Neurosteroid Analogues. 13. Synthetic methods for the preparation of 2β-hydroxygonane derivatives as structural mimics of ent-3α-hydroxysteroid modulators of GABA_A receptors

Abstract

Many different 3α-hydroxysteroids in the androstane and pregnane steroid series enhance the actions of γ-aminobutyric acid (GABA) at GABA type-A (GABA_A) receptors in the mammalian central nervous system. Recent studies have shown that (3α,5α)-3-hydroxyandrostan-17-one (androsterone) is less active at these receptors than its enantiomer ent-androsterone. Further structure–activity relationship (SAR) studies are needed to explore the structural features of ent-androsterone that are important for its enhanced action at these receptors. Molecular modeling shows that 2β-hydroxysteroids are similar in three-dimensional shape to the enantiomers of 3α-hydroxysteroids. The development of synthtetic methods to gain access to C₁₇-substituted analogues of 2β-hydroxygonanes for SAR studies is demonstrated with the synthesis of (2β,5α,13β,14β)-2-hydroxygonan-17-one.

I. Introduction

The steroid (3α,5α)-3-hydroxypregnan-20-one (allopregnanolone, Figure 1) and many analogues of it are known to be potent enhancers of γ-aminobutyric acid (GABA) at GABA Type A (GABA_A) receptors.^1–4 These neuroactive GABAergic steroids have activity as general anesthetics, anticonvulsants, sedative hypnotics and anxiolytics; and there is considerable current interest in the development of new analogues as pharmaceuticals having these activities.

Figure 1.

Structures of steroid modulators of GABA_A receptor function. Allopregnanolone and ent-androsterone 17-spiroepoxide potently enhance the actions of GABA at GABA_A receptors. Androsterone is a weak enhancer of GABA action. ent-Androsterone has activity higher than that of androsterone but less than that of the other two steroids.

As part of our ongoing studies of the enantioselectivity of neurosteroid action at GABA_A receptors, we recently investigated the enantioselectivity for (3α,5α)-3-hydroxyandrostan-17-one (androsterone, Figure 1) effects at these receptors.⁵ Androsterone has only weak actions on GABA_A receptor function^6,7 and, based on our previous results from enantioselectivity studies of allopregnanolone,⁸ we expected that ent-androsterone (Figure 1) would have even weaker actions than androsterone. Unexpectedly, we found that ent-androsterone was more active than androsterone. Moreover, a 17-spiroepoxide derivative of ent-androsterone (Figure 1) was shown to have actions comparable to those of allopregnanolone.⁵

Additional steroid analogues are needed for future structure–activity relationship (SAR) studies of ent-androgen action at GABA_A receptors. In this regard, we were intrigued by the structural similarity between 2β-hydroxysteroids and the enantiomers of 3α-hydroxysteroids. Figure 2A shows the structure of (2β,5α,13β,14β)-2-hydroxygonan-17-one (1) and ent-androsterone. Figure 2B shows a three-dimensional overlay of molecular models of steroid 1 and ent-androsterone. Both steroids are shown with their β face down because in this orientation the compounds most closely resemble the highly active 3α-hydroxysteroid allopregnanolone. As presented in Figure 2, each of the four steroid rings of one compound is proximate to the corresponding ring of the other compound. The O₂ and O₃ atoms of the two molecules are 0.68 Å apart and the O₁₇ atoms are 0.54 Å from each other in this overlay.

Figure 2.

Panel A: Structures of (2β,5α,13β,14β)-2-hydroxygonan-17-one (1) and ent-androsterone. Panel B: An overlay of steroid 1 (blue structure) and ent-androsterone (orange structure). The structures were overlaid using a RMS fit of atoms C₉, C₁₁, C₁₂, C₁₄, O₃ and O₁₇ in each structure for the alignment. The O₂–O₁₇ (9.37 Å) distance in steroid 1 is shorter than the O₃–O₁₇ distance (9.67 Å) in ent-androsterone.

Two additional design criteria were considered in the decision to select steroid 1 as an initial potential structural mimic of ent-androsterone. Previous SAR studies have shown that having methyl groups on the steroid α face decreases the activity of 3α-hydroxysteroid modulators of GABA_A receptors.^9,10 When aligned as shown in Figure 2B, the C₁₈ and/or C₁₉ methyl groups of steroids in the androstane, estrane (19-norandrostane) and 18-norandrostane series would be located below the plane of the steroid rings (i.e. they would occupy positions similar to those occupied by methyl groups on the α face of 3α-hydroxysteroids, and therefore could be expected to negatively affect pharmacological activity). By choosing a steroid in the gonane class as a synthetic target, worries about unfavorable steric effects of these methyl groups is avoided.

The decision to have the 13β,14β cis C,D-ring fusion present in steroid 1 was made because this cis ring fusion places the C₁₇ carbonyl group further above the plane of the steroid rings than it would be in the corresponding 13β,14α trans ring fusion. This was considered to be desirable since previous SAR studies indicate that the D-ring hydrogen bonding groups needs to be above the plane of the steroid rings for high pharmacological activity.^1,6,7

2. Results and Discussion

The starting material, (3β,5α)-3-hydroxyestran-17-one (3), was prepared in three steps by known methodology^11,12 from commercially available 19-nortestosterone (2) in 85% total yield (Scheme 1). Steroid 3 was brominated selectively in the C₁₆ position using CuBr₂^13,14 to give compound 4 in 69% yield (Scheme 2). HBr was eliminated from steroid 4 using LiBr/Li₂CO₃¹⁵ to give compounds 5a (33%) and 5b (39%). For characterization purposes, some spectroscopic data was collected on each of these products after their separation by chromatograpy. For synthetic purposes, products 5a and 5b were not separated and the mixture was subjected to catalytic hydrogenation to give compound 6 in 87% yield. The 17-keto group was ketalized in the standard way¹⁶ to afford compound 7 in 85% yield. Oxidation of the 3β-hydroxyl group of compound 7 using PCC in the presence of NaOAc¹⁷ gave compound 8 (75%).

Scheme 1.

Scheme 2.

When treated with 10% ethanolic KOH at room temperature for 24 h, compound 8 underwent an aldol condensation¹⁸ to give compound 9 in 74% yield (Scheme 3). Reduction of the 3-ketone group with NaBH₄ at room temperature led to the 3β-hydroxysteroid 10 (99%), and acetylation of the hydroxyl group gave the acetate derivative 11 in 90% yield. Compound 11 was treated with ozone in the usual manner¹⁸ to give steroid 12 (80%). Samarium(II) iodide-THF mediated reductive removal of the 3-acetyloxy group from compound 12 gave 2-ketosteroid 13 (41%), 2β-hydroxysteroid 14a (20%) and 2α-hydroxysteroid 14b (26%). Reduction of compound 13 with K-Selectride in THF at −78 ºC gave additional amounts of compound 14a (85%).

Scheme 3.

The hydroxyl group of 2β-hydroxysteroid 14a was reacted with acetic anhydride and pyridine in the presence of a catalytic amount of 4-dimethylaminopyridine to afford 2-(acetyloxy)steroid 15 (92%) after 5 min at room temperature. The 17-ketal of compound 15 was readily removed using p-TsOH in acetone at room temperature for 12 h to give, in quantitative yield, steroid 16. The reaction of compound 16 with NH₂OH·HCl and NaOAc gave oxime 17 (97%). Oxime 17 underwent an abnormal Beckmann rearrangement¹⁹ upon treatment with CH(OCH₃)₃/TFA in THF to give carbonitrile 18 (79%). Ozonolysis of compound 18 at −78 °C yielded ketone-nitrile 19 (58%). Treatment of compound 19 with SmI₂ in THF and irradiation with a 500 W lamp²⁰ at 0–10 °C for 8 h gave crude 13-hydroxysteroid 20. After isolation, crude product 20 was again treated with SmI₂ in THF at room temperature for 10 min to give compound 21 (17%, from compound 19). Finally, base-catalyzed hydrolysis of the 3-acetyloxy group of compound 21 and column chromatography to remove the presumed 13α,14β stereoisomer gave compound 1 (82%). The structure of steroid 1 was confirmed by single crystal X-ray diffraction analysis (Figure 3).²¹

Figure 3.

Projection view of one of the two unique molecules of steroid 1 shown with 50% thermal ellipsoids for non-hydrogen atoms.

Using methods reported previously,²² the actions of compound 1 as a modulator of GABA_A receptor function were compared to those of ent-androsterone. Whereas ent-androsterone allosterically displaced 50% of [³⁵S]-t-butylbicyclophosphorthionate bound to the picrotoxin binding site on GABA_A receptors at a concentration of 0.31 μM,⁵ no displacement of [³⁵S]-t-butylbicyclophosphorthionate occurred at concentrations up to 30 μM of compound 1. ent-Androsterone (10 μM) enhances 2 μM GABA-mediated chloride currents at rat α₁β₂γ_2L GABA receptors expressed in Xenopus laevis oocytes.⁵ Compound 1 at concentrations up to 10 μM does not enhance 2 μM GABA-mediated chloride currents at these expressed receptors. Finally, ent-androsterone causes 50% loss of righting reflex for tadpoles at a concentration of 3.38 μM.⁵ By contrast, compound 1 did not cause loss of righting reflex in tadpoles at a concentration as high as 10 μM. Thus, despite the similarities in the overall shapes of the two steroids, only ent-androsterone is effective as a positive modulator of GABA_A receptors at concentrations below 10 μM.

3. Conclusion

We have described the synthesis and crystal structure of (2β, 5α,13β,14β)-2-hydroxygonan-17-one (1) from commercially available 19-nortestosterone. Novel features of the synthetic route developed include the first use of a SmI₂-promoted reaction to remove a 3-acetyloxy group from a 2-keto-3-(acetyloxy)steroid, the first report of the abnormal Beckmann rearrangement on steroids having the 13β,14β cis C,D-ring fusion and the first use of SmI₂-promoted reactions to prepare 18-norsteroids having a 13β,14β cis C,D-ring fusion. Omission of the part of the reported synthetic sequence that was used to construct the 13β,14β cis C,D-ring fusion would also allow other 2β-hydroxygonane analogues with the 13β,14α trans C,D-ring fusion to be prepared using methods reported previously for the synthesis of (13β,14α)-18-norsteroids.^20,23

4. Experimental

4.1 General Methods

Melting points were determined on a Kofler micro hot stage and are uncorrected. NMR spectra were recorded in CDCl₃ at 300 MHz (¹H) or 75 MHz (¹³C). IR spectra were recorded as films on a NaCl plate. Elemental analyses were carried out by M-H-W Laboratories, Phoenix, AZ.

4.1.1 (3β,5α,16ββ))-16-Bromo-3-hydroxyestran-17-one (4)

A mixture of known compound 3²⁴ (1.40 g, 5.07 mmol) and copper bromide (3.06 g, 13.7 mmol) in methanol (35 ml) was stirred under reflux for 12 h. Then the solvent was removed under reduced pressure to give a residue and water (25 mL) was added. The product was extracted with CH₂Cl₂ (25 mL), the organic layer was washed with water (2 × 10 mL), brine (20 mL) and dried over Na₂SO₄. After the solvent was removed under reduced pressure, the residue was purified by column chromatography (silica gel; CH₂Cl₂/EtOAc, 10:1) to give compound 4 (1.24 g, 69%) as white crystals: mp 108–110 °C (EtOAc-hexanes); [α] ²⁰_D = +59.1 (c = 0.30, CHCl₃); ¹H NMR δ 4.56 (m, 1H), 3.56 (brs, 1H), 3.05 (s, 1H), 2.18 (m, 2H), 0.91 (s, 3H); ¹³C NMR δ 13.6, 24.4, 27.7, 29.0, 31.7, 32.5, 33.3, 34.9, 39.2, 40.4, 42.4, 45.4, 46.0, 46.5, 47.2, 47.3, 69.3, 213.0; IR ν_max 3400, 2920, 2853, 1748, 1448, 1023 cm⁻¹. Anal. Calcd. For C₁₈H₂₇BrO₂: C 60.85, H 7.66; Found C 60.78, H 7.87.

4.1.2 (3β,5α)-3-Hydroxyestr-14-en-17-one (5a) and (3β,5α,14ββ))-3-Hydroxyestr -15-en-17- one (5b)

The mixture of compound 4 (0.65 g, 1.8 mmol), LiBr (0.42 g, 5.0 mmol) and Li₂CO₃ (0.35 g, 5.0 mmol) in DMF (30 mL) was stirred under reflux for 4 h. Afterwards, the reaction mixture was chilled to room temperature and was poured into water (50 mL), the mixture was extracted with EtOAc (2 ×3 0 mL), the combined organic extract was washed with saturated NaHCO₃ (10 mL), water (10 mL), brine (10 mL) and dried over Na₂SO₄. The solvent was removed under reduced pressure. The products were purified by column chromatography (silica gel; CH₂Cl₂/EtOAc, 10:1) to give compound 5a (0.17 g, 33%; R_f = 0.47, CH₂Cl₂/EtOAc, 10:1) and compound 5b (0.20 g, 39%; R_f = 0.33, CH₂Cl₂/EtOAc, 10:1). Compound 5a was obtained as an oil: ¹H NMR δ 5.47 (d, J = 1.8 Hz, 1H), 3.59 (m, 1H), 2.86 (m, 2H), 2.29 (s, 1H), 1.12 (s, 3H); ¹³C NMR δ 19.7, 25.2, 27.8, 28.0, 32.9, 33.0, 35.4, 40.8, 41.0, 41.3, 43.0, 46.5, 48.6, 50.7, 70.0, 112.3, 152.8, 222.7; IR ν_max 3400, 3059, 2921, 2855, 1744, 1641, 1449, 1046 cm⁻¹.

Compound 5b was obtained as an oil: ¹H NMR δ 7.64 (dd, J = 6.0, 2.4 Hz, 1H), 6.15 (dd, J = 6.0, 2.4 Hz, 1H), 3.57 (m, 1H), 2.63 (m, 1H), 1.11 (s, 3H); ¹³C NMR δ 21.3, 24.8, 28.0, 30.1, 31.7, 33.4, 35.5, 39.7, 39.8, 40.8, 42.9, 47.3, 47.5, 54.4, 70.2, 132.2, 163.3, 215.2. IR ν_max 3400, 2921, 2854, 1705, 1585, 1449, 1052 cm⁻¹. Compound 5a and 5b were not purified further and were used in the next step directly.

4.1.3 (3β,5α,14β )-3-Hydroxyestran-17-one (6)

A mixture of compounds 5a and 5b (3.90 g, 14.2 mmol) was dissolved in EtOAc (50 mL) and hydrogenated under H₂ (50 psi) in the presence of 5% Pd-C (400 mg) at room temperature overnight. The reaction mixture was filtered through a pad of Celite 545^® to remove catalyst and the solvent was removed under reduced pressure. The residue was purified by flash column chromatography (silica gel; EtOAc/hexanes, 1:1) to give compound 6 (3.81 g, 97%) as white crystals: mp 116–118 ºC (EtOAc); [α] ²⁰_D = +92.0 (c = 0.27, CHCl₃); ¹H NMR δ 3.56 (m, 1H), 3.27 (brs, 1H), 2.45 (dd, J = 19.2, 8.1 Hz, 1H), 2.13 (dd, J = 19.2, 9.9 Hz, 1H), 1.07 (s, 3H); ¹³C NMR δ 18.1, 19.3, 24.6, 27.6, 27.7, 30.0, 33.4, 35.1, 35.6, 39.1, 40.0, 40.6, 42.7, 46.2, 47.5, 47.8, 69.6, 222.8; IR ν_max 3401, 2917, 2854, 1732, 1448, 1048 cm⁻¹. Anal. Calcd. For C₁₈H₂₈O₂: C 78.21, H 10.21; Found C 78.42, H 9.97.

4.14 (3β,5α,14β)-3-Hydroxyestran-17-one cyclic-(1,2-ethanediyl acetal) (7)

A solution of compound 6 (0.41 g, 1.49 mmol), ethylene glycol (2.2 g, 35 mmol) and p-TsOH (60 mg, 0.32 mmol) in benzene (35 mL) was refluxed using a Dean-Stark trap for 12 h. The mixture was cooled to room temperature, diluted with ether (50 mL), washed with saturated NaHCO₃ (2 × 20 mL) and brine (2 × 20 mL). The organic phase was dried over Na₂SO₄, and the solvent was removed under reduced pressure to give a crude product which was purified by column chromatography (silica gel; EtOAc/hexanes, 1:1) to yield compound 7 (406 mg, 85%) as an oil: [α] ²⁰_D = +73.9 (c = 0.065, CHCl₃); ¹H NMR δ 3.86 (m, 4H), 3.55 (m, 1H), 0.92 (s, 3H); ¹³C NMR δ 16.1, 19.7, 25.3, 27.8, 28.9, 30.3, 32.1, 33.6, 35.3, 39.4, 39.7, 40.8, 43.0, 45.1, 46.4, 46.8, 63.7, 65.0, 69.9, 120.4; IR ν_max 3368, 2920, 2856, 1447, 1367, 1031 cm⁻¹. Anal. Calcd. For C₂₀H₃₂O₃: C 74.96, H 10.06; Found C 75.12, H 9.86.

4.1.5 (5α,14β )-estran-3,17-dione cyclic-17-(1,2-ethanediyl acetal) (8)

To a solution of compound 7 (406 mg, 1.27 mmol) in CH₂Cl₂ (25 mL) was added NaOAc (0.46 g, 5.6 mmol) and PCC (0.72 g, 3.35 mmol) at 0° C. The mixture was stirred at 0° C for 2 h and allowed to stand overnight. Then the reaction mixture was diluted with Et₂O (100 mL) and the mixture was filtered through a pad of silica gel. Removal of solvent gave a residue which was purified by column chromatography (silica gel; hexanes/EtOAc, 5:1) to give compound 8 (302 mg, 75%) as white crystals: mp: 85–86 °C (hexanes/EtOAc); [α] ²⁰_D = +103.9 (c = 0.28, CHCl₃); ¹HNMR δ 3.90 (m, 4H), 2.29 (m, 4H), 0.94 (s, 3H); ¹³CNMR δ 16.3, 19.8, 25.7, 29.0, 30.0, 30.1, 32.2, 34.2, 39.4, 39.7, 41.1, 43.4, 45.2, 46.1, 46.9, 48.5, 63.9, 65.2, 120.4, 211.6; IR ν_max 2935, 2880, 1714, 1470, 1448 cm⁻¹. Anal. Calcd. For C₂₀H₃₀O₃: C 75.43, H 9.50; Found C 75.63, H 9.38.

4.1.6 (5α,14β)-2-(Phenylmethylene)estran-3,17-dione cyclic-17-(1,2-ethanediyl acetal) (9)

A mixture of compound 8 (1.60 g, 5.03 mmol), benzaldehyde (1.75 g, 16.5 mmol) and KOH (400 mg, 7.14 mmol) in EtOH (30 mL) and water (5 mL) was stirred at room temperature in the dark for 24 h. Then the reaction mixture was poured into ice-water (50 mL), and the product was extracted with CH₂Cl₂ (2 × 25 mL), the organic phase was washed with water (15 mL),brine (15 mL) and dried over Na₂SO₄. The solvent was removed under reduced pressure to give a residue which was purified by column chromatography (silica gel; hexanes/EtOAc, 6:1) to give compound 9 (1.51 g, 74%) as light yellow crystals: mp: 153–155 °C (EtOAc); [α] ²⁰_D = +43.0 (c = 0.33, CHCl₃); ¹H NMR δ 7.47 (d, J = 2.1 Hz,1H), 7.40 (m, 5H), 3.89 (m, 4H), 3.36 (dd, J = 16.2, 2.7 Hz, 1H), 2.63 (dd, J = 16.8, 4.2 Hz, 1H), 0.91 (s, 3H); ¹³C NMR δ 16.4, 19.9, 25.7, 29.0, 29.9, 32.3, 33.2, 33.7, 39.0, 39.1, 40.8, 44.4, 45.3, 47.0, 47.4, 64.0, 65.3, 120.5, 128.3 (2 × C), 128.5, 130.3 (2 × C), 135.4, 135.6, 135.8, 201.5; IR ν_max 2918, 2860, 1682, 1594, 1572, 1491, 1085, 735 cm⁻¹. Anal. Calcd. For C₂₇H₃₄O₃: C 79.76, H 8.34; Found C 79.66, H 8.35.

4.1.7 (3β,5α,14β)-3-Hydroxy-2-(Phenylmethylene)estran-17-one cyclic-(1,2-ethanediyl acetal) (10)

To a solution of compound 9 (1.51 g, 3.72 mmol) in THF (40 mL) and MeOH (25 mL) was added NaBH₄ (70 mg, 1.85 mmol) at room temperature. The mixture was stirred for 30 min. Most of solvent was removed under reduced pressure and EtOAc (35 mL) was added to the residue. The organic phase was washed with water (20 mL), brine (20 mL) and dried over Na₂SO₄. After the solvent was removed under reduced pressure, the residue was purified by column chromatography (silica gel; hexanes/EtOAc, 3:1) to give compound 10 (1.50 g, 99%) as a thick oil: [α] ²⁰ _D = +67.2 (c = 0.49, CHCl₃); ¹H NMR δ 7.34 (m, 5H), 6.57 (s, 1H), 4.12 (m, 1H), 3.88 (m, 4H), 3.10 (dd, J = 13.5, 2.4 Hz, 1H), 2.43 (brs, 1H), 0.91 (s, 3H); ¹³C NMR δ 16.4, 19.9, 25.3, 29.0, 30.3, 31.2, 32.3, 33.2, 39.3, 40.3, 41.4, 44.7, 45.3, 47.0, 48.5, 63.9, 65.2, 72.4, 118.3, 120.6, 125.9, 128.0 (2 × C), 128.8 (2 × C), 137.9, 144.1; IR ν_max 3435, 2917, 2859, 1445, 1087 cm⁻¹; Anal. Calcd. For C₂₇H₃₆O₃: C 79.37, H 8.88; Found C 79.54, H 8.96.

4.1.8 (3β,5α,14β)-3-(Acetyloxy)-2-(phenylmethylene)estran-17-one cyclic-(1,2-ethanediyl acetal) (11)

A mixture of compound 10 (1.50 g, 3.67 mmol), acetic anhydride (4.5 mL, 480 mmol), pyridine (4.5 mL) and 4-dimethylaminopyridine (10 mg, 0.082 mmol) was stirred at room temperature for 5 min. The reaction mixture was poured into ice-water (50 mL) and was extracted with EtOAc (2 × 30 mL). The combined organic extract was washed with water (2 × 20 mL), 10% NaHCO₃ (10 mL), brine (20 mL) and dried over Na₂SO₄. After solvent removed under reduced pressure, the residue was purified by column chromatography (silica gel; hexanes/EtOAc, 4:1) to give compound 11 (1.48 g, 90%) as white crystals: mp 141–143 °C (hexanes/EtOAc); [α] ²⁰_D = +105.0 (c = 0.20, CHCl₃); ¹H NMR δ 7.33 (m, 5H), 6.36 (s, 1H), 5.32 (m, 1H), 3.88 (m, 4H), 3.24 (dd, J = 13.5, 3.3 Hz, 1H), 2.16 (s, 3H), 0.91 (s, 3H); ¹³C NMR δ 16.4, 19.9, 21.2, 25.3, 29.1, 30.3, 31.5, 32.3, 33.1, 39.5, 40.3, 41.0, 41.2, 45.3, 47.0, 48.3, 64.0, 65.3, 73.8, 118.9, 120.6, 126.2, 128.1 (2 × C), 128.8 (2 × C), 137.5, 139.3, 170.1; IR ν_max 2931, 2860, 1738, 1599, 1241, 1046 cm⁻¹. Anal. Calcd. For C₂₉H₃₈O₄: C 77.30, H 8.50; Found 77.50, H 8.61.

4.1.9 (3β,5α,14β)-3-(Acetyloxy)estran-2,17-dione cyclic-17-(1,2-ethanediyl acetal) (12)

A solution of compound 11 (1.48 g, 3.29 mmol) in MeOH (60 mL) and EtOAc (30 mL) was treated with ozone at −78 °C until a blue color persisted (ca 30 min). Oxygen was passed through the solution for 20 min until the blue color disappeared, and Me₂S (10 mL) was added to the solution, the reaction mixture was stirred overnight from −78 °C to room temperature. Solvent was removed under reduced pressure and the residue was purified by column chromatography (silica gel; hexanes/EtOAc, 3:1) to give compound 12 (0.98 g, 80%) as white crystals: mp 188–189 °C (hexanes-EtOAc); [α] ²⁰_D = +125.0 (c = 0.45, CHCl₃); ¹H NMR δ 5.20 (m, 1H), 3.88 (m, 4H), 2.70 (d, J = 13.2 Hz, 1H), 2.13 (s, 3H), 0.91 (s, 3H); ¹³C NMR δ 16.1, 19.6, 20.3, 25.0, 28.6, 29.8, 32.0, 32.1, 38.5, 38.7, 39.9, 40.6, 43.3, 45.0, 46.5, 48.0, 63.7, 65.0, 75.5, 120.1, 169.5, 203.8; IR ν_max 2933, 2863, 1748, 1728, 1238, 1092 cm⁻¹. Anal. Calcd. For C₂₂H₃₂O₅: C 70.18, H 8.57; Found C 69.93, H 8.80.

4.1.10 (5α,14β)-estran-2,17-dione cyclic-17-(1,2-ethanediyl acetal) (13), (2β,5α,14β)- 2-hydroxyestran-17-one cyclic-(1,2-ethanediyl acetal) (14a) and (2α,5α,14β)-2-hydroxyestran-17-one cyclic-(1,2-ethanediyl acetal) (14b)

Iodine (2.06 g, 8.12 mmol) in dried THF (30 mL) was added to samarium filings (1.50 g, 10 mmol) by syringe under Ar. The mixture was stirred at room temperature for 30 min to give a black blue solution. Then compound 12 (0.92 g, 2.45 mmol) in dried THF (15 mL) and MeOH (5 mL) was added to the SmI₂-THF solution and the reaction mixture was stirred for 15 min. The reaction mixture was poured into 10% Na₂CO₃ (70 mL) and was extracted with EtOAc (3 × 30 mL), the combined organic extract was washed with water (20 mL), brine (20 mL) and dried over Na₂SO₄. After the solvent was removed under reduced pressure, the residue was purified by column chromatography (silica gel; CH₂Cl₂/EtOAc, 10:1) to give compound 13 (0.32 g, 41%), compound 14a (0.16 g, 20%) and compound 14b (0.20 g, 26%).

Compound 13 was obtained as white crystals: mp 143–145 °C (hexanes/EtOAc); [α] ²⁰_D = +91.4 (c = 0.45, CHCl₃); ¹H NMR δ 3.89 (m, 4H), 2.59 (dd, J = 15.6, 2.7 Hz, 1H), 2.35 (m, 2H), 0.91 (s, 3H); ¹³C NMR δ 16.2, 19.8, 24.9, 28.8, 30.0, 32.2, 32.6, 33.3, 38.8, 41.0, 41.1, 41.2, 45.0, 45.1, 46.7, 47.7, 63.9, 65.1, 120.4, 211.7; IR ν_max 2948, 2858, 1714, 1455, 1088 cm⁻¹. Anal. Calcd. For C₂₀H₃₀O₃: C 75.43, H 9.50; Found C 75.29, H 9.70.

Compound 14a was obtained as white crystals: mp 136–138 ºC (hexanes/EtOAc); [α] ²⁰_D = +105.5 (c = 0.12, CHCl₃); ¹H NMR δ 4.13 (m, 1H), 3.89 (m, 4H), 0.91 (s, 3H); ¹³C NMR δ 16.4, 19.9, 25.1, 27.6, 29.1, 30.5, 32.3, 32.5, 33.8, 36.3, 39.7, 40.1, 40.8, 42.6, 45.2, 47.2, 64.0, 65.2, 66.6, 120.8; IR ν_max 3436, 2918, 2860, 1305, 1095 cm⁻¹. Anal. Calcd. For C₂₀H₃₂O₃: C 74.96, H 10.06; Found C 74.94, H 9.94.

Compound 14b was obtained as white crystals: mp 141–143 °C (hexanes/EtOAc); [α] ²⁰_D = +81.2 (c = 0.07, CHCl₃); ¹H NMR δ 3.89 (m, 4H), 3.55 (m, 1H), 0.91 (s, 3H); ¹³C NMR δ 16.4, 20.0, 25.3, 29.2, 30.5, 32.0, 32.4, 33.3, 35.4, 39.2, 39.5, 40.0, 41.9, 45.3, 45.8, 47.1, 64.0, 65.3, 71.2, 120.8; IR ν_max 3368, 2920, 2857, 1305, 1097, 1031 cm⁻¹. Anal. Calcd. For C₂₀H₃₂O₃: C 74.96, H 10.06; Found C 74.92, H 10.18.

14.1.11 (2β,5α,14β)-2-hydroxyestran-17-one cyclic-(1,2-ethanediyl acetal) (Compound 14a)

To a solution of compound 13 (406 mg, 1.28 mmol) in dried THF (35 mL) was added 1M K-Selectride (6.5 mL, 6.5 mmol) in THF at −78 °C under N₂ and the mixture was stirred for 1 h at this temperature. Then the reaction was quenched by adding 10% NaOH (8 mL) and 30% hydrogen peroxide (8 mL), and the reaction was continued for other 30 min. The mixture was extracted with EtOAc (3 × 30 mL). The combined organic phase was washed with water (10 mL), brine (2×10 mL) and dried over Na₂SO₄. After the solvent was removed under reduced pressure, the residue was purified by column chromatography (silica gel; CH₂Cl₂/EtOAc, 10:1) to give compound 14a (346 mg, 85%) whose properties were identical to those for this product when isolated directly from the SmI₂ reduction.

14.1.12 (2β,5α,14β)-2-(Acetyloxy)estran-17-one cyclic-(1,2-ethanediyl acetal) (15)

A mixture of compound 14 (476 mg, 1.49 mmol), acetic anhydride (2 mL), dry pyridine (2 mL) and 4-dimethylaminopyridine (10 mg) was stirred at room temperature for 5 min. The reaction mixture was poured into ice-water (15 mL) and was extracted with EtOAc (2 × 15 mL). The organic phase was washed successively with saturated NaHCO₃ (5 mL), water (5 mL), 10% aqueous HCl (5 mL), water (5 mL), brine (5 mL) and dried over Na₂SO₄. The solvent was removed under reduced pressure to give oily compound 15 (496 mg, 92%) that was not purified and was directly converted to compound 16.

14.1.13 (2β,5α,14β)-2-(Acetyloxy)estran-17-one (16)

The mixture of compound 15 (496 mg, 1.37 mmol), p-TsOH (50 mg, 0.26 mmol) and water (36 mg, 2 mmol) in acetone (15 mL) was stirred at room temperature overnight. After the solvent was removed under reduced pressure, EtOAc (35 mL) was added to the residue. and the organic phase was washed with saturated NaHCO₃ (10 mL), water (10 mL), brine (10 mL) and dried over Na₂SO₄. After the solvent was removed under reduced pressure, the residue was purified by re-crystallization (hexanes/EtOAc) to give compound 16 (430 mg, 99%) as white crystals: mp 144–146 °C (hexanes/EtOAc); [α] ²⁰_D = +92.3 (c = 0.12, CHCl₃); ¹H NMR δ 5.10 (s, 1H), 2.44 (dd, J = 18.6, 7.2 Hz, 1H), 2.05 (s, 3H), 1.08 (s, 3H); ¹³C NMR δ 18.2, 19.4, 21.1, 24.3, 27.7, 28.0, 29.4, 30.0, 33.1, 33.4, 35.6, 39.2, 40.2, 41.3, 41.9, 47.4, 48.1, 69.6, 170.1, 222.3; IR ν_max 2910, 2850, 1727, 1250, 1020 cm⁻¹. Anal. Calcd. For C₂₀H₃₀O₃: C 75.43, H 9.50; Found C 75.27, H 9.39.

14.1.14 (2β,5α,14β)-2-(Acetyloxy)estran-17-one oxime (17)

The mixture of compound 16 (460 mg, 1.47 mmol), hydroxylamine hydrochloride (1.00 g, 14.4 mmol) and NaOAc (1.15 g, 14.0 mmol) in MeOH (35 mL) was refluxed for 1.5 h. The solvent was removed partially under reduced pressure. Water (20 ml) was added to the residue. Then the mixture was extracted with EtOAc (2 × 20 mL), the combined organic extract was washed with water (20 mL), brine (20 mL) and dried over Na₂SO₄. After the solvent was removed under reduced pressure, the residue was purified by re-crystallization (hexanes/EtOAc) to give compound 17 (476 mg, 97%) as white crystals: mp 186–188 °C (hexanes/EtOAc); [α] ²⁰_D = +76.4 (c = 0.89, CHCl₃); ¹H NMR δ 9.44 (s, 1H), 5.10 (s, 1H), 2.06 (s, 3H), 1.18 (s, 3H); ¹³C NMR δ 20.7, 21.5, 21.8, 25.2, 25.3, 28.2, 29.6, 31.0, 32.0, 33.5, 33.8, 39.1, 40.3, 41.7, 42.2, 43.9, 50.3, 70.1, 173.0, 176.4; IR ν_max 3305, 2918, 2857, 1737, 1369, 1242, 937 cm⁻¹. Anal. Calcd. For C₂₀H₃₁NO₃: C 72.04, H 9.37, N 4.20; Found C 72.18, H 9.12, N 4.10.

14.1.15 (1R,4aR,4bS,6S,8aS,10aS)-6-(Acetyloxy)tetradecahydro-2-methylene-1-phenanthrenepropanenitrile (18)

To a solution of compound 17 (476 mg, 1.43 mmol) and trimethyl orthoformate (2.8 mL, 25.6 mmol) in dried THF (35 mL) was added dropwise trifluoroacetic acid (0.26 mL, 35 mmol) at 60 °C for 10 min, and the reaction was continued for another 30 min. Then saturated Na₂CO₃ (4.5 mL) was added, the solvent was partially removed under reduced pressure and EtOAc (50 mL) was added. The organic phase was washed with saturated NaHCO₃ (10 mL), water (10 mL), brine (10 mL) and dried over Na₂SO₄. After the solvent was removed under reduced pressure, the residue was purified by column chromatography (silica gel; hexanes/EtOAc, 4:1) to give compound 18 (356 mg, 79%) as white crystals: mp 103–105 °C (EtOAc); [α] ²⁰_D = +21.5 (c = 0.13, CHCl₃); ¹H NMR δ 5.10 (s, 1H), 4.74 (t, J = 2.1 Hz, 1H), 4.72 (d, J = 9.0 Hz, 1H), 2.03 (s, 3H); ¹³C NMR δ 15.3, 21.5, 22.0, 28.2, 29.7, 29.9, 30.2, 31.7, 33.6, 33.7, 40.2, 42.1, 42.4, 46.7, 47.8, 70.0, 109.7, 112.0, 149.2, 170.6; IR ν_max 2920, 2856, 2244, 1733, 1647, 1246 cm⁻¹. Anal. Calcd. For C₂₀H₂₉NO₂: C 76.15, H 9.27, N 4.44; Found C 76.13, H 9.32, N 4.19.

14.1.16 (1R,4aR,4bS,6S,8aS,10aR)-6-(Acetyloxy)tetradecahydro-2-oxo-1-phenanthrenepropanenitrile (19)

A solution of compound 18 (356 mg, 1.13 mmol) in MeOH (60 mL) and EtOAc (35 mL) was treated with ozone at −78 °C until a blue color persisted (ca 30 min). Oxygen was passed through the solution for 20 min until the blue color disappeared, and Me₂S (4.5 mL) was added at −78 °C and the resultant mixture was stirred overnight while warming to room temperature. Solvent was removed under reduced pressure and the residue was purified by column chromatography (silica gel; hexanes/EtOAc/CH₂Cl₂, 4:1:1) to give compound 19 (206 mg, 58%) as white crystals: mp 157–159 °C (hexanes/EtOAc); [α] ²⁰_D = +24.4 (c = 0.13, CHCl₃); ¹H NMR 5.11 (s, 1H), 2.46 (m, 1H), 2.04 (s, 3H); ¹³C δ NMR δ 15.1, 21.2, 22.6, 27.7, 29.2, 29.4, 30.4, 33.0, 33.6, 37.2, 39.1, 41.5, 41.7, 45.8, 54.5, 69.5, 118.8, 170.3, 213.1; IR ν_max 2922, 2850, 2244, 1727, 1699, 1379, 1241 cm⁻¹. Anal. Calcd. For C₁₉H₂₇NO₃: C 71.89, H 8.57, N 4.41; Found C 71.64, H 8.73, N 4.49.

14.1.17 (2β,5α,14β)-2-(Acetyloxy)-13-hydroxy-18-norestran-17-one (20) and (2β,5α,14β)-2-(Acetyloxy)gonan-17-one (21)

Iodine (240 mg, 0.95 mmol) in dried THF (30 mL) was added to samarium filings (225 mg, 1.50 mmol) by syringe under Ar. The mixture was stirred at room temperature for 30 min to give a black blue solution. Then a solution of compound 19 (93 mg, 0.30 mmol) and 2-methyl-2-propanol (38 mg, 0.51 mmol) in THF (10 mL) was added to the freshly made SmI₂-THF solution under Ar at 0° C. The reaction mixture was stirred at 0–10 °C while irradiated with a 500 W tungsten lamp for 8 h and then the reaction mixture was poured into 5% aqueous HCl (10 mL), and extracted with EtOAc (2 × 15 mL). The combined organic extract was washed with water (10 mL), 10% NaHCO₃ (10 mL), brine (10 mL), and dried over Na₂SO₄. Solvent removal gave crude compound 20, (46 mg) which was used without purification or characterization.

Under Ar, a solution of crude compound 20 in dried THF (5 mL) and MeOH (0.5 mL) was added to a freshly made SmI₂-THF (0.1 M, 15 mL) solution by syringe. The reaction mixture was stirred at room temperature for 10 min, quenched by adding 5% aqueous HCL (10 mL) and extracted with EtOAc (2 × 10 mL). The combined organic extract was washed with water (10 mL), 10% NaHCO₃ (10 mL), brine (10 mL) and dried over Na₂SO₄. After solvent was removed under reduced pressure, the residue was purified by column chromatography (silica gel; CH₂Cl₂/EtOAc, 20:1) to give compound 21 (15.6 mg, 17% from compound 19) as white crystals: mp 118–120 °C (EtOAc); ¹H NMR δ 5.11 (s, 1H), 2.38 (dd, J = 18.9, 8.1Hz, 1H), 2.05 (s, 3H). From the ¹³C NMR data this product was determined to be a 9:1 mixture of epimers at C₁₄. The ¹³C NMR resonances for the major isomer were δ 20.7, 21.5, 22.9, 28.0, 28.3, 29.7, 30.2, 33.5, 33.7, 37.1, 40.6, 41.5, 41.7, 41.8, 42.3, 49.2, 70.0, 170.6, 221.4; IR ν_max 2918, 2855, 1738, 1245 cm⁻¹.

14.1.18 (2β,5α,13β,14β)-2-Hydroxygonan-17-one (1)

The mixture of compound 21 (15.6 mg, 0.05 mmol), NaOH (2 mg, 0.05 mmol) and water (18 mg, 1 mmol) in MeOH (12 mL) was refluxed for 2 h. The reaction mixture was chilled, and most of the MeOH was removed under reduced pressure to give a residue to which was added EtOAc (20 mL). The organic phase was washed with 10% aqueous HCl (5 mL), water (5 mL), brine (5 mL) and dried over Na₂SO₄. After the solvent was removed under reduced pressure, the residue was purified by column chromatography (silica gel; hexanes/CH₂Cl₂/EtOAc, 3:1:1) to give compound 1 (11 mg, 82%) as white crystalline needles: mp 123–124 °C (EtOAc/hexanes); [α] ²⁰_D = +140 (c = 0.30, CHCl₃); ¹H NMR δ 4.16 (m, 1H), 2.38 (dd, J = 18.9, 8.1Hz, 1H); ¹³C NMR δ 20.7, 23.0, 27.6, 28.0, 30.3, 32.5, 33.7, 36.3, 37.2, 40.6, 40.8, 41.7, 41.8, 42.6, 49.2, 66.7, 221.5; IR ν_max 3565, 2923, 2851, 1720 cm⁻¹. Anal. Calcd. For C₁₇H₂₆O₂: C 77.82, H 9.99; Found: C 77.68, H 10.02.

Scheme 4.