelly 283 the 10-HDA-mediated suppression was likely to occur through blocking p38 kinase and c-Jun N-terminal kinase–AP-1 signaling pathways. The results suggested that 10-HDA may be of potential therapeutic value in inhibiting joint destruction in RA [100]. Cytotoxic Activities Older investigations have shown that whole RJ and 10-hydroxy-2-decenoic acid (10-HDA) as well as certain closely related dicarboxylic acids could inhibit the development of transplantable AKR leukemia and ascites tumors (Ehrlich ascites cells) when added in vitro just prior to transfer. This activity was associated mainly with 9- and 10-carbon straight-chain monocarboxylic acids, particularly decanoic acid in the ethyl ester form. These compounds showed no activity in vivo [101]. Protective Activities The physiological effects of 57 kDa protein (among the most important ones, from RJ) in primary cultured rat hepatocytes in the absence of serum have been investigated. The assayed 57 kDa protein and RJ stimulated significantly hepatocyte DNA synthesis. Therefore, 57 kDa protein is likely to promote liver regeneration and may have a cytoprotective action on hepatocytes [102]. Protective effects of RJ on the cecal ligation and puncture (CLP)induced sepsis in X-ray-irradiated mice were also investigated and were approached through the augmentation of phagocytic activity of intestinal macrophages [53,103]. Properties in Dentistry It was hypothesized recently that RJ could have beneficial effects on the prevention or treatment of periodontal diseases, which are chronic inflammatory diseases, caused by bacterial infection that results in resorption of the toothsupporting bone. After appropriate experiments, it was suggested that the osteoinductive and anti-inflammatory effects of RJ could provide valuable benefits for the treatment and prevention of periodontal diseases [104]. Allergic Reactions and Hypersensitivity Several cases of allergic reactions and hypersensitivity through intake of RJ have been reported in the community since 1983. A woman who had ingested RJ showed an exacerbation of dermatitis when it was applied to her feet [105]. In another case report, it was the first described case of IgE anaphylactic reaction due to RJ to a 15-year-old atopic woman who presented 15 min after the intake of RJ, local angioedema, generalized urticaria, dysphonia, and 284 Studies in Natural Products Chemistry bronchospasm. She was given antihistaminic and corticosteroids and responded well, while prick test to common food allergens hymenoptera venoms and pollens was negative [106]. In another study, asthma, together with anaphylaxis, was observed in seven subjects following ingestion of RJ, used as a health tonic. A total of 18 different IgE-binding components were detected on blots following electrophoretic separation of RJ. The clinical significance of the antibodies found in the sera of control subjects is not known, but it may arise in response to common inhalant allergens that show allergenic cross-reactivity with RJ [107]. In a cross-sectional survey (clinical study) conducted to determine the prevalence and the relationship between RJ consumption and hypersensitivity reactions among 1472 hospital employees at Hong Kong, it was showed that atopic individuals are at high risk of sensitization to RJ but the precise relationship between RJ’s use, positive RJ skin test, and clinical manifestations of adverse reactions to RJ remains to be further defined [108]. Another case report was also published in 1997, as the first reported case of hemorrhagic colitis associated with RJ intake. A 53-year-old woman with abdominal pain and bloody diarrhea has been described. Prior to the onset of symptoms the patient had taken RJ for 25 days. Colonoscopy revealed that the mucosa was hemorrhagic and edematous throughout the 20 cm long sigmoid colon. Platelet aggregation in 30% of capillaries in the mucosal lesions was also revealed. Moreover, the drug-induced lymphocyte stimulation test was slightly positive for RJ compared with the control. The patient’s signs and symptoms disappeared within a few days as well as the colonic lesions after 2 weeks of conservative therapy [109]. Another two cases of severe systemic reactions (anaphylaxis and generalized urticaria/angioedema) due to honey and RJ ingestion were published, in patients sensitized to Compositae (Asteraceae) plants and especially to mugwort (Artemisia vulgaris). Both patients had a skin and RAST positivity to mugwort and a positive prick by prick to the offending foods. Both the clinical data and the laboratory analyses supported the hypothesis of a strict link between sensitization to Compositae (Asteraceae) plants and adverse reactions to honey and RJ [110]. Very recently, two young women suffered from allergic symptoms after the intake of RJ at their first time, and according to the positive skinprick test reactions of raw RJ, RJ allergy was diagnosed. As the symptoms appeared at the time of their first RJ intake, it was speculated that there was potential mechanism of cross-reaction of RJ with pollens [111], as previously reported [110]. CONCLUDING REMARKS RJ has been used worldwide for many years in medical products, health foods, and cosmetics in various parts of the world for its pharmacological properties. Different pharmacologically active components have been isolated from this natural source possessing numerous functions. RJ is traditionally thought Chapter 8 Chemistry and Bioactivities of Royal Jelly 285 and in the recent years was scientifically proved to improve menopausal symptoms and has been used as a general tonic to prevent the damage of aging and to reduce lesions from chronic degenerative diseases, as the maintenance of health, immune potentiation, and age retardation, and even as effective and safe skin-whitening agent. RJ possesses the ability to prevent osteoporosis in rats and anti-inflammatory effects; antioxidative, neuroprotective, antimicrobial, and cytotoxic properties; and immunomodulatory activity (mainly due to its protein components). As it contains natural testosterone, it is also widely used to improve male reproductive functions. In conclusion, RJ attracted scientific interest since 1852 when the first scientific studies for RJ were published and it still remains “hot” natural product. Numerous extremely interesting scientific results that appear in the international literature every year from scientific groups all over the world show that it will be kept under investigation for many more years in the future. ABBREVIATIONS 10-HDA 10-HDAA 3,10-DDA AA AA-2G ACE AFB CAT CLP CP DCs DG DPPH DTH ER FAs gtfs HDL i.m. IL-2 IY LCT LDL MDA MMP MoDCs 10-hydroxy-2-decenoic acid 10-hydroxydecanoic acid 3,10-dihydroxy-decanoic acid ascorbic acid ascorbic acid 2-O-alpha-glucoside angiotensin I-converting enzyme American Foulbrood catalase cecal ligation and puncture cisplatin dendritic cells dentate gyrus 1,1-diphenyl-2-picrylhydrazyl delayed type hypersensitivity estrogen receptor fatty acids glucosyltransferases high-density lipoprotein intramuscularly interleukin-2 Ile-Tyr lamda-cyhalothrin low-density lipoprotein malondialdehyde matrix matalloproteinase monocyte-derived dendritic cells 286 MRS NS/NPCs OGTT OVA PFE ProRJ PTH RA RASFs RCD RJ SCI SHR SOD SRBC TC TGF TMT VY VY Studies in Natural Products Chemistry menopause rating scale Neural stem/progenitor cells oral glucosetolerance test ovalbumin perilla frutescens leaf extract protease N-treated RJ parathyroid hormon rheumatoid arthritis rheumatoid arthritis synovial fibroblasts red cell deformability royal jelly spinal cord injury spontaneously hypertensive rats superoxide dismutase sheep red blood cells total cholesterol transforming growth factor Trimethyltin Ile-Val-Tyr Val-Tyr REFERENCES [1] J.R. 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Allergol. 60 (2011) 708–713. Chapter 9 Synthetic Cannabinoids: Synthesis and Biological Activities Joel Schlatter Laboratory of Forensic Toxicology, Department of Biology, University Hospital of Jean Verdier—APHP, Bondy, France Chapter Outline Introduction Distribution of Synthetic Cannabinoids Legality and Regulation Synthesis of Synthetic Cannabinoids 291 292 293 294 Biological Activities of Synthetic Cannabinoids 295 Epidemiological Data 305 Current Medicinal Purposes 305 Discussion and Conclusion 307 References 309 INTRODUCTION Synthetic cannabinoid products (SCPs), marketed as “Spice,” “K2,” and others, have been sold in retail outlets and via the Internet as early as 2004. These products are marketed as herbal incense; however, when smoked, they produce psychoactive effects similar to cannabis. Spice first started appearing on internet sites and in specialized shops under multifarious names such that Spice Gold, Spice Silver, Spice Diamond, Yucatan Makes, Yucatan Fire, Sence, Chill X, Smoke, Genie, Shunk, Algerian Blend [1]. Warning messages on the product stating it was not intended for human consumption contrasted with sophisticated packaging and marketing, promoting the product as a cannabis alternative which was undetectable by automated drug testing methodology. Synthetic cannabinoids can be easily detected by conventional drug testing methodology such as GC–MS analysis after solvent extraction. But, until 2010, there were little literatures and also there was not any commercial or free library containing the spectra of synthetic cannabinoids. With this reason, most of the forensic laboratories cannot confirm the suspicious Studies in Natural Products Chemistry, Vol. 43. http://dx.doi.org/10.1016/B978-0-444-63430-6.00009-6 © 2014 Elsevier B.V. All rights reserved. 291 292 Studies in Natural Products Chemistry compounds found in the chromatogram of the herbal mixtures. It was not until December 2008 that researchers reported Spice had been “laced” with undeclared synthetic cannabinoids JWH-018 and CP 47,497 homologue [2]. Biochemical analyses have revealed that these psychoactive properties are not because of the herbal ingredients listed on product labels, but really the addition of synthetic cannabinoids [1,2]. These are groups of structurally diverse molecules with functional similarity to tetrahydrocannabinol (THC), the psychoactive compound in cannabis [3]. These products have become popular among adolescent users because of their cannabis-like effects, easy accessibility via Internet sites or head shops (specialty shops that sell drug paraphernalia) [2]. Since being identified in “herbal” products, JWH-018 and CP 47,497 have been banned in a number of European countries and some American States [4]. Since 2008, the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) tries to control the exportations and consumptions of Spice in the European Union. EMCDDA is focusing its efforts to identify the active composites of Spice and to submit analytical methods [5]. Promptly, second generation products appeared on the market, suggesting the manufacturers had anticipated prohibition and had already synthesized an array of alternatives [6]. Since 2012, identification of newly distributed designer drugs have been reported that include synthetic cannabinoids as QUBIC, ADBICA, ADB-FUBINACA, APICA series, UR series, and others [7–9]. Currently “headshops” and the internet offer an expanding array of synthetic cannabinoids originating from various chemically distinct groups including more than 400 active molecules [3,6,10–12]. This rapid proliferation of SCPs over the past 4 years has been labeled the “Spice phenomenon” which accounted for a case study of how existing models of drug control and response are being challenged by globalization, internet technology, and innovation in the drug market [3,13]. Increasing emergency room visits and poison control center calls has led and (DEA) after U.S. Drug Enforcement Agency to exercise permanently scheduling authority to designate the most common chemicals found in synthetic cannabinoids (JWH-018, JWH-073, JWH-200, CP 47,497) as Schedule I drugs for the next year to “avoid an imminent threat to public safety” [14–16]. A recent CESAR FAX states that poison control center calls have recently decreased [17]. In light of this development, and because paucity of literature describing the psychoactive and physical effects of synthetic cannabinoid use, a greater understanding of these products is important for the development of public policy and the identification and treatment of persons who use synthetic cannabinoids. DISTRIBUTION OF SYNTHETIC CANNABINOIDS Spice represents a relatively new type of “designer drug” that has recently emerged on the recreational drug use market. It has been suggested that some Spice may have been manufactured in China, but it remains unclear where Chapter 9 Synthetic Cannabinoids: Synthesis and Biological Activities 293 TABLE 1 Vegetable Ingredients in Spice [3,19] Name of the Ingredients Specie Origin Bay bean Canavalia maritima The local people of Gulf of Mexico smoked sheets to replace marijuana. Water lily Nymphaea alba Water lotus with blue flowers. Skullcap Scutellaria nana Herb native to California, well-known American tribes (Cherokee) Indian Warrior Pedicularis densiflora Herb traditionally used by Indian tribes of North America. The buds and flowers are often smoked their relaxing properties. Lion’s tail Leonotis leonurus Long grass with yellow, orange, and red tubular flowers, evoking the tail of a lion. It is traditionally used by the Xhosa and Hottentot tribes of South Africa. The leaves were smoked for its euphoric effects. Maconha brava Zornia latifolia Native to tropical South America to northern Caribbean plant. The dried leaves are traditionally used by Indian tribes. Pink lotus Nelumbo nucifera Flower of India. Siberian Motherwort Leonurus sibiricus Herb commonly used in Brazil, Mexico, and China where it is highly valued for its psychedelic properties. and how the production of these herbal mixtures actually takes place [3,18]. Package information typically lists plant ingredients considered inert (white and blue water lily, blue and pink lotus, etc.), and less frequently lists plants that naturally contain potentially psychoactive alkaloids (Table 1) [1,3,19]. Cannabinoid constituents and dosages can vary greatly between products, lots, and within the same package [3,20]. Variations observed within the same package are often referred to as “hot spots” (Fig. 1). First identified Spice included fatty acids (linoleic acid, palmitic acid, oleamide), plant-derivate products (thymol, vanillin), preservatives (paraben family), and vitamins (alpha-tocopherol) [21–24]. Modern Spice products contain only synthetic cannabinoids and plant-derivate substances explaining by home-made manufacturing. LEGALITY AND REGULATION The emergence of a new trend involving synthetic cannabinoids contained in herbal mixtures presented technical and legal challenges. The structural 294 Studies in Natural Products Chemistry FIGURE 1 Representative packaging typically found. differences between these new drugs and natural cannabinoids including delta9-tetrahydrocannabinol (D-9-THC) analogs complicated classification of the synthetic cannabinoids by the regulation organs [25]. Regulatory aspects might entail a lag in the fight against drug abuse and distribution of synthetic cannabinoids and say that extensive regulatory efforts should to take into account not only their chemical structure [26]. Since 2009, some European countries (Austria, Germany, France, Luxembourg, Poland, Lithuania, Sweden, UK, and Estonia) warned to the Narcotics Law all products containing synthetic cannabinoids with the aim of reduce their accessibility in head shops and online stores [27]. Local governments adopted specific measures to ban products that have effects similar to synthetic cannabinoids [28]. As of March 2011, the most widely abused synthetic cannabinoids (JWH-018, JWH-073, JWH-200, CP 47,497, and (C8)-CP-47,497) were scheduled by the DEA and placed on the Schedule 1 list under 21 U.S.C.811(h) of the Controlled Substances Act [15,16]. Several individual states have also taken action under existing statutes or emergency scheduling rules. Despite this, Spice drugs are still readily available on the Internet with manufacturers continually making slight structural modifications to continue circumventing legal actions. Broad legislation may seem like a feasible solution, but care must be taken to prevent unintended consequences. A major concern is the possibility that excessive regulation could slow the development of new drugs and hamper particularly the capacity of research of therapeutic agents [29–31]. SYNTHESIS OF SYNTHETIC CANNABINOIDS Some of the first synthetic cannabinoids detected in Spice were synthesized and named after John W. Huffman, a professor emeritus of organic chemistry Chapter 9 Synthetic Cannabinoids: Synthesis and Biological Activities 295 at Clemson University, who focused his research on making a drug to target endocannabinoid receptors in the body [32]. The “JWH” series of synthetic cannabinoids were the dominant cannabinoids detected in Spice [33,34]. The chemical structure of JWH-018 is based upon that of the prototypic aminoalkylindole cannabinoid [35,36]. In addition to the JWH compounds, other synthetic cannabinoids were developed including the “HU” (Hebrew University) and the “CP” (Pfizer) series with the well-known HU-210 and CP-47,497. HU-210 is structurally very similar than D-9-THC but difficult to synthesize [37]. CP-47,497 is easier to produce. A. Makriyannis have also been detected corresponding to the “AM” series [37,38]. There has been an emergence. Based on the chemical structures of the molecules, a classification of the synthetic cannabinoids has be suggested by Howlett et al. and Thakur et al. [4,39,40]. This classification includes the groups below: classical cannabinoids (THC, HU, and AM series, e.g., HU-210, AM-906), cyclohexylphenols (CP series, e.g., CP-47,497), aminoalkylindoles (JWH series, e.g., JWH-018, JWH-073, JWH-250, and some AM series, e.g., AM-694), eicosanoids (anandamide, methanandamide), hybrid cannabinoids (combination of structural features of classical and nonclassical cannabinoids, e.g., AM-4030), others (diarylpyrazoles, e.g., rimonabant, naphthoylpyrroles, e.g., JWH-307, naphthylmethylindenes). The Tables (2–6) show the structural skeleton of principal groups, the substitution pattern, and mean affinity constants (Ki) for the CB1 receptor [19]. High affinity is defined here as Ki < 100 nM. For example, THC (Ki ¼ 10.2 nM) is approximately 10 more potent than a substance with Ki ¼ 100 nM [41]. Since 2012, new types of synthetic cannabinoids emerged rapidly, and the combinations in illegal products can be expected to become more and more diverse. In recent survey publications, the following synthetic cannabinoids QUPIC (PB-22), QUCHIC (BB-22), ADBICA, ADB-FUBINACA, AB-PINACA, AB-FUBINACA, APICA, APINACA, UR-144, JWH-122, AM-2201, and AB-001 have also been reported (Table 7) [7–9]. Because of ban or otherwise control, the majority of the tabulated substances was not detected in smoking mixtures. Many new synthetic cannabinoids were developed and synthesized in anonymous laboratories without reports or patents. BIOLOGICAL ACTIVITIES OF SYNTHETIC CANNABINOIDS Synthetic cannabinoids refer to substances with structural features that allow binding to one of the known cannabinoid receptors, that is, CB1 or CB2, present in human cells and compounds with similar chemical structures. The CB1 receptors are among the most abundant G-protein coupled receptors (GPCRs) expressed in the brain and play a significant role in the modulation of GABA and glutamate neurotransmission [42]. The CB2 receptors are predominantly expressed on immune cells and are thought to mediate immunosuppression by inducing apoptosis, inhibition of proliferation, and suppression of cytokine 296 Studies in Natural Products Chemistry TABLE 2 Group of Naphtoylindoles Substance R1 R2 R3 R4 Ki (nM) JWH-004 Hexyl Methyl H H 48 JWH-007 Pentyl Methyl H H 2.9 JWH-009 Heptyl Methyl H H >1000 JWH-015 Propyl Methyl H H 165 JWH-016 Butyl Methyl H H 22 JWH-018 Pentyl H H H 2.9 JWH-019 Hexyl H H H 9.8 JWH-020 Heptyl H H H 128 JWH-046 Propyl Methyl H Methyl 343 JWH-047 Butyl Methyl H Methyl 59 JWH-048 Pentyl Methyl H Methyl 10.7 JWH-049 Hexyl Methyl H Methyl 55 JWH-050 Heptyl Methyl H Methyl 342 JWH-070 Methyl H H H >1000 JWH-071 Ethyl H H H >1000 JWH-072 Propyl H H H >1000 JWH-073 Butyl H H H 8.9 JWH-076 Propyl H H Methyl 214 JWH-079 Propyl H Methoxy H 63 JWH-080 Butyl H Methoxy H 7.6 Chapter 9 297 Synthetic Cannabinoids: Synthesis and Biological Activities TABLE 2 Group of Naphtoylindoles—Cont’d Substance R1 R2 R3 R4 Ki (nM) JWH-081 Pentyl H Methoxy H 1.2 JWH-082 Hexyl H Methoxy H 5.3 JWH-094 Propyl Methyl Methoxy H 476 JWH-096 Butyl H Methoxy H 34 JWH-098 Pentyl Methyl Methoxy H 4.5 JWH-116 Pentyl Ethyl H H 52 JWH-120 Propyl H Methyl H >1000 JWH-122 Pentyl H Methyl H 0.69 JWH-148 Propyl Methyl Methyl H 123 JWH-149 Pentyl Methyl Methyl H 5 JWH-180 Propyl H Propyl H 26 JWH-181 Pentyl Methyl Propyl H 1.3 JWH-182 Pentyl H Propyl H 0.65 JWH-189 Propyl Methyl Propyl H 52 JWH-193 MPE H Methyl H 6 JWH-198 MPE H Methoxy H 10 JWH-200 MPE H H H 42 JWH-210 Pentyl H Ethyl H 0.46 JWH-211 Propyl Methyl Methyl H 70 JWH-212 Propyl H Ethyl H 33 JWH-213 Pentyl Methyl Ethyl H 1.5 JWH-234 Pentyl H H Ethyl 8.4 JWH-235 Propyl H H Ethyl 338 JWH-236 Propyl Methyl H Ethyl >1000 JWH-239 Propyl H Butyl H 342 JWH-240 Pentyl H Butyl H 14 JWH-241 Propyl Methyl Butyl H 147 JWH-242 Pentyl Methyl Butyl H 42 JWH-262 Pentyl Methyl H Ethyl 28 Continued 298 Studies in Natural Products Chemistry TABLE 2 Group of Naphtoylindoles—Cont’d Substance R1 R2 R3 R4 Ki (nM) JWH-386 Propyl H Br H 161 JWH-387 Pentyl H Br H 1.2 JWH-394 Pentyl Methyl Br H 2.8 JWH-395 Propyl Methyl Br H 372 JWH-397 Pentyl Methyl Cl H 8.9 JWH-398 Pentyl H Cl H 2.3 JWH-399 Propyl Methyl Cl H 187 JWH-400 Propyl H Cl H 93 JWH-412 Pentyl H F H 7.2 JWH-413 Pentyl Methyl F H 14 JWH-414 Propyl H F H 240 JWH-415 Propyl Methyl F H 530 and chemokine production [43]. But, their presence is not limited to peripheral tissues, while their presence has been clearly detected in the brain [44]. CB2 receptors can form heteromers with CB1 receptors in neuronal cells and in rat brain pineal gland, nuvleus accumbes, and globus pallidus [45]. Cannabinoid receptors might be complexed as heterodimers with other receptors that would explain the well-established crosstalk between cannabinoids and several other signaling pathways [45]. As such, the interplay between cannabinoid and opioid receptors is a target of pharmaceutical strategies aimed at new, effective pain control in humans, but the combined effects of opioid/ Spice are unknown [46]. Therefore, Spice effects are described as marijuana-like after smoking or ingestion. Data available indicate that these compounds produce a collection of effects resembling D-9-THC intoxication, although structure–activity relationship analyses reveal that some compounds may exhibit higher potency and affinity for cannabinoid receptors [47,48]. Pharmacokinetic and pharmacodynamic profiles of most synthetic cannabinoids in humans are largely unknown. Generally, synthetic cannabinoids are administered by smoking as a joint or in a water pipe and by ingestion without any indication of the degree of bioavailability. Currently, no cases of parenteral or rectal routes of administration have been published. After smoking, onset of action usually occurs within minutes, similar to cannabis use. This is due to instant absorption via the lungs and redistribution into other organs Chapter 9 299 Synthetic Cannabinoids: Synthesis and Biological Activities TABLE 3 Group of Naphthylmethylindoles Substance R1 R2 R3 Ki (nM) JWH-175 Pentyl H H 22 JWH-184 Pentyl H Methyl 23 JWH-185 Pentyl H Methoxy 17 JWH-192 MPE H Methyl 41 JWH-194 Pentyl Methyl Methyl 127 JWH-195 MPE H H 113 JWH-196 Pentyl Methyl H 151 JWH-197 Pentyl Methyl Methoxy 323 JWH-199 MPE H Methoxy 20 like the brain within minutes after use. There is a delay in absorption following oral consumption due to food intake and digestion activity [49]. Synthetic cannabinoid metabolites retain varying amounts of biologic activity and can act as agonists, neutral antagonists, or inverse agonists at CB1 receptors [50]. Importantly, some synthetic cannabinoids such as JWH-015 show affinity not only for the CB1, but also for the CB2 receptors which are highly expressed on the marginal zone of the spleen, tonsils and immune cells, especially on macrophages, B cells, natural killer cells, monocytes, T-lymphocytes, polymorphonuclear neutrophils [51–53]. Thus, it can be anticipated that Spice drugs containing synthetic cannabinoids with affinity for the CB2 receptor may also affect the immune system by modulating chemotaxis of T-lymphocytes, or inducing thymic atrophy and apoptosis [54,55]. However, most behavioral research is relatively limited. Further, human data concerning the induction and duration of adverse effects remains limited. Single case reports in adults describe an assortment of psychoactive effects ranging from 300 Studies in Natural Products Chemistry TABLE 4 Group of Naphthoylpyrroles Substance R1 R2 Ki (nM) JWH-030 Pentyl H 87 JWH-145 Pentyl Phenyl 14 JWH-146 Heptyl Phenyl 21 JWH-147 Hexyl Phenyl 11 JWH-150 Butyl Phenyl 60 JWH-156 Propyl Phenyl 404 JWH-243 Pentyl 4-Methoxyphenyl 285 JWH-244 Pentyl 4-Methoxyphenyl 130 JWH-245 Pentyl 4-Chlorophenyl 276 JWH-246 Pentyl 3-Chlorophenyl 70 JWH-292 Pentyl 2-Methoxyphenyl 29 JWH-293 Pentyl 3-Nitrophenyl 100 JWH-307 Pentyl 2-Fluorophenyl 7.7 JWH-308 Pentyl 4-Fluorophenyl 41 JWH-346 Pentyl 3-Methylphenyl 67 JWH-348 Pentyl 4-Trifluoromethylphenyl 218 JWH-363 Pentyl 3-Trifluoromethylphenyl 245 JWH-364 Pentyl 4-Ethylphenyl 34 JWH-365 Pentyl 2-Ethylphenyl 17 JWH-367 Pentyl 3-Methoxyphenyl 53 JWH-368 Pentyl 3-Fluorophenyl 16 Chapter 9 301 Synthetic Cannabinoids: Synthesis and Biological Activities TABLE 4 Group of Naphthoylpyrroles—Cont’d Substance R1 R2 Ki (nM) JWH-369 Pentyl 2-Chlorophenyl 7.9 JWH-370 Pentyl 2-Methylphenyl 5.6 JWH-371 Pentyl 4-Butylphenyl 42 JWH-373 Pentyl 2-Butylphenyl 60 JWH-392 Pentyl 2-Trifluoromethylphenyl 77 pleasant, desirable euphoria to anxiety, psychosis, and alterations in cognitive abilities [56,57]. Furthermore, development of tolerance and physical abstinence syndrome has been described after protracted use of high Spice doses [58]. Physical effects were reported ranging in severity from nausea to more serious sympathomimetic-like symptoms such as psychomotor agitation, diaphoresis, and palpitations [56,58]. After consumption of Spice, some users report sedation while others relate agitation, sickness, hot flushes, burning eyes, and xerostomia along with tachycardia [2,59]. Tremors and palpitations have also been described after consumption of “Banana Cream Nuke”, a spice blend containing JWH-018 and JWH-073 [14]. Severe toxicity including generalized seizures and tachycardia has been described including JWH products [24,60]. While the acute adverse effects of synthetic cannabinoids are recognized and documented, there is little information about the chronic use and toxicity of synthetic cannabinoids. However, speculations can be proposed based on the long-term effects of heavy marijuana use. Consumption of the Spice product is associated with psychotic symptoms, ranging from auditory and visual hallucinations to paranoid delusions, from thought blocking to disorganized speech, from anxiety and insomnia to stupor and suicidal ideation [57,61,62]. These clinical reports led to the suggestion that synthetic cannabinoids could precipitate psychosis in vulnerable individuals, similarly to marijuana. Notably, the first withdrawal syndrome with Spice use was described in a 20-year-old male who smoked “Spice Gold” daily over an 8-month period. He found Spice relaxing, sedative, and with cannabis-like psychoactive effects, but requested medical treatment after experiencing internal unrest, profuse sweating, drug craving, nocturnal nightmares, tremor, and headache after a period of abstinence [58]. A recent study described acute adverse reactions after recreational use of herbal mixtures enriched with synthetic cannabinoids in 29 patients [63]. The most frequently observed symptoms were restlessness and agitation, somnolence, hallucination, anxiousness, tachycardia, hypertension, nausea, mydriasis, and hypokalaemia. 302 Studies in Natural Products Chemistry TABLE 5 Group of Phenylacetylindoles Substance R1 R2 Ki (nM) JWH-167 H Phenyl 64 JWH-201 H 4-Methoxyphenyl >1000 JWH-202 Methyl 4-Methoxyphenyl >1000 JWH-203 H 2-Chlorophenyl 8 JWH-204 Methyl 2-Chlorophenyl 13 JWH-205 Methyl Phenyl 124 JWH-206 H 4-Chlorophenyl 389 JWH-207 Methyl 4-Chlorophenyl >1000 JWH-208 H 4-Méthylphenyl 179 JWH-209 Methyl 4-Méthylphenyl 746 JWH-237 H 3-Chlorophenyl 38 JWH-248 H 4-Bromophenyl >1000 JWH-249 H 2-Bromophenyl 8.4 JWH-250 H 2-Methoxyphenyl 11 JWH-251 H 2-Methylphenyl 29 JWH-252 Methyl 2-Methylphenyl 23 JWH-253 Methyl 3-Methoxyphenyl 62 JWH-302 H 3-Methoxyphenyl 17 JWH-303 Methyl 3-Chlorophenyl 117 JWH-304 Methyl 4-Bromophenyl >1000 JWH-305 Methyl 2-Bromophenyl 15 JWH-306 Methyl 2-Methoxyphenyl 25 TABLE 5 Group of Phenylacetylindoles—Cont’d Substance R1 R2 Ki (nM) JWH-311 H 2-Fluorophenyl 23 JWH-312 H 3-Fluorophenyl 72 JWH-313 H 4-Fluorophenyl 422 JWH-314 Methyl 2-Fluorophenyl 39 JWH-315 Methyl 3-Fluorophenyl 430 JWH-316 Methyl 4-Fluorophenyl >1000 TABLE 6 Group of Cyclohexylphenols Substance R1 R2 Ki (nM) CP-55,940 1,1-Dimethylheptyl Hydroxypropyl 0.35 CP-47,497 1,1-Dimethylheptyl H 9.54 Analogue VII 1,1-Dimethyloctyl H 4.7 Analogue I 1,1-Dimethylethyl H >1000 Analogue II 1,1-Dimethylpropyl H >1000 Analogue III 1,1-Dimethylbutyl H >1000 Analogue IV 1,1-Dimethylpentyl H 735 Analogue IX 1,1-Dimethyldecyl H 163 Analogue X 1,1-Dimethylundecyl H 381 Analogue XI 1,1-Dimethylheptyl Methyl 6.2 Analogue XII 1,1-Dimethylheptyl Methyl 7.7 Analogue XIII H Hydroxypropyl >1000 Analogue XV 1,1-Dimethylheptyl Hydroxypropyl 62 Analogue XVI 1,1-Dimethylheptyl Hydroxybutyl 1.6 304 Studies in Natural Products Chemistry TABLE 7 New Types of Synthetic Cannabinoids Substance AM-2202 (1-(4-Pentenyl)-1H-indol-3-yl) (naphthalen-1-yl)methanone AM-1220 (1-((1-Methylpiperidin-2-yl)methyl)-1H-indol-3-yl)(naphthalen1-yl)methanone AM-2233 1-[(N-methylpiperidin-2-yl)methyl]-3-(2-iodobenzoyl)indole AM-1241 (2-Iodo-5-nitrophenyl)-[1-[(1-methylpiperidin-2-yl)methyl]indol3-yl]methanone AM-1248 1-[(N-methylpiperidin-2-yl)methyl]-3-(adamant-1-oyl)indole URB-754 6-Methyl-2-[(4-methylphenyl)amino]-1-benzoxazin-4-one AM679 1-Pentyl-3-(2-iodobenzoyl)indole QUPIC Quinolin-8-yl 1-pentyl-(1H-indole)-3-carboxylate QUCHIC Quinolin-8-yl 1-(cyclohexylmethyl)-1H-indole-3-carboxylate ADB-FUBINACA N-(1-Amino-3,3-dimethyl-1-oxobutan-2-yl)-1-(4-fluorobenzyl)1H-indazole-3-carboxamide ADBICA N-(1-Amino-3,3-dimethyl-1-oxobutan-2-yl)-1-pentyl-1Hindole-3-carboxamide APICA N-(5-fluoropentyl)analog APINACA N-(5-fluoropentyl)analog UR-144 N-(5-chloropentyl)analog Some of these symptoms are similar for symptoms of intoxication with natural cannabis. Some synthetic cannabinoids appear to be more toxic. The central nervous excitation observed in this study was due to strong stimulation of CB1 receptor, because the synthetic cannabinoids are high affinity and high efficacy agonists of this receptor [63]. If poor information exists about the metabolism of the new synthetic cannabinoids, data provide information related to the metabolism properties of Spice parent compounds and their human metabolites including the “JWH” series [64,65]. In Chimalakonda et al. study, the hypothesis that JWH-018 and AM-2201 are subject to cytochrome P450 oxidation was evaluated [64]. Kinetic analysis using human liver microsomes identified CYP2CP and CYP1A2 as major P450 involved in the oxidation of both products. In Grigoryev et al. publication, 22 metabolites of JWH-250 were identified in human urine and serum samples [65]. Metabolites were products of mono- and polyhydroxylation. The monohydroxylated metabolite was the most convenient for diagnosis of drug intoxication. Chapter 9 Synthetic Cannabinoids: Synthesis and Biological Activities 305 EPIDEMIOLOGICAL DATA Recent published surveys of synthetic cannabinoids users reported users were primarily male and Caucasian with a long time natural cannabis education [66–68]. In Hu at al. study, 8% of college students stated using K2 products associated with other drugs [66]. K2 use was more common in males and first or second year college students. Spice products were frequently obtained from retail vendors and smoked with cigarettes or pipe. In Winstock et al. survey, 14,966 participants were interviewed about our consumption of synthetic cannabinoids and 17% of responds reported using them [68]. They reported ever use of natural cannabis which was preferred to synthetic cannabis by 93% of users rated having pleasurable effects. In this study, synthetic cannabinoids were associated with negative effects as paranoia. In Barrat et al. survey, 316 Australian synthetic cannabinoid users were interrogated by an online questionnaire and 35% reported use weekly and 7% reported daily use [67]. They indicated to consume for the first time by curiosity (50%), legality (39%), availability (23%), recreational effects (20%), and therapeutic effects (9%), nondetection in standard drug screening assays (8%) and to aid the reduction or cessation of cannabis use (5%). They reported buying of about 3 g and paying of about 60 dollars. They observed side effects during their last session of use, including decreased motor coordination (39%), fast or irregular heartbeat (33%), dissociation (22%), dizziness (20%), paranoia (18%), and psychosis (4%). A greater number of side effects were reported by males, those aged 18–25 years, water-pipe (bong) users and concurrent alcohol drinkers [67]. A more recent study reviewed about the consequences of the synthetic cannabis users by increasing reports of emergency department presentations [69]. The most common presentations were panic and anxiety, paranoia and breathing difficulties and concerned users with age range of 18–28. Despite side effects and drugs banned by local authorities, many synthetic cannabinoid users continued use of Spice. The frequency and consequences of use varied widely, and ranged from those who engage in sporadic use to those who develop significant chronic use. Reported data in their surveys suggest an evaluation of synthetic cannabinoid use in humans. CURRENT MEDICINAL PURPOSES Many of these synthetic cannabinoids are used in pharmacological studies involving structure–activity relationships, receptor binding studies, and detailed mechanisms of action of these drugs. Some synthetic cannabinoids have been used for medicinal purposes including rimonabant, nabilone, and dronabinol. Rimonabant is a selective CB1 receptor antagonist which was used to treat obesity for some time, but was withdrawn from the market because it showed severe side effects such as severe depression [70]. Rimonabant was the first selective CB1 receptor blocker to be approved for use 306 Studies in Natural Products Chemistry anywhere in the world. As of 2008, the drug was available in 56 countries. Since, nabilone, a potent agonist for the CB1 cannabinoid receptor, was approved by the FDA in 1985. The drug only began being marketed in the United States in 2006. It is approved for treatment of chemotherapy-induced nausea and vomiting (CINV) that has not responded to conventional antiemetics and for treatment of anorexia and weight loss in patients with AIDS. Clinical trials have demonstrated the effectiveness of nabilone in treating anxiety, CINV, and pain associated with fibromyalgia [71–73]. The most common side effects of nabilone are negative (drowsiness, dizziness, and dry mouth), while positive side effects (euphoria, mood elevation) are reported less frequently [74,75]. In terms of the euphoric side effects of nabilone, 3–5 mg of nabilone was shown to induce minimal to moderate euphoria in all subjects [76]. Medical professionals and patients report that nabilone, in comparison to smoked cannabis, has a slower onset of action, more variable efficacy, is harder to titrate to effect, and has more side effects and less overall effectiveness for symptom relief [77]. Regarding the abuse potential of nabilone, the publications reported a low abuse potential [78–80]. Another oral active cannabinoid, dronabinol, was commercialized as name Marinol®. It is synthetic D-9-THC. D-9-THC is also a naturally occurring component of Cannabis sativa (Marijuana). Dronabinol is indicated for the treatment of anorexia associated with weight loss in patients with AIDS; and for the treatment of nausea and vomiting associated with cancer chemotherapy in patients who have failed to respond adequately to conventional antiemetic treatments. The appetite stimulant effect of dronabinol in the treatment of AIDS-related anorexia associated with weight loss was studied in a randomized, doubleblind, placebocontrolled study involving 139 patients. The initial dosage of dronabinol in all patients was 5 mg/day, administered in doses of 2.5 mg 1 h before lunch and 1 h before supper [81]. As compared to placebo, dronabinol treatment resulted in a statistically significant improvement in appetite as measured by visual analog scale. Trends toward improved body weight and mood, and decreases in nausea were also seen. Dronabinol treatment of chemotherapy-induced emesis was tested in combination with prochlorperazine in a randomized, double-blind, parallel group, multicenter study [82]. This study showed the duration of episodes of nausea and vomiting was shorter for patients in the combination group than for those receiving either single agent. In addition, the combination markedly decreased the severity of nausea when compared with single-agent therapy. Although response to the combination is better than that to prochlorperazine alone, side effects with the former were more common. Neurologic and psychotropic effects were experienced by 62% of patients on dronabinol and 55% of those on the combination, as compared with 29% of those receiving prochlorperazine alone. However, only 14% of patients receiving dronabinol alone and 5% of those receiving the combination experienced severe side effects. In addition, dysphoric symptoms (depersonalization, depression, paranoia) were only half as Chapter 9 Synthetic Cannabinoids: Synthesis and Biological Activities 307 common in patients receiving the combination as in those taking dronabinol alone. Combination antiemetic therapy with dronabinol and prochlorperazine may result in synergistic or additive antiemetic effects and attenuate the toxicities associated with each of the agents. Most patients respond to 5 mg 3 or 4 daily. Dosage may be escalated during a chemotherapy cycle or at subsequent cycles, based upon initial results. Therapy should be initiated at the lowest recommended dosage and titrated to clinical response. Some medicinal products containing natural cannabinoids were also available as commercial forms. Sativex®, a combination of D-9-THC 27 mg/ml and cannabidiol 25 mg/ml, is a buccal spray indicated as adjunctive treatment for the symptomatic relief of neuropathic pain in multiple sclerosis in adults [83,84]. To determine the efficacy and safety of Sativex® on spasticity to people with multiple sclerosis, three randomized, placebocontrolled, double-blind, parallel group studies (666 patients) were combined for analysis [84]. A 0- to 100-mm visual analog scale (VAS, transformed to a 0- to 10-scale) or a 0- to 10-numerical rating scale (0–10 NRS) was used to measure spasticity. Patients achieving a 30% improvement from baseline in their spasticity score were defined as “responders.” A statistically significant greater proportion of treated patients were responders (odds ratio (OR) ¼ 1.62, 95% CI 1.15, 2.28; p ¼ 0.0073) and treated patients also reported greater improvement: odds ratio 1.67 (95% CI 1.05, 2.65; p ¼ 0.030). High numbers of subjects experienced at least one adverse event, but most were mild to moderate in severity and all drug-related serious adverse events resolved. The meta-analysis demonstrates that Sativex® is well tolerated and reduces spasticity. However, administration site irritation was very common during short-term use of Sativex®. Regular inspection of the oral mucosa is advised. Patients should be advised not to continue spraying on to sore or inflamed mucosa. A recent systematic review of safety studies of medical cannabinoids identified 8371 adverse events related to medical cannabinoid use, 4779 of which were reported in 23 randomized controlled trials and 3592 in 8 observational studies [85]. Most of the events were not serious. The rate of nonserious adverse events was 1.86 higher among medical cannabinoid users than among controls. However, there is not a higher incidence rate of serious adverse events associated with medical cannabinoid use. The fact that 99% of the serious adverse events from randomized controlled trials were reported in only two trials suggests that more studies with long-term exposure are required to further characterize safety issues. Such studies are crucial to detect rare adverse events and to address specific concerns regarding the development of tolerance and the development of cognitive and behavioral effects of medical cannabinoid use. DISCUSSION AND CONCLUSION According to the United Nations Office of Drugs and Crime, marijuana is the most widely used illicit drug, with 165 million users worldwide [86]. 308 Studies in Natural Products Chemistry Compared to marijuana, synthetic cannabinoid exposures occurred significantly more through inhalation and less via ingestion. This may be because the synthetic cannabinoids are primarily marketed as herbal incense, which may predispose users to smoke the products, while marijuana has a long history of being ingested as well as inhaled. Despite synthetic cannabinoids being available through the Internet, in gas stations, and specialized stores such as head shops, long established marijuana sources may enable adolescents to still be able to more easily obtain marijuana. So, herbal mixtures containing nondeclared synthetic cannabinoids will be a continuous challenge in the next years which requires appropriate reaction by legislation, police, and customs authorities as well as clinical and forensic toxicologists. The monitoring of these herbal mixtures reveals that the active compounds of many products were changed as soon as the first synthetic cannabinoids were put under control in various European countries. Apparently, the producers of those mixtures react in a very fast manner to changes of the legal status of those compounds. They violate national laws, as without analysis of each batch it cannot be excluded that already controlled compounds or other pharmacologically active synthetic compounds have been added to the mixtures. Consumers are also affected as they cannot rely on experience with a certain brand and take additional health risks due to the alteration of kind and amount of undeclared potentially bioactive or toxic additives. Incomplete information regarding the use and toxicity of these compounds has led to a generalized belief that synthetic cannabinoids are marijuana-like and safe for consumption; however current data clearly demonstrate the toxic nature of these new drugs of abuse. Sadly, morbidity and mortality reports continue to increase as these new drugs gain popularity worldwide [87–89]. The biomedical research response has rapidly achieved several technological advances and is now answering questions regarding safety. This information is leading to better protection of public health, but comprehensive legislation with better educational, deterrent, and monitoring programs are still lacking. Limited data are currently available on the pharmacodynamics and pharmacokinetics of synthetic cannabinoids. While it is widely known that most Spice drugs are potent CB1 agonists, exact molecular mechanisms underlying their toxic effects remain to be determined. These compounds and their metabolites have been found to possess higher binding affinity for cannabinoid receptors than marijuana, which implies greater potency, greater adverse effects, and perhaps a longer duration of action. Clearly, Spice is not a safe alternative to marijuana. The wide abuse of Spice highlights the urgent need for further evaluating the synthetic cannabinoids effects in the brain and periphery, characterizing the pharmacology and toxicology, and developing treatments for intoxication. ABBREVIATION LC–MS liquid chromatography–mass spectrometry Chapter 9 Synthetic Cannabinoids: Synthesis and Biological Activities 309 REFERENCES [1] S. Dresen, N. Ferreirós, M. Pütz, F. Westphal, R. Zimmermann, V. Auwärter, J. Mass Spectrom. 45 (2010) 1186–1194. [2] V. Auwärter, S. Dresen, W. Weinmann, M. Müller, M. Pütz, N.J. Ferreirós, J. Mass Spectrom. 44 (2009) 832–837. 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This page intentionally left blank Chapter 10 New Strategies for Identifying Natural Products of Ecological Significance from Corals: Nondestructive Raman Spectroscopy Analysis Lenize Fernandes Maia*, Beatriz Grosso Fleury{, Bruno Gualberto Lages{, Joel Christopher Creed{ and Luiz Fernando Cappa de Oliveira* *Núcleo de Espectroscopia e Estrutura Molecular, Departamento de Quı´mica, Instituto de Cieˆncias Exatas, Universidade Federal de Juiz de Fora, Juiz de Fora, Minas Gerais, Brazil { Departamento de Ecologia, IBRAG, Rua São Francisco Xavier, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, RJ, Brazil { Programa de Pós-Graduação em Ecologia e Evolução, Instituto de Biologia Roberto Alcântara Gomes, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, RJ, Brazil Chapter Outline Introduction 313 Natural Products from Cnidaria 314 A New Method for Identifying Natural Products from Cnidaria 327 Raman Spectroscopy: Basic Principles 328 Instrumentation 334 Application of Raman Scattering to Marine Natural Products: An Overview 335 Characterization of Metabolites from Marine Organisms 335 Raman Spectroscopy Applied to Biologically Relevant Natural Products 341 Concluding Remarks 343 Acknowledgments 344 References 344 INTRODUCTION Most marine invertebrates are delicate animals and are relatively vulnerable to predators. Overall, there are more than 20,000 marine natural products described to date, and almost half of those produced by invertebrates Studies in Natural Products Chemistry, Vol. 43. http://dx.doi.org/10.1016/B978-0-444-63430-6.00010-2 © 2014 Elsevier B.V. All rights reserved. 313 314 Studies in Natural Products Chemistry (subsequently termed new marine natural products of invertebrates NMNPI) have been discovered since 1990, with a pronounced increase in recent decades [1]. The search for new bioactive molecules allied to the structural diversity of marine natural products led to the discovery of compounds with biological and pharmacological properties [2,3]. However, the biosynthesis of such compounds is stimulated by the biological requirements of each species driven by its ecological interactions. The development of chemical ecological assays has demonstrated that structural defenses may be combined with chemical ones to produce coupled physical and chemical mechanisms of deterrence against predators, antifoulers, and competitors [4–6]. Laboratory and field experiments have demonstrated that bioactive secondary metabolites enhanced the survivorship of marine organisms; their chemical properties may be used to avoid microbial infection and predation or enable them to compete for space, nutrients, or other resources in the complex and competitive marine ecosystem [5]. Moreover, in some cases, secondary metabolites can influence the community structure of entire ecosystems [5,7,8], which demonstrates the importance of ecological aspects in understanding the function of each species in the dynamic community. Ecological factors such as predation contribute to marine diversity of natural products due to evolution through natural selection. The symbiosis between the microbial community and invertebrates is another factor that probably contributes to the chemical diversity of substances at the species level. There is evidence from recent studies suggesting that microbes associated with marine invertebrates may be the true producers of some of the natural products that were previously assumed to be produced by their invertebrate host [9–11]. The identification of marine natural products involved in biological interactions has been performed by spectroscopic and spectrometric techniques. The aim of this chapter is to demonstrate the application of an alternative technique, Raman spectroscopy, as a complementary tool for analyzing natural ecologically relevant products from corals. The strength of this technique compared to others is that it allows for the possibility of analyzing small amounts of biological samples in situ and in vivo. NATURAL PRODUCTS FROM CNIDARIA Invertebrates comprise about 60% of all marine animal diversity [12], the phyla Porifera and Cnidaria are the two dominant sources of NMNPI worldwide together responsible for 72% of the compounds, and 77% of the compounds are isolated from marine invertebrates. Within each phylum above the majority of substances belong to a single class of sponges (Porifera: Demospongiae) and one class of corals (Cnidaria: Anthozoa; Fig. 1) [5,13–15]. The majority of coral compounds are derived from the isoprenoid Chapter 10 315 Nondestructive Raman Spectroscopy Analysis Marine natural products (12322)* Others (5342) Invertebrates (6980) Phylum Porifera (3550) Phylum Cnidaria (1530) Others (1900) Class: Anthozoa (1492) Class: Hydrozoa (38) Class: Scyphozoa (0) Class: Cubozoa (0) Subclass: Octocorallia (1324) Subclass: Hexacorallia = Zoantharia (154) • Order: Alcyonacea (664) Family: Alcyoniidae (434) Genus: Alcyonium (40) Lobophytum (80) Sarcophyton (93) Sinularia (145) Family: Nephtheidae (96) Genus: Lemnalia (33) Order: Actiniaria (44) Family: Actinnidae (37) Genus: Anthopleura (25) Order: Zoanthidea (62) Family: Zoanthidae (41) Genus: Palythoa (19) Zoanthus (22) Order: Scleractinia (48) Family: Dendrophylliidae (23) Genus: Tubastraea (17) Nephthea (34) Family: Xeniidae (112) Genus: Xenia (90) • Suborder: Scleraxonia (167) Family: Briareidae (139) Genus: Briareum (102) • Suborder: Holaxonia (334) Family: Gorgoniidae (102) Genus: Pseudopterogorgia (74) Family: Plexauridae (101) Genus: Eunicea (59) Plexaura (12) FIGURE 1 Distribution of natural products from invertebrates with emphasis on the phylum Cnidaria. Numbers in parenthesis indicate number of compounds for each taxonomic group. Biological classification of the anthozoans from subclass Octocorallia is according to Bayer [13] and from subclass Hexacorallia is according to Daly [14]. 316 Studies in Natural Products Chemistry biosynthetic pathway (82%) and the remainder split between acetogenin and amino acid (Figs. 2 and 3) [5,15]. Cnidarians are a diverse group of organisms known to produce nematocysts, which are cellular structures that release proteinaceous toxins that act as a defense against potential predators or competitors [16]. During the 1980s and 1990s, marine natural products were investigated for a broad 6 Alcyonium Isoprenoid (100%) 6 Lobophytum Isoprenoid (90%) Acetogenin (9%) Amino acid (1%) 1 Cnidaria Isoprenoid (82%) Acetogenin (9%) Amino acid (7%) others (2%) 2 Anthozoa Isoprenoid (84%) Acetogenin (8%) Amino acid (7%) Shikimate (1%) others (1%) 4 Alcyonacea Isoprenoid (96%) Acetogenin (3%) Amino acid (1%) 5 Alcyoniidae Isoprenoid (95%) Acetogenin (4%) Amino acid (1%) 6 Sarcophyton Isoprenoid (98%) Acetogenin (2%) 6 Sinularia Isoprenoid (93%) Acetogenin (3%) Amino acid (3%) Carbohydrate (1%) 3 Octocorallia Isoprenoid (92%) Acetogenin (7%) Amino acid (1%) 5 Nephtheidae Isoprenoid (99%) Shikimate (1%) 6 Lemnalia Isoprenoid (100%) 6 Nephthea Isoprenoid (97%) Shikimate (3%) 4 Scleraxonia and Holaxonia Isoprenoid (93%) Acetogenin (6%) Amino acid (1%) 5 Xeniidae Isoprenoid (99%) Acetogenin (1%) 6 Xenia Isoprenoid (100%) 5 Briareidae Isoprenoid (99%) Acetogenin (1%) 6 Briareum Isoprenoid (99%) Acetogenin (1%) 5 Gorgoniidae Isoprenoid (89%) Acetogenin (9%) Amino acid (2%) 6 Pseudopterogorgia Isoprenoid (97%) Amino acid (3%) 5 Plexauridae Isoprenoid (88%) Acetogenin (11%) Shikimate (1%) 6 Eunicea Isoprenoid (100%) 6 Plexaura Isoprenoid (39%) Acetogenin (61%) FIGURE 2 Distribution of metabolites from main groups of octocorals: 1, phylum; 2, class; 3, subclass; 4, order; 5, family; 6, genus. Chapter 10 317 Nondestructive Raman Spectroscopy Analysis 1 Cnidaria Isoprenoid (82%) Acetogenin (9%) Amino acid (7%) others (2%) 4 Actiniaria Isoprenoid (30%) Acetogenin (5%) Amino acid (60%) Nucleic acid (5%) 5 Actiniidae Isoprenoid (36%) Acetogenin (6%) Amino acid (56%) Nucleic acid (3%) 6 Anthopleura Isoprenoid (38%) Amino acid (62%) 4 Zoanthidea Isoprenoid (12%) Acetogenin (13%) Amino acid (75%) 5 Zoanthidae Isoprenoid (13%) Acetogenin (20%) Amino acid (67%) 6 Palythoa Acetogenin (37%) Amino acid (63%) 6 Zoanthus Isoprenoid (24%) Acetogenin (5%) Amino acid (71%) 2 Anthozoa Isoprenoid (84%) Acetogenin (8%) Amino acid (7%) Others (1%) 3 Zoantharia Isoprenoid (19%) Acetogenin (19%) Amino acid (58%) Shikimate (2%) Nucleic acid (1%) 4 Scleractinia Isoprenoid (19%) Acetogenin (40%) Amino acid (34%) Shikimate (6%) 2 Hydrozoa Isoprenoid (82%) Acetogenin (9%) Amino acid (7%) Others (2%) 4 Athecata Isoprenoid (26%) Acetogenin (62%) Amino acid (12%) 4 Thecata Isoprenoid (9%) Acetogenin (18%) Amino acid (73%) 5 Dendrophylliidae Acetogenin (30%) Amino acid (61%) Shikimate (9%) 6 Tubastraea Acetogenin (41%) Amino acid (47%) Shikimate (12%) 5 Bougainvillidae Acetogenin (100%) 6 Garveia Acetogenin (100%) 5 Eudendriidae Isoprenoid (100%) 6 Eudendrium Isoprenoid (100%) 5 Hydractiniidae Amino acid (100%) 6 Hydractinia Amino acid (100%) 5 Polyorchidae Amino acid (100%) 6 Polyorchis Amino acid (100%) 5 Tubulariidae Amino acid (100%) 6 Tubularia Amino acid (100%) 5 Stylasteridae Isoprenoid (100%) 6 Allopora Isoprenoid (100%) 5 Aequoreidae Amino acid (100%) 6 Aequoreae Amino acid (100%) 5 Plumaridae Amino acid (100%) 6 Aglaophenia Amino acid (100%) 5 Sertulariidae Isoprenoid (17%) Acetogenin (33%) Amino acid (50%) 6 Abietinaria Acetogenin (100%) FIGURE 3 Distribution of metabolites from main groups of zoantharians and hydrozoans: 1, phylum; 2, class; 3, subclass; 4, order; 5, family; 6, genus. spectrum of pharmacological activity, and following from that, known compounds from Cnidaria increased substantially (72.0%; from 1031 to 1773 new natural products between 1999 and 2009) and are now one of the dominant sources of NMNPI from temperate (19.6%) and tropical regions (35.4%) 318 Studies in Natural Products Chemistry [1]. Subsequently, ecological activities produced by their chemicals became as important a research focus as the biological one (Table 1). Considering the phylum Cnidaria the class Anthozoa comprises 99.0% of NMNPI and the subclass Octocorallia account for the most NMNPI in the Anthozoa (95.5%); the order Alcyonacea account for 98.1% of new natural products from Octocorallia and 26.8% of all NMNPI. The most product-rich genera are Sinularia (11.9%), Briareum (11.2%), Pseudopterogorgia (6.6%), Sarcophyton (6.1%), and Nephthea (5.2%). The genera in which most new natural products were reported between decades were Nephthea (+468.2%; from 22 to 125 natural products), Clavularia (+255.2%; from 29 to 103 natural products), Junceella (+229.2%; from 24 to 79 natural products), and Sarcophyton (+89.8%; 59 to 112 natural products). Of Cnidaria species, Clavularia viridis, Pseudopterogorgia elisabethae, and Briareum excavatum account for the highest number of NMNPI reported since 1990, and each accounts for approximately 3% of all cnidarian’s NMNPI [1]. Terpenoids and alkaloids are the most diverse substances isolated from marine invertebrates [5,15]. Almost 98% of metabolites extracted from cnidarians are from the class Anthozoa where terpenoids dominate across the subclass Octocorallia [5,15] (Figs. 2 and 3). The order Alcyonacea (mainly the families Alcyoniidae and Xeniidae) produce half of the metabolite richness within the subclass (Fig. 1). The genus Sinularia (Alcyoniidae), for example, produces a great variety of chemical compounds ranging from sesquiterpenes to diterpenes [93]. The suborder Stolonifera (order Alcyonacea) and the orders Pennatulacea and Helioporacea all produce isoprenoids as predominant compounds. The suborder Holaxonia (order Alcyonacea) is the second richest order of subclass Octocorallia with the families Gorgoniidae and Plexauridae accounting for almost half of the metabolites within the order (Fig. 1). The chemistry of this order has been well documented since the 1960s when Ciereszko and collaborators reported the presence of biologically active diterpenes crassin acetate from Pseudoplexaura crassa and eunicin from Eunicea mammosa [94]. Diterpenoids, sesquiterpenoids, and steroids are known to act as feeding deterrents [55,93,95–97] and as allelopathic agents acting during interspecific competition for space between certain soft corals and gorgonians [29,97]. The genus Pseudopterogorgia has been the subject of the greatest number of investigations. For instance, the Caribbean gorgonian Pseudopterogorgia americana contains two secosterols that deter fish feeding [40]. However, the genera Briarium and Junceella have also received considerable attention. A range of bioactive diterpenes have been reported derived from gorgonians over the past 13 years, including those with anticancer (most common), anti-inflammatory, antiplasmodial, antibacterial, antiviral, antimalarial, and antioxidant properties, as well as activities of ecological importance such as fish-feeding deterrence [98]. TABLE 1 Biological and Ecological Activities Produced by Extracts and/or Isolated Substances from Some Coral Species Coral Species Class of Substances Extraction Media and Extracted Substances l Biological and References ▪ Ecological Activity Tubastraea aurea Alkaloids Aplysinopsins and tubastrine Tubastraea faulkneri Polyketides Mycalolide C and D and methanol extract Tubastraea coccinea Alkaloids Aplysinopsins l Astroides calycularis Alkaloid Aplysinopsins l l l a Cytotoxicity to cell cancer ; inhibit the first cleavage of fertilized sea urchin eggs and antiviral [17–22] Cytotoxic and antimicrobial activity against the genus Vibrio, Photobacterium, Alteromonas, and Staphylococcus. Toxic to larvae of scleractinian corals Platygyra sinensis, Acropora formosa, A. millepora, A. tenuis, A. pulchra, Goniastrea aspera, Montipora digitata, Fungia fungites, Favia pallida, Oxypora lacera, and Platygyra daedalea [23–25] Antidepressant in mammals, antineoplastic, antiplasmodial, antimicrobial [18,26,27] Antidepressant in mammals, antineoplastic, antiplasmodial, antimicrobial [18,27,28] Continued TABLE 1 Biological and Ecological Activities Produced by Extracts and/or Isolated Substances from Some Coral Species— Cont’d Coral Species Sinularia flexibilis Class of Substances Extraction Media and Extracted Substances Terpenoids (diterpenes) Flexibilide; sinulariolide; dihydroflexibilide; episinulariolide; episinulariolide acetate and aqueous extract; lipophilic extract; 11-episinulariolide l Biological and ▪ Ecological Activity l ▪ Briareum sp. Terpenoids (diterpene) Stecholide L l Briareum excavatum Briarane-type diterpenoids Excavatolides F–M l Briareum excavatum Polyoxygenated briarane-type diterpenoids Briaexcavatolides P l Junceella fragilis Diterpenoids Junceellolides A–D l References Antifouling activity against Ectocarpus sp. and pinnate diatoms; antimicrobial activity against Bacillus subtilis and Staphylococcus aureus; algacidal properties Deterrent to Gambusia affinis; flexibilide is allelopathic against corals Acropora formosa and Porites andrewsi; Sarcophyton glaucum and Alcyonium molle presented necrosis and moved away from S. flexibilis [29–33] Cytotoxic activity [34] Cytotoxicity toward various cancer cell lines [35] Cytotoxicity toward P-388 and HT-29 cancer cells [36] Anti-inflammatory activity [37] gorgonians Briarane diterpenoids Nui-inoalide A, gemmacolides A, B, and D Briareum asbestinum Diterpenoid Brianthein V Pseudopterogorgia americana Secosterols 9,11-Secogorgosterol and 9,11-secodinosterol Pseudopterogorgia elisabethae Diterpene glycoside Pseudopterosinsa from endosymbionts Chromonephthea braziliensis Hemiketal steroid n-Hexane extract and 23-keto-cladiellin-A Gorgonia cf. mariae and G. ventalina – Dichloromethane extracts Siderastrea siderea – Methanol–dichloromethane extract (1:1) Junceella juncea Briarane diterpenoids Juncins R–ZI; ZII, gemmacolide A, B; junceellolide Leptogorgia virgulata and L. setacea Homarine N-Methyl-2-carboxypyridine, nicotinic acid, picolinic acid, and pyridine Lobophytum pauciflorum Terpenoids (diterpenes) 14-Hydroxycembra-1,3,7,11tetraene; 15-hydroxycembra-1,3,7,11tetraene Pseudopterogorgia elisabethae Alkaloids Pseudopteroxazole; biflorane l Immunomodulatory activity [38] l Cytotoxic and antiviral activity [39] ▪ Deterrent against fish Thalassoma bifasciatum [40] Potential anti-inflammatory activities [41] l ▪ Potent feeding deterrent against fish assemblage. [42,43] Antifungal activity against Aspergillus sydowii [44] Antimicrobial activity against two strains of Gram-positive bacteria [45] Antifouling activity against barnacle larvae Balanus amphitrite [46,47] Antifouling activity against diatom Navicula salinicola [48] ▪ Antifouling activity against the [49] l l l l algae Ceramium flaccidum l Antimicrobial activity against M. tuberculosis [50,51] Continued TABLE 1 Biological and Ecological Activities Produced by Extracts and/or Isolated Substances from Some Coral Species— Cont’d Coral Species Class of Substances Extraction Media and Extracted Substances Litophyton viridis Steroid Litosterol Plexaura homomalla Terpenoid Aqueous extracts; lipid extracts; prostaglandin ▪ Toxic to goldfish; deterrent [53] Nephthea chabrolii Terpenoid Hydroxycolorenone ▪ Antifeedant activity against the [54] Pseudopterogorgia rigida Terpenoid (sesquiterpenes) Crude extract, curcuquinone, curcuhydroquinone ▪ Deterred the fish Thalassoma [55] Parerythropodium fulvum fulvum – Crude extract l Biological and ▪ Ecological Activity l Antimicrobial activity against M. tuberculosis against killifish pest insect Spodoptera littoralis bifasciatum l ▪ References [52] Antimicrobial activity against the bacteria Vibrio sp. Antifeeding by generalist reef fish Thalassoma klunzingeri and T. lunare [56,57] Sinularia dura – Crude extract l Cytotoxicity [58] Nephthea sp. – Crude extract l Cytotoxicity [58] Dendrophyllia cornigera Steroid Cholesta-4,22-diene-3,6-dione l Cytotoxicity against cancer cells [59] Nephthea sp. Terpenoid Nephtheoxydiol l Cytotoxicity against melanoma cells [60] Anti-inflammatory activity; cytotoxicity against cancer cells Nephthea chabroli Steroid Nebrosteroids I–M and R–S l Erythropodium caribaeorum Terpenoid Erythrolide B ▪ Deterrent to natural assemblage [63] Pterogorgia anceps – Polar crude extract and unnamed compound indentified as 5 ▪ Deterrent against Thalassoma [64] Sinularia maxima, S. polydactyla and Sinularia sp. – Crude extracts ▪ Deterrent against generalist fish [65,66] Nephthea erecta Steroid Crude extract; bsterols l Lophogorgia violacea Terpenoid (diterpenes) Lophotoxin; deoxylophotoxin; 13-acetoxy-11b,12bepoxypukalide; 7-acetoxy-8-hydroxylophotoxin; 3-methoxy-8-hydroxylophotoxin ▪ Chemical deterrence against [68] Phyllogorgia dilatata Terpenoid (diterpene) 11b,12b-Epoxypukalide; crude extract ▪ Chemical deterrence against [69] Pterogorgia citrina; Briareum asbestinum – Crude extracts ▪ Deterrence against blue head [70] Sarcophyton spp. Terpenoids (diterpene) Sarcotol; sarcotol acetate; and sarcotal acetate ▪ Ichthyotoxic activity against [71] Xenia elongata Terpenoids (diterpene) Deoxyxeniolide ▪ Ichthyotoxic activity against [72] of reef fish bifasciatum in lab. and natural assemblage of reef fish Cytotoxicity against cancer cells generalist fish generalist fish wrasse Thalassoma bifasciatum Japanese killifish Oryzias latipes Japanese killifish Oryzia latipes [61,62] [67] Continued TABLE 1 Biological and Ecological Activities Produced by Extracts and/or Isolated Substances from Some Coral Species— Cont’d Class of Substances Extraction Media and Extracted Substances Lobophytum schoedei Terpenoids (diterpene) Lobophynin C Heterogorgia uatumani Terpenoids (di- and sesquiterpene) (6E)-2R,9R-Epoxyeunicella-6,11 (12)-dien-3b-ol, heterogorgiolide Plexaura homomalla – Crude extract Xenia macrospiculata Terpenoids (diterpenes) Crude extract and desoxyhavannahine Briareum polyanthes Terpenoids (diterpenes) Eunicellin-type; asbestinane-type; briarane-type Lemnalia sp. Terpenoids (diterpenes) Crude extract; lemnalosides A and B Leptogorgia virgulata – Fractions of crude extract Coral Species l Biological and References ▪ Ecological Activity ▪ Ichthyotoxic activity against [73] ▪ Chemical deterrence against [74] Japanese killifish Oryzias latipes and lethal activity toward brine shrimps carnivorous reef fish species l l l l l Active against fungal infection caused by Aspergillus sydowii [75] Antimicrobial activity against Arthrobacter sp. [76] Activity against pathogenic microbes of human infectious diseases [77] Inhibitory activity against hypha formation of the genus Streptomyces [78] Growth inhibition to Escherichia coli, Vibrio harveyi, Micrococcus luteus, and Bacillus sp. isolated from scleractinian coral Acropora cervicornis [79] Alcyonium paessleri, Gersemia antarctica Steroids Crude extracts; cholesterol; 22-dehydrocholesterol/24methylenecholesterol; 22-dehydro-7b-hydroxycholesterol ▪ Antifouling activity against marine bacteria and diatoms; predator deterrent against the sea star Odontaster validus l [80,81] Antibacterial action against Alteromonas sp.; Moraxella sp. and the genus Psychrobacter Dendronephthya sp. Secosteroids Isogosterones A–D ▪ Inhibited larval settlement of the Litophyton arboretum; Rythisma f. fulvum; Xenia macrospiculata – Crude extracts l Subergorgia suberosa and Scripearia gracilis Terpenoids (sesquiterpene) steroids Crude extract; subergorgic acid; pregn-4-ene-3,20-dione; 5bpregn-3,20-dione ▪ Inhibited the larval settlement of [84] Tubastrea coccinea, T. tagusensis – Crude extract ▪ Inhibited predation by generalist [85] Sarcophyton glaucum; Lemnalia sp. and Sinularia sp. – Aqueous extracts ▪ Rejected all low concentration by [86] Nephthea brassica Terpenoids (di- and sesquiterpenes) Crude extract; brassicolene; brassicolide; brassicolide acetate; ( )-4a-O-Acetyl-selin-11-en; ( )selin-11-em-4a-ol; nephthenol; cembrene A; epoxycembrene A and ( )-b-elemene barnacle Balanus Amphitrite Inhibited the growth of microbial strains Balanus amphitrite and Bugula neritina and growth of marine microbial strains fish and algae of genera Cladophora and Lithophyllum mosquito fish Gambusia affinis l Cytotoxicity against tumor cells [82] [83] [87,88] Continued TABLE 1 Biological and Ecological Activities Produced by Extracts and/or Isolated Substances from Some Coral Species— Cont’d Coral Species Class of Substances Extraction Media and Extracted Substances l Biological and ▪ Ecological Activity References Lemnalia and Sarcophyton Aqueous extract High toxicity against the mosquito fish Gambusia affinis [89] Goniopora tenuidens Aqueous extract ▪ Larvotoxic effects against [90] Pocillopora damicornis; Platygyra daedalea; Fungia fungites and Oxypora lacera Sinularia flexibilis and Lobophytum hedleyi Diterpenoids Flexibilide; dihydroflexibilide eudesmanoid ▪ Necrosis on tissues of Acropora [91] Pseudopterogorgia rigida; Erythropodium caribaeorum; Pterogorgia anceps; and Eunicea asperula – Crude extracts ▪ Inhibited fish deterrence for [92] a formosa and Porites cylindrica Caribbean wrasse Thalassoma bifasciatum Not isolated from corals. 24-Methylcholesta-5,24(28)-diene-3b,15b,19-triol; 24-methylcholesta-5,24(28)-diene-3b,19-diol-7-on[84]e; 24-methylcholesta-5,24(28)-diene-3b,19-diol; 24-methylcholesta-5,24(28)-diene-3b,19-diol-7b-monoacetate; 24-methylcholesta-5,24(28)-diene-3b,7b,19-triol; 24-methylcholesta-5,24(28)-ene-3b,5a,6b,19-tetraol. b Chapter 10 Nondestructive Raman Spectroscopy Analysis 327 The subclass Hexacorallia (zoantharians) shows different chemical structure from metabolites of the subclass Octocorallia (Fig. 3). The order Zoanthidea produces a high proportion of amino acid derivatives and low proportions of isoprenoids and acetogenins (Fig. 3). The order Actiniaria (sea anemones) is reported to also produce a high proportion of amino acids derivatives and low proportions of isoprenoid and acetogenin derivatives. Different proportions of compound classes are observed within the order Scleractinia (stony corals) where acetogenins slightly dominate over amino acids and isoprenoids [5]. Studies have demonstrated that the genus Tubastraea (order Scleractinia) mainly produces steroids and alkaloids and some are bioactive and toxic to cells [17,18,23,99,100], while others may act against competitors and predators [24,85,101]. According to Lages and collaborators (2012), some of the compounds produced by the invasive species Tubastraea coccinea and T. tagusensis varied in concentration with proximity to the endemic native scleractinian coral Mussismilia hispida and the sponge Desmapsamma anchorata. In general, for both species of Tubastraea, a variety of substances were found in this study, where sterols represented 40.5% of all substances, on average. Fatty acids, hydrocarbons, alkaloids, esters, and alcohols represented 28.4%, 19.4%, 3.5%, 2%, and 1.7% of substances found, respectively, for both species. A NEW METHOD FOR IDENTIFYING NATURAL PRODUCTS FROM CNIDARIA In the past, the chemical composition of biologically relevant compounds has been investigated by chromatographic methods and mainly characterized by mass spectrometry, nuclear magnetic resonance spectroscopy, infrared spectroscopy, and ultraviolet spectroscopy. A new alternative is to combine Raman spectroscopy, which is a light-scattering technique that provides information about molecular vibrations [102,103], with other traditional techniques. It is a nondestructive method of analysis suitable for use in situ in studies of biomaterials as well as chemical compounds. The technique also offers the advantage of short measurement times, requires low amounts of material, and is of low sensitivity to water content present in biological samples. It has been extensively used in analyzing molecules ranging from proteins to small molecules as well as some secondary metabolites [104–107]. Raman spectroscopy allows in situ analysis of inorganic and organic samples as well as in vivo analysis of live tissues [106,108]. The technique has been successfully applied to medical diagnoses [109,110], identification of chemical composition of pathogenic and nonpathogenic microorganisms [111], plant tissues [106,112], quality control of food [113,114], investigation of textiles [106,115,116], works of arts [106,115,116], gemstones [106,117,118], astrobiological and mineralogical analysis [119–121], and forensic material [122]. 328 Studies in Natural Products Chemistry RAMAN SPECTROSCOPY: BASIC PRINCIPLES When light interacts with matter, several different events at the molecular level can happen, all of them at the same time. Matter can absorb light, if there is the correct energy, which is equal to the difference between two electronic levels of the system, or even if the energy is located at the infrared part of the electromagnetic radiation, with the system experiencing a vibrational or a rotational transition. Another type of interaction can be seen when matter emits radiation, after the interaction with the incident light; this phenomenon, known as luminescence, results from the emission of radiation with lower energy than the incident and can be divided in at least two different types: phosphorescence and fluorescence, the difference being the time the effect takes place [102]. However, matter can scatter electromagnetic radiation, this being one of the most beautiful natural phenomena in nature. If light is monochromatic, that is, only one exact wave number or frequency value is emitted by the source, the most well-known phenomenon to occur is the so-called elastic scattering, or Rayleigh scattering. Light scattered by matter can include some different phenomena, such as light diffraction. This is the main explanation why the sky seems blue in color and the blue color changes in intensity and tones, since the gas molecules that compose the atmosphere, as well as the water and pollution particles, scatter the light with frequency similar or lower than the average size, allowing only the high-frequency energy to come directly to Earth, being composed mainly by the blue, violet, and ultraviolet frequencies. In 1929, C.V. Raman published the most complete investigation proving that light could also be inelastically scattered, that is, the scattered light would present a different energy from the incident one; the difference in energy between the incident and the scattered beams would be involved in a vibrational movement, similar to the one observed in the infrared region, and this phenomenon could also be used exactly as in the infrared spectroscopy technique, to perform a molecular investigation based on the assignments of the vibrations presents as bands in the recorded spectra of molecules. The theoretical basis of the inelastic scattering, or Raman effect, as it is known nowadays, can be understood involving physical approaches: the classical, where the contributions of the elastic and the inelastic effects can be seen clearly, and the quantum approach, where it seems very clear that the effect can be explained on the basis of a mixture of electronic and vibrational states of the matter, giving rise to the vibronic model to explain the physical vision. The classical explanation for the Raman effect, also known as the Placzeck theory, takes into account a monochromatic incident radiation acting on the matter. During the time that radiation interacts with matter, both are known as a new system, where the total energy is the matter plus radiation Chapter 10 Nondestructive Raman Spectroscopy Analysis 329 energies. This is very important to understand, since there is no absorption of energy by the system, because the energy level originated by the interaction is called the virtual (r) level and exists only during the time energy is acting on matter. Physically it can be described as ! ! P¼ a E (1) where it can be seen that the electric field of the electromagnetic radiation has to generate a dipole moment; this relation is linear, and the coefficient a is the polarizability tensor, which can be physically described as the way the electronic cloud of the system behaves during the time a very strong electromagnetic field (from the incident light) is acting on matter. The electric field of the incident light can be described according to the following equation: Eðx, tÞ ¼ E0 cos ð2pn0 tÞ (2) where n0 is the frequency of the incident light. Substituting (2) in (1), ! P ¼ aE0 cos ð2pv0 tÞ (3) The normal coordinate mode q (equivalent to the molecular vibration) can be understood as an oscillatory function, depending on the frequency vn: q ¼ q0 cos ð2pvn tÞ (4) The polarizability tensor depends on the molecule geometry, and the normal coordinate mode q can change the value of a during the molecular vibration; using the Taylor’s expansion: @a a ¼ a0 + q + (5) @q where (@a/@q) means the change in polarizability by the molecular vibration. Substituting Eqs. (4) and (5) in Eq. (3), we get ! 1 @a P ¼ a0 E0 cos ð2pv0 Þ + q0 E0 fcos ½2pðv0 + vn Þt + cos ½2pðv0 vn Þg (6) 2 @q It can be clearly seen that the induced dipole moment by an incident light with frequency n0 oscillates with three different frequency values, n0, n0 + nv, and n0 nv, which correspond to the elastic scattering (Rayleigh), and the two inelastic scattering frequencies, one with high frequency, when compared to the excitation, and the other with lower frequency; these values are commonly called by anti-Stokes and Stokes frequencies, respectively, and are also known as the Raman frequencies. It is important to note that in the case of the Raman scattering, there is a shift in the frequency of the scattered light when compared to the incident, and this is commonly called the Raman shift; 330 Studies in Natural Products Chemistry Virtual levels hnL hnL hnL h(nL−nM) Stokes (nL−nM) hnL Rayleigh nL h(nL+nM) Anti-Stokes (nL+nM) FIGURE 4 Three different schemes for the light scattering: Rayleigh (elastic) and Raman (inelastic Stokes and anti-Stokes). Adapted from Wartewig [123]. experimentally, the Raman spectrum of a substance goes from the zero value in wave numbers (the absolute value is equal to the wave number of the monochromatic excitation source), and the Stokes Raman spectrum goes to the positive values of wave numbers, whereas the anti-Stokes spectrum goes to the negative values of wave numbers. Figure 4 depicts the schemes for the three discussed phenomena of light scattering, involving the elastic (Rayleigh) and inelastic (Stokes and anti-Stokes Raman); it is very clear to note that the virtual states in all the drawings are related to any type of monochromatic lasers that can be used, far from any lowest electronic energy level for the studied system, just to avoid any kind of absorption from the chemical system. The intensity of the Raman scattering (Stokes and anti-Stokes) spectra can be obtained from the following equations: 2 4 @a IStokes ðv0 vn Þ I0 ðv0 Þ (7) @q 2 @a IStokes ðv0 vn Þ4 I0 ðv0 Þe ðhvn =kT Þ (8) @q where it seems clear from Eqs. (7) and (8) that the Stokes spectrum is much more intense than the anti-Stokes and this difference becomes small in value when temperature increases. However, a trouble with this approach is that Chapter 10 Nondestructive Raman Spectroscopy Analysis 331 there is no basic formalism to understand the relative intensity of each one of the bands present in the Raman spectrum, Stokes or anti-Stokes, and for this understanding, we need to take into account the quantum approach in the explanation. According to the scheme presented on Fig. 4, the virtual state is not an actual (or stationary) state of the chemical system (molecule), but this situation is clearly dependent on the energy of the incident light we are using to obtain the Raman spectrum. For example, if the molecule presents an electronic absorption closer to the energy of the incident light, not only all the photons will be scattered, but some of them could be absorbed by the system. This is the main problem in explaining the basics of the Placzeck theory, where there is no explanation for the intensity of each one of the Raman bands observed on the spectrum. Let us try to understand the principles of the absorption of energy in spectroscopy, in a general way. A transition must occur when energy is offered to the chemical system and is of the same value as the difference between the initial and the final molecular states; there are some rules to regulate this process, but for now, we are ignoring them. It is important to note that this idea is the basis of quantum mechanics, introduced by M. Planck in 1900 and developed in the 1920s by I. Schrödinger: there is a wave function for each one of the energy states of the system, and this wave function is a contribution of each one of the individual energies of the molecule: C ¼ Ctranslational + Ctotational + Cvibrational + Celectronic (9) where Cvibrational is the energy associated to a molecule vibration, which is related, in a first approach, to the harmonic oscillator theory: En ¼ hðv + 1=2Þ, v ¼ 0,1,2 ... sﬃﬃﬃ 1 k m1 m1 , m¼ v¼ 2p m m1 + m2 DE ¼ En¼1 En¼0 (10) (11) (12) The energy radiation involved in the electronic absorption spectrum (which usually happens in the ultraviolet-visible region) corresponds to the transition between electronic energy levels of the molecule and can also happen in other transitions between vibrational levels of the ground and excited electronic states of the molecule. The pattern of this vibronic structure is given by the Franck–Condon factors [102], which are integrals involving vibrational wave functions of ground and excited electronic states. From the vibrational level vi ¼ 0 of the ground electronic level, the intensity of the transitions to excited state vibrational levels is proportional to the overlap integral value, hf |ti, between the correspondent functions: 332 Studies in Natural Products Chemistry ð h f jii ¼ Cf Ci dt (13) where |ii and hf | are the bra–ket notations for the initial vibrational wave functions, Ci, and the final (conjugated complex), Cf*. The intensity of an absorption band in function of the radiation frequency, IA(o), is given by [3–5] IA ðoÞ∝ X f ½hef jmjgii2 2 Eef Eei o + G2 2 (14) where m is the electric dipole moment operator, Egi is the initial vibrational level (i) energy of the ground electronic state (g), Eef is the correspondent energy of the excited state, and G is a dumping factor that results in the broadening of the vibrational band; in several cases, only a broad band where the vibronic structure is not resolved can be observed. The intensity of a Raman band is given by an analogous expression of Eq. (14), the Kramers– Heisenberg–Dirac (KHD) equation: IR ðoÞ ¼ X hgf jmjer iher jmjgii f Eer Egi o + iG 2 (15) In this case, there are two transition moments: one between the initial vibrational level of the ground electronic state and a vibrational level of the excited electronic state, her|m|gii, and another between this intermediate state and the final vibrational level of the ground electronic state, hgf |m|eri. It is worth mentioning that in the equation for the absorption (Eq. 14), the initial vibrational state |ii belongs to the ground electronic state |gi and the final vibrational state |f i belongs to the excited electronic state |ei, where in the KHD equation, both |f i and |ii belong to |gi and the vibrational states of |ei are the intermediate states |ri. In the preceding equations, and in all this work, the energy notation is permutated with frequency, because there is a relationship between them, provided by the Planck equation, E ¼ hn. Equation (3) is the starting point on the resonance Raman effect approach, which has been developed by Albrecht and others [102,124]. In this theory the Born–Oppenheimer approach is assumed, that is, the total wave function is considered as a product between an electronic and a vibrational function, for example, |gii ¼ |gi|ii. The dependence of the transition moment with the vibrational coordinates is included by a Taylor’s expansion in the normal modes: X m ¼ m0 + m0k Qk + (16) r mk0 ¼ (@m/@Qk)0. where Substitution of Eq. (4) into the numerator of Eq. (3) leads to several terms involving m0m0, m0m0 , and others. These terms are called Albrecht A term, B term, and so on. For example, the Albrecht A term is given by Chapter 10 Nondestructive Raman Spectroscopy Analysis IR ðoÞ∝ m0ge 4 X hf jr ihr jii Eer Egi + iG r 333 2 (17) For the enhancement of a normal mode by Albrecht A term, first of all, it is necessary that m0ge be different from zero, that is, the electronic transition must be electric dipole allowed. In a practical way, there will be a good chance to study the resonance Raman profile of chemical systems with an electronic transition, which presents a very high value of molar absorptivity. Furthermore, it is also necessary that the overlap integralshf |rihr|ii be different from zero. The bigger the horizontal displacement of the potential energy surface of the excited electronic state related to the fundamental one (the Dk parameter), the higher the vibrational quantum number r that results in bigger values for these integrals. From a physical point of view, the Dk displacement means that the equilibrium distance along the vibrational coordinate Qk is bigger in the excited state than in the ground electronic state, being one of the main values that we can obtain with the calculations via resonance Raman profiles because this is information about how much the molecule is distorted when the electronic transition is going on. The potential energy surfaces of the two states are arbitrarily designed with the same curvature, that is, we assume that the vibrational frequency in the excited state, oek, is the same of the ground state, ogk. Since this is not the general case, we can also consider oek as an additional parameter. Therefore, the more complex is the theoretical models to the involved electronic states, more parameters are needed to be incorporated to the correspondent expressions, and a more detailed picture of the states can be obtained. If the normal mode Qk is a non-totally symmetrical mode, the Dk displacement implies that the symmetry of the molecule in the excited electronic state is different from the ground state. In the case that we assume the same symmetry, Dk is zero for the non-totally symmetrical modes, and the two potential energy surfaces are identical; the vibrational wave functions are an orthogonal set hm|ni ¼ dmn, where dmn ¼ 1 if m ¼ n and dmn ¼ 0 if m 6¼ n, and the product of the overlap integrals is zero. For example, consider the vi ¼ 0 ! vf ¼ 1 transition: The products h1|0ih0|0i, h1|1ih1|0i, etc., in this case are zero for all the intermediate levels. The enhancement of non-totally symmetrical modes can be explained analyzing the Albrecht B term, where the KHD equation numerator contains the term m0mk0 hf|rihr|Qk|ii. The integrals are different of zero with a combination of vibrational quantum numbers, for example, h1|1ih1|Qk|0i, because |0i is even, |1i is odd, and Qk is also even, so the product between them will be odd and the integral does not void. The transition moment derivative related to the normal coordinate, mk0 , can be treated as another adjustable parameter to obtain the best agreement between theoretical and experimental fits. In the Albrecht theory, it is given a more detailed meaning to mk0 present in the B term when we use the 334 Studies in Natural Products Chemistry so-called Herzberg–Teller coupling, which is a perturbation theory treatment where one electronic wave function is expanded in all the others. The expansion coefficients involve Hamiltonian derivatives related to the normal coordinate, and then the Herzberg–Teller expansion looks like a type of vibronic coupling. As a general result in quantum chemistry perturbation theory, the several electronic states are coupled with a weight factor that is given by the inverse of the difference between them, leading B term to be smaller than A term. The Albrecht C term includes the coupling between the ground electronic state and more than one excited state and is smaller than the B term due to the great energy difference between them for the majority of the systems. The D term is also so small because it includes the terms with two derivative products of the transition moment mk0 mk0 . However, the calculation of resonance Raman profiles by the direct use of the KHD equation is so difficult in the case of polyatomic molecules due to the sum of a great number of intermediate states, even using the A term or using the great order terms B, C, and D of Albrecht theory. In a polyatomic molecule with N atoms, there will be 3N 6 normal modes, and the |vi term is an abbreviate notation used for the vibrational quantum number set of all modes, that is, |v1v2v3 . . . v3N 6i. Each possible combination of the vi vibrational quantum numbers is an intermediate state to be considered in the KHD equation, easily leading to millions of terms! However, experimentally, what can be done is to obtain the Raman excitation profile of the investigated chemical system and, after the observation of the enhanced vibrational modes, try to identify which molecule groups are directly involved in the electronic transition. Both ordinary and resonance Raman techniques have been used to characterize a diverse array of biological systems, from proteins and amino acids, lipids and fatty acids, and carbohydrates to phenolic substances, terpenoids, alkaloids, and polyacetylenes [112]. It is a nondestructive technique, which when coupled with microscopy can be very useful for qualitative and quantitative analyses. INSTRUMENTATION The modern Raman spectroscopy uses a monochromatic laser beam as a source of electromagnetic irradiation; the instruments can be equipped with dispersive elements (diffracting light) operating with argon ion laser at 457.9, 488.0, or 514.5 nm wavelength (excitation line from blue to green), krypton ion laser at 530.9, 647.1, or 676 nm (excitation lines from yellow to red), helium–neon laser line at 632.8 nm, or even solid state diode lasers operating at 785 or 830 nm (red and infrared). Instruments can also be equipped with interferometers operating in the infrared region with Nd:YAG diode laser at 1064 nm. There are other types of Raman instruments, besides the benchtop spectrometers, such as the fiber-optic spectrometers, where the Chapter 10 Nondestructive Raman Spectroscopy Analysis 335 laser arrives at the sample by an optical fiber, which can be several meters long, and Raman microscopes, where it is possible to obtain Raman spectra with microns of optical resolution. Raman spectra may be obtained for a wide range of compounds, including solid, liquid, or gaseous samples, in the pure form, or as a part of a complex matrix, such as in marine organisms. Raman microscopy is a highly selective and sensitive method of studying the distribution, concentration, and molecular structure of in situ and in vivo samples. APPLICATION OF RAMAN SCATTERING TO MARINE NATURAL PRODUCTS: AN OVERVIEW The main advantage of using Raman spectroscopy in natural products is the versatility of the technique in analyzing samples in situ as well as crude extracts and pure compounds. It has been extensively applied to terrestrial plants and animals for the identification of carotenoids [125–127], diterpene acids [128], sesquiterpenes, monoterpenes [112,129], essential oils [106,129–131], fatty acids, sterols [132–134], polyphenols [135], flavonoids [112,129,136,137], alkaloids [112,127,138], polyacetylenes [127,129,131], and wood resins [133,139]. Studies involving marine organisms are emerging, and Raman analysis has been applied to animals, algae, and dinoflagellates, which have identified carotenoids, sterols, nonsubstituted conjugated polyenals, chromophores of green fluorescent proteins (GFPs), chlorophylls, melanins, and mycosporine-like amino acids (MAAs). Carotenoids have been identified from corals [140–142], mollusks [143,144], lobster carapace [145,146], fish [147], brown algae [148] and dinoflagellates [149]. Polyenals are a recently described class of polyenes identified from shells of mollusks [143,144] and octocorals [140–142,150–153]. Pigments such as melanin from cuttlefish ink sacs of Sepia officinalis [148], chromophore of GFPs from anthozoan corals [154], and chlorophylls [155,156] have also been characterized. The ubiquitous mycosporine and MAAs are UV-absorbing molecules present among aquatic organisms, particularly coral reef algae and corals [157]. They are key biomolecules that have been detected by Raman spectroscopy in living and fossil organisms that lived in suitable and extreme environments [158]. CHARACTERIZATION OF METABOLITES FROM MARINE ORGANISMS Carotenoids are tetraterpenes widely distributed in marine animals [159], algae, and dinoflagellates [160]. The most common carotenoids found in marine animals are b-carotene, astaxanthin (Fig. 5), and their derivatives [161]. However, they are not endogenously biosynthesized but are incorporated from dietary intake or from the coral-associated microbiota. Another source of carotenoids in animals is derived from symbiotic 336 Studies in Natural Products Chemistry FIGURE 5 Carotenoids from marine organisms identified by Raman spectroscopy. Chapter 10 Nondestructive Raman Spectroscopy Analysis 337 relationship with dinoflagellates, which are known to produce xanthophylls with acetylenic, allenic, epoxy and acetoxy structural elements [162]. The molecular arrangement of isoprenic units in tetraterpenes results in symmetrical compounds with an extended conjugated double-bond system, which acts as a light-absorbing chromophore responsible for the yellow, orange, or red color. The scattering efficiency of conjugated system of p electrons in such polyenes has been particularly well characterized by Raman spectroscopy, which became a technique of choice for identification of different types of carotenoids (Table 2). Strong bands appear in the Raman spectrum within the 1500–1550 and 1150–1170 cm 1 ranges, due to in-phase n1(C]C) and n2(CdC) stretching vibrations of the polyenic chain. Additionally, a band of medium intensity is seen in the 1000–1020 cm 1 region, due to in-plane rocking mode of r3(CdCH3) groups attached to the polyenic chain. The wave number position of CdC stretching modes of a particular molecule is influenced by the individual number of conjugated double bonds, by the terminal groups of the polyene chain, and by their interaction with other compounds. For instance, astaxanthin (Fig. 5 and Table 2) identified from crude extracts and tissues of octocorals Muricea atlantica, Leptogorgia punicea, and Carijoa riisei [142] and from lobster carapace [146] presented vibrational bands at ca. 1520 n1(C]C), 1159 n2(CdC), and 1008 cm 1 r3(CdCH3). However, in the blue a-crustacyanin and b-crustacyanin, which are carotenoproteins containing astaxanthin from lobster carapace, the n1 mode is shifted from 1523 to 1492 and 1498 cm 1, respectively [146]. Unidentified carotenoids presenting Raman bands at ca. 1516 n1(C]C), 1159 n2(CdC), and 1014 cm 1 r3(CdCH3) were reported for hydrocoral Stylaster spp. [119,131], which are known to contain astaxanthin and zeaxanthin [167,168]. Most of the zooxanthellate corals accumulate carotenoids typical of dinoflagellates, as the allenic oxo-carotenoid peridinin (Fig. 5) with a unique C37 skeleton, identified by Raman spectroscopy (Table 2) from extracts of the dinoflagellate Amphidinium carterae [149] and from the zooxanthellate octocoral Phyllogorgia dilatata [141]. The characterization in P. dilatata was performed by analysis of major bands at 1929 nas(C]C] C), 1527 n1(C]C), 1185 d(CdH), 1156 n2(CdC), 1145 d(CdH), and 1008 cm 1 r3(CdCH3) [141]. A C40 allenic carotenoid fucoxanthin (Fig. 5 and Table 2) was identified from the brown algae Laminaria saccharina [146,164] and the freshwater diatom Cyclotella meneghiniana [163] due to major bands around 1532 n1(C]C), 1156 cm 1 n2(CdC), 1003 r3(CdCH3), and 1020 cm 1 n3(CdCH3). The acetylenic carotenoid diadinoxanthin identified from C. meneghiniana [163] and P. dilatata was characterized due to Raman bands at 2173 n(C^C), 1537 n1(C]C), 1159 n2(CdC), and 1019 r3(CdCH3). Similar light-scattering properties have also been observed in the unmethylated polyenic pigments identified from parrot’s feathers [169], mollusks [143,144,170], and octocorals [140–142,150,152,153]. The main 338 Studies in Natural Products Chemistry TABLE 2 Raman Wave Numbers (cm 1) and Vibrational Assignments of Compounds Identified from Marine Organisms Compounds Raman bands (cm 1) Tentative assignment Occurrence Carotenoids b-carotene (Fig. 5) 1515–1529 1156–1158 1004–1012 n1(C]C) n2(CdC) r(CdCH3) Microalgae and mollusks Astaxanthin (Fig. 5) 1512–1521 1156–1160 1005–1015 n1(C]C) n2(CdC) r(CdCH3) Octocorals [142,146] and crustaceans Peridinin (Fig. 5) 1929 1522–1526 1181–1184 1147 nas(C]C]C) Dinoflagellates n1(C]C) and octocorals d(CdH) n2(CdC) [141,149] Diadinoxanthin (Fig. 5) 2173 1531–1537 1159–1160 1015–1019 n(C^C) n1(C]C) n2(CdC) r(CdCH3) Dinoflagellates and octocorals [142,163] Fucoxanthin (Fig. 5) 1532–1536 1150–1156 1000–1003 1018–1020 n1(C]C) n2(CdC) r(CdCH3) n3(CdCH3) Brown algae [148,163,164] Polyenals (Fig. 6) 1500–1530 1116–1130 n1(C]C) n2(CdC) Octocorals and mollusks [140–143,150, 152,153] Sterol 1716, 1606 (23-Keto-cladiellin-A) 1663, 1606 (Fig. 7) 1452 n(C]O) n (C]C) d(CdH) Octocoral [152] References [143,156] Green fluorescent protein (GFP) (Fig. 7) 1641,1560,1603 n(C]C) 1560, 1446 n(C]N) Octocorals [154] and Hydrozoans Eumelanin (Fig. 4) 1580 1400 n(C]C) n(CdN) Mollusks Chlorophyll a (Fig. 7) 1670 1605, 1495 1398 1325 915 744 n(C]O) Microalgae n(CdC) d(CH3) n(CdN) d(NdCdC) d(HdCdO) [165,166] [156] Chapter 10 339 Nondestructive Raman Spectroscopy Analysis o o o o o n=8 n=9 n = 10 n = 11 n = 12 FIGURE 6 Polyenals from octocorals identified by Raman spectroscopy. differences between polyenals and carotenoids that can be observed from Raman spectroscopic analysis are the red-shifted wave number positions of the CdC stretching mode of polyenals by ca. 30 cm 1 when compared to carotenoids, as well as the absence of the deformation mode related to the CdCH3 group in the spectra at ca. 1000 cm 1 [169]. Major Raman bands from in situ analysis ranging from 1500 to 1530 n1(C]C) and 1120 to 1130 cm 1 n2(CdC) (Fig. 6 and Table 2) have been identified in pink and purple tissues of the octocorals Renilla muelleri, Muricea flamma, L. punicea, L. violacea, P. dilatata [141,142,152], Chromonephthea braziliensis [142,152], and Corallium rubrum [140,150,151,153]. Yellow polyps of C. braziliensis and yellow tissues of L. setacea presented a mixture of pigments ranging from 1538 to 1520 n1(C]C) and 1135 cm 1 n2(CdC) observed by analysis in different laser lines [142]. Colored shells of several species of mollusks showed the same patterns of Raman bands ranging from 1500 to 1530 n1(C]C) and 1120 to 1130 cm 1 n2(CdC) [143,144]. Indeed, coloration due to extended conjugation of polyenes is known to be dependent of the number of CdC double bonds present in the chain. The purple pigment from R. muelleri and P. dilatata showed major vibrational modes at ca. 1500 n1(C]C) and 1116 cm 1 n2(CdC), which has been attributed to 12 carbon double bonds [170,171]; however, the yellow L. setacea present a mixture of pigments with n1(C]C) bands at 1538 and 1520 and n2(CdC) band 1135 cm 1 [142] corresponding to a range of 8–10 carbon double bonds (Fig. 6). Experimental data have already demonstrated that in a homologous series of linear polyenes, there is a linear dependence of Raman shifts of resonantly coupled modes with inverse conjugation length; longer polyenic chains present lower Raman shifts [144,151,171]. Distinct classes of compounds have also been successfully identified from corals (Table 2), as the characterization of the antifeedant hemiketal sterol 23-keto-cladiellin A (Fig. 7) from the exotic octocoral C. braziliensis (Southwest Brazil), which presented main bands at 1716 n(C]O), 1663 n(C]C), 1606 n1(C]O)/n1(C]C), and 1452 d(CdH), also identified in 340 Studies in Natural Products Chemistry O HO O O OCH3 H HO HO H NH H COOH O Cladiellin-A Mycosporine-gly OH + CH3 N HO HN + CH3 4-Hydroxybenzylidene-2,3-dimethyl-imidazolinone (HBDI) N ++ N O Mg N N O O O O O COOH N H O HO COOH N H HO O COOH NH O OH Chlorophyll a Eumelanin FIGURE 7 Ecologically relevant molecules from marine organisms identified by Raman spectroscopy. the lipophilic crude extracts [152]. Pigments as chlorophylls from macroalgae [148], microalgae [156], and dinoflagellates [172] present bands at about 1670 n(C]O), 1605 n(CdC), 1495 n(CdC)/d(CH3), 1389 d(CH3)/d(CdH)/ n(CdN), 1348 d(CH3)/d(CdH)/n(CdN), 1325 n(CdN)/d(CdH), 988 d(CH3), 915 d(NdCdC)/d(CdCdC), and 744 cm 1 d(HdCdO)/d(NdCdC). Raman spectroscopy applied to photoprotective compounds from corals and associated organisms comprised FPs, melanins, and MAAs (Fig. 7). The corals Renilla reniformis and Aequorea victoria produce GFPs, which are best characterized by Raman analysis from their chromophore Chapter 10 Nondestructive Raman Spectroscopy Analysis 341 (4-hydroxybenzylidene-2,3-dimethyl-imidazolinone) due to vibrational modes at 1641 n(C]C), 1603 n(C]C), 1560 n(C]N)/n(C]C)), and 1446 cm 1 n(CdN) [154]. The well-known pigment melanin, typical of vertebrates and invertebrates, presented characteristic Raman bands near 1580 n(C]C) and 1400 n(CdN) identified from the cuttlefish S. officinalis [165,166]. The UV filters MAAs are ultraviolet-absorbing molecules widely distributed in marine organisms [173] that have been characterized due to Raman wave numbers at 1493, 1414, 1340, 1293, 1215, 1181, 1150, 920, 845, and 485 cm 1 [158]. Regardless of the organic composition, corals, shells, and pearls of mollusks share a common fingerprint in Raman bands attributed to CaCO3. Carbonates are biogenic minerals that compose skeletal structure of a vast array of marine organisms. Biomineralization is a dynamic and physiological process by which organisms transform ions in a solution into a solid structure. The polymorphs calcite and aragonite are the most commonly found and can be differentiated by Raman spectroscopy due to vibrational modes near 1089 n1(CO3) and 717 cm 1 n4(CO3) and near 1085 n1(CO3) and 705 cm 1 n4(CO3), respectively [174]. Data shown in Table 2 are the main Raman bands and vibrational assignments of compounds identified from different marine organisms. RAMAN SPECTROSCOPY APPLIED TO BIOLOGICALLY RELEVANT NATURAL PRODUCTS Corals produce natural products derived from primary and secondary metabolism, which are used in survival, maintenance, and chemical defense [93,175,11]. Diverse ecological roles have been attributed to crude extracts and pure compounds based on field and laboratory assays [11]. The chemical composition of biologically relevant samples has been investigated by chromatographic methods and characterized by mass spectrometry, nuclear magnetic resonance spectroscopy, infrared spectroscopy, and ultraviolet spectroscopy. A new alternative to the conventional techniques is to combine Raman spectroscopy, which is a nondestructive method of analysis suitable for in situ studies of biomaterials as well as chemical compounds. These features make the technique attractive for application in ecological experiments aimed at observations and comparisons between undisturbed and disturbed ecosystems. Climate changes, eutrophication, coastal development and sedimentation, and overfishing are major problems in marine communities. One of the most studied events is the warming of seawater temperature, which promotes an expulsion of coral endosymbionts called zooxanthellae, causing a paling or whitening termed “bleaching” in the affected coral and loss of their associated pigment [176]. Zooxanthellae are unicellular photosynthetic organisms that are known to produce chlorophylls, xanthophylls [177], and MAAs [178] responsible for the construction and growth of tropical reefs through their symbiotic association with stony corals. In the mutualistic relationship, 342 Studies in Natural Products Chemistry zooxanthellae also provide the host with amino acids and fixed organic carbon, whereas corals provide zooxanthellae with nitrogen and phosphorus. Bleached corals are more susceptible to invasion by parasites and competitors [179], and the levels of chlorophylls, carotenoids [177], and MAAs in tissues and cells [180,181] are indicative of the degree of vulnerability to stressors. One particular feature of Raman spectroscopy is the analysis in situ and in vivo cells by Raman microspectroscopy and Raman microimaging as a method of studying location, distribution, and concentration of chemical compounds [103]. The distribution of carotenoids and chlorophylls in plants [112,125] and microalgae [182] has been well characterized. Studies performed with Raman mapping demonstrated in vivo measurement of nutrient status of the marine microalgae Dunaliella tertiolecta cells; the method was able to identify different populations of N-starved cells from those of N-replete ones, based on analysis of the levels of chlorophyll a and b-carotene compounds [156]. Raman microimaging has similarly been used for investigating the variation on lipid content in situ in cell analysis from two species of microalgae under normal growth and nutrient-depleted conditions [183]. Healthy cells from both species showed only the carotenoid component. On the other hand, nitrogen-starved cells showed the presence of signals from carotenoids, chlorophylls, and triglycerides. Differentiation of toxic and nontoxic strains of the marine diatom Pseudonitzschia was performed by analysis of domoic acid content [132,184], which is a neurotoxin that contaminates shellfish and causes human poisoning. Raman analysis has also been applied to chemotaxonomy; for example, it indicated a separation of clones from cyanobacteria and three classes of algae from phytoplankton (Bacillariophyceae, Chlorophyceae, and Prymnesiophyceae). The distinction was based on Raman bands observed in spectra of each clone cell [132]; taxonomic affinities of algae were observed due to different pigments associated with the different algal classes. The speed of running for detection and identification of toxic microorganisms makes the technique of interest from both an ecological and a public health viewpoint [132]. Raman spectroscopy for monitoring allelopathy is a promising technique that has been used for evaluating defensive secondary metabolites in the feeding deterrent hemiketal sterol from C. braziliensis: This is the first example of a nonpolyenic compound of ecological relevance identified by the technique. Experiments investigating allelopathy involving the exotic C. braziliensis and the endemic octocoral P. dilatata have showed that chemical substances induced necrosis on P. dilatata tissues, which may be a real threat to the local ecosystem [42]. Interestingly, damaged tissues of P. dilatata present purple pigments identified by Raman spectroscopy as polyenals inserted into sclerites [142]. This pattern of pigmentation, “purpling,” has also been observed in Caribbean Sea fans Gorgonia spp. and is produced in response to multiple biotic agents including aspergillosis [185,186]. Purpling surrounding necrotic tissues as reported in the gorgonians Gorgonia ventalina, G. flabellum [185], and P. dilatata [142] has been considered to be an inflammatory signaling of infection [187]. Immune Chapter 10 Nondestructive Raman Spectroscopy Analysis 343 response of G. ventalina to pathogen involved a melanization process coupled with an increase of abundance of purple sclerites associated with tissue necrosis due to aspergillosis or response to invading agents [185]. In corals and sponges, melanins are primary compounds of the innate immune system, being laid down as a physiochemical barrier in cellular response to infection [188]. Increase of melanin production in a non-normal pigmented tissue has also been observed in scleractinians Acropora millepora and Porites sp., which suggested the presence of generalized defense against localized stress [189]. Pigments derived from fluorescent proteins (FPs) are also implied in chemical defense of some scleractinians, which presented significant H2O2-scavenging activity and play a role in coral stress and immune responses [190]. One particular case of physiological response to bleaching of anemones is the correlation of loss of zooxanthellae with reduction of green FPs [191]. However, one of the primary functions of FPs is as a visual sign for other organisms; GPF-like proteins may confer colors perceivable to reef fish [192]. Besides organic molecules, characterization by Raman spectroscopy of biogenic minerals is also possible and important since they influence the chemistry of seawater, the composition of the sediment and reefs accumulating in the oceans, and the nature of diagenesis [174]. Raman studies have provided information about early steps of diagenesis, revealing changes in both organic and mineral composition of scleractinians [193]. Raman mapping microspectroscopy has been also used to evaluate cyclic changes between aragonite and organic matrix in the blue coral Heliopora coerulea, where each growth layer acts as an environment recording unit [194]. Raman has also demonstrated to be useful in evaluating the formation of phosphorus records on daily environmental variation [194]. In summary, Raman spectroscopy has proved to be a technique of choice to identify pigments, so it is suitable to monitor the presence and variations of FPs, polyenals, melanins, and carotenoids, among others, in coral tissues. It is also valuable in detecting MAAs, absorbing in the range if 310–360 nm, which are transparent molecules to visible light, that act as sunscreens to reduce UV-induced damage from marine organisms to high alpine lakes to polar seas [157]. They take part in an important photoprotective mechanism in the marine ecosystem that has been evolutionarily conserved [178]. Coral holobionts and symbiotic dinoflagellates are a rich source of a diverse structural suite of MAAs, which has been hypothesized to function as a source of intracellular nitrogen, play a role in stress responses, and have also been implied as active in photosynthetic processes [178]. CONCLUDING REMARKS We have demonstrated that the technique of Raman spectroscopy can be used as a complementary tool in taxonomy, monitoring nutrient availability (which play major hole in primary production and algal blooms), and investigation of 344 Studies in Natural Products Chemistry antioxidants and defense compounds. The input of nutrients by corals during skeleton formation and the constitution of both mineral and organic matrix are especially important in management of population growth of endangered species versus regular community in different sites. Coastal eutrophication by analyzing phosphorous records and other elements could be monitored, since nutrient enrichment and pollution can lead to deleterious effects on reef health. 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This page intentionally left blank Chapter 11 Insulin Resistance as a Target of Some Plant-Derived Phytocompounds Mohamed Eddouks, Amina Bidi, Bachir EL Bouhali and Naoufel Ali Zeggwagh Moulay Ismail University, Errachidia, Morocco Chapter Outline Introduction Insulin Resistance Phytocompounds Targeting of Insulin Resistance Amorfrutins Bassic Acid Caffeic Acid Christinin-A Cinnamaldehyde Cryptoleptine Diosgenin Epicatechin Galactomannan 4-Hydroxybenzaldehyde 351 352 355 355 355 356 357 357 358 359 359 360 361 Lagerstroemin and Flosin B Mangiferin Marsupin and Pterostilbene Myricetin Oleanolic Acid Oleuropeoside Paeoniflorin Stevioside Ursolic Acid Vanillin Conclusion Acknowledgment References 361 362 363 364 364 365 366 366 367 368 368 370 370 INTRODUCTION Actually, diabetes mellitus represents a serious health care problem. More than 371 million people have diabetes and this number is expected to increase dramatically [1], this explosive increase has already imposed a huge burden on health care systems and this will continue to increase in the future. People suffering from diabetes are not able to produce or properly use insulin. There are two types of diabetes, namely type 1 and type 2. Type 1 Studies in Natural Products Chemistry, Vol. 43. http://dx.doi.org/10.1016/B978-0-444-63430-6.00011-4 © 2014 Elsevier B.V. All rights reserved. 351 352 Studies in Natural Products Chemistry diabetes accounts for 5–10% of diabetes. Type 2 is the most common form of the disease, accounting from 90% to 95% of diabetes [1]. Emerging data suggest that both adipose tissue and islets isolated from both type 1 and type 2 diabetics are characterized by abnormally autoimmunological representation in human subjects and animal models [2]. Reduction in b cell glucose sensitivity after chronic exposure to hyperglycemia and/or islets death is the two causes of the reduction in insulin secretion in type 2 diabetes [3]. Type 2 diabetes is communally characterized by impaired insulinstimulated glucose disposal [3]. This phenomenon is well known as insulin resistance which occurs in muscle and adipose tissue. These tissues are known to be responsible for the insulin-mediated glucose uptake [4]. The aim of this review is to provide the available data about phytocompounds targeting insulin resistance associated with diabetes. The data information were collected from the available literature at various databases, including PubMed, Science Direct, and Scopus till September 2013. Key search words included: medicinal plants, phytochemicals, insulin resistance, antidiabetic plants, glucose uptake, and insulin sensitivity. Moreover, the antidiabetic activity of these phytocompounds, their chemical structure, the plant origin, and the mechanisms of action are discussed in the present review. INSULIN RESISTANCE Insulin resistance, a reduced biological effect of endogenous or exogenous insulin, is a common biochemical entity which is associated, either directly or indirectly, with a range of noncommunicable human diseases. The main sites of insulin resistance are known to be the decreased peripheral glucose uptake by muscle and adipose tissue and/or increase of hepatic glucose production (Fig. 1). Insulin resistance may be entirely genetically determined (as in rare syndromes of severe insulin resistance) or acquired, either during intrauterine development or during adolescence and adult life [5–8]. Relative insulin resistance is also a transient feature of a number of physiological states in human [9]. The clinical impact of insulin resistance ranges from subclinical hyperinsulinemia to major life-limiting disturbances of carbohydrate and lipid metabolism [10–13]. The main clinical concern derives from the association between impaired insulin action and the development of vascular disease [14–17]. Microvascular disease is a complication of type 2 diabetes mellitus, in which insulin resistance is a prominent feature. Moreover, atherosclerotic macrovascular disease has a more complex association with insulin resistance that extends beyond hyperglycemia [18]. Due to the unknown causes and development mechanisms, insulin resistance represents the main challenge in both type 2 and long-standing type 1 diabetes pharmacotherapy. Essentially, thiazolidinediones are used to improve insulin resistance via activation of a nuclear factor regulating the transcription Chapter 11 Insulin Resistance as a Target of Phytocompounds 353 FIGURE 1 Schematic representation of diabetes showing the role of the organs (pancreas, liver, muscle, and adipose tissue) in the pathophysiology of this disease. of genes involving in lipid and glucose metabolism known as peroxisome proliferator-activated receptor gamma (PPARg). Several sites of thiazolidinediones action have been identified such as improvement of muscle and liver insulin sensitivity and decreasing hyperinsulinemia [19]. In addition, thiazolidinediones failed to control insulin resistance complications such as diabetic dyslipidemia, hypercoagulation, fibrinolysis, hypertension, and some thiazolidinediones are now withdrawn from the Food and Drug Administration list due to serious side effects [19]. In this context, new generation of pharmacological agents has been investigated in order to renew hopes to control insulin resistance [20]. Consequently, interest was focused on the potential use of plants or their constituents in the treatment of type 2 diabetes. Some plants used in the folk medicine have been shown to contain some compounds able to stimulate the activity of PPARa and g [21]. Protein tyrosine phosphatases are known to be expressed in insulin-sensitive tissues and can act as negative modulators in insulin signal transduction by dephosphorylation of tyrosyl residues [22]. Protein tyrosine phosphatase 1B (PTP1B), a prototypical member of the protein tyrosine phosphate superfamily, is known to be a key negative regulator of both insulin- and leptin-signaling pathway by dephosphorylating the insulin receptor, insulin receptor substrates, and Janus kinase 2 [23]. Inhibition of PTP1B may improve type 2 diabetes by increasing insulin sensitivity and resistance in obesity [24]. In this view, some synthetic inhibitors were discovered; however, few was subject of clinical trials [25]. Trodusquemine, pentacyclic triterpenoids including oleanolic acid and its derivatives are some natural products which may act as PTP1B inhibitors [26–28]. Other signaling 354 Studies in Natural Products Chemistry pathways are involved in improving insulin sensitivity. In this view, Resveratrol (trans-3,40 ,5-trihydroxystilbene), a phytoalexin first isolated from the roots of white hellebore (Veratrum grandiflorum O. Loes) in 1940, has been shown to improve insulin sensitivity in type 2 diabetic patients. This effect seems to be mediated via phosphorylated protein kinase (Akt) pathway [29]. In addition, this famous natural product is known to upregulate the sirtuins (SIRTs) (SIRT1 and SIRT2) involved in many cellular processes such as fatty acid metabolism [30]. The insulin resistance syndrome, also known as the metabolic syndrome, is a common pathophysiological condition which is implicated in the development of type 2 diabetes, atherosclerosis, dyslipidemia, and hypertension [31–34]. The leading cause of mortality in people with the metabolic syndrome is cardiovascular disease (CVD). The close association between the metabolic syndrome and CVD may be due to the dyslipidemia [35–38]. The dyslipidemia observed in the metabolic syndrome is characterized in part by high plasma triglycerides and low high-density lipoprotein—cholesterol concentrations [39–41]. Although we have made major strides in treating the dyslipidemia associated with type 2 diabetes with both lipid-lowering and antidiabetic drugs, the optimal diet for treating this disorder remains controversial [42–44]. Other than unsaturated fats and fiber, little is known about what nutrients may be beneficial in treating metabolic dyslipidemia [45]. Insulin sensitivity may be affected by many circulating lipids, which included hypertriglyceridemia and increased free fatty acids (FFAs). Raised plasma FFA level is an important inducer of both peripheral and hepatic insulin resistance because it inhibits insulin signaling [46–50]. In addition, hypertriglyceridemia is also an important marker of insulin resistance. The traditional use of medicinal plants can lead to the discovery of new potent botanical agents in the treatment of several diseases. Some 7000 natural compounds are currently used in modern medicine most of these had been used for centuries by traditional healers [51]. In spite of the development of pharmacological agents for the treatment of diabetes, the use of medicinal plants continues to flourish. The WHO estimates that more than 1200 plants species are used to treat diabetes around the world representing more than 725 genera in 183 families ranging from marine algae to higher plants [51]. The phylogenetic distance between families is a strong indication of the varied nature of the active constituents and mechanism of actions. Approximately 80% of these plants have been reported to exhibit antidiabetic activity [52]. Thus, the study of traditional remedies for diabetes might yield an excellent return in potential sources of antidiabetic drugs [52]. Although various synthetic drugs were developed to treat diabetes, but few number of drugs is available for the treatment of this chronic pathology. In addition, about 200 pure compounds derived from plant sources are reported to exhibit blood glucose-lowering activity. The phytocompounds are known Chapter 11 Insulin Resistance as a Target of Phytocompounds 355 to include flavonoids, alkaloids, steroids, carbohydrates, glycosides, terpenoids, peptides and amino acids, lipids, phenolics, glycopeptides, iridoids, etc. [53]. However, not all the plants reported to be useful in the treatment of diabetes around the world are safe, they emphasize the need for carefully planned scientific research to identify those hypoglycemic plants with true therapeutic efficacy and safety [53]. It is worth bearing in mind that the use of Galega officinalis from ancient time for the treatment of diabetes has led to the discovery of guanidine. The synthesis of biguanidine, a third generation of guanidine derivatives, was efficacious as a new class of antidiabetic agent actually known as Metformin [53]. PHYTOCOMPOUNDS TARGETING OF INSULIN RESISTANCE Amorfrutins Amorfrutins, a family of isoprenoid-substituted benzoic acid derivatives without any stereocenters, were investigated in vitro by binding and cofactor recruitment assays and by transcriptional activation assays in primary human adipocytes and murine preosteoblasts, as well as in vivo using insulin-resistant high-fat diet-fed C57BL/6 mice [54]. The amorfrutins, amorfrutin 1, amorfrutin 2, amorfrutin 3, and amorfrutin 4, have been identified from edible parts of two legumes, Glycyrrhiza foetida and Amorpha fruticosa (Fabaceae). This family of natural products has been demonstrated to improve insulin resistance in diet-induced obese and db/db mice [55]. Amorfrutin 1 considerably enhanced glucose tolerance (19%) during oral glucose tolerance tests [55]. In addition, the amorfrutins have been shown to bind and activate selectively PPARg, an important gene regulator in glucose and lipid metabolism, without any undesirable side effects on osteoblastogenesis and fluid retention while PPARg-activating drugs of the thiazolidinedione class are known to provoke adverse side effects including weight gain [55]. Amorfrutin B has been identified as a novel partial agonist of PPARg with a considerably higher affinity than that of previously reported amorfrutins, similar to that of rosiglitazone [56]. Moreover, in insulin-resistant mice, amorfrutin B considerably improved insulin sensitivity, glucose tolerance, and blood lipid variables after several days of treatment [55]. Finally, amorfrutins treatment strongly improves insulin resistance and other metabolic and inflammatory parameters without concomitant increase of fat storage or other unwanted side effects such as hepatoxicity (Fig. 2) [55]. Bassic Acid Bassic acid is an unsaturated triterpene isolated from an ethanolic extract of Bumelia sartorum (Sapotaceae). This plant is known in Brazilian folklore 356 Studies in Natural Products Chemistry O O OH O OH OH O Amorfrutin 1 OH Amorfrutin 2 O O OH OH OH OH OH O Amorfrutin 4 Amorfrutin 3 FIGURE 2 Amorfrutins 1, 2, 3, and 4. HO H COOH HO HO FIGURE 3 Bassic acid. for the treatment of diabetes mellitus and inflammatory disorders. The hypoglycemic activity of B.melia sartorum has been demonstrated using the ethanol extract of root bark in normal and alloxan-induced diabetic rats [57]. The underlying mechanism of action seems to be the increase of glucose uptake in skeletal muscle and the inhibition of glycogenolysis in the liver [57]. A per os administration of bassic acid induced a significant hypoglycemic activity in alloxan-diabetic rats and altered the pattern of glucose tolerance [58]. Additionally, bassic acid treatment increased significantly the glucose uptake process and glycogen synthesis in isolated rat diaphragm ameliorating the insulin sensitivity of peripheral tissues. An increase of plasma insulin levels has been also reported in alloxan-diabetic rats (Fig. 3) [58]. Caffeic Acid This phenolic compound, present in Xanthium strumarium (Compositae), has been demonstrated to exhibit antidiabetic activity in streptozotocin-induced Chapter 11 Insulin Resistance as a Target of Phytocompounds 357 HO OH HO O FIGURE 4 Caffeic acid. diabetic rats with a dose-dependent manner and this effect seems to be the result of the elevated glucose utilization induced by caffeic acid [59]. More recently, caffeic acid from Prunus mume (Rosaceae) fruits has been identified and seems to be involved in the antidiabetic effect of this plant, either in vitro (high-fat diet mice) or in vivo. In db/db mice, treatment with caffeic acid caused 20% of decrease in blood glucose levels [60]. This blood glucoselowering activity may be mediated, at least in part, by the activation of PPARg. Indeed, treatment with an extract of this plant increased glucose uptake in C2C12 myotubes and also increased PPARg activity or PPARg mRNA expression [61]. In addition, caffeic acid phenethyl ester induced antidiabetic activity in the special model of CsA/STZ. This antidiabetic effect may be related to its antiinflammatory and angiostatic effects [62]. Finally, caffeic acid was shown to represent a possible candidate involved in the glucose transport-lowering activity elucidated in the effect of a polyphenol-rich herbal extract in Caco-2 intestinal model (Fig. 4) [63]. Christinin-A Christine-A is the major saponin glycoside present in Zizyphus spina-christi (Rhamnaceae), a commonly used plant in the folk medicine especially in North Africa. This compound has been shown to exert hypoglycemic activity in streptozotocin-induced diabetic rats. The oral administration of christinin-A (100 mg/kg) to type 2 diabetic rats reduced the serum glucose level after 60 and 120 min of administration. The maximal percentages of reduction which occurred after 60 min of administration were 20% and 19% in control rats as well as 24% and 22% in type 2 diabetic rats, respectively [64]. This effect is the result of many cellular changes such as a reduction of liver phosphorylase and glucose-6-phosphatase activities, and a significant increase on serum pyruvate level, liver glycogen content, serum insulin, and pancreatic cAMP levels [65]. Additionally, in type 2 but not in type 1 diabetic rats, pretreatment with christinin-A improved the oral glucose tolerance and potentiated glucose-induced insulin release (Fig. 5) [64]. Cinnamaldehyde Cinnamaldehyde (CA) has been reported to be the major component of volatile oils from cinnamon (Lauraceae). CA has been demonstrated to exert 358 Studies in Natural Products Chemistry OH O O O OH OH OH O HO OH O O HO O O OH OH FIGURE 5 Christinin-A. O FIGURE 6 Cinnamaldehyde. antihyperglycemic and antihyperlipidemic actions in C57BLKS/J db/db mice. On day 28 of treatment fasting blood glucose levels in the CA group showed a significant reduction compared with the control group [66]. The beneficial property of this compound on insulin resistance has been explained by the upregulation of mRNA expression of glucose transporter GLUT-4 in skeletal muscle and the inhibition of mRNA expression of tumor necrosis factor-alpha in adipose tissue [66]. Moreover, CA has been demonstrated to exert antidiabetic effects through modulation of the PPARg and AMP-activated protein kinase signaling pathways [67]. No toxicity of CA was found in either acute or subchronic toxicity studies. Methanol extract of Cinnamomum burmannii (containing 0.07% and 0.20% of coumarin and trans-cinnamaldehyde, respectively), which was given orally at doses of 500, 1000, and 2000 mg/kg caused neither visible signs of toxicity nor mortality (Fig. 6) [68]. Cryptoleptine Cryptolepine is an indoloquinolone alkaloid isolated from Cryptolepis sanguinolenta (Asclepiadaceae) which is widely used traditionally in folklore medicine in many parts of the world for the management, and/or treatment of a plethora of human ailments, including diabetes mellitus. This compound Chapter 11 Insulin Resistance as a Target of Phytocompounds 359 was found to significantly lower blood glucose level when given per os to a mouse model of diabetes [69]. The percentage of reduction on blood glucose levels was near 20% [70]. The antihyperglycemic activity of cryptolepine is mediated by the elevation of glucose uptake. This mechanism of action has been demonstrated in vitro by the increase of glucose transport in 3T3-L1 cells. Cryptolepine caused a significant decline in plasma insulin concentration [69]. However, this finding is not in accordance with a recent study showing that the C. sanguinolenta enhanced the structural and functional abilities of the b cells [70]. The treatment of mammalian cells with cryptolepine can lead to DNA damage and suggest that the routine use of C. sanguinolenta and the potential use of Cryptolepine derivatives in malaria chemotherapy could carry a genotoxic risk (Fig. 7) [71]. Diosgenin Isolated from Trigonella foenum-graecum (fenugreek) (Fabaceae), diosgenin is a steroidal sponin, which has been demonstrated to contain antidiabetic and hypolipidemic activities [72]. In streptozotocin-induced diabetic rats, supplementation of commercial diosgenin resulted in about sixfold decrease in fasting blood glucose compared to the control group [73]. Diosgenin miniaturized the adipocytes and increased the mRNA expression levels of differentiation-related genes in adipose tissues. Fenugreek also inhibited macrophage infiltration into adipose tissues and decreased the mRNA expression levels of inflammatory genes. In addition, diosgenin promote adipocyte differentiation and inhibit expressions of several molecular candidates associated with inflammation in 3T3-L1 cells [74]. A recent study confirmed the antidiabetic activity of diosgenin in high-fat diet mice through the modulation of PPARs [75]. These results suggest that diosgenin may be beneficial in ameliorating insulin resistance associated with diabetes and obesity. A study revealed that diosgenin has potent anticlastogenic effects on 7,12dimethylbenz(a)anthracene-treated hamsters (Fig. 8) [76]. Epicatechin Epicatechin, a flavonoid isolated from Pterocarpus marsupium L. (Fabaceae), has been shown to exert antidiabetic activity especially in type 2 diabetes situation [62]. The beneficial effect of this compound on insulin resistance has been evoked as an underlying mechanism of hypoglycemic activity. In N N FIGURE 7 Cryptoleptine. 360 Studies in Natural Products Chemistry O O H H H H HO FIGURE 8 Diosgenin. OH HO OH O OH OH FIGURE 9 Epicatechin. addition, this flavonoid may also inhibit a-glucosidase in vitro [77]. A significant reduction (about 60%) of blood glucose level was noticed in type 2 diabetic rats after the oral administration of aqueous extract of Terminalia paniculata bark (Combretaceae)-containing epicatechin when compared to diabetic control rats [78]. More recently, a study has demonstrated that cacao liquor procyanidin extract, which consists of 4.3% catechin, 6.1% epicatechin, 39.4% procyanidins, and others, ameliorated hyperglycemia and obesity in C57BL/6 mice through many molecular mechanisms including suppression of high-fat diet-induced hyperglycemia, glucose intolerance, and fat accumulation in white adipose tissue. The extract also promoted translocation of glucose transporter 4 and phosphorylation of AMP-activated protein kinase a in the plasma membrane of skeletal muscle and brown adipose tissue. Finally, phosphorylation of AMP-activated protein kinase was also enhanced in the liver and white adipose tissue [79]. It has been noticed that catechins, a family of polyphenols found in tea, evoke various responses, including cell death (Fig. 9) [80]. Galactomannan Galactomannan is a polysaccharide isolated from T. foenum-graecum (fenugreek) (Fabaceae). This compound has been shown to be involved in the antidiabetic and hypolipidemic activities of fenugreek in diabetic rats [72]. After the oral glucose tolerance test, plasma glucose concentrations in type 2 diabetic patients receiving fenugreek were significantly lowered (about 20%) at 120 min as compared to the control groups [72]. This effect was due to the Chapter 11 361 Insulin Resistance as a Target of Phytocompounds presence of galactomannans. The beneficial effect of this compound on insulin resistance resulted from the promotion of adipocyte differentiation and inhibition of several molecular candidates associated with inflammation in 3T3-L1 cells [74]. No eventual toxicological effect of galactomannan has been reported (Fig. 10). 4-Hydroxybenzaldehyde 4-Hydroxybenzaldehyde, one of the three isomers of hydroxybenzaldehyde isolated from Gastrodia elata Blume (Orchidaceae), has been involved in the beneficial effect on insulin resistance in a type 2 diabetic animal model (rats fed a high-fat diet). The mechanism of action of this compound includes the potentiation of glucose uptake demonstrated in 3T3-L1 adipocytes as well as the stimulation of glucose metabolism and the inhibition of hepatic glucose production [81]. Moreover, G. elata Blume water extract mainly as a result of the action of 4-hydroxybenzaldehyde, reduced insulin resistance by decreasing fat accumulation in adipocytes by activating fat oxidation and potentiating leptin signaling in diet-induced obese rats [81]. Additional studies are needed to eventual the efficacy and safety of 4-hydroxybenzaldehyde (Fig. 11). Lagerstroemin and Flosin B The leaves of Lagerstroemia speciosa (Lythraceae), a Southeast Asian tree more commonly known as Banaba, have been traditionally consumed in various forms by Philippinos for treatment of diabetes. The antidiabetic effects of OH OH H HO H H OH H OH OH O O HO H H H O O O H H H H H OH OH OH O OH HO H H H H FIGURE 10 Galactomannan. O OH FIGURE 11 Hydroxybenzaldehyde. H OH O H H n 362 Studies in Natural Products Chemistry L. speciosa L. (Lythraceae) were studied using hereditary diabetic mice (type II, KK-Ay/Ta Jcl) [82]. Furthermore, a study revealed the stimulation of glucose uptake by lagerstroemin and Flosin B, two active polyphenols of L. speciosa in isolated adipocytes [83]. A significant decrease (16.6%) in fasting blood glucose levels was observed in individuals with fasting blood glucose levels greater than 110 mg/dL [84]. After both 6 months and 1 year, significant improvements were observed with respect to glucose tolerance and glycated albumin following treatment with the Banaba extract. No adverse effects have been observed or reported in any studies involving human subjects receiving Banaba (Fig. 12) [84]. Mangiferin Isolated from the rhizome of Anemarrhena asphodeloides (Asparagaceae), mangiferin is a xanthonoid known in traditional oriental medicine for its antidiabetic property and has been used for treatment of diabetes as well as the related symptoms such as polyuria and polydipsia. The antidiabetic activity of the rhizome of A. asphodeloides was investigated in KK-Ay mice, an animal model of genetic type 2 diabetes [85]. The water extract of the rhizome induced a decrease of blood glucose levels when administered per os and also reduced the insulinemia in the diabetic mice. Especially, the water extract of A. asphodeloides reduced significantly the blood glucose levels in an insulin OH OH OH HO OH HO R1 O HO O HO OH H R2 O O H H O H O O O OH OH OH O CH2 OH HO OH O O HO O O O O OH OH 1. R1 = OH, R2 = H 2. R1 = H, R2 = OH FIGURE 12 Lagerstroemin (1) and Flosin B (2). Chapter 11 Insulin Resistance as a Target of Phytocompounds 363 tolerance test. Therefore, this finding indicates that A. asphodeloides exerted the antidiabetic activity by decreasing insulin resistance. Moreover, recent studies have demonstrated that upregulation of glyoxalase 1 by mangiferin improved impaired glucose intolerance and prevented diabetic nephropathy progression in streptozotocin-induced diabetic rats [86,87]. In addition, treatment of the 3T3-L1 cells with mangiferin increased the glucose utilization in a dose-dependent manner [88]. In addition, mangiferin concentration has been demonstrated to be safe within 12.5–100 mmol/L (Figs. 13 and 14) [89]. Marsupin and Pterostilbene Marsupin and Pterostilbene (stilbenoid) are isolated from P. marsupium L. (Fabaceae). This plant is known for its antidiabetic property and the beneficial role of marsupin and pterostilbene on insulin resistance as antidiabetic agents has been demonstrated [77]. Treatment with the P. marsupium extract for 30 days significantly lowered the serum glucose levels in comparison with control group in diabetic rats feeding an adequate diet, with fructose as sole carbohydrate [90]. Chronic treatment with pterostilbene remarkably reduced the pathological changes observed in liver and kidney of diabetic rats indicating an antioxidant property [91]. Moreover, the activities of the hepatic enzymes such as hexokinase were significantly increased, whereas glucose6-phosphatase and fructose-1,6-bisphosphatase were significantly decreased by the administration of pterostilbene in streptozotocin diabetic rats [92]. OH O OH OH O HO OH HO OH O OH FIGURE 13 Mangiferin. OH OH OH HO O OH O OH OH O O HO FIGURE 14 Mangiferin-7-O-betaglucoside. OH O OH OH 364 Studies in Natural Products Chemistry OH O HO O O OH OH Marsupin O O Pterostilbene FIGURE 15 Marsupin and pterostilbene. Recently, a double-blind placebo-controlled intervention trial, enrolling patients with hypercholesterolemia, has shown that pterostilbene is generally safe for use in humans up to 250 mg/day (Fig. 15) [93]. Myricetin Myricetin is a phenolic compound isolated from the aerial part of Abelmoschus moschatus (Malvaceae). The hypoglycemic activity of myricetin has been investigated in streptozotocin-induced diabetic rats [94] using an i.v. injection which produced a significant decrease of the plasma glucose concentrations in a dose-dependent manner. The mechanism of action of myricetin seems to be the enhancement of glucose utilization [94]. Recently, EmulinTM, a patented blend of chlorogenic acid, myricetin, and quercetin has shown efficacy in reducing background blood glucose levels of type 2 diabetic patients [95]. In addition, myricetin purified from the aerial portion of the A. moschatus induced a dose dependent decrease in the plasma glucose concentration in obese Zucker rats when administered intravenously. Two weeks of treatment with myricetin provoked about 10% of blood glucose decrease in fructose-fed rats [96]. Moreover, myricetin improved insulin sensitivity through increased postreceptor insulin signaling mediated by enhancements in IRS-1-associated PI3-kinase and GLUT-4 activity in muscles of obese Zucker rats (Fig. 16) [97]. Oleanolic Acid This triterpenoid is a constituent of Cornus officinalis Sieb (Cornaceae) and may be involved in the lowering glucose activity in diabetic rats [98]. A study has revealed that this compound increased the glucose transport activity and then the insulin action in peripheral target tissues [99]. A study designed to evaluate the effects of oleanolic acid on hepatic insulin resistance Chapter 11 Insulin Resistance as a Target of Phytocompounds 365 OH OH HO O OH OH OH O FIGURE 16 Myricetin. H OH H O HO H FIGURE 17 Oleanolic acid. and underlying mechanisms in db/db obese diabetic mice revealed that oleanolic acid decreased fasting blood glucose, improved glucose and insulin tolerance, enhanced insulin signaling, and inhibited gluconeogenesis [100]. Oral administration of oleanolic acid (100 mg/kg/day) caused about 10% of decrease on blood glucose levels in C57BL/6J mice fed a high-fat diet followed by low doses of streptozotocin to generate a type 2 animal diabetic model [101]. The limit of oleanolic acid safety has not been determined clinically (Fig. 17). Oleuropeoside The hypoglycemic activity of olive leaves is well known [90]. Oleuropeoside, a phenylethanoid isolated from Olea europaea (Oleaceae), demonstrated a significant hypoglycemic activity in alloxan-induced diabetes. Oleuropeoside showed the antihyperglycemic activity at a dose of 16 mg/kg [90]. Additionally, the hypoglycemic activity of this compound may result from both the increased peripheral uptake of glucose and potentiation of glucose-induced insulin secretion [102]. Extracts from O. europaea have been found to exhibit cytotoxic effects at concentrations higher than 500 mg/mL in cells from the liver hepatocellular carcinoma cell line (HepG2) and cells from the rat L6 muscle cell line (Fig. 18) [103]. 366 Studies in Natural Products Chemistry O O COOCH3 O HO O OH OH HO HO O OH H FIGURE 18 Oleuropeoside. Paeoniflorin Paeoniflorin, a monoterpene glucoside isolated from the root of Paeonia lactiflora Pall. (Panunculaceae), reduced the glucose levels in streptozotocininduced diabetic rats [104]. The use of P. lactiflora in Chinese medicine is very ancient especially as antispasmodic, tonic, astringent, and analgesic agent. Additionally, the above-mentioned compound has been demonstrated to decrease blood glucose levels in streptozotocin diabetic rats by increasing the glucose utilization and then the amelioration of insulin sensitivity [105]. However, paeoniflorin had no effect on phosphoenopyruvate carboxykinase expression in streptozotocin-induced diabetes and db/db mice [106]. Streptozotocin-induced diabetic rats treated with 5, 10, or 20 mg/kg paeoniflorin in drinking water once daily showed about 35% on blood glucose levels reduction (Fig. 19) [107]. Stevioside Stevioside is a glycoside present in Stevia rebaudiana Bertoni (Compositae). In type 2 diabetic patients, this compound has been shown to reduce postprandial blood glucose levels [108]. Medium-polar extract from leaves of S. rebaudiana, when administered orally (200 and 400 mg/kg) for 10 days, produced a significant dose-dependent reduction in blood glucose levels which were normalized at the 9th day of treatment [109]. The antidiabetic effect of this extract may be due to the presence of stevioside. The hypoglycemic effect of this compound may result from two mechanisms: (a) stimulation of insulin secretion and (b) improvement of glucose utilization in peripheral tissues and inhibition of hepatic glucose production mediated via inhibition of phosphoenolpyruvate kinase expression [110]. Stevioside might also ameliorate insulin resistance in HFD-fed mice by attenuating adipose Chapter 11 Insulin Resistance as a Target of Phytocompounds 367 OH O HO HO O O OH O OH O O FIGURE 19 Paeoniflorin. O O OH HO HO O OH H O H OH H O HO HO H O H H H OH H O HO HO H H OH H FIGURE 20 Stevioside. tissue inflammation and inhibiting the nuclear factor-kappa b pathway [111]. Acute toxicological study of S. rebaudiana extracts has shown a low toxicity of S. rebaudiana (Fig. 20) [110]. Ursolic Acid C. officinalis Sieb (Cornaceae) is used traditionally as a tonic. The antidiabetic activity of this plant has been reported in streptozotocin-induced diabetic rats when given per os [112]. Ursolic acid (0.14%), a pentacyclic triterpene acid, caused about 30% of reduction on blood glucose levels when administered for 6 weeks in high-fat diet mice [113]. The main underlying mechanism of action seems to be mediated by the increase of the insulin-sensitive glucose 368 Studies in Natural Products Chemistry H OH H O HO FIGURE 21 Ursolic acid. transporter GLUT-4 mRNA as well as its protein expression in diabetic rats and the amelioration of glucose transport [99]. The main chemical entities responsible for this activity are ursolic acid and oleanolic acid. Recently, ursolic acid has been shown to prevent high-fat diet-induced obesity in mice possibly by inhibiting pancreatic lipase activity [114] and to improve the glycometabolism and differentiation of 3T3-L1 adipocytes with insulin resistance by upregulating the expression of c-Cbl-associated protein [115]. Finally, the effects of ursolic acid in improving hepatic insulin resistance in KK-Ay mice with spontaneous type 2 diabetes may be closely related to: (1) the change in the contents of FFAs, tumor necrosis factor-alpha and adiponectin, (2) improvement of the expression of PPARa protein, (3) regulation of the transcription of phosphoenolpyruvate carboxykinase protein, and (4) induction of phosphorylation of insulin receptor substrate-2 (Fig. 21) [115]. Vanillin Vanillin is a phenolic aldehyde isolated from G. elata Blume (Orchidaceae) which has been demonstrated to improve insulin resistance in male Sprague-Dawley rats fed a high-fat diet. This compound induced an increase in whole-body glucose disposal rates and decreased hepatic glucose output. Moreover, vanillin decreased triglyceride accumulation by modulating the expression of genes involved in fat metabolism in 3T3-L1 adipocytes, activating fat oxidation, and potentiating leptin signaling in diet-induced obese rats. The resulted increased insulin-stimulated glucose uptake may explain the reduction of insulin resistance (Fig. 22) [81]. CONCLUSION In the present review, we focused on scientific studies of selected phytocompounds and their ability to target insulin resistance. Twenty-six selected phytocompounds have been investigated either in vivo using various animal models: streptozotocin-induced diabetic rats, high-fat diet mice and rats, and C57BLKS/J db/db mice or in vitro using some cellular lines essentially Chapter 11 Insulin Resistance as a Target of Phytocompounds 369 O HO O O OH FIGURE 22 Vanillin. FIGURE 23 Schematic illustration of the beneficial action of phytocompounds on insulin resistance. 3T3-L1 cells. Their beneficial action in terms of improving insulin resistance associated with diabetes has been elucidated. It has been shown that the potential phytocompounds targeted insulin resistance globally via several pathways: inhibition of hepatic glucose production and/or potentiation of peripheral glucose utilization in the muscles and adipocytes by regulating the activity and expression of key enzymes and glucose transporter GLUT-4 (Fig. 23). More detailed studies at molecular and cellular levels as well as in animal models are required to elucidate antidiabetic activity of phytocompounds and further clinical as well as toxicological studies of a lot of these compounds are needed to develop antidiabetic medicines with satisfactory efficacy and no severe undesirable adverse effects. 370 Studies in Natural Products Chemistry ACKNOWLEDGMENT Conflict of Interests. The authors report no conflicts of interest. REFERENCES [1] Annual report of International Diabetes Federation, 2012. www.idf.org. [2] S.J. Richardson, A. Willcox, A.J. Bone, A.K. Foulis, N.G. Morgan, Diabetologia 52 (2009) 1686–1688. 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Imam, F. Anwar, Eur. J. Pharmacol. 709 (2013) 28–36. [115] D. Li, G.L. Wang, M.Y. Shan, J.H. Liu, L. Wang, D.Z. Zhu, Zhong Xi Yi Jie He Xue Bao 10 (2012) 886–893. This page intentionally left blank Chapter 12 Saponins Produced by Gypsophila Species Enhance the Toxicity of Type I RibosomeInactivating Proteins Idris Arslan Biomedical Engineering, Faculty of Technology, Pamukkale University, Denizli, Turkey Chapter Outline Saponins 375 Gypsophila Saponins 376 Ribosome-Inactivating Proteins 377 Saponins as Cytotoxic Agents 378 Acknowledgment 379 References 380 SAPONINS Saponins are a widespread class of bioactive compounds produced by many plant species. Saponins consist of a hydrophobic polycyclic triterpenes C-30 or steroidal C-27 sapogenin (aglycone/genin) and one or two (rarely three) hydrophilic glycoside moieties attached to backbone. Because of their amphiphilic properties, saponins are amphiphilic glycoconjugates which are able to interact with cell membranes and are also able to decrease the surface tension of an aqueous solution. This activity is the reason for the name saponin, derived from the Latin word sapo, which refers to the formation of a stable soap-like foam in aqueous solution [1,2]. According to the chemical character of the aglycone (known as sapogenin), the saponins are divided into steroidal and triterpenoid saponins. Steroidal saponins from plants are mainly compounds containing 27C atoms forming the core structures, spirostan, and furostan. Triterpenoidal saponins mainly contain aglycones with 30C atoms or their nonderivatives (Fig. 1) [3]. Also reported are the presence of epoxy groups, keto functions, and double bonds between C-12 and C-13. The hydroxyl groups can be acylated, and this leads to the formation of ester saponins. Acidic components in such cases are very Studies in Natural Products Chemistry, Vol. 43. http://dx.doi.org/10.1016/B978-0-444-63430-6.00012-6 © 2014 Elsevier B.V. All rights reserved. 375 376 Studies in Natural Products Chemistry 29 A O 25 12 17 1 O H H 15 H H 1 H 2 30 B HO 4 H HO FIGURE 1 Chemical structure of the main aglycones steroidal (A) and triterpenic (B) saponins. often formic, acetic, n- and iso-butyric, isovalerianic, a-methyl butyric, angelic, tiglic, benzoic, cinnamic, ferulic acid, and in some cases, sulfuric acid [4–6]. The monosaccharide moieties of saponins include a broad spectrum of simple sugars, like D-glucose, D-galactose, D-fructose, 3-methyl-D-glucose, D-xylose, L-arabinose, L-rhamnose, L-fucose, D-apiose, and D-chinovose, in addition to D-glucuronic acid and D-galacturonic acid. The sugars often bind in position C-3 via the hydroxyl group as glycosides, or as esters bound via the carboxylic moiety in position C-28 [7]. Saponins present a broad spectrum of biological uses such as permeabilizing of the cell membrane, lowering of serum cholesterol levels, stimulation of luteinizing hormone release leading to abortifacient properties, immune stimulation, antitrombotic and hypocholesterolemic, ion channel blocking, immunomodulatory potential via cytokine interplay, cytostatic and cytotoxic effects on malignant tumor cells, adjuvant properties for vaccines as immunostimulatory complexes, and synergistic enhancement of the toxicity of immunotoxins [8–15]. Saponins have additionally been reported to exhibit adjuvant-active properties. An open cage-like immunostimulating complex of cholesterol, lipid, immunogen, and saponins from bark of Quillaja saponaria MOL. (soap bark tree) has found successful application as an active adjuvant for vaccination [16]. GYPSOPHILA SAPONINS Triterpenoid saponins are mainly found in dicotyledons in the plant kingdom. Approximately, 60 families of this taxon produce this type of saponin, including Apiaceae, Araliaceae, Caryophyllaceae, Fabaceae, Primulaceae, Ranunculaceae, and Theaceae [17]. As it can be clearly seen Fig. 2 that Gypsophila species (Caryophyllaceae) are an especially rich source of triterpenoidal saponins [18–20]. Gypsophila saponins are of interest in terms of their applications in vaccines [21]. Saponinum album (Merck) is a complex mixture of triterpenoid saponins from Gypsophila paniculata and Gypsophila arrostii which used to be commercially Chapter 12 377 Saponins Produced by Gypsophila Species O HO O HO HO O O O O O O R1 O HO O OH OH O O HO OH O OH HO HO OR2 O OH O O O OH HO OH OH HO OR3 Compound R1 R2 R3 Nebuloside A Nebuloside B Gypsophila saponin 1 Gypsophila saponin 2 Gypsophila saponin 3 -OH -H -OH -H -H -Xyl -Xyl -OH -OH -Xyl -OH -Gal -OH -OH -OH FIGURE 2 The most common saponin compounds found in Gypsophila genus. available. Also, they are exploited commercially for a variety of purposes including medicines, detergents, adjuvants, and cosmetics [20,22]. As it can be clearly seen Fig. 3, the majority of saponins in the Gypsophila genus possess gypsogenin, gypsogenic acid, hederagenin, or quillaic acid as aglyconic components [23,24] (Fig. 3). RIBOSOME-INACTIVATING PROTEINS Ribosome-inactivating proteins (RIPs) are EC 3.2.32.22 N-glycosidases that recognize a universally conserved stem-loop structure in 23S/25S/28S ribosomal ribonucleic acid (rRNA), depurinating a single adenine (A4324 in rat), and irreversibly blocking protein translation, leading finally to cell death of intoxicated mammalian cells [25]. RIPs are widely distributed in nature but are found predominantly in plants, bacteria, and fungi. Besides their activity on rRNA, certain RIPs display a variety of antimicrobial activities in vitro, such as antifungal, antibacterial, and broad-spectrum antiviral activities against both human and animal viruses, including the human immunodeficiency virus (HIV) [26]. 378 Studies in Natural Products Chemistry O R2 OH HO R1 R1 R2 CHO H COOH H Quillaic acid CHO OH Hederagenin CH2OH H Gypsogenin Gypsogenic acid FIGURE 3 Structures of gypsogenin, gypsogenic acid, quillaic acid, and hederagenin. Basically, the plant RIPs can be subdivided into holoenzymes and chimero-enzymes. Holoenzymes or type I RIPs consist solely of a RIP domain, whereas the chimero-enzymes are built up of an N-terminal RIP domain linked (at least in the gene) to an unrelated C-terminal domain. Depending on the nature of the latter chain, the chimeric forms are referred to as type II RIPs (with a lectinic B-chain) and type III RIPs (with an unidentified C-terminal domain). Both type I and type II RIPs are quite common in plants whereas hitherto only a single type III RIP has been isolated and characterized, namely the barley JIP60 [27]. SAPONINS AS CYTOTOXIC AGENTS Cytotoxic activity has been described for a number of saponins, and numerous reports of cytotoxic saponins continue to appear in the literature every year [18,19]. Moreover, Gypsophila saponins showed the ability to amplify the toxicity of type I RIPs, lectins like saporin and agrostin in a synergistic manner. It has been reported that Gypsophila saponins enhanced the cytotoxicity of saporin protein, a type I RIP, from Saponaria officinalis L. 100,000-fold [28]. The combination of the saporin-based toxin (SA2E) with Gypsophila saponins (saponinum album) resulted in 94% tumor regression in mice, compared to treatment with pure SA2E [29]. A number of type I RIPs, usually unable to penetrate the cellular membrane, remove adenine residues from the 28S ribosomal RNA as part of a process, which leads to inhibition of protein synthesis. The synergistic amplification of toxicity was not based on the damage of cellular membranes by the saponins. Moreover, it was not based on a simple increase in endocytosis for the type I RIPs, but a triggering of clathrin-mediated endocytosis by the saponins [30,31]. This brings up a strong enhancement of toxicity for the naturally Chapter 12 379 Saponins Produced by Gypsophila Species O C o OH GlcA HO HO Xyl HO HO O O OH HO HO Fuc HO O CHO O O O O O O HO OH Rha O OH HO Gal HO Xyl1 HO HO O O OH O O Glc OH OH HO FIGURE 4 Structure of a typical Gypsophila saponin. Note that amphiphilic structure includes a hydrophobic aglycone (30C) and two hydrophilic sugar chains attached at carbon positions 3 and 28 of the aglycone. The most crucial difference between Gypsophila saponins and other saponins is the formyl group at the position C-4. membrane-impermeable type I RIPs, making them as toxic as the membranepermeable type II RIPs, like viscumin and ricin at same concentrations. Saponins that provided the highest synergistic toxicity were those, whose triterpenoid components were gypsogenin or quillaic acid—both of oleanane type. On the other hand, saponinum album formed a standard in activity for hemolytic test in the German pharmacopeia DAB 7 [32]. Herein, it was discussed that Gypsophila saponins in particular with 4C formyl group in aglycone part showed cytotoxicity enhancing properties on type I RIPs. It is known that the formyl group is crucial for interactions with the cell membrane to support the uptake of the toxin into the cell (Fig. 4). It has also been unambiguously stated by many authors which aglycone structures are in general more cytotoxic on cell lines. 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Chapter 13 Biologically Active Compounds from the Genus Uncaria (Rubiaceae) Anjaneya Swamy Ravipati*, Narsimha Reddy* and Sundar Rao Koyyalamudi*,{ *School of Science and Health, University of Western Sydney, Locked Bag 1797, Penrith South DC NSW 1797, Australia { Departments of Biochemistry, The Children’s Hospital at Westmead, Sydney, NSW 2145, Australia Chapter Outline Introduction 382 Ethnobotany of Uncaria spp. 384 Herbal Formulations Containing Uncaria spp. 384 Chotoko 385 Gou-teng 385 Kampo 385 Gud 385 Kuiyangling 385 Phytochemistry of Uncaria spp. 386 Alkaloids 386 Terpenoids 389 Flavonoids 389 Extraction, Isolation, Purification, and Identification of Novel Compounds 390 Bioactivities 392 Cytotoxicity 392 Antiinflammatory Activities 397 Antibacterial Activity 397 Antiviral Activity Antimutagenic Activity Activity Against Vascular Diseases Immunostimulation Activity Hypotensive Effects CNS-Related Activity and Effects on Locomotion Response Activity Against Vascular Dementia and Ischemia Structural Diversity of Compounds from Uncaria spp. Structure and Activity Relationship of Bioactive Compounds from Uncaria spp. Alkaloid Biosynthesis in U. tomentosa Concluding Remarks References Studies in Natural Products Chemistry, Vol. 43. http://dx.doi.org/10.1016/B978-0-444-63430-6.00013-8 © 2014 Elsevier B.V. All rights reserved. 398 398 398 399 400 400 401 401 402 403 404 404 381 382 Studies in Natural Products Chemistry INTRODUCTION In modern medicine, the medicinal plant-derived products are increasingly being sought after as pharmaceuticals and nutraceuticals for the treatment of several ailments due to their efficacy and cost-effectiveness. Rubiaceae family, consisting of 13,200 species, is of special interest as they contain economically and medicinally important members [1]. Coffee from Coffea spp. is considered as second most economically precious commodity after oil worldwide. Furthermore, Rubiaceae family is also often regarded as coffee family. Quinine is the first and most effective antimalarial which is also used as antipyretic and analgesic and antiinflammatory agent, discovered from Cinchona spp. provides evidence for the crucial role of Rubiaceae family members in the medicinal field [2]. Yohimbe is an aphrodisiac derived from Pausinystalia yohimbe available as prescribed medicine and recently received an exploration as remedy for type 2 diabetes [3]. Rubiaceae family plant species play a significant role in day-to-day life being source of many products including, Uncaria gambir (source of tannin), Calycophyllum, and Neolamarckia chinensis as sources of timbers [4], Gardinea spp. as source of perfume and ornamentals [5]. The plant species of this family are accessible on almost every region of world except Antarctic continents. Uncaria Schreb. (Rubiaceae) spp. are other major sources for the discovery of novel medicinal natural compounds [6]. Species of Uncaria are commonly woody climbers, shrubs, or small trees with characteristic hooks on either side of the shoots [7]. Most of the species of Uncaria are native to Asia and some of them are also distributed in Africa and South America [7]. The species of Uncaria have significant history of traditional medicinal use, for the treatment of many illnesses such as wounds, ulcers, fevers, headaches, gastrointestinal illness, microbial infections, hypertension, and nervous disorders [6]. Many species of the genus Uncaria, including U. gambir, U. guianensis, U. hirsuta, U. glabrata, U. macrophylla, U. quadrangularis, U. rhynchophylla, U. sinensis, and U. tomentosa have been used as traditional medicine for the treatment of above-mentioned diseases [6]. In traditional medicines of China and Japan, the extracts of many Uncaria species have been used in the herbal formulations to treat various ailments including vascular dementia, epilepsy, and arthritis (Table 1) [8,9]. In 1978, Ridsdale [7] systematically revised the genus Uncaria and included 34 species in the genus. In the same year, Phillipson et al. [10] extensively studied the alkaloids of Uncaria spp. from approximately 400 herbarium samples collected from across their range of distribution and identified 40 different alkaloids. Subsequently, several classes of compounds including alkaloids, flavonoids, and terpenoids were isolated and identified from this genus [10]. Among these, alkaloids are, however, recognized as the most prominent class of compounds. The phytochemical studies of Uncaria spp. for isolation of bioactive compounds started several decades ago [11]. Chapter 13 383 Biologically Active Compounds from Uncaria TABLE 1 The Distribution and Medicinal Use of Various Uncaria spp. Name of the Species Distribution Medicinal Uses U. gambir Malaysia, Singapore, and Indonesia Aqueous extract of this plant is used as astringent and tanning material [10]. U. guianensis Southern America To treat cancer, arthritis, diabetes, and inflammation [13,14]. U. hirsuta Central and northern Taiwan Used to treat primarily hypertension [15]. U. glabrata Malaysian peninsula, Sumatra, Java, and Borneo Used as remedy for food poisoning [16]. U. macrophylla Yunnan province, China Component of gambir plant (Gouteng), a traditional Chinese medicine used to treat ailments in the cardiovascular and central nervous systems [17]. Especially, it was used for sleep disturbance in treating vascular dementia [17]. U. perrottetii Kanawan, Morong, in the province of Bataan, Philippines To treat hematuria as well as a remedy during the 6-week period of postnatal care to prevent puerperal fever [18]. U. tomentosa Central and South America To treat abscesses, arthritis, asthma, cancer, chemotherapy side effects, contraception, disease prevention, fevers, gastric ulcers, hemorrhages, inflammations, menstrual irregularity, recovery from child birth, rheumatism, skin impurities, urinary tract inflammation, weakness, and wounds [19]. U. sinensis China and Japan It is a major constituent of the Chinese drug “Chotoko” used against fever and nervous disorders. The hooks of this plant were used as analgesic, spasmolytic, and in the treatment of hypertension [20]. U. sessilifructus Guangxi, Yunnan (Bangladesh, Bhutan, India, Laos, Myanmar, Nepal, and Vietnam [21]) Used in the treatment of high blood pressure, giddiness, bellyache, hysteritis, rheumatoid arthritis, arthritis, hemiplegia, sciatica, injuries from falls, ulcer [22]. Continued 384 Studies in Natural Products Chemistry TABLE 1 The Distribution and Medicinal Use of Various Uncaria spp.—Cont’d Name of the Species Distribution Medicinal Uses U. elliptica Mainly in wetlands of Sri Lanka As a folk medicine [23]. U. rhynchophylla Temperate and tropical Asia (China, Japan, Bangladesh, India, Laos, Myanmar, Thailand, and Vietnam [GRIN database, 2013]) To treat convulsive disorders, such as epilepsy [24]. Rhynchophylline (1.1) is one of the earliest reported compounds from U. rhynchophylla [11]. The present review also presents a comprehensive phytochemical and pharmacological data on this genus. The aim of the present review is to systematically summarize the therapeutic importance of different species of Uncaria, their derived phytochemicals and structure–activity relationship of the isolated compounds in order to explore further research on Uncaria species. ETHNOBOTANY OF Uncaria spp. Uncaria spp. have traditionally been widely used for the treatments of wounds, ulcers, fevers, headaches, and gastrointestinal illnesses [8,9]. Dried hooks of Uncaria spp. are used as major components of traditional medicines and have therapeutic applications as analgesics and sedatives for the nervous system-related disorders [8,9]. One of the important uses of Uncaria Spp. is their use in the treatment of hypertension [12]. Table 1 summarizes the list of ethno-medicinally important Uncaria species that are commonly used for the treatment of various diseases. HERBAL FORMULATIONS CONTAINING Uncaria spp. Traditionally, herbal medicine has been consumed as either individual herbs or combination of herbs in the form of formulations for synergetic effects. The traditional Chinese practitioners have studied the synergistic effects of biochemical complexes of herbal mix than the individual herbs. For instance, studies carried out by Wang et al. [25] has demonstrated that the net synergistic effect of Sheng Mai, a traditional Chinese medicine (TCM), showed the free radical scavenging activity that is significantly higher than the individual Chapter 13 Biologically Active Compounds from Uncaria 385 components [25]. The following are some of the herbal formulations that are commonly used in the TCM. Chotoko This is a widely used crude drug in China and Japan prepared from the hooks of U. sinensis. In TCM, Chotoko is mainly used against fever and nervous disorders. In addition, the hooks of plant were used as spasmolytic, analgesic, and for the treatment for hypertension [26]. Gou-teng This is also identified as “Chotoko” in Japanese traditional medicine. It is composed of several Uncaria spp. including U. rhynchophylla, U. macrophylla, U. sinensis, and U. sessilifructus; however, U. rhynchophylla is a major component. Gou-teng is commonly used for its sedative, antispasmodic, analgesic, anticonvulsive, hypertensive, antiepileptic, and antiviral properties [12]. The crude drug is used to relieve headaches and dizziness caused by hypertension and infantile nervous disorders. Recent studies have evaluated that the active ingredients of Gou-teng are the alkaloids of U. rhynchophylla [12]. Kampo The term Kampo is known as traditional Japanese herbal medicine. This formulation mainly constitutes hooks of U. rhynchophylla and nine other medicinal herbs. Traditionally, this formulation was used in the treatment of behavioral and psychological symptoms of dementia. Kampo is usually prescribed for patients with chronic headache, painful tensions in shoulder and cervical muscle, vertigo, morning headache, tinnitus, and insomnia [27]. This formulation has been approved by the Ministry of Health, Labor, and Welfare of Japan for the treatment of neurosis, insomnia, and irritability in children [28]. Gud It is a Gastrodia elata and U. rhynchophylla decoction that has been used for centuries as TCM for the treatment of hypertension, convulsions, and epilepsy [29]. Clinical studies carried out by Chen-guang et al. showed its potential antihypertensive effect [30] and antiproliferative effect [31]. Kuiyangling This formulation is prepared based on the knowledge of TCM theory. It is mainly used in the treatment of “stomach duct pain” and known to be involved in muco-protection with efficacy and low side effects. The major 386 Studies in Natural Products Chemistry composition of this formulation is leaf and stem of U. gambir and other species including Chinese licorice root (Glycyrrhiza uralensis) and bletilla root (Bletilla striata) [32]. PHYTOCHEMISTRY OF Uncaria spp. The phytochemical studies of Uncaria spp. have begun in early 1900s [11]. Rhynchophylline (1.1) is one of the earliest reported compounds from the hooks of U. rhynchophylla [11]. Latter studies on these plants reported over 150 compounds that belong to mainly three classes of compounds: (i) alkaloids, (ii) terpenoids, and (iii) flavonoids. In addition, several unusual structures were also identified [6]. Major emphasis of this chapter is on the occurrence of these compounds among various species, their structural diversity and pharmacological activities. Alkaloids Alkaloids are the most abundant bioactive compounds among this genus plants that attained intense focus due to their pharmacological importance. Till date, about 40 different alkaloids of biological importance have been identified from this genus [6,33]. In 1978, Phillipson et al. [10] reviewed the alkaloids of this genus extensively from approximately 400 samples mainly obtained from herbaria. These samples represented a wide geographical range and 40 different alkaloids were identified (Fig. 1) including some of the pharmacologically important alkaloids found in different species of Uncaria. The most prominent alkaloid is mitraphylline (1.10) which was found in 20 of 34 species of Uncaria. Other alkaloids including rhynchophylline (1.1), isorhynchophylline (1.4), and isomitraphylline (1.11) were identified in 18 species. In addition, uncarines are a group of oxindole alkaloids containing a spiro-cyclic ring on the indole ring and varying stereochemistry H H R N C 3 20 OCH3 R2 D B O OCH3 3 H A 20 N R1 H H O CO2CH3 NH H CO2CH3 NH 1.1 Rhynchophylline: (R = Et, 3α-H, 20β-H) 1.4 Isorhynchophylline (R1 = H, R2 = Et, 3α-H, 20β-H) 1.2 Corynoxiene: (R = vinyl, 3α-H, 20β-H) 1.5 Isocorynoxiene (R1 = H, R2 = vinyl, 3α-H, 20β-H) 1.3 Corynoxine B: (R = Et, 3α-H, 20α-H) 1.6 Corynoxine (R1 = H, R2 = Et, 3α-H, 20α-H) FIGURE 1—CONT’D Chapter 13 387 Biologically Active Compounds from Uncaria CH3 CH3 H H N 19 20 O 19 N 3 20 3 O H H H O H O CO2CH3 CO2CH3 NH NH 1.7 Formosanine (uncarine B) (3α-H, 19β-Me, 20β-H) 1.11 Isomitraphylline (3α-H, 19β-Me, 20β-H) 1.8 Pteropodine (uncarine C) (3α-H, 19α-Me, 20α-H) 1.12 Speciophylline (uncarine D) (3β-H, 19α-Me, 20α-H) 1.9 Uncarine F (3β-H, 19α-Me, 20α-H) 1.13 Isopteropodine (3α-H, 19α-Me, 20α-H) 1.10 Mitraphylline (3α-H, 19α-Me, 20β-H) H COOH R HO 1.14 Uncarinic acid A R = E-feruloyl 1.15 Uncarinic acid B R = Z-feruloyl 1.16 Uncarinic acid E R = E-coumaroyl H H COOH N H N H R HO 1.17 Uncarinic acid C R = E-feruloyl 1.18 Uncarinic acid D R = Z-feruloyl FIGURE 1—CONT’D COOCH3 1.19 Villocarine A OCH3 388 Studies in Natural Products Chemistry OH N H H N O O H OCH3 O- H O N H COOCH3 N COOCH3 OCH3 1.21 Villocarine C 1.20 Villocarine B H N+ H N OCH3 H O N H OCH3 COOCH3 OCH3 O O N N OCH3 H H O OCH3 H OCH3 H N H O H OCH3 O 1.24 Macrophyllines A N H O 1.23 Macrophyllionium 1.22 Villocarine D N H H O- N O 1.25 Macrophyllines B N H OMe O OMe OMe 1.26 Geissoschizine methyl ether FIGURE 1 Novel alkaloids and their structures identified from various Uncaria spp. over the years. Chapter 13 Biologically Active Compounds from Uncaria 389 in the ring skeleton. Uncarine D (1.12) and uncarine B (1.7) are quite prevalent and found in 16 and 7 species of Uncaria, respectively. Recent bioassay guided fractionation resulted in the discovery of indole alkaloids, villocarines A–D (1.19–1.22), with vasorelaxant activity [34]. Arbain and coworkers [35] discovered unusual indole monoterpenoid gluco-alkaloid, characterized by a glucose moiety on the benzenoid ring. Recent studies reported an unusual oxindole alkaloid, macrophyllionium (1.23), pair of new tetracyclic oxindole alkaloids, macrophyllines A and B (1.24 and 1.25) with significant vasodilating activity [36]. Terpenoids The species of Uncaria also contain different pentacyclic triterpenoids with structural diversity. These are mainly ursane, oleanolic, and quinovic acid structures. The group of uncarinic acids A–E (1.14–1.18), which include ursane- and oleanane-type skeletons and are represented in over 16 different species of Uncaria [37,38]. Quinovic acid is another ursane-type pentacyclic triterpene, reported from U. guianensis. The quinovic acid skeleton has carboxylation at both C27 and C28. In addition to carboxylation at C27, there is hydroxylation at C28 with further esterification at this position with ferulic or coumaric acid [37–39]. Two novel triterpenoids of the ursolic acid series including uncaric acid (2.1) and floridic acid methyl ester (2.2) were isolated from U. florida. Recently, two new 27-nor-triterpene glycoside derivatives of pyroquinovic acid, tomentosides A and B (2.3 and 2.4) are isolated from U. tomentosa [39]. Studies conducted by Diyabalanage et al. [23] discovered that the presence of different triterpenoids in samples collected from different locations demonstrates that these could be of chemotypes [23] (Fig. 2). Flavonoids Gambier is a natural product from the leaves of U. gambir and it has economic importance as astringent and tanning agent. Many other Uncaria spp. are also found with similar use on the basis of their polyphenolic content [10]. Flavonoids are another major class of compounds mostly found in U. elliptica. Rutin (3.3) and ( )-epicatechin (3.2) are considered to be highly abundant flavonoids [40]. Tissue distribution studies carried out by Law and Das [41] showed that leaves possess more rutin (3.3) than the woody parts. Similar studies carried out by Balz and Das [40] showed the difference in abundance of rutin (3.3) and ( )-epicatechin (3.2) in young and old leaves [40,42]. Other flavonoids including hyperin (3.5) and trifolin (3.6) were isolated from the leaves of U. rhynchophylla [43], while quercetin (3.4) and catechins (3.1 and 3.2) were isolated from hooks and stems [43,44] of this plant. In addition, afzelin (3.7) and neohesperidin (3.8) were isolated from leaves of U. hirsuta collected in Taiwan [15] (Fig. 3). 390 Studies in Natural Products Chemistry R3 Me OH COOH COOMe R1 R1 CH2 R2 2.1 Uncaric acid 2.2 Floridic acid methyl ester H COOR2 H OR COOR2 H H 1 OH 1 H OR H 2.4 Tomentosides B 2.3 Tomentosides A Me Me Me Me H COOH Me OH Me Me H 2.5 Ursolic acid FIGURE 2 Novel terpenoids and their structures identified from various Uncaria spp. over the years. EXTRACTION, ISOLATION, PURIFICATION, AND IDENTIFICATION OF NOVEL COMPOUNDS Extraction procedure is the key step in the separation of medicinally active constituents from the plant or animal tissues using different solvents. Conventionally, the medicinal plants were consumed in the form of decoction prepared by using either water or water–alcohol mixture. Over the years, several novel extraction procedures have been developed including infusions [45]. With years of experience, the following extraction strategy is employed in our laboratory (Fig. 4) for the isolation of bioactive compounds from the Chapter 13 391 Biologically Active Compounds from Uncaria HO HO OH OH O OH O OH OH OH OH OH 3.1 (+)-Catechin 3.2 (-)-Epicatechin OCH3 OH O OH R1 Glucose O O OH Rhamnose O OH O R2 OH 3.3 Rutin (R1 = OH, R2 = rutinose) 3.4 Quercitrin (R1 = OH, R2 = rhamnose) 3.5 Hyperin (R1 = OH, R2 = galactose) 3.6 Trifolin (R1 = H, R2 = galactose) 3.7 Afzelin (R1 = H, R2 = rhamnose) O 3.8 Neohesperidin FIGURE 3 Novel flavonoids and their structures identified from various Uncaria spp. over the years. Ethanol extract residue CHCl3 fraction Add methanol and hexane Add 90% MeOH + hexane in equal partition Hexane (fraction 1) Water fraction Extract with n-butanol (fraction 3) Methanol (fraction 2) FIGURE 4 Flowchart showing the partition of different solvent soluble compounds from ethanol extract of U. rhynchophylla. 392 Studies in Natural Products Chemistry crude extracts. Initially, the ground material is extracted with 95% ethanol for about 72 h on hot water bath at approximately 95 C. After extraction, the ethanol is removed using rotary evaporator under reduced pressure at low temperature (30–35 C). The concentrated crude extract is then partitioned with chloroform–water (1:1, v/v). The chloroform soluble is concentrated and then partitioned between hexane (fraction 1) and 90% aqueous methanol (fraction 2) (1:1, v/v). The water fraction is further extracted with n-butanol and collected as fraction 3. In the early 1900s, the phytochemical analysis and identification of compounds from Uncaria spp. was carried out by thin layer chromatography (TLC). The emergence of high-performance liquid chromatographic (HPLC) and spectroscopic techniques (MS and NMR) has improved the identification of novel compounds form Uncaria spp. In the recent years, natural product laboratories adopted the combination of HPLC, MS, and NMR techniques for the purification, identification, and discovery of novel bioactive compounds from plants [46]. TLC technique also plays a crucial role in the separation and identification of compounds from plant crude extracts. As shown in Table 2, rhynchophylline, isorhynchophylline, corynoxeine and isocorynoxeine, corynoxeine B, uncarinic F N-oxide, speciophylline N-oxide, pteropodine N-oxide, and gambirine possess same molecular weight of 384. These are separated and identified by TLC technique. The capillary electrophoresis method is also used in the identification of alkaloids in Uncaria spp. [47]. Solution NMR spectroscopy has been employed for structural and conformational studies [42–44]. BIOACTIVITIES Uncaria spp. has been widely used in the form of decoctions made from single species or from herbal formulations in traditional systems of medicine to treat several disorders as discussed in previous sections. Among all, the species of Uncaria, U. tomentosa is the widely studied species which is followed by U. rhynchophylla. The pharmacological activities of crude extracts and isolated compounds are discussed in the following subsections. Cytotoxicity In traditional systems of medicine, species of Uncaria have been used for the treatment of neurotoxicity. Bioactivity-guided fractionation of U. rhynchophylla led to the discovery of pentacyclic triterpene esters, namely uncarinic acid A and B [38,37]. Literature demonstrates that these compounds inhibit the growth of human cancer cell lines A-549, HCT-15, MCF-7, HT-1197, and phospholipase Cg1, an enzyme which induces proliferation of human cancer cells [38,37]. The studies on cDNA microarray revealed that hyperin (3.5) isolated from stems of U. rhynchophylla, down regulated SNU-668 human gastric cancer cells. TABLE 2 The Chromatographic, Mass Spectrometric, and Structural Studies of Alkaloids from Uncaria spp. Compound TLC HPLC MS NMR References 1 Dihydrocorynantheine A*, B*, C, and D* (Dragendorff’s reagent with 0.2 M FeCl3 in 35% HClO4) LiChrospher-100, RP18, 70% methanol in 0.01 M aqueous ammonium acetate (pH 8.04), 245 nm 368 (100) and 367 (90), 353 (55), 251 (10), 239 (14), 225 (24), 213 (25), 184 (70), 170 (39), 169 (25), 156 (30) H, NOESY (conformational studies) [48–50] Hirsutine A, B, C, and D (Dragendorff’s reagent with 0.2 M FeCl3 in 35% HClO4) L-6200A Intelligent pump, 10 mM phosphate buffer as solvent A and acetonitrile– methanol (1:1) as solvent II, at 245 nm 368 (100) and 367 (68), 353 (100),311 (19), 251 (13), 239 (13), 225 (28), 197 (17), 184 (70), 170 (27), 169 (28), 156 (29) 1 H and 2DNMR spectra (C–H COSY, HMBC) (spectral assignment and structure confirmation) [50–53] Hirsuteine A, B, C and D (Dragendorff’s reagent with 0.2 M FeCl3 in 35% HClO4) L-6200A Intelligent pump, 10 mM phosphate buffer as solvent A and acetonitrilemethanol (1:1) as solvent II, at 245 nm 366 351 237 184 169 (89) and 365 (69), (100), 335 (36), (22), 223 (51), (78), 170 (53), (42), 156 (53) 1 H and 2D NMR spectra (C–H COSY, HMBC) (spectral assignment and structure confirmation) [50,52,53] Mitraphylline A, B, C, and D (Dragendorff’s reagent with 0.2 M FeCl3 in 35% HClO4) L-6200A Intelligent pump, 10 mM phosphate buffer as solvent A and acetonitrilemethanol (1:1) as solvent II, at 245 nm 368 (40) and 351 (3), 337 (4), 223 (100), 222 (13), 208 (11), 146 (6), 145 (10), 144 (6), 130 (11), 69 (27) 1H, 2D NMR (COSY and DEPT) (spectral assignment and structure confirmation) [50,53,54] Continued TABLE 2 The Chromatographic, Mass Spectrometric, and Structural Studies of Alkaloids from Uncaria spp.—Cont’d Compound TLC HPLC MS NMR References Isomitraphylline A, B, C, and D (Dragendorff’s reagent with 0.2 M FeCl3 in 35% HClO4) L-6200A Intelligent pump, 10 mM phosphate buffer as solvent A and acetonitrilemethanol (1:1) as solvent II, at 245 nm 368 (64) and 351 (5), 337 (7), 223 (100), 222 (10), 208 (11), 146 (6), 145 (5), 144 (7), 130 (11), 69 (23) 1H, 2D NMR (COSY and DEPT) (spectral assignment and structure confirmation) [50,53,54] Pteropodine A, B, C, and D (Dragendorff’s reagent with 0.2 M FeCl3 in 35% HClO4) L-6200A Intelligent pump, 10 mM phosphate buffer as solvent A and acetonitrilemethanol (1:1) as solvent II, at 245 nm 368 (100) and 351 (6), 337 (8). 223 (86), 222 (33), 208 (25), 180 (21), 146 (8), 145 (11), 144 (11), 130 (19), 69 (40) 1H, 2D NMR (COSY and DEPT) (spectral assignment and structure confirmation) [50,53–55] Isopteropodine A, B, C, and D (Dragendorff’s reagent with 0.2 M FeCl3 in 35% HClO4) L-6200A Intelligent pump, 10 mM phosphate buffer as solvent A and acetonitrilemethanol (1:1) as solvent II, at 245 nm 368 (100) and 351 (8), 337 (8), 223 (77), 222 (24), 208 (19), 180 (19), 146 (6), 145 (9), 144 (8), 130 (14), 69 (41) 1 H and 13C NMR (spectral assignment and structure confirmation) [50,53,56] Speciophylline A, B, C, and D (Dragendorff’s reagent with 0.2 M FeCl3 in 35% HClO4) L-6200A Intelligent pump, 10 mM phosphate buffer as solvent A and acetonitrilemethanol (1:1) as solvent II, at 245 nm 368 (100) and 351 (7), 337 (8), 223 (78), 222 (28), 208 (25), 180 (11), 146 (9), 145 (12), 144 (12), 130 (21), 69 (78) 1 [50,53–55] H, 2D NMR (COSY and DEPT) (spectral assignment and structure confirmation) Uncarinc F A, B, C, and D (Dragendorff’s reagent with 0.2 M FeCl3 in 35% HClO4) L-6200A Intelligent pump, 10 mM phosphate buffer as solvent A and acetonitrile– methanol (1:1) as solvent II, at 245 nm 368 (100) and 351 (8), 337 (10), 223 (70), 222 (20), 208 (15), 180 (13), 146 (5), 145 (7), 144 (6)., 130 (11), 69 (45) 1 H NMR spectra (structure confirmation) [50,53,55] Uncarinc A A, B, C, and D (Dragendorff’s reagent with 0.2 M FeCl3 in 35% HClO4) Not available 368 (50) and 351 (5), 337 (6), 223 (100), 222 (12), 208 (13), 146 (8), 145 (11), 144 (10), 130 (19), 69 (41) 1 H NMR spectra (structure confirmation) [50,55] Uncarinc B A, B, C, and D (Dragendorff’s reagent with 0.2 M FeCl3 in 35% HClO4) Not available 368 (85) and 351 (6), 337 (8), 223 (l00), 222 (13), 208 (l6), 146 (6), 145 (7), 144 (8), 130 (15), 69 (35) 1 H NMR spectra (structure confirmation) [50,55] Isorhynchophylline A, B, C, and D (Dragendorff’s reagent with 0.2 M FeCl3 in 35% HClO4) L-6200A Intelligent pump, 10 mM phosphate buffer as solvent A and acetonitrile– methanol (1:1) as solvent II, at 245 nm 384 (100) and 369 (5), 367 (6), 355 (5), 353 (9), 239 (80), 238 (38),224 (29), 210 (17), 208 (21), 146 (6), 145 (9), 144 (10), 130 (17), 69 (85) 1 H and 13C NMR (structure confirmation) [50,53,57,58] Rhynchophylline A, B, C, and D (Dragendorff’s reagent with 0.2 M FeCl3 in 35% HClO4) L-6200A Intelligent pump, 10 mM phosphate buffer as solvent A and acetonitrile– methanol (1:1) as solvent II, at 245 nm 384 (100) and 369 (7), 367 (6), 355 (5), 353 (10), 239 (95), 238 (43), 224 (31), 210 (19), 208 (24), 146 (8), 145 (12), 144 (13), 130 (20), 69 (>100) 1 [50,53,57,58] H and 13C NMR (structure confirmation) Continued TABLE 2 The Chromatographic, Mass Spectrometric, and Structural Studies of Alkaloids from Uncaria spp.—Cont’d Compound TLC HPLC MS NMR References Corynoxeine A, B, C, and D (Dragendorff’s reagent with 0.2 M FeCl3 in 35% HClO4) L-6200A Intelligent pump, 10 mM phosphate buffer as solvent A and acetonitrile– methanol (1:1) as solvent II, at 245 nm 382 (100) and 367 (8), 365 (5), 351 (14), 237 (6), 236 (9), 222 (7), 206 (7), 192 (61), 146 (3), 145 (3), 144 (5), 130 (8), 108 (30), 69 (15) 1 13 H and C NMR (structure confirmation) [17,50,53,59] Isocorynoxeine A, B, C, and D (Dragendorff’s reagent with 0.2 M FeCl3 in 35% HClO4) L-6200A Intelligent pump, 10 mM phosphate buffer as solvent A and acetonitrile– methanol (1:1) as solvent II, at 245 nm 382 (100) and 367 (9), 365 (5), 351 (18), 237 (15), 236 (20), 222 (23), 208 (15), 206 (19), 159 (18), 146 (17), 145 (20), 144 (30), 130 (52), 108 (56), 69 (46) 1 H and 13C NMR (structure confirmation) [17,50,53,60] Corynoxine A, B, C, and D (Dragendorff’s reagent with 0.2 M FeCl3 in 35% HClO4) Not available 384 (100) and 369 (9), 367 (9), 355 (5), 353 (12), 239 (90), 238 (42), 224 (35), 210 (20), 208 (26), 146 (8), 145 (11), 144 (17), 130 (25), 69 (>100) 1 H and 13C NMR (structure confirmation) [17,50,60] Corynoxine B A, B, C, and D (Dragendorff’s reagent with 0.2 M FeCl3 in 35% HClO4) L-6200A Intelligent pump, 10 mM phosphate buffer as solvent A and acetonitrile– methanol (1:1) as solvent II, at 245 nm 384 (100) and 369 (9). 367 (9). 355 (5), 353 (10), 239 (84), 238 (39), 224 (30), 210 (17), 208 (24), 146 (7), 145 (8), 144 (12), 130 (20), 69 (85) 1 [17,50,53,61] H and 13C NMR (structure confirmation) A* (chloroform–acetone (5:4)), B* (chloroform–ethanol (95:5)); C* (ether–ethyl acetate (1:1)); D* (ethyl acetate–isopropanol–conc. ammonia (100:2:1)). Chapter 13 Biologically Active Compounds from Uncaria 397 The aqueous extracts of U. tomentosa showed the significant inhibitory activity against human leukemia cancer cell lines (K562 and HL 60) and a human EBVtransformed B lymphoma cell line. Literature revealed that the extract of this plant-induced apoptosis, in conjugation with DNA fragmentation, which evidences the antitumor activity [38,37]. The aqueous extract of this plant had also been tested for toxicity against Chinese hamster ovary cells, which revealed that it is nontoxic at low concentrations [62]. Bioactivity-guided fractionation of other plant extracts targeting cytotoxicity led to the discovery of novel compounds. Uncarine C (1.8) and E isolated from U. guianensis exhibited cytotoxicity and DNA-damaging activity by RS321 and RS322 yeast assays. Rhynchophylline (1.1), which is one of the widely found alkaloids among Uncaria spp., showed neurotoxicity against methamphetamine and also attenuated intracellular calcium overload triggered by methamphetamine [63]. Antiinflammatory Activities Various parts of Uncaria plants are commonly used in the traditional medicine for the treatment of inflammatory diseases. In a study, hydro-alcoholic extract (EtOH–H2O) and an aqueous extract of bark of U. tomentosa have been analyzed for their antiinflammatory activity [64]. The hydro-alcoholic extract exhibited higher activity in mouse paw edema model and suppressed NF-kB [64]. Phytochemical analysis of these extracts revealed that hydroalcoholic extract possesses higher oxindole alkaloid content than the aqueous extract. It is believed that the antiinflammatory activity of U. tomentosa is attributed to the synergistic effect from a combination of these compounds [64]. A clinical trial was conducted to evaluate the therapeutic potential of U. tomentosa in the treatment of rheumatoid arthritis [65]. After 24 weeks of treatment, joint pain was reduced by 53.2% in the treated group, indicating its potent activity, while control group only reduced by 24.1% [65]. The phytochemical analysis of this extract showed the presence of immunemodulating pentacyclic oxindole alkaloids (POAs). A clinical investigation was undertaken by Passos et al. aiming to evaluate the efficiency of a cream from U. tomentosa for the treatment of the herpes labials. The assessment of clinical efficiency of U. tomentosa was evaluated against acyclovir, a control drug. The study has shown that the cream of U. tomentosa is significantly more potent than acyclovir with no adverse reactions [66]. Antibacterial Activity Traditionally, Uncaria spp. were not extensively used for the treatment of bacterial infections. In traditional medicine, U. glabrata has been used to treat microbial-related infections including food poisoning. Studies conducted by 398 Studies in Natural Products Chemistry Arret et al. [67] showed that the extract of U. glabrata inhibits the growth of Staphylococcus aureus and Escherichia coli. An antimutagenicity study of U. tomentosa was conducted aiming at evaluating the activity against Salmonella typhimurium TA98 and TA100 strains. The study revealed that aqueous extract of U. tomentosa showed no activity up to the concentration of 100 mg/mL [68]. Recent studies conducted by Wolska et al. [69] showed significant antibacterial activities of oleanolic and ursolic acid isolated from Uncaria spp. [69]. The study revealed that both the compounds displayed significant activity against many bacterial strains particularly Gram-positive bacteria including mycobacteria. Both the compounds inhibited the bacterial growth with minimum inhibitory concentration [66]. Antiviral Activity A quinovic acid glycoside (28)-b-D-glucopyranosyl-ester that was isolated from U. tomentosa exhibited antiviral activity against rhinovirus type 1B infection in HeLa cells. Cinchonain-Ia and cinchonain-Ib and epicatechin isolated from the bark of this plant showed significant antiviral and antiinflammatory activity [70]. Antimutagenic Activity In vivo studies of the decoction and fractions of U. tomentosa showed significant antimutagenic activities against S. typhimurium [9]. The same authors conducted tests on smokers and showed that the mutagenicity of smokers was significantly decreased after the daily consumption of decoction of U. tomentosa for 15 days [9]. The in vitro and in vivo studies conducted by Wurm et al. [71] on the bark extracts of U. tomentosa showed the antimutagenic activity. It was also suggested that this effect is associated with antioxidant activity [6,71]. Activity Against Vascular Diseases Uncaria spp. are commonly used in the treatment of vascular diseases. Kim et al. [59] isolated corynoxeine (1.2) from hooks of U. rhynchophylla, that inhibited rat aortic vascular smooth muscle cells (VSMCs) [59]. The study revealed that, corynoxeine (1.2) exerted inhibitory effect on platelet-derived growth factor–BB-induced rat aortic VSMCs proliferation. Pretreatment of VSMCs with 5–50 mM of corynoxeine for 24 h decreased the cell proliferation without cytotoxicity [59]. Dihydrocorynantheine isolated from the dried leaves and stems of U. calophylla was tested in both conscious and anesthetized normotensive rats. The results of these experiments showed that the arterial pressure in both Chapter 13 Biologically Active Compounds from Uncaria 399 types of rats decreased substantially [17]. Rutin (3.3) is a flavonoid found to be active toward cardiovascular system. It is commonly found in the leaves of U. hirsuta as well as in various parts of U. elliptica [40]. The hydroxyl ethyl derivative of rutin (3.3) including “venoruton” and “paroven” are used as drugs to treat blood capillary ailments [41]. Qian Yang He Ji is a TCM composed of U. gambir and other medicinal herbs and has shown significant activity in improving the arterial functionality of hypertension patients treated with antihypertensive angiotensin II receptor blocker. In addition, it also showed significant functionality in the hypertensive patients with diabetes and coronary heart disease [72]. Immunostimulation Activity The hydro-alcoholic extract of U. tomentosa has shown to be a potential immunostimulant [73]. The extract of this plant was given to BALP/c mice seven times intragastrically with formalin-inactivated whole Sandai virus (SV) [67]. It was noticed that the animals inoculated with 5.6 mg of the dry extract of U. tomentosa induced higher saliva IgA antibodies. In addition, the same amount of extract had significantly higher IgA, IgG, and HI antibody responses to SV than did those administered with the SV alone [74]. The water extracts of U. tomentosa have been shown to induce apoptosis and inhibit proliferation of tumor cells in vitro and to enhance DNA repair in vivo [75,76]. U. tomentosa has been demonstrated to display strong immunostimulant activity through in vitro and in vivo phagocytosis tests. Isopteropodine (1.13), pteropodine (1.8), isomitraphylline (1.11), isorhynchophylline (1.4), rhynchophylline (1.1), and mitrophylline (1.10) showed the enhancement of phagocytosis [77]. In another study, the extracts of U. tomentosa showed activity toward the production of cytokines including interleukin-1 (IL-1) and interleukin-6 (IL-6). The results indicated that the cytokines production/ secretion by macrophages is dose dependent and exposure of the same macrophages to lipopolysaccharide increased the levels of ILs [78]. Immunomodulatory activity of U. tomentosa is also likely due to its ability to suppress TNF-alpha production. In an in vivo animal study, the mice were fed with the C-Med 100, a commercial extract of U. tomentosa which does not contain the high-molecular weight compounds such as tannins [59]. It was observed that there was an increment in the number of immune cells including B, T, and NK cells, granulocytes, and memory lymphocytes. The authors concluded that the prolonged survival of the mice was mainly due to the ability of U. tomentosa to decrease oxidative stress and to activate NF-kB that counteract apoptosis and increase DNA repair [79]. POAs from U. tomentosa have shown to be a potent immunostimulants that increased the phagocytosis of human granulocytes and macrophages 400 Studies in Natural Products Chemistry and blocked the proliferation of myeloid cell lines [71]. The tested POAs including speciophylline (uncarine D) (1.12), uncarine F (1.9), mitraphylline (1.10), isomitraphylline (1.11), pteropodine (1.8), and isopteropodine (1.13) are considered to be active immunostimulants, while the tetracyclic compounds including rhynchophylline (1.1) and isorhynchophylline (1.4) were not active [78]. Hypotensive Effects Traditional medicine derived from Uncaria spp. are commonly used for the treatment of hypertension. For instance, Gou-teng is a Chinese traditional medicine that is used for the treatment of hypertension and its associated affects. However, the literature reported that isorhynchophylline (1.4) isolated from Gou-teng showed hypotensive activity in spontaneous hypertensive rats [80]. Bioactivity-guided fractionation of another Choto-san led to the discovery of two other compounds including 3a-dihydrocadambine and 3bisodihydrocadambine with hypotensive activity [81]. Studies carried out by Shimada et al. [81] demonstrated that treatment of hypertensive rats with 3a-dihydrocadambine and rhynchophylline resulted positive changes in blood pressure, heart rate, electrocardiogram, and respiratory rate [81]. Hemodynamic studies carried out on dogs demonstrated that 3a-dihydrocadambine exhibits significant hypotensive and antihypertensive activities in vitro [82]. Furthermore, geissoschizine methyl ether is an indole alkaloid isolated from U. ramulus acts as vasorelaxant [83]. CNS-Related Activity and Effects on Locomotion Response Aqueous extracts of U. rhynchophylla showed the significant binding activity to adrenoceptor, 5-HT, dopamine, and GABA receptors. In a study conducted on the aqueous extracts of U. rhynchophylla, U. sinensis, and U. macrophylla for the locomotive activity, the highest activity was observed with U. macrophylla while least was observed with U. rhynchophylla. Furthermore, nine compounds isolated including rhynchophylline (1.1), isorhynchophylline (1.4), corynoxeine (1.2), isocorynoxeine (1.5), corynoxine B (1.3), corynoxine (1.6), geissoschizine methyl ether (1.26), hirsuteine, and hirsutine were analyzed for their ability to depress the locomotion response [17]. The studies reported that corynoxine (1.6), corynoxine B (1.3), and isorhynchophylline (1.4) significantly depressed the locomotive activity. Pharmacological studies carried out on geissoschizine methyl ether (1.26) revealed its specific activity as agonist and blocking agent to 5-HT1A and 5-HT2A receptors, respectively [84]. Another study revealed that oxindole alkaloids pteropodine (1.8) and isopteropodine positively modulated the 5-HT2 and muscarinic M1 receptors [76]. It was demonstrated that these activities are due to their interaction with central cholinergic system [85]. Chapter 13 Biologically Active Compounds from Uncaria 401 Activity Against Vascular Dementia and Ischemia Choto-san is a traditional Chinese and Japanese medicine prepared from U. sinensis. Choto-san is mainly used for the treatment of vascular diseases and psychiatric symptoms associated with dementia, mainly associated with sleep disturbance. Corynoxine (1.6), corynoxeine B (1.3), rhynchophylline (1.1), and isorhynchophylline (1.4) were isolated from U. macrophylla, have been evaluated for their ability to perpetuate the state of hypnosis in mice. Oral administration of 100 mg/kg of each compound resulted in prolongation of thiopental-induced hypnosis [17]. Isorhynchophylline (1.4), corynoxine (1.6), and corynoxine B (1.3) significantly prolonged the sleeping time compared to the controls. It has been well stated that reactive oxygen species are known to involve in ischemia, inflammation, and aging processes. In this regard, antioxidants play a protective role in the regulation of reactive oxygen species [17]. A study carried out on rat blood cells showed that ingestion of an aqueous extract of U. sinensis caused a dose dependent decrease in susceptibility of RBC to lysis. Phenolic antioxidants including procyanidin B-1, catechin, epicatechin, and caffeic acid were evaluated for their antioxidant activity. These results showed that they exhibit a strong and dose-dependent protection of the cell membrane [86]. Another study was conducted to evaluate the neuroprotective effects of methanol extracts of U. rhynchophylla in rats upon transient global ischemia [78]. The study revealed that the extract significantly reduced the death of CA1 hippocampal neurons by 72% at a dose of 100 mg/kg body weight [87]. In another study, hexane, ethyl acetate, and methanol extracts were analyzed for cerebrovascular effect on photothrombic ischemic injury in mice [79]. It was found that the hexane extract significantly decreased infarct volume and edema size and improved neurological function in a dose-dependent manner [88]. Furthermore, hexane extract produced a concentration-dependent relaxation in mouse aorta and rat basilar artery, which suggests that this extract causes vasodilation via eNOS-dependant mechanism [88]. STRUCTURAL DIVERSITY OF COMPOUNDS FROM Uncaria spp. Natural products are source of several biologically active molecules with unique structural variations. The structural variations are responsible for diverse biological activities. As modern drug discovery is mainly based on structure–activity relationship, understanding the natural derivatives would lead to the discovery of potential therapeutic molecules. The chemical armory of Uncaria spp. is diverse. Cat’s claw is a Peruvian U. tomentosa that has been identified as the richest source of chemical compounds. Approximately, 50 different compounds with varying structural diversity have been isolated [6]. Fifteen compounds have been identified as 402 Studies in Natural Products Chemistry novel constituents from this plant. A new glucoindole alkaloid, 3,4dehydro-5-carboxystrictosidine was isolated from stems of U. tomentosa. This alkaloid is the first example from the nature with 3,4-dihydro-b-carboline ring system [39]. A study was carried out with 16 individual plants of this genus, in order to determine alkaloid distribution in various parts [80]. Two new chemotypes were discovered which were found among the progeny from seeds of individual plants [89]. Interestingly, the young leaves were predominant with oxindole alkaloids including pteropodine (1.8), isopteropodine (1.13), and speciophylline (1.12), while the mature leaves have speciophylline (1.12) in abundance. This clearly indicates that younger leaves have more alkaloids than the mature leaves. The structural diversity has been found in many other species of Uncaria including U. elliptica (44 compounds), U. attenuata (34 compounds), and U. rhynchophylla (33 compounds). The chemical diversity of these species is as complex as U. tomentosa. For instance, U. elliptica possesses several ajmalicinine derivatives, corynoxines, rauniticines, and D-secoalkaloids. In addition to the compounds present in U. tomentosa, U. elliptica also possesses uncarinic acids and uncaric acids. STRUCTURE AND ACTIVITY RELATIONSHIP OF BIOACTIVE COMPOUNDS FROM Uncaria spp. Structure-based drug design plays a crucial role in the development of potent therapeutic agents in modern medicine. Uncaria spp. are sources of diverse range of bioactive compounds such as oxindole alkaloids. However, variation in the conformational structures of tetracyclic oxindole alkaloids such as rhynchophylline (1.1) and POAs mitraphylline (1.10) showed significant difference in their pharmacological activity. X-ray crystallographic studies carried out by Laus and Wurst [90] revealed that the higher potency of tetracyclics as antagonists and immunostimulants than that of the pentacyclics is mainly due to the difference in the position of side chain relative to the ring core of the molecule [90]. For instance, tetracyclic alkaloids including rhynchophylline (1.1) and isorhynchophylline (1.4) adopt normal configuration with 18,19-seco ring, and the side chain is perpendicular to the ring plane and also contains an additional methyl group. While pentacyclic alkaloids such as mitraphylline adopt a conformationally rigid tricyclic core due to the trans D/E ring junction and exhibit weaker antagonism. The experimental data suggest that these conformational variations between tetracyclic and pentacyclic alkaloids are responsible for their convincing antagonism. Uncaria spp. also contain pentacyclic triterpenoids, mostly of the ursane type. Uncarinic acids A–E (1.14–1.18) which include both ursane- and oleanane-type skeletons are prevalent among 16 different species that have important pharmacological activities. Phosphatidylinositol-specific phospholipase C plays a crucial role in DNA synthesis and cell proliferation. This Chapter 13 Biologically Active Compounds from Uncaria 403 enzyme is an active target for pharmacological intervention especially abnormal cell proliferation. Uncarinic acids A–E (1.14 to 1.18) isolated from CHCl3 extract of U. rhynchophylla, showed significant inhibitory activity against cancer cell lines over expressing PLCg1. Synthesis of analogs with structural modification has been used in the literature to discover novel bioactive compounds with enhanced activity [37,91]. For instance, 3b-hydroxy27-p-E-coumaroyloxyurs-12-en-28-oic acid is a triterpene ester that contains an ursane moiety. The trans configuration and the p-coumaroyloxy group showed significant inhibitory activity. Structure–activity relationship studies have been carried out in the literature aiming to attain enhanced activity [91,37]. It was reported that 3-OH and 27-esterification is essential and that 28-COOH and double bond appear to be important for the activity [91,37]. In addition, the compound possessing a p-coumaroyloxy group at position 27, instead of at positions 2 and 28, showed enhanced inhibitory activity against PLCg1 [91,37]. Quercetin is a commonly available flavonoid in fruits and vegetables and is also found in Uncaria spp. including U. elliptica, U. hirsuta, and U. rhynchophylla [92]. Several studies have demonstrated the neuroprotective ability of this compound to improve the memory and dementia [93–96]. Studies also have demonstrated that this compound possesses scavenging ability toward reactive oxygen species including superoxide, hydroxyl, and peroxyl [97,98]. Wang and Joseph [99] have conducted a study aiming to demonstrate the antioxidant activity and its relationship with structure [99]. In that study, flavonoids including catechin, kaempferol, cyanidin, and taxifolin have been considered which are structurally similar to quercetin [99]. It was concluded that: (i) 30 ,40 -hydroxyl (OH) groups in the B ring, and (ii) a 2,3-double bond in conjugation with a 4-oxo group in the C ring, along with polyphenolic structure are important for protection. In agreement with the identified structural features, quercetin showed strong antioxidant property by reducing the H2O2-induced Ca2+ dysregulation and oxidative stress [99]. ALKALOID BIOSYNTHESIS IN U. tomentosa U. tomentosa is a significant traditional medicinal plant that is an important source of monoterpenoid oxindole alkaloids (MOAs) with bioactivities including immunomodulatory, cytotoxic, anti-HIV, and antileukemic activities [100]. It will be of interest to understand biosynthetic insights of bioactive compounds of this plant as an example here. Studies have reported the regulation of biosynthesis of sterols and triterpenes in U. tomentosa [100]. Administration of pectin to U. tomentosa cell suspension cultures increased the activity of isopentyl diphosphate isomerase of ursolic and oleanolic acid. The treated cells also transformed a higher percentage of labeled mevalonic acid precursors into triterpenes and resulted in the decrease in activity of farnesyl diphosphatase by a factor of two when compared to the control. The 404 Studies in Natural Products Chemistry results suggest that the biosynthetic flux for sterols and triterpenes are controlled by enzymatic complexes involving IPP isomerase and squalene synthase [100]. Another study carried out to examine the effect of oxidative stress on indole alkaloids accumulation, by cell suspensions and root cultures of U. tomentosa [100–102], revealed an increased production of MOAs up to 40 mg/L when 200 mM H2O2 was added to the cell suspension culture. It was concluded that when the roots of U. tomentosa in the bioreactor were exposed to an oxidative stress, the antioxidant system is active allowing them to develop and produce alkaloids [100–102]. CONCLUDING REMARKS Over the past few decades, much progress has been made on research on Uncaria spp. This genus plants are considered as a valuable source for the discovery and application of medicinal plant-derived natural products such as alkaloids and pentacyclic triterpenes as pharmaceutical agents. In vitro and in vivo studies evidenced the biological activity and therapeutic potential of the compounds isolated from this genus. However, further research is required for the development of potential drug leads from the compounds of this genus in the area of inflammatory, immunomodulatory, and vascular-related disorders. Many clinical trials have been undertaken on the crude extracts and pure compounds derived from this genus plants. For instance, products developed from bark and other parts of U. tomentosa have been commercially available in the form of nutraceutical supplements. For instance in Austria, preparations from root are accepted for the prescription for rheumatoid arthritis. However, unauthorized products are being used worldwide without appropriate Good Manufacturing Practice. For this reason, the Council of Experts of the United States Pharmacopeial Convention prepared a monograph on the U. tomentosa in order to establish specifications for further improvement in the quality of nutraceutical supplements in this country. 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Gururaja*,1 and Mario Waser* *Institute of Organic Chemistry, Johannes Kepler University Linz, Altenbergerstr. 69, 4040 Linz, Austria Chapter Outline Introduction 409 PTCs Derived from Cinchona Alkaloids 414 Michael Addition Reactions 414 a-Alkylation Reactions 415 Epoxidations 422 Alkylative Dearomatization/ Annulation 423 PTCs Derived from Binaphthol 424 a-Alkylation Reactions Mannich Reactions Michael Reactions PTCs Derived from Tartaric Acid Mannich Reactions Michael Reactions a-Alkylation Reactions Synopsis References 425 427 428 429 429 430 430 431 433 INTRODUCTION The introduction of the concept “Phase Transfer Catalysis” by Starks in 1971 [1] to explain the beneficial effect of tetraalkylammonium (or phosphonium) salts for reactions between two reaction partners that are present in two immiscible phases has significantly influenced and widened the field of organic synthesis, and the use of achiral ammonium salts as phase-transfer catalysts (PTCs) has attracted the attention as catalyst of choice for many 1. Present Address: Department of Chemistry, National Institute of Technology Karnataka, Surathkal, Mangalore, India. Studies in Natural Products Chemistry, Vol. 43. http://dx.doi.org/10.1016/B978-0-444-63430-6.00014-X © 2014 Elsevier B.V. All rights reserved. 409 410 Studies in Natural Products Chemistry fundamental reactions [2,3]. The pioneering work by Stark, Makosza, and Brändström laid a foundation for the growth of phase-transfer catalysis [1–8]. Some of the most important benefits of phase-transfer catalysis are simple experimental conditions, which usually allow for an easy scalability, mild reaction conditions, and the use of inexpensive and environmentally friendly reagents and solvents. Although the extensive use of achiral onium species dates back to the 1960s, the development of asymmetric versions progressed surprisingly slowly [9]. However, over the last decades, wide varieties of different highly important reactions have been successfully carried out in an asymmetric fashion in the presence of chiral PTCs, thus illustrating the versatile nature of asymmetric PTCs to prepare complex chiral compounds in an efficient manner [9]. Among the different commonly employed catalytically active structural motives, chiral quaternary ammonium salts have found the most widespread applications so far [9]. Following the seminal reports of Wynberg [10] and a group of Merck scientist [11] employing Cinchona alkaloid-derived quaternary ammonium salts for asymmetric epoxide formation [10a] and methylation of a phenylindanone derivative [11], Cinchona alkaloids remained the privileged source of chiral information for syntheses and investigations concerning novel PTCs and applications thereof until the beginning of the twenty-first century. Pioneering work by the groups of O’Donnell [12], Lygo [13], and Corey [14] resulted in the development of several highly stereoselective applications using a variety of structurally carefully optimized Cinchona alkaloid-based PTCs 1–5 (Fig. 1). Catalysts based on this easily obtained naturally occurring chiral starting material still belong to the most commonly employed and most thoroughly investigated ones which was impressively demonstrated in recent reports by the groups of Deng [15], Jørgensen [16], and others [17,18]. The introduction of a new catalyst system by Maruoka and coworkers using C2-symmetric binaphthyl-based chiral spiro ammonium salts 6 in 1999, paved the way for a new era in asymmetric phase-transfer catalysis. This PTC system was found to be highly effective for a variety of asymmetric transformations (e.g., Michael additions, a-amino acid syntheses, epoxidations, X OR N OH N N 1 (R = H, X = Cl) 2 (R = Allyl, X = Br) O'Donnell N N N N Br Cl Cl OH O N 3 4 5 Lygo Lygo Corey FIGURE 1 Representative examples for powerful Cinchona alkaloid-based PTCs. Chapter 14 411 Asymmetric Phase-Transfer Catalysis aldol-type reactions, isoxazoline syntheses, etc.), even using only minimum amounts of catalysts (<1 mol%) [9,19,20], thus belonging to the most powerful and versatile PTCs known to date. In addition to these classical monofunctional ammonium salts 6 and numerous other derivatives thereof, Maruoka et al. also successfully introduced bifunctional ammonium salt PTCs of the general structure 7 (Fig. 2). In addition, Lygo’s biphenyl-based spirocyclic catalysts 8 [21], Shibasaki’s tartaric acid-derived bidentate PTCs 9 [22], our group’s TADDOL-derived N-spiro catalysts 10 [23], or Denmark’s tricyclic ammonium salts 11 [24] have been carefully investigated in the past (Fig. 3). Apart from chiral ammonium salt catalysts, chiral phosphonium salts 12 developed by Manabe et al. have shown catalytic activity [25] (Fig. 4). Although Manabe’s work paved the way for new catalysts, the use of phosphonium-based catalysts was significantly less exhaustively investigated and used for asymmetric transformations, especially due to the formation of the corresponding ylide under the basic conditions that are usually required for most of the PT-catalyzed reactions [26]. Nevertheless, there has been significant progress in overcoming these limitations recently by carefully designing phosphonium salts that are stable under mild basic reactions conditions [27]. Maruoka et al. have proven that under base free neutral conditions quaternary phosphonium salts 13a show catalytic activity and reported Br Ar R Ar Br Ar OH N N OH R Ar Ar 6 Ar 7 FIGURE 2 Selected examples of chiral PTCs developed by Maruoka et al. OMe R tBu Ar X NR2 R R O Ar Ar 2X O N Ar R tBu Ar N R R Ar O X N O Ar Ar R R O R N H Me OMe 8 9 10 Lygo Shibasaki Waser FIGURE 3 Examples for PTCs based on alternative chiral backbones. 11 Denmark X R H 412 Studies in Natural Products Chemistry R OH HO R O NH HN O Ar P PR3 12 Br Ar R Br PPh2 R OH Br Ar 13a 13b FIGURE 4 Phosphonium salt derived PTC 12 developed by Manabe et al. and catalysts 13 introduced by Maruoka’s group. several asymmetric reactions catalyzed by these chiral phosphonium salts [28]. In addition, they recently introduced the bifunctional chiral phosphonium salt catalysts 13b and successfully demonstrated the utility of these catalysts under base free phase-transfer condition [29]. The asymmetric a-alkylation of glycine Schiff base 14 with various electrophiles for the preparation of natural and unnatural a-amino acids upon hydrolysis of the alkylated product was initially reported by O’Donnell and coworkers [12] and has emerged as one of the most powerful and important reactions for the field and also as the classical benchmark reaction for new chiral ammonium salt catalysts [14,19,24,30]. The commonly accepted mechanism for the stereo-differentiation suggests formation of a contact ion pair between the cationic chiral ammonium species and the enolate. As illustrated in Scheme 1, the key step in this reaction is a cation exchange of the initially formed achiral enolate (which would lead to formation of racemic 16 upon alkylation) with the PTC to give the chiral ion pair with the sterically defined ammonium group. The exact activation mechanisms of these catalysts have been extensively investigated in the past [12,14,19,24,30,31]. However, it must also be admitted that further investigations in this field are still urgently required to gather a better understanding of this powerful methodology and to facilitate the development of even more potent catalysts. Depending on the catalyst system and the reaction conditions, both the (E)- and the (Z)-enolate have been postulated to be involved in these types of reactions. This, together with the spatial arrangement of the catalyst results in the formation of a structurally defined chiral ion pair where only one face of the enolate is exposed toward the electrophile (for some well-investigated examples see Scheme 1, lower part). Noteworthy, the positive charge of the ammonium group can be expected to be delocalized to the a-carbon, thus resulting a strong H-bonding moiety as demonstrated for Shibasaki’s catalyst system [31]. Accordingly, it was proven in numerous case studies that chiral PTCs (especially ammonium salt-based ones) are among the privileged classes of catalysts in organic synthesis. The application scope includes a wide range of different asymmetric reactions where other activation modes give less Chapter 14 Ph N 413 Asymmetric Phase-Transfer Catalysis O Ph O Q X + RX OtBu Solvent, MOH 14 Ph N * Ph 15 α-Amino acids OtBu R 16 O OtBu Ph N OQ RX Ph MX Ph N * OtBu Ph R 16 (Asymmetric induction) Q X OtBu 14 Ph N RX OM Ph rac-16 Organic phase interface aqueous phase MOH H2O Cinchona catalysts pairing with (E)- or (Z)-enolate (O'Donnell, Lygo, and Corey): Coordination of Shibasaki's catalyst to the (Z)-enolate: Me HO N N O Ph N Ph Ot-Bu Ph Ph N N t-BuO N O OR Me tBu Bn N Bn O O H H H H O Bn N Me H H Ph Ph N Ph Ot-Bu SCHEME 1 Phase-transfer-catalyzed syntheses of (non-)natural amino acids starting from the achiral glycine Schiff base 14 and the commonly accepted activation modes of exemplified ammonium salts. satisfying selectivities only (e.g., a-alkylation; aldol reaction; Michael addition; Mannich, Darzens, and Strecker reactions; and also Neber rearrangement, epoxidation, fluorination, aziridination, and dihydroxylation reactions), thus representing a highly powerful and complementary activation mode to other strategies [9]. As shown in the next chapters, some of these reactions were also successfully employed to access the chiral skeletons of complex natural products or biologically active molecules. The following chapters will therefore highlight the successful use of the three most prominent chiral ammonium salt PTC classes (Cinchona alkaloids, Maruoka’s catalysts, and Shibasaki’s catalysts) to facilitate demanding stereoselective key steps in complex natural product syntheses and in the synthesis of biologically active (either natural or synthetic) compounds. 414 Studies in Natural Products Chemistry PTCs DERIVED FROM CINCHONA ALKALOIDS Cinchona alkaloids gained importance as chiral catalysts in various asymmetric reactions [32] and it was already mentioned above that Cinchona alkaloidbased PTCs are by far the most commonly used chiral ammonium salt catalysts. Accordingly, it also comes as no surprise that PTCs derived from Cinchona alkaloids have been systematically exploited in the syntheses of many (biologically active) natural products. Michael Addition Reactions Asymmetric Michael addition reactions have been among the most widely employed strategies when using chiral PTCs for the synthesis of bioactive molecules. Shishido et al. [33] reported the total synthesis of (+)triptoquinone A (17), a diterpenoid quinone isolated from the Chinese herb Tripterigium wilfordii var. regelli, possessing interleukin-1 inhibitory properties [34]. The key step in this elegant total synthesis was a stereoselective Michael addition of tetralone derivative 19 to Michael acceptor 18 using the Cinchona alkaloid-derived PTC 20 to install the quaternary stereogenic center of intermediate 21. This intermediate was subsequently converted to the annulation product 22 and further manipulations then gave (+)-triptoquinone A (17) (Scheme 2). Br N OH N CF3 20 20 (10 mol%) KOH aq (60%) O O + O O O Toluene O O O 19 18 21 [18]Crown-6 O O O HOOC H (+)-Triptoquinone A (17) O O 22 (81%, 81% ee) SCHEME 2 Stereoselective synthesis of (+)-triptoquinone (17). Chapter 14 415 Asymmetric Phase-Transfer Catalysis Park and coworkers [35] reported the efficient synthesis of (+)-polyoxamic acid (23), a key amino acid moiety of a family of peptidyl nucleoside antibiotics (polyoxins). The key step in this procedure was a stereoselective conjugate addition of glycine Schiff base 14 to acceptor 24 catalyzed by the chiral PTC 25. This gave the key intermediate 26 in quantitative yield and with very high enantioselectivity. The intermediate 26 was then successfully transferred further to yield (+)-polyoxamic acid (23) in seven steps with a high overall yield of 46% (Scheme 3). a-Alkylation Reactions The Park group explored the effective use of Cinchona alkaloid PTCs for the synthesis of (+)-hygrine (27) using an a-alkylation—ring-closing metathesis strategy [36]. (+)-Hygrine 27 is a key precursor for the synthesis of tropinone (28), the advanced intermediate in the (bio)-synthesis of the tropane alkaloids. Asymmetric methallylation of 14 in the presence of the dimeric dihydrocinchonidium salt 30 was the early key step to install the stereogenic center in this sequence. The successful total synthesis involved a total of 12 steps and proceeded in an exceptionally high overall yield of 29% (97% ee) (Scheme 4). The Park group also reported the total synthesis of ( )-cis-claviciptic acid (32), an ergot alkaloid with a tricyclic azepinoindole skeleton [37]. The synthetic analogues of claviciptic acids show activity on central nervous system in treatment of migraine attacks and have proven useful for the treatment of circulatory and digestive tract disorders [38]. Again, the dimeric dihydrocinchonidium salt 30 was found to be the catalyst of choice for the key a-alkylation of glycine Schiff base 14 with 4-iodo-N-Boc-3-bromomethylindole 33. Br N O N 25 O Ph N Ph O OtBu 25 (10 mol%) Ph 14 CO2Et N Ph 50% aq KOH CH2Cl2, −20 °C, 1 h OtBu SePh CO2Et 26 (99%, 96% ee) SePh 24 SCHEME 3 Stereoselective synthesis of (+)-polyoxamic acid (23). NH2 OH OH HO2C OH (+)-Polyoxamic acid (23) 416 Studies in Natural Products Chemistry N 2 Br N O N O N 30 30 (1 mol%) 50% aq KOH 14 + Br Toluene/CHCl3 -20 °C, 7 h O Ph N O OtBu Ph N 31 (95%, 97% ee) 29 (R)-(+)-Hygrine (27) Me N O Tropinone (28) SCHEME 4 Stereoselective synthesis of (+)-hygrine (27). Br 14 + CPh 2 I 30 (10 mol%) 50% aq KOH Toluene/CHCl3 –20 °C, 7 h N Boc 33 I N H N CO 2tBu N Boc N Boc 34 (98%, 99% ee) CO 2 H (−)-cis -Claviciptic acid (32) SCHEME 5 Stereoselective synthesis of ( )-cis-claviciptic acid (32). After installation of the stereogenic center and further manipulations, ( )-cisclaviciptic acid 32 was obtained in 20% yield and with 99% ee (Scheme 5). In 1991, Lee et al. reported the synthesis of ( )-esermethole (35) [39], which is an important precursor for the synthesis of the naturally occurring anticholinesterase agent ( )-physostigmine (36). Again a phase-transfercatalyzed enantioselective a-alkylation was the key step in this procedure. Using the quaternary ammonium salt 39, a reasonably enantioselective cyanomethylation of indole 37 to obtain intermediate 40 could be carried out. Cyclization and subsequent reactions lead to the synthesis of ( )-esermethole (35) (Scheme 6). Chapter 14 417 Asymmetric Phase-Transfer Catalysis Br OH N Cl N 39 Cl Me MeO 39 (15 mol%) N Me O + Cl 38 37 Me HN O CN O Toluene NaOH (50%) rt Me MeO N H (−)-Physostigmine (36) CN O N 40 (83%, 73% ee) MeO N Me Me N N H (−)-Esermethole (35) SCHEME 6 Stereoselective synthesis of esermethole (35). The outstanding potential of asymmetric phase-transfer catalysis to install stereogenic centers via an asymmetric a-alkylation reaction was also demonstrated by Andrus et al. in the total synthesis of hydroxyl ketones kurasoin A (41) [40] and kurasoin B (42) [41]. These are potential anticancer drugs and were isolated during a search for protein fernesyltransferase inhibitors from the fungus Paecilomyces sp. [42]. PTCs 30 and 45 derived from dihydrocinchonidine catalyzed the asymmetric alkylation of the starting materials 43 and 47 to set up the correct configuration of the protected secondary alcohol intermediates 46 and 49 with high enantioselectivities. Subsequent standard functional group manipulations then gave kurasoin A (41) and kurasoin B (42) (Scheme 7). An asymmetric a-alkylation strategy has been employed by the same group for the efficient enantioselective synthesis of naproxen (50) [43]. Naproxen is a nonsteroidal antiinflammatory drug used against pain, fever, and inflammation [44]. The highly enantioselective a-methylation of aryl acetate 51 has been achieved by using PTC 52 to give the naproxen precursor 53 straightforwardly (Scheme 8). Castle et al. reported the efficient synthesis of tryptophan derivatives with different substituents on the indole ring [45]. Their strategy involves a PTC 45 mediated alkylation of 14 with propargyl bromide 54 first. The key intermediate 55 was then transformed into the central tryptophan residue 58 of celogentin C (59) in two more steps, involving a hydrolysis of the Schiff base and amine-protection first, followed by a palladium-catalyzed heteroannulation reaction (Scheme 9). 418 Studies in Natural Products Chemistry Br N O F N O Ph F O F 45 O Ph O 45 (10 mol%) 43 + O CsOH.H2O Br CH2Cl2 hexanes −40 °C PivO O O OPG PivO OH HO O 46 (95%, 83% ee) 44 Kurasoin A ( 41) O BnO N 47 + 30 (10 mol%) N O CsOH.H2O Br O N CH2Cl2 −40 °C OBn N N Boc N Boc OH N H 49 (98%, 99% ee) Kurasoin B ( 42) 48 SCHEME 7 Asymmetric PT-catalyzed stereoselective syntheses of kurasoin A (41) and kurasoin B (42). N 2 Br O H N O N N 52 MeO O O 51 MeO 52 (10 mol%) O CsOH.H2O CH2Cl2 −40 °C, 28 h O 53 (71%, 92% ee) Ph Ph MeO O OH Naproxen (50) SCHEME 8 Stereoselective synthesis of naproxen (50). Chapter 14 419 Asymmetric Phase-Transfer Catalysis SiEt3 14 Ph 45 (10 mol%), 50% aq KOH Br N Toluene/CHCl3 (7:3), −20 °C 54 55 TBDMSO TBDMSO NH2 57 CbzHN Pd(OAc)2 (5 mol%) N H 58 (58%) SiEt3 SiEt3 (1) 1N HCl,THF (2) NaHCO3, Cbz-Cl I CO2tBu CO2tBu Ph CbzHN CO2tBu LiCl, Na2CO3, DMF, 90 °C, 23 h SiEt3 56 NH2 O HN N H O H N O H N HN NH O HN O O N H HN N H N O N NH CO2H O Celogentin C (59) SCHEME 9 Phase-transfer-catalyzed synthesis of the tryptophan residue of celogentin C (59). Me O Br(CH2)5Br ( 62) 20 (10 mol%) CH2Br (CH2)3 O NH2 Toluene/ aq NaOH rt, 48 h 61 63 (70%, 60% ee) (−)-Wy-16225 ( 60) SCHEME 10 Stereoselective synthesis of the potent analgesic agent ( )-Wy-16225 (60). An asymmetric PT-catalyzed alkylation strategy has also been the method of choice employed by Nerinckx et al. [46] for the enantioselective synthesis of the potent analgesic agent ( )-Wy-16225 (60) in 1990 already. The cinchonidine-derived PTC 20 has been employed for the asymmetric alkylation of isotetralone derivative 61 with 1,5-dibromopentane 62 under oxygen-free conditions to give key intermediate 63 with modest enantioselectivity. This compound then allowed for the straightforward synthesis of the target compound ( )-Wy-16225 (60) (Scheme 10). In 2002, Boeckman et al. reported the stereoselective total syntheses of bengamides B (64) and Z (65) [47]. These are naturally occurring 420 Studies in Natural Products Chemistry caprolactams belonging to a larger family of 24 members, which have been isolated from coral species and marine sponges [48]. Bengamide B (64) showed to possess effective antitumor activity [47]. The Cinchona-based Corey catalyst 5 was successfully used to promote the alkylation of the glycine Schiff base 14 with the chiral epoxide-containing alkylating agent 66. The key intermediate 67 was obtained as a single diastereomer and was successfully employed for the synthesis of bengamides B (64) and Z (65) (Scheme 11). Some years ago, Huffman et al. [49] reported an efficient synthesis of the estrogen b-modulator 68 using an asymmetric a-alkylation of the indanone derivative 70 (obtained from 2-fluoroanisole 69) with 1,3-dichlorobut-2-ene 71 in the presence of PTC 72. This resulted in the key intermediate 73 and following subsequent reactions then yielded the tricyclic estrogen b-modulator 68 in 34% overall yield (Scheme 12). The successful total synthesis of ( )-antofine (74), a naturally occurring phenanthroindolizidine alkaloid, was reported by Kim et al. in 2003 [50]. This alkaloid is known to be a highly potent cancer cell growth inhibitor with IC50 values in the low nanomolar range [51]. As so often, an asymmetric PT-catalyzed a-alkylation of glycine Schiff base 14 (with electrophile 75 in the presence of the dimeric catalyst 30) was the method of choice to install the stereogenic center with high enantioselectivity. The key intermediate 76 was then subjected to subsequent reactions to get the natural product in a straightforward manner with high efficiency (Scheme 13). Lygo et al. reported the asymmetric a-alkylation of 14 with allyl bromide 77 to obtain the almost enantiopure 78 as a key intermediate for the synthesis of aroylalanine derivatives like kynurenine (79) [32a]. This approach illustrates the high potential of asymmetric PTCs as it represents a powerful and direct protocol to access important naturally occurring L-tryptophan metabolites like kynurenine (79). Interestingly, the oxidative cleavage of Br N O N 5 (10 mol%) CsOH.H2O 14 + I O 66 CH2Cl2 −60 °C O Ph N OH O OtBu Ph H N O N OH OH O O 67 (83%) OR Bengamide B (R = H) (64) Bengamide Z (R = CO(CH2)12CH3) (65) SCHEME 11 Stereoselective syntheses of bengamides B (64) and Z (65). Chapter 14 421 Asymmetric Phase-Transfer Catalysis OH Br N 72 F F MeO HO Cl Bu Cl 69 Bu HO Cl Cl 73 (95%, 76% ee) 71 70 O 72 (5 mol%) 50% aq NaOH, Toluene, 10 °C, 30 h Cl Cl CF3 F O Br O F Bu HO Cl Estrogen β-modulator 68 SCHEME 12 Asymmetric synthesis of the estrogen b-modulator 68 using an early stage phasetransfer-catalyzed a-alkylation. O O O Br 30 (2 mol%) 50% aq NaOH O CO2tBu 14 N PhMe/CHCl3 (7:3) 0 °C O Ph Ph O 76 (97%, 96% ee) 75 O O N O (−)-Antofine (74) SCHEME 13 Stereoselective synthesis of ( )-antofine (74). 422 Studies in Natural Products Chemistry Br Br 14 77 H2N O (1) 5 (10mol%) KOH aq BocHN Toluene/CH2Cl2 RT (2) 15% Citric acid (Boc)2O, Et3N COOH O OtBu H2N Br 78 (75%, 94% ee) Kynurenine (79) SCHEME 14 Enantioselective total synthesis of kynurenine (79). 14 MeO Br F18 MeO O Ph 5 (4.2 equiv) 81 C N OtBu Ph Toluene CsOH.H2O 0 °C, 10 min O 57% HI OMe 18 F OMe 82 (90%, 96% ee) H2N OH OH 200 °C, 20 min 18 F OH [18F] Fluoro-L-Dopa (80) (90%, 96% ee) SCHEME 15 Stereoselective synthesis of the PET agent [18F]fluoro-L-Dopa (80). L-tryptophan to metabolites like kynurenine (79) is supposed to play a very important role in a variety of fundamental biological processes like cell growth and cell division, thus making it a promising target with respect to drug development [52] (Scheme 14). The efficient synthesis of [18F]fluoro-L-Dopa (80) was reported by Lemaire et al. [53] [18F]fluoro-L-Dopa (80), a radiopharmaceutical for positron emission tomography (PET), was synthesized by asymmetric PT-catalyzed alkylation of 14 with 2-[18F]fluoro-4,5-dimethoxybenzyl bromide 81. An excess of the catalyst was employed in the key step of this reaction to give key intermediate 82 in very short time with high yield and enantioselectivity followed by a final very fast hydrolysis to give [18F] fluoro-L-Dopa (80) to be readily used for PET diagnostics (Scheme 15). The Lygo–Corey-type catalyst 5 was successfully employed by Zhu and coworkers to facilitate an early stage a-alkylation in the synthesis of lemonomycinone amide 83 [54], a precursor for the synthesis of ( )-lemonomycin (84). Hereby an enantioselective alkylation of 14 with benzyl bromide 85 was employed to obtain the intermediate 86 in high yield and with excellent enantioselectivity (Scheme 16). Epoxidations Besides stereoselective a-alkylation and Michael addition reactions, the epoxidation of a,b-unsaturated carbonyl acceptors using either hydrogen peroxide or hypochlorite as the oxygen source has emerged as a powerful methodology under chiral phase-transfer conditions. As an example for the potential of this methodology, the PTC-mediated stereoselective synthesis of loxistatin (88) was reported by Lygo et al. in 2006 [55]. Herein, the diastereoselective epoxidation of enone 89 has been employed as a key step in this sequence (Scheme 17). Chapter 14 C N Br 5 (10 mol%) MeO O Ph 14 OTBS OMe 423 Asymmetric Phase-Transfer Catalysis OMe Me OtBu Ph CO2Me OTBS CsOH. H2O, CH2Cl2 24 h, −78 °C NH MeO MeO OBn OMe Me Me 86 (85%, 98% ee) 85 HO O Me 87 OH HO H NH O N MeO O O OBn Me OH OH H NH N MeO O OH O OH NMe2 (−)-Lemonomycin (84) Lemonomycinone amide (83) SCHEME 16 Stereoselective synthesis of lemonomycinone amide (83). Br N O N Ph 90 90 (5 mol%) O Ph N H O CO 2 Me O Ph 15% aq NaOCl toluene, 25 °C O N H O CO 2 Me 91 (70%, 5:1 dr) 89 O EtO O N H O H N O Loxistatin (88) SCHEME 17 Lygo’s stereoselective synthesis of loxistatin (88). Alkylative Dearomatization/Annulation Porco and coworkers [56] reported the use of the Cinchona alkaloid-derived dimeric ammonium salt 92 to catalyze an alkylative dearomatization/annulation process to build up the highly functionalised adamantane core of 424 Studies in Natural Products Chemistry N H N H BnO 2 Br N OBn N 92 OH O O + HO OH Clusiaphenone (94) 5 O 92 (25 mol%) O CHO CsOH.H O, MS 2 CH2Cl2 −50 °C 95 OH O O O 96 (71%, 90% ee) O O O O Hyperibone K (93) SCHEME 18 Total synthesis of hyperibone K (93). hyperibone K (93). Hyperibone K (93) is known for its moderate activity as an inhibitor of human tumor cell replication [57]. It was found that the dimeric catalyst 92 is the best suited among a series of different Cinchona alkaloid-based ammonium salts for the dearomatization/annulation reaction between the prenylated benzophenone clusiaphenone B (94) and enal 95. This protocol gave access to the highly functionalized adamantane-based key intermediate 96 in good yield and with high enantiomeric excess of 90% (Scheme 18). PTCs DERIVED FROM BINAPHTHOL In addition to PTCs derived from Cinchona alkaloids, chiral PTCs derived from (R)- or (S)-binaphthol (the so-called Maruoka catalysts) have emerged as one of the most powerful catalyst classes in organocatalysis. Without exaggeration it can be said that the Maruoka group has really pioneered a whole field herein by carefully developing these catalysts and by introducing a variety of complex and extraordinarily stereoselective transformations facilitated by these highly potent catalysts [9,20]. These catalysts have also recently Chapter 14 425 Asymmetric Phase-Transfer Catalysis proven their high potential to facilitate the synthesis of natural products or biologically active molecules. a-Alkylation Reactions Very recently, Maruoka and coworkers reported the efficient asymmetric synthesis of chiral 2,4-substituted piperidine derivatives as exemplified in the synthesis of selfotel (CGS-19755) (97) [58], a potent NMDA receptor antagonist. Hereby, a phase-transfer-catalyzed asymmetric alkylation followed by a ring-closing reductive amination was used as the key step to access the target piperidine key intermediate 101 in good yield and with high stereoselectivity. This compound was then subsequently transformed into selfotel (CGS-19755) (97) by standard functional group manipulations (Scheme 19). The Maruoka group also showed the potential of this methodology to access cis-configured 2,6-disubstituted chiral piperidine derivatives like the naturally occurring alkaloid (+)-dihydropinidine (102) in the same publication [58]. Park and coworkers reported the synthesis of ( )-paroxetine (103) [59], an antidepressant drug from the class of drugs known as selective serotonin reuptake inhibitors (SSRIs) [60]. In their approach, Park and coworkers synthesized 103 by an asymmetric alkylation of malonic ester 104 in the presence of PTC 105. The key intermediate 106 was obtained in 92% yield and 95% ee. Ar Br Bu N Bu Ar 99 (Ar = 3,4,5-F3C6H2) O Ph 14 Br 99 (2 mol%) O BnO O N OH OtBu Ph CsOH Toluene −40 °C, 5 h OBn O 1. TFA, H2O EtOH, RT, 1 h 2. Pd/C, H2 40 °C, 2 4 h N H O 100 (87% 97% ee) 98 CO2tBu 101 (65%, 10:1 dr) PO(OH)2 14 N H Pr N H HCl (+)-Dihydropinidine.HCl (102) CO2H Selfotel (97) SCHEME 19 PT-catalyzed asymmetric synthesis of chiral piperidine derivatives. 426 Studies in Natural Products Chemistry Ar Br N Ar 105 (Ar = 3,4,5-F3C6H2) MeO MeO O N CO2tBu 77 OMe O CO2tBu N 105 (2 mol%) HN CsOH toluene −40 °C, 5 h O O OMe 106 (92%, 95% ee) 104 F O Br (−)-paroxetine (103) SCHEME 20 Stereoselective total synthesis of paroxetine (103). Ph Ph O N 108 + 77 CO2tBu 105 (5 mol%) KOH, toluene −40 °C, 24 h Ph Ph O HO CO2tBu N HN Br 109 (95%, 98% ee) (+)-Isonitramine (107) SCHEME 21 Enantioselective total synthesis of (+)-isonitramine (107). Compound 106 was then subsequently converted to ( )-paroxetine (103) without any loss of optical purity (95% ee of the target product) (Scheme 20). Park et al. also reported an analogous strategy for the synthesis of (+)isonitramine (107) [61]. A phase-transfer catalyzed enantioselective a-alkylation of lactam 108, followed by subsequent manipulations, was employed for the successful total synthesis of the Nitraria alkaloid (+)isonitramine (107) (Scheme 21). The asymmetric alkylation of 14 with o-bromobenzyl bromide 110 catalyzed by the (R,R)-enantiomer of ammonium salt 105 was employed by Maruoka and coworkers for the efficient asymmetric synthesis of (S)-N-acetyl indoline-2-carboxylate 111. Following the initial asymmetric alkylation, hydrolysis, and N-acetylation gave the key intermediate 112, which could be further converted into 111 [20f]. (S)-N-acetyl indoline-2-carboxylate 111 is the key intermediate in the synthesis of the ACE inhibitor 113 (Scheme 22). A highly enantioselective PT-catalyzed alkylation mediated by Maruoka’s catalyst was also successfully employed for the synthesis of the Parkinson therapeutics L-Dopa ester and its analogues [20f,62]. Hereby, minute amounts of catalyst 105 were used to catalyze the alkylation of 14 with benzyl bromides 114 and 115 followed by hydrolysis to give the corresponding amino esters 116 and 117, respectively. The corresponding L-Dopa tert-butyl ester Chapter 14 (R,R)-105 (1 mol%) Br 14 427 Asymmetric Phase-Transfer Catalysis 1 M citric acid, THF, rt, 3 h AcCl, Et3N, CH2Cl2 toluene 50% aq KOH 0 °C, 24 h Br 110 CO2tBu Br NHAc 112 (86%, 99% ee) Pd2(dba)3, P(o-tol)3 Cs2CO3, toluene 100 °C, 10 h N O CO2H Me CO2Et N Ac Me CO2tBu 111 (94%, 99% ee) ACE inhibitor 113 SCHEME 22 Stereoselective synthesis of (S)-N-acetyl indoline-2-carboxylate (111). 14 R (R,R)-105 (1 mol%) Br BnO 114 (R = OBn) 115 (R = H) Toluene 50% aq KOH 0 °C, 1–2 h 1 M citric acid, THF, rt, 10 h H2N CO2tBu R OBn 116 (R = OH, 94%, 98% ee) 117 (R = H, 93%, 98% ee) 10% Pd/C,H2 THF, rt, 5 h H2N CO2tBu R OH 118 (R = OH, 81%, 98% ee) 119 (R = H, 83%, 98% ee) SCHEME 23 Stereoselective synthesis of L-Dopa ester 118 and its analogue 119. 118 and the derivative 119 were then obtained by a hydrogenative debenzylation of amino esters 116 and 117 (Scheme 23). The Maruoka catalyst 105 was also the catalyst of choice for the efficient alkylation of glycine amide derivative 120 in the synthesis of levobupivacaine (121) as reported by Kumar et al. [63]. The hereby obtained key intermediate 123 was then successfully used for the synthesis of the local anesthetic drug levobupivacaine 121 (Scheme 24). Mannich Reactions Mannich-type reactions are another important class of transformations that have very successfully been carried out using asymmetric phase-transfer 428 Studies in Natural Products Chemistry Me O Me O Ph N Ph N Bn (R,R)-109 (1 mol%) Cl I Me 120 Ph 122 CsOH.H2O toluene/CH2Cl2 (7:3) −40 °C, 18–20 h N N Bn Me Ph Cl 123 (57%, 86% ee) N Bu O H N Me Me Levobupivacaine (121) SCHEME 24 Phase-transfer catalyzed a-alkylation in the synthesis of levobupivacaine (121). O PMP O HN 105 (2 mol%) 1 N HCl N OEt 14 O 124 Mesitylene 17% aq NaOH −20 °C, 6 h THF PMP CO2Et tBuO NH2 126 (91% ee, syn) HN N PMP H H OH O N PMP 125 SCHEME 25 Stereoselective synthesis of an advanced precursor for the synthesis of streptolidine lactam. catalysis. For example, the direct Mannich reaction of 14 with a-amino esters 124 has been successfully employed by Maruoka et al. for the efficient synthesis of 125 [64], which is an important precursor of streptolidine lactam (streptolidine lactam constitutes the core structure of the streptothricine antibiotics) (Scheme 25). Michael Reactions Maruoka and coworkers [65] also reported the total synthesis of (+) monomorine (127) using a phase-transfer-catalyzed conjugate addition of glycine ester 128 to Michael acceptor 129 as an early key step in the synthesis sequence. Monomorine (127) is a bicyclic amine, known to be the trail pheromone of Monomorium pharanois [66]. The conjugate addition product 131 was subjected to an intramolecular reductive amination and acetal hydrolysis in one pot reaction with Hantzsch ester 132 and trifluoroacetic acid in aqueous Chapter 14 429 Asymmetric Phase-Transfer Catalysis F F Ar N F Br O Ar = F Ar F 130 F O Ph N EtO2C OCHtBu2 Ph Ph 130 (1 mol%) 128 O O O CsCl, K2CO3 CPME O 0 °C, 12 h N OCHtBu2 Ph O O O CO2Et N H 132 H N CF3COOH EtOH-H2O CO2CHtBu2 133 (52%, 93% ee) 131 129 H N (CH2)3CH3 (+)Monomorine (127) SCHEME 26 Asymmetric PT-catalyzed synthesis of (+)-monomorine (127). ethanol to give the octahydropyrrolizine core structure 133 with a high enantiomeric excess of 93% (Scheme 26). PTCs DERIVED FROM TARTARIC ACID Tartaric acid belongs to the most easily available natural chiral sources, benefiting from the fact that both enantiomers are easily available in pure form and thus finds wide applicability either to access chiral ligands or to obtain organocatalysts [67]. With respect to the application of tartaric acid-derived PTCs [22,23] for natural product synthesis, the work of Shibasaki’s group should be highlighted herein. Using his powerful bidentate TaDiAS PTCs, asymmetric phasetransfer-catalyzed alkylations, Michael addition reactions, and Mannich-type reactions have been systematically carried out. Mannich Reactions After a thorough screening of different TaDiAS for the Mannich reaction of glycine Schiff base 14 with Boc-protected imines 134 it was found beneficial 430 Studies in Natural Products Chemistry Ph 14 N Boc R 2 BF4 O N O N Ar Ar Ar Ar Ph 135 (Ar = 4-MePh) 135 (10 mol%) R PhF/pentane, −20 °C 134 NHBoc CO2tBu N Ph Ph 136 (95%, 99:1 dr, 93% ee) O Cl MeHN NBn N H OMe (+)-Nemonapride (137) SCHEME 27 Stereoselective total synthesis of nemonapride (137) using a phase-transfer-catalyzed Mannich reaction. to introduce a 2,6-disubstituted cyclohexane-based ketal group and employ tetrafluoroborates as the counter anions (catalyst 135) to obtain the Mannich products 136 in high enantio- and diastereoselectivity even when using enolizable aliphatic imines [22]. This powerful Mannich strategy was then successfully employed in the short total synthesis of the antipsychotic agent (+)-nemonapride (137) [22c] as shown in Scheme 27. Michael Reactions The Shibasaki group also demonstrated the potential of their C2-symmetric diammonium salt catalyst 135 for the syntheses of the alkaloids (+)cylindricine C (138) and ( )-lepadiformine (139) [22c,68,69]. By applying a PT-catalyzed addition of Schiff base 140 to Michael acceptor 141, the key intermediate 142 was obtained in good yield and with good enantiomeric excess. Compound 142 could then be used to obtain selectively either the cylindricine C precursor 143 or the lepadiformine synthon 144 in a very efficient tandem cyclization reaction by choosing the optimum reagents. The impressively short total synthesis of (+)-cylindricine C (138) could be achieved in only two additional steps, whereas the synthesis of the tricyclic intermediate 144 represents a formal total synthesis of ( )-lepadiformine (139) (Scheme 28). a-Alkylation Reactions Shibasaki’s group also reported an elegant synthesis of aeruginosin 298-A (145), a potent serine protease inhibitor isolated from a blue-green alga [70], using chiral phase-transfer-catalyzed alkylations in the syntheses of three fragments [71]. Aeruginosin 298-A (145) has a tetrapeptide-like structure including nonstandard amino acids, thus presenting an appropriate motif for Chapter Ph 14 N 431 Asymmetric Phase-Transfer Catalysis CO2Bn + O Ph 135 (10 mol%) O 5 C6H13 Cs2CO3 (1.5 eq.) 3-fluorotoluene −40 °C CSA (3 eq.) MgCl2 (3 eq.) 1,2-dichloroethane 50°C O BnO2C N O C6H13 Ph Ph O N BnO2C N C6H13 CSA (3 eq.) HO C6H13 (+)-Cylindricine C (138) 143 142 DMSO 50 °C 5 142 (84%, 82% ee) 141 140 O O N N BnO2C C6H13 144 HO C6H13 (−)-Lepadiformine (139) SCHEME 28 Stereoselective of cylindricine C (138) and lepadiformine (139). phase-transfer-catalyzed amino acid syntheses. As depicted in Scheme 29, the two-center tartaric acid-derived catalysts 146 and 147 worked well for the installation of the stereogenic centers of the amino acid fragments 150, 151, and 152 by appropriate alkylation of Schiff base 14 with the corresponding electrophiles in the presence of the matching enantiomer of the C1-symmetric catalyst. One interesting fact concerning this methodology is the importance of the catalyst counter anion X in these alkylation reactions. While catalyst 146 with X ¼ I gave fragment 152 in 79% yield and 91% ee, the use of BF4 as the counter anion (catalyst 147) gave an even better yield (85%) and enhanced the enantioselectivity slightly (93% ee). These amino acid-based fragments could then be assembled successfully to obtain aeruginosin 298-A (145) in a short and high yielding sequence (Scheme 29). SYNOPSIS Asymmetric phase-transfer catalysis is a unique method that has for almost three decades proven its high utility. Although its typical application is for (nonnatural) amino acid synthesis, over the years, other types of applications have been reported. The unique capability of quaternary ammonium salts to form chiral ion pairs with anionic intermediates gives access to stereoselective 432 Studies in Natural Products Chemistry tBu O Ph N Ph OtBu O N O N Ar Ar Ar 2 X Ar 146 (Ar = C6H4-4-OMe; X = I) 147 (Ar = C6H4-4-OMe; X = BF4) 14 Br Br (S,S)-146 (10 mol%) 29 Br (R,R)-146 (10 mol%) O O (R,R)-147 (10 mol%) 149 148 O Ph N O OtBu Ph Ph N O Ph OtBu Ph N OtBu Ph O O 150 (93%, 91% ee) 152 (85%, 93% ee) 151 (80%, 88% ee) HO O HO OH H N H O N H H N O NH N H NH2 OH Aeruginosin 298-A (145) SCHEME 29 Shibasaki’s phase-transfer-catalyzed stereoselective total synthesis of aeruginosin 298-A (145). transformations that are otherwise very difficult to conduct using metal catalysts or other organocatalysts. Thus, this catalytic principle has created its own very powerful niche within the field of asymmetric catalysis. As illustrated in this review, the high potential of this methodology to facilitate asymmetric a-alkylation reactions, Michael additions, epoxidations, or Mannich-type reactions has been exhaustively used by organic chemists to access a variety of structurally different (biologically active) (natural) products. Based on the unique potential of this methodology, it is therefore without doubt that these catalysts will be systematically used to access even more complex natural products in the future. Chapter 14 Asymmetric Phase-Transfer Catalysis 433 REFERENCES [1] C.M. Starks, J. Am. Chem. Soc. 93 (1971) 195–199. [2] E.W. Dehmlow, S.S. Dehmlow, Phase Transfer Catalysis, third ed., VCH, Weinheim, 1993. [3] C.M. Starks, C.L. Liotta, M.E. Helpern, Phase Transfer Catalysis, Chapman and Hall, New York, 1994. [4] M. Makosza, Tetrahedron Lett. 7 (1966) 4621–4624. [5] M. Makosza, Tetrahedron Lett. 7 (1966) 5489–5492. [6] M. Makosza, Tetrahedron Lett. 10 (1969) 673–676. [7] M. 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This page intentionally left blank Chapter 15 Challenges of Biopesticides Under the European Regulation (EC) No. 1107/2009: An Overview of New Trends in Residue Analysis Juan José Villaverde, Beatriz Sevilla-Morán, Pilar Sandı́n-España, Carmen López-Goti and José Luis Alonso-Prados Plant Protection Products Unit, DTEVPF, INIA, Madrid, Spain Chapter Outline Introduction 438 European Legislation for Sustainable Use of Pesticides and Challenges that Biopesticides Have to Face 440 Evolution of Regulation at the European Level 440 Regulation (EC) No. 1107/2009: Data Requirements and Uniform Principles 443 Directive 2009/128/EC: IPM Practices and Current Barriers for Biopesticide Registration 445 Advantages and Disadvantages of “Biopesticides” and the Controversial Use of this Term 448 Requirements in Analytic Methods to Fulfill the Actual European Legislative Framework Analytic Techniques Applied to the Analysis of Biopesticide Residues Sample Preparation Techniques Chemical Analysis: Instrumental Techniques and Future Trends Analysis of Biopesticide Residues in Environmental Matrices Conclusions References Studies in Natural Products Chemistry, Vol. 43. http://dx.doi.org/10.1016/B978-0-444-63430-6.00015-1 © 2014 Elsevier B.V. All rights reserved. 449 451 452 467 473 475 478 437 438 Studies in Natural Products Chemistry INTRODUCTION There is not a formally agreed definition of a biopesticide at the European level. Regulation (EC) No. 1107/2009 [1], concerning the placement of plant protection products on the market, shall be applied to substances, including microorganisms, with general or specific action against harmful organisms or plants, parts of plants or plant products. This regulation defines substances as those chemical elements and their compounds that occur naturally or are produced by manufacturer. Therefore, biopesticides are in the scope of this regulation. According to the definition given by the U.S. Environmental Protection Agency (U.S. EPA), biopesticides are defined as crop protection agents derived from natural materials such as animals, plants, bacteria, and certain minerals [2]. Comparing the U.S. EPA definition with the Regulation (EC) No. 1107/2009, it can be observed that the latter includes biopesticides defined by the U.S. EPA. These definitions from both respective territorial agencies include living organisms that are grown in bulk and used on a seasonal basis as a pesticide (including active agents of bacteria, fungi, nematodes, protozoa, virus, and beneficial insects) and natural products that are introduced into an ecosystem on a single occasion getting the suppression of a pest on a long-term basis [3]. The biological activity spectrum of biopesticides is extremely variable and has a range of attractive properties via novel mechanisms of action [4–6]. These properties make them good to manage the appearance of resistance to the compounds currently available. At the beginning of this century, about 1400 biopesticide products have been sold in the world [7,8], accounting for about 2.5% of the total pesticide market [8,9]. According to MarketsandMarkets [10], the demand for biopesticides is expected to grow at the faster compound annual growth rate of 16.1% (compared with 3% for synthetic pesticides) from 2012 to 2017 [10], which is expected to produce a global market of $5.2 billion in 2017 [10,11]. Currently, North America consumes about 40% of global biopesticide production, with Europe, Oceania, and Latin America accounting for 20%, 20%, and 10%, respectively [12]. Therefore, Europe is an important market for the promotion of these products, with the fastest growing [12] and a modern regulation [1] that supports and regulates their use: any plant protection product, before being marketed, must pass a rigorous evaluation process and prove that there is no risk on human and animal health and the environment. Regulation (EC) No. 1107/2009 [1] describes residues as substances present in or on plants or plant products, edible animal products, drinking water, or elsewhere the environment and resulting from the use of a plant protection product, including their metabolites, and breakdown or reaction products (e.g., substances resulting from water treatment). Society has a general false perception that biopesticides are safe due in part to their natural origin. However, they also generate residues that may be toxic to humans. Based on the Chapter 15 Biopesticides, EU Regulation and Residue Analysis 439 definition of residues fixed by plant and animal metabolism studies, adequate analytic methods must be validated to analyze the residue of field trials, carried out for setting maximum residue levels (MRLs) in crops and animal products (for risk assessment purposes), so as to supervise in field (for monitoring purposes). The knowledge and elucidation of the biopesticides’ behavior in the different compartments of nature represent difficulties, and the definition of residue for risk assessment and monitoring is not easy to establish. The determination of these residues at very low concentrations or very complex samples is not possible in many cases without the use of previous treatment and/or chromatographic techniques combined with spectroscopy/spectrometry. All these analytic methods should be validated to obtain consistent, reliable, and accurate data and satisfy the actual legislative framework. It is not an exaggeration to say that the whole analytic method, from pretreatments to final determination by modern instrumental techniques, has a decisive influence on the reliability of the data measured. About 25 years ago, many practices in sample preparation were based on traditional technologies, such as solid–liquid extraction [13] or liquid–liquid extraction [14], which need huge quantities of solvent and sample. Today, in most modern methodologies, the main purposes are to minimize the use of solvents and to achieve a greater automation of the process in minor time (e.g., by solid-phase extraction (SPE)). In the 1990s, gas chromatographs (GCs) equipped with element-selective detectors (e.g., electron capture detector (ECD), flame photometric detector, and flame thermionic detector) and high-performance liquid chromatographs (HPLCs) equipped with UV detectors were mainly used. Nowadays, liquid chromatography (LC) and gas chromatography (GC) coupled to mass spectrometry (MS) are among the most important techniques to determine the trace levels of pesticides and their transformation products [15,16]. In fact, today, both chromatographic techniques can be equipped with several mass analyzers, each of which provides unique features capable to identify, quantify, and solve ambiguities by selecting appropriate ionization/acquisition parameters. This technological progress each day achieves lower limits of quantification (LOQs) and limits of detection (LODs), so that time to time, the legal requirements for pesticide residues in several matrices (e.g., foodstuff of animal origin, soil, water, air, and body fluids and tissues) are hardened with the objective of reducing the maximum risk on human and animal health and the environment. The purpose of this chapter is to provide a detailed study about the impact of bioactive natural products on the progress of new pesticides and their future projection. Furthermore, it is also intended to carry out a state of the art about the European legislation for the sustainable use of plant protection products from natural origin, highlighting the importance of analytic method validation on the registration of these products and focusing the attention on spectroscopic and chromatographic equipments for risk assessment and monitoring of residues from these compounds to comply with the legislative framework. Finally, the controversial use of the term biopesticide will be clarified. 440 Studies in Natural Products Chemistry EUROPEAN LEGISLATION FOR SUSTAINABLE USE OF PESTICIDES AND CHALLENGES THAT BIOPESTICIDES HAVE TO FACE Evolution of Regulation at the European Level The potential scope of this revision could be vast due to all existing world regulations for pesticides. However, we have decided to focus the attention on the novel EU regulation due to the important market of this economic and political union and because in the last years, this regulation has encouraged the development of integrated pest management (IPM) practices and has provided incentives that could support the entry of biopesticides in the market. The first attempts to harmonize national laws were produced in the 1970s, but failed due to the existence of two major obstacles: The first, of a political nature, was the reluctance of member states to cede sovereignty plots of the community institutions, and the second, of a practical nature, was the difficulty to provide an authorization with all variants needed for the diversity of environmental conditions, plant health, and cultural conditions in the different regions of Europe. In the late 1980s, the objective of achieving the unique market, established a few years later by the birth of the European Union with the Maastricht Treaty [17], determined the immediate need for harmonization of national legislation on the marketing of plant protection products. The commission presented in 1989 a draft directive and after being discussed for more than 2 years, it was adopted by the Council Directive 91/414/EEC of 15 July 1991 [18]. The need to avoid such difficulties determined that this directive could not establish a true European unique registration system, but a mixed solution of unique positive list for active substances and a unique registration system, applicable to all member states, for sold authorizations of plant protection products in their respective territories. The unique market was ensured with the requirement of mutual recognition by establishing that any authorization granted by a member state must be recognized by others. The great breakthroughs of this directive, concerning the placement of plant protection products on the market, urged member states that plant protection products must be sufficiently effective. Furthermore, they must have no unacceptable effect on plants or plant products, no harmful effect on human or animal health (directly or indirectly) or on groundwater, and no unacceptable influence on the environment. Special regard should be taken into account to the fate and distribution in the environment, particularly the contamination of water including drinking water and groundwater, together with its impact on nontarget species. This directive also establishes that only plant protection products, whose active substances are listed in its Annex I, can be commercialized in EU, together with the requirements for inclusion in this unique positive list. In parallel, a review program of active substances already in the EU market was developed, as referred to in Article Chapter 15 Biopesticides, EU Regulation and Residue Analysis 441 8 (point 2) of the Council Directive 91/414/EEC, to determine their inclusion or not in such Annex I [18] and to ensure the safety of all active substances used throughout the EU by modern standard evaluations. This review program was divided into four stages implemented by the following commission regulations: No. 3600/92 (first stage) [19], No. 451/2000 (second and third stages) [20], and No. 1112/2002 (fourth stage) [21]. At present, the EU portfolio comprises 422 approved active substances, of which 321 are existing substances before the review program in 1992 (first stage, 55; second stage, 34; third stage, 120; and fourth stage, 112) and 101 are new substances [22] (Fig. 1). The fourth (and final) stage of the EC pesticide review programme was started in 2004 and was completed in 2009. This was different from the first three because the substances involved were different in nature: While the first three stages were focused on conventional chemicals, the fourth stage was focused on various groups of substances that do not fit precisely under this description such as animal products, attractants or repellents, commodity substances, disinfectants, microorganisms, pheromones and other semichemicals, plant extracts, rodenticides, substances authorized in human food or animal feed, substances for treating stored plants or plant products, and substances on the market in one of the new member states but not approved in the existing member states. It could be said that biopesticides are included within this group of substances, being the objective of the fourth stage to maintain essential health and environmental protection safeguards while trying to avoid a situation in which many substances are lost, because the data requirements cannot be fulfilled economically. The inclusion directives for the existing active substances set out a program to review the authorizations of plant protection products by member states, according to Annex III (requirements for plant protection products) and Annex VI (uniform principles) of the Council Directive 91/414/EEC [18]. This process is called reregistration [23]. The European Commission has adopted the precautionary principle [24], already referenced and adumbrated in the EC Treaty [17], which establishes a risk management strategy that is applied if there are reasonable grounds FIGURE 1 Status under Regulation (EC) No. 1107/2009 of active substances intended to be commercialized in the EU by 2013 (left) and distribution of approved active substances according to their stage (right). 442 Studies in Natural Products Chemistry for concern that potential dangerous effects may affect the environment or human, animal, or plant health and when the available data are insufficient for a detailed risk evaluation. But these restrictions continued being based on undesirable risks, necessarily increasing the safety level by introducing cutoff criteria related to specific properties. In July 2011, the Regulation (EC) No. 1107/2009 [1] replaced the Council Directive 91/414/EEC and brought up some significant modifications to solve the identified problems, although in much, it was operated in the same way as under the Council Directive 91/414/EEC. The main differences within this regulation regarding the directive are those related with the active substances: Now, approval conditions are specified in Article 4 and Annex II, including new hazard criteria based on the substances’ intrinsic properties [1]; additionally, substances may be approved and listed in a separate commission regulation (Article 13) in various categories that include standard, low-risk, and basic substances, together with those candidates for substitution, being approved for up to 10 years (Article 5), 15 years (Article 22), without specific time limit (Article 23), and 7 years (Article 24), respectively. This fact clearly favors the development and use of those substances that comply with Section 5 of Annex II (low-risk substances) and commodity chemicals (basic substances), regarding those that meet any of the conditions of Section 4 of Annex II (candidates for substitution) and standard substances. Approvals may be renewed for up to 15 years in the case of standard and low-risk substances or up to 5 years in the case of those approved under the derogation in Article 4 (Article 14) or up to 7 years in the case of candidates for substitution (Article 24). There are three main differences observed for the marketing authorization proceedings after the adoption of the Council Directive 91/414/EEC and kept within the Regulation (EC) No. 1107/2009 [1,18]: First, a uniform time limit, across the European Union, of 120 days to decide regarding a mutual recognition of authorizations is set, instead of being governed by national legislations; second, applicants (manufacturers of plant protection products) are not required to demonstrate compatibility of conditions among different countries, being the registration authority responsible for providing grounds to the European Commission for refusal of marketing authorization from a member state belonging to the same zone; and third, applicants are entitled to apply to official, scientific, or professional organizations focused in agricultural activities for mutual recognition of a product. The Regulation (EC) No. 1107/2009 is now in place, cooperation between member states has enormously increased, and several pilot schemes of zonal collaboration have been carried out in the last years. However, it is necessary to continue working in the reregistration process of harmonization to obtain, with this zonal evaluation system, a more homogeneous market for the plant protection products. Chapter 15 Biopesticides, EU Regulation and Residue Analysis 443 Regulation (EC) No. 1107/2009: Data Requirements and Uniform Principles The first thing to highlight about the Regulation (EC) No. 1107/2009 is that it is a regulation, not a directive as the previous Council Directive 91/414/EEC and as such is directly applicable in all member states without necessarily being transposed into each national legislation. This regulation recognizes that the use of plant protection products is one of the most important ways of protecting plants and plant products against harmful organisms (including weeds) and of improving agricultural production. It is also recognized that the use of these products can have nonbeneficial effects on plant production and their use may involve risks and hazards for humans, animals, and the environment. For those reasons, they have been officially assessed according to the uniform principles and authorized. The following are the three basic objectives of the regulation: first, to ensure a high level of protection for both human and animal health and the environment and at the same time to safeguard the competitiveness of community agriculture; second, to safeguard the protection of vulnerable groups of the population (pregnant women, infants, and children); and third, to increase the free movement of these products and their availability in member states by laying down harmonized rules, including the rules on the mutual recognition of authorizations and on parallel trade. Particular attention will also be paid to the competitiveness of community agriculture [1]. Therefore, the Regulation (EC) No. 1107/2009 has been promoted with the aim of not approving the compounds with unacceptable risk, so that several plant protection products will have difficulties to renew their authorization (most taking place between 2016 and 2019) due to the new registration requirements (cutoff criteria). This will happen without considering their potential benefits and the potential to handle them safely, purely as a result of their classification (Table 1). In fact, the Kemikalieinspektionen Swedish TABLE 1 Cutoff Criteria for the Approval of Active Substances, According to Annex II of the Regulation (EC) No. 1107/2009 [1] Human Health Environmental Carcinogen (1A or 1B) PBT (persistent, bioaccumulative, and toxic) Mutagen (1A or 1B) POP (persistent organic pollutant) Toxic for reproduction (1A, 1B or 2) vPvB (very persistent and very bioaccumulative) Endocrine disruptor Endocrine disruptor 444 Studies in Natural Products Chemistry Chemicals Agency [25,26], using the formerly applied national criteria and principles, found that 23 active substances (8 herbicides, 11 fungicides, 3 insecticides, and 1 plant growth regulator) will be withdrawn under the Council Common Position cutoff criteria. These 23 active ingredients constitute approximately 8% of the 271 substances (included in Annex I of the Council Directive 91/414/EEC and a number of substances with decision pending). However, according to the Pesticides Safety Directorate [26,27] of the United Kingdom, the number of substances falling under the cutoff criteria would be 46 (16%) for the original commission proposal and 118 (41%) for the European Parliament first reading proposal, of 286 evaluated. There are two scenarios under which is possible the approval of an active substance that fails the cutoff criteria: if the exposure to humans, under realistic proposed conditions of use, is negligible (see Annex II, point 3 of Regulation (EC) No. 1107/2009 for more details) and if it is necessary to control a serious danger to plant health, which cannot be contained by other available means, including nonchemical methods (see Article 4, point 7 of Regulation (EC) No. 1107/2009 for more details). Another special group for active substances is that of candidates for substitution and recognized in Article 24 of Regulation (EC) No. 1107/2009. Active substances, which meet one or more of the additional criteria laid down in point 4 of Annex II, will be only approved for seven years. Furthermore, any pesticide containing that active substance will be required to undergo comparative assessment at member state-level (see Article 50 of Regulation (EC) No. 1107/2009 for more details), comparing this pesticide with other approved ones or nonchemical methods of control/prevention to substitute the more hazardous with the safer option [1]. Regulation No. 283/2013 [28] implements Regulation (EC) No. 1107/2009 and establishes the data requirements for active substances. It specifies the information that applicants must submit in its dossier for approval of the active substance and the tests and analyses that must be carried out. Part B of this regulation concerns requirements relating to microorganisms including virus. Regulation 284/2013 [29] sets out the applicable data requirements, for the authorization at the national level of plant protection products referred to in Regulation (EC) No. 1107/2009 (Article 8.1, Letter c) after the approval of the active substance contained in the plant protection product. This means that it must include a technical file with the information needed to evaluate the efficacy and the foreseeable risks that may involve the product for humans, animals, and the environment, including the results of studies explicitly described in the annex of the regulation. Microorganisms are also regulated under a separate part B. While active substances should be approved at the EU level, formulated products are authorized at the national level based on uniform principles laid down in the Commission Regulation No. 546/2011 [30]. These uniform principles aim to ensure the implementation by all member states of the requirements of Article 29.1 (Letters e–h) of Chapter 15 Biopesticides, EU Regulation and Residue Analysis 445 Regulation (EC) No. 1107/2009: The action of the product should be evaluated from the point of view of efficacy and phytotoxicity in each use for which authorization is sought, determined its dangerousness and importance, and will rule on the likely risks to humans, animals, and the environment. After performing each evaluation, member states could impose conditions or restrictions with the authorizations granted or simply refuse when there are grounds that justify it. The actual EU regulation establishes three climatically similar zones within EU (northern, central, and southern) for the authorization of plant protection products to share workload versus the previous scheme of individual assessment by countries and the mutual recognition of authorizations already granted and tight deadlines for all these mechanisms (for more details, see Articles 7–13 of Regulation (EC) No. 1107/2009). This should accelerate the arrival of new solutions to the market [1]. In fact, Regulation (EC) No. 1107/2009 guarantees a national provisional authorisation (NPA) from 30 months after the submission of the active substance dossier to the rapporteur member state (RMS), as long as an appropriate MRL has been set, in the event that evaluation has not been completed within these months (for more details, see Article 30 of Regulation (EC) No. 1107/2009) [1]. However, it should be noted that the time line for this NPA is longer than under the previous Council Directive 91/414/EEC, where NPA was already granted from 12 months of the active substance dossier submission to the RMS [18]. In short, today, Europe is evaluated by modern standards through the Regulation (EC) No. 1107/2009. This regulation encourages the development of less harmful substances, being its main challenges the application of cutoff criteria without losing tools for the future agriculture (especially for the minor uses) and the implementation of zonal evaluation and mutual recognition on deadlines. In order to achieve this, it is important that any national specific data requirements and national risk assessments are minimized. Directive 2009/128/EC: IPM Practices and Current Barriers for Biopesticide Registration The registration of low-risk substances for pest control has been enacted by the European Union through regulations that provide incentives for their use [1] and the required implementation of IPM practices by 2014 [31]. In October 2009, the Directive 2009/128/EC on the sustainable use of pesticides [31] was approved to cover a gap in the EU legislative outline related to pesticide management, providing clear references on how to manage pesticide applications in the field to reduce the use of plant protection product and prevent the potential environmental contamination. This directive, together with the Regulation (EC) No. 1107/2009, is an essential element of the actual EU Thematic Strategy on the sustainable use of pesticides [32–34]. It covers the 446 Studies in Natural Products Chemistry use of pesticides in the EU and will come into force in stages from 2011 to 2020 [35,36]. The general objectives of the Thematic Strategy and of Directive 2009/128/EC include the following: (a) the promotion of National Action Plans, aimed at diminishing and optimizing the pesticides’ usage (e.g., adoption of IPM plans), in order to reduce the impacts of pesticide use on human health and the environment; (b) the improvement of awareness and training of end users (professional users and general public), with the purpose of avoiding the inappropriate use of these compounds; (c) the improvement of the spraying equipments, together with the prohibition of aerial spraying (except in special cases), to limit the contamination risks for the operators and environment and maximize the efficacy of the treatment; (d) the measuring progress in risk reduction through appropriate indicators; and (e) the establishment of a system of information exchange at the community level [31–34,37]. Furthermore, Article 11 of the Directive 2009/128/EC requires member states to ensure that appropriate measures are adopted in order to protect the aquatic environment and drinking water supplies. This article links into the Water Framework Directive 2000/60/EC [38] and it is likely that measures taken could define areas of strongly reduced or zero pesticide use. Eight general principles of IPM are described in Annex III of the Directive 2009/128/EC (Table 2), their aim being to satisfy the full meaning of IPM defined in Article 3 of this directive as “. . .careful consideration of all available plant protection methods and subsequent integration of appropriate measures that discourage the development of populations of harmful organisms and keep the use of plant protection products and other forms of intervention to levels that are economically and ecologically justified and reduce or minimise risks to human health and the environment. IPM emphasises the growth of a healthy crop with the least possible disruption to agro-ecosystems and encourages natural pest control mechanisms.” Against this background, it seems that biopesticides may play an important role in the implementation of the IPM, replacing traditional plant protection products, serving as a base for the synthesis of new ones, or integrating the use of biopesticides with traditional plant protection products. Although the European pesticide regulation encourages the development of less harmful substances than conventional chemicals for pest control, together with alternative management techniques, the way for these biopesticides will not be easy. The reason is that natural products and biologically derived pest management agents differ notably from conventional plant protection products in their mode of action, chemistry, and complexity. In fact, such differences are difficult to categorize in terms of relative risk that generates regulatory barriers to biopesticides’ commercialization. Additionally, new legislative requirements for low-risk substances that are still to be elaborated (e.g., half-life in the soil should be less than 60 days [7]) may cause problems for some agents (e.g., living organisms). Chapter 15 Biopesticides, EU Regulation and Residue Analysis 447 TABLE 2 Integrated Pest Management (IPM) Principles Proposed for Being Implemented in the Thematic Strategy [31] Principles Methods/Tools 1 Harmful organisms must be prevented and/or suppressed Crop rotation, adequate cultivation techniques, resistant/tolerant cultivars and standard/certified seed and planting material, balanced fertilization, liming and irrigation/drainage practices, hygiene measures, protection and enhancement of important beneficial organisms 2 Harmful organisms must be monitored Observations in the field; scientifically sound warning, forecasting, and early diagnosis systems; use of advice from professionally qualified advisors 3 The professional user has to decide whether and when to apply plant protection measures based on the results of the monitoring Robust and scientifically sound threshold values. For harmful organisms, threshold levels defined for the region and specific areas, crops, and particular climatic conditions must be taken into account 4 Nonchemical methods are preferred if they provide satisfactory pest control Sustainable biological, physical, and other nonchemical methods 5 The pesticides applied shall be specific for the target and shall have the least side effects – 6 The professional user should keep the use of pesticides and other forms of intervention to levels that are necessary Reduced doses, reduced application frequency, or partial applications 7 Antiresistance strategies should be applied to maintain the effectiveness of the products Use of multiple pesticides with different modes of action 8 The professional user should check the success of the applied plant protection measures Records on the use of pesticides and on the monitoring of harmful organisms However, the new EU legislation gives a specific status to nonconventional pesticides, so that those substances that demonstrate their low risk are granted initial approval for 15 years rather than the standard 10 years and a reduced dossier for their registration, including a demonstration of sufficient efficacy, could be submitted [1,7]. Nowadays, the European Commission and member states are working in a list of compounds for possible categorization as basic substances. Among 448 Studies in Natural Products Chemistry them are extracts, substances destined for feeding, organic acids, organic salts, inorganic materials, and some ferric citrates. ADVANTAGES AND DISADVANTAGES OF “BIOPESTICIDES” AND THE CONTROVERSIAL USE OF THIS TERM Bio means life or related with life. In this sense, biopesticide means pesticide that is respectful with the environment, human, animal, and nontarget plant health. As all EU market active substances, they are evaluated according to the uniform principles guaranteeing a high degree of protection for both human and animal health and the environment. Many biopesticides have gained favor in recent years, due in part to the perception that, because they originate in nature, they are safer than the synthetic ones, being used for growing crops organically, according to guidelines set forth by certification programs. However, it is important to be aware that they are pesticides and fall under the same state and regulations as synthetic pesticides. Although actual EU regulation encourages the development of pesticides more respectful with the environment and less dangerous for human and animal health, they have to be used only as indicated on the label, which provides critical information about how to safely handle and use plant protection products. In fact, a critical aspect for registering a pesticide product is the approval of the product label, so only following its instructions strictly, the use of the biopesticide is safe. Maybe the main advantage of biopesticides is found in the vast biological activity spectrum of these compounds, exceptionally variable, with potential functional properties against pests via new mechanisms of action [4–6] that make them good to combat the evolution of resistance to the compounds currently available as was mentioned earlier in the text. The rest of their properties are more particular characteristics than advantages regarding conventional chemical pesticides. Between their characteristics, it could be highlighted that excellent substances are to be used within an IPM program due to their multimode of action; the majority of natural plant protection products have low restricted entry intervals (around 0–4 h) with no preharvest interval and their cost of registration is usually lower than for a chemical pesticide. Therefore, biopesticides (e.g., neem pesticides [39]) could be toxic for human [40,41] or for nontarget organisms [39,42], and both events are the main base for most of the legislation that aims to ban or restrict the use of certain products. In fact, the first conclusions of the European Food Safety Authority following the peer review of the initial risk assessments about the fourth stage substances, which adjust to the meaning of biopesticide defined at the beginning of this chapter and approved within the EC pesticide review programme, usually are very cautious indicating that the risk for human, animals, and nontarget organisms is assessed as low. However, in several cases, the operator and consumer risk assessment could not be performed Chapter 15 Biopesticides, EU Regulation and Residue Analysis 449 due to different causes such as lack of toxicity information of metabolites (e.g., fatty acids C7–C18, citronella oil, and pyrethrins). As usually happens with conventional pesticides, undesirable side effects of using natural plant protection products include secondary infestation, development of resistance, damage to nontarget species, pollution of surface and groundwater, and human health risks. Consequently, the use of biopesticides causes a residue in the environment, being necessary to monitor and evaluate its risk. The benefits of using plant protection products (both conventional and natural) are clear and their use in many cases is necessary and justifiable. However, its use has a cost that is necessary to assess and evaluate. In fact, in recent decades, the cost of the indiscriminate and massive use of plant protection products has been seen in terms of impact on the environment, human health and animal wildlife, ecosystems, and food security. The society of developed countries increasingly demands less use of pesticides, to achieve the reduction in the level of residues in food and the environment, but really what should be used and done is a rational use of plant protection products integrated with other practices and control measures, what is called IPM. REQUIREMENTS IN ANALYTIC METHODS TO FULFILL THE ACTUAL EUROPEAN LEGISLATIVE FRAMEWORK Biopesticide residues (not included biological agents such as fungi, bacteria, or virus) shall be analyzed by analytic methods that use instrumentation regarded as commonly available and that have sufficient sensitivity and specificity to assess the magnitude and nature of all significant residues remaining in plants, plant products, foodstuff (of plant and animal origin), feedingstuff (of plant origin), soil, water, air, and body fluids and tissues. In fact, each applicant who wants to introduce a new active substance in the market has to provide adequate analytic methods for three purposes: first, to identify and quantify all active substances present in the technical material and formulated product [43]; second, to generate residue data on which consumer dietary exposure assessments are based and to support studies on the fate and behavior of the active substance in foodstuff, the environment, ecotoxicology, and toxicology (preregistration) [44]; and third, to be used in postregistration monitoring residues and control by the competent authorities in member states [45]. According to the guidance document 7109/VI/94 rev. 6 [46] (which provides detailed guidance for the applicability of the principles of good laboratory practice (GLP) to data requirements according to individual studies of Annexes II and III (part A) of the Council Directive 91/414/EEC [18]), the development and validation of an analytic method for monitoring purposes and postregistration control are not subject to GLP. However, when the method is used to generate data for registration purposes, for example, residue data, these studies must be conducted to GLP. 450 Studies in Natural Products Chemistry To satisfy the actual EU legislative framework, each analytic method shall be provided in sufficient detail, in a stepwise fashion, so a competent analyst can apply it, even though he or she may be unfamiliar with the procedure. The submitted studies during the evaluation process of the plant protection product or active substance must include full description of the analytic method, itemization of the fortified compounds and the analytes that are quantified, and validation data. Multiresidue methods cover large numbers of analytes and are preferred than single-residue methods. In fact, these last methods should only be provided if data show that multiresidue methods involving GC and HPLC techniques cannot be used. In these cases, the method should be appropriate for the determination of all compounds included in the residue definition. Common moiety methods may also be used in agreement with the residue definition, but shall be avoided whenever possible. Likewise, where a derivatization method is used, this must be properly justified. The efficiency and precision of the derivatization step and storage stability of the derivative should be demonstrated. Where the derivative formed is a common derivative of several active substances or their metabolites or is classed as another active substance, the method should be considered nonspecific and may be deemed unacceptable. Selective methods for isomers would only be required if a single isomer is included in the residue definition. In the case of technical material and preparations where is known the presence of more than one isomer, the method should distinguish individual isomers where they are not equally active. Furthermore, impurities above 0.1%, impurities of toxicological/ ecotoxicological or environmental significance, and relevant impurities must also be determined by appropriate analytic methods. In a general way, validation is the documentary evidence process that an analytic method will lead, with a high degree of security, to obtain precise and accurate results, within specifications and quality attributes previously established. Validation data must be submitted for all analytes included in the residue definition for all representative sample matrices to be analyzed at adequate concentration levels. The five fundamental parameters required for the European Commission for analytic validations of methods used with pesticides are as follows: linearity and range, accuracy, precision, selectivity, and LOD and LOQ. These are collected in the SANCO guidelines together with their well-detailed requirements [43–45]. Confirmatory methods are also required to demonstrate the selectivity of the method. This is favoring the use of MS techniques in combination with the equipments of chromatography (GC and LC), because they allow the confirmation simultaneous to primary detection (by monitoring several selected reaction monitoring transitions) without the need to use an independent analytic technique, saving time and effort. Methods validated according to all these requirements must be submitted for representative matrices of the next four matrix groups: dry matrices Chapter 15 Biopesticides, EU Regulation and Residue Analysis 451 (e.g., wheat, barley, and rice), matrices with high water content (e.g., lettuce, apples, and tomatoes), matrices with high oil content (olives, nuts, and rapeseed), and matrices with high acid content (e.g., lemons, oranges, and grapes). For matrices that are difficult to analyze (e.g., coffee beans, cocoa beans, herbal infusions, hops, spices, tea, and tobacco), full validation data for each specific matrix shall be presented to prove the suitability of the method. The sample preparation and the analytic method selected for analysis could also determine whether a matrix is considered difficult to analyze. An independent laboratory validation (ILV) must be submitted for a primary method with monitoring purposes and used to determine residues in plants, plant products, foodstuff, and feedingstuff. The LOQ of the primary method shall be confirmed by the ILV, but at least the MRL. If validation data for the residue analytic method of an analyte in at least one of the commodities of the respective matrix group have been provided by an international official standardization body and if these data have been generated in more than one laboratory with the required LOQ, acceptable recovery, and acceptable relative standard deviation (RSD) data, no additional ILV is required [45]. In summary, the purpose of any validated analytic method is to obtain consistent, reliable, and accurate measurements. The results from method validation can be used to judge the quality, reliability, and consistency of analytic results, which is an integral part of any good analytic practice and the base of which is supported by the identification and quantification of active substances, studies of their fate and behavior, and studies of their residue. Validation of analytic methods is required by actual EU legislative framework and has received considerable attention in the literature from industrial committees and regulatory agencies. ANALYTIC TECHNIQUES APPLIED TO THE ANALYSIS OF BIOPESTICIDE RESIDUES Pesticides and their transformation products are usually present at parts per million or even parts per billion (ppb) levels in several matrices. This implies that the sample analysis is not a simple task, involving two main steps: a previous sample preparation (which includes in general sampling, extraction, concentration, and isolation of analytes) and a later analysis by the adequate analytic techniques. This section refers to both steps, giving an exhaustive summary of the trend related to the development of biopesticide residue analysis. One example that reflects perfectly the need to improve and develop both steps is the growing evidence for the declines in bee populations, where neonicotinoid residues are presented at a very low trace level [47]. In fact, several studies seem to indicate that, in many cases, there are negative impacts on bee populations at levels even below the LOQs achieved currently [47–49]. In this sense, the trends should also be to select natural substances for treating crops and beehives that do not carry high risk for the consumer 452 Studies in Natural Products Chemistry and bees. Notwithstanding, it is also necessary to dispose of simple and sensitive methods. These methods should allow the determination of residues from these natural substances, to which bees are exposed, in apiary products [50–53]. Overall, validation data for biopesticide products are still relatively scarce. Table 3 summarizes a validation data review for the main natural active substances used as biopesticides, where different analytic methods were used in numerous matrices. Figure 2 shows the structures of the main active substances. Almost all analytic methods shown in Table 3 reached correlation coefficients (r) 0.995, within their respective linearity studies and proper linearity range. In most cases, the whole analytic method was validated following a similar methodology to that mentioned earlier in the text. The objective of these research works is not always to satisfy the actual EU legislative framework. In fact, their aims are usually focused in developing analytic methodologies to detect and quantify residues from these natural plant protection products at trace levels. However, applicants take advantage of these new research developments, so this revision is an excellent way to see the trends of future legal requirements. Sample Preparation Techniques Today, sample preparation is maybe the step that most influences the accuracy of the whole analytic method, with the extraction of pesticide residues from environmental matrices the key factor for achieving it. There is no question that in the first decade of the century, SPE technique [72] is the most employed alternative to the classical solid–liquid [13] and liquid–liquid [14] extractions. These classical techniques present multiple disadvantages such as the low recovery of polar pesticides and transformation products (in the case of liquid–liquid extractions) and use of large volumes of solvents. Furthermore, several variants emerged based on the SPE technique: solid-phase microextraction (SPME) [72–74], in-tube solid-phase microextraction [72,75,76], matrix solid-phase dispersion [72,77,78], and stir-bar sorptive extraction [72,79]. In the last years, due to the new legislative requirements and the concern about the harmful effects that pesticide residues may have, even at trace levels, there is a growing interest in the development of methods to determine pesticide residues from environmental matrices. The aim is to extract the pesticide residues with a higher and predetermined selectivity, removing matrix interferences to result both in an excellent chromatographic resolution and in an increase in sensitivity of the analysis. Selective tailor-made sorbent materials have been developed, being the most interesting those based on immobilized receptors or antibodies (immunosorbents) [80–82], restricted access materials (RAMs) [80,83], and molecularly imprinted polymers (MIPs) [80,84], all with specific recognition for analytes of interest. In the TABLE 3 Data Review for the Main Natural Active Substances Used as Biopesticides for Which Developed Analytic Methods were Properly Validated Matrix Sample Preparationa Analysis Methodb Fortification Level (mg kg 1) Recovery % (RSD) LOQ (mg kg 1)c Ref. 9,21Dehydroryanodine Tomato LSE HPLC–DAD-MS 10 89 (2) 10 [54] Cevadine Wheat QuEChERS UPLC–MS/MS 10 109 (14) 4.2 [55] Cucumber 76 (4) 5.7 Wine 104 (18) 3.9 Soil 59 (16) 4.5 [56] 79 (12) 5.1 [55] Cucumber 72 (18) 3.3 Wine 71 (25) 10.0 Soil 30 (20) 6.0 [56] 70.2 (3.5) 10 [57] Chemical Class Compound Alkaloids Nicotine Wheat Apricot QuEChERS QuEChERS (PSA) UPLC–MS/MS HPLC–MS 10 10 Lemon 70.2 (6.6) Grape 71.6 (5.6) Ryanodine Tomato LSE HPLC–DAD-MS 10 87 (3) 10 [54] Sabadine Soil QuEChERS UPLC–MS/MS 10 31 (18) 7.0 [56] Continued TABLE 3 Data Review for the Main Natural Active Substances Used as Biopesticides for Which Developed Analytic Methods were Properly Validated—Cont’d Chemical Class Compound Matrix Sample Preparation Analysis Method Fortification Level (mg kg 1) Recovery % (RSD) LOQ (mg kg 1) Ref. Veratridine Soil QuEChERS UPLC–MS/MS 10 77 (9) 4.0 [56] Wheat 112 (7) 4.8 [55] Cucumber 97 (12) 5.1 Wine 78 (14) 4.5 Aminoglycosides Streptomycin Honey SPE (SCX and C18) HPLC–FL 38 91 (3.6) 10 [50] Maize LSE–LSE (silica) GC-ECD 50–500 94–106 (–) 50 [58] LSE–SPE (Florisil®) HPLC–MS/MS 10 96 (7) 2.0 [59] 5 87 (10) 10 [57] Anthraquinones 9,10Anthraquinone Peas Soil Avermectins Abamectin B1a Lemon Apple Peas 85 (15) Tomato 90 (7) Apricot QuEChERS (PSA) HPLC–MS 10 107 (7.1) Lemon 79.7 (8) Grape 96.9 (4.4) Abamectin B1b Tomato LSE–SPE (Florisil®) HPLC–MS/MS 5 90 (11) 5.0 [59] Abamectin (B1a + B1b) Orange LSE HPLC–MS/MS 10 96 (10) 10 [60] Soil QuEChERS UPLC–MS/MS 10 115 (19) 8.0 [56] Tomato LSE HPLC–DAD-MS 100 101 (5) 100 [54] d 50 [61] Pyrethrins Cinerin I Apricot QuEChERS (PSA) HPLC–MS d 50 106.7 (9.5) Lemon Cinerin II Jasmolin I Jasmolin II 95.2 (8.3) Soil QuEChERS UPLC–MS/MS 10 93 (18) 6.0 [56] Tomato LSE HPLC–DAD-MS 100 104 (8) 100 [54] Soil QuEChERS UPLC–MS/MS 10 85 (22) 10.0 [56] Tomato LSE HPLC–DAD-MS 100 89 (5) 100 [54] Soil QuEChERS UPLC–MS/MS 10 82 (14) 10.0 [56] Tomato LSE HPLC–DAD-MS 100 84 (6) 100 [54] d 10 [61] 116 (10) 6.0 [56] 99 (3) 50 Apricot QuEChERS (PSA) HPLC–MS d 10 99.2 (4.6) Lemon Pyrethrin I 106.2 (9.8) Soil QuEChERS UPLC–MS/MS 10 Tomato LSE HPLC–DAD-MS 50 Apricot Lemon QuEChERS (PSA) HPLC–MS d 200 98.6 (4.3) [54] d 200 [61] 101.2 (5.2) Continued TABLE 3 Data Review for the Main Natural Active Substances Used as Biopesticides for Which Developed Analytic Methods were Properly Validated—Cont’d Chemical Class Compound Matrix Sample Preparation Analysis Method Fortification Level (mg kg 1) Recovery % (RSD) LOQ (mg kg 1) Ref. Pyrethrin II Soil QuEChERS UPLC–MS/MS 10 86 (15) 5.5 [56] Tomato LSE HPLC–DAD-MS 5 97 (2) 50 [54] Apricot QuEChERS (PSA) HPLC–MS 140d 101.2 (6.5) 140d [61] Lemon S Pyrethrinse Water 108.2 (5.1) Soxhlet (XAD2)–SPE (Florisil® + NH2) GC–MS 6.3 10 Soxhlet (filter)–SPE (Florisil® + NH2) S Pyrethrinsf 4 137 (22) 6.3 10 4 [62] 126 (6.1) Sediment Soxhlet–SPE (Florisil® + NH2) GC–MS 10 154 (29) 10 [63] Wheat QuEChERS UPLC–MS/MS 10 95 (20) 7.0 [55] Cucumber 97 (6) 9.8 Wine 76 (15) 7.5 Rotenones Deguelin Tomato LSE HPLC–DAD-MS 10 109 (4) 10 [54] Wheat QuEChERS UPLC–MS/MS 10 90 (16) 3.0 [55] Cucumber 104 (9) 5.1 Wine 79 (16) 3.0 Soil 95 (3) 5.5 [56] Rotenolone Rotenone Tomato HPLC–DAD-MS 10 106 (9) 10 [54] HPLC–MS/MS 1 89.3 (9.8) 1 [64] 2 [64] 5 [64] 104 (15) 3.3 [55] Cucumber 93 (9) 5.4 Wine 70 (14) 4.8 Soil 90 (8) 4.0 [56] 106 (8) 8 [65] Cabbage LSE ® LSE–SPE (Florisil ) Potato 99.6 (11.2) Banana 79.3 (11.6) Carrot 88.2 (10.3) Apple 112.3 (11.6) Orange Onion 80.2 (12.6) ® LSE–SPE (Florisil ) HPLC–MS/MS 2 Lychee Tea 109.6 (10.3) ® LSE–SPE (Florisil ) HPLC–MS/MS 5 Mushroom Wheat 118.3 (14.8) 89.3 (9.8) 112.6 (10.3) QuEChERS UPLC–MS/MS UPLC–MS/MS 10 Tomato QuEChERS (PSA) 10 Cabbage QuEChERS (PSA + GCB) 71 (3) 3 Soil QuEChERS (PSA + C18) 104 (7) 7 Continued TABLE 3 Data Review for the Main Natural Active Substances Used as Biopesticides for Which Developed Analytic Methods were Properly Validated—Cont’d Chemical Class Compound Matrix Sample Preparation Analysis Method Fortification Level (mg kg 1) Recovery % (RSD) LOQ (mg kg 1) Ref. Apricot QuEChERS (PSA) HPLC–MS 10 78.6 (10.1) 10 [57] Lemon 78 (7.9) Grape 93.6 (6.8) Tomato LSE HPLC–DAD-MS 10 88 (11) 10 [54] Honey Paper filter HPLC-DAD 100 109.3 (4.1) 50 [52] Raw honey LLE HPLC-UV 100 61 (6.3) 100 [66] SPE (ODS) HPLC-UV SPE (C18)–acetonitrile elution HPLC–MS 4 [67] 106 (14) 4.5 [55] Cucumber 80 (4) 5.1 Wine 83 (7) 3.0 Water 80 (6.9) 20 SPE (C18)–SFE elution 97.8 (7.5) 95.8 (4.1) Spinosyns Spinosad (spinosyn A + D) Wheat QuEChERS UPLC–MS/MS 10 Spinosyn A Soil UPLC–MS/MS 10 68 (9) 5.0 [56] HPLC–MS/MS 2 84 (15) 1.0 [59] Lemon 4 79 (9) Peas 2 84 (11) Tomato 2 94 (7) 10 101 (6) 6 [65] Apple Spinosyn D QuEChERS ® LSE–SPE (Florisil ) Tomato QuEChERS (PSA) UPLC–MS/MS Cabbage QuEChERS (PSA + GCB) 83 (4) 4 Soil QuEChERS (PSA + C18) 92 (8) 8 Apricot QuEChERS (PSA) 91.5 (11.2) 10 [57] 1.0 [59] [65] HPLC–MS 10 Lemon 88.2 (8.5) Grape 98.7 (5.6) Apple LSE–SPE (Florisil®) HPLC–MS/MS 2 90 (14) Tomato 94 (11) Peas 99 (7) Lemon UPLC–MS/MS 4 88 (14) 10 101 (6) 6 Tomato QuEChERS (PSA) Cabbage QuEChERS (PSA + GCB) 87 (7) 7 Soil QuEChERS (PSA + C18) 96 (6) 6 Apricot QuEChERS (PSA) 92.2 (13.0) 10 HPLC–MS 10 Lemon 90.3 (7.8) Grape 96.8 (6.8) [57] Continued TABLE 3 Data Review for the Main Natural Active Substances Used as Biopesticides for Which Developed Analytic Methods were Properly Validated—Cont’d Chemical Class Compound Spinosyn J Matrix Vegetables Sample Preparation SPE (NH2) Analysis Method HPLC–UV–MS/MS Recovery % (RSD) LOQ (mg kg 1) Ref. 3 98.87 (4.13) 30 [68] 3 Fortification Level (mg kg 1) 1 10 Spinosyn L Vegetables SPE (NH2) HPLC–UV–MS/MS 1 10 89.09 (4.14) 30 [68] Spinosyn (J + L) Tomato QuEChERS (PSA) HPLC-DAD 40 88.5 (10) 40 [69] Honey SPE (C18) GC-FID 500 89.3 (14.8) 200 Terpenoids Camphor Honey Eucalyptol Honey Honey Menthol Honey Honey Solvent extraction GC-FID d 200 106.7 (3.5) Distillation–SPE (LiChrolut® RP-18) 107.8 (1.6) SPE (LiChrolut® RP-18) 106.3 (3.8) SPE (C18) Solvent extraction GC-FID GC-FID 500 d 200 89.6 (17.0) 120.5 (7.5) Distillation–SPE (LiChrolut® RP-18) 108.3 (1.8) SPE (LiChrolut® RP-18) 108.4 (2.8) SPE (C18) Solvent extraction GC-FID GC-FID 500 d 200 83.9 (10.9) 115.8 (4.3) Distillation–SPE (LiChrolut® RP-18) 108.4 (2.8) SPE (LiChrolut® RP-18) 107.4(1.6) d [51] 150 [53] 500 [51] d 100 [53] 300 [51] d 100 [53] Thymol Honey Honey SPE (C18) GC-FID Solvent extraction GC-FID 500 d 200 78.1 (9.4) 117.8 (5.5) Distillation–SPE (LiChrolut® RP-18) 109.1 (2.3) SPE (LiChrolut® RP-18) 105.1 (3.3) Honey SPE (C18)-NF Soil QuEChERS-NF 200 d [51] 100 [53] HPLC-DAD 200 101.0 (8.8) 200 [52] UPLC–MS/MS 10 99 (10) 5.0 [56] HPLC–MS/MS 10 88 (20) 5.0 [59] Lemon 20 96 (16) Peas 10 100 (13) Tomato 10 91 (10) 10 83 (10) 10 [65] Tetranortriterpenoids Azadirachtin A Apple ® LSE-SPE (Florisil ) Tomato QuEChERS (PSA) UPLC–MS/MS Cabbage QuEChERS (PSA + GCB) 67 (12) 12 Soil QuEChERS (PSA + C18) 90 (9) 9 Tomato LSE HPLC–DAD-MS 50 80 (1) 20 [54] Wheat QuEChERS UPLC–MS/MS 10 109 (12) 4.8 [55] Cucumber 98 (6) 3.5 Wine 93 (6) 5.1 Continued TABLE 3 Data Review for the Main Natural Active Substances Used as Biopesticides for Which Developed Analytic Methods were Properly Validated—Cont’d Chemical Class Compound Matrix Brinjal Sample Preparation LSE–LLE Analysis Method TLC-DIAS Fortification Level (mg kg 1) 3 1 10 92 (2.7) LOQ (mg kg 1) 3 Ref. 1 10 [70] 82 (14.6) 400 [71] Oak foliage 78 (12.8) 400 Soil (sandy) 74 (13.5) 400 Soil (clay) 82 (14.6) 400 Litter 66 (18.2) 500 Tomato 87 (1.2) Coffee 77 (3.6) Cotton Fir needles a Recovery % (RSD) 67 (3.1) ® LSE–LLE–SPE (Florisil ) HPLC–UV 500 Water LLE HPLC–UV 10 94 (8.5) 5 [71] Orange LSE HPLC–MS/MS 10 103 (10) 10 [60] GCB, graphitized carbon black; LLE, liquid–liquid extraction; LSE, liquid–solid extraction; PSA, primary secondary amine; QuEChERS, quick, easy, cheap, effective, rugged, and safe; SFE, supercritical fluid elution. DAD, diode array detector; DIAS, digital image analysis system; ECD, electron capture detector; FID, flame ionization detector; FL, fluorescence detector; GC, gas chromatography; HPLC, high-performance liquid chromatography; MS, mass spectrometry; TLC, thin-layer chromatography; UPLC, ultra-performance liquid chromatography; UV, ultraviolet detector. c LOQ, limit of quantification. d Concentration in mg L 1. e Cinerin I and II, jasmolin I and II. f Pyrethrin I and II, cinerin I and II, jasmolin I and II. b R O HO O O O O O O H O O OH H O O O H R⬙ OR O N OH O H O H OH O OH OH R⬘O O O RO HO O H OH Azadirachtin A (R= tigloyl, R⬘=acetyl , R⬙=OH) Azadirachtin B (R=H, R⬘= tiglaldehyde, R⬙=H) H Abamectin B1a (R=CH2CH3) Abamectin B1b (R=CH3) OH OH O O Cevadine (R= angelic acid) Veratridine (R= veratric acid) N N O OCH3 N O R O O O O CH3O H O O H Pyrethrin I (R=CH3, R⬘=CHCH2) Pyrethrin II (R=COOCH3, R⬘=CHCH2) Cinerin I (R=CH3, R⬘=CH3) Cinerin II (R=COOCH3, R⬘=CH3) Jasmolin I (R=CH3, R⬘=CH2CH3) Jasmolin II (R=COOCH3, R⬘=CH2CH3) R⬘O O Nicotine R⬘ OH O O O O O H H H H R O H Rotenone O H Spinosyn A (R=H, R⬘=CH3) Spinosyn D (R=CH3, R⬘=CH3) Spinosyn J (R=H, R⬘=H) Spinosyn L (R=CH3, R⬘=H) FIGURE 2 Structures of some of the most common bioactive constituents isolated from natural sources and used in crop protection. 464 Studies in Natural Products Chemistry immunosorbent, the antibody is immobilized onto a silica support or polymeric adsorbents and used as an affinity ligand to extract the target analyte and other compounds with similar structures. Immunosorbent-based procedures involve analytic separation and detection of the individual analytes after a desorption step, accomplishing extraction, trace enrichment, and cleanup. On the other hand, RAMs have a biocompatible surface and a pore size that restrict the accessibility of interaction sites within the pores to small compounds only. Simultaneous to this size-exclusion process, an extraction phase located on the inner pore surface is responsible for the isolation of specific analytes. MIPs are maybe the SPE sorbents that more have increased their popularity in recent years. These materials are obtained by creating three-dimensional polymer networks that have a memory of the shape and functional group positions of the template molecule so that the resulting MIPs can selectively recognize and retain the analyte used in the imprinting process. In recent years also have begun to appear new hybrid materials made of RAMs combined to molecularly imprinted polymers [85,86], representing a very promising polymeric device to work with analytes that are strong and negatively adsorbed to the MIP surfaces [87]. However, the general commercial utilization of MIP-SPE, using a variety of MIP preparation techniques, is pending in the near future, so today, SPE remains the technique most employed with pesticides and their transformation products. In fact, to our knowledge, no studies about biopesticides and their determination by RAMs and MIPs have been published until now in the open literature. However, the potential of these techniques is clear and research works as that of Shi et al. [88], in which was carried out the determination of pyrethroid insecticides (similar to the natural pyrethrins; Fig. 2) via MIPs, opened the path for their future application with biopesticides. Nowadays, a large number of SPE materials are available for the analysis of pesticide residues [80,89,90], covering a wide range of selectivity and, thus, a large variety of applications. The choice of sorbent is the key factor in SPE, because this can control parameters such as selectivity, affinity, and capacity. This choice depends strongly on the analytes and their physically chemical properties, which should define the interactions with the chosen sorbent. However, results also depend on the kind of sample matrix and interactions with both the sorbent and the analyte [91]. Sorbents can be classified generally as silica-based sorbents, oxides of metals, graphitized carbon, or carbon-based and polymer-based sorbents [92]. The most common are the silica-based (especially C18), consisting of siloxane chemically modified with molecules having functional groups suitable for the desired application. However, silica-based sorbents have some limitations such as the instability in a broad pH range, containing silanol groups on their surface that can cause the irreversible binding of some groups of compounds [91], and reversedphase silica must be conditioned with a wetting solvent and remain wetted before sample application [93]. Current technologies have overcome the Chapter 15 Biopesticides, EU Regulation and Residue Analysis 465 presence of residual silanols on the silica surface, and today, there are endcapped packings that exhibit negligible absorption due to free hydroxyls, by attaching a blocking group commonly referred to as an endcap. However, it should be kept in mind that this does not eliminate minor absorptive effects due to the oxygen in the siloxane structure (SidOdSi) [94]. Polymer-based sorbents offer solutions to those drawbacks [95]: they can be used over the whole pH range, do not have high active sites, and are less sensitive to drying out after conditioning. In Table 3 are schematized several sample preparation procedures by SPE technology with biopesticides. Most of them use a simple step to carry out the extract cleanup and/or the trace enrichment. However, Edder et al. [50] and Woudneh and Oros [62,63] had developed respective procedures for streptomycin and pyrethrin (Fig. 2) residues, respectively, where two steps are involved. Thanks to these SPE steps, each one with different affinities for analytes/impurities, were allowed to reach LOQs until 10 mg kg 1 [50,63], in difficult matrices such as honey and sediments, and even 6.3 10 4 mg kg 1 in water [62]. The most common SPE sorbents reported in the recent literature to develop analytic methodologies for the analysis of biopesticides continue being both hydrophilic adsorbents, such as normal-phase-activated magnesium silicate (Florisil®) cartridges [59,62–64], and hydrophobic stationary phases, such as LiChrolut®, C18, or ODS cartridges [50–52,66,67]. The former are used to extract hydrophilic analytes from nonpolar matrices (e.g., organic solvents), while the latter are used to extract hydrophobic analytes from hydrophilic mobile phases (e.g., methanol/water and acetonitrile/water). Other sorbents are also applied with other purposes, such as SCX cartridges, which allow cation-exchange extractions [50], and SidNH2 cartridges [62,63,68], which can act as either a polar phase (interacting with any molecule containing dOH, dNH, or dSH functional groups) or weak anion exchanger in an aqueous environment. Jimenez et al. [66] reported that an inadequate choice of the SPE sorbent leads to unsatisfactory recoveries for reasons such as the strong interaction between the active substance and the sorbent (e.g., rotenone (Fig. 2) and Florisil®). Other efforts are aimed to nearly completely eliminate the use of organic solvents to elute biopesticides from the SPE sorbents by supercritical fluid elution (SFE) [67], avoiding concentration of the organic solvent extract and achieving their analysis in a minor time frame. Several authors have carried out the validation of analytic methods for the analysis of terpenoids and aminoglycosides used in beekeeping, with honey as a matrix and different ways of sample preparation [50–53]. Studies of Nozal et al. [53] showed that a simple trapping on ODS cartridges allows one to obtain excellent, reliable, and precise determinations of traces of thymol, menthol, eucalyptol, and camphor in honey, using low amount of sample and elution solvent. Despite these good results, trace levels of any pesticide can have strong negative consequences in population level of bees, so the LOQ with this matrix must be improved until levels are achieved with other 466 Studies in Natural Products Chemistry matrices (1–10 ppb) (Table 3). Meanwhile, streptomycin is an aminoglycoside antibiotic, also used in beekeeping, for which Edder et al. [50] reached an LOQ of 10 ppb in honey using SPE to trace enrichment and cleanup: first with a cation-exchange cartridge (Si-SCX) and second with an octadecyl cartridge (C18). Anastassiades et al. [96] had developed the dispersive solid-phase extraction (dSPE) based on the SPE technique. dSPE is a novel cleanup technique that was included within the quick, easy, cheap, effective, rugged, and safe (QuEChERS) method, also introduced by these authors [96]. QuEChERS was successfully applied to determine several biopesticides such as alkaloids [55–57], avermectins [57], pyrethrins [55,56,61], rotenones [55–57,65], spinosyns [55–57,65,69], and tetranortriterpenoids [55,65] (Table 3, Fig. 2). In dSPE, the crude extract is cleaned up by the addition of a small amount of SPE sorbent material (primary secondary amine (PSA) is the most frequently used sorbent in the study of biopesticides; Table 3) to an aliquot of the extract to remove the matrix coextractives (interferences), which is then separated from the extract bulk by centrifugation avoiding passing the extract through an SPE column. The main advantages of the method include high recoveries from a wide range of polarities of biopesticides and other types of samples and analytes, high sample throughput, and inexpensive procedures that require less labor, time, and organic solvents and their simplicity, which minimize the systematic and random errors [96]. For example, Prestes et al. [56] carried out a comparison of several extraction procedures for the determination of biopesticides in soil samples, concluding that QuEChERS (with modified extraction and combined with fast separation step (<9 min)) provided better results than other extraction procedures evaluated, increasing significantly sample throughput. However, the QuEChERS method cannot be used for concentrating the target analytes. Therefore, to achieve sufficient sensitivities for the analysis of trace levels, additional concentration procedures and strategies are required (e.g., large-volume injection [97] and solvent evaporation [98][99]). It is also important to highlight the use of liquid membrane-based extractions as an alternative to conventional sample preparation procedures [100]. These techniques are in principle variants of liquid–liquid extraction (LLE) but various shortcomings of LLE are overcome as membrane extraction techniques use none or very little organic solvents, high enrichment factors can be obtained, and there are no problems with emulsions. They can also provide high cleanup efficiency and extraction selectivity [100]. There are two main variants of these techniques: three(aq/org/aq)- and two(aq/org)-phase systems. The former of these systems is called supported liquid membrane (SLM) extraction, while the latter is called microporous membrane liquid–liquid extraction (MMLLE). In the SLM extraction, analytes are extracted from the aqueous sample into an organic liquid, immobilized in a porous hydrophobic membrane support and further to a second aqueous phase. This technique is suitable for the extraction of polar compounds and it is compatible with Chapter 15 Biopesticides, EU Regulation and Residue Analysis 467 reversed-phase HPLC. In the MMLLE, analytes are extracted from the aqueous sample into an organic solvent by a hydrophobic porous membrane. This technique is suitable for more hydrophobic analytes and is compatible with GC. The two main formats are based on either flat membranes or hollow fiber membranes in various ways. Traditional pesticides were already extracted and analyzed using MMLLE [101] and SLM extraction [102], predicting a promising future in the analysis of biopesticides. Finally, sometimes, biopesticides cannot be analyzed without a previous derivatization that implies a modification of their chemical structure [103–106]. This is because they are not in a suitable form for the analytic technique (e.g., nonvolatile compounds and insoluble compounds for GC and HPLC analysis, respectively, or instable materials under the conditions of the technique). Most of the analytic derivatization reactions used for GC and HPLC fall into three general reaction types [103]: alkylation, acylation, and silylation. In GC, compounds containing functional groups with active hydrogens (e.g., dCOOH, dOH, dNH, and dSH) tend to form intermolecular hydrogen bonds with column packing materials, which affect compounds’ volatility. Furthermore, thermal stability of compounds with groups of this kind is lower regarding other derivatives such as TMS. Therefore, TMS derivatives could be analyzed at temperatures above 300 C. Detection enhancement is by far the most common use of derivatization in HPLC, but derivatives may also be used to alter separation properties. However, the derivatization is rather time-consuming and potentially introduces additional experimental error. As a consequence, only few works with biopesticides are found in the literature with a derivatization step [104–106] and analytic methods without this step are preferred. Chemical Analysis: Instrumental Techniques and Future Trends Chromatographic methods are the most commonly chosen for the analysis of pesticide residues. As we know today, chromatography consists of a mobile phase that carries the sample through a stationary phase getting separation of complex mixtures due to the different affinities of the sample molecules for the stationary media. As a result, the time for a particular molecule to travel through the chromatographic medium will depend on its physicochemical properties. A convenient classification of chromatographic techniques can be made in terms of the physical state of the phases employed in the separation process (Fig. 3): If the mobile phase is a gas, the separation techniques are known as gas–liquid chromatography (GLC) or gas–solid chromatography (GSC) when the stationary phase is a liquid or solid, respectively. GLC is the more popular separation mode and is often simply referred to as GC. If the mobile phase is a supercritical fluid, the separation technique is known as supercritical fluid chromatography (SFC) whether the stationary phase is an 468 Studies in Natural Products Chemistry FIGURE 3 Family of column chromatographic methods. GLC, gas–liquid chromatography; GSC, gas–solid chromatography; SFC, supercritical fluid chromatography; LLC, liquid–liquid chromatography; MEKC, micellar electrokinetic chromatography; LSC, liquid–solid chromatography; AC, affinity chromatography; IEC, ion-exchange chromatography; SEC, size-exclusion chromatography; RPC, reversed-phase chromatography; CEC, capillary electrochromatography. immobilized liquid or a solid. If the mobile phase is a liquid and (a) the stationary phase is a liquid, the separation technique is known as liquid–liquid chromatography (LLC); (b) if it is a solid, the chromatographic techniques are subdivided into six types according with the dominant separation mechanism (liquid–solid chromatography (LSC), affinity chromatography (AC), ion-exchange chromatography (IEC), size-exclusion chromatography (SEC), reversed-phase chromatography (RPC), and capillary electrochromatography (CEC)); and (c) if it is with ionic surfactants that can form micelles as a continuous phase dispersed throughout a buffer, the separation technique is known as micellar electrokinetic chromatography (MEKC). Nowadays and as summarized in Table 3, LC is the most used technique for the analysis of natural plant protection products for crop protection (far ahead of GC), and reversed-phase chromatography (C8 and C18), the most appropriate/employee chromatographic technique. Traditionally, GC was the most used technique to determine pesticides and their transformation products. However, today, LC is found to be more adequate. The reason is found in the low volatile capacity and high thermolability of the majority of these new compounds, due to the new and more stringent regulations that favor Chapter 15 Biopesticides, EU Regulation and Residue Analysis 469 the less persistent ones. In this sense, the agrochemical industry also tends to develop effective pesticides at a low application rate, with a minimal toxicity for nontarget organism and a more polar character to prevent bioaccumulation. Since these new pesticides usually show high efficacy at low doses, it is expected that their trace concentrations could be found in the environment. Thus, for example, due to high phytotoxicity of new active substances, small amounts of residual herbicides in soil may affect sensitive succeeding crops, neighboring crops, and nontarget plants. Also, their polar character makes them easily leach to groundwater and potentially contaminate at levels above 0.1 mg L 1 [107]. Therefore, analytic methods must provide high sensitivities. The situation becomes more complex when transformation products are present due to the lack of analytic standards and scarce information available [108]. Qualitative analysis is the precursor for quantitative analysis. Literature contains a great amount of data on qualitative analysis with MS with excellent books recognized as standard references in the field [109–112]. GC–MS has been used as a routine technique to carry out the easy screening of unknown pesticides in different matrices using conventional library-searching routines. However, no adequate searchable libraries currently exist for LC–MS. Therefore, today, it is still difficult to identify and detect unknown nontarget pesticides and transformation products without resorting to their isolation and identification by other techniques (e.g., NMR). Nevertheless, great efforts are being made to the routinization of LC–MS as GC–MS (e.g., by the combination of LC–MS and several databases [113]). A known alternative to give LC mass spectra compatibility with commercial spectrum libraries (electron impact (EI) spectra; see succeeding text) is the particle beam (PB) interface. This device was designed for ionizing a sample, released from an LC column, with a beam of high energetic electrons. However, this interface has been rarely used because only a collection until m/z ¼ 600 can generate highly informative, reproducible, library-matchable EI mass spectra [114]. Nevertheless, some HPLC-PB/MS methods have been described for the study of biopesticides with good results. For example, Ho and Budde [67] developed an analytic method for rotenone (Fig. 2) in water based on SPE, modified SFE, and HPLC-PB/MS. Using this method, it was possible to analyze water from river in a reasonable time frame and measure rotenone concentrations above 4 mg kg 1 (LOQ). Furthermore, if it were the case, it would be also possible to look for unknown pesticides present in samples. Over the last decade, several developments in chromatographic packing materials and instrumentation for liquid chromatography (LC) have enabled rapid and highly efficient separations. The three main modern developments in LC methods are the following: the use of monolithic columns, hightemperature liquid chromatography (HTLC), and ultra-performance liquid chromatography (UPLC) [115,116]. Monoliths can support high flow rates (up to 10 mL/min) in conventional column lengths without generating high back pressures (difference between the pressure at the column inlet and that 470 Studies in Natural Products Chemistry at the outlet [117]), which is their main advantage. Monolithic rods with solgel technology enable the formation of highly porous material, containing both macropores and mesopores in its structure. LC columns consist of a single rod of silica or polymer-based material where the large pores (typically 2 mm) enable low flow resistance and therefore allow the application of high eluant flow rates, while the small pores (about 12 nm) ensure sufficient surface area in order to reach high separation efficiency [91]. HTLC refers to any separation carried out at temperatures above room temperature with a mobile phase in a liquid state. Work at high temperatures (60 C < T < 200 C) presents several advantages like a significant reduction of mobile-phase viscosity, which leads to a decrease in column pressure, a reduction of eluant strength, the change in selectivity, the increase in diffusivity, and the change in the dissociation rate for ionizable compounds [118]. Even though this is an interesting analytic technique, to our knowledge, no studies about biopesticides have been carried out with HTLC. The usual high-thermolability character of biopesticides, already mentioned earlier in the text, makes that this analytical technique maybe does not reach a great development in the study of these substances. The opposite occurs with UPLC. Waters was the first producer that coined the term UPLC by the introduction of a new category of LC technology [119], which permitted the use of sub-2 mm particle-packed columns [115,116] with a high back-pressure requirement (DP > 400 bar) [115]. Today, there is a wide variety of stationary phases packed with sub-2 mm particles [120] and UPLC instruments achieving maximum pressures between 600 and 1200 bar [121]. In fact, gradually, UPLC is displacing HPLC in the study of biopesticides (Table 3) due to its major advantages over HPLC: speed of analysis, improved resolution, and sensitivity. In this sense, Romero-González et al. [55] concluded that a QuEChERS-based method combined with UPLC can be used for the routine simultaneous analysis of several biopesticides, at trace levels in organic samples, with running analysis times less than 13 min. All the previously mentioned processes are examples of column chromatography because the stationary phase is contained in a column. On the other hand, if the stationary phase is distributed as a thin layer on a flat support and the mobile phase is allowed to ascend through the layer by capillary forces, then this technique is referred as planar or thin-layer chromatography (TLC), which has almost replaced paper chromatography (PC) in the current practice due to the inferior separation capacity of the latter. High-performance thin-layer chromatography (HPTLC) is an enhanced form of TLC. Although this is a technique that today is gaining some relevance, its use for quantitative analysis is still low. Sucrose esters from the surface of leaves of tobacco plant (Nicotiana tabacum L.) have been shown to possess biological activities as insecticides. HPTLC silica gel plates were used for their separations and off-line TLC–MS for the detection and identification [122]. On the other hand, capillary electrophoresis–mass spectrometry (CE-MS), in which the Chapter 15 Biopesticides, EU Regulation and Residue Analysis 471 components of the analyzed samples are separated according to mass-tocharge ratios, is an alternative to GC–MS and LC–MS [123]. This technique has a great potential for metabolism studies. However, from a practical standpoint, nowadays, it is not being used for environmental samples due to its complexity, time-consuming sample preparation, and requirement for expensive apparatus and trained persons to operate. Something similar happens with the HPLC-NMR-MS [124]: it has been proved as a powerful tool in several areas of the pharmaceutical industry [125–128], chemical reactivity [129], and polymer research [130]. However, this is not an usual technique in labs due to its high cost (equipment and solvents) and the necessity of very specialized technicians. However, it could be a promised technique in the future if costs decrease considerably. As for the detection techniques, MS has become one of the most important techniques in analytic chemistry for the study of biopesticides. In fact, it has almost displaced other detection devices in the development of analytic methods properly validated for the study of biopesticides (Table 3). The combination of chromatographic techniques and an MS detector appears to be a suitable approach to satisfy the actual EU legislative requirements in terms of sensitivity, selectivity, and peak-assignment certainty for the rapid determination of analytes at low concentrations in complex matrices. A mass spectrometer is an analytic instrument that can separate charged molecules according to their mass-to-charge ratio (m/z). There are three main parts/critical components of a mass spectrometer: the ion source, the mass analyzer, and the detector [131]. In a mass spectrometer, the role of the ion source is to create gas-phase ions and transfer them into the mass analyzer. They are separated according to their m/z in the mass analyzer and then counted by the detector. The choice of ion source depends on the application, with EI and electrospray ionization (ESI) as the preferred ones for GC–MS and LC–MS equipments, respectively. The EI source utilizes energetic electron beams (negative charges), which are electrically accelerated and directed to collide with a vaporized sample, causing electron expulsion from the analytes and subsequent formation of positively charged radical cations M+ [109,132]. For all practical purposes, EI is basically a positive ion source. The electron energy is in most cases set to 70 eV. Since most organic compounds have ionization potentials of 7–20 eV, the energy transferred on collision between the electron and a neutral molecule is sufficient to cause both ionization and extensive fragmentation [133]; thus, an EI mass spectrum shows the molecular ions and also fragment ions. Large spectral libraries, created thanks to EI fragment patterns, are relatively reproducible and instrument-independent. On the other hand, ESI source is encompassed within the named soft ionization techniques because minimum internal energy is transmitted to the analytes during the ionization process. ESI-MS is currently used for qualitative and quantitative studies of a wide variety of nonvolatile and thermally labile structures [134]. 472 Studies in Natural Products Chemistry Briefly, the sample is solubilized in a polar solvent, which is infused under atmospheric pressure into the ionization source via a thin needle in the form of a spray. A high electric potential is applied at the needle, resulting in the formation of highly charged droplets, which are then driven electrically and vaporized with the aid of a warm neutral gas (e.g., nitrogen) [109,132]. If molecules ionize forming positively charged ions [M+H]+ and/or [M+X]+ (X ¼ Na, NH4 + , and others), the acquisition of a mass spectrum is carried out in positive mode, while negative mode is used in the opposite case. In recent literature about the validation of analytic methods for biopesticides (Table 3), ESI in LC and EI in GC are almost the only used ion sources with few exceptions (e.g., atmospheric pressure chemical ionization [54] and PB interface [67] for LC). This is in accordance with the previously mentioned text and what happens in other research areas [135]. As a curiosity, all MS references from the data review for biopesticides (Table 3) were used in positive ionization mode, maybe because most analytes can form positive ions. However, negative ion formation is selective and can be very efficient, providing very high sensitivity, which could be a good option in many cases. After ionization, the ions formed are carried to the analyzer. All mass analyzers presently in use are based on electromagnetism, so ions are required to obtain separation. Therefore, an ion source has to be coupled to the analyzer. Nowadays, the more common three basic types of mass analyzers [135] are quadrupole (Q) [136], ion trap (IT) [137], and time of flight (TOF) [137], although they usually are associated with several other ones forming hybrid mass spectrometers, such as triple quadrupole (QqQ), ion trap–time of flight, and quadrupole–time of flight [135]. The orbitrap is one of the most recently developed mass analyzers [138–140], and although it is still not very common to find it in the laboratories for analysis, its use is increasing [135]. Finally, the function of the detector is to convert the energy of incoming particles into a current signal that is registered by the electronic components and transferred to the computer of the acquisition system of the mass spectrometer. It is not the purpose of this chapter to go deeper into the issue of these devices, especially when there are abundant specialized books on this subject [109–112]. Until now and to our knowledge, QqQ [55,56,59,60,64,65,68] and IT [57,61] are the most used analyzers to validate methods for biopesticides. LC–MS with QqQ and IT have been compared in several works with pesticides [135,141,142]: QqQ provides better linear dynamic range, higher precision and sensitivity, less matrix interferences, and better robustness, while IT provides an excellent sensitivity for product ion measurement (MSn experiments). On the contrary, TOF and orbitrap are still analyzers that have not been used with this purpose. The price is surely one of the parameters that have most influenced this tendency. Table 3 shows that UPLC–MS achieves LOQs lower than 10 ppb in almost all examples and regardless of the matrices. Instead, HPLC–MS only has succeeded these LOQ in studies with rotenone [64,67] and spinosyns [59] (Fig. 2). It is very likely that as the prices of these devices begin to be more Chapter 15 Biopesticides, EU Regulation and Residue Analysis 473 accessible, they will be commonly available in all analytic laboratories. Therefore, in a near future, lower LOQs should be met, so each applicant who wants to introduce a new active substance in the market will have provided adequate analytic methods probably with UPLC coupled to MS. Despite the increasingly widespread use of UPLC–MS within studies with validated analytic methods, today, there is still research with classical chromatographic techniques. For example, Adamczyk et al. [51] and Nozal et al. [53] developed methods based on GC with flame ionization detector (FID) for the analysis of thymol in honey. These methods allow obtaining similar LOQs than with most modern techniques, such as HPLC with diode array detector (DAD) [52]. This fact is due to the greater sensitivity of GC. However, these are particular cases with low-molecular-weight biopesticides. Similarly, other authors still continue using LC systems with UV and DAD detectors, the most universal detectors for pesticides, which also gives some kind of structural confirmation by UV spectra. However, each day will be more difficult to reach LOQs as those with MS, especially with complex matrices and the presence of interferences. Analysis of Biopesticide Residues in Environmental Matrices Studying the behavior and impact of biopesticides in different environmental compartments is one of the main objectives for which validation of analytic methods is carried out. The control of residue levels in the matrices of interest is also important to evaluate the efficacy of different treatments used. However, monitoring data for biopesticides and their transformation products in field are generally poor and there are still scarce full research studies. Environmental and toxicological studies begin to employ enzyme-linked immunosorbents for the analysis of biopesticide residues [143–146]. For example, Xu et al. [144], Hemalatha et al. [143], and Zhang et al. [146] developed enzyme-linked immunosorbent assays (ELISAs) for the determination of podophyllotoxin, azadirachtin A (also named simply as azadirachtin) (Fig. 2), and toosendanin, respectively. In these studies, ELISAs were validated and evaluated by quantitating respective biopesticides in water, five (tomato, brinjal, coffee, tea, and cottonseed) and three (cabbage, tomato, and apple) spiked agricultural commodities. The recoveries of podophyllotoxin from tap water samples (spiked between 20 and 2 104 mg kg 1) by ELISA were in the range of 72–115%. Azadirachtin was spiked in the agricultural commodities at 500 and 1 103 mg kg 1 and the recoveries were between 62% and 100%. In ELISAs with toosendanin, the food matrices were spiked between 100 and 1 104 mg kg 1, reaching recoveries between 76.4% and 113.2%. ELISA results with podophyllotoxin and toosendanin were compared with those obtained by HPLC, and the results showed good stability, recovery, and accuracy. Therefore, these highly specific and reliable ELISAs are suitable for the sensitive analysis of water and agricultural samples. 474 Studies in Natural Products Chemistry Thoeming et al. [147] studied the translocation and persistence of active neem ingredients (azadirachtins A and B) in bean plants. The residues of these active substances from substrates with different contents of organic matter and from various plant parts were quantified by HPLC–MS after carrying out previous liquid–solid and liquid–liquid extractions. Residue analysis of the bean plants showed that only small proportions of the initial amounts of azadirachtins A and B were present in the plant (0.3–8.18%). Variable amounts of residues of the active components in relation to plant parts and time of analysis indicated a different translocation pattern for the two active ingredients. Rotenone (Fig. 2) is extracted with trichloroethylene from the roots of some Leguminosae (Derris elliptica, Lonchocarpus nicou, and Tephrosia vogelii) to obtain cubè resin that shows four rotenoids as major ingredients: rotenone, deguelin, b-rotenolone, and tephrosin. This resin is used to prepare rotenonebased insecticide formulations. Although at this moment, rotenone is not approved according to the Regulation (EC) No. 1107/2009, MRLs and import tolerances can be established according to the Regulation No. 396/2005, and plant protection products with this substance could be used in case of emergency. However, only rotenone is taken into consideration in the legal determination of the residue in case of using cubè resin. A study, conducted by Cabizza et al. on the residues on olives by HPLC-DAD, was carried out to assess not only the rotenone (Fig. 2) content but also the main rotenoids [148]. The residues of deguelin, tephrosin, and b-rotenolone were 100, 60, and 100 mg kg 1, respectively. These contents were very similar to rotenone (80 mg kg 1) and few data indicate similar acute toxicity values for deguelin [149]. The most important information that can be extracted from this research work are not only the excellent recoveries (above 80%) and low residual standard deviations (lower 3%) reached with the analytic method but also the importance of establishing an appropriate residue definition according to the plant metabolism studies and toxicological studies. On the other hand, the field data obtained by Cabras et al. [150], using an analytic method by HPLC–MS, demonstrate that rotenone on olives decay more slowly than rotenone on other crops, such as lettuce and tomato. These results agree with those also showed by Cabizza et al. [148], so the low MRL fixed in Europe (10 mg kg 1) makes very difficult to obtain olives with lower residues at a preharvest interval of 10 days. Pyrethrins (Fig. 2) could be analyzed by GC [151] and HPLC [148,152–154]. The former study described a fast and simple method for the sampling and analysis of insecticides’ indoor air, based on sampling through Tenax and XAD2 tubes, ultrasonic extraction, and GC–MS separation and analysis. The latter methods by HPLC were used for vegetable matrices and soil. From these results, it obviously seems that more studies are needed to assess the overall impact of biopesticides and their transformation products in different environmental compartments. Chapter 15 Biopesticides, EU Regulation and Residue Analysis 475 CONCLUSIONS The strict safety criteria on conventional chemical pesticides by EU and other developing countries are creating an opportunity to introduce novel biopesticides in the market. Today, the Regulation (EC) No. 1107/2009 encouraged the development of less harmful substances like biopesticides. The adoption of real IPM tactics is the key to favor their usage. It is necessary to highlight that conventional pesticides will remain a vital part of crop protection within an IPM framework, using them sparingly to minimize the evolution of resistance in target pest populations. However, using conventional synthetic chemical pesticides applied on a calendar basis can be difficult to replace in favor of an IPM portfolio of alternative tactics including biopesticides. Therefore, IPM practices require two conditions: the existence of a range of crop protection strategies and the knowledge on how to integrate them by farmers (training). Scientific advances in biopesticides will be achieved because the Regulation (EC) No. 1107/2009 has been promoted with the aim of prohibiting the compounds with unacceptable risk. In fact, several plant protection products will have difficulties to renew their authorization because of the new cutoff criteria. Furthermore, the evolution of resistance to the conventional chemical pesticides may stimulate challenges in the market and R&D strategies by companies, which will surely be involved in the adoption of future specific policies for these natural substances. Biopesticides shall be monitored by analytic methods with sufficient sensitivity and specificity to assess the magnitude and nature of all significant residues remaining in plants, plant products, foodstuff, feedingstuff, soil, water, air, and body fluids and tissues. Furthermore, these analytic methods must be properly validated. This is fundamental to satisfy the actual EU legislative framework and guarantee minimal adverse effects of biopesticides on human health, nontarget organisms, and the environment. In this sense, the most relevant studies in the recent literature about sample preparation and analytic techniques (together with the trends) to determine these bioactive natural compounds have been compiled and discussed. In the most cases, the whole analytic method was properly validated according to the requirements mentioned earlier in the text. However, the objective of these research works was not always to satisfy the actual EU legislative framework, but develop analytic methodologies to detect and quantify residues from these biopesticides at the lowest trace level. Today, sample preparation is maybe the step that most influences the accuracy of the whole analytic method, with the extraction of pesticide residues from environmental matrices the key factor to get it. SPE is a fully accepted technique that allows sample preparation time to be halved or better and reduces solvent usage by up to 90% compared to liquid–liquid 476 Studies in Natural Products Chemistry extractions. Each day, sorbents are more adapted to the extraction of polar biopesticides and degradation products and emerging techniques such as SPME have high potential for the cleanup step. Several other cleanup methods based on the SPE technique, such as dSPE, have been developed and successfully applied to determine different biopesticides. GC could be chosen for analyzing a wide range of low-molecular-weight biopesticides. However, its use is declining significantly in favor of LC techniques that have been used widely during the last 15 years. Furthermore, UPLC is gaining more strength for the analysis of these compounds. On the other hand, an increase in the use and development of MS techniques has been taking place in the last years for the detection of environmentally important compounds. Today, GC–MS accomplishes the main points desired in an analytic technique (selectivity, sensitivity, and reproducibility), while LC–MS permits sensitivity limits that approach those of GC. Great efforts are being made to routinization of LC–MS as GC–MS. The trends in the analysis of biopesticides are aimed at analytic methods that allow the detection of trace amounts of these compounds in a very short time. In this sense, the best-positioned techniques at this moment are as follows: (a) SPE (with novel selective tailor-made sorbent materials) and QuEChERS (with its variants) for sample preparation and (b) ultra-performance LC coupled to MS with an electrospray source and a triple quadrupole analyzer (UPLC-ESI-QqQ-MS/MS) for identification/quantification. However, more improvements are still needed to allow a full understanding of the fate and behavior of biopesticides in the environment. ABBREVIATIONS AC CE CEC DAD DIAS dSPE EC ECD EEC EI ESI ELISA EU FID FL GC affinity chromatography capillary electrophoresis capillary electro-chromatography diode array detector digital image analysis system dispersive solid-phase extraction European community electron capture detector European economic community electron impact electrospray ionization enzyme-linked immunosorbent assay European union flame ionization detector fluorescence detector gas chromatography Chapter 15 GCB GLC GLP GSC HPLC HPTLC HTLC IEC ILV IPM IT LC LLC LLE LOD LOQ LSC LSE MEKC MIP MMLLE MRL MS m/z NMR NPA ODS PB PBT PC POP ppb PSA QqQ QuEChERS r RAMs RMS RPC RSD SANCO SEC SFC SFE Biopesticides, EU Regulation and Residue Analysis 477 graphitized carbon black gas–liquid chromatography good laboratory practice gas–solid chromatography high-performance liquid chromatography high-performance thin-layer chromatography high-temperature liquid chromatography ion-exchange chromatography independent laboratory validation integrated pest management ion trap liquid chromatography liquid–liquid chromatography liquid–liquid extraction lower limit of detection lower limit of quantification liquid–solid chromatography liquid–solid extraction micellar electrokinetic chromatography molecularly imprinted polymer membrane liquid–liquid extraction maximum residue level mass spectrometry mass-to-charge ratio nuclear magnetic resonance national provisional authorisation octadecylsilane particle beam persistent, bioaccumulative, and toxic paper chromatography persistent organic pollutant parts per billion primary secondary amine triple quadrupole quick, easy, cheap, effective, rugged, and safe correlation coefficients restricted access materials rapporteur member state reversed-phase chromatography relative standard deviation Health & Consumers—European Commission size-exclusion chromatography supercritical fluid chromatography supercritical fluid elution 478 Studies in Natural Products Chemistry SLM SPE SPME TLC TMS TOF UPLC U.S. EPA UV vPvB supported liquid membrane solid-phase extraction solid-phase microextraction thin-layer chromatography trimethylsyl time-of-flight ultra-performance liquid chromatography United States Environmental Protection Agency ultra-violet very persistent and very bioaccumulative REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] Regulation (EC) No. 1107/2009, Off. 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A a-Alkylation reactions, 412, 415–422, 425–427 Abacavir, 202–203 Absolute configurations, 2–3, 4, 12, 13–14, 16–18, 20, 22–24, 25–27, 44, 45–49, 47f, 56 of vibsanin F, 42, 43f, 47f Acetyl-CoA, 85–88 Acetyl coenzyme A acetyltransferase 1 (ACAT-1), 104–105 Acetyl coenzyme A acetyltransferase 2 (ACAT-2), 104–105 Acetylinic lipids, 230–232 Acid acetic, 71–72, 101–102, 106, 109, 165t, 224–225, 273 alkyl, 80 amino, 85–88, 167, 261–262, 269, 269t, 270, 327, 334, 341–343, 354–355, 412, 413s, 430–431 arabinoic, 269 ascorbic (aa), 281–282 bassic, 355–356 benzoic, 225, 265–266, 355 boric, 166 caffeic, 265–266, 356–357, 401 10-camphorsulfonic, 71–72 3-chloroperoxybenzoic (mCPBA), 22–24, 24s cinnamic, 88 coumaric, 161, 389 decanoic, 283 deoxyribonucleic, 201, 245–246 diffractaic, 250, 252t 3,10-dihydroxy-decanoic, 267f, 273–274, 277–278 evernic, 235 fumarprotocetraric, 237 g-aminobutyric (GABA), 101, 207–209, 295–304, 400 gyrophoric, 238, 246, 247t, 251–253 homosekikaic, 226–227 10-hydroxydecanoic, 262, 263t, 273–274, 276 10-hydroxy-2-decenoic, 262, 271–272, 273–274, 276, 277–278, 279–280, 283 hyperhomosekikaic, 227–229 isothreonic, 269 keto, 85–88 lactic, 203, 269 lecanoric, 249–253 lobaric, 241t, 246–249, 250–251 malic, 269 m-chloroperbenzoic, 46–47 monocarboxylic, 283 mycosporine-like amino (MAAs), 335, 339–343 nitric, 164 oleanolic, 352–354, 364–365, 367–368, 399 orsellinic, 249–250, 251–253 pannaric, 226–227 picrolichenic, 226–227 protolichesterinic, 226–227, 237, 241t, 246 pulvinic, 226–227 quinic, 269 rhizocarpic, 230 ribonic, 269 ribonucleic, 201, 377 sakikaic, 252t salazinic, 226–227, 237, 246–249 sebacic, 271 shikimic, 227, 228s, 230 tartaric, 411, 429–431 threonic, 269 trans-2-decenoic, 273–274 umbilicaric, 232–233, 250–251 uncarinic, 389, 392–397, 402–403 ursolic, 367–368, 389, 397–398, 403–404 ()-usnic, 235, 240–246 virensic, 253–254, 254t vulpinic, 226–227 Acid hydrolysis, 20, 30 Acremonium sp., 2 Activity agonistic, 97–98 antidiabetic, 352, 354, 356–357, 362–363 antiinflammatory, 397 antimutagenic, 398 483 484 Activity (Continued ) catalytic, 409–410 contractile, 253–254 estrogenic, 273–274 hypolipidemic, 359, 360–361 immunomodulatory, 213–214, 270, 277–278, 284–285, 399 locomotive, 400 outgrowth-promoting, 62, 72 Acute rejection, 176–178 Acyl-ACP thioesterase, 85–88 Acyl carrier protein (ACL), 85–88, 87f 1,4-Addition, 60–61 S-Adenosyl-methionine, 87f Adipocytes, 355, 359, 360–361 Adipose tissue, 99–100, 203, 352, 353f, 357–358 Aeruginosin, 430–431, 432s Aglycones, 161–162, 375, 376f, 377 AIDS, 197–198, 199, 201–202, 204–205, 206, 212–213, 305–307 AIDS-associated neuropathologies, 212–213 Albophoma sp., 2 Alcohol addiction, 7 Aldol condensation reaction, 44 Aldol-type coupling reaction, 4–6, 25, 32–33 Aldovibsanins, 50 Aliphatic amine residue, 79–80 Aliphatic unsaturated fatty acid residue, 79–80 Alkaline hydrolysis, 34–35 Alkaloids, 124, 127–140, 318, 319t, 327, 334, 335, 382–384, 385, 386–389, 392, 393t, 397, 400, 409–410, 410f b-carboline, 128–131 natural, 79–80, 110, 127–128 pentacyclic, 397 piperidine, 124, 137–138 Alkamides, 79–83, 80f, 81f, 83f, 84, 84f, 85, 86f, 88–89, 88f, 92, 93–94, 99, 102, 102f, 103, 104, 105f, 106, 109–111 natural, 85, 90, 91, 102, 104–105, 106, 110 producing, 89 Alkenylamides, 79–80, 110 Allogeneic transplant, 178–179 Allograft rejection, 176–178 Allyl alcohol, 4–6, 7–9, 10–11, 25–27, 46–47 5-Allyl-L-cysteine, 209–210 p-Allylpalladium, 48 Amentoflavone, 213 4-Aminomethyl-TMP, 184–185 Ammi majus, 147, 150t, 158t Amorfrutins, 355, 356f Amorpha fruticosa, 355 L-amphetamine, 126 Index Amprenavir, 204, 208t, 211 Analgesic, 79–80, 89, 90–91, 92, 93–95, 100–101, 102, 103, 111, 232–233, 255, 366, 382, 385, 419 Angelica archangelica, 158t Angelicin, 146–147, 146f, 150t, 158t, 168, 184 Angular furanocoumarins, 149, 149f, 158t, 161, 164 ANOVA, 64f Anthelmintic activities, 80–82 Anthozoa, 314–316, 315f, 316f, 317f, 318 Antiapoptotic proteins, 126 Antibacterial activity, 79–80, 232–233, 235, 270–271, 397–398 Anticonvulsants, 106–107, 204–205 Antidepressant activity, 106–107, 126, 209 Antidepressant effect, 125–126, 138 Antidepressants, 106–107, 125–126, 207–209 Antidiabetic drugs, 354 Antidiabetic effect, 356–357, 361–362, 366–367 Antidiabetic plants, 352 Antiepilepsirine, 138 Antifungal activities, 184, 233–240 Antigen-presenting cells, 169–170, 178–179 Antihyperglycemic activity, 358–359, 365 Antihypertensive, 2, 279–280, 399 Anti-inflammatory, 2, 79–80, 89, 90, 100–101, 102–103, 106–107, 109, 179, 182–183, 211–212, 270, 281–282, 318 Antimetastatic effects, 182 Antimicrobial activities, 209–210, 211, 233–234, 237, 238, 270–272, 377 Antimicrobial properties, 184, 235 Antimycobacterial activity, 184 Antioxidant, 79–82, 89, 90, 106–107, 209–210, 232–233, 249–251, 252t, 272–273, 318, 343–344, 397, 401 Antiproliferative effects, 181–182, 183, 245–246, 385 Antiretroviral drugs, 198, 199–200, 202–207, 209, 213 Antiretrovirals, 199, 200, 210–211 Antiretroviral therapy, 197–198, 205, 206–207 Anti-tuberculosis drugs, 184 Antitumor activities, 245–249, 392–397 Antiviral activity, 205–206, 240–245, 377, 398 Anxiety, 279, 295–304, 305–307 Anxiousness, 295–304 Apiaceae, 146, 149, 150t, 158t, 161, 376 Apigenin, 213 Apium graveolens, 150t 485 Index Apoptosis, 169–170, 179, 245–246, 273, 295–304, 392–397, 399 Araliaceae, 376 Aromatic amine residue, 79–80 Aromatic b-carbolines, 128 Arthralgias, 173 Arthritis, 2–3, 92, 109, 169, 173, 175–176, 270, 282–283, 397, 404 Aspergillus niger, 184 Astaxanthin, 335–337 Asteraceae, 80, 81f, 85, 86f, 100–101, 110, 211–212, 283–284 Asteraceae plants, 80, 86f, 110 Asthma, 93–94, 223–224, 283–284, 383t Asymmetric alkylation, 412, 417, 419, 420–422 Asymmetric catalysis, 431–432 Asymmetric phase-transfer catalysis, 410–411, 427–428, 431–432 Atazanavir, 204 Atopic dermatitis, 169, 172–173 ATP-binding cassette (ABC), 2–3, 4, 200 Azadirachtin, 453t, 463f, 473, 474 2,20 -Azobisisobutyronitrile, 13s B Bacillus licheniformis, 235 Bacillus megaterium, 235 Bacillus subtilis, 235, 237, 270–271 (+)-Bakuchiol, 67–69, 69s Banisteriopsis caapi, 128 Barton–McCombie procedure, 12, 16–18 Baylis–Hillman reaction, 66 BD Gentest Supersomes, 129np, 133np Bean plants, 474 Berberine, 134–135 Bergamot oil, 147 Bergamottin, 150t, 161, 208t, 211 Bergapten, 148f, 149, 161, 162, 164 Bergaptol, 148, 148f, 149, 150t, 161 Bioactive compounds, 375, 382–384, 386–389, 390–392, 402–403 isolation of, 375, 386–389 Biopesticide residue analysis, 449, 473–474 Biopesticides, 438–439, 440–449, 451–474, 453t Biosynthesis, 60–61, 85–89, 102–103, 110, 224–225, 227–232, 228s, 313–314, 403–404 of alkamide, 82–83 of polyphenols, 228s ()-4,5-Bis-epi-neovibsanins A, 64–65 ()-5,14-Bis-epi-spirovibsanin A, 64–65 bis-(2-ethyl-hexyl)-phthalate, 225 bis-TBS ether, 10–11 Blood-brain barrier, 124–125 Blood glucose levels, 175, 354–355, 356–360, 361–363 Botrytis cinerea, 270–271 Brain-derived neurotrophic factor (BDNF), 62 Brain mitochondria, 131–132, 133np, 137, 138 Breast adenocarcinoma, 245–246 Breast cancer cells, 182 Briareum, 315f, 316f, 318, 319t 3-Bromo-2-methoxy-5,6-dimethyl-4H-pyran4-one, 12 Bromophenyl carbamate, 44 Bronchitis, 223–224, 232–233 Bronchospasmolytic, 2 C Caenorhabditis elegans, 275–276 Caffeic acid O-methyltransferase, 85–88, 126 Calcium/calmodulin dependent kinase II, 93–94 Cancer cell lines, 2, 182–183, 245–246, 392–397 (–)-Candelalide A, 14–18, 19–20, 21–24 Candelalides, 2–3, 14–24, 23s, 24s, 29–30, 35 Candida albicans, 184, 233, 271–272 Capillary electrophoresis (CE), 163, 166, 186, 470–471 Capsaicin, 85–88, 90, 91–100, 101–102, 107–109, 110–111 Capsaicinoids, 80, 82f, 85–88, 87f, 89–91, 92–93, 95–97 natural, 90, 95–96 Capsaicinol, 95–96 Capsaicin synthase, 85–88 Capsicum genus, 80, 82f b-Carboline system, 128 Carcinogenicity, 147 b-Carotene, 335–337, 338t, 341–343 Carotenoids, 335–339, 338t, 343 Caryophyllaceae, 376–377 Catalyst, 33, 409–413, 414, 415–416, 419–420, 422, 423–425, 426–427 Catalytic by-products, 125 Catechin, 359–360, 389, 391f, 401, 403 Catechol-O-methyl transferase (COMT), 141 CB1 receptor, 294–304, 305–307 CB2 receptors, 295–304 CD4 cells, 200–202, 206 Cell membrane, 96–97, 375, 376, 379, 401 486 Cells, 167, 168, 169–170, 182, 197–198, 275, 276, 277–279 apoptotic, 169–170 host, 201, 202–203, 205 mast, 169, 277 Central nervous system (CNS), 92–93, 124–126, 141, 175, 201, 383t, 415–416 Cetraria islandica, 237, 246 Cheese reaction, 125–126 Chemokine receptors, 201, 205 Chemotaxonomic markers, 80 Chiral PTCs, 409–410, 411f, 412–413, 414, 424–425 Chlorophylls, 162, 335, 339–343 Cholesterol levels, 279–280, 376 Chromatographic techniques, 438–439, 467–469 Cimetidine, 182 Cinchona, 139, 382, 398, 409–410, 410f, 414, 419–420 Cinchona alkaloids, 409–410, 413, 414–424 Cinchonae cortex, 139 Cinchonaminone, 139 Cinchona succiruba, 139 Cinchonicinol, 139 Cinnamaldehyde, 357–358 Cinnamic acid 4-hydroxylase (CA4H), 85–88 c-Jun, 99–100, 282–283 Cladosporium cucumerinum, 184 Clorgyline, 124, 126–127, 138 Cnidaria, 314–327 Cnidium monnieri, 182–183 Coenzyme A (CoA), 85–88 (+)-Columbianetin, 147–148 Column chromatography, 20, 70–71, 163, 470–471 Community agriculture, 443 competitiveness of, 443 Complementary and alternative medicines (CAMs), 199 Corals, 314–316, 318, 319t, 327, 335, 337–343 Coronidium scorpioides, 161 Corynoxine, 392, 398, 400, 401 Coumaric acid 3-hydroxylase, 85–88, 87f Coumarins, 146–147, 161, 181–183, 357–358 extraction of, 161–162 4-Coumaroyl-CoA ligase, 85–88, 87f Coupling reaction, 7–9, 12, 14–15, 19, 29–30, 35–37 Coxiella burnetii, 184 Cross-conjugated diene, 49 Crotalaria juncea, 161 Index Cryptococcus neoformans, 184 Cucumber, 452, 453t Cu(I)-mediated intermolecular, 25–27 Cutaneous lymphocyte antigen (CLA), 171 Cutaneous pruritus, 182–183 Cutaneous T-cell lymphoma, 169, 170, 180–181 Cutaneous ulcers, 173 Cyanide displacement 7, 7 Cyclization reaction, 16–18, 22–24, 30–31, 430 Cyclooxygenase 1 (COX-1), 106 Cyclooxygenase 2 (COX-2), 102–103 Cyclosporine A, 4 Cyclovibsanins, 51, 52, 53s A, 51 B, 51 Cytochrome P450, 106, 147–148, 200 Cytotoxic activities, 2, 283 Cytotoxicity, 61–62, 72–74, 245–249, 378, 392–397, 398 D Deamination, oxidative, 123–124, 125, 129 Decalin aldehyde, 7–9, 14–15, 29–30, 35–37 Decarboxylation, 85 Dehydration, 14–15, 16–18, 20, 25–27, 52, 60–61, 228s 2,3-Dehydro-piperidinyl, 80 Delta-9-tetrahydrocannabinol, 293–294 Delviradine, 203–204 Dementia, 199, 212–213, 382, 383t, 385, 401, 403 Dendritic cells (DCs), 169–170, 179, 277–278 De-O-acetylsesquicillin, 13–14 Deoxygenation, 25 Deoxyribonucleic acid (DNA), 101–102, 147, 168, 169–170, 183, 201, 202, 203, 245–246, 283, 358–359, 392–397, 399, 402–403 L-deprenyl, 126 Depression, 93–94, 123–124, 125–126, 138, 199, 207–209, 208t, 279, 305–307 Deprotection, 7, 10–11, 12, 13–14, 15–18, 19, 21–22, 25–27, 34–35, 66–67 Depsidones, 224–225, 226–229, 230, 245–246, 253–254 Dess–Martin oxidation, 7, 12, 16–18, 20, 25–27, 71s Diabetes, 2–3, 100, 169, 175, 179, 198, 232–233, 251–253, 351–356, 353f, 358–360, 361–363, 365, 366, 367–369 Diabetes mellitus, 169, 175, 351, 352 487 Index Diabetic rats, streptozotocin-induced, 355–356, 357, 359–361, 362–363 Diarrhea, 201–202, 232–233, 283–284 Diastereomeric mixture, 46–47, 67–69, 70–71 1,8-Diazabicyclo[5.4.0] undec-7-ene (DBU), 7 Dibenzofurans, 226–227 Didanosine, 202–203 Dietary supplements, 199, 209–210, 212–213, 282 Dihydro-b-carbolines, 128, 401–402 Dihydrocapsaicin, 82f, 90, 91f, 93–94 Dihydrofuranocoumarins, 147 6a,7a-Dihydroxyannonene, 62 7a,20-Dihydroxyannonene, 62 6,7-Dihydroxycoumarin, 183 3,10-Dihydroxydecanoic, 263t, 267f 5,8-Dihydroxypsoralen, 148 Diisobutyl aluminum hydride (DIBAL), 7, 8s 2,4-Dimethoxybenzyl (2,4-DMPM), 66–67 6,7-Dimethoxycoumarin, 185 5,7-Dimethoxy-8-(30-hydroxy-30methyl-10butene)-coumarin, 184 b,b-Dimethylacryl ester group, 56 3,3-Dimethylacryloyl chloride, 66–67 Dimethylallyl diphosphate, 147–148 4-Dimethyl-aminopyridine, 8s Dinoflagellates, 335–337, 339–341 Diosgenin, 359, 360f 2-(1,3-Dioxolan-2-yl)ethyl group, 25 2,2-Diphenyl-1-picrylhydrazil (DPPH), 249–250, 272 Disease Alzheimer’s (AD), 62, 72, 123–124, 126 autoimmune, 2–3, 169, 170, 171, 172–173, 174–176, 277–278 autoimmune mucocutaneous blistering, 172–173 Crohn’s, 169, 174 cutaneous, 169, 180–181 disabling, 172–173 graft versus host (gvhd), 169, 178–179 heart, 223–224 high risk of coronary artery, 204–205 Huntington’s, 123–124, 126 multifocal, 169–170 neurodegenerative, 72, 123–124, 125, 126–127 neurological, 124–127 Parkinson’s, 123–124, 125, 126, 254–255 renal, 198 T cell-mediated autoimmune, 2–3 Disproportional autoimmune responses, 173 Diterpenes, 53–56, 230s, 254–255, 318, 319t, 335 Diterpenoid pyrones, 2, 4, 35 candelalides A, 2–3 nalanthalide, 35 Diterpenoids, 41–63, 64–74, 75, 318, 319t 1,3-Dithiane moiety, 25–27 2,4-DMPM-ether, 71–72 2,4-DMPM-trichloroacetoimidate, 71–72, 71s DNA polymerases, 203 2D NMR experiments, 2–3 Dolutegravir, 205–206 Dopamine, 123–124, 125, 126, 135–136, 207–209, 400 Dopaminergic agonists, 126 Dronabinol, 305–307 Drug–drug interactions (DDIs), 198–199, 200, 203–205, 207, 210 Drug interactions, 198–199, 200, 203–204, 205, 207, 210 Dyslipidemia, 204–205, 352–354 Dysphagia, 173 Dyspnea, 173 E Eczema, 174t, 182–183 Efavirenz, 203–204, 208t, 213 Electronic states, 167, 331–332, 333 b-Elemine, 213–214 Elephantiasis, 185 Enantiomers, 98–99, 131, 135–136, 423–424, 426 Endo-peroxide group, 56 Endophytic fungus, 4 Enzyme-linked immunosorbent assays (ELISAs), 473 E-olefin, 43–44, 48–49 Epicatechin, 359–360, 389, 391f, 398 Epilepsy, 232–233, 382, 383t 14-epi-neovibsanin, 57f 7-epi-neovibsanin D, 56, 57f, 58s 14-epi-18-oxoneovibsanin, 57f, 58 5-epi-vibsanin, 43–44, 50, 51f, 53–56, 61–62 Epoxidation, 20, 22–24, 46–47, 410–411, 412–413, 422, 431–432 Epstein–Barr virus, 184 Eschenmoser–Claisen rearrangement, 6–7 Esophageal tumors, 183 Estrogen receptor, 273–274 Ethambutol, 184 Etravirine, 203–204 488 European monitoring centre for drugs and drug addiction, 291–292 Evodia, 80–82, 139–140 Excitotoxicity, 126 6-exo cyclization, 19 of epoxy alcohol, 19 Extra-corporeal photopheresis (ECP), 169–170, 173, 174t, 177t Extractable lichen compounds, 230–232 F Fabaceae, 355, 359–361, 363–364, 376 Fatigue, 173, 276 Fatty acid metabolism, 85–88, 352–354 Fatty acids (FAs), 85–88, 95–97, 110–111, 262–270, 263t, 273–274, 276, 277–278, 293, 327, 334, 335 Fatty acid synthase, 85–88 Fevers, 184, 201–202, 255, 382, 383t, 384, 385, 417 Fibrinolysis, 352–354 Fibrotic processes, 173 Ficus carica, 158t Filamentous fungi, 240 Flavin adenine dinucleotide, 125f Flavonoids, 208t, 209–210, 211, 213–214, 335, 354–355, 359–360, 382–384, 386, 389, 391f, 398–399, 403 Florisil, 163, 453t, 464–465 Fluorescent proteins (FPs), 341–343 Fortification, 453t Fosamprenavir, 204 Fucaceae, 146 Fumulane-like skeleton, 49 Furanocoumarin, 146–166, 146f, 150t, 158t, 167–185, 208t, 211 Furanovibsanin, 53, 54f, 55s A, 53, 54f B, 53, 54f C, 53, 54f D, 54f E, 54f F, 54f G, 54f Fusarium subglutinans, 4 G Galactomannan, 351, 360–361 Garlic extracts, 209–210 Gas chromatography (GC), 163, 166, 262–265, 450, 466–468, 470–471, 476 Index Gas-solid chromatography (GSC), 467–468 Gastrointestinal illness, 382, 384 Gastrointestinal tract, 173, 178–179, 201 Gene expression, 99–100, 167, 183, 273–274 Ginsenosides, 208t, 213–214 Glatiramer acetate, 175 Glial cell line-derived neurotrophic factor (GDNF), 62 Glucocorticoid-mediated signal transduction, 2 Glucose uptake, 352, 355–356, 358–359, 368 Glucose utilization, 356–357, 362–363, 364, 366–367 Glucuronidation enzymes, 205–206 Glycine Schiff base, 412, 415, 420, 429–430 Glycosides, 146–147, 161–162, 230–232, 357, 366–367, 375 Glycosmis, 82–83, 88–89, 110 Glycosmis genus, 84f, 88f Glycosylated hemoglobin (HbA1c), 175, 186 Glycyrrhiza foetida, 355 Good laboratory practice (GLP), 449 Gram-positive bacteria, 270–271, 397–398 Granulocytes, 399–400 Green fluorescent proteins (GFPs), 335, 338t Grubbs second-generation catalyst, 33 GVHD, chronic, 178–179 Gypsophila arrostii, 376–377 Gypsophila saponins, 376–377, 378, 379, 379f Gypsophila species, 376–377 H Hallucination, 295–304 Harmala alkaloids, 128, 129t, 131, 132–134 HDL and LDL, 279–280 Headache, 100, 170–171, 295–304, 384, 385 HeLa cells, 398 Helicobacter pylori, 237 Heliopsis longipes, 100–102, 102f Hematopoietic stem cell transplantation, 178–179 Hepatitis, 178, 198, 211–212 Hepato-cellular carcinoma, 182–183 Heracleum, 150t, 158t, 161, 165t High-density lipoprotein (HDL), 279–280, 284–285 Highly active antiretroviral therapy (HAART), 198–199, 206–209, 211, 214–215 High-risk hematologic malignant disorders, 178–179 High-temperature liquid chromatography (HTLC), 469–470 Histaminic disorders, 169 Index HIV-infected patients, 198, 209–211, 212–213 HIV infection, 197–198, 205–206, 207–209 primary, 201–202 HIV replication cycle, 197–198, 200–202 1 H NMR NOESY spectra, 16–18 Homocapsaicin, 82f, 90, 96–97 Human brain metathesis, 245–246 Human breast cancer cell lines, 2, 182–183 Human cervical carcinoma cells proliferation, 183 Human chronic myelogenous leukemia, 245–246 Human glioblastoma cell lines, 245–246 Human immunodeficiency virus (HIV), 100, 197–198, 199, 200, 205–207, 211–212, 377 Human monoamine oxidase A (hMAO-A), 141 Human monoamine oxidase B (hMAO-B), 132–134, 133t, 137, 141 Human monoamine oxidases (hMAO), 128, 132–134, 137, 141 Human papillomavirus, 183 Hydroboration, 10–11, 15–16 Hydrophilic properties, 161–162 Hydrophobic properties, 161–162 Hydroxycinnamoyl-CoA (HCHL), 87f 4-Hydroxycoumarin, 181–182 Hydroxydihydropyran, 147 3-Hydroxy-15-O-methylcyclovibsanin, 51, 52f 8-Hydroxypsoralen, 148 6-Hydroxytrypargine, 131 5-Hydroxytryptamine, 123–124, 141 16-Hydroxyvibsanin, 51 3-Hydroxyvibsanin E, 53 Hypercoagulation, 352–354 Hypertension, 179, 198, 279, 295–304, 352–354, 383t, 385, 399, 400 Hypertriglyceridemia, 204–205, 206–207, 354 Hypoglycemic activity, 355–356, 357, 359–360, 364, 365 Hypokalaemia, 295–304 I Ichthyotoxic activity, 316–318, 319t Imagawa–Nishizawa’s intermediate, 71–72 Immune system, 174, 176, 199, 202, 277–278, 295–304, 341–343 Immunodeficiency syndrome, 197–198, 213–214 Immunoglobulin, 175 Immunostimulant, 79–80, 89, 399–400, 402 Immunostimulatory drugs, 184 489 Immunosuppressive activities, 2, 4 Immunosuppressive agents, 4 Immunosuppressive diterpenoid pyrones, 4 Immunosuppressive drugs, 172–173, 176, 177–178, 179 Inconsistent gastrointestinal absorption, 180–181 Indinavir, 204, 208t, 209, 211, 212 Induced nitric oxide synthase (iNOS), 102–103 Inhibition, 98–99, 104–105, 109, 124–127, 128, 129–130, 129t, 131–137, 133t, 135t, 138, 139–140, 141, 167, 169, 175, 182–183, 203, 246, 249–250, 272, 355–356, 366–367, 368–369 competitive, 131, 135–136, 137 of hepatic glucose production, 361, 366–367, 368–369 Inhibitors, 123–124, 125, 127–140, 141 of monoamine oxidases, 123–124, 125, 126, 127, 135–136, 138, 139, 141 nucleoside reverse transcriptase, 202–203 Insecticidal activities, 2, 79–80, 82–83, 89, 106, 110 Insomnia, 170–171, 295–304, 385 Insulin-dependent diabetes, 2–3 Insulin resistance, 204–205, 351, 352–355, 357–358, 359–360, 361, 362–365, 366–367, 368–369 Insulin sensitivity, 352–354, 355–356, 366 Integrase, 201, 205–206, 253–254, 254t Integrase inhibitors, 205–206 Integrated pest management (IPM), 440, 445–448, 447t, 449 Interferon-1b, 175 Interferon-gamma (IFN-g), 170, 186 Interleukin 6, 99–100 Interleukin 1b, 99–100 Intramolecular Diels–Alder reaction, 66 Intramolecular hemiacetal formation, 14–15 Ion-exchange chromatography (IEC), 467–468 Iridoids, 41–42, 62, 354–355 Ischemia, 401 Isidiophorin, 250, 252t Isidorov, V.A., 265–266, 269 Isoenzyme, 124, 128, 132–134, 204, 207–209 Isomitraphylline, 386–389, 393t, 399–400 Isoniazid, 184 Isopimpinellin, 149, 150t, 161, 164, 166, 186 Isoprenoid chains, 146–147 Isoprenoids, 318, 327 Isopteropodine, 386f, 393t, 399–400 490 Isoquinoline, 124, 127–128, 134–137 Isorhynchophylline, 386–389, 392, 393t, 399–400 Isoxazoline syntheses, 410–411 K Keratinocytes, 169, 171, 172, 230 b-Ketoester, 25–27 b-Ketoester moieties, 48 Kynuramine, 129t, 134–135, 138 L Lactic acidosis, 203 g-Lactones, 226–227 Lagerstroemin, 351, 361–362 Lamivudine, 202–203, 209 Langerhans cells, 169, 172 Larvae, 261–262, 269, 270–271, 272, 275–276, 280–281 Leguminosae, 146, 149, 158t, 161, 165t, 474 Lemieux-Johnson oxidation, 20, 30 Leprosy, 223–224 Lewis lung carcinoma, 245–246 Lichen chemistry, 224–225 Lichen compounds, extractable, 230–232 Lichen-derived substances, 230–232 Lichen planus, 172–173 Lichens, 223–225, 227–233, 235, 238, 240–246, 247t, 251–253, 255–256 secondary metabolites of, 224–225, 240–245 Lichen species, 224, 226–227, 232–233, 235, 237–238, 241t, 243t, 245–246, 247t, 252t, 253t, 254t, 255–256 Lichen substances, 224–225, 227–232 Lichexanthone, 226–227, 230–232 Ligand-binding assay, 273–274 Linear furanocoumarins, 147–148, 149, 161, 164, 165–166 5-Lipoxygenase (5-LOX), 106, 246, 253–254 Listeria monocytogenes, 235–237, 270–271 3-Lithio-g-pyrone, 7–9, 12, 14–15, 16–18, 19, 20, 29–30 Lithium diisopropylamide (LDA), 7, 37 Liver transaminases, 168–169 Low-density lipoprotein (LDL), 212–213, 280 Lung cancer cell lines, 182–183, 245–246 Lung transplantation, 173, 177–178 Lung transplant recipients, 177–178 Lyaloside, 132–134, 133t Lymphokine release, 2–3 Lymphoproliferative disorders, 180 Index M Maceration, 161–162 Macrophage inflammatory protein 1 (MIP-1), 99–100 Macrophages, 99–100, 102–103, 295–304, 399 Malaise, 201–202 Malignant melanoma, 181–182 Mangiferin, 362–363, 363f Mannich reactions, 427–428, 429–430, 430s Marijuana, 293t, 295–304, 305–307 Marine chemical ecology, 313–314 Marine natural products, 313–314, 315f, 316–318 Marine organisms, 313–314, 334–341, 343 (+)-Marmesin, 147–148 Marmesin synthase, 147–148 Marsupin, 359–360, 363–364 Maruoka catalysts, 413, 424–425, 426–427 Mass analyzers, 439, 471–472 Mass spectrometry (MS), 163, 164–166, 325, 327, 341–343, 393t, 439, 462np, 469 Matrix metallo-proteinase (MMP), 282–283 Melanin, 270, 282, 335, 339–341, 343 Memory lymphocytes, 399 Merck research group, 2 Meristematic cells, 167 Mesylate, 10–11, 49 Mesylation, 7, 15–16 Methicillin-resistant Staphylococcus aureus, 184 7-Methoxy-8-(3-methyl-2-butenyl) coumarin, 182–183 5-Methoxypsoralen (5-MOP), 147, 161, 168–169, 172, 181–182 8-Methoxypsoralen (8-MOP), 161, 168–172, 173, 175–177, 179, 183–184 24-Methylenecholesterol, 266–267, 273–274 3,4-Methylenedioxyphenyl group, 80 4-Methylmorpholine N-oxide, 23s, 37 3-Methyl-2-oxobutanoate dehydrogenase (BKDH), 87f ()-5-Methyl-Wieland–Miescher ketone, 4–6 Michael reactions, 344, 414–415, 422 Microalgae, 223–224, 230–232, 338t, 339–343 Microbial infections, 382 Microporous membrane liquid-liquid extraction (MMLLE), 466–467 Microvasculature abnormalities, 173 Mitochondria, 123–125, 126–127, 131–135, 133np, 135np, 137, 138, 139–140, 167, 172, 203 Mitochondrial DNA polymerase g, 203 Mitoxantrone hydrochloride, 175 Index Mixed lymphocyte reaction (MLR) assay, 4 Modality, 168–169, 171, 173 Molecularly imprinted polymers (MIPs), 452–464 Monoamine oxidase (MAO) activity, 124, 126–127 Monoamine oxidases (MAOs), 123–125, 126–127, 129, 135–136, 139, 141 Monoamine oxidases (MAO) inhibition, 125–126, 127, 135–137, 139 Monoamine oxidases (MAO) inhibitors, 124, 125–126, 135–136 Monocyte-derived dendritic cell (MoDCs), 179, 277–278 Monocytes, 170, 174, 295–304 5-Monooxygenase, 148 Monooxygenases, 167 Monoterpene indole alkaloids (MIAs), 131–134 Monoterpenoid oxindole alkaloids (MOAs), 131–134, 403–404 Multiple ion monitoring-informationdependent acquisition-enhanced product ion, 165–166 Multiple sclerosis, 2–3, 169, 175, 305–307 Mutagenicity, 147, 398 Mycobacterium aurum, 235–237 Mycobacterium avium, 184 Mycobacterium tuberculosis, 184, 237 Mycobacterium vaccae, 235 Mycoplasma pneumoniae, 184 Mycosis fungoides, 169, 180–181 Mydriasis, 295–304 Myricetin, 213, 364, 365f N Nalanthalide, 2–3, 13–15, 35 (–)-Nalanthalide, 7–15 Nalanthalide synthesis, 13–15 Nalanthamala sp., 2 Naphtoylindoles, 296t National provisional authorisation (NPA), 445 Natural health products (NHPs), 199–200, 204–205, 207–214 Natural killer cells, 295–304 Natural substance, 91, 103, 451–452, 475 Nausea, 168–169, 170–171, 180–181, 182, 295–304, 305–307 Necrosis, 179, 272–273, 319t, 341–343, 357–358, 367–368 Nelfinavir, 204 Neovibsanin, 41–42, 56–61, 62–63, 72–74 491 A, 41–42, 56, 58–60, 62–63, 63f, 64f, 65f, 73f, 75 B, 56, 57f, 58–60, 58s, 62–63, 63f, 64–65, 64f, 65f, 66–67, 66f, 71–72 F, 56, 58 J, 57f, 58 K, 57f P, 57f ()-Neovibsanin B, 64–65, 66–67, 71–72 Neovibsanin-framework, 58–60 Neovibsanin skeleton, 72 Neovibsanins, outgrowth-promoting activities of, 62 Nephritis, 173 Nephthea, 315f, 316f, 318 Nerve growth factor (NGF), 62–63, 63f, 72, 74f Nervous disorders, 382, 383t Neurite outgrowth activity, 62, 72–74 Neurites, 62–63, 72 Neurological disorders, 124–127, 212–213 Neuronal cells, 62, 295–304 Neuropsychiatric disorders, 124 Neurotransmitters, 66–69, 123–124, 207–209 Neurotrophic activity, 41–42, 62–63, 72–74, 75 Neurotrophic properties, 62 Neurotrophin 3 (NT-3), 62 Neurotrophins, 62 Nevirapine, 203–204 NGF-mediated PC12 cells, 62 NHPs and antiretroviral drugs, 198, 199–200, 202–214 Nicotine, 125, 127, 274–275, 279–280, 453t Nobiletin, 209–210 Nodakenetin, 146f, 147–148 NOE correlated spectroscopy, 75 Non-nucleoside reverse transcriptase inhibitors (NNRTIs), 200, 203–204, 205–207 Noradrenaline, 106–107, 123–124, 125, 207–209 Noradrenergic neurons, 125 Norcapsaicin, 82f, 90 Nordihydrocapsaicin, 82f, 90, 96–97 Nuclear magnetic resonance (NMR), 2, 43–44, 48–49, 70t, 327, 341–343, 393t, 469 Nuclear overhauser effect, 37, 75 Nuclear overhauser effect spectroscopy, 37 Nucleophiles, 30, 48, 53–56, 60–61, 66–67, 229s Nucleoside reverse transcriptase inhibitors (NRTIs), 200, 202–204, 205–207 492 O Octocorals, 315f, 335, 337–339 Olefin cross-metathesis, 25–27, 32–33 Olefin cross-metathesis reactions, 33 Oleuropeoside, 365, 366f 3-O-Methyl congener, 53 2-O-Methyl congeners, 58 15-O-Methyl congeners, 50 18-O-Methyl congeners, 50 15-O-Methylcyclovibsanin, 50, 51 ()-2-O-Methylneovibsanin H, 64–65 18-O-Methylvibsanin, 50, 51f One-carbon homologation, 4–6 Oral glucose tolerance test (OGTT), 280–281, 355, 360–361 ortho-prenylated phenol, 147–148 Osteogenic activity, 4 Osteoporosis, 198, 275, 284–285 Osthenol, 147–148 Osthole, 166, 182–183 Outgrowth-promoting activities of neovibsanin, 63, 64f, 65f, 72–74 Oxidation, 6–7, 10–11, 12, 16–18, 20, 25–27, 30, 34–35, 56, 66–67, 71–72, 88–89, 100, 131–132, 295–304 b-Oxidation, 85, 99–100 Oxidative phosphorylation, 167 Oxidative stress, 125, 126–127, 170, 250–251, 272–273, 399, 403, 404 Oxindole alkaloids, 386–389, 397, 400, 401–402 Oxocarbenium ion, 25 oxy-Cope rearrangement, 43–44, 58–60 Oxygen-based free radicals, 126–127 Ozonolysis, 25 P Paeoniflorin, 351, 366 Pancreas, 175, 176, 177t, 178, 246–249, 353f Paper chromatography (PC), 224–225, 470–471 Parathyroid hormone (PTH), 275 Parietin, 226–227, 240–245, 243t Parmelia sulcata, 233–234 Pathogenesis, 171, 175, 178–179, 180, 198, 271–272 Pathways, 53–56, 146, 200, 227, 352–354, 366–367, 368–369 calcium-dependent, 2–3 phenylpropanoid, 85–89, 146 polymalonate, 223–224, 227–229 shikimic acid, 223–224, 227, 230 Index Peganum harmala, 128, 138 Pemphigus vulgaris, 169, 172–173 Pentacyclic oxindole alkaloids (POAs), 397, 399–400 Percutaneous transluminal coronary angioplasty, 185 Peroxisome proliferator-activated receptor gamma (PPARg), 352–354, 355, 356–357 Pertusaria pertusa, 233–234 Pesticide, 438, 439, 440–449, 464–466, 467, 468–469, 472, 475 Pesticide residues, 439, 452–465, 467, 475–476 PGE2, 109 P-glycoprotein (p-gp), 200, 204, 209, 210–211 Phagocytosis, 399 Pharmacokinetic, 110, 138, 199–200, 202–203, 207–211, 295–304, 307–308 Pharyngitis, 201–202 Phase-transfer catalysts (PTCs), 409–412, 414–431 Phellopterin, 150t, 162 Phenylalanine ammonia lyase, 87f 2-Phenylethylamine, 124 Phosphatidylinositol system, 168 Photochemical reaction, 58–60 Photosensitized oxidation, 56 Photosensitizer, 167, 171 Photosensitizer compounds, 167 Phytomedicines, 164 Pimpinella, 150t, 161 Piperaceae families, 80, 110 Piper alkamides, 88, 106, 109–110 Piperidinyl (or piperidide), 80 Piperine, 79–80, 83f, 88f, 93, 106–110, 137 Piper species, 88, 106–107, 110 Plasma glucose concentrations, 360–361, 364 Pleiospermium, 80–82 Polyacetylenes, 213–214, 334, 335 Polyenals, 335, 337–339, 338t, 341–343 Polyketide synthases (PKSs), 227–229 Polymorphism, 127, 205–206 Polymorphonuclear neutrophils, 295–304 Polysaccharides, 209–210, 230–232, 360–361 Prangos, 161, 163 Precursor scan information-dependent acquisition-enhanced product ion (PRECIDA-EPI), 165–166 Pressurized capillary electrochromatography (pCEC), 163, 186 Primary secondary amine (PSA), 453t, 466 Primulaceae, 376 Proinflammatory cytokines, 169–170, 171 493 Index Proliferation, melanocyte, 169 Prostate carcinoma, 245–246, 249 Protease, 200, 201, 204–205, 275–276, 279, 430–431 Protease inhibitors (PTIs), 200, 204–205, 207, 209, 210–211 Protein biosynthesis, 168, 183 Protein kinases (PK), 93–94, 168, 352–354, 357–358, 359–360 A, 93–94 C, 168 Protein tyrosine phosphatase 1B (PTP1B), 253–254, 352–354 Protousnea poeppigii, 240 Pseudoaxial methyl group, 48 Pseudoequatorial C-6 unit, 48 Psoralea corylifolia, 147, 166, 168 Psoralen, 146–148, 161, 164, 165–166, 168–169, 170–171, 172–173, 175, 176–177, 179, 180–182 Psoralen synthase, 147–148 Psoriasis, 169, 171–173 Psychiatric illness, 198 Psychotropics, 204–205 PTCs, alkaloid-based, 409–410, 413 Pterostilbene, 351, 363–364 Pulmonarianin, 250, 252t Putative aminotransferase (pAMT), 87f PUVA, 168–169, 170–174, 180–181, 184, 186 PUVA therapy, 168–169, 171, 172, 181–182 Pyranocoumarins, 146–147 Pyridinium chlorochromate (PCC), 6–7, 37 Pyridinium 4-toluenesulfonate, 6s, 37 g-Pyrone, 2, 7–9, 12, 13–14, 16–18, 19, 20, 29–31, 35–37 g-Pyrone moiety, 12, 13–14, 29–31, 35–37 Pyrone ring, 2, 4–6, 13–14, 25, 35–37, 164 Pyron-ring substituted coumarins, 146–147 Q Quercetin, 209–210, 213, 364, 403 Quinazoline, 124, 127–128, 138 Quinine, 139, 382 R Raltegravir, 205–206, 208t, 213 Raman bands, 331, 332, 337, 338t, 339–341 Raman spectroscopy, 314, 327, 328–337, 336f Raman spectrum, 329–331, 335–337 Ranunculaceae, 376 Rapporteur member state (RMS), 445 Rash, 201–202 Raynaud’s phenomenon, 173 Reactive oxygen species, 172, 401, 403 Reagent Dragendorff’s, 163 Grignard, 20, 25–27, 30 Jones, 25–27 Marqui’s, 164 Organolithium, 66–67 Tollen’s, 164 Wagner’s, 164 Rearranged vibsane-type diterpenoids, 56–61 Reductive demesylation, 48–49 Regioselective epoxidation, 46–47 Relative standard deviation (RSD), 451, 453t Reproductive system, 274–275 Residue definition, 438–439, 449, 473–474 Response immune, 169–170, 172–173, 184, 276, 277–278, 341–343 Retinoids, 180–181 Retrosynthetic plan, 4–6, 5s, 7–9, 9s, 14–15, 16s, 19s, 21–22, 23s, 25, 26s, 29–30, 29s, 32–33, 33s, 34, 35s Reverse transcriptase, 200, 202–203 Rheumatoid arthritis, 2–3, 89, 92, 93–94, 109, 169, 175–176, 270, 282–283, 383t, 397, 404 Rheumatoid arthritis synovial fibroblasts (RASFs), 282–283 Rhizonaldehyde, 250 Rhizonyl alcohol, 250 Rhynchophylline, 382–384, 386, 392, 397, 399–400, 401 Ribosome-inactivating proteins (RIPs), 377–378 Rifampicin, 184 Rilpivirine, 203–204 Ritonavir, 204, 210–211, 213 Rodgersia pinnata, 161 Rotenone, 464–465, 466, 469 Royal jelly, 261–262, 270 Rubiaceae, 139, 382 Rutaceae, 80–82, 103, 110, 146, 149, 161, 211 Rutaceae family, 80–82 Ruta graveolens, 149, 161, 162 S Saccharomyces cerevisiae, 184 Salivary glands, 185 Salsolinol, 135–136 Sandai virus, 399 494 Saponins, 209–210, 357, 375–376 Saporin-based toxin (SA2E), 378 Saquinavir, 204, 210, 211 Sarcina lutea, 270–271 Sarcophyton, 318 SCoA, 88, 88f, 228s L-selectride, 15–16 Selegiline, 124, 127 Sensory neurons, 90–91, 92, 93–94, 104–105 Serositis, 173 Serotonergic neurons, 125 Serotonin syndrome, 124 Sesquicillin, 2, 3f, 4–7, 10–11, 12–14, 14s, 15s, 25, 35 ()-Sesquicillin [()-2], 4–6, 5s, 7, 8s Sesquicillin [()-2], 4–7 Sesquicillin A, 2 Sesquicillin analogues, 2 Sesquicillins B–E, 2 Sesquicillium candelabrum, 2–3 Sesquiterpene, 318, 335 Sézary syndrome, 169, 180, 181 Shibasaki’s catalysts, 411 Shikimate, 146 Silica gel, 163, 225, 470–471 SN2-type cyclization, 29–31, 34–35 Solanaceae, 80, 110, 111 Solanaceae family, 80 Solid organ transplant rejection, 147, 169 Solid-phase extraction (SPE), 162, 439, 452, 464–465, 469 Solid-phase microextraction (SPME), 452 Somnolence, 295–304 Speciophylline, 392, 399–400, 401–402 SPE sorbents, 452–464 Spilanthol, 100–103 Spinal cord injury (SCI), 278–279 Spinosyns, 472–473 Spirovibsanin, 58, 64–65 Spleen enlargement, 223–224 Stannylmethyl ether, 7–9, 11s, 14–16, 19, 20, 30, 35–37 Staphylococcus aureus, 235, 270–271 Stavudine, 202–203, 209 Stereoselective reduction, 6–7, 15–16, 34–35 Steroids, 170, 174–175, 176–177, 318, 327, 354–355 Sterols, 213–214, 266–267, 318, 327, 335, 403–404 Stevioside, 366–367 (–)-Subglutinol A, 25–28, 28s, 29–31, 32–33, 32s (–)-Subglutinol B, 32–33, 34–35 Index Subglutinols, 25–28, 29–31, 34–35 Subglutinols A, 25–28, 29–31, 34–35 Supercritical fluid chromatography (SFC), 166, 467–468, 468f Supercritical fluid extraction (SFE), 162, 166, 464–465, 469 Superoxide dismutase, 272–273 Synovial cells, 175–176 Synovial fluid, 175–176 Synthesis, 421s, 422 collagen, 281–282 complex natural product, 413 of levobupivacaine, 427 Synthetic cannabinoid products (SCPs), 291–292 Synthetic cannabinoids, 292–293, 294–304 Systemic lupus erythematosus, 169, 173 T Tachycardia, 295–304 Tangeretin, 209–210 3-(60 -TBDPS-30 -methylhex-20 -enoyl)-4phenyloxazolidin-2-one, 67–69 TBS group, 7, 10–11, 16–18, 66–67 T-cell lymphoma, 169, 180–181 Tenofovir, 202–203 Terpenes, 209–210, 229 Terpenoids, 41–42, 318, 334, 354–355, 386, 389, 465–466 tert-butyldimethylsilyl (TBS), 5s, 6–7 tert-butyldimethylsilyl chloride, 48–49 tert-butyl hydroperoxide, 21s Tetrachloromethane, 161–162 Tetrahydro-b-carbolines (THbCs), 128, 129, 131 Tetrahydroisoquinoline (TIQ), 124, 127–128, 134–136 Tetrahydropyran ring, 20, 21–24 Tetra-n-butylammonium fluoride (TBAF), 16–18, 17s, 20, 22–24, 48s, 66–67, 68s Tetra-n-propyl ammonium perruthenate (TPAP), 20, 23s, 34–35 Theaceae, 376 Thin-layer chromatograms, 163–164 Thymocyte proliferation (TP) assay, 4 Tin/lithium exchange, 11–12 Tipranavir, 204 TIQ alkaloids, 134–135, 136–137 T-lymphocytes, 295–304 Tobacco mosaic virus, 240–245 Toddalia asiatica, 184 Tomentosides, 389 495 Index Toothache, 80, 89, 92, 101, 102, 104–105, 111, 232–233 Toxoplasma gondii, 184 Toxoplasmosis, 184 Traditional Chinese medicine (TCM), 182–183, 384–385, 401 Trans-decalin aldehyde, 7–9 Trans-decalin skeleton, 2 Trans-decalone, 4–6, 15–16 Transforming growth factor-beta, 169–170 Trans-fused AB rings, 4 Transplantation, 173, 176–178, 198 Transplanted organ, 176 Treatment anorexia, 305–307 diabetes, 354–356 hypertension, 382, 384, 385, 399 immunosuppressive, 172–173 levodopa, 126 oxidative, 6–7 prostatic carcinoma, 182 psoriasis, 169, 171 Treatment of Alzheimer’s disease (Treatment of AD), 62 T regulatory cell response, 176 Trichloroethylene, 161–162, 474 Trichomonas vaginalis infection, 182–183 Tricyclic antidepressants, 125–126 Tricyclic dodecahydronaphtho[2,1-b]furan, 4 Tricyclic lactone, 66–67 Tricyclic 7-membered vibsanins, 51 Tricyclic pyrido[3,4-b]indole ring structure, 128 Triethysilyl (TES), 15–16, 20, 30 Trifluoromethanesulfonate, 6–7, 6s Triisopropylsilyl, 25, 26s 3,4,5-Trimethoxyphenyl, 80 4,50,8-Trimethoxypsoralen, 168–169 Trimethylsilyl, 7, 8s Triterpenes, 229, 230s, 240, 375, 402–404 Tropane, 124, 126–127, 139, 415 TRPA1 activation, 105–106, 276 TRPV1, 92–99, 104–106, 107–109, 276 TRPV1 receptor, 92–94, 98–99, 104–105, 107, 110–111 Tryptamines, 124, 127–128 Tumor, 61–62, 169–170, 182–183, 201–202, 246–249, 270, 283, 357–358, 367–368, 376, 399, 423–424 Tumor necrosis factor-alpha (TNF-a), 102–103, 169–170, 171, 277–278, 357–358, 367–368, 399 Two-dimensional thin-layer chromatography (2D TLC), 163 Type I ribosome-inactivating proteins (Type I RIPs), 378–379 Tyramine, 125, 131–132 U Ubiquitin-proteasome system, 126 UDP-glucuronosyl transferase (UGT), 212 Ultra-performance liquid chromatography (UPLC), 164, 453t, 469–470, 472–473, 476 Ultrasonic-aided extraction, 161–162 Ultraviolet A (UVA) radiation, 169, 172, 174, 175, 176–177, 183–185 Umbelliferone, 146f, 147–148 Uncaria guianensis, 382, 389, 397 Uncaria hirsuta, 382, 389, 398–399, 403 Uncaria quadrangularis, 382 Uncaria rhynchophylla, 382–384, 385, 386, 392–397 Uncaria sinensis, 382, 385, 400, 401 Uncaria spp., 382–389, 383t, 391f, 392, 397, 398, 400, 401–402 Uncarine, 386–389, 397, 399–400 Urease enzyme, 255 Usnea glabrata, 382, 397–398 Uvaria macrophylla, 382, 385, 400, 401 UVB radiation, 180–181 V Validation data, 450–451 Vanillylamine, 85–88, 90, 95 Vascular smooth muscle cells (VSMCs), 398 Vibsane, 41–42, 64–74, 75 Vibsane-type diterpenes, 53–56 Vibsane-type diterpenoids, 42–63, 64–74, 75 Vibsanin, 43–44, 45–49, 62–63, 72–74 A, 41–42, 49, 60–61 B, 41–42, 43–44, 45, 47f, 49, 53–56, 58–62 C, 43–44, 52, 53–56, 60, 61–62 D, 56 E, 42, 51, 52 F, 45–49, 47f, 64–65 7-membered ring, 42, 50–56, 54f, 58–60 11-Membered ring, 42, 49 Viburnum, 41–42, 50, 56, 75 Viburnum odoratissimum, 42, 45, 49, 50, 51, 56, 61–62 Viburnum opulus L, 41–42 Viburnum prunifolium L, 41–42 Viburnum species, 41–42, 50, 56, 75 Viburnum suspensum, 49, 50, 56, 58 496 Vinylogous methyl ester, 12 Viola sieboldi, 49, 56 Vitamins, 213–214, 261–262, 267, 293 Vitiligo, 147, 168, 169 Voltage-gated potassium channel Kv1.3, 2 W Wittig olefination, 10–11, 22–24, 71–72 Wittig reaction, 6–7 [2,3]-Wittig rearrangement, 7–9, 10–12, 11s, 14–15, 16–18, 19, 20, 30, 35–37 X Xanthones, 226–227 Xanthoparmelia scabrosa, 226–227 Index Xanthotoxin, 149, 161, 164 Xanthotoxol, 148, 149 X-ray crystallographic analysis, 44 X-ray diffraction analysis, 4 Y Yeast, 147–148, 184, 240, 271–272, 397 Z Zalcitabine, 202–203 Zanthoxylum, 80–82, 84f, 103, 104, 110 Zanthoxylum genus, 80–82, 103 Zanthoxylum species, 103 Zidovudine, 202–203 Zooxanthellae, 337, 341–343

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