226

A New Route to Steroid Ring C Aromatization from Readily Available Precursors MARIO ANASTASIA*, PIETRO ALLEVI, ALBERTO FIECCHI and ANTONIO SCALA, Institute of Chemistry, School of Medicine, University of Milan, Via Saldini 50, 1-20133, Italy

ABSTRACT

3#-Acetoxy-8~,9a-epoxy-5,*-cholest-14-ene (1); 3#-acetoxy-14a,15~-epoxy-5~-cholest-8-ene (2); 3fl-acetoxy-5~-cholest-8(14)-ene-9~, 15a-diol (3); and 3/~-acetoxy-5a-cholesta-8(14),9(11 )-dien-I 5a-ol (4) have been aromatized to a 9:1 mixture of 3/~-hydroxy-12-methyl-18-nor-5c(,17#(H)-cholesta-8,11, 13-triene (5a) and 3#-hydroxy-12-methyl-18-nor-5~,l 7a(H)-cholesta-8,11,13-triene (5b) in ethanol solution by using hydrochloric acid. The aromatization by action ofp-toluenesulfonic acid gave main- ly the epimer with the natural C-17 configuration as the acetate 5c at the appropriate p-toluenesul- fonic acid concentration. 3#-Acetoxy-5a-cholesta-7,9(ll),14-triene (7a)and 3t~-hydroxy-5a-cholesta- 8,11,14-triene (Sa), 2 intermediary compounds in the aromatization, were isolated and characterized. Lipids 17:226-229, 1982.

INTRODUCTION

The synthesis of aromatic ring C steroids is of recent interest (1) because some of them are analogous to naturally occurring estrogens and have potential as a new class of agents useful in the antifertility field (2). We now report a novel molecular rearrangement leading to ring C aro- matic steroids from readily available steroids.

MATE RIALS AND METHODS

Reagents

3 ~Acetoxy-8ot, 9~- epoxy- 5rv-cholest- 14-ene (1); 3~-acetoxy- 14~, 15ot-epoxy-5rv-cholest-8-ene (2); 3~acetoxy-5c~-cholest-8(l 4)-ene-9~, 15a- diol (3); and 3~-acetoxy-5~-cholesta-8(14),9- (11 )-dien-I 5~-ol (4) were prepared as described previously (3).

Reactions with Hydrochloric Acid at Reflux

A solution of the steroid (300 mg) in eth- anol (33 ml) and hydrochloric acid (2 rnl, 37%) was refluxed under nitrogen for 8 hr. The solu- tion was concentrated under reduced pressure, diluted with water and extracted with diethyl ether. The combined extracts were washed suc- cessively with aq. NaHCO3 solution and water, dried over anhydrous sodium sulfate and evapo- rated to dryness under reduced pressure to yield a residue which was then chromato- graphed (4) on silica (40-63/.tm) with hexane/ ethyl acetate (80: 20, v/v) to separate aromatic sterols (Sa) and (5b) from 3~hydroxy-5a-cho- lest-8(l 4)-en- 15-one (6a). The aromatic fraction was then rechromatographed on Silica Gel G/ Celite/AgNO3 (1 : 1 :.3, w/w) with hexane/ethyl acetate (100:5, v/v) to separate the 2 epimeric aromatic compounds.

Reactions with Hydrochloric Acid at Room Temperature

A solution of the steroid (300 mg) in eth- anol (33 ml) and hydrochloric acid (2 ml, 37%) was kept at room temperature for 30 min. At this time, the solvent was reduced to half of its volume under reduced pressure and cooled. Under these conditions, 3~-acetoxy-5ot-cholesta- 7,9(11),14-triene (7a) crystallized. The mother liquors, after filtration, were evaporated and chromatographed on Silica Gel G/Celite (1:1, v/v, eluting with hexane]ethyl acetate, 90:10, v/v) to afford 3/3-acetoxy-5ot-cholest-8(14)-en- 15-one (6b).

Reactions with Excess p-Toluenesulfonic Acid

A solution of p-toluenesulfonic acid (0.5 g) in toluene (70 ml) was refluxed and part of the solvent (20 ml) was distilled off. The steroid (800 mg) was added and the solution refluxed for 30 rain under nitrogen. The solution was washed successively with aq. NaHCO3 and water, and evaporated to afford a residue which was purified by chromatography as just de- scribed for the reactions with hydrochloric acid at reflux.

Reactions with Catalytic Quantity of p-Toluenesulfonic Acid

The reaction was done as already described except that a catalytic quanti ty of p-toluene- sulfonic acid (30 rag) was used for the same amount of sterol (.800 mg).

Analysis of the Steroids

Ultraviolet (UV) spectrometry was per- formed using a Varian Model 635 UV-visible spectrophotometer. Infrared (IR) spectra were recorded for solutions in chloroform or for

LIPIDS, VOL. 17, NO. 3 (1982)

STEROID RING C

nujol mulls using a Perkin-Elmer Model 157 IR spectrophotometer . Gas liquid chromatographic (GLC) examination of the sterols was on I% SE-30 on Gas-Chrom Q (80-120 mesh) at 200 C on a Carlo Erba Model Fractovap 2101 gas chromatograph. Mass spectral analysis was per- formed on a Varian Mat 112 S spectrometer by direct inlet. Proton magnetic resonance (1H NMR) spectra were obtained at 60 MHz at am- bient temperature on a Perkin Elmer instru- ment, Model R-24, in CDC1 a with Si(CHa)4 as internal standard. Routine optical rotations were recorded with a Perkin-Elmer Model 141 spectropolarimeter for 1% solutions in chloro- form. The progress of all reactions was moni- tored by thin layer chromatography (TLC) on Silica Gel G (HF2s4) microplates.

RESULTS

Treatment of 3~-aeetoxy-5c~-cholcsta-8(14), 9(11)-dien-15t~-ol (4) with hydrochloric acid at reflux after double chromatography gave (Fig. 1): (a) 3~-hydroxy-12-methyl-18-nor-5a,17/~- (H)-cholesta-8,11,13-triene (5a) in which the original 3~-acetoxy group has been hydrolyzed, the 15t~-hydroxy group has been eliminated, the angular methyl group (C-18) has migrated from C-13 to C-12; and the 17/~-configuration of the side-chain has been inverted. Compound (5a) (178 mg from 300 mg of 4) resisted all efforts to crystallize and showed: IR 3500, 3330 e r a - l ; UV ;kma x (cyclohexane) 225 nm (log e 4.07); IH NMR 8 0.55 (d, 3H, J = 6 Hz, 21-CH3), 1.10 (s, 3H, 19-CHa), 2.26 (s, 3H, 18-CHa), 3.3 (m, 1H, w l / 2 ca. 12 Hz, 17~-H), 3.67 (m, 1H, w l / 2 ca. 20 Hz, 3~_H), 6.94 (s, IH, l l -H) : MS m/e 382 (M+). Anal. calcd, for C27H420: C, 84.8; H, 11.1. Found: C, 84.6;H, 11.0.

(b) 3~Hydroxy-12-methyl-18-nor-5a, 17t~(H)- cholesta-8,11,13-triene (5b) (20 rag) epimer at C-17 of 5a. The compound crystallized from methanol and showed mp 97-99 C (lit. [5] gum) and showed: IR 3500, 3330 e m - l ; UV Xmax (cyclohexane) 225 nm (log e 4.07); IH NMR 5 1.10 (s, 3H, 19-CHa) , 2.26 (s, 3H, 18-CHa), 3.15 (m, IH, w l / 2 ca. 12 Hz, 17tx-H), 3.67 (m, 1H, w l / 2 ca. 20 Hz, 30t-H), 6.94 (s, IH, 1 I-H); MS m/e 382 (M+). Anal. calcd, for C27H420: C, 84.8; H, 11.1. Found: C, 84.5; H, 11.0. These chemicophysical properties, apart from the mp, agree well with those reported (5).

(c) 3/~-Hydroxy-5ot-eholest-8(14)-en- 15-one (6a) (25 mg) which, upon crystallization from methanol, melted at 145-146 C and showed identical chemicophysical properties with an authentic sample (6).

AROMATIZATION 227

2 1

I I

/

m R 6

S

3

FIG. 1. Formation of compound 5a-r and 6a,b from 1-4.5a, R = H, R 1 = H, R 2 = CsH17.5b, R = H, R 1 = CsH17 , R 2 = H. 5e, R = Ac, R 1 = C8H17, R 2 = H. 5(t, R = Ac, R t = H, R 2 = CsHIT. 5e, R = Ae, R t = C9HI~Br2, R 2 = H. 6a, R = H. 6b, R -- Ac.

When compounds 1, 2 and 3 were subjected to the action of hydrochloric acid at reflux, essentially the same results were obtained. The yields of 5a were 70-80%, those of 5b 6-8%, and those of 6a 7-10%.

In order to isolate possible intermediates of the aromatization, 4 was treated with hydro- chloric acid at room temperature. In these con- ditions, 3~-acetoxy-5a-cholesta-7,9(11), 14-tri- ene (7a) was obtained in 70% yield accompa- nied by a minor amount (10%) of 3~-acetoxy- 5a-cholesta-8(14)-en-15-one (6b) (Fig. 2). 7a showed: mp 90-92 C (from methanol); [a] ~ - 89; UV ~max (cyclohexane) 228 (log e 3.99), 236 (4.01), and 268 nm (3.96); 1H NMR5 2.1 (s, 3H, OCOCH3), 4.7 (m, IH, w l / 2 ca. 20 Hz, 3a-H), 5.5, 5.8, (m, 3H, 7-, 11-, and 15-H);MS m/e 424 (M+). Anal. calcd, for C29I-I4402: C, 82.0; H, 10.4. Found: C, 81.8; H, 10.2.

When the triene (7a) was refluxed for 30 min in hydrochloric acid, 3/g-hydroxy-5tx-cho- lesta-8,11,14-triene (8a) was obtained in 65% yield, accompanied by a trace amount of 5a and 5b (Fig. 2). Triene (8a), and oil, showed: UV Xmax 314 nm (log e 3.8); IH NMR 5 3.6 (m, IH, w l / 2 ca. 20 Hz, 3a-H_), 6.0 (m, 1H, 15-H), 6.1 (dd, 2H, J = 11 Hz, 11-and 12-H); MS m/e 382 (M+).

LIPIDS, VOL. 17, NO. 3 (1982)

228 M. ANASTASIA, P. ALLEVI, A. FIECCHI AND A. SCALA

When 1, 2, 3, 4 and 7a were treated with excess p-toluenesulfonic acid at reflux, after the described work-up, 3fl-acetoxy-12-methyl- 18-nor-5a,17a(H)-cholesta-8,11,13-triene (5e) was obtained in 70% ~r The compound, oil, showed IR 1730 c m - ' ; UV hma x (cyclohexane) 225 nm (log e 4.07); IH NMR ~ 1.1 (s, 3H, 19-CH3) , 2.02 (s, 3H, OCOCH_3), 2.26 (s, 3H, 18-CH3) , 3.15 (m, 1H, wl/2 ca. 12 Hz, 17r 4.75 (m, 1H, w l [2 ca. 20 Hz, 30t-H_), 6.92 (s, IH, 1 l-H); MS m/e 424 (M+). Anal. calcd, for C29H4402: C, 82.0; H, 10.4. Found: C, 82.2; H, 10.5.

When catalytic amounts of p-toluenesulfonic acid were used in the aromatization reaction, a mixture of 5e and 3~-acetoxy-12-methyl-18- nor-5ot,17/~(H)-cholesta-8,1 I , I 3-triene (Sd) was obtained in 1:1 ratio in 75% total yield. Com- pound 5(!, oil, showed: UV Xmax (cyclohexane) 225 nm (log e 4.07); IH NMR5 0.56 (d, 3H, J_ = 6 Hz 21-CH3), 1.08 (s, 3t l , 19-CH3), 2.00 (s, 3H, OCOCH_a), 2.20 (s, 3H, 18-CH_3) , 3.3 (m, IH, w l / 2 ca. 12 Hz, 17fl-_H), 4.8 (m, 1H, w l /2 ca. 20 Hz, 3~-H), 6.94 (s, IH, 11-H_); MS m/e 424 (M+). Anal. calcd, for C29H4402: C, 82.0; H, 10.4. Found: C, 81.8; H, 10.3.

DISCUSSION

Considerable work has boen reported on the aromatization of ring A of steroids by molecu- Jar rearrangement or by elimination of the C-19 methyl group. On the contrary, relatively few methods have been reported on the aromatiza- t ion of ring C of steroids having a 17j3 side chain. The original method of Margulis et al. (7) starting from a 7~,1 la-dibromo-8-unsatu- rated sterol, was improved by Edmunds et al. (5), who demonstrated that, with an acidic catalyst, the dibromo unsaturated sterol is aromatized in high yield. Until now, this method represents the best route to ring C aromatic sterols.

We recently obtained (3) 2 monoepoxida- l ion compounds, 3/$-acetoxy-8~,9a-epoxy-Sa- cholest-14-ene (1) and 3[]-acetoxy-140~,150t- epoxy-5--eholest-8-ene (2) by action of per- acids on 313-acetoxy-5a-cholesta-8,14-diene. Compound 2 was then transformed by silica in a mixture of 3/3-acetoxy-Sa-cholest-8(l 4)-ene- 9oqlS~-diol (3) and 3j3-acetoxy-5~-cholesta-8- (14),9(1 l)-dien-I 5a-ol (4).

As i t was considered probable that dehydra- tion of the allylic hydroxy group present in compound 4 would yield molecular rearrange- m~nt able to produce aromatization of the ring C, we treated compound 4 with hydrochloric acid in ethanol at reflux; under these condi- tions, we obtained as a major product 3~

R

7 B

/ 1'

1 I

FIG. 2. Intermediates in the formation of ring C aromatic steroids: 7a, R = Ac, R~ = CsHtT. 7b, R = Ac, R l = CgHITBr 2. 8a, R = H, R 1 = (;5H17. 8b, R --- Ac, R I = CsH17.

hydroxy-12-methyl-18-nor-5a, 1713 (H)-cholesta- 8,11,13-triene (5a, 72%) accompanied by a minor amount of 3fl-hydroxy-12-methyl-18- nor-5cx, 17r 1,13-triene (5b) (8%) and 3fl-hydroxy-5cx-cholesta-8(14)-en-15- one (6a, 10%). The success of this reaction in giving the aromatization of ring C prompted us to subject the other products obtained in the epoxidation of 3fl-acetoxy-50l-cholesta-8,14- diene and structurally related to compound 4 to the same acidic treatment. In all cases, the same pat tern of reaction products was observed.

In order to clarify the mechanism of this new rearrangement leading to ring C benzenoid steroids, we treated the same sterols with hy- drochloric acid at room temperature. Under these conditions, 30-acetoxy-5~-cholesta-7,9- ( l l ) ,14- t r iene (7a) was obtained in crystalline form. The formation of 7a was also observed in the reaction b y GLC-mass spectrometry. A similar triene (7b) was also obtained by Ed- rounds et al. (5) in the ring C aromatization of 7c~,110t-dibromo unsaturated steroids. So, it is clear that despite the different starting com- pound, the aromatization involves a similar intermediate.

Treatment of the triene (7a) with hydro- chloric acid at reflux afforded, after 30 min, a new conjugated triene (8a) which was isolated and characterized by its chemicophysical prop-

LIPIDS, VOL. 17, NO. 3 (1982)

STEROID RING C AROMATIZATION 229

erties. Pursuing the ref lux wi th h y d r o c h l o r i c acid, the t r i ene (8a) was comple t e ly t rans- fo rmed i n t o the a roma t i c s terols 5a and 5b in a 9:1 rat io.

In view of the fac t t h a t E d m u n d s et al. (5) o b t a i n e d the co r r e spond ing r ing C a roma t i c c o m p o u n d (5e) wi th the " n a t u r a l " side chain by t r e a t m e n t o f 7b wi th excess of p - to luene- sul fonic acid, we cons idered the poss ibi l i ty o f ob t a in ing c o m p o u n d (5e) by t r e a t m e n t of 4 wi th excess p - to luenesu l fon i c acid. U n d e r these cond i t ions , c o m p o u n d 5e was o b t a i n e d as a ma jo r p roduc t , a ccom pan i ed by a m i n o r a m o u n t o f 5d and 6b. When a ca ta ly t ic a m o u n t of p - t o luenesu l fon i c acid was used, c o m p o u n d s 5e and 5d were o b t a i n e d in a b o u t the same rat io. The same results were o b t a i n e d when c o m p o u n d s 1, 2 and 3 were sub jec ted to these react ions . Moni to r ing the reac t ions by GLC-MS, the i n t e rmed ia ry f o r m a t i o n of t r ienes 7a and 8b was observed. F r o m these results , i t is clear t h a t the a r o m a t i z a t i o n wi th h y d r o c h l o r i c acid and p - to luenesu l fon i c acid p roceeds t h r o u g h the f o r m a t i o n o f t r ienes as 7 and 8. However , hy - d roch lor ic acid a romat i zes 8 wi th ep imer i za t i on of t he side chain whereas p - to luenesu l fon i c acid causes a r o m a t i z a t i o n wi th p reva len t r e t e n t i o n of the side chain conf igu ra t ion , the e x t e n t of the ep imer i za t ion be ing d e p e n d e n t o n the con- c e n t r a t i o n of p - to luenesu l fon i c acid (5) . A reasonable m e c h a n i s m for the a r o m a t i z a t i o n o f a t r iene as 7 was f o r m u l a t e d by E d m u n d s et al. (5) (Fig. 2). The i so la t ion of t r i ene 8a s t rongly suppor t s this m e c h a n i s m , which appears the mos t reasonable for the f o r m a t i o n of the aro- ma t i c c o m p o u n d wi th a na tu ra l s ide-chain s t e r eochemis t ry . However , f o r m a t i o n o f the 17-

ep imer ic c o m p o u n d could be more complex . In fact , some years ago, we (8) and A b e r h a r t e t al. (9) d e m o n s t r a t e d t h a t h y d r o g e n chlor ide t rea t - m e n t of 3 ~ a c e t o x y - 5 a - c h o l e s t - 1 4 - e n e causes f ide-chain invers ion t h r o u g h t he f o r m a t i o n of a s p i r o c o m p o u n d . Accord ing ly , such a mecha- n i sm could be supposed to expla in the side- chain invers ion in the p resen t a roma t i za t i on . Work is in progress to evaluate this possibi l i ty .

ACKNOWLEDGMENTS

This research was supported by Ministero della Pubblica Istruzione. We thank Professor G. Galli, Institute of Pharmacology and Pharmacognosy, Uni- versity of Milan, for mass spectra.

REFERENCES

1. Khanapure, S.P., Hazra, B.G., a n d Das, K.G. (1981) J. Chem. Soc. Perkin Trans. I, 1360-1362.

2. Dalzell, H.C., Manmade, A., Mastrocola, A.R., and Razdan, R.K. (1979) J. Org. Chem. 44, 2457- 2461.

3. Anastasia, M., Allevi, P., Fiecehi, A., and Scala, A. (1981) J. Org. Chem. 46, 3265-3267.

4. Still, W.C., Kahn, M., and Mitra, A. (1978) J. Org. Chem. 43, 2923-2925.

5. Edmunds, R., Midgley, J.M., Tagg, L.G., Wilkins, B.J., and Whalley, W.B. (1978) J. Chem. Soc. Perkin Trans. I, 76-80.

6. Anastasia, M., Fiecchi, A., and Scala, A. (1979) J. Chem. Soc. Perkin Trans. I, 182 I-1824.

7. Margulis, T.N., Hammer, C.F., and Stevenson, R. (1964) J. Chem. Soc. 4396-4400.

8. Anastasia, M., Fiecchi, A., and Scala, A. (1978) J. Org. Chem. 43, 3505-3508.

9. Aberhart, D.J., Cahn, T.Y., and Caspi, E. (1979) J. Chem. Soc. Perkin Trans. 1,220-224.

[Received Augus t 17, 1981]

LIPIDS, VOL. 17, NO. 3 (1982)