1. Introduction
Progesterone is a precursor to steroids with potent effects on multiple ion channels [1]. One of these ion channels, the GABAA receptor, is gated by the neurotransmitter γ-aminobutyric acid (GABA), the major inhibitory transmitter in mammalian brain. Positive allosteric modulation of GABAA receptors by allopregnanolone derived from progesterone augments the actions of GABA and increases neuronal inhibition. Positive allosteric modulation of GABAA receptors by intravenously administered brexanolone (an FDA approved formulation of allopregnanolone) and the newly FDA approved orally active drug zuranolone is accepted as their major mechanism of action for the treatment of postpartum depression [2,3].
Although it is well established that exogeneously administered progesterone can be converted endogenously to allopregnanolone, questions related to how exogenously administered progesterone is metabolized under various conditions and how endogenous levels of other endogenous steroids produced in the brain are affected by exogenously administered progesterone remain. In this regard, progesterone with C18 and/or C19 trideuterated methyl groups can be useful reagents for distinguishing by mass spectrometry between steroids produced from endogenous progesterone and those generated from exogenously administered progesterone.
Multiple syntheses of testosterone, androstenedione and progesterone with trideuterated 19-methyl groups have been reported previously [4-8]. These syntheses either use a steroid precursor to directly introduce the 19-trideuterated methyl group or construct it from a steroid containing a functionalized C19 substituent. A total synthesis of enantiomeric androgens with 19-trideuterated methyl groups has been reported by us previously [9]. A Chemical Abstracts search revealed only one previous report of steroids with an 18-trideuterated methyl group. Progesterone with an 18-trideuterated methyl group was prepared from a steroid containing a 18-hydroxyl group and progesterone side chain [8]. This synthetic route to 18-trideuterated progesterone will only lead to androgens with this trideuterated methyl group after removal of the progesterone side chain. No references with synthetic routes to androstane or pregnane classes of steroids with both trideuterated 18- and 19-methyl groups were identified in Chemical Abstracts.
Because of our previous experience with the a total synthesis of enantiomeric androgens with a 19- trideuterated methyl group as well as difficulties we encountered in trying to use the approach previously reported for the synthesis of 18-trideuterated progesterone, we carried out a total synthesis of steroids with either 18-trideuterated or 19-trideuterated methyl groups (Figure 1). The synthetic routes to the trideuterated progesterones and androstenediones proceed through the intermediacy of the corresponding trideuterated testosterones. This unified total synthetic approach to 18- and 19-trideuterated methyl steroids can also be used to produce steroids with both 18- and 19-trideuterated methyl groups.
Figure 1. Structures of prepared 18 or 19-trideuterated Steroids.
Trideuterated testosterone (1a,1b), trideuterated androstenedione (2a,2b) and trideuterated progesterone (3a,3b).
2. Experimental
2.1. General Methods
The 1,3-cyclopentane was purchased from Aaron Chemicals, Ltd. (San Diego, CA). Iodomethane-d3 was purchased from MilliporeSigma (St. Louis, MO) as were all solvents and other reagents. Solvents were either used as purchased or dried and purified by standard methodology. Flash column chromatography was performed using silica gel (32–63 μm) purchased from Scientific Adsorbents (Atlanta, GA). Optical rotations were measured on a Perkin-Elmer Model 341 Polarimeter in the solvent indicated. NMR spectra were recorded on a Varian 400 MHz spectrometer in CDCl3 at ambient temperature at 400 MHz (1H) or 100 MHz (13C), Chemical shifts are reported as δ values relative to internal chloroform (δ = 7.27) for 1H and chloroform (δ = 77.0) for 13C. Compound 5 was referenced to DMSO-d6 (1H, δ = 2.50; 13C = 39.5). Mass spectrometry analysis were performed by Washington University in St. Louis Mass Spectrometry Facility.
2.2. Synthesis of 2-(Methyl-d3)-1,3-cyclopentanedione (5).
1,3-Cyclopentanedione (4, 49 g, 500 mmol) was dissolved in 5 N aqueous NaOH (100 mL, 500 mmol) at 0 °C for 1 h. Iodomethane-d3 (100 g, 690 mmmol) was added to the resulting red/brown solution in one portion. The ice bath was removed and the reaction mixture was heated at 65 °C for 72 h. After cooling, the orange solid product 5 was filtered, and washed with hexanes and a minimum amount of water until a pale beige solid (20.7 g, 36%) was obtained: 1H NMR (400 MHz, DMSO-d6 δ 11.4 (s, br, 1H), 2.31 (s, 4H); 13C NMR (100 MHz, DMSO-d6 δ 111.5, 30.1 (The resonance for the ketone groups, which exist in the conjugated keto-enol form, were not observed due to the poor solubility of the compound in the solvent.); MS (ESI) calcd for [C6H5D3O2 + H]+ : 116.1, found: 116.2.
2.3. Synthesis of 2-(Methyl-d3)-2-(3-oxobutyl)-1,3-cyclopentanedione (6).
Compound 5 (20.7 g, 180 mmol), water (41.4 mL), acetic acid (0.54 mL), and methyl vinyl ketone (30.9 mL, 360 mmol) were combined in a 250 mL flask covered with aluminum foil. The reaction was stirred under N2 at 70 °C for 3 h. After cooling, the product was extracted into CH2Cl2 (2 x 150 mL). The combined extracts were washed with brine (2 x 30 mL), dried over anhydrous Na2SO4 and the solvent removed. The residue was purified by flash column chromatography (silica gel, 25-50% EtOAc in hexanes) to afford product 6 (30.2 g, 91%): 1H NMR (400 MHz, CDC13) δ 2.81-2.63 (m, 4H), 2.39 (t, J = 7.4 Hz 2H), 2.01 (s, 3H), 1.80 (t, J = 7.4 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 215.8 (2 x C), 207.7, 54.8, 37.3, 34.6 (2 x C), 29.9, 27.6.
2.4. Synthesis of (7aS)-2,3,7,7a-Tetrahydro-7a-(methyl-d3)-1H-Indene-1,5(6H)-dione (7).
L-Proline (75.2 mg, ground into a fine powder with a mortar and pestle) dissolved in DMF (125 mL) was added to a water jacketed flask covered with aluminum foil and connected to a circulating water bath set to 15 °C. The flask was degassed and refilled with N2 several times. Then stirred for 45 min to allow the reaction to cool down to 15 °C. Trione 6 (30.2 g, 163 mmol) dissolved in DMF (40 mL) was added to the reaction flask and the degassing procedure was repeated three times. The reaction was stirred at 15 °C for 72 h. The flask contents were transferred to a standard 500 mL flask and heated to 70 °C. A solution of H2SO4 in DMF was prepared before use by adding concentrated H2SO4 (1.8 mL) to DMF (32 mL) chilled on ice. Part of this solution (14 mL) was added to the reaction and heated to 95 °C for 1 h. An additional portion of H2SO4 in DMF solution (16 mL) was added and the reaction was stirred at 95 °C for 2 h. The dark reaction solution was then cooled to 60 °C and the solvent (DMF) was removed under reduced pressure on a rotary evaporator. The residue was dissolved in CH2Cl2 (400 mL) and washed with aqueous NaHCO3 (3 x 200 mL) and brine (150 mL). The aqueous layers were combined and extracted with CH2Cl2 (2 x 250 mL). After being combined, the CH2Cl2 was removed and the residue was purified by flash column chromatography (silica gel, 25-50% EtOAc in hexanes) to afford product 7 which was recrystallized from Et2O (18 g, 66%): [α]D25 = +356.7° (c = 1.04, toluene); 1H NMR (400 MHz, CDCl3) δ 5.74 (s, 1H), 2.85-2.52 (m, 3H), 2.37-2.17 (m, 3H), 1.91-1.82 (m, 1H), 1.67-1.59 (m, 1H); 13C NMR (100 MHz, CDCl3) δ 216.0, 197.4, 169.5, 123.1, 48.0, 35.3, 32.3, 28.5, 26.3.
2.5. Synthesis of (1S,7aS)-1,2,3,6,7,7a-Hexahydro-1-hydroxy-7a-(methyl-d3)-5H-inden-5-one (8).
Dione 7 (18 g, 108 mmol) was dissolved in absolute EtOH (100 mL) and chilled to −10 °C in a NaCl/ice bath. NaBH4 (1.25 g, 33 mmol) dissolved in EtOH (125 mL) was added dropwise over 30 min maintaining the temperature below −5 °C during addition. The solution turned orange and then green. The reaction was allowed to warm up to 5 °C for 1 h, re-cooled to −10 °C and the pH was adjusted to 6 using 1 N HCl. The solvent was removed, and the product extracted from the orange residue with EtOAc (5 x 100 mL). The combined extracts were washed with brine (2 x 100 mL), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to give crude product 8 (18 g , 99%): 1H NMR (400 MHz, CDCl3) δ 5.74(s, 1H), 3.82 (t, J = 8.1, 1H), 3.07 (s, br, 1H), 2.78-1.69 (m, 8H); 13C NMR (100 MHz, CDCl3) δ 199.6, 175.9, 123.1, 80.2, 45.0, 33.8, 33.2, 28.8, 26.4.
2.6. Synthesis of (1S,7aS)-1-(1,1-Dimethylethoxy)-1,2,3,6,7,7a-hexahydro-5H-inden-5-one (9).
Crude compound 8 (18 g, 106 mmol) dissolved in CH2Cl2 (200 mL) was added to a flask equipped with a mechanic stirrer and gas condenser. The flask was then cooled to −78 °C using a dry ice/acetone bath. In a separate flask, P2O5 (2 g) was added to 85% H3PO4 (5.6 mL) to obtain dry H3PO4. Dry H3PO4 (4.3 mL) and BF3·Et2O (10 mL) were added to the reaction flask while stirring at −78 °C and isobutylene (150 mL) was condensed in the flask. The flask was warmed up to −5 °C for 6 h and 23 ° for 12 h. Saturated aqueous NH4Cl (300 mL) was added and the product was extracted into CH2Cl2 (5 x 100 mL) and EtOAc (100 mL). The combined extracts were dried over anhydrous Na2SO4, filtered, the solvents removed and the residue was purified by flash column chromatography (silica gel, eluted with 25-50% EtOAc in hexanes) to afford product 9 (16.8 g, 70%): 1H NMR (400 MHz, CDCl3) δ 5.57 (s, 1H), 3.44 (t, J = 8.5, 1H), 2.56-2.48 (m, 1H), 2.37-2.13 (m, 3H), 1.88-1.79 (m, 2H), 1.67-1.51 (m, 2H), 1.02 (s, 9H); 13C NMR (100 MHz, CDCl3) δ 198.6, 174.9, 122.4, 79.1, 72.5, 44.1, 33.8, 32.9, 29.1, 28.2 (3 x C), 26.4.
2.7. Synthesis of (3S,3aS,5aS,6R,9aS,9bS)-3-(1,1-Dimethylethoxy)dodecahydro3a-(methyl-d3)-6-(methyl)-6-[2-(2-methyl-1,3-dioxolan-2-yl)ethyl]-7H-benz[e]inden-7-one (15a) from indenone 9.
To a dry flask was added compound 9 (16.8 g, 74.7 mmol) and magnesium methyl carbonate (150 mL, 2 M in DMF, 300 mmol) at room temperature. The reaction was heated to 120 °C for 3 h and after cooling, it was poured into stirred concentrated HCl and ice (1:1). Crude product 10 was extracted into EtOAc (3 x 200 mL). The combined extracts were made basic (pH 9) with saturated Na2CO3, causing the deprotonated acid 10 to partition into the aqueous phase. The aqueous phase was then acidified (pH 3) with concentrated HCl to protonate the acid, allowing extraction of carboxylic acid 10 into EtOAc (3 x 150 mL). The combined extracts were dried over anhydrous Na2SO4, filtered, and concentrated to give the crude unsaturated acid 10 (18.2 g, 91% crude) as a light yellow solid which was used without further purification.
The crude unsaturated acid 10 (18.2 g) was dissolved in MeOH (100 mL) and Pd/BaSO4 (3 g) was added. The mixture was cooled to 0 °C, warmed to 23 °C and hydrogenated (55 psi, H2) for 3 h. The catalyst was removed by filtration through Celite. MeOH was removed in vacuo (10 °C water bath, high vacuum pump) to give product 11 as a brown viscous oil (18.2 g, 99% crude), which was immediately used without purification.
The crude saturated ketoacid 11 (18.2 g) was added a ice cooled solution of the following: 37% aqueous formaldehyde (24 mL), piperidine (0.65 mL) and DMSO (30 mL). After stirring for 2 h, the reaction was poured into a slurry of ice and saturated NaCl (1:1). Product 12 was extracted into Et20 (3 x 100 mL). The combined organic extracts were washed with saturated NaHCO3 (2 x 100 mL), dried over anhydrous Na2SO4 and filtered. Solvent removal (25 °C, water bath) gave compound 12 as a brown oil (15.8 g, 98% crude). This unstable compound was used immediately without purification.
A 0.1 N solution of sodium methoxide was prepared by adding sodium (450 mg) slowly to MeOH (190 mL) under N2. Part of this NaOMe solution (120 mL) was added to methyl 6-(2-methyl-1,3-dioxolan-2-yl)-3-oxohexanoate (18.6 g, 81 mmol) which was prepared according to the literature [10]. Crude compound 12 (15.8 g) was dissolved in MeOH (60 mL), chilled to 0 °C and then added dropwise to the methyl 6-(2-methyl-1,3-dioxolan-2-yl)-3-oxohexanoate solution. The reaction was stirred at room temperature for 16 h and then 5 N NaOH (40 mL, 200 mmol) was added dropwise. After 1 h, most of the MeOH was removed (25 °C, water bath) and the remaining aqueous MeOH was extracted with EtOAc (3 x 200 mL). The aqueous phase was then chilled and acidified to pH 3 with 6 N HCl. The product was extracted with EtOAc (3 x 150 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered, and the solvent removed. The remaining oil was heated in vacuo (80 °C, vacuum pump) for 2 h at which time a constant weight was obtained to afford crude benz[e]indenone 14a (20.2 g, 77% crude), a 16 g portion of which was used without purification.
A dry flask was equipped with a mechanic stirrer with a Teflon blade, addition funnel, and dry ice/acetone cooled gas condenser and cooled to −78 °C in a dry ice/acetone bath. Anhydrous NH3 (300 mL) was condensed in the flask. Small pieces of Li wire (700 mg, 100 mmol) were added, and the resultant blue solution was stirred for 30 min. Crude benz[e]indenone 14a (16 g) was dissolved in THF (100 mL) and added dropwise to the flask over 30 min. The reaction was kept at −78 °C for 1 h and the blue color persisted throughout. CH3I (60 mmol) in THF (20 mL) was added and the reaction turned yellow. The cooling bath and gas condenser were allowed to warm to 23 °C and the reaction flask was stirred overnight to allow NH3 to evaporate. NH4Cl (40 g) and water (300 mL) were added, and the product was extracted into EtOAc (3 x 250 mL). The combined extracts were washed with brine, dried over anhydrous Na2SO4, filtered, and the solvent removed. The residue was purified by flash column chromatography (silica gel, eluted with 10-25% EtOAc in hexanes) to afford product 15a (3.45 g, 20.6% from crude compound 14a): 1H NMR (400 MHz, CDCl3) δ 4.08-3.81 (m, 4H), 3.35 (t, J = 8.2, 1H), 2.53-0.84 (m, 22H), 1.31 (s, 3H), 1.09 (s, 9H); 13C NMR (100 MHz, CDCl3) δ 214.6, 110.0, 80.3, 72.0, 64.3, 64.2, 50.3, 50.2, 47.4, 42.1, 37.9, 36.4, 34.6, 32.8, 30.8, 30.6, 28.8, 28.5 (3 x C), 23.6, 23.2, 21.0, 20.7.
2.8. Synthesis of 17-Hydroxyandrost-4-en-3-one-18,18,18-d3 (1a).
Benz[e]indenone 15a (3.45 g, 8.44 mmol) in MeOH (80 mL) and 3 N HCl (40 mL) were refluxed for 24 h. After cooling, most of MeOH was removed, water (50 mL) was added and the product was extracted into CH2Cl2 (3 x 100 mL). The combined organic extracts were washed with NaHCO3 (1 x 100 mL) and brine (1 x 100 mL), dried over anhydrous Na2SO4, filtered, and the solvent removed. The residue was purified by flash column chromatography (silica gel, eluted with 10-25% EtOAc in hexanes) to give a white solid which was recrystallized from acetone and water (1:1) to afford white crystals of 1a (1.51 g, 61%): [α]D23 +118 (c 0.32, CHCl3); 1H NMR (400 MHz, CDCl3) δ 5.73 (s, 1H), 3.65 (t, J = 8.6 Hz, 1H), 2.46-0.89 (m, 20H), 1.19 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 199.7, 171.4, 123.8, 81.5, 53.8, 50.4, 42.5, 38.6, 36.3, 35.7, 35.6, 33.9, 32.7, 31.5, 30.4, 23.3, 20.6, 17.4; HRMS (ESI) calcd for [C19H25D3O2 + Na]+ : 314.2175, found: 314.2184.
2.9. Synthesis of Androst-4-ene-3,17-dione-18,18,18-d3 (2a).
To a solution of steroid 1a (28 mg, 0.096 mmol) in CH2Cl2 (5 mL) was added Dess-Martin periodinane (167 mg, 0.4 mmol) at 23 °C. After 2 h, water (20 mL) was added and the product was extracted into CH2Cl2 (2 x 50 mL). The solvent was removed and the residue was purified by flash column chromatography (silica gel, eluted with 10-25% EtOAc in hexanes) to afford steroid 2a as a white solid (27 mg, 96%): 1H NMR (400 MHz, CDCl3) δ 5.74 (s, 1H), 2.50-0.95 (m, 19H), 1.20 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 220.5, 199.3, 170.3, 124.1, 53.7, 50.7, 47.3, 38.6, 35.7, 35.6, 35.1, 33.8, 32.5, 31.1, 30.7, 21.7, 20.2, 17.3; HRMS (ESI) calcd for [C19H23D3O2 + Na]+: 312.2019, found: 301.2024.
2.10. Synthesis of (3S,3aS,9aS,9bS)-3-(1,1-Dimethylethoxy)-1,2,3,3a,4,5,8,9,9a,9b-decahydro-3a-(methyl-d3)-6-[2-(2-methyl-1,3-dioxolan-2-yl)ethyl]-7H-benz[e]inden-7-one (15b) from indenone 13.
Crude indenone 13 (25 g) was prepared according to the literature procedure [11]. Crude benz[e]indenone 14b (16g, 39% crude) was prepared from crude indenone 13 by the same procedure described for the preparation of crude benz[e]indenone 14a. Crude compound 14b (16 g) was then converted to benz[e]indenone 15b (9.58 g, 57% from crude compound 14b) as described for the similar 14a to 15a conversion except that CD3I was used in the alkyation step. Compound 15b had: 1H NMR (400 MHz, CDCl3) δ 3.90-3.93 (m, 4H), 3.32 (t, J = 8.2, 1H), 2.492.15 (m, 2H), 1.87-0.84 (m, 17H), 1.28 (s, 3H), 1.06 (s, 9H), 0.70 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 214.6, 110.0, 80.3, 72.0, 64.3, 64.2, 50.2, 50.1, 47.3, 42.3, 37.9, 36.5, 34.5, 32.8, 30.8, 30.6, 28.7, 28.5 (3 x C), 23.6, 23.2, 20.7, 11.4.
2.11. Synthesis of 17-Hydroxyandrost-4-en-3-one-19,19,19-d3 (lb).
Benz[e]indenone 15b (9.58 g, 23.6 mmol) was converted to steroid 1b (5.9 g, 86%) using the same procedure reported for the preparation of steroid 1a from benz[e]indenone 15a. Steroid 1b was crystallized from acetone and water (1:1) and had: [α]D23 +116 (c 0.12, CHCl3); 1H NMR (400 MHz, CDCl3) δ 5.58 (s, 1H), 3.51 (t, J = 8.6 Hz, 1H), 2.85 (s, br, 1H), 2.32-0.73 (m, 19H), 0.65 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 199.6, 171.5, 123.6, 81.2, 53.7, 50.3, 42.6, 38.3, 36.2, 35.4, 35.4, 33.7, 32.6, 31.3, 30.1, 23.1, 20.5, 10.9; HRMS (ESI) calcd for [C19H25D3O2 + Na]+ : 314.2175, found: 314.2175.
2.12. Synthesis of Androst-4-ene-3,17-dione-19,19,19-d3 (2b).
Steroid 1b (100 mg, 0.34 mmol) was converted to steroid 2b (96 mg, 97%) using the same procedure reported for the conversion of steroid 1a to steroid 2a. Steroid 2b was obtained as a white solid and had: 1H NMR (400 MHz, CDCl3) δ 5.73 (s, 1H), 2.49-0.94 (m, 19H), 0.90 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 220.2, 199.1, 170.2, 124.1, 53.7, 50.7, 47.4, 38.3, 35.6, 35.5, 35.0, 33.8, 32.5, 31.2, 30.6, 21.6, 20.2, 13.6; HRMS (ESI) calcd for [C19H23D3O2 + Na]+ : 312.2019, found: 312.2020.
2.13. Synthesis of 17-Hydroxyandrost-5-en-3-one-18,18,18-d3, cyclic 1,2-ethanediyl acetal (16a).
To a stirred solution of steroid 1a (265 mg, 0.91 mmol) in toluene (150 mL) was added ethylene glycol (2 mL) and PTSA (50 mg) at 23 °C. The mixture was refluxed in a flask equipped with a Dean-Stark apparatus for 16 h. After cooling, solid NaHCO3 (400 mg) was added and stirring continued for 30 min. Water was added and the product was extracted into EtOAc (350 mL). The organic layer was washed with brine (3 x 100 mL), dried over anhydrous Na2SO4 and the solvent removed. The residue was purified by flash column chromatography (silica gel, eluted with 25-40% EtOAc in hexanes) to afford steroid 16a (225 mg, 74%) and recovered steroid 1a (42 mg). Steroid 16a had: 1H NMR (400 MHz, CDCl3) δ 5.32-5.30 (m, 1H), 3.95-3.88 (m, 4H), 3.62 (t, J = 8.5, Hz, 1H), 2.55-2.51 (m, 1H), 2.11-0.88 (m, 19H) 1.01 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 140.1, 121.8, 109.3, 81.6, 64.3, 64.1, 51.1, 49.6, 42.4, 41.6, 36.6, 36.4, 36.2, 31.8, 31.2, 30.9, 30.3, 23.3, 20.5, 18.8.
2.14. 17-Hydroxyandrost-5-en-3-one-19,19,19-d3, cyclic 1,2-ethanediyl acetal (16b).
Steroid 1b (1.5 g, 5.15 mmol) was converted to steroid 16b (1.6 g, 93%) using the same procedure reported for the preparation of steroid 16a from steroid 1a. Steroid 16b had: 1H NMR (400 MHz, CDCl3) δ 5.34-5.33 (m, 1H), 3.97-3.89 (m, 4H), 3.65 (t, J = 8.6, Hz, 1H), 2.56-2.53 (m, 1H), 2.12-0.90 (m, 19H) 0.74 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 140.1, 121.8, 109.3, 81.7, 64.3, 64.1, 51.2, 49.6, 42.6, 41.7, 36.5, 36.4, 36.2, 31.9, 31.2, 30.9, 30.3, 23.4, 20.5, 10.9.
2.15. Androst-5-ene-3,17-dione-18,18,18-d3, cyclic 3-(l,2-ethanediyl acetal) (17a).
To a stirred solution of steroid 16a (225 mg, 0.67 mmol) in CH2Cl2 (10 mL) was added NaHCO3 (300 mg) and Dess-Martin periodinane (568 mg, 1.34 mmol) at 23 °C. After 1 h, water was added and the product was extracted into CH2Cl2 (2 x 100 mL). The combined extracts were washed with brine (2 x 50 mL), dried over anhydrous Na2SO4 and the solvent removed. The residue was purified by flash column chromatography (silica gel, eluted with 25% EtOAc in hexanes) to afford steroid 17a (225 mg, ~100%): 1H NMR (400 MHz, CDCl3) δ 5.33-5.31 (m, 1H), 3.93-3.85 (m, 4H), 2.54-2.36 (m, 2H), 2.10-1.04 (m, 17H) 1.00 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 220.7, 140.1, 121.1, 109.0, 64.2, 64.0, 51.4, 49.5, 47.1, 41.5, 36.5, 36.0, 35.6, 31.3, 31.1, 30.7, 30.4, 21.6, 20.1, 18.7.
2.16. Androst-5-ene-3,17-dione-19,19,19-d3, cyclic 3-(1,2-ethanediyl acetal) (17b).
Steroid 16b (1.6 g, 4.78 mmol) was converted to steroid 17b (1.12 g, 70%) using the same procedure reported for the preparation of steroid 17a and had: 1H NMR (400 MHz, CDCl3) δ 5.28-5.27 (m, 1H), 3.88-3.81 (m, 4H), 2.48-2.32 (m, 2H), 2.05-0.82 (m, 17H), 0.79 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 220.6, 140.1, 121.0, 108.9, 64.1, 63.9, 51.4, 49.5, 47.2, 41.5, 36.2, 35.9, 35.5, 31.2, 31.1, 30.7, 30.3, 21.6, 20.0, 13.2.
2.17. (17Z)-Pregna-5,17(20)-dien-3-one-18,18,18-d3, cyclic 1,2-ethanediyl acetal (18a).
To a stirred suspension of ethyl triphenylphosphonium bromide (2.6 g, 7 mmol) in THF (30 mL) was added potassium tert-butoxide (672 mg, 6 mmol) at 23 °C. The reaction was refluxed for 1 h and then steroid 17a (225 mg, 0.67 mmol) in THF (10 mL) was added and reflux was continued for 16 h. After cooling, water was added and most of the THF was removed on a rotary evaporator under reduced pressure. Water was added and the product was extracted into EtOAc (2 x 100 mL). The combined extracts were washed with brine (2 x 50 mL), dried over anhydrous Na2SO4 and the solvent removed. The residue was purified by flash column chromatography (silica gel, eluted with 10% EtOAc in hexanes) to afford steroid 18a (212 mg, 91%): 1H NMR (400 MHz, CDCl3) δ 5.37-5.35 (m, 1H), 5.16-5.11 (m, 1H), 3.98-3.91 (m, 4H), 2.39-1.08 (m, 22H) 1.05 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 150.0, 140.0, 121.9, 113.3, 109.3, 64.3, 64.1, 56.3, 49.5, 43.7, 41.7, 36.8, 36.5, 36.1, 31.4, 31.3 (2 x C), 30.9, 24.4, 21.1, 18.7, 13.0.
2.18. (17Z)-Pregna-5,17(20)-dien-3-one-19,19,19-d3, cyclic 1,2-ethanediyl acetal (18b).
Steroid 17b (1.12 g, 3.36 mmol) was converted to steroid 18b (928 mg, 80%) using the same procedure reported for the preparation of steroid 18a and had: 1H NMR (400 MHz, CDCl3) δ 5.34-5.33 (m, 1H), 5.13-5.10 (m, 1H), 3.96-3.89 (m, 4H), 2.56-2.52 (m, 1H), 2.38-1.06 (m, 21H) 0.87 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 150.2, 140.1, 122.1, 113.4, 109.4, 64.4, 64.2, 56.4, 49.6, 44.0, 41.8, 36.9, 36.4, 36.2, 31.5, 31.4, 31.4, 31.0, 24.5, 21.2, 16.6, 13.1.
2.19. (20S)-20-Hydroxypregn-5-en-3-one-18,18,18-d3, cyclic 1,2-ethanediyl acetal (19a).
To a stirred solution of steroid 18a (212 mg, 0.61 mmol) in THF (20 L) was added 9-BBN (0.5 N, 3 mL, 1.5 mmol) at 23 °C and the reaction was stirred for 16 h. 3 N NaOH (10 mL) and H2O2 (5 mL) were then added and stirring continued for 1 h. The product was extracted into EtOAc (3 x 100 mL) and the combined extracts were washed with brine (3 x 100 mL), dried over anhydrous Na2SO4 and the solvent removed. The residue was purified by flash column chromatography (silica gel, eluted with 25-40% EtOAc in hexanes) to afford steroid 19a (207 mg, 93%): 1H NMR (400 MHz, CDCl3) δ 5.32-5.31 (m, 1H), 3.95-3.88 (m, 4H), 3.67-3.63 (m, 1H), 2.55-2.51 (m, 1H), 2.11-1.02 (m, 23H) 1.00 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 129.9, 121.9, 109.3, 70.1, 64.3, 64.1, 58.2, 56.3, 49.5, 41.6, 41.2, 38.6, 36.5, 36.2, 31.6, 31.4, 30.9, 25.7, 24.1, 23.4, 20.6, 18.7.
2.20. (20S)-20-Hydroxypregn-5-en-3-one-19,19,19-d3, cyclic 1,2-ethanediyl acetal (19b).
Steroid 18b (928 mg, 2.69 mmol) was converted to steroid 19b (850 mg, 87%) using the same procedure reported for the preparation of steroid 19a and had: 1H NMR (400 MHz, CDCl3) δ 5.29-5.28 (m, 1H), 3.92-3.84 (m, 4H), 3.65-3.58 (m, 1H), 2.51-2.47 (m, 1H), 2.07-0.93 (m, 23H) 0.58 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 139.8, 121.8, 109.2, 70.0, 64.2, 64.0, 58.2, 56.3, 49.4, 41.5, 41.3, 38.6, 36.2, 36.0, 31.5, 31.3, 30.8, 25.7, 24.0, 23.4, 20.5, 12.2.
2.21. Pregn-5-ene-3,20-dione-18,18,18-d3, cyclic 3-(1,2-ethanediyl acetal) (20a).
To a stirred solution of steroid 19a (207 mg, 0.57 mmol) in CH2Cl2 (10 mL) was added NaHCO3 (300 mg) and Dess-Martin periodinane (483 mg, 1.14 mmol) at 23 °C. After 1 h, water was added and the product was extracted into CH2Cl2 (2 x 100 mL). The combined extracts were washed with brine (2 x 50 mL), dried over anhydrous Na2SO4 and the solvent removed. The residue was purified by flash column chromatography (silica gel, eluted with 20% EtOAc in hexanes) to afford compound steroid 20a (192 mg, 93%): 1H NMR (400 MHz, CDCl3) δ 5.28-5.27 (m, 1H), 3.92-3.84 (m, 4H), 2.50-2.46 (m, 2H), 2.14-1.04 (m, 18H), 2.06 (s, 3H), 0.96 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 209.2, 139.9, 121.6, 109.1, 64.2, 64.0, 63.3, 56.6, 49.3, 43.5, 41.5, 38.5, 36.4, 36.1, 31.6, 31.4, 31.3, 30.8, 24.2, 22.6, 20.8, 18.7.
2.5. Synthesis of Pregn-4-ene-3,20-dione-18,18,18-d3 (3a).
To a stirred solution of steroid 20a (192 mg, 0.53 mmol) in acetone (20 mL) and water (0.2 mL) was added PTSA (50 mg) at 23 °C. After 16 h, solid NaHCO3 (200 mg) was added and stirring continued for 20 min. The solvent was removed, water was added and the product was extracted into EtOAc (2 x 100 mL). The combined extracts were washed with brine (2 x 50 mL), dried over anhydrous Na2SO4 and the solvent removed. The residue was purified by flash column chromatography (silica gel, eluted with 30% EtOAc in hexanes) to afford steroid 3a (155 mg, 92%): [α]D23 +194 (c 0.14, CDCl3); 1H NMR (400 MHz, CDCl3) δ 5.64 (s, 1H), 2.46-0.87 (m, 20H), 2.05 (s, 3H), 1.11 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 209.2, 199.3, 171.0, 123.8, 63.3, 55.9, 53.5, 43.6, 38.5, 38.5, 35.6, 35.4, 33.9, 32.7, 31.8, 31.5, 24.3, 22.7, 20.9, 17.3; HRMS (ESI) calcd for [C21H27D3O2 + Na]+ : 340.2332, found: 340.2332.
2.22. Pregn-5-ene-3,20-dione-19,19,19-d3, cyclic 3-(1,2-ethanediyl acetal) (20b).
Steroid 19b (850 mg, 2.34 mmol) was converted to steroid 20b (785 mg, 93%) using the same procedure reported for the preparation of steroid 20a and had: 1H NMR (400 MHz, CDCl3) δ 5.32-5.31 (m, 1H), 3.96-3.88 (m, 4H), 2.54-2.49 (m, 2H), 2.16-1.08 (m, 18H), 2.09 (s, 3H), 0.60 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 209.5, 140.0, 121.8, 109.3, 64.3, 64.1, 63.5, 56.8, 49.4, 43.9, 41.6, 38.7, 36.3, 36.1, 31.7, 31.5, 31.5, 30.9, 24.4, 22.7, 20.9, 13.1.
2.23. Synthesis of Pregn-4-ene-3,20-dione-19.19.19-d3 (3b).
Steroid 20b (785 mg, 2.17 mmol) was converted to steroid 3b (615 mg, 89%) using the same procedure reported for the conversion of steroid 20a to steroid 3a. Steroid 3b was obtained as a white solid and had: [α]D23 +195 (c 0.13, CHCl3); 1H NMR (400 MHz, CDCl3) δ 5.61 (s, 1H), 2.47-0.87 (m, 20H), 2.02 (s, 3H), 0.56 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 209.0, 199.1, 170.7, 123.6, 63.1, 55.7, 53.2, 43.6, 38.3, 38.1, 35.3, 35.2, 33.6, 32.5, 31.6, 31.2, 24.0, 22.5, 20.7, 13.0; HRMS (ESI) calcd for [C21H27D3O2 + Na]+ : 340.2332, found: 340.2345
3. Results and Discussion
The synthesis of the 18-trideuterated steroids 1a-3a begins with the preparation of 2-(methyl-d3)-1,3-cyclopentanedione 5 as summarized in Scheme 1. This procedure is an adaptation of the total synthetic route originally reported for the synthesis of 19-nortestosterone [11]. Commercially available cyclopentane-1,3-dione 4 was reacted with CD3I using a one step procedure previously reported for the synthesis of natural abundance 2-methyl-1,3-cyclopentanedione [12] to obtain compound 5 in 36% yield. Reaction of compound 6 with methyl vinyl ketone yielded trione 6 (91%) and its cyclization in the presence of l-proline yielded trideuterated Hajos-Parrish indenone 7 (66%). Reduction of the ketone group on the five-membered ring of indenone 7 gave indenone 8 (99%) and conversion of the resultant hydroxyl group to the corresponding tert-butyl ether gave indenone 9 (70%). A five step sequential procedure (9→10→11→12→14a→15a) performed without purification after each step converted indenone 9 to compound 15a (11%, calculated directly from compound 9). The first three steps of this sequential procedure are shown in Scheme 1 and the remaining two steps are shown in Scheme 2. This reaction sequence completes the synthesis of the B, C and D rings of the final 18-trideuterated steroid structures 1a-3a.
Scheme 1a.
aReagents: (a) 0 °C, 1 h then 65 °C, 72 h (36%); (b) H2O/AcOH, methyl vinyl ketone, under N2, 70 °C, 3 h (91%); (c) L-proline, DMF, under N2, 15 °C, 72 h followed by aqueous H2SO4, 95 °C, 3h (66%); (d) NaBH4, EtOH, −5 to 5 °C, 1 h (99%); (e) H3PO4. BF3·Et2O, isobutylene, −78 °C then −5 °C, 6 h and 23 °C, 12h (70%); (f) methyl magnesium carbonate, DMF, 120 °C, 3h (91% crude); (g) Pd/BaSO4, MeOH, 23 °C, H2, (55 psi) (~100% crude); (h) aqueous HCHO, DMSO, piperidine, 0 °C, 2 h (98% crude).
Scheme 2a.
aReagents: (a) i: NaOMe, MeOH, 23 °C, 16 h; ii: 5 N NaOH, 1 h; iii: 6 N HCl, 80 °C, 2h (14a, 77% crude from 12; 14b, 39% crude from 13); (b) anhyd. NH3, THF, −78 °C,1 h, then CD3I or CH3I addition (15a, 21% from crude 14a); 15b, (57% from crude 14b); (c) 3 N HCl, MeOH, reflux, 24 h (1a, 61%; 1b, 86%); (d) NaHCO3, Dess-Martin periodinane, CH2Cl2, 23 °C, 2h (2a, 96%; 2b, 97%).
Also shown in Scheme 2 is compound 13 which was prepared as described previously [11]. Compound 13 was similarly converted (13→14b→15b) to the trideuterated benz[e]indenone 15b (22%, from crude compound 13), thus completing the synthesis of the B, C and D rings of the final 19-trideuterated steroids (1b,2b). Cyclization of benz[e]indenones 15a and 15b under conditions that also removed the t-butyl ether group on the resultant steroid yielded the corresponding trideuterated steroids 1a (61%) and 1b (86%), and oxidation of the 17-hydroxyl groups yielded trideuterated steroids 2a (96%) and 2b (97%), respectively.
The overall yield for the synthesis of steroid 1a from 2-(methyl-d3)-1,3-cyclopentanedione 5 was ~3%. We are unable to calculate the overall yield of steroid 1b from 2-methyl-1,3-cyclopentanedione because the indenone 13 used to prepare compound 15b was from pooled material from several indenone 13 preparations carried out in our lab previously.
In the 1H-NMR spectrum of testosterone the chemical shifts of the 18-methyl and 19-methyl group proton resonances are δ 0.80 and δ 1.20 ppm, respectively, and in the 13C-NMR the carbon-13 resonances are δ 11.03 and δ 17.39 ppm, respectively. In the 18-trideuterated testosterone 1a the 18-methyl group proton resonance at δ 0.80 ppm is absent and the carbon-13 resonance at δ 11.03 ppm is absent. In the 19-trideuterated testosterone 1b the 19-methyl group proton resonance at δ 1.20 ppm is absent and the carbon-13 resonance at δ 17.39 ppm is absent. The reason the carbon-13 resonances of the trideuterated methyl carbons are not observed is as follows. The off-resonance decoupling of the proton-carbon coupling used to simplify the carbon-13 resonances so that these resonances appear as singlets does not decouple the deuterium-carbon coupling. The deuterium-carbon scalar (spin-spin) couplings (~20-25 Hz apart) of the trideuterated methyl group remain. These couplings result in the carbon resonances appearing as a septet (7 lines spaced ~20-25 Hz apart) with intensity ratio 1:3:6:7:6:3:1. These multiplet components that each have significantly reduced intensity relative to that which would be observed for the carbon resonance in the absence of deuterium coupling, result in a trideuterated methyl carbon of undetectable intensity.
The conversions of steroids 1a and 1b into the trideuterated progesterones 3a and 3b are shown in Scheme 3. The 3-ketone groups of 1a and 1b are first converted into their corresponding Δ5-3-ketals 16a (74%) and 16b (93%) and then oxidized to the 17-ketosteroids 17a (~100%) and 17b (70%). A Wittig reaction is then used to introduce a two carbon side-chain at the 17 position yielding steroids 18a (91%) and 18b (80%). A hydroboration reaction was used to covert the side chain to the (20S)-hydroxysteroids 19a (93%) and 19b (87%). The (20S)-hydroxyl groups were then oxidized to the 20-ketone groups of steroids 20a (93%) and 20b (87%). Removal of the 3-ketal groups from steroids 20a and 20b yielded the trideuterated progesterones 3a (92%) and 3b (89%).
Scheme 3a.
aReagents: (a) toluene, HOCH2CH2OH, PTSA, reflux, 16 h (16a, 74%; 16b, 93%); (b) Dess-Martin periodinane, CH2Cl2, 23 °C, 1h (17a, ~100%; 17b, 70%); (c) ethyl triphenylphosphonium bromide, THF, KOBut, reflux, 16 h (18a, 91%; 18b, 80%); (d) 9-BBN, THF, 23 °C, 16 h (19a, 93%; 19b, 87%); (e) NaHCO3, Dess-Martin periodinane, CH2Cl2, 23 °C, 2 h (20a, 93%; 20b, 93%); (f) acetone, H2O, PTSA, 23 °C, 16 h (3a, 92%, 3b, 89%).
In conclusion, total steroid synthesis provides a unified approach for the preparation of either 18- or 19-trideuterated steroids. Although not performed herein, the trideuterated benz[e]indenone 14a could also be used to prepare 18,19-hexadeuterated steroids by using CD3I instead of CH3I in the reaction shown for the conversion of benz[e]indenone 14a to benz[e]indenone 15a. To the best of our knowledge steroids with both trideuterated 18- and 19-methyl groups have not been synthesized previously from steroid starting materials.
Supplementary Material
A unified total synthetic route to 18- and 19-trideuteromethyl steroids is described.
The method can also be used to prepare 18,19-hexadeuteromethyl steroids.
Compounds are useful for preparing calibration curves for mass spec analysis of these steroid hormones.
Compounds are useful for distinguishing between these endogenous and exogenous steroids under various conditions.
Acknowledgments
This work was supported by NIH grant 1 P50 MH122379 and The Taylor Family Institute for Innovative Psychiatric Research. We thank the Washington University Biomedical Mass Spectrometry Resource facility (NIH grant 8P41GM103422) for the mass spectrometry analyses.
Footnotes
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Appendix A. Supplementary data
Supplementary data to this article can be found online.
References
- [1].Kudova E, Rapid effects of neurosteroids on neuronal plasticity and their physiological and pathological implications, Neurosci. Lett 750 (2021) 135771. [DOI] [PubMed] [Google Scholar]
- [2].Edinoff AN, Odisho AS, Lewis K, Kaskas A, Hunt G, Cornett EM, Kaye AD, Kaye A, Morgan J, Barrilleaux PS, Lewis D, Visanath O, Urits I, Brexanolone, a GABAA modulator, in the treatment of postpartum depression in adults: a comprehensive review, Front. Psychiatry 12 (2021) 699740. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [3].Deligiannidis KM, Meltzer-Brody S, Maximos B, Peeper EQ, Freeman M, Lasser R, Bullock A, Kotecha M, Li S, Forrestal F, Rana N, Garcia M, Leclair B, Doherty J, Zuranolone for the treatment of postpartum depression, Am. J. Psychiatry, 180 (2023) 668–675. [DOI] [PubMed] [Google Scholar]
- [4].Baba S, Shinohara Y, Kasuya Y, Synthesis of trideuterated testosterone labeled selectively at the carbon-19 angular methyl group, J. Labelled Comp. Radiopharm 14 (1978) 783–791. [Google Scholar]
- [5].Holland HL, Taylor GJ, Enzymatic aromatization of deuterium labeled testosterone and androst-4-ene-3,17-dione, Can. J. Chem 59 (1981) 2809–2819. [Google Scholar]
- [6].Dyer RL, Harrow TA, Synthesis of C-19 deuterium labeled steroids, Steroids 33 (1979) 617–624. [DOI] [PubMed] [Google Scholar]
- [7].Jacolot F, Berthou F, Picart D, Le Gall JY, Floch HH, Preparation of deuterated analogs of 5α-androstane-3ξ,17β-diol, J. Labelled Comp. Radiopharm 19 (1982) 553–572. [Google Scholar]
- [8].Kirk DN, Smith CZ, Varley MJ, Honour JW, Synthesis of [19-2H3]progesterone and [18-2H3]progesterone, J. Chem. Soc. Perkin Trans I (1990) 2745–2748. [Google Scholar]
- [9].Komarapuri S, Krishnan K, Covey DF, Synthesis of 19-trideuterated ent-testosterone and the GABAA receptor potentiators ent-androsterone and ent-etiocholanolone, J. Labelled Comp. Radiopharm 51 (2008) 430–434. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [10].Kirar EP, Drev M, Mirnik J, Groseli U, Golobic A, Dahmann G, Pozgan F, Stefane B, Svete J, Synthesis of 3D-rich heterocycles: hexahydropyrazolo[l,5-a]pyridine-2(1H)-ones and octahydro-2H-2a,2a1-diazacyclopenta[cd]inden-2-ones, J. Org. Chem 81 (2016) 8920–8933. [DOI] [PubMed] [Google Scholar]
- [11].Micheli RA, Hajos ZG, Cohen N, Parrish DR, Portland LA, Sciamanna W, Scott MA, Wehrli PA, Total synthesis of optically active 19-norsteroids. (+)-Estr-4-ene-3,17-dione and (+)-13β-ethylgon-4-ene-3,17-dione, J. Org. Chem 40 (1975) 675–68. [DOI] [PubMed] [Google Scholar]
- [12].Lertpibulpanyaa D, Marsden SP, Concise access to indolizidine and pyrroloazepine skeleta via intramolecular Schmidt reactions of azido 1,3-diketones, Org. Biomol. Chem, 4 (2006) 3498–3504. [DOI] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.