Method for Making 3α-Hvdroxγ.3ι8-Substituted-Pregnanes

This application is a continuation-in-part of copend- ing application Serial No. 745,216 filed August 13, .1991, which is in turn a continuation-in-part of copending application Serial No. 521,724, filed May 10, 1990, which is in turn a continuation-in-part of copending application Serial No. 379,047, filed July 13, 1989, which in turn is a continuation-in-part of application Serial No. 089,362 filed August 25, 1987, now abandoned. These applications and their claims and figures are incorporated herein by reference.

Background of the Invention

The present invention is directed to a method for making compositions and compounds useful for modulating animal brain excitability via the gamma-aminobutyric acid (GABA) receptor-chloride ionophore complex (GR complex) .

A variety of steroid derivatives, such as 3α- hydroxy,3/3-methyl-5α-pregnan-20-one, have been shown to be effective in stimulating the GR complex, with a variety of physiological effects. See U.S. Serial No. 07/745,216, 07/521,724, and 07/379,047, incorporated herein by refer¬ ence. The standard procedure for making this compound and other 3α-hydroxy,3/3-substituted-pregnanes includes a step wherein compounds with two keto groups have one group protected prior to reaction at the other keto group to produce a 3/S-substituted pregnane. During this process, one method of protecting the 20-keto group is to change it to an ethylene ketal group. See U.S. Patent No. 3,953,429. The drawbacks of this method include the requirement for the use of two steps—protection before reaction at the 3-keto position and deprotection after reaction— ith their accompanying loss in material.

We have discovered a novel method for production of 3α-hydroxy,3/3-substituted-pregnanes which does not require protection of the 20-one group. By this method, each ketone group is treated independently. A variety of groups can be substituted into the 3/3 position. The ketone at the 20 position can also be modified .in an independent manner.

Summary of the Invention

This invention provides a simplified method for con- vertingpregnan-3,20-dione compounds to 3α.-hydroxy,3j3-sub- stituted-pregnanes. By selective use of reagents the unprotected dione is converted chemoselectively and dias- tereoselectively into a 3(R)-pregnan-3-spiro-2*oxirane-20- one intermediate. This intermediate can then be converted regioselectively by a second set of reactions to the 3α- hydroxy,3jS-substituted-20-one form, which can be further modified at the 20-keto position.

Through this method, each ketone group is indepen¬ dently treated. By modifying the ketones one at a time, one can obtain the desired stereo-specificity at each site.

Detailed Description

The starting materials for this reaction are pregnan- 3,20-diones. The first step converts the ketone at the 3 position into an oxirane.

For this reaction, Corey's reagent and base are mixed in a suitable aprotic polar solvent to form an ylide. Corey, et al., "Dimethylsulfoxonium Methylide and Dimethylsulfonium Methylide Formation and Application to Organic Synthesis," J. Amer. Chem. Soc. 7:1354-1364, 1965. The ylide is mixed with the pregnan-3,20-dione which has been suspended or dissolved in the solvent of choice. Sufficient reagent is provided to produce an amount of ylide that will give complete reaction of the ketone. The amount of base used to produce the ylide

should be chosen so as to leave no unreacted base after formation of the ylide. The reaction is performed in a dry atmosphere, such as under nitrogen or argon gas, with dry solvents (in the absence of water) . The time and temperature of ylide formation and subsequent reaction with ketone are determined by monitoring loss of ketone starting material or formation of oxirane product using a suitable analytical technique such as TLC or HPLC.

The reagent can be any Corey's reagent which reacts chemoselectively so as to selectively convert only the 3-keto group to an oxirane. The reagent is also chosen for the ability to diastereoselectively convert the 3-keto group to the desired oxirane, in this case a 3(R)-pregnan- 3-spiro-2'oxirane-20-one. This reagent is preferably tri- methyl sulfoxonium iodide, but any equivalent reagent which will react with the appropriate selectivity will do.

The reagent is dissolved in an appropriate aprotic, polar solvent, such as polyethers, amides, phosphoric amides, sulfoxides, sulfolanes. Specific examples are dimethylsulfoxide (DMSO) , tetrahydrofuran (THF) , hexa- methylphosphoric triamide (HMPT) , sulf lane, N-methyl- pyrolidone, dioxane, dimethoxyethane (DME) , and dimethyl- formamide (DMF) .

The base is chosen so that its basicity is suffi- ciently high to remove a proton from Corey's reagent in order to form the ylide. Examples of appropriate bases are NaH, potassium t-butoxide, and NaNH ₂

The oxirane group is then regioselectively opened to form the 3α-hydroxy,3/3-substituted compounds. The reac- tion is performed in a dry atmosphere, such as under nitrogen or argon gas, with dry solvents (in the absence of water) . Time of reaction and temperature are suffi¬ cient to insure complete reaction, as monitored by analytical techniques such as HPLC or TLC. Opening of the oxirane is performed by reaction with an appropriate nucleophile which will open the oxirane without affecting the remaining 20-keto group. Preferred

nucleophiles include alkoxides, thioalkoxides, azides, cyanide, isocyanide, amines, and halide anions such as iodide. Bonini et al., "A Facile Che o and Regioselective Reductive Opening of 1,2 Epoxides Via Free Radical Reac- tion," Tetrahedron Letters, 29_:819-822, 1988.

The oxirane can optionally be opened and subsequently converted to the 3j8-methyl compound. Hydrogenolysis can be performed with hydrogen gas and a suitable catalyst. Examples of such a catalyst are transition metals such as palladium and platinum, dissolved in the appropriate sol¬ vent. The catalysts can be provided complexed to acti¬ vated carbon. Other reagents for the reduction of the C-X bond (where X stands for halide) include trialkyl tin hydrides. Optionally, a weak base such as sodium acetate can be added to the reaction after the oxirane ring is opened in order to remove acid formed as a byproduct during hydrogenolysis.

A nucleophile can also add to the oxirane at the 3' position. For example, a 3 _/ S-ethyl compound can be pro¬ duced using dimethyl lithium cuprate; -a 3/3-bromomethyl compound can be produced using sodium bromide;, a 3/3- azidomethyl compound can be produced using sodium azide or trimethylsilyl azide; a 33-propyl compound can be produced using diethyl lithium cuprate. The nucleophile is sel¬ ected for its ability to regioselectively provide the desired group upon opening of the ring.

Any solvent.capable of dissolving a sufficient quan¬ tity of the reagents to promote the reaction is appro- priate. Examples of appropriate solvents are 1,2- dimethoxyethane, and a combination of THF and methanol. Aprotic polar solvents enhance the reaction by stabilizing the transition state and are therefore preferred.

The following examples of this procedure are provided for illustrative purposes. One skilled in the art will recognize that other reagents, solvents and bases can be substituted. Additionally one skilled in the art will

recognize that similar starting compounds can also be used.

Example 1

(3R>-5Q.-preσnan-3-spiro-2'oxirane-20-one To a stirred solution of trimethyl sulfoxonium iodide (5.290 g, 24.04 mmol) in DMSO (75 mL) at room temperature was added NaH (97%; 488 mg, 19.72 mmol) in one portion. The resulting mixture that became a clear solution after ~ 10 min was stirred at room temperature under a nitrogen atmosphere for 1 h. Then a suspension of 5α-pregnan-3,20- dione (1.538 g, 4.860 mmol) in DMSO (40 mL + 10 mL for the rinse) was added dropwise through a pressure-equalizing funnel. The mixture so obtained, which was not completely clear, was stirred at room temperature under a nitrogen atmosphere for a total of 2.5 h although TLC (3:1 hexane/ EtOAC) after 1.5 h showed complete disappearance of the starting material. The mixture was then poured into ice/water and extracted with ether (x3) . The combined organic phase was washed with water (x3) and brine, dried (MgS0 ₄ ) , filtered, and evaporated under reduced pressure to give a solid. Finally, recrystallization from ^' hot 1:1 methanol/acetone ( ^" 50 mL) gave the epoxide (1.176 g, 73%) as white crystals.

Example 2 (3R)-5o.-prec,nan-3-spiro-2'oxirane-20-one

A dry 3-neck. flask with argon atmosphere, fitted with a mechanical stirrer, was charged with 400 mL of dry tetrahydrofuran and 34.0 g (0.29 moles) of potassium t-butoxide. After stirring the solution for thirty minutes, 63.1 g (0.29 moles) of trimethyl sulfoxonium iodide was added and the suspension stirred at ambient temperature for 2.0 hours, resulting in a heterogenous mixture. In a separate flask cooled to 15-20°C was slurried 80.0 g (0.26 moles) of powdered 5α-pregnane-3,20- dione in 400 mL dry tetrahydrofuran. The ylid was then

slowly added to the steroid slurry, which was stirred for 1.5 hours at ambient temperature. The reaction was fol¬ lowed by TLC. The solids, potassium iodide and unreacted trimethylsulfoxonium iodide, were then removed by filtra- tion (fast paper) or decantation. The solids were washed with ethyl acetate and the organic layers combined, or washed with dichloromethane and the washes added to the dichloromethane extract below. The reaction solvent was removed on an evaporator to afford a solid material. The solids were then washed with phosphate buffer (pH 7.5) (1.2 g (8.7 mmol) KH ₂ P0 ₄ and 3.55 g (30.4 mmol) Na ₂ HP0 ₄ (anh) in 1 L water) , then taken up in 1.5 L dichloromethane, and 500 mL water. Both layers were separated and saved. The organic layer was washed successively with phosphate buf- fer (2X, pH 7.5) , and 300 mL saturated brine. If an emul¬ sion formed, more dichloromethane (or methanol) was added to help break the emulsion. The combined aqueous washes were back-extracted with dichloromethane (2X 200 mL) . The combined organic fractions were dried over anhydrous sodium sulfate and the solvent removed on an evaporator to afford an off-white solid that could be recrystallized from methanol or ethanol. ^' ,

Example 3 3α-hvdroxy-3β-methyl-5o:-preqnan-20-one To a solution (light yellow) of the (3R)-5α-pregnan- 3-spiro-2'-oxirane-20-one (101 mg, 0.305 mmol) and Nal (115 mg, 0.767 .mmol) in anhydrous 1,2-dimethoxyethane (DME) (5mL) at room temperature was added τ7-Bu ₃ SnH (0.22 mL, 0.238 g, 0.818 mmol). The reaction solution became colorless. Azobisisobutylnitrile (AIBN) (10 mg, 0.061 mmol) was then added. The resulting solution was refluxed under a nitrogen atmosphere for 21 h. at which point TLC

(3:1 hexane/acetone) indicated completion of the reaction.

The reaction was quenched with methanol; the mixture was stirred at room temperature for a while. The solvent was removed in vacuo to give an oil which did not dissolve in

ether. Addition of CH ₂ C1 ₂ gave a solution which was washed with water, IN- HCl, and saturated NaHC0 ₃ . The organic layer was dried (MgS0 ₄ ) , filtered, and concentrated under reduced pressure to a white solid. Purification by gra- dient flash chromatography (hexane, 7:1 hexane/acetone, 5:1 hexane/acetone) furnished the titled compound (93mg, 92%) .

Example 4

Preparation of 3a-hydroxy-3?-methyl-5o--preqnan-20-one A suitable sized flask was charged with 100 g (0.316 mole) of 5α-pregnan-3-spiro-2'-oxirane-20-one, 70 g (0.47 mol) sodium iodide, 40 ml (0.7 mol) glacial acetic acid, and 1.5 L of 50:50 tetrahydrofuran/methanol. The solution was stirred at 65°C for 1-2 hr. The reaction was followed by TLC (25% EtOAc/Hexanes, n-phase silica; develop iodine; approx. Rf epoxide 0.75, iodide 0.65). After complete consumption of starting epoxide, 70 g (0.85 mol) of anhy¬ drous sodium acetate was added. After flushing the reac¬ tion flask with nitrogen 20 g (20% by wt) of 5% Pd/Carbon was carefully added. The reaction flask-was flushed with nitrogen (2x) and hydrogen (2x) , then charged with-hydro¬ gen (45 psia) . The reaction was shaken for 16 hours at ambient temperature, then checked for completeness by TLC (25% EtOAc/Hexanes, n-phase silica; develop iodine; approx. Rf iodide 0.65, hydroxy 0.25). After purging the reaction flask well with an inert gas, the catalyst was removed by filtration through a bed of celite. When fil¬ tering out the catalyst, care was taken to keep the cata¬ lyst wet. Also, due to the large loading of catalyst, an alternative method of filtration, with the elimination of celite was sometimes used to allow for recycling the cata¬ lyst. The filter cake was rinsed several times with tetrahydrofuran or methylene chloride. The reaction mix¬ ture was then concentrated to near dryness, taken up in methylene chloride, rinsed with distilled water (2x) , and dried over anhydrous sodium sulfate. After removal of the

drying agent, the solvent was removed on a rotoevaporater. The pinkish product was purified by recrystallization from ethanol. The material was then bleached with decolorizing carbon to remove colored impurities.