Phytochemistry, Vol. 29, No. 4, pp. 1293.~1296, 1990. Printedin Great Britain. THREE STEROIDAL ATTA-UR-RAHMAN, 003 l-9422/90 $3.00 + 0.00 (C_Z 1990 PergamonPressplc ALKALOIDS MUZAFFAR FROM BUXUS ALAM, HABIB NASIR, ERMIAS DAGNE*~ zyxwvutsr HILDEBRANDTZI and ABIY zyxwvutsrqponmlkjihgfedc YENEsEwt H.E.J. Research Institute of Chemistry, University of Karachi, Karachi 32, Pakistan; TDepartment of Chemistry, Addis Ababa University, P.O. Box 1176, Addis Ababa, Ethiopia zyxwvutsrqponmlkjihgfedcbaZYXWVUTS (Received in revised form 6 September 1989) Key Word Index-Buxus hildebrandtii; Buxaceae; steroidal alkaloids; 0(30)-benzoyl-16-deoxybuxidienine-C, 30hydroxybuxamine-A; 30-norbuxamine-A; cyclomicrobuxamine; buxamine-A; buxamine-C, cyclobuxoviridine; moenjodaramine; cyclorolfeine. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Abstract-From the leaves of Buxus hildebrandtii three new steroidal alkaloids have been isolated and their structures determined by spectroscopic analysis. The following derived names have been suggested for these new alkaloids: 0(30)benzoyl-16-deoxybuxidienine-C, 30-hydroxybuxamine-A and 30-norbuxamine-A. In addition the known alkaloids cyclomicrobuxamine, buxamine-A, cyclobuxoviridine, moenjodaramine, buxamine-C and cyclorolfeine were also isolated. INTRODUCTION The family Buxaceae is known to contain several species occurring in temperate regions of both hemispheres and at only higher elevations in the tropics. Hutchinson in his most recent work merged the purely African genus Notobuxus Oliv. containing eight species with Buxus Cl]. Buxus hildebrandtii Baill. is the only indigenous member of the family known to occur in Ethiopia. Chemical studies on Buxus species occurring in Asia, in particular B. papilosa Schneider and B. sempervirens L. have resulted in the isolation and characterization of several but closely related series of steroidal alkaloids [2]. Some of these alkaloids are known to exhibit antimalarial and antituberculosis properties [2]. Except for a report on B. madagascarica [Z], it appears that none of the African species belonging to the genus Bums have been subjected to a phytochemical study. Our investigation of the leaves of B. hildebrandtii, collected from Ethiopia, has resulted in the isolation and structure elucidation of several steroidal alkaloids. We report here three novel and some known steroidal alkaloids. The type of alkaloids present indicate the close relationship of this African species with other members of the genus Buxus. RESULTS AND DISCUSSION An ethanol extract of the leaves of B. hildebrandtii proved to be a rich source of alkaloids, which after separation by chromatographic techniques yielded several known as well as some hitherto undescribed Buxus alkaloids. It was possible by means of spectroscopic methods and comparison of physical data with literature reports [3-83 to identify the following known alkaloids: cyclomicrobuxamine (l), buxamine-A (2), buxamine-C (3), cyclobuxoviridine (4), moenjodaramine (5) and cyclorolfeine (6). *Author to whom correspondence should be addressed. We describe below the structure elucidation of three novel steroidal alkaloids isolated from B. hildebrandtii. 0(30)-Benzoyl-16-deoxybuxidienine-C (7) gave by HR mass spectrometric analysis a CM]’ at 518.3860 which suggested the molecular formula C,,H,,N,02. The UV spectrum showed absorption maxima at 238 and 246 nm with shoulders at 225 and 254 nm, characteristic of a 9(10+19)abeo-diene system [9]. The presence in the mass spectrum of a base peak at m/z 72 due to CGH,,NI+ clearly indicated that the dimethylamino group is located at C-20. Furthermore, a peak at m/z 105 [C,H,O]+ suggested the presence of a benzoyl substituent. The ‘H NMR (CDCI,, 400 MHz) showed three Cmethyl singlets at 60.67, 0.69 and 0.72 corresponding to the tertiary methyl protons at C-32, C-31 and C-18, respectively. A doublet integrating for three protons at 60.85 was due to the C-21 secondary methyl protons. A multiplet at 65.52 and a broad singlet at 66.00 were assigned to the vinylic protons at C-l 1 and C-19, respectively, and the two AB doublets centred at 64.11 and 4.56 (J = 11.4 Hz) were due to the geminal methylene protons at C-30. The N,-methyl protons resonated at 62.37 while the N,-dimethyl protons appeared as 6H singlet at 62.19. Three sets of multiplets centred at 67.43, 7.54 and 8.03 resulted from C-3’/C-5’, C-4’ and C-2’/C-6’ protons of the benzoyl group, respectively. The assignments of the remaining protons, established by means of ‘H-‘H COSY and hetero-COSY are given in the Experimental section. The benzoyl group in the compound was placed at position 30 based on the following observations. The IR spectrum suggested the presence of a secondary amine (3400 cm-‘) and an ester carbonyl(l710 cm- ‘) ruling out an alternative structure in which the benzoyl group would be attached to N, and a hydroxyl group at C-30. Further the ‘HNMR spectrum of compound 7a, the product obtained upon cleavage of the benzoyl moiety, showed an upfield shift of 0.48 and 0.82 ppm for each of the methylene protons of C-30, respectively, clearly indicating the attachment of the benzoyl moiety on the 1293 1296 ATTA-UR-RAHMAN et ul. s, Me), 2.14 (3H. s. 21-Me), 2.43 (3H, s, N-Me), 3.00 (1H. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFE d, 428 [MI’ (4), 413.3551 (C,,H,,N,O), 369, 357. 344, 342, 314. J 1,=,,hb=h.6Hz. H-17x), 4.88 (1H. m, H-168). 13CNMR: 631.5 282, 183, 157. 143, 91. 73. 72.0814 (C,H,,N) (100). (C-l), 26.9 (C-2), 68.6 (C-3). 39.9 (C-4). 47.5 (C-5). 21.2 (C-6). 26.1 30-Yorhtrwamine-,4 (9). C,,H,,NL. Amorphous. [r& - 13’ 398.3666; CZTH,,N2 398.3661. UV (C-7). 48.4 (C-8). 19.2 (C-9), 26.2 (C-l l), 32.6 (C-12). 47.7 (C-13), (CHCI,). Found: [M]’ 45.9(C-15). 72.O(C-16),70.6(C-17), li.O(C-18),30.1 (C-19). 209.9 I.,,,.~ nm: 238.246 with shoulders at 228 and 254. IR vzyF’lcm ‘: (C-20). 31.2 (C-20, 25.8 (C-30). 20.7 (C-31). 20.7 (C-32), 35.6 (N; 1596 (C=C). ‘H NMR: 67.52 (11-1. m, H-3x). 5.54 (IH, m, H-11), Me). EIMS m:z (xl. int.) 387 (40). 372.2899 (C,,H,,N02) (50). 5.88 (IH. hr s. H-19). 2.40 (II-i, m, H-20). 0.87 (3H, (1, J,,:,, 30 (0)-Bm;oyl-16-deoxyhuxidienine-C (7). Gum [alo -75” =6.4 Hz, Me-21).0.72(3H.s. Me).0.75(3H.s, Me), l.O2(3H,d, J (CHCI,). Found: [M] ’ 518.3860; C,+,H,,,NZOZ requires =6.4 Hz), 2.23 (6H. .v, N,-(MejZ), 2.21 (6H. s, N,-(Me12). EIMS 518.3872. UV i.,,, nm (log c): 225sh (4.49). 238 (4.53). 248 (4.49). nt! z (rel. int.): 39X LM] ‘, 383.3405 (C,,H,,N,). 369,356.354,308. 254sh (4.23). IR I’,,, cm I: 3400 (N--H), 2900, 17 10 (ester C=O), 279, 239. 225. 183. 169. 167. 157. 129, 119, 91. X3, 72.0813 1600 (C=C). ‘H NMR: 62.24 (IH, m, H-l/Q, 2.28 (IH. m, H-lx), (C&H, ,,N. 1.42 (IH, m. H-‘/l). 2.17 (IH, M. H-23). 2.54 (1H. dd. J,,,zz = 11.8 Hz, J 31 ,,=4.2 Hz. H-3x) 1.84 (IH, nr, H-5~) 1.28 (1H. m, Ackno~lmyc~mt~~.s~ A.Y. is grateful to the International ProH-6[1), 1.54(lH, m. H&). 1.56(lH, m. H-7/1), 1.89(lH,m, H-7r), gram m the Chemical Sciences (IPICS, Sweden) for a fellowship 2.13(1H,m.H-8~),5.52(1H,~n.H-I1~.2.00(1H,m,H-12~~),2.09 that enabled him to spend two months at H.E.J. E.D. is grateful (lH,m.H-12r)l.27(lH,m,H-l5~~),1.39(IH,m,H-l5r).l,45(lH, to SAREC (Sweden) for a research grant. Dr Sebsebe Demissew (National Herbarium. Addis Ababa University) is thanked for m, H-16/?), 2.05 (lH, m, H-16%), 2.47 (IH, m, H-17%).0,72(3H, s, authentication of the plant material. Me-18). 6.00 (IH. br s. H-19). 2.49 (IH, m, H-20). 0.85 (3H, d, -67Hz. Me-21), 4.11 (lH, d, J,,,=l1.4Hz H-30), 4.56 ;:1;;=, , 4cm=11.4 Hz. H-30). 0.69 (3H. s, Me-3 I ).0.67 (3H. s, Me32). 2.37 (3H. s. N,-Me). 2.19 (6H. A, N,-(Me),), 8.03 (2H, m. HREFERENCES 2’!6’). 7.43 (ZH, m, H-3,/5’). 7.54 (1H. m, H-4’). ‘“CNMR [see 1. Hedberg. I. and Edwards. S.. (eds), (1988) Flora c$Ethiopia Table I]. EIMS m/z 518 [Ml+ 497,447.396.369,367, 354,326. Vol. 3. Addis Ababa. Ethiopia. 268. 211, 185. 183, 171. 119, 105. 84. 73, 72. 2. Nisa, M. (1985) Ph.D. thesis. H.E.J. Research Institute of Hydrolwis of compound7. Compound 7 (6 mg) was dissolved Chemistry. University of Karachi. in 3 ml of methanolic NaOH (1 “A) and the soln allowed to stand 3. Atta-ur-Rahman, Ahmed. D., Choudhary, M. I., Sener, B. for 12 hr at room temp. Solvent was removed and the product and Turdoz. S. (1989) I;ilorerapia LX, 439. partitioned between H,O and CH,CI,. The debenzoyl product 4. Glasby, J. S. (1975) I<‘nc)c,/opedicl r$ rhc AIknloids, Plenum 7a (4 mg) was obtained from the CHZCI, layer. 7a: amorphous, Press. N.Y. [a], +84 (CHCI,). Found: [M]’ 414.3611; C2,!H,,N,0 rc5. Shamma. M.. Georgiev. V. St., Miana. G. A. and Khan, F. S. quires 414.3610. UV ;.~~~” nm: 238, 246, with shoulders at 228 (1973) Ph~rochemrsrr~ 12. 205 I. and 254. IR ~~~~ilcm~‘: 3650 (OH) and 3400 (NH). ‘H NMR: 6. Nakano. T., Terao, S. and Saehi, Y. (1966) J. Chem. Sot. (C), 5.53 (IH, m, H-II), 5.99 (IH, hr s, H-19), 0.86 (3H, d, Jzo,zl 141’. =6.4 Hz. H-21). 0.69 (3H. s. Me), 0.74 (3H. s. Me), 0.X8 (3H, s, 7. Atta-ur-Rahman. Nisa, M. and Farhi, S. (19X3) PlantaM ed. Me). 3.74 (IH, d, J,,.,=9.4 Hz, H-30) 3.63 (IH. d. J,,,=9.4 Hz. 49, 126. H-30), 2.21 (6H. \. N,-(Me),). 2.45 (3H. s. N,,-Me). EIMS m.‘z(rel. 8. (rern$. V. and Sorm. F. (1967) in The Alkaloids (Manske, R. int.): 414 CM]’ (lOO), 399 (IO), 344 (5). 343 (I 5). 328 (5). 768 (5). 253 (3), 95 (38), 91 (44). H. F.. ed.), Vol. XI. p. 305. Academic Press. New York. 9. Atla-ur-Rahman. Alam, M. and C’houdhary (1988) J. Nar. 30-Hydro.uyhuxamine-A (8). Amorphous, [z]~, + 61 (CHCI,). Prod. 51, 309. Found: [Ml’ 428.3766; C,,H,,N,O requires 428.3767. UV IO. Atta-ur-Rahman, Nisa. M. and Jahan. K. (1985) Ph~tochernA,,,,, nm: 225 (sh), 238, 248, 254 (sh). IR vmar cm I: 3345 (OH). istry 24, 1398. ‘HNMR:62.l6(lH,m.H-1/3),2.28(IH,m,H-lr).1.59(1H,m.HIl. Kupchan, S. M., Kennedy. R. M., Schlcigh. W. R. and Ohta, 2/j), 1.73 (lH, m, H-21), 2.62 (lH, dd, J=3.7. 12.5 Hz. H-3x). 5.52 G. (1967) Tetrahedron 23, 4586. (1H.m.H-11),2.00(1H,m,H-12~),2.10(1H.m.H-12c(),2.56(1H. m, H-20/Q 0.89 (3H. d, J 21,z0=6.4 Hz, Me-2l), 3.60 (IH, d, J,,, 12. Sangare. M., Kuong-Huu. F.. Herlem, D., Milliet, A., Septe, B., Berenger, G. and Lukacs. G. (1975) Tcwuhedron Lerters = 10.4 Hz, H-30), 3.71 (lH, d, J,,,= 10.4 Hz. H-30). 0.74 (3H. s, 1791. Me), 1.00 (3H. s, Me), 0.68 (3H. s. Me). 2.25 (6H, s. N,-(Me),), 2.30 13. Guilhem. J. (1975) T~rruh~~dron f.c~tters 2937. (6H. s. N,-(Me),). “C NMR (see Table I ). EIMS m/z (rel. int.):