Epoxidation of Diosgenin, 25(R)-1,4,6-Spirostatrien-3-one and 25(R)-4,6-Spirostadien-3β-ol

Abstract

Upon treatment of 25(R)-spirost-5-en-3β-ol (diosgenin), 25(R)-1,4,6-spirostatrien-3-one and 25(R)-4,6-spirostadien-3β-ol with 30% H₂O₂ and 5% NaOH in methanol, diosgenin did not react, 25(R)-1,4,6-spirostatrien-3-one was converted to 25(R)-1α,2α-epoxy-4,6-spirostadien-3-one and 25(R)-4,6-spirostadien-3β-ol was oxidized to give a small amount of 25(R)-4,6-spirostadien-3-one, while most of the original starting material remained unchanged. On the other hand, reactions of diosgenin, 25(R)-1,4,6-spirostatrien-3-one and 25(R)-4,6-spirostadien-3β-ol with m-chloroperoxy-benzoic acid in chloroform yielded 25(R)-5α,6α-epoxyspirostan-3β-ol, 25(R)-6α,7α-epoxy-1,4-spirostadien-3-one and 25(R)-4β,5β-epoxy-6-spirosten-3β-ol, respectively.

Introduction

Diosgenin (25(R)-spirost-5-en-3β-ol) is the steroidal saponin which is isolated from the Mexican yam (Dioscorea). Estrogenic, progesterogenic and anti-inflammatory effects have been hypothesized for diosgenin due to its structural similarity to estrogen and progesterone precursors [1]. Diosgenin has been reported to lower serum cholesterol in chicken and rabbits fed cholesterol and to decrease liver cholesterol in cholesterol-fed rats, an action associated with increased rates of cholesterol synthesis, increased secretion of cholesterol into bile acid, and increased fecal excretion of neutral sterols [2,3,4,5,6].

Diosgenin was used commercially to produce steroid hormones such as cortisone, estrogen, and progesterone by in vitro chemical modification [7]. 1α-Hydroxydiosgenin and 1β-hydroxydiosgenin (ruscogenin) were used as starting materials in the synthesis of 1α-hydroxycholesterol and 1β-hydroxycholesterol by chemical removal of its side chain [8,9]. The study showed that 1β-hydroxydiosgenin could be used as a potential starting material for 1α- or 1β-hydroxy vitamin D analogs [10,11]. Diosgenin was also used as the natural source to synthesize dehydroepiandrosterone (DHEA), a steroid that declines with age, but has significant activities as an antioxidant to modify serum lipid levels and which inhibits proliferation of cancer and noncancer cells in both in vitro and in vivo studies [12,13,14].

Since 1α- or 1β-hydroxydiosgenin is of great interest as a starting material in the synthesis of various active compounds, it is desirable to make hydroxydiosgenin derivatives more available for biological experimentation. The efficient stereoselective synthesis of hydroxydiosgenin derivatives requires a stereoselective epoxidation of double bond as an important intermediate step. Consequently we have examined position selective and reagent selective epoxidations of the olefinic position of diosgenin, 25(R)-1,4,6-spirostatrien-3-one [9] and 25(R)-4,6-spirostadien-3β-ol using 30 % H₂O₂ and m-chloroperoxybenzoic acid as epoxidizing agents, respectively.

Results and discussion

In this paper, we report the results of a study of the reagent selective and regioselective epoxidation reactions of diosgenin derivatives. The syntheses were accomplished by the procedures described in Scheme 1. 25(R)-Spirost-5-en-3β-ol (diosgenin) was epoxidized with 30% H₂O₂ and 5% NaOH in methanol, but it did not react, whereas when diosgenin was treated with m-chloroperoxy-benzoic acid (mCPBA) in CHCl₃ it gave 25(R)-5α,6α-epoxy-spirostan-3β-ol (1). In this case, the equatorial attack of the mCPBA to the double bond of diosgenin is destabilized by the steric interaction between the reagent and the axial CH₃ group (number 19), therefore the downside axial attack is preferred for reagent approach in the transition state. The oxidative dehydrogenation of diosgenin with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone in refluxing 1,4-dioxane gave 25(R)-1,4,6-spirostatrien-3-one (2). Only the 1,2-double bond of compound 2 was selectively epoxidized with 30% H₂O₂ and 5% NaOH in methanol to yield 25(R)-1α,2α-epoxy-4,6-spirostadien-3-one (3) [9,15]. In the absence of directing effects, electrophilic epoxidation of the double bond takes place predominantly from the α-face of the molecule.

Scheme 1.

Scheme 1.

On the other hand, when compound 2 was epoxidized with mCPBA, 25(R)-6α,7α-epoxy-1,4-spirostadien-3-one (4) was obtained. This result is basically caused by the attack of the mCPBA directed on the less hindered α-face of 6,7-double bond. 25(R)-1,4,6-Spirostatrien-3-one (2) was reduced with NaBH₄ in absolute ethanol to give 25(R)-4,6-spirosta-dien-3β-ol (5) [11]. The pπ-electronic system of compound 5 is more stable than 25(R)-1,4- spirostadien-3β-ol [16]. When compound 5 was epoxidized with 30% H₂O₂ and 5% NaOH in methanol, the 3-hydroxy group was dehydrogenated to yield only 25% of 25(R)-4,6-spirostdien-3-one (6) while most of the original starting material was recovered. In the case of H₂O₂, epoxidation seems to only occur in the presence of a carbonyl group conjugated with the double bond. Compound 5 was epoxidized with mCPBA to form 25(R)-4β,5β-epoxy-6-spirosten-3β-ol (7). 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 [16,17]. All synthesized compounds were identified by IR, ¹H-NMR, ¹³C-NMR and mass spectra, respectively.

Acknowledgements

The authors wish to thank the Catholic University of Daegu for financial support.

Experimental

General

Reactions were performed under nitrogen. 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. ¹H- and ¹³C-NMR spectra were obtained on Gemini 200 MHz or 50MHz spectrometers in CDCl₃, chemical shifts δ are in ppm are relative to tetramethylsilane, coupling constants J in Hz. IR spectra were determined for KBr pellets on a Jasco FT-IR 300E spectrometer. FAB mass spectra were recorded on a Tandem mass spectrometer. Analytical TLC was performed on Merck precoated silica gel 60 F₂₅₄ plates. Column chromatography was carried out on Merck silica gel 9385 (230-400 mesh).

25(R)-5α,6α-Epoxy-spirostan-3β-ol (1). m-Chloroperoxybenzoic acid (480 mg, 2.78 mmol) was added at room temperature to a solution of 25(R)-spirost-5-en-3β-ol (diosgenin, 1 g, 2.4 mmol) in CHCl₃ (100 mL). The mixture was stirred under nitrogen at room temperature for 18 hours. Water was added and the aqueous layer was extracted with several portions of CHCl₃ and the organic extracts were combined, dried with MgSO₄, filtered and evaporated to leave a crude solid that was recrystallized from ethyl acetate and n-hexane (1:5) to afford 1 as white crystals. Yield: 960 mg (93 %); mp : 202.3-204.5°C; IR (cm^-1): 3513, 3401; ¹H-NMR δ: 0.71 (3H, s, H-27), 0.78 (3H, s, H-18), 0.94 (3H, d, J=6.8Hz, H-21), 1.07 (3H, s, H-19), 2.90 (1H, d, J=14.0Hz, H-6), 3.30-3.52 (2H, m, H-26), 3.87-3.96 (1H, m, H-3), 4.35-4.44 (1H, m, H-16); ¹³C-NMR δ: 109.3, 80.7, 68.7, 66.9, 65.7, 61.9, 59.2, 56.7, 42.6, 41.7, 40.4, 39.9, 39.5, 35.1, 31.2, 30.4, 29.6, 29.1, 28.9, 20.6, 17.2, 16.4, 16.1,14.6; FABMS (M+H)⁺ : 431.2.

25(R)-1,4,6-Spirostatrien-3-one (2). A solution of diosgenin (5 g, 12 mmol) and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (10 g, 88 mmol) in dioxane (125 mL) was refluxed for 24 hours under nitrogen. The reaction mixture was cooled down and the resulting precipitate was filtered off and washed with dichloromethane. The filtrate was rotary evaporated to dryness and the dark brown residue was purified by column chromatography on silica gel (ethyl acetate/n-hexane=1:7) to afford white crystals of 2. Yield: 3.04 g (62 %); mp: 207.1-209.2°C; IR (cm^-1): 1661, 1452; ¹H-NMR δ: 0.80 (3H, d, J=3.1Hz, H-27), 0.90 (3H, s, H-18), 1.00 (3H, d, J=3.4Hz, H-21), 1.21 (3H, s, H-19), 3.36-3.51 (2H, m, H-26), 4.38-4.49 (1H, m, H-16), 6.00 (2H, d, J=9.4Hz, H-6, H-7), 6.23 (2H, dd, J=2.0, 12.0Hz, H-2, H-4), 7.05 (1H, d, J=10.0Hz, H-1); ¹³C-NMR δ: 162.5 152.9, 138.1, 128.3, 127.7, 123.9, 109.3, 80.5, 67.0, 62.0, 53.4, 48.5, 41.7, 41.1, 39.5, 37.8, 31.4, 31.3, 30.3, 29.8, 28.8, 21.8, 20.9, 17.2, 16.4, 14.5; FABMS (M+H)⁺ : 409.2.

25(R)-1α,2α-Epoxy-4,6-spirostadien-3-one (3). A mixture of 5 % NaOH-MeOH (0.6 mL) and 30 % H₂O₂ (6 mL) was added to a solution of the trienone 2 (3 g, 7.34 mmol) in methanol (60 mL). The mixture was stirred at room temperature for 22 hours, at which time TLC indicated reaction completion, then extracted with dichloromethane. The organic layers were combined and washed with brine and H₂O, dried over MgSO₄, filtered, and concentrated to leave a crude solid. Column chromatography on silica gel, eluting with ethyl acetate/n-hexane (1:5), gave pure white crystals of epoxide 3. Yield: 2.53 g (81 %); mp: 219.2-220.8°C; IR (cm^-1): 1669, 1459; ¹H-NMR δ: 0.73 (3H, d, J=6.2Hz, H-27), 0.88 (3H, s, H-18), 0.92 (3H, d, J=6.6Hz, H-21), 1.13 (3H, s, H-19), 3.27 (1H, br d, J=10.8Hz, H-26), 3.35-3.46 (2H, m, H-26, H-2), 3.51 (1H, d, J=4.2Hz, H-1), 4.43-4.46 (1H, m, H-16), 5.58 (1H, d, J=1.4, H-4), 6.01 (2H, m, H-6, H-7); ¹³C-NMR δ: 193.6, 157.5, 138.8, 126.9, 118.6, 108.3, 79.3, 65.9, 58.4, 53.7, 52.0, 45.1,40.7, 40.0, 38.2, 37.0, 36.1, 30.3, 30.2, 29.2, 27.8, 20.0, 17.5, 16.1, 15.1, 13.4; FABMS (M+H) ⁺ : 425.2.

25(R)-6α,7α-epoxy-1,4-spirostadien-3-one (4). mCPBA (490 mg, 2.84 mmol) was added to a solution of trienone 2 (1 g, 2.45 mmol) in CHCl₃ (100 mL) at room temperature. The reaction mixture was stirred at the same temperature until no starting material could be detected by TLC. The mixture was stirred with H₂O and then extracted with dichloromethane. The extract was dried over MgSO₄, filtered, and evaporated to give a pale yellow precipitate. Column chromatography on silica gel, eluting with ethyl acetate/n-hexane (1:5) gave the pure white crystals of epoxide 4. Yield: 655 mg (63 %); mp: 264-265.5°C; IR (cm^-1): 1653, 1448; ¹H-NMR δ: 0.79 (3H, d, J=6.2Hz, H-27), 0.88 (3H, s, H-18), 0.98 (3H, d, J=6.6Hz, H-21), 1.21 (3H, s, H-19), 3.31-3.50 (2H, m, H-26, H-7), 3.64 (1H, d, J=3.6Hz, H-6), 4.50-4.67 (1H, m, H-16), 6.24 (1H, dd, J=1.6, 10.2Hz, H-2), 6.48 (1H, d, J=1.8Hz, H-4), 7.0 (1H, d, J=16.4Hz, H-1); ¹³C-NMR δ: 185.2, 159.7, 153.2, 131.2, 127.8, 101.4, 80.7, 67.6, 61.9, 53.9, 51.8, 50.9, 41.8, 41.2, 41.1, 39.3, 38.4, 35.1, 31.5, 31.3, 30.3, 28.9, 22.0, 20.9, 17.2, 16.3, 14.6; FABMS (M+H) ⁺: 425.2.

25(R)-4,6-Spirostadien-3β-ol (5). To a solution of compound 2 (1 g, 2.45 mmol) in absolute ethanol (100 mL), NaBH₄ (370 mg, 9.8 mmol) was added portionwise at room temperature. The resulting mixture was stirred overnight at this temperature, then concentrated by rotary evaporation to afford a white solid. This was stirred with water for 30 minutes, then extracted with dichloromethane. The organic layer was washed with water and dried with MgSO₄, filtered, and concentrated to give a white solid, which was recrystallized from ethyl acetate and n-hexane to afford white crystals. Yield: 900 mg (89 %); mp: 184-186°C; IR (cm^-1): 3418, 2929; ¹H-NMR δ: 0.77 (3H, d, J=2.4Hz, H-27), 0.81 (3H, s, H-18), 0.97 (3H, d, J=6.8Hz, H-21), 1.01 (3H, s, H-19), 3.34 (1H, br d, J=9.0Hz, H-26), 3.37-3.52 (1H, m, H-26), 4.23-4.31 (1H, m, H-3), 4.36-4.37 (1H, m, H-16), 5.37 (1H, br s, H-6), 5.62 (1H, br d, J=10.0Hz, H-4), 5.92 (1H, dd, J=2.8, 9.8Hz, H-7); ¹³C-NMR δ: 144.7, 131.0, 128.2, 126.4, 109.3, 80.8, 68.1, 67.0, 62.2, 54.0, 51.6, 41.7, 41.5, 40.0, 37.1, 35.3, 33.7, 31.5, 30.4, 29.2, 28.9, 20.7, 18.3, 17.2, 16.5, 14.6; FABMS (M+H) ⁺ : 413.2.

25(R)-4,6-Spirostadien-3-one (6). To a solution of compound 5 (500 mg, 1.2 mmol) in methanol (10 mL) was added a mixture of 5 % NaOH-MeOH (0.25 mL) and 30 % H₂O₂ (0.75 mL) and then it was treated as described for the synthesis of compound 3. Pure compound 6 was obtained as white crystals by column chromatography (ethyl acetate-n-hexane=1:5). Yield: 123 mg (25 %); mp: 108-110°C; IR (cm^-1): 2940, 1664 ; ¹H-NMR δ: 0.80 (3H, d, J=6.4Hz, H-27), 0.87 (3H, s, H-18), 0.98 (3H, d, J=6.4Hz, H-21), 1.26 (3H, s, H-19), 3.37-3.50 (1H, m, H-26), 4.40-4.51 (1H, m, H-16), 5.67 (1H, s, H-4), 6.11 (2H, d, J=2.4Hz H-6, H-7); ¹³C-NMR δ: 163.4, 140.6, 127.8, 123.6, 109.2, 80.4, 66.8, 62.0, 53.1, 50.7, 41.6, 41.3, 39.5, 37.2, 36.1, 33.9, 33.8, 31.3, 31.2, 30.2, 29.6, 28.7, 20.4, 17.1, 16.3, 16.2, 14.4; FABMS (M+H) ⁺ : 411.2.

25(R)-4β,5β-Epoxy-6-spirosten-3β-ol (7). m-Chloroperoxybenzoic acid (486 mg, 2.82 mmol) was added to a solution of dienol 5 (1 g, 2.43 mmol) in CHCl₃ (100 mL) at room temperature and then the reaction mixture was treated as described for synthesis of compound 1. Pure compound 7 was obtained as white crystals by column chromatography (ethyl acetate-n-hexane=1:3). Yield $:$ 603mg (58 %); mp : 114-116 °C; IR (cm^-1): 3500, 2950; ¹H-NMR δ: 0.74 (3H, d, J=6.0Hz, H-27), 0.84 (3H, s, H-18), 0.96 (3H, d, J=6.8Hz, H-21), 1.28(3H, s, H-19), 3.23-3.45 (2H, m, H-26), 3.55-3.65 (1H, m, H-3), 4.15 (1H, d, J=3.4Hz, H-4), 4.60-4.70 (1H, m, H-16), 5.60 (1H, dd, J=2.2, 8.0Hz, H-7), 5.66 (1H, d, J=2.2Hz, H-6); ¹³C-NMR δ: 149.4, 134.6, 127.6, 125.0, 109.4, 80.6, 71.9, 69.3, 66.9, 61.9, 49.3, 44.0, 41.8, 40.3, 39.4, 37.2, 36.6, 35.9, 31.8, 31.4, 30.3, 28.8, 25.2, 20.2, 19.5, 17.2, 16.0, 14.6; FABMS (M+H) ⁺ : 429.2.