Pergamon 0031-9422(95)00415-7 Phytocheraistry,-Vol. 40, No. 5, pp. 1485-1487, 1995 Copyright © 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0031-9422/95 $9.50 + 0.00 TIRUCALLANE TRITERPENES FROM THE STEM BARK OF A G L A I A LEUCOPH YLLA ABDELILAH BENOSMAN, PASCALRICHOMME,* THIERRY SEVENET,Jf GUILLAUME PERROMAT,~ A. HAMID A. HADI~ and JEAN BRUNETON CEPM, 16, Bd Daviers, Angers, F-49100, France; tlCSN, Gif-sur-Yvette, F-91191, France; :~Department of Chemistry, The University of Malaya, 59100 Kuala-Lumpur, Malaysia (Received 11 April 1995) Key Word Index--Aolaia leucophylla; Meliaceae; stem bark; tirucallane triterpenes. Abstract--A new tirucallane triterpene, ( - )-leucophyllone, was isolated from the stem bark of Aglaia leucophylla and its structure was elucidated from spectral data as 23(Z)-25-methoxy-tirucall-7,23-diene-3-one. In addition, ( - )-caryophyllene oxide, ( - )-niloticin, ( - )-bourjotinolone and ( - )-piscidinol were isolated from the same plant. INTRODUCTION We recently showed that ( + )-ocotillone was responsible for the cytotoxic activity of an ethanolic extract of Aglaia leucophylla King against KB cells [1]. It then became of interest to evaluate further the bioactivity of this compound in different in vitro test procedures. However, to the small amount of ( + )-ocotiUone isolated in our previous work, it was necessary to collect a new specimen of the Malaysian Aglaia in order to extract further amounts of the bioactive compound. In this paper, we report on the isolation of the new compound 1 and the known compounds 2-5 from dried stem bark of A. leucophylla. RESULTS AND DISCUSSION The dried powdered stem bark of A. leucophylla was extracted and chromatographed as previously described [1]. Five compounds were isolated and identified by means of spectroscopic methods as new ( - )-caryophyllene oxide (2), ( - )-niloticin (3), ( - )-bourjotinolone (4), ( - ) - p i s c i d i n o l (5) and the new compound ( - ) - l e u c o phyllone (1). Compound 1 was assigned the molecular formula C31H5oO2 (HR EI-MS: caled: 454.83811; found: 454.3821). An IR absorption band at 1709 era- ~ suggested it to be a keto-triterpenoid [--2].The mass spectrum of 1 also exhibited fragment ions characteristic of a tirucallane skeleton [3] at m/z 439 [ , M - M e ] + , 271 [ C 1 9 8 2 7 0 ] + and 138 ['C9Ht40] +, the latter being attributable to a retro Diels-Alder cleavage of ring B (Scheme 1). The 1HNMR spectrum of 1 showed methyl resonances at 60.82 (3H, s, Me-18), 0.88 (3H, d, J = 6.0 Hz, Me-21), 1.01 (6H, s, Me-19 and Me-30), 1.12 (3H, s, Me-28) and 1.26 (6H, s, Me-26 and Me-27). Signals for olefinic protons were observed at 65.30 (1H, m, H-7) [4], 5.40 (1H, d, J = 16.0 Hz, H-24) and 5.60 (1H, ddd, J = 16.0, 8.0 and 5.6 Hz, H-23) whereas the signal of a methoxyl group was seen at 63.16 (3H, s, OMe-25). The large coupling constant (16.0 Hz) between H-23 and H-24 was indicative of a Z-geometry of the side-chain double bond. Based on the 13C and DEPT N M R spectra, it was also shown that this triterpenoid possessed eight methyl groups, eight methylenes, seven methines and one carbonyl group. The signals for C-8 and C-7 appeared at c5145.8 and 117.8, respectively [5]. These chemical shifts are characteristic of the ALeuphane and the AT-tirucallane series [6]. The strongly negative optical rotation of 1 indicated that it belonged to the tirucallane series [-7] 21 18 ~ .= 29 ..-"2o 21t Scheme 1 * Author to whom correspondence should be addressed. 1485 22 24 23 2o ~'OM® A. BENOSMANet al. 1486 Table 1. 13CNMR spectral data and atom type from DEPT for 1 (CDC13, 67.5 MHz) Carbon Atomtype 6(ppm) Carbon Atomtype 6(ppm) no. no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 CH2 CH 2 C C CH CH2 CH C CH C CH2 CH 2 C C CH2 CH2 38.5 34.9 216.9 47.8 52.3 24.3 117.8 145.8 48.4 34.9 18.2 34.0 43.4 51.1 33.5 28.0 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 CH CH 3 CH3 CH CH3 CH2 CH CH C CH 3 CH3 CH 3 CH3 CH 3 OCH3 52.5 21.9 12.7 36.3 18.4 39.1 128.6 136.6 74.8 25.7 26.2 24.5 21.6 27.4 50.2 and assignment of each carbon resonance of the molecule was completed through IH-13C COSY and HMBC experiments (Table 1). In this way, 1 was identified as 25-methoxy-tirucall-7-23 (trans)-diene-3-one, which we have named ( - )-leucophyllone. Thus, this plant only contains tirucallane derivatives, whereas secotirucallane and dammarane derivatives were isolated from our first specimen of A. leucophylla. This difference between the two specimens collected in the Malaysian peninsula prompted us to reinvestigate the botanical identity of each plant. Careful examination of the fruit from samples of each specimen recollected during fructification showed that the former specimen actually belonged to a genus closely related to Aglaia in the Meliaceae family--Dysoxylum--and this plant was finally identified as Dysoxylum cauliflorum Hiern. This name should thus replace the erroneous Aglaia leucophylla used in our earlier paper [1-l. As far as biological properties are concerned, it should also be noted that the ethanolic extract of A. leucophylla does not exhibit cytotoxic activity, unlike D. cauliflorum, nor anti-HIV, antifungal or antibacterial activity. EXPERIMENTAL Extraction and fractionation. The plant material was collected by one of us (G.P.) in Lumut, Perak, Malaysia, in November 1992. A herbarium specimen is deposited at the Laboratoire de Phanrrogamie, MNHN, Paris, and at the University of Malaya, Kuala-Lumpur, under the reference KL4175. The stem bark (3.0 kg) was air-dried, powdered and allowed to stand in MeOH at room temp. The extract was filtered and evapd to dryness to yield an oil (222.6 g). This extract was then partitioned between aq. MeOH and hexane. The hexane extract (19.3 g) on MPLC on silica gel (40-63 p.m mesh) gave caryophyllene oxide (2.16 g; fr 1-13, n-hexane-EtOAc 47:3), niloticin (7.85 g; frs 20-49, 3: 1), leucophyllone (497 mg; frs 50-55, 4:1), bourjotinolone (325 rag; frs 56-61, 3:2) and piscidinol (193 mg; frs 62-67, CHC13-MeOH, 99:1). Compound (1) (25-methoxy-tirucall- 7-23 (trans)-diene3-one. [Ct]n-95 (CHC13; ci); HR-MS 70ev, re~z: 454.3821 [M] + (calc. for C31H5oO2, 454.3811); IR vtBr cm-l: 1709 (CO, carbonyl), 1076 (CO, ether); El-MS 70 ev, m/z (rel. int.): 454 (10) [M] +, 439 (10) [M - Me] +, 423 (2) [M - OMe] +, 407 (100), 325 (25) [C23H330 ] +, 271 (9) [C19H2vO] +, 175 (16), 138 (2), 123 (22), 113 (8), 81 (37) [C6H91 +, 73 (23) [C4H50] +, 1HNMR (270 MHz, CDCI3, TMS = 0): 85.50 (ddd, J = 15.7, 8, 5.6 Hz, H-23), 5.40 (d, J = 16 Hz, H-24), 5.30 (m, H-7), 3.16 (s,OMe), 2.76 (ddd, J = 14, 5.4 Hz; H-2ax), 1.26 (s, Me-26 and Me-27), 1.12 (s, Me-28), 1.05 (s, Me-29), 1.01 (s, Me-19 and Me30), 0.88 (d, J = 6 Hz, Me-21), 0.82 (s, Me-18); laC NMR: Table 1. Caryophyllene oxide (2). EI-MS 70 ev, m/z: 220 [M] ÷ C15H240; I H N M R (270MHz, CDCI3, T M S = 0 ) : 84.96-4.85 (2br s, 2H, C =CH2), 2.88 (dd, J = 10.6, 4 Hz, 1H, c/°\cn), 1.2 (S, 3H, c/°\c-cn3) 1.00 and 0.98 (2s, 6H, C(CH3)2. Niloticin (3). ['ct]D- 60 (CHC13; cl); IR vKBr cm-1: 3395 (OH), 1707 (CO); El-MS 70 ev, m/z (rel. int.): 456 (12) [M] +, 441 (5) [M - Me] +, 369 (82) I-C25H3702] +, 325 (56) [M-CrH1202-Me] +, 143 (15), 138 (6), 71 (24). Bourjotinolone (4). [~t]D- 60 (CHC13; cl); IR vgs~ cm- 1:3404 (OH), 1707 (CO); El-MS 70 ev, m/z (rel. int.): 472 (19) [M] +, 457 (9) [M - Me] +, 439 (22) [M-MeH20] +, 413 (9) [M-C3H70] +, 396 (39) [M-CaHvO-OH] +, 381 (100) [M-C3HTO-Me-OH] +, 271 (5) [C19H270-I +, 159 (11) [CsH1503] +, 59 (44) [C3H70] +. Piscidinol (5). [~t]D- 76 (CHC13; cl); IR vKar cm-l: 3450 (OH), 1710 (CO); El-MS 70 ev, m/z (rel. int.): 474 (10) [M-] +, 459 (3) [M - Me] +, 441 (12) [M-Me-H20] +, 423 (7) [M-Me-2H20] +, 369 (100), 325 (53) [C23H330-1 +, 271 (12.5) [C19H2703] +, 161 (23) ~'C8Hl1703] +, 143 (10) [CsH1703 - H20] +, 59 (40) [-C3H70 ] + 41 (68) [CaH7O - H2O ] +. 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