Antimalarial Agents from Plants. III. Trichothecenes from Ficus fistulosa and Rhaphidophora decursiva Original Paper
Abstract Bioassay-directed fractionation of an extract prepared from the dried leaves and stem barks of Ficus fistulosa Reinw. ex Blume (Moraceae) led to the isolation of verrucarin L acetate (1), together with 3a-hydroxyisohop-22(29)-en-24-oic acid, 3b-gluco-sitosterol, 3,4-dihydro-6,7-dimethoxyisocarbostyril, 3,4,5trimethoxybenzyl alcohol, a-methyl-3,4,5-trimethoxybenzyl alcohol, indole-3-carboxaldehyde, palmanine, and aurantiamide acetate. Roridin E (2) was identified in a subfraction
Introduction
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Two plants with antimalarial activity, Ficus fistulosa Reinw. ex Blume (Moraceae) and Rhaphidophora decursiva Schott (Araceae), were collected in the Cuc Phuong National Park (Nho Quan District, Ninh Binh Province, Vietnam) as part of our International Cooperative Biodiversity Group (ICBG) project [1], [2]. Our initial study of F. fistulosa led to the identification of two new and five known triterpenes, none of which demonstrated antimalarial activity [3]. A continued investigation of the active fraction of F. fistulosa unexpectedly yielded a trichothecene, verrucarin L acetate, as the active antimalarial compound. Our previous chemical and biological investigation of R. decursiva led to the isolation of several antimalarial compounds having IC50 values in the range of 0.35 ± 6.5 mg/mL with the W2 (chloroquineresistant) and D6 (chloroquine-sensitive) clones of Plasmodium
Hong-Jie Zhang1 Pamela A. Tamez1 Zeynep Aydogmus1 Ghee Teng Tan1 Yoko Saikawa2 Kimiko Hashimoto2 Masaya Nakata2 Nguyen Van Hung3 Le Thi Xuan3 Nguyen Manh Cuong4 D. Doel Soejarto1 John M. Pezzuto1 Harry. H. S. Fong 1
from the dried leaves and stems of Rhaphidophora decursiva Schott (Araceae). Verrucarin L acetate and roridin E were characterized as macrocyclic trichothecene sesquiterpenoids and found to inhibit the growth of Plasmodium falciparum with IC50 values below 1 ng/ml. Key words Ficus fistulosa ´ Moraceae ´ Rhaphidophora decursiva ´ Araceae ´ antimalarial activity ´ bioassay-directed fractionation ´ trichothecene sesquiterpenoids ´ verrucarin L acetate ´ roridin E
falciparum [4], [5]. However, these levels of activity did not correlate proportionally with the activity of a minor fraction (F87), which inhibited the growth of D6 by 99.4 % at a concentration of 5 ng/mL. A comparison of the spectral data of verrucarin L acetate isolated from F. fistulosa led to the identification of another trichothecene, roridin E, as the primary active antimalarial compound in fraction F87 of Raphidophora decursiva. The current paper describes the isolation, identification and biological evaluation of these active trichothecenes.
Materials and Methods Plant material The initial collection of leaf + stem bark samples of Ficus fistulosa Reinw. ex Blume (Moraceae) and leaf + stem samples of
Affiliation 1 Program for Collaborative Research in Pharmaceutical Sciences, m/c 877, Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, the University of Illinois at Chicago, Chicago, Illinois, USA 2 Faculty of Science and Technology, Department of Applied Chemistry, Keio University, Kohoku-ku, Yokohama, Japan 3 Institute of Chemistry, National Center for Science and Technology, Nghia Do, Tu Liem, Hanoi, Vietnam 4 Cuc Phuong National Park, Nho Quan District, Ninh Binh Province, Vietnam Correspondence Professor Harry H. S. Fong ´ Program for Collaborative Research in the Pharmaceutical Sciences, M/C 877 ´ College of Pharmacy ´ University of Illinois at Chicago ´ 833 S. Wood St. ´ Chicago, IL 60612 ´ USA ´ E-mail:
[email protected] ´ Fax: +1-312-413-5894 Received April 25, 2002 ´ Accepted July 28, 2002 Bibliography Planta Med 2002; 68: 1088±1091 ´ Georg Thieme Verlag Stuttgart ´ New York ´ ISSN 0032-0943
Rhaphidophora decursiva (Araceae) was made at the Cuc Phuong National Park (CPNP), with voucher herbarium specimens represented by living collections in the park. The leaves + stem bark sample of Ficus fistolusa (SVA5015, 11.8 kg) was collected in the park for isolation work (Voucher specimen KO1029A), and the leaves + stems sample of R. decursiva (SVA5005, 5.03 kg) was made outside of the CPNP, in Hatinh, Central Vietnam, on December 3, 1999 (Voucher specimen Soejarto et 11220). Both voucher specimens are on deposit at the herbaria of CPNP, Institute of Ecology and Biological Resources (National Center for Science and Technology, Hanoi), and the Field Museum of Natural History (Chicago, IL, USA).
Antimalarial assay and cytotoxicity assays Antimalarial assays conducted with cultured Plasmodium falciparum clones W2 and D6, and cytotoxicity assays conducted wth cultured KB cells, have been described previously [4]. Extraction and isolation The dried, milled leaf + stem bark samples of F. fistulosa (11.8 kg) was extracted with MeOH/H2O (9 : 1), concentrated, defatted with petroleum ether, and partitioned with CHCl3. The CHCl3-soluble fraction (65 g) was chromatographed over a Si gel column (1.0 kg), which was developed by gradient elution with CHCl3 and increasing concentrations of MeOH [CHCl3-MeOH/100 : 0 (eluates F1 and F2, each 2.5 l), 99 : 1 (eluate F3, 3.0 l), 97: 3 (eluate F4, 3.0 l), 95 : 5 (eluate F5, 3.0 l), 90 : 10 (eluate F6, 3.0 l), 85 : 15 (eluate F7, 3.0 l), 80 : 20 (eluate F8, 3.0 l), 0 : 100 (eluate F9, 5.0 l), respectively] to afford nine fractions. 3a-Hydroxyisohop-22(29)-en-24-oic acid ([a]D20: ±32.18, c 0.39, MeOH, 7.0 mg) [6] and 3b-gluco-sitosterol ([a]D20: ±47.68, c 1.32, pyridine, 25.3 mg) [7] were obtained from fractions F-5 and F-8, respectively, by crystallization. Bioassay localized the antimalarial activity in fractions F-1, F-2, and F-3. Fractions F-1 to F-3 were pooled (9.5 g) and subjected to flash column chromatography on Si gel (400 g, gradient elution with petroleum ether and EtOAc) to yield 24 fractions [PE-EtOAc/100 : 0 (eluate F10, 2.0 l), PE-EtOAc/95 : 5 (eluates F11 and F12, each 0.5 l), PE-EtOAc/90 : 10 (eluates F13, F14, F15, each 0.5 l), PE-EtOAc/85 : 15 (eluates F16, F17 and F18, each 0.5 l), PE-EtOAc/80 : 20 (eluates F19, F20 and F21, each 0.5 l), PE-EtOAc/70 : 30 (eluates F22 and F23, each 0.5 l), PE-EtOAc/ 0 : 100 (eluate F24, 3.0 l), respectively]. The active fractions F-19, F-20 and F-21 were subsequently combined (0.72 g) and subjected to a C-18 reversed phase (RP-18, 30 g) column, eluting with MeOH-H2O/7: 3 (0.5 l) to yield an active fraction F-25 (0.42 g).
Bioassay-directed extraction and fractionation leading to a very active fraction F-87 (0.49 mg ) containing rhaphidecurperoxin and another minor compound from R. decursiva has been fully described in our previous paper [4]. Fraction F-87 showed inhibition of parasite growth (P. falciparum chloroquine-sensitive clone D6) by 99.4 % at a concentration of 5 ng/mL.
Original Paper
General experimental procedures NMR spectra were recorded on a Bruker DRX-500 MHz spectrometer. All NMR experiments were obtained by using standard pulse sequences supplied by the vendor. Column chromatography was carried out on Si gel (200 ± 400 mesh, Natland International Corporation). Reversed-phase flash chromatography was accomplished with RP-18 Si gel (40 ± 63 mm, EM Science), and reversed-phase HPLC was carried out on a Waters 600E Delivery System pump, equipped with a Waters 996 photodiode detector, and a GROM-SIL ODS 4-HE column (120 , 5 mm, 300 20 mm). Thin-layer chromatography was performed on Whatman glassbacked plates coated with 0.25 mm layers of Si gel 60. Mass spectra were recorded on a Micromass QTOF-2 spectrometer or a Finnigan LCQ.
Repeated chromatography of F-25 on a C-18 reversed phase (RP18, 107 g) resulted in 4 fractions [MeOH-H2O/45 : 55 (eluate F30, 0.5 l), MeOH-H2O/7: 3 (eluate F31 and F32, each 0.5 l), MeOHH2O/1 : 0 (eluate F33, 2.0 l), respectively]. The active fraction F30 (114 mg) was subjected to a series of semi-preparative HPLC separation on a GROM-SIL ODS 4-HE column (solvent system: either MeCN/H2O or MeCN/H2O) to afford 3,4-dihydro-6,7-dimethoxyisocarbostyril (0.67 mg), 3,4,5-trimethoxybenzyl alcohol (0.55 mg), a-methyl-3,4,5-trimethoxybenzyl alcohol ([a]D20: 0, c 0.06, MeOH, 0.99 mg), indole-3-carboxaldehyde (1.29 mg), and palmanine ([a]D20: + 24.2, c 0.02, MeOH, 0.79 mg). Work-up of active fraction F-31 (177 mg) in a similar manner yielded aurantiamide acetate ([a]D20: ±28.8, c 0.41, CHCl3, 6.73 mg) and verrucarin L acetate (1, [a]D20: ±19.6, c 0.03, CHCl3, 0.36 mg).
Results and Discussion The dried leaves + stem bark sample of F. fistulosa (11.8 kg) was milled and extracted with MeOH. Subsequent partitioning with petroleum ether and CHCl3 afforded an active antimalarial CHCl3 extract (65 g). Bioassay-directed fractionation of the CHCl3 extract by repeated flash column chromatography on silica gel and RP-18, followed by preparative HPLC on GROM-SIL ODS 4-HE column, afforded a trichothecene sesquiterpene, verrucarin L acetate (1) [8]; as well as two benzenoid derivatives [3], [4], 5trimethoxybenzyl alcohol [9] and a-methyl-3,4,5-trimethoxybenzyl alcohol [10]; an isoquinoline alkaloid, 3,4-dihydro-6,7-dimethoxyisocarbostyril [11]; an isoindolobenzazepine alkaloid, palmanine [12]; an indole alkaloid, indole-3-carboxaldehyde [13]; and a dipeptide, aurantiamide acetate [14]. Compound 1, C29H34O10, ESI m/z 565 [M+Na]+, was shown to generate characteristic trichothecene sesquiterpenoid 1H-NMR signals. Comparison of the NMR data of 1 to those published by Jarvis et al. [8] permitted identification of the isolate as the fungal macrocyclic trichothecene, verrucariin L acetate. This isolate was found to be active in the anti-malarial assay, with IC50 values of 0.6 and 0.7 ng/mL against the D6 and W2 clones of Plasmodium falciparum, respectively. Coincidentally, we noted that the highly active fraction F-87 of R. decursiva was composed of only two compounds in an approximate ratio of 4 : 1, according to the 1H-NMR spectra of the mixture. The major compound in F-87 was identified as rhaphidecurperoxin (IC50 values of 540 and 420 ng/ml with the D6 and W2 clones of P. falciparum, respectively) [4], with the minor constituent having 1 H-NMR spectral signals reminiscent of a macrocyclic trichothecene. Discovery of the presence of verrucarin L acetate (1) from F. fistulosa prompted us to inspect the 1H-NMR data of the R. decursiva F87 mixture more thoroughly to determine if the minor constituent was another fungal trichothecene. The 1H-NMR chemical shifts of this minor compound in F-87 were found to be Zhang H-J et al. Antimalarial Agents from ¼ Planta Med 2002; 68: 1088 ± 1091
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Original Paper 1090
very similar to those of 1. The characteristic 1H-NMR signals of trichothecene sesquiterpenes were observed at dH 6.19 (1H, dd, J = 8.04, 3.83 Hz), 5.45 (1H, brd, J = 4.80 Hz), 4.29 (1H, ABd, J = 12.53 Hz), 3.92 (1H, ABd, J = 12.36 Hz), 3.83 (1H, d, J = 4.43Hz), 3.89 (1H, brd, J = 5.82 Hz), 3.12 (1H, ABd, J = 4.06 Hz), 2.80 (1H, ABd, J = 4.11 Hz), 1.69 (3H, s), and 0.77 (3H, s), and a macrocyclic lactone ring was also discerned with 1H-NMR signals at dH 7.50 (1H, dd, J = 15.66, 11.76 Hz), 6.55 (1H, t, J = 11.49 Hz), 5.94 (1H, brs), 5.88 (1H, dd, J = 15.85, 3.27 Hz), 5.73 (1H, d, J = 11.15 Hz), 3.68 (1H, m), 3.63 (1H, m), 3.50 ± 3.62 (2H, m), 2.51 (2H, m), 2.24 (3H, s), and 1.18 (3H, d, J = 6.23 Hz). The minor compound in F-87 was therefore determined to be an analogue of compound 1. A search of the literature revealed a recent publication describing the isolation and structural elucidation of a series of macrocyclic trichothecenes from the poisonous mushroom Podostroma cornu-damae [15]. A comparison of the 1H-NMR data of the F-87 minor component to those published [15] for roridin E (2) showed them to be identical. Thus, the antimalarial activity of R. decursiva may be due to the presence of roridin E. To further confirm this suggestion, samples of pure roridin E (2), along with structurally related satratoxin H derivatives (3 ± 5)
Table 1
obtained from Podostroma cornu-damae [15] were evaluated for antimalarial activities. As a result, these trichothecenes, along with verrucarin L acetate, were found to demonstrate significant antimalarial potency (Table 1). This activity level strongly supports our suggestion that roridin E (2) is the primary compound responsible for the antimalarial activity exhibited by R. decursiva, and that verrucarin L acetate (1) is the constituent responsible for the biological effect of F. fistulosa. This conclusion is not without precedent as the antimalarial activities of a series of trichothecenes isolated from the fungus Myrothecium verrucaria have been reported [16]. Trichothecenes are mycotoxins found in fungal organisms [17], but some have been reported to exist in higher plants such as Baccharis cordifolia [18]. The presence of 1 and 2 in the unrelated plant species F. fistulosa and R. decursiva indicates that they are either metabolites of epiphytic/endophytic fungi or that the two plant materials, collected in the same general area of Vietnam, may have been contaminated with fungal organisms during field collection. However, the fact that the antimalarial activities were observed in multiple recollections would suggest contamination is not likely.
Bioactivities of extracts of F. fistulosa and R. decursiva and compounds 1 ± 5 KB
W2
IC50 (ng/mL)
SIa
IC50 (ng/mL)
SIa
MeOH ext. of R. decursiva
> 20 000
2 106 89
>6
6 840 43
>3
CHCl3 ext. of F. fistulosa
> 20 000
7 330 2 480
>4
3 760 690
>5
0.6 0.04
158
0.7 0.05
135
Name of Compounds
1
Verrucarin L acetate
2
Roridin E
0.21
0.2 0.08
1
0.6 0.11
0.4
3
Satratoxin H
2.9
0.8 0.04
4
0.8 0.02
4
4
Satratoxin H 13¢-acetate
1.9
1.3 0.03
2
1.9 0.27
1
5
Satratoxin H 12¢,13¢-diacetate
2.1
3.7 0.47
0.6
5.4 0.80
0.4
F87
a
D6
ED50 (ng/mL)
Samples
94.5
31
8.0
4
11.1
3
chloroquine
17 400
3.9
4 462
49.3
353
artemisinin
> 20 000
3.2
> 6 666
3.1
quinine
> 20 000
10.6
> 1 886
73.8
SI = Selectivity Index = ED50 KB/IC50 P. falciparum.
Zhang H-J et al. Antimalarial Agents from ¼ Planta Med 2002; 68: 1088 ± 1091
> 6 450 > 270
Trichothecenes are known to mediate a range of biological effects, most notably, inhibition of protein synthesis [19]. These compounds bind to eukaryotic ribosomes and block either the initiation step of translation or the elongation-termination step, depending on the A/B ring substituents [20]. In addition to the well-documented inhibitory effect in human beings, inhibition of translation in yeast and protozoa has been reported [21]. Because 1±5 inhibited the growth of P. falciparum, a eukaryotic protozoan, we postulate that growth arrest results through inhibition of plasmodial protein synthesis.
Acknowledgements This work was supported by a grant administered by the Fogarty International Center, NIH (U01-TW01015-01), as part of an International Cooperative Biodiversity Group (ICBG) (2). The authors are grateful to the staff of the Cuc Phuong National Park for assistance with the collections of F. fistulosa and R. decursiva samples, and to the Research Resources Center, University of Illinois at Chicago for the acquisition of MS data with verrucarin L acetate and for access to the Bruker DRX 500 MHz instrument used for this study. P.A.T. is the recipient of a pre-doctoral fellowship by the American Foundation for Pharmaceutical Education.
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Zhang HJ, Tamez PA, Hoang VD, Tan GT, Hung NV, Xuan LT et al. Antimalarial compounds from Rhaphidophora decursiva. Journal of Natural Products 2001; 64: 772 ± 7 5 Zhang HJ, Qiu SX, Tamez PA, Tan GT, Aydogmus Z, Hung NV et al. Antimalarial agents from plants. II. Decursivine, a new antimalarial indole alkaloid from Rhaphidophora decursiva. Pharmaceutical Biology, 2002; 40: in press 6 Kitajima J, Kimizuka K, Arai M, Tanaka Y. Constituents of Ficus pumila leaves. Chemical Pharmaceutical Bulletin 1998; 46: 1647 ± 9 7 Faizi S, Ali M, Saleem RI, Bibi S. Complete 1H- and 13C-NMR assignments of stigma-5-en-3-O-b-glucoside and its acetyl derivative. Magnetic Resonance Chemistry 2001; 39: 399 ± 405 8 Jarvis BB, Midiwo JO, DeSilva T, Mazzola EP. Verrucarin L, a new macrocyclic trichothecene. Journal of Antibiotics 1981; 34: 120 ± 1 9 Patra A, Mukhopadhyay PK. Carbon-13 NMR spectra of some benzene derivatives. Journal of Indian Chemical Society 1983; 60: 265 ± 8 10 Koul S, Koul JL, Taneja SC, Dhar KL, Jamwal DS, Singh K, et al. Structureactivity relationship of piperine and its synthetic analogues for their inhibitory potentials of rat hepatic microsomal constitutive and inducible cytochrome P450 activities. Biorganic and Medicinal Chemistry 2000; 8: 251 ± 68 11 Kametani T, Ohsawa T, Ihara M, Fukumoto K. Studies on the syntheses of heterocyclic compounds. DCCLV. Iminoketene cycloaddition. (4). Alternative syntheses of 5,6,7,8-tetrahydro-2,3-dimethoxy-8-oxoisoquinolo[1,2-b]quinazoline and rutecarpine. Chemical Pharmaceutical Bulletin 1978; 26: 922 ± 6 12 Valencia E, Weiss I, Firdous S, Freyer AJ, Shamma M. The isoindolobenzazepine alkaloids. Tetrahedron 1984; 40: 3957 ± 62 13 Sammes MP, Katritzky AR, Law KW. 13C-NMR chemical shift assignments for some 1H-pyrrole-2-carboxylic acid derivatives. Magnetic Resonance Chemistry 1986; 24: 827 ± 33 14 Wahidulla S, D'Souza L, Kamat SY. Dipeptides from the red alga Acanthophora spicifera. Phytochemistry 1991; 30: 3323 ± 5 15 Saikawa Y, Okamoto H, Inui T, Makabe M, Okuno T, Suda T et al. Toxic principles of a poisonous mushroom Podostroma cornu-damae. Tetrahedron 2001; 57: 8277 ± 81 16 Isaka M, Punya J, Lertwerawat Y, Tanticharoen M, Thebtaranonth Y. Antimalarial activity of macrocyclic trichothecenes isolated from the fungus Myrothecium verrucaria. Journal of Natural Products 1999; 62: 329 ± 31 17 Tamm C, Breitenstein W. The biosynthesis of trichothecene mycotoxins: Study of Secondary Metabolism. Editor(s): Steyn PS. Academic Press, New York: 1980: 69 ± 104 18 Jarvis BB, Midiwo JO, Bean GA, Aboul-Nasr MB, Barros CS. The mystery of trichothecene antibiotics in Baccharis species. Journal of Natural Products 1988; 51: 736 ± 44 19 Ueno Y, Hosoya M, Morita Y, Ueno I, Takashi T. Inhibition of the protein synthesis in rabbit reticulocyte by nivalenol, a toxic principle isolated from Fusarium nivale-growing rice. Journal of Biochemistry 1968; 64: 479 ± 85 20 Cundliffe E, Davies JE. Inhibition of initiation, elongation, and termination of eukaryotic protein synthesis by trichothecene fungal toxins. Antimicrobial Agents and Chemotherapy 1977; 11: 491 ± 9 21 Hernandez F, Cannon M. Inhibition of protein synthesis in Saccharomyces cerevisiae by the 12,13-epoxytrichothecenes trichodermol, diacetoxyscirpenol and verrucarin A: Reversibility of the effects. Journal of Antibiotics 1982; 35: 875 ± 81
Zhang H-J et al. Antimalarial Agents from ¼ Planta Med 2002; 68: 1088 ± 1091
Original Paper
Given the structural similarity of compounds 1±5, preliminary structure-activity relationships can be considered. For instance, verrucarin L acetate (1), the only trichothecene of the group having an acetyl moiety at C-8, showed much higher selectivity indices (158 and 135) than compounds 2 ± 4 (Table 1). Increasing the number of -OAc groups on the macrocyclic ring decreased activity by 2±9-fold, as observed with 4 and 5. Thus, it may be surmised that adding substituents to rings A and B could increase selectivity, while adding substituents to the macrocyclic ring could decrease antimalarial potency. Additional chemical and biological studies of structurally related or chemically modified trichothecenes might lead to antimalarial compounds with greater therapeutically desirable selectivity indices.
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