Phyfochem~srry, Vol 30. No. 4. pp. 1239 1243, 1991 0031-9422p1 s3.oo+o.cll Prmed m Great Britain. Q 1991 Pcrgamon Press plc

STEROIDAL DERIVATIVES FROM THE ROOTS OF MANDEVZUA PENTLANDIANA

GABRIELA CABRERA, JORGE A. PALERMO, ALICIA M. SELDES, EDUARDO G. GROS and JUAN CARLOS OBERTI*

Departamento de Quimica Orginica, Facultad de Ciencias Exactas y Natwales, Universidad de Buenos Aires, Pabellbn 2, Ciudad Universitaria, 1428 Buenos Aires, Argentina; l Departamento de Quimica Orglnica, Facultad de Ciencias Quimicas, Universidad

National de Cbrdoba. Sucursal 16, Casilla 61, 5016 Cbrdoba, Argentina

(Received in revised form 7 September 1990)

Key Word hiex-Mandevilla pentlandiana; Apocynaceae; roots; A ‘*“‘-diunsaturated sterols; 6-keto-5a-stanols.

Abstract-The steroids from the roots of Mandeuilla pentlandiuna were analysed. Six AzO-steroids and four 6-keto-Sa- stanols, novel as natural products, were isolated and characterized by spectroscopic methods. The plant also produces cardenolide glycosides, for which 20-hydroxysterols are known to be biosynthetic precursors. The hydroxylated derivatives have not been detected, but AZ0-derivatives, closely related to them, may be involved in that metabolic pathway.

INTRODUCTION

The family Apocynaceae is well known for the presence of cardenolides, iridoids and pregnanes Cl]. In view of this, and continuing with our interest in the characterization of steroidal derivatives from natural sources [2] we undertook the analysis of the less polar fractions of the ethanolic extract of the roots of the liana Mandeuilla pentlandiana. We report the isolation and character- ization of six A5s 20-diunsaturated steroids and four 6- keto-Sa-stanols which were present as esters of CLeLO, C,6:3, C,sZ1 and C,,:, acids. To the best of our knowl- edge all these compounds are described here for the first time as natural products.

RESULTS AND Dl!XU!WON

The ethanolic extract of the roots of M. pentlandiana displayed a positive Kedde test even at high dilutions. Fractionation of this extract led to the isolation of complex mixtures of mono, tri and more highly gly- cosidated cardenolides, which are under study in our laboratory. The less polar fraction of this extract was further purified by column chromatography to obtain two subfractions, A and B, which showed the character- istic ‘H NMR spectra of steryl esters. In particular, the spectrum of the crude ester subfraction A displayed four signals at 64.650 which were of importance in ascer- taining the origin of some of the compounds identified in the present work.

Saponification of subfractions A and B afforded two steroidal mixtures, A2 and B2, respectively, which were further separated into pure components by HPLC (Table I). Fraction A2 contained besides the common sterols, cholest-S-en-3b-ol (7). 24(-methylcholest-5-en- 38-01 (lo), 24(-methylcholesta-5,22,-dien-3/3-ol(1 l), 245- ethylcholest-5-en-3/l-ol (12), cholesta-5,24-dien-3B-ol (8) and 24-methylcholesta-5,24(24’)-dien-3fi-ol(9) a group of six compounds with lower HPLC RR, (0.560.73, sex? Experimental). These compounds (l-6), showed two com-

mon features in their ‘H NMR spectra:a pair of broad singlets (1H each) at 64.8 and 4.9 and a pronounced upfield shift (60.60) for the C-18 methyl. These features have been described for synthetic A”-steroids [3].

Compound 1 displayed ‘HNMR and mass spectra similar to those reported for synthetic cholesta-5,20,24- trien-3j?-ol [3]. This structural characterization was also confirmed by assignment of its “CNMR spectrum (see Experimental).

Compound 2 could not be fully resolved by HPLC from compound 3 and for this reason it was only isolated as a pure compound in small amounts. Its mass spectrum showed [M]’ at m/z 396 and fragments at m/r 273,271, 255, 229 and 213, indicative of a C,,-steroid with a monounsaturated nucleus and diunsaturated side chain. This was confirmed by the fragments at m/z 150,123 and 107, characteristic of A”-C,,-steroids with disubstituted side chains [3]. The ‘H NMR spectrum of 2 showed the characteristic broad singlets at 64.78 (1H) and 4.87 (1H) corresponding to vinylic C-21 protons, a singlet at 0.60 for the C-18 methyl protons together with a broad singlet at 4.66 (2H), a doublet at 1.02 (3H) and a broad singlet at 1.66 (3H), which could correspond to a methyl group linked to a double bond. The signal at 65.36 (IH) corresponded to H-6 of a A’-nucleus. These data, which strongly resembled the spectrum described for codisterol [4], indicated a 24-methyl-A’. ‘O* 25-steroidal structure for 2. The *‘C NMR spectrum, although it was performed with a very small amount of sample, clearly showed the presence of two sp2 methylenes (6 109. I and I 11.3). Thus, 2 is 24-methylcholesta-5,20,25-trien-3/?-ol.

The most abundant component of this group of ster- oids, compound 3, showed [M]’ at m/z 396 and fragment ions at m/z 273,271 (lOO%), 255,229 and 213, indicating that compounds 2 and 3 are isomeric steroids with a monounsaturated nucleus and a diunsaturated side chain. The fragments at m/z 150, 123 and 107 were again consistent with a A20-diunsaturated side chain. This, and the presence of a A5-nucleus, were confirmed by its ‘H NMR spectrum. Two broad signals at 64.70 and 4.77

1239

1240 G. CABRERA et al.

Table 1. Steroidal composition of Mandeoilla penthdiana roots

Compound % GC (RR,)+ HPLC(RR,)t

Cholesta-5,20,24-trien-38-01 (1) 9.6 1.01 0.56 24~-Methylcholesta-5,20,25-trien-3fi-ol (2) 0.6 1.03 0.57 24~-Methylcholesta-5,20,24(24’)-trien-3j?-oi (3) 25.6 1.04 0.57 Cholesta-5,20-dien-3p-ol (4) 6.8 0.99 0.65 24~-Methylcholesta-5,2O-dien-3~-ol (5) tr. 1.04 0.70 24~-Ethylcholesta-5,2O-dien-3~-ol(6) 2.6 1.09 0.73 Cholest-5-en-3/I-ol (7) 3.4 1.00 1.00 Cholesta-5,24-dien-3/?-ol (8) 0.6 1.02 0.78 24~-Methylcholesta-5,24(24r~dien-3/3-ol (9) 0.5 1.05 0.80 24~-Methylcholest-5-en-3~-ol (10) 15.5 1.06 1.09 245-Ethylcholesta-5,22-dien-3/I-o] (11) 2.1 1.08 1.07 24~-Ethylcholest-5-en-3/I-ol (12) 28.8 1.1 I 1.14 3/?-Hydroxy-5x-cholestan&one (13) tr. 1.15 0.39 3/?-Hydroxy-24~-ethyl-5a-cholest-22-en-6-one (14) 0.9 1.26 0.40 3/?-Hydroxy-24<-methyI-5a-cholestan-6one (15) 0.4 1.23 0.40 3~-Hydroxy-24[-Ethyl-5a-cholestan&one (16) 2.5 1.30 0.43

l RR, relative to cholest-5en-3fi-ol (see Experimental). IRR, to cholest-5-en-3/?-o] (Column Alltech 10 m, eluant: MeOH, see Experimental).

were indicative of a Azqz4’) double bond while the doublet at 6 1 .OS was characteristic of the C-26 and C-27 methyls of a A24(24’) steroid. Further evidence of this structure was obtained by 13C NMR: two sp’ methylenic carbons (C-21 and C-24’) and two sp2 quaternary carbons (C-20 and C-24). Chemical shifts of C-25, C-26 and C-27 of 3 were almost identical to those of the correspond- ing carbons of 24-methylenecholesterol. Thus, 3 is 24 methylcholesta-5,20,24(24’)-trien-3j?-01.

Compound 4 showed only the signals due to A’ and AZ0-unsaturations in its ‘HNMR spectrum. By com- parison of these data, and also those of its mass spec-

trum with published data [3], and interpretation of its “CNMR spectrum compound 4 was identified as cholesta-5,20-dien-3/?-01.

Compounds 5 and 6 were isolated in very small amounts which prevented determination of their respect- ive 13C NMR spectra. Thus, these compounds were characterized by only their ‘H NMR and mass spectra. Compound 5 showed CM] + at m/z 398 and fragment ions at m/z 314 (base peak), 299, 271, 229 and 213 which indicated the presence of a C,,-diunsaturated steroid. The presence of A5 and AZ0 unsaturations was evident from the ‘H NMR spectrum as well as a signal for the C-

Steroids from Mondt~illa pentlandiana 1241

24 methyl group at 60.80. The base peak of the mass spectrum, m/z 314, probably arises by a McLafferty rearrangement via a six-membered transition state with transference of H-24 to C-21. This mechanism was dem- onstrated by deuteration [3] in the case of Sz-cholest-20- ene-3p-ol. This fragment appeared in low abundance in the mass spectrum of compound 4, in good agreement with values published by Djerassi [3]. Hence, compound 5 was identified as 24-methylcholesta-5,20-dien-3fl-ol.

Compound 6 presented CM]’ at m/z 412 and frag- ments indicating a C,,-diunsaturated steroid. The unsat- urations were characterized as A5 and AZ0 by the ‘H NMR spectrum, and the presence of a 24-ethyl group was also evident in the spectrum which closely resembled that of sitosterol. The base peak in the mass spectrum was m/z 314, in analogy to compound 5, and presumably produced by the same type of rearrangement. Therefore compound 6 is 24-ethylcholesta-5,20-dien-38-01.

Separation of fraction B2 by HPLC afforded, besides common phytosterols as the main components, a fraction containing pure 16 and another containing a mixture of 1>15 which, due to the small quantity obtained, was analysed by GC-MS.

the greatest structural variety, in recent years there has been an upsurge in the identification of new steroids from terrestrial sources, probably due to the use of modern separation techniques [9-l 11. Steroids with A20(22) and A’7(2o’ double bonds have been isolated from marine organisms [12], but to the best of our knowledge no report has been made on the isolation of A20 derivatives from natural sources. Some derivatives of this type had been synthesized [3] in order to accumulate spectro- scopic data in view of their possible future isolation from marine organisms. 20_Hydroxysteroids, possible biosyn- thetic intermediates on the way to cardenolide glycosides produced by this plant, were not detected as free or esterified derivatives. Thus, the possibility that A2’-ster- oids may be artifacts formed by dehydration of a 20- hydroxysteroid during work-up of the esters can be ruled out. Moreover, the ‘H NMR spectrum of the crude steroidal ester fraction showed, before saponification, the signals at 64.8e5.00 characteristic of the A2’-double bond. The possible role of the A2’-steroids as precursors of cardenolides must be demonstrated. It is noteworthy that the new compounds described here appear only as ester derivatives.

Compound 16 showed in its ‘HNMR spectrum the presence of a 3B-hydroxy group and no vinylic protons. The mass spectrum showed [M] + at m/z 430 correspond- ing to a molecular formula C,,H,,O,. Some diagnostic fragments were m/z 359 (loss of ring A of a 6-keto-steroid bearing a C,,H2, side chain), m/z 290 (loss of ring A plus ring B of the same type of steroid) and m/z 95 (ring A) [S, 63. The structure was confirmed by the “CNMR spectrum, which clearly showed the presence of 29 sig- nals, a keto group and no olefinic carbons. The presence of a 3/I-hydroxy group could be ascertained as well as the signals corresponding to the side chain of sitosterol. The carbonyl group was assigned to position C-6 in view of the downfield shifts of C-S and C-19 [7J The spectrum showed a great resemblance with that of 3/I?-hydroxy-5a- cholestan-6-one [8], thus enabling the identification of compound 16 as 38-hydroxy-24<-ethyl-5r-cholestan-6- one.

EXPERIMENTAL

General. HPLC separations were performed on Alltech R-W C-18 10pm (500x 10 mm; flux 8 mlmin-‘) and Altex Ultra- sphere ODS 5 m (250 x IO mm; flux 3 ml min _ I) columns, using MeOH as eluant. CC: HP-5 (25 m x 0.32 mm) capillary col- umn (1X1-280”, 10” min-‘) unless otherwise stated. ‘H NMR: 100.1 MHs 13C NMR: 25.2 MHt

Plant material. Ma&villa pentiandiana was collected at Agua de Oro (Cbrdoba, Argentina) and identified by Dr Luis Arita Espinar (Muse0 de Botlnica de C6rdoba).

Compounds 1%15 showed similar mass spectral frag- mentation patterns to that of 16, being thus also charac- terized as 6-ketostanols. Compound 13 showed [Ml’ at m/z 402 and fragments at mJz 331,289 [M-side chain]‘, 262 and 95, indicating the presence of a C,H,, side chain. Thus, compound 13 is identified as 3B-hydroxy-5a- cholestan&one.

The mass spectrum of compound 15 showed [M]’ at m/z 418 and fragments at m/z 345,289,276 and 95 which indicated a &HI, side chain and it was identified as 3/?- hydroxy-24<-methyl-5a-cholestan-6-one.

The mass spectrum of 14 showed CM] + at m/z 428 and fragments at m/z 357,288 and 95, corresponding to a C,, compound with a monounsaturated side chain and it was characterized as 3/I-hydroxy-24[-ethyl-cholest-22-en-6- one. The low abundance of fragments at m/z 330 and 287 reinforced the characterization of A22-unsaturation.

Extraction und isolation. Air-dried roots (2.7 kg) were ex- tracted at room temp. with E1OH. The extract was concd at red. pres. to afford 180 g ofcrude material, which was fractionated by dry column flash chromatography on silica gel (300 g) using CH,C12 and mixtures of CH,CI,-MeOH of increasing polarity. The fraction eluted with CH,CI, (5 g) was further purified by the same technique using mixtures of petrol-EtOAc of increasing polarity. Subfractions A and B (ca 1 g each) were eluted with petrol-EtOAc (49: 1) and (19: 1) respectively and saponified with 5% KOH in MeOH at reflux for 2 hr. The acidic fractions were methylated in Et,0 using CH,NI and analysed by GC (SP-2330 column, 60-260”; 8” min- ‘) and CC-MS. Fatty acids from fraction A (%): C,, I (5.6). C,,:, (16.7A C,,:o (0.5), C16.3 (34.7), C,szO (3.8), C,,:, (6.8), C,,:, (23.8), C,s,3 (1.0); unidentified: 7,1%.Fattyacidsfromfraction B:C,,., (4.9),C,,:,(1.6XC16:o (17.8), C,b:s (17.1)s C,,., (3.9A Cl*:1 (15.3). C,s:z (30.0X C,,., (5.3). Unidentified: 8.0%. The unsaponifiables A2 and B2 were chromatographed on silica gel (petrol-EtOAc) to obtain the crude steroidal mixtures, which were purified by HPLC. Frac- tion A2 yielded compounds 1-12 while compounds 7 and 10-16 were obtained from fraction 82. The total sterol content of the dried plant material was 0.19 mg g- ’ root.

The acidic components obtained by saponification of ChoIesta-5,20,24-trien-3B_ol (1). MS m/z (rel. int.): 382 [Ml’ fractions A and B were methylated with diazomethane (75X 367 (35). 364 (8). 349 (35). 273 (22). 272 (84), 271 (100). 258 and analysed by GC-MS; the most important compon- (lo), 256 (lo), 255 (36). 253 (26), 229 (20). 213 (41). 159 (45). 145 ents were identified as the fatty acids C16:o, C16:3r C,s:, (62), 136 (11). 109 (79). 93 (85). 13CNMR (CDCl,, TMS): 637.2 and CI s: 2. The free sterol fraction of the ethanolic extract (C-l), 31.8 (C-2*), 71.7 (C-3). 42.3 (C-4). 140.6 (C-5), 121.4 (C-6). contained the common phytosterols 10-12. Compounds 31.6 (C-7*), 32.3 (C-8), 50.3 (C-9), 36.5 (C-lo), 21.2(C-l l), 38.7 (C- l-9 and W16 were not detected as free steroids. 12), 43.0 (C-13), 56.6 (C-14), 24.2 (C-15). 25.8 (C-16), 56.0 (C-17),

Although sterols from marine organisms have shown 12.8 (C-18). 19.4 (C-19), 149.1 (C-20). 109.3 (C-21), 37.6 (C-22).

I

2

3

4

5

6

I6

G. CAHRERA cf ul.

Table 2. lH NMR spectra of compounds Id and 16 (CDCI,. TMS)

H-3 H-6 H-18 H-19 H-21 H-24 H-26, 27 H-24’ H-29

3.50 5.36 m. IH m. I H

3.SO 5.36 tn. IH m. I H

3 50 s 35 m. IH m. I H

3.50 5.36 m. 1 H m. I H

3.50 5.36

w. I H m. I H

3 50 5.36

m. I I I m. IH

3.55

m. 1 H

0.60 1.02

5. 3H .s. ?H

0.58 \. 3t1

I 02

s, 3H

0.60

.\. 3H

I.01

.s. 3H

0.w

z. 3H

1 .n2

>. 3H

0.5Y

s. 3H

I .02

.t. 3H

0 59

s. 3H

I .02

.s. 3H

0.67

s. 3H

0.76

s. 3H

4.X0

hr s. IH

4.8X

hr s, IH

4.7X br .s, I H

4.87

br s. 1 H

4.111 hr s. IH

4.90 hr s. IH

4.7X

hr s, IH

4.X5 hr s, 1 H

4.17

hr s, 1 H

4.x7 hr s, I H 4 77

hr s, 1 H

4.XX

hr .s. I H

0.92

d, 3H (6)

5.12 1.61

m, IH hr s, 3H

I .69

hr s, 3H

4.66

br s, 2H

I .66

5. 3H

I .05

d. 6H (6)

0.88

d. 6H (6)

_.. 0.86

d. 6H (6)

0.84

d. 6H (6)

0.83

d, 3H (7) 0.8 1

d, 3H (6)

1.02

d, 3H (7)

4.70

m, IH

4.77

m, IH

0.80 d, 3H (6)

0.85

t. 3H (7)

0.85

r. 3H (7)

‘7.1 (C-23). 124.2 (C-24). 131.2 (C-25). 25.6 (C-26), 17.7

(C-27) (*assignments may he Interchanged). ‘H NMR: see

‘Table 2. 24;:-:Mt,rh~l~hol~,,sr~-S.20,25-trlen/ (2). MS m:; (rel. mt.):

396 [M] ’ (100). 381 (20). 37X (8). 363 (21). 273 (8). 272 (33). 271

(4X). 255 (IO). 229 (24). 213 (36). I.59 (27). 150 (IX). 145 (38). 123

(201. IO? (3X). 93 (37). ‘H NMR. see Table 2.

?41-.\~~~f/1~1~~/1~,lrslu-5.20.~4(24’)-rri~t~-3/1-o/ (3). Mp X3--85

(from IitOH H,O). MS n1.z (rcl. int.): 396 [.MJ’ (83). 381 (46).

37X(X).363l.131.273(?1).272(X7),271(1(x)),’55(39),229(19),213 (3h). I.59 (3.5). 150 (13). 145 (46). I23 (26). 107 (3X). 93 (37).

IV NMR K:DCl.,. TMS): ($37 3 ((‘-I 1. 31.x (C-2*), 71.6 (C-3).

42 3 (C-4). 14O.b(c‘-5). 121.3 (C-6). 31.h (C-7*). 32.3 (C-X), 50.3 (C-

9). 36.5 (C-IO). ‘I.2 (<‘-I I). 3X.7 (C-12). 43.0 (C-13). 5b.b (C-14).

24.2(c‘-15).2S.9(C-lbl.56.O(C-l7), 12.8(<‘-IX), 19.4(C-19). 149.1

(C-20). IOY.3 (C-21). 36 4 (C-22’). 33.4 (C-23*). 155.7 (C-24). 34.0

(C-25). 21.X (C-26). 21.X (C-27). lob.1 ((‘-2X) ‘HNMR. see

Table 2.

(‘ho/esro-5.20-drrn-3P_ol (4). MS m;; (rcl. ml.): 3X4 LMJ ’ (7X).

369(Y).366(6).351l17).314(l0),299(7).281(4),272(13),271(12),

229 (21). 213 t23), I59 (24). I45 (40). I33 (34). Xl (X5), 55 (100). ‘Q‘NMR K‘DC‘I,. TM% ($37.3 (c-l). 31.x (C-2*). 71.7 (C-3),

42.3K-4). I40.7(c‘-5). 12l.4(C-6).3l.b(C-7*).32.3((‘-X).50.3(C-

0). 36.5 (C-10). 21.1 (<‘-I I). 38.7 (C-12). 43.0 (C-13). 56.6 (C-14).

24.:!(c‘~l5)~25.Y(C~lb)~ SS.X((:-17). l2.7(C-IX). 19.3(C-19). 149.5

(C-20). 109.1 (C-21). 37.9 (C-22). 26.2 (C-23). 3X X (C-24), 27 8 (C-25). 22.6 (C-26’). 22.5 ((‘-27’). ‘H NMR: see Table 2.

24 5-.hfefh~lcholc,.sra-5.2O-dien-3P_ol (5). MS rn!: (rel. mt.): 398

~M]+(99).383(18),380(12).365(41),314(100).299(34).2XI(1X),

272 (12). 271 (26). 25X (IO). 255(S). 253 (101,229 (52). 213 (44). I59

(30). 145 (44). I38 (4). 133 (30), Xl (59). 69 (65). 55 (6X). 43 (59). ‘H NMR: see Table 2.

24~-E~hylchole.stu-5.20-dirn-3S_ol (6). MS m/z (rel. int.): 412

(651. 397 (IO). 394 (71, 379 (22). 314 (100). 299 (30). 2X1 (14). 271

(22), 229 (48). 213 (42). I59 (41). 145 (59). 133 (43), 95 (69). 81 (74).

69 (75). 55 (76). 43 (6X). ‘H NMR: see Table 2.

38-HJ‘dro.~y-Sa-cholrsran-6-onr (13). MS m;: (rcl. int.): 402

[M] - (6X), 387 (8). 3X4 (I 7). 369 (I I), 331(32), 289 (23). 288 (9). 2X7 (II), 271 (16). 262(ll). 247 (33). 95 (100).

3~-Ilydroxy-24~-e~hy/-5a-cholest-22-en-6-one (14). MS miz

(rel. mt.): 428 ([M] ., 28),413 (5),410(6). 395 (Z), 367(10). 357 (8).

289 (15). 288 (18). 287 (2X), 271 (22). 247 (17). 95 (96). 55 (100).

3B-Hydroxy-245-methyl-Sa-cholestan-6-t (15). MS m!:z (rel.

int.): 416 [MJ’ (15). 401 (2). 398 (6). 385 (12), 383 (4). 345 (6). 289

(15). 276 (6). 271 (22). 247 (17). 95 (96). 55 (100). 3/I-Hydroxy-24(-erhy/-k-cholesrun-b-one (16). MS m/z (rel.

mt.):430 [M]’ (100),415(5),412(5), 397(4), 359(2), 340(20), 290

(4), 289 (13). 271 (5). 247 (IX), 95 (2l), X6 (27). X4 (40). “CNMR (CDCI,. TMS) 636.7 (C-l), 30.7 (C-2*), 70.6 (C-3). 30.1 (C-4’).

56.7 (C-S), 210.3 (C-6). 46.7 (C-7). 37.9 (C-8). 54.0 (C-9). 40.8 (C-

IO), 21.5 (C-l I). 39.5 (C-12). 42.9 (C-13). 56.7 (C-14). 24.0 (C-IS),

28.O(C-16),56.O(C-17). 12.O(C-18). 13.1 (C-19). 36.O(C-20). IX.7

(C-21). 33.9 (C-22x 26.2 (C-23). 45.X (C-24). 29.2 (C-25). 19.0 (C-

26). 22.7 (C-27). 23.1 (C-28). 12.0 (C-29). ‘H NMR, see Table 2.

Acknowledgemenrs -We thank UMYMFOR (CONICET-

FCEN) for spectroscopx analysis and CONICET and the

Organization of the American States for partial financial sup

port. We greatly appreciate the technical aswtance of Mr Adrian Bcsso.

Steroids from Mandeuilla pentlandiana I243

REFERENCES 7. Wherli, F. and Nishida, T. (1979) Prog. Chem. Org. Nat.

1. Yamauchi, T. (1985) Yakugoku Zusshi 10!!,3345. 2. Seldeq A. M., De.luca, M., Gros, E. G., Rovirosa, J, San

Martin, A. and Darias, J. (1990) Z. Naturforsch. 4Sb. 83. 3. Sheikh, Y. and Djerassi, C. (1973) J. Org. Chem. 38, 3545. 4. Rubinstein, I. and Goad, L. J. (1974) Phytochemistry 13,

481. 5. Djerassi, C., Shapiro, R. and Vandewalle, M. (1965) J. Am.

Gem. Sot. 87.4892. 6. Zaretskii, Z. (1976) Mass Spectrometry o/ Steroids (John

Wiley, New York) p. 11. Israel University Press, Jerusalem.

8. 9.

10.

11.

12.

Prod. 3&l. Blunt, J. W. and Stothers, J. (1977) Org. Mogn. Reson. 9,439. Akihisa, T., Tamura, T., Matsumoto, T., Kokke, W. and Yokota, T. (1989) J. Org. Chem. 54,606. Akihisa, T., Yokota, T., Takahashi, N., Tamura, T. and Matsumoto, T. (1989) Phymchemistry 28, 1219. Akihisa, T., Kanari, M., Tamura, T. and Matsumoto, T. (1989) Phytochemisrry 28, 1271. Sheikh, Y., Tursch, B. and Djerassi, C. (1972) J. Am. Chem. Sot. 94, 3278.