© Copyright 2002 by the American Chemical Society and the American Society of Pharmacognosy Volume 65, Number 10 October 2002 Full Papers Antiplasmodial Activity of Alkaloids from Various Strychnos Species Michel Frédérich,*,†,‡ Marie-José Jacquier,§ Philippe Thépenier,§ Patrick De Mol,‡ Monique Tits,† Geneviève Philippe,† Clément Delaude,† Luc Angenot,† and Monique Zèches-Hanrot§ University of Liège, Natural and Synthetic Drugs Research Center, Laboratory of Pharmacognosy, Avenue de l’Hôpital 1, B36, B-4000 Liège, Belgium, University of Liège, Laboratory of Medical Microbiology, Liège, Belgium, Laboratoire de Pharmacognosie, UMR 6013 CNRS, Bâtiment 18, Moulin de la Housse, 51687 Reims Cedex 2, France Received February 28, 2002 The in vitro antiplasmodial activities of 69 alkaloids from various Strychnos species were evaluated against chloroquine-resistant and chloroquine-sensitive lines of Plasmodium falciparum. The compounds, comprising mainly indolomonoterpenoid alkaloids, exhibited a wide range of biological potencies in the antiplasmodial assays. The most active alkaloids were also tested for cytotoxicity against HCT-116 colon cancer cells to determine their antiplasmodial selectivity. As a result of these studies, structure-activity relationships for these alkaloids have begun to emerge. Alkaloids presenting four types of bisindole skeleton exhibited potent and selective activities against Plasmodium. They were sungucine-type (IC50 values ranging from 80 nM to 10 µM), longicaudatine-type (IC50 values ranging from 0.5 to 10 µM), matopensinetype (IC50 values ranging from 150 nM to 10 µM), and usambarine-type alkaloids. Within the last structural type, isostrychnopentamine (49) and ochrolifuanine A (46) were found to be active against chloroquinesensitive and -resistant strains (IC50 values of 100-150 and 100-500 nM, respectively), and dihydrousambarensine (51) exhibited a 30-fold higher activity against the chloroquine-resistant strain (IC50 ) 32 nM) than against the chloroquine-sensitive one. Malaria is the major parasitic infection in many tropical and subtropical regions, leading to more than one million deaths (principally among young African children) out of 400 million cases each year.1 More than half of the world’s population lives in areas where they remain at risk of malaria infection. During recent years, the situation has worsened in many ways, mainly due to malarial parasites becoming increasingly resistant to several antimalarial drugs.2 This resistance concerns numerous drugs, but is thought to be most serious with chloroquine, the cheapest and most widely used drug to treat malaria. Furthermore, the control of malaria is becoming more complicated by the parallel spread of resistance of the mosquito vector to * To whom correspondence should be addressed. Tel: + 32 4 366 43 38. Fax: + 32 4 366 43 32. E-mail: M.Frederich@ulg.ac.be. † Laboratory of Pharmacognosy, University of Liège. ‡ Laboratory of Medical Microbiology, University of Liège. § Laboratoire de Pharmacognosie, Reims. 10.1021/np020070e CCC: $22.00 currently available insecticides. Urgent efforts are therefore necessary to identify new classes of antimalarial drugs.3 The in vitro antiplasmodial, antiamebic, and cytotoxic activities of several indole alkaloids, particularly those isolated from various Strychnos species, have been previously investigated.4-9 To complement these studies it was considered to be of interest to test the antiplasmodial activity of other Strychnos alkaloids available in our laboratories. In total, 69 alkaloids with various structures have been tested against three lines of P. falciparum, thus allowing us to determine preliminary structure-activity relationships. Results and Discussion Among a total of 69 alkaloids examined, compounds 1-4 were of the monoterpenoid type, compounds 5-45 were monoindole alkaloids, and compounds 46-69 were bisindole alkaloids. Compounds exhibiting IC50 values higher than 10 µM, between 1 and 10 µM, and lower than 1µM © 2002 American Chemical Society and American Society of Pharmacognosy Published on Web 08/16/2002 1382 Journal of Natural Products, 2002, Vol. 65, No. 10 Frédérich et al. Figure 1. Structures of alkaloids 29-31 and 41-56. were respectively considered as weak, moderately active, and potent antiplasmodial agents. Compounds 1-40 were inactive or showed less than 30% growth inhibition at a concentration of 50 µM (Supporting Information). The results obtained for compounds 5-40 were largely confirmatory of previous studies where other monoindole alkaloids (akagerine, retuline, strychnine, icajine, vomicine, novacine derivatives, diaboline, holstiine) were shown to be devoid of any antiplasmodial activity.7-9 However, five monoindole alkaloids evaluated in the present study showed a weak activity, with antiplasmodial IC50 values of 10-30 µM: ngouniensine (41), epingouniensine (42), akagerine lactone (43), demethoxycarbonyl 3,14-dihydrogambirtanine (44), and 9-methoxy 16(R)-E-isositsirikine (45) (Table 1). Since the compounds possess highly varied structures, no structure-activity relationships could be deduced from these data. The most active of the monomeric compounds was 45, with an IC50 value of approximately 10 µM against all P. falciparum strains tested. This compound contains an additional methoxy substituent on the aromatic ring, compared to its inactive analogues 29-31. All the other active alkaloids were bisindole derivatives (Table 1). Twelve compounds (48, 53, 54, 57, 59, 63, 64, 65, 66, 67, 68, 69) exhibited moderate to weak activity, with antiplasmodial IC50 values of 2-20 µM in at least one of the Plasmodium lines. The remaining 12 compounds (46, 47, 49, 50, 51, 52, 55, 56, 58, 60, 61, 62) showed IC50 values of <2 µM against all Plasmodium lines tested. Of these, the two compounds isostrychnopentamine A (49) and ochrolifuanine A (46) demonstrated very potent activity of Alkaloids from Strychnos Species Journal of Natural Products, 2002, Vol. 65, No. 10 1383 Figure 2. Structures of alkaloids 57-69. <500 nM against all lines tested, and one compound, dihydrousambarensine (51), was selectively highly active (39 nM) against the chloroquine-resistant strain W2. Among these 24 bisindole compounds it was possible to distinguish four principal structural classes: usambarinetype or quasi-dimeric-type (i.e., made of two tryptamine units with a single iridoid unit) (46-51), matopensine-type (52-55), longicaudatine-type (56-58), and sungucine-type (59-62) alkaloids. Among alkaloids of the usambarine-type (46-51), most of the compounds evaluated exhibited potent antiplasmodial activity (IC50 below 1 µM). The most interesting compound was isostrychnopentamine (49), which possessed an IC50 value of about 100 nM against all plasmodial lines tested. The absence of the hydroxyl substituent at C-11 and of the pyrrolidine ring at C-12 in its analogue usambarine (50) led to a 20-fold reduction in activity. As previously reported,7 the 11-hydroxy derivative of usambarine was also 3 times more active than usambarine. Ochrolifuanine A (46) also exhibited potent activity against the three strains tested (100-500 nM). This compound was also of interest, as its total synthesis has been recently described.10 Ochrolifuanine E (47), exhibiting the H-3β and H-3’R configuration, was clearly less potent than ochrolifuanine A (46) (H-3’β, H-3R and an ethyl side chain). Furthermore, within this group, two compounds (50, 51), presenting a fully or partially aromatized lower portion of the molecule, were clearly more potent against the chloroquine-resistant strain, particularly dihydrousambarensine (51), which showed a 30-fold lower IC50 value for the chloroquine- 1384 Journal of Natural Products, 2002, Vol. 65, No. 10 Frédérich et al. Table 1. In Vitro Activities of Some Strychnos Alkaloids Against Three Lines of P. falciparum FCA 20/ Ghana (chloroquine-sensitive line) compound monoindole alkaloids ngouniensine (41) epingouniensine (42) akagerine lactone (43) demethoxycarbonyl 3,14dihydrogambirtannine (44) 9-methoxy 16(R) E-isositsirikine (45) usambarine-type bisindole alkaloids ochrolifuanine A (46) ochrolifuanine E (47) usambarine (48) isostrychnopentamine (49) usambarensine (50) dihydrousambarensine (51) matopensine-type bisindole alkaloids matopensine (52) matopensine-N-oxide (53) 18-hydroxymatopensine (54) 16-methoxyisomatopensine (55) longicaudatine-type bisindole alkaloids longicaudatine (56) longicaudatine F (57) tetradehydrolongicaudatine Y (58) sungucine-type bisindole alkaloids sungucine (59) isosungucine (60) 18-hydroxyisosungucine (61) strychnogucine B (62) various bisindole alkaloids strychnofuranine (63) janussine A (64) janussine B (65) S-panganensine (66) panganensine Y (67) panganensine X (68) 16,17-dehydroisostrychnobiline (69) reference compounds chloroquine (70) quinine (71) artemisinin (72) IC50 µM (SDa IC90 µM FCB1-R/Colombia (moderately chloroquine-resistant line) nb IC50 µM (SDa IC90 µM W2/Indochina (chloroquine-resistant line) nb IC50 µM (SDa IC90 µM nb 26.8 7.8 17.0 11.3 71.0 26.9 60.7 42.2 1 1 1 1 17.8 16.3 27.5 12.9 62.8 49.7 55.2 47.4 2 1 1 2 20.2 15.8 25.9 18.3 70.1 52.8 53.3 89.5 1 1 1 1 13.6 ( 5.9 37.8 3 12.5 36.3 2 9.9 34.6 1 3 1 1 2 6 5 0.266 ( 0.185 0.702 ( 0.431 ND 0.104 ( 0.036 ND ND 1.02 2.42 3 6 0.386 3 0.492 ( 0.145 1.90 2.36c 0.152c 0.594 ( 0.052c 0.032 ( 0.002c 4.11 24.8 11.8 3 2 4 0.243 8.66 0.121 9.52 3 3.64 33.4 2 2 0.118 ( 0.024 0.276 2.50c 0.120c 1.52 ( 0.031c 0.857 ( 0.061c 0.495 1.49 9.21 0.450 4.41 2.49 1.25 13.2 5.11 6.63 73.3 12.3 1 1 2 0.362 ( 0.035 4.67 2.43 ( 0.37 0.54 2.53 2 1.54 ( 0.21 0.986 7.70 1.236c 3.87 26.7 7.970 2 1 1 0.560 11.7 ND 7.82 ( 1.14d 1.32 ( 0.25d 0.847 ( 0.141d 26.3 7.06 4.35 3 3 4 ND ND 0.207 ( 0.126 1.47 3 10.1 d 0.265d 0.140d 3.79 2 0.529 ( 0.038 2.24 3 0.085d 33.5 28.3 33.1 12.7 43.7 26.1 7.13 2 2 2 2 2 2 2 0.617d 4.11 9.55 12.6 9.77 10.4 14.9 4.58 0.011 ( 0.005 0.269 ( 0.006 ND 15.0 30.9 34.9 17.7 68.2 34.8 18.7 0.071 1.91 2 2 1 1 1 1 6 3 10.8 15.9 17.0 5.27 13.5 12.9 2.12 0.032 ( 0.019 0.200 ( 0.033 0.005 0.084 2.74 0.013 3 4 2 1.38 6.76 7.29 0.628 4.83 4.68 3 1 2 2 3 4 7.74 86.7 2.26 1 1 2 0.122 ( 0.076 2.84 4 0.569 ( 0.228 9.58 0.958c 4.83 28.1 12.087 3 2 2 33.2 1.72 1.35 2 2 2 2.80 12.7 4.75 > 20 17.4 17.0 3.31 0.284 ( 0.017 0.413 ( 0.011 0.002 0.358 16.0 27.6 27.8 78.7 85.8 18.5 1.75 1.72 0.017 2 2 2 2 1 1 1 1 5 3 1 a Values are expressed as mean ( standard deviation (for n > 2). ND ) not determined. b n ) number of independent experiments. All tests were realized in duplicate. c Data from ref 7. d Data from ref 5. resistant line W2 (32 nM). Its IC90 value, however, was similar to that obtained with the chloroquine-sensitive line, FCA20. For alkaloids of the matopensine-type (52-55), the N-oxide derivative (53) was clearly less potent than the other compounds in this class. This may be attributed to the ionic nature of the N-oxide, since quaternary alkaloids have consistently been inactive in our assays (data not published). The three alkaloids 52, 54, and 55 were slightly more potent against the chloroquine-resistant strain, but no clear structure-activity relationships could be deduced for these three compounds. For alkaloids of the longicaudatine-type, only three compounds were available (56-58). The most interesting was longicaudatine itself (56), which exhibited an IC50 of 0.5-1 µM. Longicaudatine F (57), whose structure differs from that of 56 only by the opening of ring G in the strychnine part of the molecule, was less active. Compound 58, which possessed a β-carboline structure in the lower part of the dimer (as in 50), demonstrated enhanced activity against the chloroquine-resistant strain. It would be interesting to test additional analogues of longicaudatine. Among alkaloids of the sungucine-type, compounds 6062 possessed potent activities against the different lines of Plasmodium falciparum. Isosungucine (60), a compound possessing a C-16′/C-17′ double bond in the lower part of the dimer, was more potent than its isomer sungucine (59). The presence, in 18-hydroxyisosungucine (61), of an additional hydroxyl substituent on the 18-19 ethylidene side chain resulted in a small increase in activity. Finally, cyclization of ring G in the lower portion of the molecule Alkaloids from Strychnos Species Journal of Natural Products, 2002, Vol. 65, No. 10 1385 Table 2. Cytotoxic Activity Against the Human Colon Cancer Cell Line HCT-116 and Antiprotozoal Selectivity Indexa of the More Potent Antiplasmodial Alkaloids compound ochrolifuanine A (46) ochrolifuanine E (47) isostrychnopentamine (49) dihydrousambarensinec (51) matopensine (52) 18-hydroxymatopensine (54) 16-methoxyisomatopensine (55) longicaudatine (56) sungucinec (59) 18-hydroxyisosungucinec (61) strychnogucine Bc (62) chloroquine (70) IC50, µMb FCA SI FCB1 SI 16.1 5.70 7.47 12.0 136 20.7 62.3 14 60.7 32.8 8.12 3.0 71.8 49.1 ND 375 12.0 42.5 9.59 8.31 24.5 45.3 4.93 6.2 16.2 4.59 0.79 19.1 15 33.7 24.3 3063 W2 SI 33.1 17.5 49.5 351 15.9 200 8.81 7.96 ND 0.611 78.3 115 28.3 1053 176.4 118.6 a Selectivity index (SI) is defined as the ratio of 50% cytotoxicity over 50% antiplasmodial activity. b Number of independent experiments ) 2. Each experiment was realized in triplicate. c Data on KB cells from refs 5 and 7. and the presence of a hydroxyl substituent on the C-18 in the upper portion led to strychnogucine B (62), the most potent derivative in this group of alkaloids. In contrast, strychnogucine A, presenting a C-16′/C-17′ double bond, with a G cyclization on the upper part of the dimer, was more active than sungucine but markedly less active than 60-62 (IC50 near 3 µM for all strains).4 Thus, strychnogucine B (62) and 18-hydroxyisosungucine (61) were found to be the most active compounds in this series, with 12120 times higher activity than sungucine (59) against the three plasmodial lines. Furthermore, the activities were several-fold higher against the chloroquine-resistant lines W2 and FCB1-R than against the chloroquine-sensitive strain FCA of Plasmodium falciparum. To further assess the clinical potential of these antiplasmodial bisindolomonoterpene alkaloids, the most active compounds were evaluated for cytotoxicity with a human colon cancer cell line (HCT-116), to distinguish general cellular toxicity from specific antiplasmodial activity (Table 2). Many cytotoxic compounds also possess antiplasmodial properties under conditions of in vitro testing. Hence, the selectivity index (SI) of a compound was used, defined as IC50(in HCT-116 cells)/IC50(in P. falciparum). This selectivity index is an attempt to estimate the potential of tested compounds to inhibit the growth of the intracellular malaria parasite without cellular toxicity. The HCT-116 cell line was selected because it demonstrated no resistance to current anticancer drugs.11 In addition, this cell line was easily cultured and exhibited comparable sensitivity to noncancer cell lines (such as WI38 fibroblasts). Most compounds evaluated (Table 2) exhibited a 5- to 400-fold higher activity against Plasmodium than against the human cancer cells used, thus indicating some antiplasmodial selectivity (except 59). The most generally selective compounds were isostrychnopentamine (49) and ochrolifuanine A (46), which showed selectivity indices of 50-70 and 30-140 times, respectively. Dihydrousambarensine (51), 18-hydroxymatopensine (54), and 16-methoxyisomatopensine (55) yielded a favorable SI only for the W2 strain (SI ) 400, 350, and 200, respectively). Even though sungucine (59) exhibited no selectivity against Plasmodium, 18-hydroxysungucine (61) and strychnogucine B (62) exhibited 20-180-fold higher activity against the P. falciparum lines than against the human cancer cell line, thus indicating a satisfactorily high selectivity. The results of the present study confirmed the previously reported antiplasmodial activities of Strychnos bisindole alkaloids8 and more particularly of four alkaloid subtypes. All active compounds were tertiary dimers. A certain degree of basicity seems to be necessary for the antiplasmodial activity of this family of compounds. For example, usambarine (48) (possessing a methyl substituent at N4′) was less active than the ochrolifuanines (46 and 47) (N4′-H being more basic) or isostrychnopentamine (49) (which possesses a third basic nitrogen in the pyrrolidine ring). Also, N-oxide or quaternary derivatives were also less active than the respective tertiary compounds. These observations were consistent with the ability of basic compounds to accumulate to higher levels in the acidic food vacuole of the parasite, as has been hypothesized for chloroquine. Chloroquine is thought to be selectively accumulated (to at least a 1000-fold level) in the parasite food vacuole, where digestion of hemoglobin takes place. The weakly basic properties of chloroquine explain its accumulation in the food vacuole: at neutral pH, chloroquine has the ability to diffuse freely through membranes, but at the acidic pH of the food vacuole, the compound is protonated and is, therefore, sequestered.12,13 In the case of the sungucine-type alkaloids, it was apparent that the antiplasmodial selectivity was clearly associated with the presence of a 16′-17′ double bond or a 17′-18′ ether link. Isostrychnopentamine (49) exhibited potent and selective activities against all three lines of Plasmodium used in this study. The activity of this compound seemed to be directly linked to the presence of the basic methylpyrrolidine substituent. It would be useful to test semisynthetic and closely related compounds possessing these kinds of structures. Dihydrousambarensine (51), has been previously tested in vivo against Plasmodium berghei but was inactive at a dose of 30 mg/kg/day.8 Nevertheless, this compound was essentially active against chloroquine-resistant strains of P. falciparum and the P. berghei strain used in this study was chloroquine-sensitive. This could explain its inactivity in vivo, in addition to the differences between the biology of the two species. It is now desirable to examine if other lead alkaloids are able to inhibit Plasmodium growth in animal models and if they possess original modes of action (in particular, if they act or not on the heme polymerization process). Experimental Section Chemicals. All alkaloids tested (1-69) were isolated and purified in our laboratories from various Strychnos species as previously described. Compounds 1, 3, and 29 were isolated from Strychnos pungens Solered.;14,15 2, 26, and 31 from S. angolensis Gilg;16 4, 11, 20, 56, and 57 from S. longicaudata Gilg;17-19 5, 6, 12, 18, 19, 41, and 42 from S. ngouniensis Pellegr.;18,20,21 7, 8, 27, and 28 from S. potatorum L.;22,23 9, 10, 53, and 69 from S. kasengaensis De Wild;24 13 from S. xantha Leeuwenberg;25 14, 15, 16, 17, 21, 22, 43, 44, 64, and 65 from S. johnsonii Hutch. et M.B. Moss;26,27 23, 24, 25, 32, 33, 34, and 37 from S. henningsii Gilg;28 30, 52, 54, 55, and 63 from S. matopensis S. Moore;29,30 39 from S. staudtii Gilg;31 45 from S. lucens Bak.;32 46 and 47 from S. potatorum L.;22 48, 49, 50, 51, and 58 from S. usambarensis Gilg;33-35 59, 60, 61, and 62 from Strychnos icaja Baill.; 4,5 and 66, 67, and 68 from S. panganensis Gilg.36 The purity (>95%) of the compounds has been determined by TLC and spectroscopic (UV, IR, MS, NMR) comparison with authentic samples. Chloroquine diphosphate (Sigma, Bornem, Belgium), artemisinin (Sigma, Bornem, 1386 Journal of Natural Products, 2002, Vol. 65, No. 10 Belgium), and quinine base (Aldrich 14590-4) were used as antimalarial references. [3H]Hypoxanthine was from NEN Life Science Products (Zaventem, Belgium). Plasmodium falciparum Lines. Three P. falciparum lines were used in this study: the W-2 chloroquine-resistant line from Indochina, the FCB1-R line from Columbia, and the chloroquine-sensitive FCA 20 from Ghana. These lines were provided by Prof. P. Grellier (Laboratoire de Biologie Parasitaire et Chimiothérapie, Muséum d’Histoire Naturelle, Paris), Prof. J. Le Bras (Hôpital Bichat-Claude Bernard, Laboratoire de Parasitologie, Centre National de Référence de la Chimiosensibilité du Paludisme, Paris), and Prof M. Wéry (Tropical Medicine Institute, Antwerp, Belgium), respectively. The FCB1-R line was described as moderately chloroquineresistant, but IC50 values near 30 nM were obtained (the chloroquine resistance threshold was set at 100 nM). In Vitro Antiplasmodial Testing. Continuous in vitro cultures of asexual erythrocytic stages of the three P. falciparum strains were maintained following the procedure of Trager and Jensen37 and as described previously.5 Each test sample was applied in a series of eight 4-fold dilutions (final concentrations ranging from 20 to 0.0012 µg/mL) and was tested in duplicate. Parasite growth was estimated by the determination of [3H]hypoxanthine incorporation as described by Desjardins et al.38 and modified by Mirovsky et al.39 The Student t-test was used to test the significance of differences between results obtained for different samples. Statistical significance was set at p e 0.05. Evaluation of Cytotoxic Potential. The HCT-116 colon cancer cell line was cultured as described previously.40 Compounds were tested in 96-well microplates using the tetrazolium salt WST-1 (Boehringer) colorimetric assay based on the cleavage of the reagent by mitochondrial succinate-tetrazolium reductase in living cells. Altogether, 5000 cells were seeded per well in 200 µL of medium supplemented with adequate concentrations of tested drugs. Corresponding controls with analogous concentrations of DMSO were included in parallel. At least two experiments were performed per cell line. After a 72 h incubation, 20 µL of WST-1 was added to the well. After 30 min at 37 °C, plates were shaken and absorbance values were recorded as described in the commercial assay, against a background control as blank (medium plus WST-1 without cells). Relative absorbance values were expressed as percent of the respective controls (100% in ordinate). Means ( SEM were calculated. IC50 values were calculated from graphs. Acknowledgment. The authors wish to thank Prof. J. Boniver (Anatomie et Cytologie Pathologique, Université de Liège) for liquid scintillation measurements, V. Bours and M. P. Merville (CTCM, ULg) for HCT-116 cell lines and access to their laboratory, and M. Bentires (CTCM, ULg) for his technical assistance. This research was supported by the Belgian National Fund for Scientific Research (FNRS) (Grant No. 3453201 and fellowship for M.F.). Supporting Information Available: Table of alkaloids exhibiting less than 30% growth inhibition at a concentration of 50 µM against P. falciparum FCA-20 Ghana line. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Greenwood, B.; Mutabingwa, T. Nature 2002, 415, 670-672. (2) White, N. J. Br. Med. Bull. 1998, 54, 703-715. (3) Ridley, R. G. Science 1999, 285, 1502-1503. Frédérich et al. (4) Frederich, M.; De Pauw-Gillet, M.; Prosperi, C.; Tits, M.; Brandt, V.; Penelle, J.; Hayette, M.; De Mol, P.; Angenot, L. J. Nat. Prod. 2001, 64, 12-16. (5) Frederich, M.; De Pauw-Gillet, M. C.; Llabres, G.; Tits, M.; Hayette, M. P.; Brandt, V.; Penelle, J.; De Mol, P.; Angenot, L. Planta Med. 2000, 66, 262-269. (6) Frederich, M.; Tits, M.; Hayette, M. P.; Brandt, V.; Penelle, J.; De Mol, P.; Llabres, G.; Angenot, L. J. Nat. Prod. 1999, 62, 619-621. (7) Frederich, M.; Hayette, M. P.; Tits, M.; De Mol, P.; Angenot, L. Antimicrob. Agents Chemother. 1999, 43, 2328-2331. (8) Wright, C. W.; Bray, D. H.; O’Neill, M. J.; Warhurst, D. C.; Phillipson, J. D.; Quetin-Leclercq, J.; Angenot, L. Planta Med. 1991, 57, 337340. (9) Wright, C. W.; Allen, D.; Cai, Y.; Chen, Z. P.; Phillipson, J. D.; Kirby, G. C.; Warhurst, D. C.; Tits, M.; Angenot, L. Phytother. Res. 1994, 8, 149-152. (10) Malhotra, I.; Malhotra, N. J. Serb. Chem. Soc. 1999, 64, 155-162. (11) Brattain, M. G.; Fine, W. D.; Khaled, F. M.; Thompson, J., Brattain, D. E. Cancer Res. 1981, 41, 1751-1756. (12) Yayon, A.; Cabantchik, Z. I.; Ginsburg, H. EMBO J. 1984, 3, 26952700. (13) Ginsburg, H.; Geary, T. G. Biochem. Pharmacol. 1987, 36, 15671576. (14) Thépenier, P.; Jacquier, M. J.; Nuzillard, J. M.; Massiot, G.; Le Men Olivier, L.; Delaude, C. Bull. Soc. R. Sc. Liège 1996, 65, 379-382. (15) Thépenier, P.; Jacquier, M. J.; Henin, J.; Massiot, G.; Le Men-Olivier, L.; Delaude, C. Phytochemistry 1990, 29, 2384-2386. (16) Delaude, C.; Thépenier, P.; Jacquier, M. J.; Nuzillard, J. M.; Massiot, G.; Le Men-Olivier, L. Bull. Soc. R. Sci. Liège 1995, 64, 243-246. (17) Thépenier, P.; Jacquier, M. J.; Massiot, G.; Le Men Olivier, L.; Delaude, C. Phytochemistry 1990, 29, 686-687. (18) Massiot, G.; Thépenier, P.; Jacquier, M. J.; Lounkokobi, C.; Mirand, C.; Zeches, M.; Le Men-Olivier, L.; Delaude, C. Tetrahedron 1983, 39, 3645-3656. (19) Massiot, G.; Zeches, M.; Mirand, C.; Le Men Olivier, L.; Delaude, C.; Baser, K. H. C.; Bavovada, R.; Bisset, N. G.; Hylands, P. J.; Strombom, J.; Verpoorte, R. J. Org. Chem. 1983, 48, 1869-1872. (20) Massiot, G.; Jacquier, M. J.; Thépenier, P.; Levy, J.; Le Men Olivier, L.; Delaude, C.; Guilhem, J.; Pascard, C. J. Chem. Soc., Chem. Commun. 1983, 1018-1020. (21) Massiot, G.; Zeches, M.; Thépenier, P.; Jacquier, M. J.; Le Men Olivier, L.; Delaude, C. J. Chem. Soc., Chem. Commun. 1982, 768-769. (22) Massiot, G.; Thépenier, P.; Jacquier, M. J.; Le Men Olivier, L.; Delaude, C. Phytochemistry 1992, 31, 2873-2876. (23) Massiot, G.; Thépenier, P.; Jacquier, M. J.; Le Men Olivier, L.; Delaude, C. Heterocycles 1989, 29, 1435-1438. (24) Thépenier, P.; Jacquier, M. J.; Massiot, G.; Le Men Olivier, L.; Delaude, C. Phytochemistry 1984, 23, 2659-2663. (25) Thépenier, P.; Jacquier, M. J.; Nuzillard, J. M.; Massiot, G.; Le MenOlivier, L.; Delaude, C. Bull. Soc. R. Sci. Liège 1996, 65, 383-386. (26) Massiot, G.; Thépenier, P.; Jacquier, M. J.; Delaude, C.; Le Men Olivier, L.; Verpoorte, R. Tetrahedron Lett. 1985, 26, 2441-2444. (27) Massiot, G.; Thépenier, P.; Jacquier, M. J.; Le Men Olivier, L.; Verpoorte, R.; Delaude, C. Phytochemistry 1987, 26, 2839-2846. (28) Massiot, G.; Thépenier, P.; Jacquier, M. J.; Henin, J.; Le Men-Olivier, L.; Delaude, C. Phytochemistry 1991, 30, 3449-3456. (29) Massiot, G.; Massoussa, B.; Jacquier, M. J.; Thépénier, P.; Le MenOlivier, L.; Delaude, C.; Verpoorte, R. Phytochemistry 1988, 27, 32933304. (30) Massiot, G.; Massoussa, B.; Thépenier, P.; Jacquier, M. J.; Le MenOlivier, L. Heterocycles 1983, 20, 2339-2342. (31) Massiot, G.; Thépenier, P.; Jacquier, M. J.; Le Men Olivier, L.; Delaude, C. Phytochemistry 1988, 27, 657-659. (32) Delaude, C.; Thépenier, P.; Jacquier, M. J.; Massiot, G.; Le MenOlivier, L. Bull. Soc. R. Sci. Liège 1992, 61, 429-440. (33) Angenot, L.; Coune, C.; Tits, M. J. Pharm. Belg. 1978, 33, 11-23. (34) Angenot, L.; Bisset, N. G. J. Pharm. Belg. 1971, 26, 585-588. (35) Frederich, M.; Quetin-Leclercq, J.; Gadi-Biala, R.; Brandt, V.; Penelle, J.; Tits, M.; Angenot, L. Phytochemistry 1998, 48, 1263-1266. (36) Nuzillard, J. M.; Thépenier, P.; Jacquier, M. J.; Massiot, G.; Le Men Olivier, L.; Delaude, C. Phytochemistry 1996, 43, 897-902. (37) Trager, W.; Jensen, J. B. Science 1976, 193, 673-675. (38) Desjardins, R. E.; Canfield, C. J.; Haynes, J. D.; Chulay, J. D. Antimicrob. Agents Chemother. 1979, 16, 710-718. (39) Mirovsky, P.; Gay, F.; Bustos, D.; Mazier, D.; Gentilini, M. Trans. R. Soc. Trop. Med. Hyg. 1990, 84, 511-515. (40) Hellin, A. C.; Calmant, P.; Gielen, J.; Bours, V.; Merville, M. P. Oncogene 1998, 16, 1187-1195. NP020070E