TETRAHYDRORHO;LIBIFOLISE, -1 FURTHER CONSTITCENT O F L U P I S U S OSCA R-HA GGHTII AND L . T R U S C A T U S \ I . ~ S U E LF. B.LL.~SDRIS and A . DOUGL.\S KISGHORS Depnrtnieiit of P h n r ? , i n c o g i i o s ~atid Piinrniiicologg, College oj' Pharmacy, L-)lizmersitg oj' Zliiiiois cit t h e -1ledical Ceizler, Ciiicogo, IL 60612
Gas chromatography, mass spectrometry (gc ms) has become a useful analytical tool for tlie rapid identification of quinolizidine alkaloids in species of the genus Lupiiiiis, since diastereomeric pairs can usually be resolved (1-6). Hon-ever, not all solute components in lupine extracts studied by gc nis may be readily identifiable (2, 4, 6),and, in one such study, n-e were initially unable to identify a minor quinolizidine alkaloid constituent occurring in extracts of both L. Oscar-hazcghtii and L.f r i t i i c a f i i s (6), as no molecular ion peak \\-as apparent in the ma. compound. I n the present work, this previously unassigned compound was isolated from L . Oscar-hazightii and tentativelJcharacterized as (+)-tetrahydrorlionibifoline, by comparison of the physical data arid pnir spectrum of t!ie isolate with literature data ( i ) . Since the detailed mass spectrum of this compound has not been published, we confirmed its identity by direct coniparison (gc nis, tlc) with an authentic sample synthesized from angustifoline using a modified Eschv-eiler-Clarke reagent (S) . Tetrahydrorhombifoline a-as detected in L. trztiicafits (.stems, roots, leares and flowers) by gc, ms and tlc only, since insufficient material 1T-a.savailable to isolate this compound and perform physical and spectroscopic measurements. Tetr:ihydrorhombifoljne has been postulated as an intermediate in bio-
synthetic pathways from lupanine to cytisine (9) and from lupanine to S-met1i)-ltetrahydrocytisine (10). Seitlier cytisine nor S-met hyltetrahydrocytisine are known to accumulate in tlie genus L i I p i i i i u , and tetrahydrorhonibifoljne appears to be rare in tlie lupines (11). IT-e have detected this compound only in L . oscarhairghtii arid L. truiicaizrs among over 25 lupines investigated. It may be pointed out that based on the relative abundance of its mass spectral fragment ions, the unidentified alkaloid A of K i n k and coworkers, observed recently in a L . polypliyllus cell suspension culture (1), is probably tetrahydrorhombifoline, a known constituent of the seeds of this species (12). I n addition, we ~vouldlike to draw attention to tke posGbility of confusion of tetral:ydror!iombifoline with ructural isomer, S-me foline, during gc, ms ana The latter compound, recently claimed to be a constitue:it of L . polypliyllus (13), was prepared from angustifoline by a literature method (6). Both alkaloids possessed the same retention time on the glc stationary phase used and exhibited identical mass spectral fragment peaks that varied only in relative abundance, although tlie compounds were separable by tlc. This example demonstrates the need to substantiate gc ms screening data of lupine quinolizidine alkaloids by tlc to prevent errors in compound identification.
Journal of Satural Products
496
EXPERIMENTALi PL.AST Y.ITERIAL.-Lyophilized 80% ethanolic extracts of t h e stems (10 g) and t h e inflorescences (in fruit, 10 g) of Lupinus oscar-hnughfii C . P . Smith (Leguminosae), collected in Colombia in hlarch, 1972, and of t h e combined stems, roots, leaves and flowers of L.truncafiis S u t t . ex Hook. 6: iirn. (Leguminosae) (3 g ) , collected in California in March, 19G5, were supplied through the Developmental Therapeutics Program ( S a t u r a l Products Branch) of the National Cancer Institute, formerly t h e Cancer Chemotherapy S a t i o n a l Service Center, Bethesda, hlaryland. J-oucher specimens representing these collections are deposited a t the Herbarium of the S a t i o n a l Arboretum, Agricultural Research Service, U.S. Department of Agriculture, Washington, D . C .
EXTRACTION OF ALKALOIDS
ASD CHROM.\METHODS.-Crude alkaloidal extracts of the two L. oscnrhnughlii plant parts, and of L.iruizcniiis, were prepared from t h e original ethanolic extracts a s described previously ( 5 , 6 ) . These were then subjected t o gclms on 3'5 O\--17 on Gas Chrom Q (100-120)) with the temperature programmed 180-310", 4' min-1, and t h e f l o ~r a t e of helium 18 ml min-'. T h e injector temperature was set a t 260", t h e interface separator temperature at 240°, and spectra were recorded with 6 see between scans. T h e alkaloidal extracts were chromatographed by tlc on silica gel G H L F Z in S1, chloroform-methanol-ammoniuni hydroxide solution (85:15:1), Sp, methanol-ammonium hydroxide solution (19:l) and Sa, diethyl ether-methanolammonium hydroxide solution ( 1 O O : l O : l ) . TOGR.IPHIC
SCREESISG
1SOL.ITION OF T E T R 1 H I D R O R H O J I B I F O L I K E . -
Tetrahydrorhombifoline was isolated by preparative tlc of the crude alkaloidal extracts of both the stems and the fruiting inflorescences of L. oscnr-hnughfii on silica gel G H L F 2 in S, (Ri! 0.82) and S?,(Ri, 0.57). This compound, which was obtained a s an oil, exhibited the following d a t a : 'Optical rotations were obtained on a Perkin-Elmer 241 polarimeter, and the ir spectrum was measured on a Perkin-Elmer 710 instrument. T h e pmr spectrum was recorded on a 1-arian T-GOA instrument, operating a t 60 MHz, with a Kicolet model TT-7 Fourier Transform attachment. Tetramethylsilane was used as t h e internal standard, and chemical shifts are reported in 6 (ppm) units. Combined gcims (70 e l - ) was performed on a T-arian M A T 112s mass spectrometer, equipped with a 1.arian 166 d a t a system, linked t o a T-arian 1400 model gas chromatograph. T h e low resolution mass spectrum was also recorded on this mass spectrometer. *Analtech, Inc., Xewark, Delaware.
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260", [a1~&,+150",[a]:&+83", [ c ~ ] ~ ~ ~ and +77" [a]$',+iO" (e 0.03, ethanol); i r ( P max) ( K B r ) 3170, 2850, 2760, 1625, 995 and 905 cm-l; p m r (CDC13) 6 1.1e3.20 (19H, m ) , 3.46 ( l H , m, H-G), 4.70 ( l H , bd, J = 1 4 Hz, H-lOeq.),4.83,5.08 ( 2 H , m , C=CHp, Hp-17) and 5.70 ( l H , bm, H-16); ms, m / z (20 eV) 248 (ML,2 5 ) , 208 (14)) 207 (loo), 136 (l), 112 (ZO), 108 ( l l ) , 94 (3), 83 ( 5 ) , 59 (3) and 58 187): ec/ms. R t . 12.7 min. ms. ntlz h I L missing, 208 (13), 207 (94), 112'(19); 108 ( l o ) , 94 (5), 58 (loo), 55 (E),42 (13) and 41 (18); tlc, S1, Rr 0.82, Sp, R I 0.57 and S3, R i 0.74. PREPAR.iTION OF TETR.IHYDRORHOMB1FOLINE A N D ~ - ~ I E T H Y L A N G U S T I ~ - O L I NFROM E
.~sGusT~FOL~~E.-Angustifoline (3 mg) was obtained as previously described (6) and divided into two portions. Treatment of angustifoline with formaldehyde and sodium borohydride in aqueous methanol under published reaction conditions (8) resulted in t h e formation of tetrahydrorhombifoline. Methyl iodide in acetone was used t o synthesize S-methylangustifoline from angustifoline (8). These reaction products were purified by preparative tlc in SI (Rr, 0.82, tetrahydrorhombifoline, 1 m g j and Sa (Rr, 0.68, S-methylangustifoline, 1 m g ) . The tetrahydrorhombifoline so produced was identical t o t h e isolate from L. oscnr-hnughtii b y gc/ms and tlc in S1-S3. S-llethylangustifoline exhibited t h e following d a t a : eclms. R t . 12.7 min. ms. nil2 hf' m k n g , 2008 (14): 207 (loo), 112 (45), 108 (8), 94 ( 7 ) , 58 (2G), 55 (14), 42 (12) and 41 (18); tlc, Si,Ri 0.82, S p , R i 0.48, Sa, Ri 0.68.
ACKNOWLEDGMEKTS We are grateful t o D r . J. L. Hartwell, formerly Chief, Natural Products Section, D r u g Research and Development Branch, Chemotherapy, National Cancer Institute, Bethesda, Maryland, for supplying t h e plant eytracts used in this study, and t o Sandra Saufferer, Economic Botany Laboratory, BARC-East, United States Department of Agriculture, Beltsville, Maryland, for checking on the identity of t h e species studied from specimens deposited in t h e Herbarium of the National Arboretum. M.F.B. is grateful t o t h e American Foundation for Pharmaceutical Education for the award of the Sydnor Barksdale Penick Memorial Fellowship (1978-81). Receaaed 8 D e c e m b e r 1980
LITERATURE C I T E D Keeler, R.F., Teratology, 7, 31 (1973). Anderson, J. S . and R. 0. Martin, J . Org. C h e m . , 41, 3441 (1976). Keller, W. J . and S. G. Zelenski, J . Pharm. Sci., 65, 430 (1978). Wink, hl., L. Witte, H . 3 1 . Schiebel and T. Hartmann, Planta 'Wed., 38, 238 (1980).
Jul-ilug 19811 5.
6.
i. 8.
9.
Balandrin et al. : Tetrahydrorhombifoline
Kinghorn, A. D . and S. J. Smolenski, Pluntn X e d . , 38, 280 (1980). Kinghorn, 8.D., 31. A . Selim and S. J. Smolenski, Phytochemistry, 19, liO5 (1980). Bohlmann, F., E. Rinterfeldt, B. Janiak, D. Schumann and H. Laurent, Chem. Ber., 96, 2254 (1963). B r a t e k - W i e ~ i 6 r o ~ - s kM. a , D., J . M o l . S t r u t . , 55, 69 (1979). Sowacki, E. K . and G. R . Waller, Phytochemistry, 14, 165 (1975).
497
10. Salatino, A. and 0. R . Gottlieb, Bzochem. System. Ecol., 8 , 133 (1980). 11. Mears, J. A. and T. J. Mabrj-, In “Chemotaxonomy of the Leguminosae” (J. A. Harborne, D . Boulter and B. Turner, e d s . ) , Academic Press, London, 1971, p . i 3 . 12. Bohlmann, F. and D. Schumann, Chem. Ber., 98, 3133 (1965). 13. Wink, M. and T . Hartmann, Planta X e d . , 40, 149 (1980).
