1976 669Reactions of Nic-I (Nicandrenone), a Naturally Occurring Ring-D-aromaticSteroidBy Erwin Glotter and Pnina Krinsky, Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot,IsraelIsaac Kirson,' Department of Organic Chemistry, The Weitmann institute of Science, Rehovot, IsraelSome reactions of Nic-1 (nicandrenone) (2a). the main steroidal component of Nicandraphysaloides (Solanaceae),and its relation to the steroidal lactones of the withanolide group are discussed.WE have previously reported the isolation and charac-terization of withanicandrin (1) from a population ofNicandra physaloides (Solanaceae) growing in SouthIndia. While work towards the elucidation of thestructure of nicandrenone,2 the main constituent of thisplant, was in progress, we learned that Crombie and hiscolleagues had characterized a series of steroidal com-ponents from N .Physaloides, including nicandrenonerenamed Nic-1 (2a)l. Similar conclusions were reachedin an independent investigation by Bates4 Thepopulations of N . Physaloides studied by these authorscontained neither withanicandrin (1) nor any othersteroidal components with the unsaturated S-lactonesystem in the side chain, as present in most of thewithanolides investigated so far.Our work on Nic-1 (2a) has been discontinued; wereport here some chemical transformations originallydirected towards the determination of its structure.Acetylation with acetic anhydride-pyridine afforded thelactol monoacetate (2b), displaying a significantly low-field n.m.r.singlet 6 6.12 (26-H). Catalytic hydrogen-ation over Pd-CaCO, resulted in the 2,3-dihydro-deriva-tive (3) (disappearance of the n.m.r. signals due to thevinylic 2- and 3-H). Jones reagent induced smoothoxidation of the epoxy-lactol system to give the corre-sponding epoxy-lactone (4), characterized by the dis-appearance of the 26-H signal and the significant shiftof a broad multiplet from 6 3.87 in (2a)J to 6 4.65 in(4). The position and the pattern (double triplet) ofthis signal, in conjunction with the signals of the sub-stituents of rings A and B, suggested the close relationshipbetween Nic-1 and withanicandrin (1). The structureof (4) was substantiated by decoupling experiments(see Experimental section). The c.d.spectrum showednegative bands at 338 nm for the 2-en-l-one (~~LHZS-AB-system) and 244 nm for the side-chain epoxy-lactone,as well as a very weak triplet of bands a t 275.5, 268,and 263 nm for the aromatic ring D absorption (opticallyactive owing to its dissymmetric environment). Cata-lytic hydrogenation of (4) resulted in the 2,3-dihydro-derivative ( 5 ) , alternatively obtained by oxidation(CrO,) of compound (3). In order better to visualizethe c.d. band of the epoxy-lactone system, compound(5) was reduced (NaBH,) to the la-ol (7) (lP-H, narrowI. Kirson, D. Lavie, S. S. Subramanian, P. D. Sethi, andE. Glotter, J.C.S. Perkin I , 1972, 1972.F. v. Gizycki and G. Kotitschke, Avch. Pharm., 1951, 284,129; 0. Nalbandov, R. T. Yamamoto, and G.S. Fraenkel,J . Agric. Food Chem., 1964, 12, 55.multiplet at 6 3.66), which showed a negative c.d. bandat 233 nm. Reduction of the epoxy-lactone (5) (CrCl,)afforded in good yield a compound (6) possessing theunsaturated 8-lactone unit normally present in thewithanolides. This conversion was indicated in then.m.r. spectrum by the shift of the 24- and 25-CH,signals from 6 1.43 and 1.55 in (5) to 6 1.89 in (6)J.The c.d. behaviour of (6) supports the proposed structurepositive band at 245 nni for the a@-unsaturated 6-lactoneOxidation of (6) by peroxy-acid proceeded in astereoselective manner, albeit slowly, t o give only theepoxy-lactone (5), with the epoxide possessing the sameconfiguration as in the natural Nic-1. Owing to theinfluence of the 5a-OH, the 6~q7ct-epoxide system in thewithanolide (10) undergoes with hydrobromic acid abidirectional opening to give mainly (70) the corre-sponding trans-diequatorial bromohydrin .5 To ascertainthe influence of the aromatic ring on one side and of thehydroxy-group on the other side of the 6a,7~-epoxy-group in Nic-1, compound (6) was treated with hydro-bromic acid, leading to a mixture of two compounds,separated by preparative layer chromatography.Theupper band was due to the trans-diequatorial bromo-hydrin (Sa), converted by acetylation into the bromo-acetate (8b), J6p,7cr 10 Hz. The lower band did notcontain the expected tram-diaxial bromohydrin (6p-bromo,7~~-hydroxy) because the axial 5a-OH attackedthe incipient C-6 carbocation to give a 5a,6a-epoxide(9a) isomeric with the starting compound (6).Acetyl-ation of (9a) afforded the corresponding monoacetate(9b), J S f l , ~ , g 5 Hz. The axial orientation of the 7a-acetatein (9b) places its methyl group below the plane of thearomatic ring, thus resulting in a sizable upfield shift ofits resonance (6 1.80), as well as that of the aromatic15-H (6 7.02). The structures assigned to the bromo-acetate (8b) and the epoxy-acetate (9b) were confirmedby spin decoupling and by high resolution mass spectro-metry.The mass spectral fragmentation of Nic-1 and deriva-tives is dominated by the cleavage of the 20,22-bond,leading to the fragments m/e 323 in the A2-l-one andm/e 325 in the l-one derivatives, and the alternativefragments m/e 141 in compounds (4) and (5) and m/ea M.J. Begley, L. Crombie, P. J. Ham, and D. A. Whiting,R. B. Bates and D. J. Eckert, J. Amer. Chem. Soc., 1972,I . Kirson, E. Glotter, D. Lavie, and A. Abraham, J . CIzem.(22R)I *J.C.S. Chem. Comm., 1972, 1250.94, 826.8.Soc. (C), 1971, 2032670 J.C.S. Perkin I0 21( Z ) a ; A 2 ; R - Hb ; A 2 ; R=Ac( 3 ) R - H(4) A'; R = O15iR :O(71R : a - O H , P - HHo OR :* b-. '.OR18la; R = H ( 9 ) a ; R:Hb; R=Ac b ; R = AcN.m.r. data *Aromatic protons Methyl groupsCompound 2-H 3-H 6-H 7-H '16-H 16-H 17a-H(Za) 5.88dt 6.621n 3.22d 4.0m 7.38d 7.05d 7.0s(10) (4) narrow (8) (8)(2b) 6.92dt 6.66m 3.28d 4.06m 7.38d 7.05d 6.97s3.27 4.05(3) 3.26d 3.98111(4) t 5.88dt 6.65m 3.25d 4.04m 7.39d 7.05d 7.01s3.23d 4.02dd 7.38d 7.05d 6.98s(6) 3.30d 4.03dd 7.43d 7.02d 7.08s4.08d 4.57dd 7.50d 7.12d 7.05s(4! narrow (8) (8) (10)( 5 )(4) (4;3) (8) (8)(4) (4;3) (8) (8)22-H 6-H i9-H 21-H 27-and28-H Other3.87m 4.98~0 1.22s 1.24d 1.33;1.354.06m 6.12s 1.26s 1.23d 1.32;1.37 2.10 (OAc)4.47 6.47 1.20 L1.261 1.25;1.32 2.073.83 5.00s" 1.27s 1.23d 1.35;1.374.65dt 1.24s 1.36d 1.50;1.404.68dt 1.30s 1.35d 1.43;1.554.52dt 1.31s 1.41d 1.894.42 1.42s 1.40d 1.85(7)(12;5) (7)(10;6) (7)(12;5) (8)(10) (10;11) (8) (8) (8)(12 ;5) (8) (10) (1O;ll)(narrow multiple ts) (8) (5)(6) (5) (narrow multiplet) (12;5) (8)5.68d 4.55dd 7.48d 7.08d 7.03s 4.47dt 1.50s 1.38d 1.85 2.21 (OAc)3.50d 4.533.55d 5.67t6.9d8'7.13 ca.4.5 1.42s 1.37d 1.867.02 4.42dt 1.42s 1.35d 1.85 1.78 (Ohc)* ,4t 60 MHz; solvent CDC1,; 6 values; data for C,D,N solutions in square brackets; coupling constants (Hz) in parentheses.0 After addition of D,O.-f Measured at 90 MHz on a Brucker instrument1976 671125 in compounds (6), (8), and (9). Further fragment-ation of the mfe 323 and 325 ions involves cleavage ofthe 7,8- and 9,10-bonds, resulting in two strong signalsat nz/e 155 and 157, containing rings c and D. Thefragmentation of the epoxy-acetate (9b) follows adifferent path owing to the ready elimination of aceticacid m / e 432 (M+ - 60), thus precluding cleavage ofthe 7,8-bond; the m/e 155 and 157 signals are thereforeof low intensity. Alternatively, this spectrum ischaracterized by the intense formation of two fragments :C21H2402 m/e 308 (M' - 125 - 59)J which results fromrings A-D and a C, side chain, and C,,H,, (m/e 221)which results from rings B-D and the same C, side chain.EXPERIMENTALM.p.s were taken with a Fisher-Johns apparatus.Optical rotations were recorded with an automatic Perkin-Elmer 141 polarimeter and refer to solutions in chloroform.C.d.measurements were performed by Mrs. B. Romanowith a Cary 60 instrument for solutions in ethanol. 1.r.spectra were recorded on a Perkin-Elmer Infracord 137spectrophotonieter and refer to solutions in chloroform;U.V. spectra were recorded on a Cary 14 instrument forsolutions in ethanol; n.m.r. spectra were determined on aVarian SV-14 instrument for ca.5 solutions in deuterio-chloroform containing tetramethylsilane as internal stan-dard. T.1.c. was carried on plates of silica gel G (Merck)and spots were developed with iodine vapour. Preparativechromatoplates (1 mm thick) were prepared from silicagel (Merck). Mass spectra were taken under thedirection of Dr. 2. Zaretskii with a Varian MAT 731 HRinstrument. Analyses were performed in the micro-analytical laboratory of the Weizmann Institute, under thedirection of Mr. R. Heller.Plant Material.-Nicandra Physaloides was raised byMr. A. Abraham in the nursery of the Agricultural ResearchOrganization, Volcani Center, Israel, from seeds receivedfrom India (courtesy of Dr. P. D. Sethi and Dr. S. SankaraSubramanian, Pondicherry) .The isolation procedure wasas describedl The amount of Nic-1 (nicandrenone) (2a)isolated following the processing of 1 kg of dry leaves was800 rng. The product was further purified by preparativelayer cIiromatograpliy (p.1.c.) in benzene-ethyl acetate(1 : 4) and recrystallization from benzene; m.p. 138-139"(lit.,, 137"); c.d. - 1.7, +6.22.Nic-1 Acetate (2b) 26-Acetoxy-6a, 7a:22,26:24,25-triepoxl,-5-Jzydvoxy-17(13 _). 18)abeo-5a-ergosta-2, 13,15,17-tetraeiz-l-one.-Xic-1 (50 mg) was acetylated with acetic anhydride(0.5 ml) and pyridine (0.5 ml), overnight a t room teni-perature. The firoduct was precipitated with ice-water,isolated by filtration, and crystallized from ethanol; m.p.180--181", R ~ +22.5" (c 0.4), v,,,. 1690 and 1740 cm-l;Amx. 275.5 (E 1 000), 265 (1 150), 217.5sh (15 500), and222 nm (16 000) (Found: C, 70.9; H, 7.1.C3,H,,0,requires C, 70.8; H, 7.1).18)abeo-5a-ergosta-13,15,17-trien-l-one (3).-Nic-1 (100 mg)in absolute ethanol was hydrogenated (10 Pd-CaCO,) atroom temperature and atmospheric pressure. After ab-6a,?a:22,26:24,25-TriePoxy-5,26-dihydroxy-17( 13 _+1705 cm-l; Lx 220sh (E 10500) and 215sh nm vm,(12 000); c.d. Aamp;Z95 3-0.73 and positive a t wavelengthsshorter than 220 nm (Found: C, 71.7; H, 7.8. C2,H,,0,requires C, 71.8; H, 7.7).6a,7a:24,25-Die~oxy-5-hydroxy-l7 (13 _t 18)abeo-5cr-ergosta-l3,15,17-t~ien-l-one 26,22-Lactone (5) .-To a solutionof compound (3) (50 mg) in acetone (25 ml), a solution ofJones reagent was added dropwise at 16-18 'C.After30 min the excess of reagent was destroyed with a few dropsof methanol, the solvent was removed under vacuum, andthe residue was crystallized (yield 40 mg) from chloroforni-ethyl acetate; m.p. 243-245'; a, +62.6" (c 0.4); v,,,1708 and 1715 cm-l; Lx. 275.5 (c 1050), 267 (1 loo),261 (950), 220sh (8 700), and 214 nm (10 000); c.d.+0.70, A ~ 2 3 4 -2.3 (Found: C, 72.0; H, 7.25; Mf, 466.C,,H,,O, requires C, 72.1; H, 7.35; .W, 466).6a,7a:24,25-DiePoxy-5-hydroxy-17( 13 ----Lc 18)abeo-5cr-ergosta-2,13,15,17-tetraen-l-one 26,22-Lactone (4) .-The oxida-tion of Nic-1 (1 g) was performed as described for compound(3) and the product was crystallized (yield 700 mg) fromacetone-hexane; m.p. 230-232', a, + 34.6' (c 0.5) ;Amx.1 698 and 1 715 cm-l; Am,. 216 nm (E 16 600); c.d.- 0.39, + 5.29; n.m.r. decoupling : irradiationof 20-H + 20-CH, singlet and 22-H double doublet;irradiation of 7P-H --+ 6P-H singlet ; irradiation of6P-H- 7P-H narrow band Wi 3 H z ; irradiation of8P-H---+ 7P-H doublet ( J 4 Hz) (Found: C, 72.5; H,7.0; M+, 464. CZ8H3,O6 requires C , 72.4; H, 6.9;M , 464).Hydrogenation of the Laclone (4).-Compound (4) (330 mg)in absolute ethanol (140 ml) was hydrogenated (10Pd-CaCO,) at room temperature and atmospheric pressure.After absorption of 1 mol. equiv. the product was crystal-lized from chloroform-ethanol. It was identical withcompound (5).Reduction of the Lactone ( 5 ) with Chronzizanz(I1) Chloride.-A solution of chromium(r1) chloride from CrC13,6H,0 6(3.2 g) was added at room temperature under CO, to asolution of compound ( 5 ) (220 mg) in acetone (15 ml) andacetic acid (10 ml).The mixture was heated during 1 ha t 50 "C, then ice-water was added and the product wasisolated by filtration. Ga,7a-Epoxy-5-hydroxy-17( 13 --+1 8)abeo-5~-ergosta-l3,15,17,24-tetraen-l-one 26,22-Lactone(6) crystallized from acetone-hexane (yield 100 mg) ; m.p.186--187", a, f136.4" (c 0.4); vmx 1 700 cm-l; Amax. 275(E 730), 266 (1 140), 261 (1 500), and 213 nm (19 000);c.d. +0.63sh, +7.OOsh, +8.17 (positive atshorter wavelengths) (Found: C, 74.5; H, 7.7; M+, 450.C2,H3,O, requires C, 74.6; H, 7.6; 144, 450).Reduction of Compound ( 5 ) wit?$ Boro?zj~dvide.-Sodiumborohydride (50 mg) was added to a solution of compound(5) (50 mg) in methanol (20 ml).After 1 h a t room tem-perature the solution was neutralized with dilute hydro-chloric acid (1 : 4), the solvent was removed, water wasadded, and the product was filtered off. Ga,7a:24,25-Die-Poxy-la,5-dihydvoxy-17( 13 18)abeo-5a-ergostu-l3,15,17-triene 26,22-Zactone (7) was purified by p.1.c. benzene-ethylacetate (1 : 4) and crystallized from ethanol; 1n.p. 185-187",a, -23.5" (C 0.3); vmak 1715 cm-l; c.d. A~233 -2.96(Found: C, 71.6; H, 7.9. C,,H,,O, requires C, 71.8; H,Aamp;338 - 2.25, AEz75.5 + 0.11, A E ~ ~ B + 0.10, AE,,~, O.O7sh,ry - o r bsol;sorption of 0.9 mol. equiv. the Product was purified by p.1.c.benzene-ethyl acetate (1 : 4 1 and from 8 L. Fieser and amp;I. Fieser, ' Reagents for Organic Synthesis,'acetone-hexane; m.p.125-127'; a, +51.8 (c 0.45); vol. I, TTiley, 1967, p. 149.' * ' -/O)672 J.C.S. Perkin IEpoxidation of Cmfiound (6) .-To a solution of (6) (35 mg)in benzene (2 ml), a solution of perbenzoic acid (30 mg) inbenzene was added and the mixture was kept for 1 week a troom temperature. The solution was then washed withdilute aqueous sodium carbonate and water, and evapor-ated. The residue was purified by p.1.c. benzene-ethylacetate (1 : 4). The main band gave the lactone (5)(30 mg), identical with the compound described above.Treatment of CowPoand (6) with Hydrobroinic A cad .-Toa solution of (6) (100 mg) in acetone (90 ml), aqueous 45hydrobromic acid (3 ml) was added. After 7 h a t roomtemperature the solution was neutralized with sodiumcarbonate, the solvent was removed, and the residue wasextracted with methylene chloride.The crude product(105 mg; two spots on a chromatoplate) was separated byp.1.c. benzene-ethyl acetate (1 : 4). The upper bandyielded 7P-bromo-5,6a-dihydro.oxy-17( 13 -t 18)abeo-5a-ergosta-13,15,17,24-tetraen-l-one 26,224actone (8a) (20 mg),m.p. 218-220' (from acetone): aD f311.5" (G 0.15); vmx.1 690 and 1 710 cm-l; A,, 213 nm (E 20 000) (Found: C,63.3; H, 6.7; M f , 530/532. C2,H,,Br0, requires C,63.3; H, 6.6; M , 530/532). Acetylation afforded the non-crystalline 6-monoacetate (8b) ; n.ni.r. decoupling : irradi-ation of 6p-H ~-- 7a-H doublet (J 11 Hz) ; irradiation of7a-H + 6p-H slightly broadened singlet (Found : M+,572.177 2/574.178 0. C,,H,,BrO, requires MI 572.177 3/574.175 4).18)abeo-5a-ergosta-l3,15,17,24-tetraen-l-one 26,22-lactone(9a) (40 mg), map. 195-197" (from aqueous methanol);a, -9' (G 0.1); v,, 1710 cm-l; A,= 216 nm (c 16 200)(Found: C, 74.5; HI 7.6. C,,H340, requires C, 74.6; H,7.6). Upon acetylation with acetic anhydride andpyridine overnight a t room temperature, the non-crystalline7-monoacetate (9b) was obtained ; n.m.r. decoupling :irradiation of 613-H 7p-H doublet (J 5 Hz) ; irradiationof 7P-H + 6B-H singlet (Found : Mf, 492.253 9. C30H3606requires iM, 492.251 2).The lower band yielded 5, 6cc-e$ox~~-7a-hydroxy-17( 13We thank Professor L. Cronibie and Dr. D. Whiting for asample of Nic-1. The work was supported in part by theCentral Fund for Research and Development of the HebrewUniversity.5/1562 Received, 7th August, 1975
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