Synthesis of 19-Trideuterated ent-Testosterone and the GABA_A Receptor Potentiators ent-Androsterone and ent-Etiocholanolone

Summary

19-Trideuteromethyl enantiomers of androgens namely ent-testosterone, ent-androsterone and ent-etiocholanolone were prepared by total synthesis. The isotope labeling at the carbon-19 angular methyl group was achieved by using deuterated methyl iodide (99.5% d₃) for introduction of C-19 before closure of the steroid A-ring. This method yields 19,19,19-trideuterated steroids without increasing the number of steps involved in the total synthesis of ent-androgens. Analysis by mass spectrometry showed no loss of deuterium during incorporation of C-19 into ent-testosterone. The availability of the compounds will enable these ent-androgens to be distinguished by mass spectrometry from their natural enantiomers in future pharmacokinetic and metabolic studies.

Introduction

Anxiolytic, anticonvulsant, sedative and anesthetic effects of neuroactive steroids are known to occur due to binding of neuroactive steroids to GABA_A receptors.¹ As part of our structure–activity studies of neuroactive steroid modulation of GABA_A receptors, we have investigated the enantioselectivity of neuroactive steroid action at these receptors.² Although we initially found that the naturally occurring enantiomers of several potent neuroactive steroids were more effective modulators of GABA_A receptors than their synthetic non-naturally occurring enantiomers (ent-steroids), we recently found that ent-androsterone and ent-etiocholanolone enhanced GABA-mediated chloride currents at these receptors more effectively than their natural counterparts.³ These previous results suggest that ent-androsterone and ent-etiocholanolone may have useful in vivo activity as GABAergic drugs of the future. In preparation for future pharmacokinetic and metabolic studies we modified the total synthesis of ent-testosterone to obtain ent-testosterone-19,19,19-d₃ (Scheme 1). Modification was easily achieved by using trideuterated methyl iodide in place of methyl iodide during introduction of the C-19 angular methyl group and subsequent closure of the ent-steroid A-ring. This trideuterated ent-testosterone was then converted into ent-androsterone-19,19,19-d₃ and ent-etiocholanolone-19,19,19,-d₃ by established literature procedures (Figure 1).^3,4

Scheme 1.

Synthesis of ent-testosterone-19,19,19-d₃ (3); Reagents: (a) i. Li/liq. NH₃, THF; ii. CD₃I; (b) 3 N HCl, MeOH. For complete description of the preparation of compound 1 see references.⁷,⁸

Figure 1.

Target compounds: 19-Trideutered Androgens.

Results and discussion

Although deuterium labeling of the C-19 position of natural testosterone has been achieved previously by several different methods,^5,6 we wanted to incorporate the trideuterated C-19 methyl group into ent-testosterone without increasing the number of synthetic steps required to prepare this ent-steroid. Accordingly, tricyclic intermediate 1 was first prepared according to the literature.^7,8

Compound 1 was then subjected to Li/ liquid NH₃ reduction and the radical anion intermediate produced in the reaction was reacted with methyl iodide-d₃ (99.5% d₃) to obtain the desired C-19 position deuterium labeling in 35% yield. Compound 2 thus obtained was then treated with MeOH under acidic conditions to obtain ent-testosterone-19,19,19-d₃ (3) in 67% yield. The ¹H-NMR of product 3 lacked the resonance at δ 1.27 ppm for the C-19 methyl group in the natural abundance ¹H-NMR of this compound. Mass spectroscopic analysis of compound 3 showed complete retention (99.5% d₃) of the three deuterium atoms derived from the methyl iodide-d₃ in the product.

The desired ent-androsterone-19,19,19-d₃ (6) was obtained from ent-steroid 3 using procedures reported previously for this transformation on natural abundance ent-testosterone (Scheme 2).⁴ Reduction of compound 3 using Li/liquid NH₃ gave ent-steroid 4 in 65% yield. Jones oxidation converted ketoalcohol 4 into dione 5 (82% yield) and regio- and stereoselective reduction of dione 5 with K-Selectride^® gave ent-androsterone-19,19,19-d₃ (6) in 47 % yield. Because the molecular ion from product 6 is weak, interference from background noise in the mass spectra of this product only permitted the d₃ content of the products to be accurately measured as >95%.

Scheme 2.

Synthesis of ent-androsterone-19,19,19-d₃ (6). Reagents: (a) Li/Li NH₃, THF, toluene, NH₄Cl (b) Jones reagent, acetone, 0° C (c) i. K-selectride^®, THF; ii. 10% NaOH, 30% aqueous H₂O₂.

The desired ent-etiocholanolone-19,19,19-d₃ (12) was also obtained from ent-steroid 3 using previously reported procedures described for this transformation on natural abundance ent-testosterone (Scheme 3).³ Catalytic hydrogenation of ent-steroid 3 using Pd/C gave three isolated products (4, 7 and 8). Compound 4 was easily separated from unseparated products 7 and 8 by column chromatography. Separation of products 7 and 8 required acetylation of the inseparable mixture and column chromatography to separate products 9 and 10. Saponification of the ketoacetate 9 gave ketoalcohol 7 (37% overall yield). Jones oxidation converted ketoalcohol 7 into dione 11 (77% yield) and regio- and stereoselective reduction of dione 11 with LiAl[OC(CH₃)₃]₃H gave ent-etiocholanolone-19,19,19-d₃ (12) in 80% yield. As was the case for product 6, mass spectrometry could only accurately determine the d₃ content of product 12 to be >95%.

Scheme 3.

Synthesis of ent-etiocholanolone-19,19,19-d₃ (12). (a) Pd/C, KOH, ⁱPrOH, H₂; (b) Acetic anhydride, Pyridine, DMAP; (c) K₂CO₃, MeOH; (d) LiAl[(OC(CH₃)₃]₃H, THF.

Experimental

Materials and instruments

All solvents were purchased or dried and purified by standard methods. All air and/or moisture sensitive reactions were carried out under N₂ using oven-dried glassware and cooled under vacuum or N₂. All extraction solvents were dried with anhydrous Na₂SO₄ and the drying agent was removed by filtration before solvent removal. Extraction solvents were removed on a rotary evaporator. Flash chromatography was performed using silica gel (32–63 μm) purchased from Scientific Adsorbents (Atlanta, GA). Optical rotations were determined on a Perkin-Elmer Model 341 polarimeter. Melting points were determined on a Kofler micro hot stage and are uncorrected. IR spectra were recorded as films on an NaCl plate with a Perkin-Elmer Spectrum One FTIR spectrophotometer. NMR spectra were recorded at ambient temperature in CDCl₃ with a 5 mm probe on a Varian Gemini 2000 operating at 300 MHz (¹H) or 75 MHz (¹³C). ¹H and ¹³C NMR spectra were referenced to CDCl₃ (δ 7.27) and (δ 77.0), respectively. Mass spectroscopic analyses were performed by the UCR Mass Spectrometry Facility (Riverside, CA). Deuterated methyl iodide (99.5% d₃) was purchased from Cambridge Isotope Laboratories (Andover, MA).

[3R-(3α,3aα,5aβ,6β,9aα,6β,9aα,9bβ)]-3-(1,1-Dimethylethoxy)dodecahydro-3a,6-dimethyl-6-[2-(2-methyl-1,3-dioxolan-2-yl)ethyl]-7H-benz[e]inden-7-one-3a,3a,3a-d₃ (2)

A dry flask was equipped with a mechanical stirrer, addition funnel and a gas bubbler. After cooling the flask to −78 °C in a dry ice-acetone bath, NH₃ (∼1 L) was condensed in the flask. Small pieces of lithium (1.56 g, 0.225 mol) were added, and the blue solution was stirred for 20 min. Enone 1^7,8 ([α]_D = -356.7 (c=0.1, toluene), 22 g, 0.056 mol) was dissolved in THF (100 mL) and added dropwise to the reaction mixture over 30 min. The reaction mixture was maintained at −78 °C for 1 h with persistence of blue color. Then, deuterated methyl iodide (14.6 mL, 0.22 mmol) in THF (20 mL) was added dropwise to the solution. This led to a rapid change from blue to yellow color. The reaction was allowed to stir overnight to evaporate the ammonia. After addition of water (1 L) and NH₄Cl (40 g), the product was extracted with EtOAc (3 × 150 mL) and the combined EtOAc extractions were washed with brine and dried. After solvent evaporation, an orange-brown oil was obtained. Chromatography (silica gel, 15% EtOAc in hexanes) gave product 2 (8.41 g, 35 %) as a white solid: mp 105–106 °C; [α]_D = −34.2 (CHCl₃, c = 1.03); IR 2966, 1696, 1358, 1201, 1078, 947 cm^-1; ¹³C-NMR δ 214.83, 110.24, 80.50, 72.20, 64.47, 64.40, 50.39, 50.31, 47.53, 42.42, 38.09, 36.68, 34.71, 32.98, 30.97, 30.72, 28.88, 28.68, 23.73, 23.35, 20.85, 11.54; ¹H-NMR δ 3.93 (4H, m); 3.38 (1H, t, J = 8.1 Hz), 2.51(1H, m), 2.22-2.39 (1H, m), 1.35 (3H, s), 1.13 (9H, s), 0.78 (3H, s); HRMS (ESI) m/z calcd for C₂₅H₃₉D₃O₄, (M+Na)⁺ 432.3169; found 432.3178.

ent-Testosterone-19,19,19-d₃ (3)

Benz[e]indenone 2 (6.12 g, 0.014 mol) in MeOH (160 mL) and 3 N HCl (40 mL) were heated to reflux for ∼ 24 h. After the reaction mixture cooled to room temperature, the MeOH was removed on a rotary evaporator and water (135 mL) was added. The water was extracted with CH₂Cl₂ (3 × 150 mL) and the combined extracts were washed with NaHCO₃ (50 mL), brine (50 mL) and dried. Solvent evaporation gave a yellow oil. Column chromatography (silica gel, 20% EtOAc in hexanes) yielded product 3 (2.91 g, 67%) as a white solid: mp 151–153 °C; [α]_D = −113 (CHCl₃, c =1.1); IR 2942, 1671, 1349, 1179, 940 cm^-1; ¹³C-NMR δ 199.70, 171.50, 123.72, 81.43, 53.76, 50.36, 42.71, 38.37, 36.32, 35.54, 35.50, 33.83, 32.72, 31.43, 30.24, 23.33, 20.55, 10.97; ¹H-NMR δ 5.73 (1H, s), 3.64 (1H, m), 0.79 (3H, s); HRMS (ESI) m/z calcd for C₁₉H₂₅D₃O₂, MH⁺ 292.2356; found 292.2358. No natural abundance MH⁺ peak for C₁₉H₂₈O₂ was detectable by MS analysis indicating that no deuterium loss accompanied incorporation of the deuterated methyl group derived from deuterated methyl iodide (99.5% d₃) into product 3.

(5β,8α,9β,10α,13α,14β,17α)-17-Hydroxyandrostan-3-one-19,19,19,-d₃ (4)

A solution of compound 3 (2.1 g, 7.2 mmol) in THF (20 mL) was added to a blue solution of liquid ammonia (500 mL) and dissolved lithium metal (0.16 g, 2.4 mol) in THF (6 mL) and toluene (60 mL) at −78 °C. After 30 min, NH₄Cl (3 g) was added portionwise to discharge the blue color and the ammonia was allowed to evaporate. The reaction mixture was diluted with 10% aqueous HCl (50 mL) and extracted with EtOAc (3 × 25 mL). The combined organic extracts were washed with water (50 mL) and brine (50 mL) and dried. The solvent was removed to give a yellow oil which was purified by column chromatography (silica gel, 20% EtOAc in hexanes to 35% EtOAc in hexanes) to give compound 4 (1.35 g, 65 % yield): mp = 174-176 °C; [α]_D = −32.6 (CHCl₃, c = 0.8); IR 2925, 1713, 1358, 1201, 1076, 945 cm^-1; ¹³C-NMR (δ 212, 81.72, 53.82, 50.77, 46.65, 44.62, 42.93, 38.44, 38.08, 36.59, 35.44, 35.39, 31.19, 30.41, 28.74, 23.32, 20.97, 11.09; ¹H-NMR δ 3.63 (1H, t, J = 8.3 Hz); 0.75 (3H, s); HRMS (ESI) m/z calcd for C₁₉H₂₇D₃O₂, MH⁺ 294.2507; found 294.2512.

(5β, 8α,9β,10α,13α,14β)-Androstane-3,17-dione (5)

Jones reagent (8 M) was titrated dropwise to a stirred solution of compound 4 (1.38 g, 4.6 mmol) in acetone (100 mL) at 0 °C until an orange color persisted. After an additional 5 min, ⁱPrOH was also titrated until the excess reagent was destroyed, and water (100 mL) was added. The mixture was extracted with EtOAc (3 × 50 ml). The combined organic layers were washed with brine (70 mL) and dried. The solvent was removed to give a solid which was recrystallized from hexanes to give the product 5 (1.10 g, 82%) as a white solid: mp 124–125 °C [α]_D = −113 (CHCl₃, c = 1); IR 2971, 1732, 1596, 1200, 1098, 940 cm^-1; ¹³C-NMR δ 220.78, 211.46, 53.74, 51.13, 47.62, 46.49, 44.50, 38.27, 37.99, 35.72, 35.50, 34.87, 31.40, 30.43, 28.53, 21.68, 20.62, 13.72; ¹H-NMR δ 0.89 (3H, s). HRMS (ESI) m/z calcd for C₁₉H₂₅D₃O₂, MH⁺ 292.2350; found 292.2352.

ent-Androsterone-19,19,19-d₃ (6)

K-selectride^® (1.0 M solution in THF, 5.2 mL, 5.2 mmol) was added to a stirred solution of compound 5 (1.10 g, 3.7 mmol) in dry THF (5.6 mL) at −78 °C. After 1.5 h, 10% aqueous NaOH (20 mL) and then 30 % aqueous H₂O₂ (7.6 mL) were added. The mixture was allowed to warm to room temperature, stirred for another 30 min, and extracted with CH₂Cl₂. The combined organic layers were washed with brine and dried. Solvent removal gave a solid which was recrystallized from EtOAc to give compound 6 as a white solid (520 mg, 47%): mp 177–178 °C; [α]_D = −92.1 (CHCl₃, c = 1.3); IR 2923, 1738, 1371, 1200, 1072, 960 cm^-1; ¹³C-NMR δ 221.44, 66.32, 54.33, 51.45, 47.76, 39.03, 35.98, 35.80, 35.74, 35.01, 32.02, 31.52, 30.79, 28.95, 28.21, 21.70, 20.0, 13.78; ¹H-NMR δ 4.05 (1H, m); 0.86 (3H, s); HRMS (ESI) m/z calcd for C₁₉H₂₇D₃O₂, MH⁺ 294.2507; found 294.2510; >95% d₃.

(5a,8α,9β,10α,13α,14β,17α)-17-Hydroxyandrostan-3-one-19,19,19-d₃ (7)

KOH (0.48 g) and ⁱPrOH (30 mL) were added to a hydrogenation bottle, the solution was stirred for 30 min to allow some of the KOH to dissolve. Pd/C (0.11 g, 5%) and ent-steroid 3 (2 g, 6.8 mmol) dissolved in ⁱPrOH (30 mL) were then added and the total volume was increased to ∼100 ml with ⁱPrOH. The solution was hydrogenated using a Parr hydrogenator for 18 h at 45 psi and the catalyst removed by filtration through a pad of Celite which was washed with MeOH. After removal of the solvent, a yellow oil remained. To this oil, brine (150 mL) and Et₂O (250 mL) were added, and the aqueous phase was neutralized with 6 N HCl. After thorough mixing, the aqueous layer was removed and the organic layer was washed with brine (3 × 200 mL) and dried. Solvent removal gave a white solid. Column chromatography (silica gel, 20% EtOAc/hexanes to 40% EtOAc/hexanes) separated ketoalcohol 4 from an inseparable mixture of ketoalcohol 7 and diol 8. Acetylation of the mixture of products 7 and 8 in the standard manner⁹ (pyridine 5.48 mL, acetic anhydride 5.35 mL, DMAP, catalytic amount) gave acetylation products 9 and 10 which were easily separated by column chromatography (silica gel, 180 gms 5-15% 3 × 500 cm³ EtOAc in hexanes). Saponification of 9 using K₂CO₃ (200 mg and MeOH 20ml, reflux for an hour and purification by column chromatography on silica gel eluted with 30% EtOAc in hexanes) gave pure product 7 as a white solid (730 mg, 37%): mp 140–141 °C; [α]_D = −28.90 (EtOH, c = 0.6); IR 2865, 1712, 1073, 855 cm^-1; ¹³C-NMR δ 213.24, 81.78, 50.96, 44.21, 43.09, 42.27, 40.87, 37.14, 36.91, 36.79, 35.57, 34.72, 30.52, 26.43, 25.33, 23.32, 20.76, 11.09; ¹H-NMR δ 3.6 (1H, t, J = 8.5 Hz), 2.67 (1H, t, J = 14.3 Hz), 0.76 (3H, s); HRMS (ESI) m/z calcd for C₁₉H₂₇D₃O₂, MH⁺ 294.2507; found 294.2513.

(5α,8α,9β,10α,13α,14β)-Androstane-3,17-dione-19,19,19-d₃ (11)

Compound 7 (730 mg, 2.48 mmol) was dissolved in acetone (20 mL) and while stirring Jones reagent (8 M) was titrated dropwise to this solution until a yellow color persisted. The solution was stirred at room temperature for 30 min after which ⁱPrOH was titrated dropwise to quench any remaining Jones reagent. The reaction mixture was poured into brine (150 mL) and the aqueous solution was extracted with EtOAc (3 × 200 mL). The organic extracts were combined and dried. After solvent removal, a blue solid remained. This solid was chromatographed (silica gel, 50% EtOAc in hexanes) to yield compound 11 (560 mg, 77%) as a white solid: mp 125–126 °C; [α]_D = −104.66 (EtOH, c = 0.4); IR 2931, 1738, 1712, 1373, 1202, 1093, 915 cm^-1; ¹³C-NMR δ 220.66, 212.60, 51.30, 47.72, 44.03, 42.13, 40.90, 37.01, 36.76, 35.75, 35.04, 34.74, 31.57, 26.25, 24.61, 21.67, 20.39, 13.70; ¹H-NMR δ 2.68 (1H, t, J = 14.2 Hz), 0.89 (3H, s); HRMS (ESI) m/z calcd for C₁₉H₂₅D₃O₂, MH⁺ 292.2350; found 292.2350.

ent-Etiocholanolone-19,19,19-d₃ (12)

To a dry flask compound 11 (560 mg, 1.92 mmol) dissolved in dry THF (30 mL) was added. While under N₂, the solution was cooled to −42 °C in a bath of acetonitrile and dry ice. Lithium tri-tert-butoxyaluminohydride (1.9 mL, 2 mmol, 1 M in THF) was added dropwise and the solution was stirred for 2 h at −42 °C under N₂. TLC after 2 h confirmed that reaction was not complete, hence it was warmed to −20 °C for another 1 h. The reaction was quenched with 3 N HCl (20 mL). EtOAc (250 mL) was added and the organic solution was washed with aqueous NaHCO₃ (150 mL) and brine (150 mL). The organic layer was dried and the solvent was removed to give a white solid. Column chromatography (silica gel, 30% EtOAc/hexanes) yielded product 12 (480 mg, 80%) as a white solid: mp 149–150 °C; [α]_D = −95.3 (EtOH, c = 0.7); IR 2929, 1738, 1370, 1089, 947 cm^-1; ¹³C-NMR δ 221.35, 71.59, 51.45, 47.82, 41.92, 40.69, 36.27, 35.89, 35.39, 35.19, 34.48, 31.69, 30.44, 26.86, 25.33, 21.78, 20.04, 13.75; ¹H-NMR δ 3.67−3.60 (1H, m), 2.44 (dd, J = 18.9, 8.7 Hz), 0.85 (3H, s); HRMS (ESI) m/z calcd for C₁₉H₂₇D₃O₂ MH⁺ 294.2507; found 294.2508; >95% d₃.

Conclusion

An efficient synthesis of ent-testosterone, ent-androsterone and ent-etiocholanolone containing 19,19,19-d₃ deuterium labeling was developed.