PERGAMON Phytochemistry 50 (1999) 75±79 Leishmanicidal alkaloids from Kopsia grithii Toh-Seok Kam a, *, Kooi-Mow Sim a, Takashi Koyano b, Kanki Komiyama c a Department of Chemistry, University of Malaya, 50603 Kuala Lumpur, Malaysia b Temko Corporation, 4-27-4 Honcho, Nakano, Tokyo 164, Japan c The Kitasato Institute, 5-9-1 Shirokane, Minato-ku, Tokyo 108, Japan Revised 16 June 1998 Abstract Thirteen alkaloids were isolated from the stem-bark extract of Kopsia grithii, of which three were new. These were the N(4)oxides of akuammiline, 16-epi-deacetylakuammiline and 11,12-methylenedioxykopsinaline. Harmane, pleiocarpine and buchtienine showed antileishmanial activity. # 1998 Published by Elsevier Science Ltd. All rights reserved. Keywords: Kopsia grithii; Apocynaceae; Stem-bark; Antileishmanial alkaloids 1. Introduction 2. Results and discussion The genus Kopsia comprises some 30 species found mostly in tropical Asia (Markgraf, 1972; Sevenet et al., 1994). There are ca. 17 species which occur in Malaysia. The Malaysian members of this genus have received considerable attention which has resulted in several novel alkaloid structures and some important bioactivities (Awang, Sevenet, Hadi, David, & Pais, 1992; Kam, Yoganathan, & Chuah, 1995; Kam, Yoganathan, & Li, 1996; Kam, Yoganathan, Koyano, & Komiyama, 1996; Kam, Yoganathan, & Chen, 1996a,b; Uzir et al., 1997). We have previously reported the alkaloidal composition of the leaf extract of Kopsia grithii King and Gamble (Kam & Sim, 1998). We now report the alkaloidal composition of the stem-bark extract, including the isolation of three new alkaloids. Preliminary screening of extracts from this species showed strong anti-leishmanial activity which was previously traced to the basic fraction from the ethanol extract of the leaves (Kam & Sim, 1998). We have now identi®ed the active principles responsible for this activity. The ethanol extract of the leaves of K. grithii furnished the following alkaloids: harmane 1, harmicine 2, kopsilongine 3, kopsamine 4, kopsamine-N(4)-oxide 5, pleiocarpine 6, 12-methoxypleiocarpine 7, leuconolam 8, leuconoxine 9, kopsinine 10, (+)-eburnamonine 11, N(1)-methoxycarbonyl-12-methoxy-D-16,17-kopsinine 12, N-carbomethoxy-11-hydroxy-12-methoxykopsinaline 13, N-carbomethoxy-11,12-dimethoxykopsinaline 14, tetrahydroalstonine 15, 12-methoxy-10-demethoxykopsidasinine 16, deacetylakuammiline (rhazimol) 17, 16-(R)-19,20-E-isositsirikine 18 and buchtienine 19 (Kam & Sim, 1998). From the stem-bark extract, a total of 13 alkaloids were isolated including 1, 8, 9, 10, 17, 19, (ÿ)-eburnamine 20 (Kam, Tan, & Chuah, 1992; Kam, Tan, & Chen, 1993), kopsinine N(4)-oxide 21 (Thomas, Achenbach, & Biemann, 1966), 16-epi-deacetylakuammiline 22 (Kan, Deverre, Sevenet, Quirion, & Husson, 1995), rhazinaline N(4)-oxide 23 (Abe & Yamauchi, 1993), akuammiline N(4)-oxide 24, 16-epideacetylakuammiline N(4)-oxide 25 and 11,12-methylenedioxykopsinaline N(4)-oxide 26; the last three are new alkaloids. Harmane 1 and buchtienine 19 are the major alkaloids in the leaves, while the major alkaloids in the stem-bark are deacetylakuammiline 17 and kop- * Corresponding author. Tel.: +60-3-759-4266; Fax: +60-3-7594193; E-mail: tskam@kimia.um.edu.my. 0031-9422/98/$ - see front matter # 1998 Published by Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 1 - 9 4 2 2 ( 9 8 ) 0 0 4 9 2 - 0 76 T. Kam et al. / Phytochemistry 50 (1999) 75±79 sinine N(4)-oxide 21. Compounds 24±26 are readily deduced to be the N(4)-oxides of akuammiline (Dugan, Hesse, Renner, & Schimd, 1969), 16-epi-deacetylakuammiline (Kan et al., 1995) and 11,12-methylenedioxykopsinaline 27 (Feng, Kan, Potier, Kan, & Lounasmaa, 1983), respectively, from the 1 H and 13 C NMR spectral data (Tables 1 and 2), in particular the characteristic down®eld shifts of the C-3, -5 and -21 resonances when compared with the parent compounds (Table 2). Kopsia grithii is notable for furnishing a remarkable array of alkaloidal types which include, simple b-carbolines (1, 2), corynantheine alkaloids (15, 18), eburnane alkaloids (11, 20), sarpagine alkaloids (17, 22, 23, 24, 25), aspidosperma alkaloids (8, 9), aspidofractinine alkaloids (3, 4, 5, 6, 7, 10, 12, 13, 14, 16, 21, 26) and the quasidimer, buchtienine 77 T. Kam et al. / Phytochemistry 50 (1999) 75±79 Table 1 1 H NMR spectral data for compounds (22, 24±26) (400 MHz, CDCl3) H 24 3 3 5 5 6 6 9 10 11 12 14 14 15 15 17 17 18 18 19 19 21 21 CO2Me OCOMe OCH2O OCH2O NH 5.05 ± 3.17 3.42 2.12 3.42 7.62 7.24 7.37 7.66 2.17 2.91 3.57 ± 3.59 3.59 1.72 ÿ 5.73 ÿ 4.07 4.53 3.79 1.59 ± ± ± 22 d (4.5) m m br dd (15, 5) m dd (7.5, 1) td (7.5, 1) td (7.5, 1) dd (7.5, 1) m ddd (15, 4.5, 2.5) br s m m dd (7,2) q (7) d (16) dd (16, 2) s s 4.87 ± 2.79 3.02 1.97 2.79 7.72 7.18 7.33 7.62 2.44 2.79 3.20 ± 4.34 4.34 1.77 ± 5.69 ± 3.33 4.30 3.11 ± ± ± ± 25 d (4.5) 5.07 ± 3.13 3.58 1.93 2.86 7.76 7.22 7.35 7.64 2.81 2.81 3.38 ÿ 4.26 4.30 1.85 ± 5.88 ± 4.60 4.28 3.18 ± ± ± ± m m br dd (15, 5) m br d (7.5) td (7.5, 1) td (7.5, 1) br d (7.5) ddd (14, 4.5, 2) m br s m m dd (7,2) q (7) d (17) dd (17, 2) s 26 br s 3.57 3.93 3.57 3.93 2.35 2.47 7.50 6.15 ± ± 1.82 2.94 1.75 2.04 1.26 3.16 1.45 1.67 1.33 1.82 3.62 ÿ 3.83 ÿ 5.51 5.71 5.29 m br dd (13, 5) br dd (15, 5) m br d (7.5) td (7.5, 1) td (7.5, 1) br d (7.5) m m br s d (12) d (12) dd (7, 2) q (7) dd (17, 2) d (17) s m m m m m br dd (15, 8) d (8) d (8) m m m m d (15) dd (15, 2) m m m m br s s d (1.5) d (1.5) br s Table 2 13 C NMR spectral data for compounds (22, 24±27) (100 MHz, CDCl3) C 24 22 25 27 26 2 3 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 CO2Me CO2Me OCOMe OCOMe OCH2O 178.4 77.1 69.0 27.6 56.2 129.5 125.8 126.7 129.0 122.2 155.0 28.7 34.5 58.0 64.8 13.7 125.1 143.0 71.8 52.1 170.5 19.9 169.0 ± 189.5 54.8 51.2 33.5 57.3 137.9 125.2 125.4 128.0 120.6 155.9 32.0 34.5 62.3 64.2 14.4 121.6 145.4 52.8 51.7 173.6 ± ± ± 178.4 77.2 67.5 25.2 55.2 128.5 125.5 126.5 128.6 121.6 155.1 29.6 31.8 62.3 62.7 14.9 126.8 144.3 70.6 52.1 171.8 ± ± ± 71.9 47.9 50.7 35.6 58.5 130.9 114.4 101.1 147.4 137.9 133.2 17.4 26.7 76.9 40.9 36.1 33.5 33.0 67.9 53.2 174.8 ± ± 100.9 71.1 64.7 64.8 32.2 58.7 131.2 117.7 100.1 147.4 134.0 131.4 19.6 25.0 75.6 40.0 33.7 34.0 34.7 84.5 53.0 175.1 ± ± 100.6 78 T. Kam et al. / Phytochemistry 50 (1999) 75±79 (19). Since antileishmanial activity was detected in preliminary screening, we have carried out bioassays on the alkaloids isolated in order to determine the compounds responsible for this activity. Only three compounds were found to show activity against Leishmania donovani, viz., harmane 1 and pleiocarpine 6 (6.25 < IC50 < 25 mg ml ÿ 1), and the quasidimer, buchtienine 19 (0.39 < IC50 < 1.56 mg ml ÿ 1), with the highest level of activity being shown by buchtienine 19. This result, which identi®es buchtienine as the principal compound responsible for the leishmanicidal activity, is in agreement with the recent isolation of buchtienine along with other indole alkaloids from the Bolivian plant Peschiera buchtieni (Syn. Tabernaemontana buchtieni), which is also used locally for the treatment of leishmaniasis (Azoug et al., 1995). 3. Experimental 3.1. Plant material Details of the collection of plant material and the deposition of voucher specimens are given in Kam and Sim (1998). C21H24N2O4, 368.1736. 1 H and and 2. 13 C NMR: Tables 1 3.5. 11,12-Methylenedioxykopsinaline N(4)-oxide 26 [a]D = ÿ 168 (CHCl3, c 0.19). UV (EtOH), lmax (log e) 221 (4.35), 246 (3.77), 282 (2.97). EIMS, m/z (rel. int.): 414 [M] + (15), 398 (100), 370 (36), 339 (22), 124 (25) and 109 (23); HREIMS, [M] + , found 414.1799, calcd for C22H26N2O6, 414.1791. 1 H and 13 C NMR: Tables 1 and 2. 3.6. Leishmanicidal assay (Roy, Bhattacharya, Siddiqui, & Bhadra, 1990) Leishmania donovani (promastigote) was maintained in 199 medium supplemented with 10% foetal calf serum and kanamycin. In a 96 well round-bottom plate were placed 200 ml each of the cell suspension (10000 cells ml ÿ 1), followed by the addition of 5 ml each of the test solns. The initial concn of the test soln was 25 mg ml ÿ 1 in a well and four di€erent concns of each sample were examined for their growth rate. Cells were cultured for 3 days and then examined using the MTT staining method (Alley et al., 1988). 3.2. Extraction and isolation Acknowledgements Extraction of alkaloids was carried out as described previously to obtain the crude alkaloidal mixt. from the stem-bark (Kam & Sim, 1998; Kam & Tan, 1990). Alkaloids were isolated by CC and centrifugal TLC on silica gel. The solvent system used for CC was CHCl3± MeOH. Solvent systems used for centrifugal TLC were Et2O, Et2O±hexane and MeOH±CHCl3. The yields (g kg ÿ 1) of the alkaloids isolated from the stem-bark were: 1 (0.01), 8 (0.01), 9 (0.003), 10 (0.002), 17 (0.02), 19 (0.001), 20 (0.001), 21 (0.027), 22 (0.0008), 23 (0.0008), 24 (0.002), 25 (0.002) and 26 (0.003). We thank the University of Malaya and IRPA for ®nancial support of this work. 3.3. 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