Epoxidation and Reduction of DHEA, 1,4,6-Androstatrien-3-one and 4,6-Androstadien-3β,17β-diol

Dehydroepiandrosterone (DHEA) reacted with m-chloroperoxybenzoic acid (m-CPBA) to form 3β-hydroxy-5α,6α-epoxyandrostan-17-one (1), but it did not react with 30% H2O2. 1,4,6-Androstatrien-3,17-dione (2) was obtained from DHEA and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone in dioxane. Compound 2 was reacted with 30% H2O2 and 5% NaOH in methanol to give 1α,2α-epoxy-4,6-androstadien-3,17-dione (3), which was stereoselectively reduced with NaBH4 to form 1α,2α-epoxy-4,6-androstadien-3β,17β-diol (7) and reacted with Li metal in absolute ethanol-tetrahydrofuran mixture to give 2-ethoxy-1,4,6-androstatrien-3,17-dione (8). Compound 2 was also epoxidized with m-CPBA in dichloromethane to afford 6α,7α-epoxy-1,4-androstadien-3,17-dione (4), which was reacted with NaBH4 to synthesize 6α,7α-epoxy-4-androsten-3β,17β-diol (9). Compound 4 was reduced with Li metal in absolute ethanol-tetrahydrofuran mixture to form 7β-ethoxy-6α-hydroxy-1,4-androstadien-3,17-dione (10). Compound 2 was reduced with NaBH4 in absolute ethanol to form 4,6-androstadien-3β,17β-diol (5), which was reacted with 30% H2O2 to give the original compound, but which reacted with m-CPBA to give 4β,5β-epoxy-6-androsten-3β,17β-diol (6).


Introduction
Dehydroepiandrosterone (3β-hydroxyandrost-5-en-17-one, DHEA) is an endogenous steroid synthesized in the adrenal cortex, gonads, brain, and gastrointestinal tract [1,2]. It is known to have chemopreventive and anti-proliferative actions on tumors [3][4][5]. DHEA is the most abundant steroid in human blood; it is not only an intermediate in the biosynthesis of testosterone and estrogens but it also exerts several physiological effects independent of the sex hormones. It is also an anti-obesity agent for genetically obese and normal animals, but does not affect food intake [6]. It decreases blood cholesterol concentration in several species [7][8][9], lessens the severity of diabetics in genetically predisposed mice [10], enhances the immune system [11,12], and improves memory in aged mice [13].
Epoxides are among the most frequently occurring functional groups found in natural products and their synthetic analogues. Not only are these compounds easily prepared from variety of starting materials, but the inherent polarity and the strain of their three-membered ring makes them susceptible to reaction with a large number of reagents -electrophiles, nucleophiles, acids, bases, reducing agents, and oxidizing agents [22,23]. Whilst the balance between the various stereoelectronic factors that control the stereochemistry of epoxidation of steroidal alkenes has been thoroughly investigated, there have been relatively few studies of the epoxidation of conjugated steroidal dienes and trienes [24].
In order to investigate the reagent selectivity and position selectivity of different epoxidizing agents towards 1,4,6-androstatrien-3,17-dione and 4,6-androstadien-3β,17β-diol as examples of trienes and dienes, 30% H 2 O 2 and m-chloroperoxybenzoic acid were used. These reagents are easily handled and inexpensive materials that did not require complicated procedures or expensive metal-catalysts, which were mostly used to control the asymmetric epoxidation. In addition, and in order to study the reagent and position selectivity of the resulting epoxy derivatives towards reducing agents, we selected NaBH 4 and Li metal in absolute ethanol-tetrahydrofuran mixture and we tried to compare the selectivity of these reagents for the cleavage of epoxide ring, and reduction of carbonyl groups and double bonds of epoxy unsaturated DHEA derivatives.

Results and Discussion
For the synthesis of dehydroepiandrosterone (DHEA) derivatives, commercially available DHEA was used as the starting material. The planned synthetic routes from DHEA are shown in Scheme 1. First, DHEA was treated with 30% H 2 O 2 and 5% NaOH in MeOH solution at room temperature, but it did not react. DHEA was then reacted with m-chloroperoxybenzoic acid to give 5α,6αepoxyandrostan-3β-ol (1), which was formed by α-face attack of the peracid to the 5,6-double bond.
The initial axial downside attack on the 5-androsten-3β-ol involves an interaction between the π-HOMO of the alkene and the σ*-LUMO of the O-O bond of the peracid. The peracid may be directed to the α-face of the steroid by unfavourable interaction with C-10 angular methyl group on the β-face [25].

Scheme 2. Formation of stable allylic carbocationic intermediate by
Compound 2 was reduced with NaBH 4 in absolute ethanol to form 4,6-androstadien-3β,17β-diol (5) as 1,2-and 1,4 addition product [29]. Metallic hydrides, such as LiAlH 4 and NaBH 4 , do not in general reduce carbon-carbon double bond, but these reagents may also double bond in conjugated with C=O bonds, as well as reducing the C=O bonds [30,31]. The attack of hydride anion of NaBH 4 to the axial direction of the carbonyl group produced the 3β-and 17β-hydroxy configuration and only the 1,2-double bond of the 1,4,6-triene 2 was reduced. The stereochemistry was established from the NOESY spectrum. No correlation between the hydrogens of the C-10 methyl group on the β-face and H-3, and C-13 methyl hydrogens and H-17 was observed in the NOESY spectrum of 5, therefore both OH groups on C-3 and C-17 are directed in the β-position. On attempted epoxidation of compound 5 in the presence of 30% H 2 O 2 and 5% NaOH in methanol, only starting material was recovered.
Compound 5 reacted with m-chloroperoxybenzoic acid to give 4β,5β-epoxy-androst-6-en-3β,17β-diol (6). In this case, the presence of the 3β-hydroxy group of the allylic alcohol directed the epoxidation exclusively to the β-face of the double bond of the molecule [32,33].

Acknowledgments
We thank the Catholic University of Daegu for financial support.

General
Reactions were performed under nitrogen gas. Solvents were purified and dried prior to use.
Melting points were measured on Thomas-Hoover melting point apparatus in open capillary tubes and are not corrected. 1 H-and 13 C-NMR spectra were obtained on Gemini 200 MHz or 50MHz spectrometers in CDCl 3 , chemical shifts δ are in ppm are relative to tetramethylsilane, coupling constants J in Hz. NOESY spectra were measured on Bruker AM-500MHz spectrometer (Rheinstetten, Germany). IR spectra were determined on a Jasco FT-IR 300E spectrometer as KBr pellets. FAB mass spectra were recorded on a tandem mass spectrometer made by Jeol Ltd (Japan). Analytical TLC was performed on Merck precoated silica gel 60 F 254 plates. Column chromatography was carried out on Merck silica gel 9385 (230-400 mesh). 5α,6α-Epoxy-anrostan-3β-ol-17-one (1) To a solution of androst-5-en-3β-ol (dehydroepiandrosterone, 1 g, 3.47 mmol) in CHCl 3 (50 mL) m-chloroperoxybenzoic acid (2 g, 6.93 mmol) was added at room temperature. The mixture was stirred at room temperature for 24 hours. The solution was diluted with CHCl 3 , treated with aqueous sodium sulfite and the aqueous layer was extracted with several portions of CHCl 3 . The combined organic extracts were washed with water, dried with anhydrous MgSO 4 , filtered and evaporated to leave a crude solid, which was column chromatographed with 1:2 ethyl acetate/n-hexane as eluent to afford white crystals. Yield: 897 mg (85 %); mp: 208. 5

1,4,6-Androstatrien-3,17-dione (2)
A solution of DHEA (5 g, 17.36 mmol) and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (11.8 g, 52.08 mmol) in dioxane (100 mL) was refluxed for 24 hours according to the procedure reported by  (5) To a solution of compound 2 (1 g, 3.55 mmol) in absolute ethanol (100 mL), NaBH 4 (537 mg, 14.2 mmol) was added at room temperature. The resulting mixture was stirred overnight at same temperature, and then was concentrated by rotary evaporation to afford white solid. It was stirred with water for 30 minutes, then extracted with dichloromethane from the aqueous layer. The organic layer was washed with aqueous sodium hydrogen carbonate, water and dried with anhydrous MgSO 4 , filtered, and concentrated to give a residue, which was recrystallized from 1:2 ethyl acetate/n-hexane to afford white crystals of the title compound. Yield 4β,5β-epoxyandrost-6-en-3β,17β-diol (6) m-Chloroperoxybenzoic acid (611 mg, 3.55 mmol) was added to a solution of dienol 5 (500 mg, 1.77 mmol) in CHCl 3 (50 mL) at room temperature and the reaction mixture was then treated in a manner similar to that described for the synthesis of compound 1. The pure compound 6 was obtained as white crystals by column chromatography on silica gel (elution with 1:3 ethyl acetate/n-hexane=). 1α,2α-Epoxy-1,4--androstadien-3β,17β-diol (7) To a solution of compound 3 (500 mg, 1.68 mmol) in absolute ethanol (100 mL) NaBH 4 (237 mg, 6.27 mmol) was added at room temperature. The resulting mixture was stirred overnight at the same temperature and then was concentrated by rotary evaporation to afford a white solid which was stirred with water and d-HCl solution for 30 minutes, then extracted with dichloromethane from the aqueous layer. The organic layer was washed with water and dried with MgSO 4 , filtered, and concentrated to give a white solid, which was purified by column chromatography (1: (8) To a solution of 3 (500 mg, 1.68 mmol) in absolute ethanol (50 mL) lithium metal (400 mg) was added slowly and the resulting mixture was vigorously stirred at the room temperature for 18 hours.

2-Ethoxy-1,4,6-androstatrien-3,17-dione
The reaction mixture was cooled down to 0°C and water was slowly added with stirring until a clear solution was obtained. The mixture was evaporated to remove the ethanol, and then the aqueous layer was extracted several times with ethyl acetate. The combined organic layer was washed with H 2 O and dried over anhydrous MgSO 4 , filtered and evaporated to give a crude oil, which was purified by column chromatography on silica gel (1:3 ethyl acetate/n-hexane 6α,7α-Epoxy-androst-4-en-3β,17β-diol (9) To a solution of 4 (500 mg, 1.68 mmol) in absolute ethanol (100 mL) NaBH 4 (177 mg, 4.70 mmol) was added and the mixture stirred at room temperature until the starting material had disappeared in a TLC test. The reaction mixture was evaporated to a dry solid and H 2 O was added to the residue with stirring. The aqueous layer was extracted with ethyl acetate, and the organic layer was dried over anhydrous MgSO 4 , filtered, concentrated to give a crude precipitate. The 7β-Ethoxy-6α-hydroxy-1,4-androstadien-3,17-dione (10) To a solution of 4 (500 mg, 1.68 mmol) in absolute ethanol (50 mL) lithium metal (300 mg) was added slowly and the mixture vigorously stirred at room temperature until the starting material had disappeared in a TLC test. The workup was as described in the preparation of compound 8. The purified product was obtained as white crystals by column chromatography on silica gel (1: