Letters Planta Med. 61 ('1995) -89 mutually coupled, high-field multiplets were also present (6 References = 1.49-2.05), accounting for a sequence of two methyl- enes. Selective decoupling and 'H-'H COSY experiments * De Mösbach, E. W. (1992) Botanica Indigena de Chile, p. 93, showed the coupling of these methylenes with the previous Fundaciòn Andes, Santiago de Chile. Soukoup, J. (1970) Vocabulario de los Nombres Vulgares de la Flora oxymethine, therefore assigning a gluc-O-CH(CH,)-CH2- Peruana, p. 211, Colegio Salesiano, Lima. CH2-sequence. Correu, D. S., Johnston, M.C. (1970) Manual of the Vascular of Texas, pp. 1083-1087, Texas Research Foundation, Renner. The presence in the 13C-NMR spectrum of El-Naggar, L. J., Beal, J. L. (1980) J. Nat. Prod. 43,649- 707. the resonances corresponding to the saturated aliphatic car- Boros, C.A., Stermitz, F. R. (1990) J. Nat. Prod. 53, 1055-1147. bonyl and the carboxyl functions, at 201.1 and 171.1 ppm, Boros, C.A., Stermitz, F. R. (1991) J. Nat. Prod. 54,1173-1247. Crombie, L., King, R. W., Whiting, D. A. (1975) J. Chem. Soc., Perlcin respectively, together with the signal of a quaternary Trans. 1,913. carbon atom at 43.9ppm, allowed us to assign to 1 the Vogeli, U., Van Philipsborn, W. (1975) Org. Magn. Res. 7,617. structure of 9-O-P-D-glucopyranoside of the 9-hydroxy- 2,2,4-trimethyl-cleca-3-en-6-~n-l-oicacid. Finally, a Z- configuration was assigned to the three-substituted double bond on the basis of the chemical shift value of the methyl group at C-4, which did not show the shielding effect ex- pected for an E-configuration (7, 8). In the NOESY experi- ment, evident correlations between the two methyl singlets and the protons of the methylene at 6 = 2.14 and 2.65 were also in accordance with these assignments.

Concerning the biosynthetic origin of the aglucone, it could be considered as derived from the junc- + Alkaloids from Leaves and Stems Of tion of three isoprenoid units and the subsequent loss of two terminal carbons. glabru

Materials and Methods M. Zèches'~~,K. Mesbah', B. Richard', C. Moretti'.', material J. M. Nusillard', and L. Le Men-Oliuier ' ' Faculté de Pharmacie, URA au CNRS n" 492,51 rue Cognacq-Jay, Mentzelia incisa Urban et Gilg. was collected in F-51096 Reims Cedex, France the Monterrey district (N.L. ) and identifed by Prof. H. z ORSTOM (UR 451,213 rue Lafayette, F-75480 Paris Cedex Sanchez at ITESM in Monterrey (voucher specimen SH-346). France Address for correspondence Isolation Received: March 21,1994; Accepted: May 21,1994 The aerial parts (350g) were exhaustively ex- tracted with MeOH at room temuerature and the extract evauo- rated to an aqueous suspension. After a further addition of water (0.511, the resulting solution was extracted with EtOAc and n- BuOH (0.5 1, each) in the sequence. The CC separation of the n- BuOH extract was performed on SiO? (ca. 100g) in CH,C1, : MeOH, 85 : 15, 1ml/min; elution vol. of 1700- 750ml. Eleven known indole alkaloids: vallesine, 'H- and I3C-NMR spectra were recorded on a aspidospermine, 11-methoxydichotine, apparicine, tu- Bruker AM-500 spectrometer. The assignments were confirmed botaïwine, vincadifformine, condylocarpine, (-)-rha- by COSY and NOESY experiments. zinilam, aspidospermatine, haplocidine, and 18-oxo- Compound 1 haplocidine, have been isolated from leaves and stems of (Cav.) Link. () from Bo- Amorphous powder; [a]:': -26.5 (c = 1.0, livia. Analysis of 2 D NMR spectra complete previous 'H MeOH). IR (KBr) v,, cm-': 3400, 1710, 1670, 1210. 'H-NMR and I3C data for vallesine, aspidospermine, and 11- i (CD30D, TMS): S= 1.01 (3H, s, H3-ll), 1.08 (3H, s, H3-12), 1.23 methoxydichotine. I (3H, d, J= 5.0Hz, &-lo), 1.49 (lH, m, H-Sb), 1.74-1.87 (ZH, m, ! ri-Sa and H-7bl2.03 (3H, d, J= 1.3 Hz, H3-13), 2.05 (lH, m, H-7a), 2.14 (lH, dd, J'= 18.0 and 1.3Hz, H-5b), 2.65 (lH, d,J= 18.0Hz, H-5a). 3.13 (lH, dd, J= 8.0 and 8.5Hz. H-2'). 3.26-3.33 (3H, m, The genus I/allesia includes 12 mecies. 8 H-4', H-5' andH-3'),3.67 (1H, dd, J= 12.0and5.8 HZ, H-6'b), 3.80 of which are spreaded out from Fiorida 'to (1H,m,H-9),3.84(1H,dd,J=12.0and3.0Hz,H-6'a),4.31(1H,d, heGCan, &gentina' To the best Of Only J = 8.0Hz, H-l'), 5.52 (IH, m, H-3). 13C-NMR (CD30D, TMS): 6 = 22.2 (C-10 or c-13h22.3 (C-13 or C-IO), 24.3 (c-il or c-12),24.7 were chemically studied: Vallesia dichotoma Ruiz. and (c-12 or C-li), 33.2 (C-8), 34.9 (C-7), 43.1 (c-2), 51.2 (c-5), 62.8 PaV. (from ) (1, 2). (from ) (3, (c-60, 71.8 (c-4'1, 75.2 (C-2'). 77.8 (C-5' or c-37, 78.0 (c-91, 78.3 41, and Vallesiaglabra (Cav.)Link. (5) from which only two IC-3' or C-5'1, 104.2 (C-1'1, 127.1 (C-31, 133.7 (C-41, 171.7 (C-l), alkaloids, vallesine and aspidospermine, were isolated. 201.0 (C-61.

Plantabled. GI (1995) 89-91 Q Georg Thieme Verlag Stuttgart. New 'íurk -90 Planta Med. 61 (1995) Letters According to some botanical authorities V.glabra and V. dichotoma are synonyms but they may represent differ- ent varieties of the same species (6).

We report here on the alkaloidal contents of leaves and stems of Vallesia glabra from . Val- lesia glabra is a small shrub common in the arid valleys of Bolivia and . Plant materials were collected in the Bolivian Province of Misque, in September 1991 during an ÒCH3 ethnobotanical field work (ORSTOM - U.M.S.S. project: 1 R = CHO, vallesine Medicinal plants of Bolivian tropical region). Voucher 2 R = COCH3, aspidospermine 3 Il-methoxydichotine specimens (Morem No 1510) are deposited in the National Herbarium of Bolivia, La Paz. Botanical identification was Vallesine (1) confirmed by L. borge (Museum National d'Histoire Na- turelle de Paris). [alD: -91" (c 1, CHCI,). 'H-NMR (300MHz, CDCl,, TMS, Gvalues inppm): 9.3 (s, CHO), 7.0 (t, J= 7.5 Hz, H-lo), The extraction of the alkaloid mixtures (A. 6.85 (d,J= 7.5Hz, H-9), 6.8 (d, J= 7.5Hz, H-ll), 4.53 (dd, J= 11, M.) was carried out according to the usual acid-base pro- 6.1 Hz, H-2),3.87 (s, OCH,), 3.1 (td, J= 9,3.3 Hz, H-5),3 (br. d, J= 11 Hz, H-3), 2.25 (s, N-CO-CH3 and H-21). 2.23 (m,H-57, 2.15 (m, cess (7), and the following yields were obtained: 2.4 % H-16), 2.08 (m, H-17), 2.03 (dd,J= 12,3.3Hz, H-6), 1.93 (dd,J= (stems), 2.9% (leaves). The purification of A.M. was per- 11, 2.8Hz, H-3'). 1.75 (qt, J= 13, 4Hz, H-141, 1.63 (br. d, J= formed by column chromatography (silica gel, CH2C12- 13HZ, H-15), 1.5 (dd, J= 12, 3HZ, H-6'), 1.45 (dd, J= 13, ~Hz, MeOH with increasing amounts of MeOH), and the isolation H-14'), 1.4 (dq, J= 14,7.3Hz, H-19), 1.27 (m, H-16'). 1.1 (dd, J= of pure alkaloids was realized by preparative TLC, CC (with 13.4, 4.4Hz, H-17'). 1.05 (dd, J= 13,4.3Hz, H-15'). 0.85 (dq,J= the same solvents), or by crystallization. Eleven known al- 14, 7.3Hz, H-19'1, 0.63 (t, J= 7.3Hz, CH3-18).'3C-NMR 6: 161.2 kaloids were obtained (Table l)and their structures were (CHO), 148.1 (C-12). 140.8 (C-13), 127 (C-8), 124.4 (C-10). 115.6 elucidated by comparing their spectra with literature data, (C-9),110.1 (C-ll), 70.7 (C-21), 63.9 (C-2). 55.2 (OCH3), 53.5 (C-3), 52.3 (C-5 and C-71, 39.6 (C-61, 35.3 (C-201, 34.1 (C-151, 29.8 (C- or with those of reference samples available in our labora- 19),24.2 (C-16), 22.5 (C-17),21.4 (C-14),6.6 (C-18). tory. Aspidospermine (2) [alo: -99" 0.5, CHC1,). 'H-NMR (300MHz, Table 1 Alkaloids from Vailesia giabra. (C CDCl,, TMS, 6 values in ppm): 7.07 (t, J= 8.7 Hz, H-101, 6.83 (d, J Alkaloids I Leaves r',Stems%' , Isolation I Spectral analysis,Ref. =8.7Hz,H-9),6.8(d.J= 8.7Hz,H-ll),3.87(s,0CH3),3.11(td,J= I I 9, 3.2Hz, H-5), 3 (br. d, J= 10.7Hz, H-3), 2.25 (m, H-5'1, 2.23 (s, Vallesine (1) I 31.91 I TLC I a+b+c (5) H-21). 2.2 (s, N-CO-CH,), 2.05 (m, H-6), 2 (m, H-17), 1.95 (m, H-161, Aspidospermine (2) 5.52 7.42 TLC aibic (5) 1.9(m,H-3'),1;73(qt,J=13,4Hz,H-14),1.6(m,H-15),1.55(m, Il-methoxydichotine(3) 0.13 crystallization a + b + c (9) H-6'). 1.5 (br. d, J= 13Hz, H-14ll.35 (m, H-lu), 1.2 (dq, J= 14, (-)-apparicine 1.02 0.13 TLC a+b (9) 7.1Hz, H-19). 1.1 (m, H-17'), 1.05 (ni, H-15'). 0.8 (dq, J= 14, Tu botaïwine" 0.22 0.16 TLC a (12) 7.1 Hz, H-19'), 0.6 (t, J= 7.1 Hz, CH,-l8). ',C-NMR 6: 160 (N-CO- Vincadifformine" 0.39 TLC a (9) CH3), 148 (C-12), 141 (C-13), 128 (C-8), 125.9 (C-lo), 115.4 (C-9), Condylocarpine 0.32 TLC a (9) (-)-rhazinilam 0.33 TLC a+b+c (14) 110 (C-11), 71 (C-21), 64 (C-2), 55.3 (OCH,), 53.5 (C-3), 52.4 (C-5 Aspidospermatine 0.28 0.64 cc a+b (13) and C-7), 38 (C-G), 35.4 (C-20), 34.1 (C-15), 29.9 (C-lg), 24.7 (C- Haplocidine 1.91 TLC a+b (11) 16), 23 (C-17), 22.9 (COCH,), 21.5 (C-14), 6.7 (C-18). The proton 18-oxohaplocidine 0.53 cc a+ b (11) H-2 did not appear on the spectra, but its presence was ascertained by the signal of a methine carbon at 6 = 64 in the I3C undecoupled .Yields obtained from alkaloid mixture. C-H spectrum. ** Alkaloids compared with reference samples. a = UV, IR, MS, and 'H-NMR: b = %NMR and COSY 'H-'H; c = HMBC, HMQC. I I-Methoxydichotine (3)

Crystallized from acetone; m.p. 121 OC, [alD: - 11" (c 1, CHClJ. 'H-NMR as in (2). 13C-NMR (CDC13, 6 values in The novel isolation of vallesine (11, aspido- ppm): 158.7 (C-13), 147.3 (C-ll), 126 (C-E), 125 (C-S), 120.3 (C- spermine (21, and 11-methoxydichotine (3) has provided lo), 114 (C-9), 99.8 (C-2), 98.3 (C-13'), 77.7 (C-19), 76.5 (C-16), 60 the opportunity to complete previous 'H- and 13C-NMR (C-7), 56.6 (OCH3-13'). 56.3 (OCHB-ll), 53.5 (C-3), 50 (C-201, 49 data, by means of 2D NMR experiments (COSY H-H, HMQC, (C-5), 42 (C-6), 41.3 (N-CH,), 39.5 (C-15), 18.6 (C-14), 17.2 (C-18). HMBC). These techniques were specially helpful to solve the problem of assignments of the eight methylene groups This study shows that V.glabra possesses a of 1 and 2. Furthermore, HMBC spectra of 1 and 2 high alkaloid content and a chemical composition similar permitted US to detect C-7 (superimposed with C-5) by its to that of V. dichotoma (9). The most significant feature of long-range correlations (3J)with H-9 and H-16. In 2, the this work is the novel isolation of the cytotoxic (-1-rha- presence of this quaternary carbon was also confirmed by zinilam, an original mitotic poison which inhibits the poly- the signal at 6 = 52.4 in the 13C-NMR spectrum using the merization of tubuline to microtubules (10). Spin Echo Broad Band Off-Resonance Decoupling (SEBBORD)technique (8). Detailed information on the work-up pro- cedure and copies of the original spectra can be obtained from the author of correspondence. Letters Planta Med. 61 (1995) -91 Acknowledgements ' Clivio, P., Richard, B., Deverre, J. R., Sévenet, T., Zèches, M., Le Men-Olivier, L. (1991) Phytochemistry 30,3785-3792. We thank Dr. S. Arrazola Rivero &om the Centro Cookson, D. J., Smith, B. E., White, N. (1981) J. Chem. Soc., Chem. de Investigaciones Botanicas y Ecologicas, C.I.B.E. - UMSS, CO". 12-13. Cochabamba Bolivia, for the botanical support. Walser, A., Djerassi, C. (1965) Helv. Chim. Acta 48,391- 403. lo Thoison, O., Guenard, D., Sevenet, T., Kan-Fan, C., Quirion, J. C., Husson, H. P., Deverre, J. R., Chan, K. C., Potier, P. (1987) C. R. References Acad. Sc. Paris 304, série II, no4,157-160. *'Brown, K. S. Jr., Budzikiewicz, H., Djerassi, C. (1963) Tetrahedron Walser, A., Djerassi, C. (1964) Helv. Chim. Acta 47,2072- 2086. Lett. 1731-1736. ' Ling, N. C., Djerassi, C. (1970) J. Am. Chem. Soc. 92,6019-6035. l2 Schripsema, J., Van Beek, T. A., Verpoorte, R., Erkelens, C., Perera, Cuellar, A., Lorenzo, M. (1977) Rev. Cubana Farm. 11,133-146. P., Tibell, C. (1987) J. Nat. Prod. 50,89-101. Cuellar, A., Padron Palomares, G. (1978) Rev. Cubana Farm. 12, l3 Biemann, K., Spiteller-Friedmann, M., Spiteller, G. (1963) J. Am. 169-176. Chem. Soc. 631-638. Hartmann, M., &chlitter, E. (1959) Helv. Chim. Acta 22,547-549. l4 Goh, S. H., RazakM0hdAli.A.. Wong, W. H.(1989) Tetrahedron24, Hollcer, J. S. E., Cais, M., Hochstein, F. A., Djerassi, C. (1959) J. Org. 7899-7920. Chem. 24,314-316.

chemical study of the plant aimed at isolating and identi- Methyl Angolensate: The Antiulcer fymg the antiulcer agent($. This paper describes the iso- Agent of the Stem Bark of lation and identification of methyl angolensate (1)from the U methanol extract of the stem bark ofË. angolense and pro- Entandrophragma angolense vides evidence that l is the antiulcer agent of the crude ex- tract of the plant. Vincent C. O. NjarlG3,Julius K. Adesanwo', and Yinusa Raji Department of Chemistry, University of Ibadan, Ibadan, Nigeria Department of Physiology, College of Medicine, University of Ibadan, Ibadan, Nigeria Address for correspondence Received: April 6,1994; Revision accepted: June 11,1994

H ;U631CHiC O OC H 3 1

Abstract Fractionation of the methanol extract of the stem bark of E, angolense led to the isolation of 1 as the Methyl angolensate (l),the major com- major compound. The identity of 1 was established by pound isolated from the methanol extract of the stem spectroscopy (UV, IR, OR, 'H-NMR, EI-mass) and by com- bark of Entandrophragma angolense produced a dose- parison of our data with those reported in the literature related inhibition of gastric ulceration, 40 mg/kg body (2- 4). Consequently, the efficacy of 1 to protect against ex- weight (B. W.) being more effective than 40 mg/kg B. W. perimental indomethacin-induced gastric mucosal damage of propranolol. The highest dose used (80 mg/kg B.W.) and to influence gastric acid secretion was investigated as completely inhibited gastric ulceration and significantly indicated in the Materials and Methods section. The results reduced gastric acidity (P < 0.05). Furthermore, 1 (40mg/ presented in Table 1 clearly show that 1 produces a dose- kg B. W.) significantly reduced gastric acid secretion in- related gastroprotective action in indomethacin-induced duced by histamine (l.Omg/kg B. W.) and carbachol ulceration in rats. The cytoprotection produced by pro- (1.0 mg/kg B. W.). These results suggest that 1 produces pranolol (40 mg/kg) was lower than that caused by 40 mg/ its antiulcer activity by inhibition of gastric acid se- kg of 1. Thus, 1 is more potent than propranolol in pro- cretion. tecting against gastric ulceration in rats. These results are identical to those that we have previously reported (2) for the methanol extract of E. angolense and they confirm that We have recentlv shown (11 that the 1 is the antiulcer agent ofthestem bark ofE. awolense. methanol extract of the stem baik of Entandrophragma angolense (Welw.) C.DC. (Meliaceac) is highly potent in in- The significant reduction in total intra- hibiting indomethacin-induced gastric ulceration in rats. gastric acidity observed in this study (see Table 1) strongly We also showed that the crude extract was non-toxic to the suggests that 1 may act by inhibiting gastric acid secretion rats at a wide range of doses (20- 200 g/kg B. W.) tested. by the parietal cells. To investigate the mechanism of ulcer These observations prompted us to undertake a phyto- inhibition, the effects of 1 (40mg/kg B. W.) on basal, hist- amine- (1mg/kg, B. W.), and carbachol- (1mg/kg, B.W.) in- Planta Med. 61 (1995) 91-92 duced gastric acid secretion in male albino rats were O Georg Thieme Verlag Stuttgart. New York studied. The results (mean of 5 experiments -t SEM) are as