(8S,9S,10R,13S,14S,17S)-17-(2-hydroxyacetyl)-10,13-dimethyl-1,2,6,7,8,9,11,12,14,15,16,17-dodecahydrocyclopenta[a]phenanthren-3-one

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: 1.0 g. of 21-hydroxy-4-pregnene-3,20-dione is dissolved in 125 ml. of methanol and combined with a solution of 250 mg. of copper(II) acetate in 125 ml. of methanol. For 30 minutes, air is passed through the solution, and the latter is then combined with a solution of 500 mg. of ethylenediaminetetraacetic acid in 50 ml. of water, adjusted to pH 9 with 1N sodium hydroxide solution. The mixture is concentrated under vacuum at 40° C. and extracted with ethyl acetate. The extract is dried over sodium sulfate and evaporated under vacuum. As the residue, 1.1 g. of 3,20-dioxo-4-pregnen-21-al is obtained as a viscous oil. The mixture is concentrated under vacuum at 40° C. extracted with ethyl acetate The extract is dried over sodium sulfate evaporated under vacuum

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: 1.0 g. of 21-hydroxy-4-pregnene-3,20-dione is dissolved in 125 ml. of methanol and combined with a solution of 250 mg. of copper(II) acetate in 125 ml. of methanol. For 30 minutes, air is passed through the solution, and the latter is then combined with a solution of 500 mg. of ethylenediaminetetraacetic acid in 50 ml. of water, adjusted to pH 9 with 1N sodium hydroxide solution. The mixture is concentrated under vacuum at 40° C. and extracted with ethyl acetate. The extract is dried over sodium sulfate and evaporated under vacuum. As the residue, 1.1 g. of 3,20-dioxo-4-pregnen-21-al is obtained as a viscous oil. The mixture is concentrated under vacuum at 40° C. extracted with ethyl acetate The extract is dried over sodium sulfate evaporated under vacuum

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: At room temperature, 1.6 g of 21-chloro-17- formyloxy-4-pregnene-3,20-dione is stirred in a mixture of 60 ml of methanol and 9 ml of water with 500 mg of potassium bicarbonate After one hour, the reaction mixture is stirred into ice water. The thus-precipitated product is suctioned off, washed with water, and dried over sodium sulfate, yielding 1.3 g of 21-chloro-17™hydroxy-4-pregnene-3,20-dione; mp 239.4° C. washed with water dried over sodium sulfate

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: 1.16 g (3.5 mmoles) of 4-pregnene-3,20-dione-21-ol [prepared as described in Step (c) above] are acetylated as described in Example 2 to give 1.16 g (89% yield) of the aimed product, m.p.: 156°-158° C., [α]D =+174° (c=1.0 in dioxane).

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A solution containing 7.5 g of potassium hydrogen carbonate in 14 ml of water is added to the suspension containing 2.5 g (5.7 mmoles) of 4-pregnene-3,20-dione-21-ol benzoate in 100 ml of methanol and the mixture is refluxed for 3 hours. Subsequently, the solvent is evaporated and the residue is taken up in 150 ml of water. The product is extracted 3 times with 80 ml of ethyl acetate each, the organic phase is washed with water, dried and evaporated. The residue is recrystallized from a mixture of ethyl acetate and diisopropyl ether to give 1.89 g 99% yield of the aimed product, m.p.: 132°-135° C., [α]D =+175.2° (c=0.5 in ethanol). is added to the suspension the mixture is refluxed for 3 hours Subsequently, the solvent is evaporated The product is extracted 3 times with 80 ml of ethyl acetate each, the organic phase is washed with water dried evaporated The residue is recrystallized from a mixture of ethyl acetate and diisopropyl ether

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A solution containing 7.5 g of potassium hydrogen carbonate in 14 ml of water is added to the suspension containing 2.5 g (5.7 mmoles) of 4-pregnene-3,20-dione-21-ol benzoate in 100 ml of methanol and the mixture is refluxed for 3 hours. Subsequently, the solvent is evaporated and the residue is taken up in 150 ml of water. The product is extracted 3 times with 80 ml of ethyl acetate each, the organic phase is washed with water, dried and evaporated. The residue is recrystallized from a mixture of ethyl acetate and diisopropyl ether to give 1.89 g 99% yield of the aimed product, m.p.: 132°-135° C., [α]D =+175.2° (c=0.5 in ethanol). is added to the suspension the mixture is refluxed for 3 hours Subsequently, the solvent is evaporated The product is extracted 3 times with 80 ml of ethyl acetate each, the organic phase is washed with water dried evaporated The residue is recrystallized from a mixture of ethyl acetate and diisopropyl ether

Conditions: temp:32.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A suspension of fission yeast (S. pombe PE1) with a cell density of 3·107 cells/ml was prepared on a freshly grown culture using fresh EMMG (pH 7.4) as modified according to Ehmer et al. (Ehmer, P. B. et al., 1. Steroid. Biochem. Mol. Biol. 81, 173-179 (2002)). 492.5 μl of this cell suspension was admixed with 5 μl of inhibitor solution (50 μM of the compound to be tested in ethanol or DMSO) and incubated at 32° C. for 15 min. Controls were admixed with 5 μl of ethanol. The enzyme reaction was started by adding 2.5 μl of 11-deoxycorticosterone (20 μM, containing 1.25 nCi of [4-14C]11-deoxycorticosterone in Ethanol), followed by horizontal shaking at 32° C. for 6 h. The test was quenched by extracting the sample with 500 μl of EtOAc. After centrifugation (10,000 g, 2 min), the EtOAc phase was removed and evaporated to dryness. The residue was taken up in 10 μl of chloroform. The reaction of the substrate to form corticosterone was analyzed by HPTLC (see below). A suspension of fission yeast (S. pombe PE1) with a cell density of 3·107 cells/ml was prepared on a freshly grown culture The enzyme reaction The test was quenched by extracting the sample with 500 μl of EtOAc After centrifugation (10,000 g, 2 min), the EtOAc phase was removed evaporated to dryness The reaction of the substrate

Conditions: temp:32.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A suspension of fission yeast (S. pombe PE1) with a cell density of 3·107 cells/ml was prepared on a freshly grown culture using fresh EMMG (pH 7.4) as modified according to Ehmer et al. (Ehmer, P. B. et al., 1. Steroid. Biochem. Mol. Biol. 81, 173-179 (2002)). 492.5 μl of this cell suspension was admixed with 5 μl of inhibitor solution (50 μM of the compound to be tested in ethanol or DMSO) and incubated at 32° C. for 15 min. Controls were admixed with 5 μl of ethanol. The enzyme reaction was started by adding 2.5 μl of 11-deoxycorticosterone (20 μM, containing 1.25 nCi of [4-14C]11-deoxycorticosterone in Ethanol), followed by horizontal shaking at 32° C. for 6 h. The test was quenched by extracting the sample with 500 μl of EtOAc. After centrifugation (10,000 g, 2 min), the EtOAc phase was removed and evaporated to dryness. The residue was taken up in 10 μl of chloroform. The reaction of the substrate to form corticosterone was analyzed by HPTLC (see below). A suspension of fission yeast (S. pombe PE1) with a cell density of 3·107 cells/ml was prepared on a freshly grown culture The enzyme reaction The test was quenched by extracting the sample with 500 μl of EtOAc After centrifugation (10,000 g, 2 min), the EtOAc phase was removed evaporated to dryness The reaction of the substrate

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A mixture of 21-hydroxyprogesterone (200 mg, 0.605 mmol), 1,5-dibromopentane (1.65 mL, 12.1 mmol), 40% KOH (300 μL), and tetrabutylammonium hydroxide (60 μL) was stirred for 16 h at room temperature. The reaction mixture was diluted in dichloromethane and washed with water three times. The organic layer was dried over sodium sulfate and concentrated by rotary evaporation. The crude residue was purified by flash chromatography with hexanes/ethyl acetate (2:1) as the eluent to afford 7 as a colorless oil (125 mg, 43%). 1H NMR (500 MHz, CDCl3) δ 5.67 (s, 1H), 3.96 (q, J=17.2 Hz, 2H), 3.41 (t, J=6.4 Hz, 2H), 3.38-3.31 (m, 2H), 2.56 (t, J=9.1 Hz, 1H), 2.40-2.09 (m, 6H), 1.96 (m, 1H), 1.90-1.74 (m, 5H), 1.72-1.16 (m, 14H), 1.12 (s, 3H), 1.05-0.86 (m, 2H), 0.65-0.58 (m, 3H). 13C NMR (125 MHz, CDCl3) δ 207.55, 198.50, 169.94, 122.92, 75.79, 70.34, 61.56, 57.43, 55.12, 52.51, 43.53, 37.54, 34.61, 32.85, 31.74, 31.47, 30.82, 28.69, 27.74, 23.77, 23.47, 21.81, 19.97, 16.34, 12.55. ESI-MS m/z [M+H]+ observed: 481.1, calculated: 481.2. washed with water three times The organic layer was dried over sodium sulfate concentrated by rotary evaporation The crude residue was purified by flash chromatography with hexanes/ethyl acetate (2:1) as the eluent

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: 1.0 g. of 21-hydroxy-4-pregnene-3,20-dione is dissolved in 125 ml. of methanol and combined with a solution of 250 mg. of copper(II) acetate in 125 ml. of methanol. For 30 minutes, air is passed through the solution, and the latter is then combined with a solution of 500 mg. of ethylenediaminetetraacetic acid in 50 ml. of water, adjusted to pH 9 with 1N sodium hydroxide solution. The mixture is concentrated under vacuum at 40° C. and extracted with ethyl acetate. The extract is dried over sodium sulfate and evaporated under vacuum. As the residue, 1.1 g. of 3,20-dioxo-4-pregnen-21-al is obtained as a viscous oil. The mixture is concentrated under vacuum at 40° C. extracted with ethyl acetate The extract is dried over sodium sulfate evaporated under vacuum

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: At room temperature, 1.6 g of 21-chloro-17- formyloxy-4-pregnene-3,20-dione is stirred in a mixture of 60 ml of methanol and 9 ml of water with 500 mg of potassium bicarbonate After one hour, the reaction mixture is stirred into ice water. The thus-precipitated product is suctioned off, washed with water, and dried over sodium sulfate, yielding 1.3 g of 21-chloro-17™hydroxy-4-pregnene-3,20-dione; mp 239.4° C. washed with water dried over sodium sulfate

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: 1.16 g (3.5 mmoles) of 4-pregnene-3,20-dione-21-ol [prepared as described in Step (c) above] are acetylated as described in Example 2 to give 1.16 g (89% yield) of the aimed product, m.p.: 156°-158° C., [α]D =+174° (c=1.0 in dioxane).