zyxw zyxw zyxw zyxwv FUROQUINOLINE AND PYRANO-2-QUINOLONE ALKALOIDS OF VEPRIS STOLZII' zyxwvutsrqponmlkjihgfedc SAMI A. KHALJD and PETER G. WATERMAN* zyxwvut zyxw zyxwv Phytochemistry Research Laboratory, Department of Pharmaceutical Chemistry, University of Strathclyde, Glusgow G1 I X W , Scotland, U.K. hSTUCr.-siX alkaloids and the pentacyclic triterpene lupeol were isolated from the stem bark of Vepris stolzii Verdoorn (Rutaceae). Three of the alkaloids were identified as the furoquinolines skimmianine (1) and gays-fagarine (3) and the pyrano-%quinolone veprisine (2) by comparison with authentic samples or literature data. The remaining three alkaloids, all pyrano-2quinolones, were novel. They were identified, on the basis of spectral data and positive Gibbs tests on their hydrolysis products, as N-methyl+(3',3 n-dimethylallyloxy)-flindersine (4), N-methyl-7methoxy-tb (3",3'-dimethy1a11y1oxy)-flindersine (5) , and N-methyl-7-methoxy-8- (2',3 'epoxy-3n,3'-dimethylallyloxy)-flindersine (7). The chemotaxonomic significance of these alkaloids was discussed. zyxwv The African species of the subfamily Toddalioideae (Rutaceae) have proved (1-5) to be a good source of the furoquinoline, pyranoquinolone and acridone alkaloids that typify the family as a whole (6). Limonoids have also been found in a number of species (1, 5, 7, 8), but coumarins have not so far been recorded from any species other than the atypical Toddalia aculeata (9). As part of our contiiuing study of the distribution of secondary compounds in this group ( 1 , 2 , 8 ) , we have investigated the stem bark of Vepris stolzii, a small tree of montane forest, indigenous to east Africa (10, 11). No previous phytochemical examination of this species has been reported. RESULTS Column chromatography of the petroleum spirit extract of the stem bark yielded the pentacyclic triterpene lupeol and six furoquinoline and pyranoquinolone alkaloids. Lupeol, skimmianine (1) and veprisine (2) were identified by direct compsrison with authentic samples (1). A third alkaloid, gamma-fagarine (3), was identified by comparison of spectral data with that published (12, 13). The remaining three alkaloids, which were all isolated as oils, exhibited the spectroscopic characteristics of AT-methylpyrano-2-quinolones. Thus, their complex uv spectra showed no shift on addition of HC1, and the ir spectra had carbonyl absorption at 1640 cm-l, typical of 2-quinolones (14, 15). Their 'H nmr spectra exhibited a sharp singlet (6H) at 6 1.50 together with an AB quartet centred at 6 5.50 and 6.75, typical of a 2,2-dimethylpyran system, and a further singlet (3H) at about 6 3.78 for N-Me. The remainder of the 'H nmr spectra of the three alkaloids varied, indicating that they differed in substitution of the benzene ring. The simplest had a MW of 325, shown by exact mass measurement to be C ~ H B N O ~Eims . showed facile loss of m/z 68 (C5Hs+)indicative of a prenyloxy side-chain. This was confirmed by the 'H nmr spectrum which exhibited resonances at 6 1.80 and 1.83 (3H each), 4.64 (2H) and 5.53 (1H) typical of a 3,3dimethylallyloxy substituent. The remaining resonances were for three aromatic protons which, from their coupling interactions, had to be adjacent to each other. One of the aromatic protons occurred as a double doublet at 6 7.87, its highly deshielded position being typical of H-5 (2, 3). After hydrolysis, the alkaloid gave a positive Gibbs test for a phenol with a free para-H. On the basis of the above, the alkaloid was identified as N-methy1-8-(3",3"-dimethy1al1y1oxy)-flindersine (4), which appears to be novel. The second novel alkaloid had a MW of 355, shown by exact mass measurement 'Paper 15 in the series "Chemosystematics in the Rutaceae." For paper 14 see W. E. Campbell, G. J. Provan and P. G. Waterman, Phytochemistry-in press. 'Author to whom correspondence should be addressed. 343 zyxwv zyx zy zyx [Vol. 45, No. 3 Journal of Natural Products 344 to be CZ1Hz6NO4.The 'H nmr spectrum differed from that of (4) by showing only two aromatic protons, as an AB quartet exhibiting ortho-coupling, and an additional OMe resonance at 6 3.93. The strongly deshielded position of one of the aromatic protons (6 7.70) required that it be assigned to G 5 permitting the assignment of eit,herstructure 5 or structure 6 to this compound. A Gibbs test on the hydrolysis product proved positive indicating a free para-H with respect to the 0-prenyl substituent of the original alkaloid and confirming its identification as N-met hyl-7-met hoxy-8- (3",3"-dimethylallyloxy)-flindersine (5). zyxwvutsrqponmlkjihgfedcbaZYXW OMe OMe 1 R=OMe 3 2 zyxwvutsrq R = R =OMe,R = H 2 3 1 ,11 4 5 6 R=H 2.. $. R =OCHZCH:C(Me)2, R1 = R2 = H 3 R3 =OCH2CH:C(Me)2, R2 =OMe, R, = H R2 =OCH2CH:C(Me)2, R3 =OMe, R, = H 7 R =OCH2CH - C(&) , R =O&, R1 = H 2 2 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQP 3 '0' 8 R 1 =R 2 =OMe,R 9 R2=O&,R 1 3 =H =R3=H The third alkaloid had a MW of 371, shown by exact mass measurement to be CzlH25RTO5. The 'H nmr spectrum was identical to 5 except for the signals for the 0-prenyl side-chain in which both Me and H-2mresonances showed considerable shielding. These changes are in accord with requirements (16) for 2,3-epoxidation of a 3,3-dimethylallyl side-chain and the presence of the oxygenated substituent was confirmed by facile loss of m/z 84 (C5HsO+) in the eims. A positive Gibbs test on the hydrolysis product confirmed a substitution pattern identical to 5 and permitted the alkaloid to be identified as N-rnethyl-7-methoxy-8-(2",3"epoxy-3m,33-dimethylallyloxy)-flindersine(7). DISCUSSION Six species of Vepris have now been reported to contain alkaloids; V. stobzii, V. Zouisii (3-5) and V . heterophylla (17) from Africa, and V. ampody (18), Y . bilocdaris (18) and V . pilosa (19) from Malagasy and the Indian subcontinent. zyx zyxwvut zyxw zyx zyxwvut zyxw zyx zyxwvu zyxw zyxw May-Jun 19821 Khalid and Waterman: Alkaloids of Vepris stolzii 345 All six species produce quinoline alkaloids, but only the latter three have so far been reported to produce acridones. According to Gilbert (20) V . louisii is most closely allied to V , stolzii. The close similarity between the alkaloids of these two taxa, they are the only two species of Vepris to produce pyrano-2-quinolones and in both substitution is restricted to C-7 and C-8, bears out this contention. However, V . louisii has a!so yielded limonin ( 5 ) , and this could not be detected in V . stolzii. Pyrano-2-quinolones are rare in the African Toddalioideae. The only other sources so far reported are the Oricia species, 0. suaveolens (I), which has yielded oricine 8, and 0. renieri (2), which has yielded 2, 8 and N-methyl-7-methoxyflindersine (9). In addition, both Oricia species have yielded acridones. In pyrano-Bquinolones from both Oricia species substitution at C-6 occurs as well as at C-7 and C-8. However, whilst this distinction holds at present for pyrano2-quinolones it should be noted that c-6 substitution occurs among the furoquinolines of all of the other four Vepris species. EXPERIMENTAL3 PLANT umm.-The zyx stem bark of Ve'epris stolzii Verdoorn was collected in montane forest a t Moo m from near Gisovu in the prefecture of Kibuye, Rwanda, in February 1980. A voucher specimen (D. Bridson 440) has been deposited a t the herbarium of the Royal Botanic Gardens, Kew, England. EXTRACTION A N D ISOLATION OF THE ALKALOIDS.-Ground stem bark (300 g) was extracted with petroleum spirit (bp 4040') and then chloroform. The petroleum spirit extract was concentrated and yielded a solid (3 g) which, after recrystallization from petroleum spirit/ ethyl acetate, was identified as lupeol (mixed mp, ir, or, tlc). Concentration of the chloroform extract gave a brown oil (7 g), which was shown by tlc (silica gel, solvent A) to contain six blue fluorescent spots that gave positive reactions for alkaloids. The oil was subjected to cc over silica gel. Elution of the column with petroleum spirit (bp S c r S o O ) containing 15% ethyl acetate gave 17 mg of N-methyl-7-methoxy~(3n,3n~imethylallyloxy)-fl~dersine (5) followed by 45 mg of veprisine, 2, (mixed mp, 1H nmr, eims, uv, ir, tlc). Further elution with petroleum spirit containing 18% ethyl acetate gave a mixture of two compounds which were separated by plc on silica gel (solvent B) to give 25 mg of N-methyl-8-(3n,3n-dimethylallyloxy)flindersine, 4, (Rf 0.40) and 18 mg of N-methyl-7-methoxy-~(2n,3n-epoxy-3n,3n-dimethylallyloxy)-flindersine, 7, (Rf 0.27). Final elution with petroleum spirit containing 35% ethyl acetate gave a mixture of the remaining two alkaloids, which were separated by plc on silica gel (solvent A) to give 32 mg of gamma-fagarine, 3, (Rf 0.17) mp 138" ('H nmr, eims, uv, ir), lit. (12) 138", and 47 mg of skimmianine, 1, (Rf 0.10), (mixed mp, 'H nmr, eims, uv, ir, tlc). IDENTIFICATION OF NOVEL A L K A L O I D S . - - I % ~ ~ ~ ~ ~ ~"3 - Ln-dimethylallyloxy)-flindersine %(~ (4) was an oil showing the following: Eims ( m / z , re1 intensity): 325(47), 310(2), 257(52), 242(100), 213(12); exact maw 325.1680, calc for CZOHZINOI: 325.1678; uv X max (EtOH) 235, 262, 270, 3%h, 338, 352, 366; fHC1-no change; ir y max (CHC13) 1640, 1620, 1600; 1H nmr (90 MHz, CDClq): 1.50 ( 8 , 6H, 2'-(CH3)2), 1.80, 1.83 (2 x S, 2 x 3H, 3"(CH3)2), 3.74 (s, 3H, N-CHI), 4.64 (d, 2H, J 7, ln-CHZ),5.53 (t, lH, J 7, 2'-CH), 5.48, 6.73 (AB quartet, 2H, J 10, 3'-H and 4'-H), 6.84 (dd, lH, J 9 and. 8, 6-H), 6.88 (dd, lH, J 8 and 2, H-7), 7.87 (dd, lH, J 9 and 2, H-5). A solutim of the alkaloid in acetic acid and HtSO, was heated to 80"for 30 min to effect hydrolysis. The hydrolysis product was dissolved in H3B03/Na2B407 buffer (pH 9) and reacted with a saturated solution of 2,6dichloro-~benzoquinone4chloraminein water. A blue color developed indicative of a positive Gibbs test. N-methyl-7-methoxy-%(3"3 n-dimethylallyloxy)-flindersine ( 5 ) was an oil showing the following: Eims ( m / e , re1 intensity): 355(9), 338(1), 303(14), 237(39), 272(100), 257(13), 250(32), 221(4), 206(1); exact mass 355.1789, calc for CI1HfSNO4: 355.1783; uv X max (EtOH) 232, 260sh, nosh, 316sh, 326, 351, 366; HCI-no change; ir y max (CHC13) 1640, 1600, 1580; 1H nmr (90 MHz, CDCIJ): 1.49 (s, 6H, ~ ' - ( ~ H I ) z1.73 ) , (broad s, 6H, 3 n - ( C H ~ ) ~3.78 ) , (s, 3H, N-CH3), 3.93 (s, 3H, 0-CHs), 4.68 (d, 2H, J 7 , l"-CH,), 5.52 (t, l H , J7,2"CH), 5.48, 6.73 (AB quartet, 2H, J 10, 3'-H and 4'-H), 6.83, 7.70 (AB quartet, 2H, J 9, 6-H and 5-H). The hydrolysis product gave a positive Gibbs test. N-methyl-7-metho~y-t+(2~,,3 n-epoxy-3n,3n-dimethylallyloxy)-flindersine(7) was an oil showing the following: Eims ( m / z , re1 intensity): 371(85), 356(100), 314(2), 305(1), 286(19), n2(73), 257(17); exact mass 371.1718 calc for C I L H ~ ~ N371.1733; O~: uv X rnax (EtOH) 235, 263, n 3 , 315sh, 336, 349, 368. ir y max (CHCls) 1640 1610, 1595; 1H nmr (90 MHz, CDC13): 1.38, 1.40 (2 x s, 2 x 3H, 3 n - ( 6 H ~ ) t )1.49 , ( s , 6H, 2'-(6H3)~),3.21 (t, 1H, J 5, 2"-CH), 3.82 (s, 3H, N-CHd, 3.93 (s, 3H, @CHI), 4.26 (d, 2H, J 5, l"-CH,), 5.50, 6.75 (AB quartet, 2H, J 10, 3'-H + zyxwvu JMelting points were determined by means of a Kofler hot plate and are uncorrected. 'H nmr spectra were run on a Perkin-Elmer R.32 instrument. Eims (probe) were obtained a t elevated temperature and 70 eV. For tlc: solvent A refers to to1uene:ethyl acetate-formic acid (5:4:1) and solvent B to benzene:ethyl acetate (3:l). 346 zyxwvu zyxwvut zyx zyxwvutsrq zyxwv Journal of Natural Products and 4’-H), 6.93, 7.72 (AB quartet, 2H, I 9, 6-H and 5-H). positive Gibbs test. [Vol. 45, No. 3 zyxwvutsrqponm The hydrolysis product gave a ACKNOWLEDGMENTS The supply of plant material by Dr. D. Bridson, Royal Botanic Gardens, Kew, is gratefully acknowledged. One of us (S.A.K.) wishes to thank the University of Khartoum for the award of a scholarship. This study was supported by an equipment grant (no. A 83754) from the Science Research Council. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Received 14 September 1981 LITERATURE CITED 1. P. G. Waterman, 1. A. Meshal, J. B. Hall and M. D. Swaine, Biochem. Syst. Ecol., 6 , 239 (1978). 2. S. A. Khalid and P. G. Waterman, Phytochemistry, 20, 2761 (1981). 3. J. F. Ayafor, B. L. Sondengam and B. Ngadjui, Tetrahedron Lett., 21,3293 (1980). 4. J . F. 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