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Phytochemistry,Vol. 34, No. 3, pp. 868-870, 1993 0031-9422/93 6.00 0.00Printed in Great Britain. 0 1993Pergamon Press Ltd

WITHANOLIDES OF DATURA FASTUOSA LEAVES*M. MANICKAM, ANJANA SINHA-BAGCHI,~ SUBHASH C. SINHA,? MOHINI GUPTA and ANIL B. RAYS

Department of Medicinal Chemistry, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, India(Recei ved10 March 1993)

Key Word Index-Datura fastuosa; Solanaceae; withanolides; withafastuosins A and B.

Abstract-The leaves of Dat ura fastuosa yielded two new withanolides, withafastuosins A and B, in addition towithametelin, previously reported from D. metel. The structures of the withanolides were established on the basis ofdetailed spectral and chemical evidence.

INTRODUCTIONIn continuation of our search for withasteroids fromDatura species [l-5], we have isolated two new with-anolides from Daturufastuosa in addition to withametelin(l), a hexacyclic withanolide previously reported from D.metel. The structures of the new compounds, namedwithafastuosins A and B, have been advanced, respect-ively, as 2 and 3 on the basis of detailed spectral andchemical evidence. This is the first report of the occur-rence of withanolides in D. astuosa.

RESULTSAND DISCUSSIONWithafastuosins A and B, isolated from the leaves of

D. astuosa, were recognized to be the close relatives of 1,the major withanolide of D. mete1 2], from their spectralanalysis, which also disclosed the relationship betweenthe two withafastuosins. H NMR spectral comparison ofthe two withanolides revealed withafastuosin A,C,sH,&, (m/z 472), to be the dihydroderivative ofwithafastuosin B, CZBH3s06 (m/z 470), mp 255, whichwas confirmed by catalytic reduction of the latter to theformer. Like 1, withafastuosin B showed in its H NMRspectrum signals for three tertiary methyls (S0.65, 1.24and 1.29, 3H, s each) and two olefinic hydrogens of asteroidal 2-en-l-one system (66.85, lH, ddd, J= 10.0, 6.4,2.4 Hz, H-3, and 6.00, lH, dd, J= 10.0, 2.4 Hz, H-2). Italso showed the diagnostic withanolide hydrogen (H-22)signal as a broad singlet at 8 4.66 and not as a doubletriplet indicating the presence of a withametelin-typebicyclic side chain. Unlike withametelin, however, with-afastuosin B failed to show in its HNMR spectrumolefinic hydrogen signals of r-methylene-&lactone andH-6, and gave a monoacetate derivative (3a) on acetyl-

*Part 24 n the series of withanolides. For part 23 see J. Chem.Res. S) 234 (1993).

PPresent address: The Scripps Research Institute, 10666North Torey Pines Road, La Jolla, CA 92037, U.S.A.$Author to whom correspondence should be addressed.

ation with acetic anhydride-pyridine. The HNMRspectrum of this acetate derivative showed, in addition toother signals, a double doublet (at-6 2.58, IH, dd, J= 5.2,3.6 Hz) for C-25 methine hydrogen and two sets of signalsfor a pair of -CH-CH,-O- groupings. While one set ofthese signals (63.87, lH, d, J= 13.6 Hz; 3.57, lH, dd, J= 13.6,3.4 Hz), which remained unaltered on acetylation,

4 6

R2 H3 H, A23a AC,A2

868

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Short Reports 869was assigned to methylene hydrogens of C-21, the other(64.68, lH, dd, J= 11.6, 3.6 Hz; 4.41, lH, dd, .I= 11.6,5.2 Hz) was obviously due to the methylene hydrogens ofC-27. Withafastuosin B contains two extra oxygen atomscompared to 1 but shows three extra oxycarbon signals(659.3,62.0,63.2) in its 13C NMR spectrum indicating thepresence of an epoxide ring. A 5/?,6@poxide structurewas considered probable from the H NMR parameters(63.11, lH, d, J = 2.4 Hz, H-6) of a carbinyl hydrogensignal. Based on these observations, the structure ofwithafastuosin B was advanced as 3 which was in perfectagreement with its 13CNMR data (Table 1). Chemicalevidence in support of the structure came from a conver-sion of 3 to its dehydrated product. Thus, withafastuosinB on treatment with acetic anhydride-pyridine at 60 for16 hr gave a product which was identical with the Sfi,6/?-epoxide of withametelin (lb) [2]. The stereochemistry atC-25 is based on the observed NOE between H-25 and H-22. The orientation of the C-25 substituent has also beenwitnessed in similar withanolides, viz., daturametelins Dand G [6-J.The most significant feature of the H and 13CNMRspectra of withafastuosin A (2) is the conspicuous absenceof signals, respectively, for olefinic hydrogens and spcarbons other than those observed for two carbonylcarbons. Its 13C NMR spectral data (Table 1) also corro-

Table 1. %NMR spectral as-signments of 2 and 3 (CDCI,)C 2 31 213.0 203.32 31.8 129.23 21.8 144.24 30.2 31.05 64.3 62.06 60.3 63.2I 35.1 29.88 29.1 29.19 42.8 43.710 52.2 48.311 21.8 23.512 39.1 39.513 42.6 42.514 55.6 55.615 23.9 23.816 26.1 26.117 47.6 44.518 12.5 12.719 18.3 18.220 39.5 39.621 60.3 60.322 76.4 76.423 32.9 32.824 70.7 70.625 51.3 51.426 174.8 174.821 59.3 59.328 26.2 26.2

borate the dihydro withafastuosin B structure 2 for themolecule.Compound 1, the major withanolide of the local var-

iety of D. meteZ[2], was isolated in a relatively poor yieldfrom this plant. Isolation of withafastuosins and with-ametelin from D. asruosa reveals that C-21 oxygenatedwithanolides are not a monopoly of D. metel only andestablishes the chemotaxonomic relationship betweenD. astuosa and Dmetel.

EXPERIMENTAL

Mps were determined in open capillary and are uncorr.CC was carried out over silica gel (60-120 mesh, Qualig-ens Fine Chemicals) and TLC over silica gel (QualigensFine Chemicals). H NMR (400 MHz) and 13CNMR(100 MHz) spectra were taken with a Bruker AMX 400Spectrometer using CDCl, solns.

PIant mat erial . Dat urafastuosa Linn was collected fromthe University campus and identified by Prof. G. N.Choudhri, Department of Botany, Banaras Hindu Uni-versity, Varanasi, India. A specimen sample is beingpreserved in the department.

Extraction and solat ion of w it hanolides -3. Dried andpowdered leaves (3 kg) of D. astuosa were extracted withMeOH, and the extract was coned to a thick syrup(330 gm), diluted with Hz0 (300 ml) and extracted succes-sively with petrol-benzene (1: 1) mixt. and CHCl,. TheCHCl, extract was freed from solvent, chromatographedover silica gel and eluted first with benzene (fr. A) andthen with benzene-EtOAc (3: 1) (fr. B). Fr. A (10 mg) onrechromatography over silica gel and elution withpetrol-EtOAc (17: 3) afforded 1 (50 mg). Fr. B yielded amixt. of withafastuosins from which 2 and 3 were sepd byrepeated CC and prep. TLC (hexane-EtOAc, 3: 7).

Wi thafastuosin A (2). H NMR: 64.65 (lH, br s, H-22),3.97 (2H, nn, H-27), 3.86 (lH, d, J= 13.6 Hz, H-21), 3.62(lH, dd, J= 13.6, 3.2 Hz, H-21), 3.11 (lH, br s, H-6), 2.45(lH, dd, 5=8.4, 3.6Hz, H-25), 1.31 (3H, s, Me-28), 1.14(3H, s, Me-19), 0.62 (3H, s, Me-18); CIMS: m/z 473 [M+H]+.Dehydration of 2 o la. A mixt. of 2 (4 mg) and Ac,O (0.1

ml) in pyridine (0.2 ml) was heated at 60 for 16 hr.Solvents were removed under vacuum to give la.HNMR:66.74(1H,s,H-27),5.99(1H,s,H-27),4.62(1H,br s, H-22), 3.86 (lH, d, J= 13.6 Hz, H-21), 3.72 (lH, dd, J=13.6,2.0Hz, H-21), 3.12(1H, brs,H-6), 1.41(3H,s, Me-28), 1.15 (3H, s, Me-19), 0.63 (3H, s, Me-18); EIMS: m/z454 [M] +.

Wi tk afastuosin B (3). Mp 255, [aID -5.87 (CHCl,;1.5). HNMR: 66.85 (lH, ddd, J= 10.0, 6.4, 2.4 Hz, H-3),6.00 lH, dd, J= 10.0, 2.4 Hz, H-2), 4.65 (lH, br s, H-22),3.87 (lH, d, J=13.4Hz, H-21), 3.63 (lH, dd, 5=13.4,3.2Hz,H-21),3.97(2H,m,H-27),3.11 (lH,d,J=2.4Hz,H-6), 2.45 (lH, dd, J=8.4, 3.6 Hz, H-25), 1.29 (3H, s, Me-28), 1.22 (3H, s, Me-19), 0.65 (3H, s, Me-18); CIMS: m/z471 [M+H]+.

Dehydration of 3 to lb. A mixt. of 3 (4 mg) and Ac,O(0.1 ml) in pyridine (0.2 ml) was heated at 60 for16 hr. Removal of solvents under vaccum afforded lb.

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870 Short ReportsH NMR: 66.83 (1H, ddd, J== 10.0, 6.4 2.4 Hz, W-3), 6.74(~H,s,~-27),6.OZ(lH,dd,~=lO.O,2.8 Hz,H~2),6.~~iH,s, H-27), 4.62 (lH, br s, H-22), 3.88 (lH, d, J= 13.6 Hz, H-21), 3.72 (IH, dd, X3.6, 3.2Hz, H-21), 3.12 (IN, d, J=2.4 Hz, H-6), 1.41 (3H, s, Me-28), 1.23 (3H, s, Me-19),0.66 (3H, s, Me-18); FABMS: mjz 585 [M cCs] *; identi-cal with ~~,6~-epoxide of withametelin (co-TLC,H NMR}.

Acety ~ffr~o~ f 3 1~ 3a. To a precooled ( - 30) soln of 3(4 mg) in dry pyridine (0.2 ml), AC& (0.1 ml) was addedand the reaction mixt. was kept at -15 for 48 hr.Addition of H,O to the reaction mixt. afforded a whitesolid. This was filtered and dried under vaccum to give(4 mg) 3a. HNMR: 86.84 (IH, ddd, J= 10.0, 6.0, 2.4 Hz,H-3), 6.02 (lH, dd, J= 10.0, 2.8 Hz, H-2), 4.68 (1H, dd, J= 11.6,3.6 Hz, H-27), 4.62 flH, br s, H-22), 4.41 (ZH, dd, J=1~.6,5.2H~,H-27~,3.87(iH,d,~=~3.6Hz,~-2~)~3.57~IH,dd,~~~3.6~3.4Hz,H-21),3,~i~lH,d,~=2.4H~,H-6),2PP~~H,d~,~= f&9,2,6 ~z,~-4),2.5g~~H,dd,~=5.~3.6 Hz, H-25), 2.05 (3H, s, -OAc), 1.29 (3H, s, Me-28), 1.24(3H, s, Me-19), 0.64 (3H, s, Me-l&; FABMS m/z 645 EMiCS].

Conversion of 3 5 2. A soln of 3 (4 mg) and 20% Pd/C(1 mg) in MeOH (1 ml) was stirred under H, atm. for 16

hr. Insoluble material was removed by f&ration oversilica gel to give a product (4 mg) identi~l with 1 (co-TLC, H NMR).Ackraowiedgements-Financial assistance from CSIR,New Delhi is gratefully ack~owled~,

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Prod. 54, 599.5. Gupta, M., Manickam, M., Sinha, S. C., S~nha-Bagchi,A. and Ray, A. B. (1992) P~y~uc~e~~~tr~ 1, 2423.6. Shingu, K., Furusawa, Y., Harnba~oshi, N., Ueda, I.,Yahara, S. and Nohara, T. (1990) C/tern. award. Bull.38, 2866.