Plonta Med. 570991)

A Novel Leishmanicidal Labdane from Polyalthia macro poda Pascal Riclzomnze1' , Marie-Chantal Godet . Fran

Foussard2, LoIc Toupef3, Thierry Sévenet4,

arid Jean Bruneton' 2

C.E.P.M., Facultd de Pharmacie, l6Bd Daviers, F-49100 Angers, France l.aboratoire de Biochimie et de Biologic Cellulaire, Facultd de Pharmacie, 16 Bd Daviers, F-491 00 Angers, France C.R.M.P.O, Campus de Beaolieu, F-35045 Rennes Cédex, France 1.C.S.N., C.N.R.S., F-91190 tiif sur Yvette, France Address for correspondence

Abstract

Plant material __________________________

A new diterpene, (4S,9R,1OR) methyl 18-

carboxy-labda-8, 1 3(E)-diene-1 5-oate has been obtained from the stem barks ofPolyalthia macropoda (Annonaceae). This labdanic derivative was identified on the

basis of spectroscopic data and is biologically active against the promastigote Leishmania donovani donovani.

The stems of Polyalfhia macropoda were collected in October 1984 at Selangor, Genting Simpah, Malaysia. A voucher specimen (lot Sdvenet-Deverre 258) has been deposited at the herbarium of the laboratoire de Phandrogamie, Museum National d'Histoire Naturelle, Paris.

Organisms and media The strain of Leishmania donovani donovoni (reL: MIIOM/IN/80/LEM 138) was cultivated at 22°C in 199 Earle

Key words

Polyalthia macropoda, Annonaceae, diterpene, labdane, leishmanicidal activity.

medium supplemented with 2% Ultroser G® and 0.6mg % Hermine. Parasites were passaged every 5 days (stationary phase) at 2 to 3.10 organisms mU1. Cultures were daily examined by an inverted microscope and compared with an untreated control test, then parasites were brought to a standstill with 2% HCHO and counted using a Malassez haematocymeter.

Biological activity of 1

Introduction Some

-

_____________

representatives of the tropical genus

Polyalthia (Annonaceae) are locally used as medicinal

Solvent toxicity test: The toxicity of DMSO was checked by adding different amounts of solvent (0.1 to 1.5%) to suspensions of promastigotes in the conditions described above. After 5 days, the value of the non-toxic dilution was found to be 0.6% and a growth inhibition (up to 15%) was observed between 0.6 and 1 %, hence we never used higher concentrations of DMSO.

agents and recent notes described the isolation of clerodan diterpenes from Polyalthia viridis (1, 2). As a part of our investigation of the phytochemical constituents of a MalayDetermination of' the leishmaniacidal activity: sian species (3) and in the wake of a large survey of the flora of this peninsula (4), we now wish to report our preliminary The drug sensitivity and the percentage of control growth vs. drug concentration were determined after five days of infection. They study of the neutral extract obtained from the stem barks of were compared with the inhibitory effect of pentamidine Polyalthia macropoda. On the basis of the chemotherapeu- methanesulfonate (Lomidine®), a toxic polyamioe known to be

tical interest of diterpenes and the amount of the major product I extracted, its in vitro leishmanicidal activity was

also studied by biological assays on a culture form of Leishmania donovani donocani, a visceral leishmaniasis agent. Materials and Methods General M.p. 's are uncorrected. 5H-NMR (300 MFIz) and 15C-NMR (75 MHz) were recorded on a Bruker AM 300 instrument in CDC15, using TMS as internal standard. High resolution El-MS were obtained at 70 eV.

strongly active against leishmaniasis, and with untreated control cultures.

Extraction and purification of the diterpene Air-dried stem barks (4200g) were powdered and defatted with n-hexane. The extract was concentrated and a precipitate was filtered off and repeatedly crystallised in methanol to give pure 1 (152 g). Prisms from MeOH, m.p. 106—108°C. [a)0: —40° (CHC13; c 1). IR (KBr) vmax cm: 3300, 3080, 1735, 1700, 1650. MS m/z (rd. mt.): 348 (Mt, 4.9), 333 (19.5), 302 (12.8), 287 (20.3), 274 (36.0), 271 (9.5), 235 (12.2), 189 (34.0), 175 (9.4), 167 (15.0), 161 (14.2), 159 (10.0), 147 (13.1), 139 (20.2), 135 (13.6),

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Received: November 30, 1990

Planta Med. 57(1991) 553

A Novel Leishmanicidal l.abdane from Polyalthia macropoda

133 (25.0), 123 (28.1), 122 (21.2), 121 (73.2), 120 (18.9), 119 (20.1), 114 (100), 109 (24.3); 'H-NMR (CDC13, 300MHz) h ppm: 5.65 (q, J= 1.1 Hz, H-14), 4.86 and 4.52 (hr. s, Ha17 and Hb-17), 3.69 (S. COOMe), 2.16(d, J= 1.1 Hz, CH3-16), 1.15 (s, CH3-19), 0.72 (s, CHi-20); 13C-NMR (CDCI3, 75 Mhz): see Fig. 1.

X-ray data: Measurements were carried out on an Enraf-Nonius CAD-4 diffractometer system using MoIç radiations (A = 0.71073 A) with an w/20 scan technique (Umax 26°). Structures were solved using a MULTAN 80 method with the EnrafNonius SPD programs. Full crystal data are deposited at the Cambridge Crystallographic Data Centre, University Chemical Laboratory, Lensfield Road, Cambridge CB2 1EW, U.K.

Results and Discussion Identification of the diterpene Compound 1, C21H3204 (found: 348.2305; required: 348.2300) showed IR bands for an exocyclic dou-

ble bond (1650, 3080cm1), for an ester function (1735cm') as well as for a carboxylic acid group

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(1700 cm1). An allylic methyl and a methoxycarbonyl appeared in the 1H-NMR spectrum (CDC13, 300MHz) at 2.16 (d, J= 1.1 Hz) and 3.69 ppm, respectively, while two quater-

nary methyls were located as sharp singlets at 0.72 and

1.15 ppm. Noteworthy were the signals at 4.52 and 4.86 ppm, for the exomethylene, and the resonance of the

olefinic side chain proton as a quartet at 5.65 ppm (J = 1.1 Hz). The assignments of the 13C-NMR spectrum (CDC13,

70MHz), summarized in Fig. 1, were supported by heteronuclear spin decouplings, by INEPT experiments and by comparison with literature data previously reported for labdanic derivatives (5, 6, 7). These features so indicated a close relationship of compound 1 with agathic acid monomethyl esters 2a and 2b already isolated from Agathis lanceolata (5) and A. australis (8, 9) respectively. However, optical rotation and m.p. values were found to be quite different for 1 and either

monomethylester-15 agathic acid (2a) or monomethyl-

ester-19 agathic acid (2b), suggesting a diastereomeric relationship between these three compounds. The structure of 1 was thus solved through a single crystal X-ray diffraction study. A perspective drawing of the molecule (Fig. 2) then showed that 1 possessed the same 4ci-carboxylic function as dehydropinifolic acid (10). Therefore, compound 1 was unambiguously identified as (4S,9R,1OR)-methyl 18carboxy-labda-8,13(E)-diene-15-oate or (—)-monomethylester-15 dehydropinifolic acid. 167.3 50.8

13

18.5 390 147.6 I 269J38.l 37.9t

2 13

10

5)

8 7)

20

Lomidi,1o

2

0

3

doses (mg/mi)

The leishmanicidal activity of 1.

Study of the leishmanicidal activity

Fig. 3 depicts graphically the growth of

185.2

1

40

H2

H3C476'000H 16.4

0

:

H3

07.1

OH2

a

Fig. 3

39.7

80

60

H 30 .2 11.

5.2

37.2

100

'0

CO OR1

H3C...,J11 160.7 148

120

15

189 C000H3

A perspective drawing (ORTEP) of the molecule of 1.

Fig. 2

R2 --

H 2 a R1 ° CH3, 2k R1 - H R2 -- H C

Fig. 1 13C-NMR (h ppm) data for compound land structures of 2a, b.

Leishmania donovani donovani in the absence or in the presence of I or Lomidine®. It should be pointed out that a diterpene concentration of 0.25mg mt1 inhibits the para-

sites division to an extent of about 15 %while the same con-

Planta Mcd. 57(1991)

References 1 Kijjoa, A., Pinto, M. M., Hers, W. (1989) Planta Med. 55, 205. 2 Kijjoa, A., Pinto, M. M., Tantisewie, B., Ilerz, W. (1990) Phytochemistry 29, 653.

'

leishmaniasis). Leishmaniasis caused by protozan Leishmania ssp is an endemic parasitic disease in Central

and South America. Chemotherapic agents against Leishmania (pentavalent antimony compounds, pen-

6

tamidine, and amphotericin B) are toxic and expensive

products and very few antiprotozoal natural products such as diospyrin are known (11). Further in vivo studies on the biological activity of I would then be worthwhile to validate any employment suggested by these preliminary results.

10 11

Lavault, M., Guinaudeau, H., Bruneton, J., Sévenet, T., Hadi, H. (1990) Phytochemistry 29, 3845. Teo, L. M., Pachiaper. G., Chan, K. C., Hadi, H. A., Weber, J. F., Dcverre, J. R., David, B., Sévenet, T. (1990) J. Ethnopharm. 28, 63. Buckwalter. B. L., Burfitt, I. B., Nagel, A. A., Wenkert, F., Nat, W. (1975) Helv. Chim. Arta 58, 1567. DoKhacManh,D.,Bastard,J.,Fétizon,M.,Sévenet,T.(1983)J.Nat. Prod. 46, 262. Bastard, J., Do Khac Manh, D., Fétizon, M., Francis, M., Grant, P., Weavers, H., Kaneko, C., Baddeley, C., Bernassan, J. M., Bnrfitt, I., Wovkulich, I., Wenkert, F. (1984) J. Nat. Prod. 47, 592. Thomas, n. B. (1966) Acta Chem. Scand. 20, 1074. Zinkel, D. F., Critchfield, W. B. (1974) Phytochemistry 13, 2876. Non T., Sudin, S., Theander, 0. (1980) Acta Chem. Scand. 34, 30. Hazra,B.,Saha,A.K.,Hay,H.,ltoy,D.K.,Sur,P.,Banerjee,A.(1987) Trans. H. Soc. Trop. Med. Hyg. 81, 738.

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centration of Lomidine® causes cells death with crenelled surfaces. The dose required to inhibit 100% of the promastigote growth is 1.5mg mL1 (LD50: 0.75mg mL1) and the lytic effect becomes very pronounced at higher concentrations. Thus, I possesses an interesting leishmanicidal activity which was furthermore confirmed on a virulent strain of Leishinania donovani infantwn (post Kala Azar cutaneous

Pascal Richomree ci aL