PROCESS FOR PREPARING HIGH PURITY ALLOPREGNANOLONE AND INTERMEDIATES THEREOF

Abstract

The invention relates to an efficient and industrially applicable process for the preparation and purification of allopregnanolone and intermediates thereof without the assistance of column chromatography.

Claims

1.-13. (canceled)

14. A process for preparing and purifying a 3-carboxylic ester of allopregnanolone which comprises: reacting isoallopregnanolone with a strong carboxylic acid having a pka≤3 under Mitsunobu conditions; precipitating the 3-carboxylic ester of allopregnanolone in a solvent system comprising water and an organic solvent; and recrystallizing the precipitate of the 3-carboxylic ester of allopregnanolone in a non-polar solvent.

15. The process for preparing allopregnanolone according to claim 14, which further comprises: subjecting the 3-carboxylic ester of allopregnanolone thus obtained to hydrolysis under neutral conditions, mild basic conditions or energetic basic conditions.

16. The process according to claim 14, wherein the carboxylic acid is selected from the group consisting of mono-, di-, and trifluoroacetic acid, mono-, di-, and trichloroacetic acid, cyanoacetic acid, and orto-nitrobenzoic- and dinitrobenzoic acid.

17. The process according to claim 14, wherein the 3-carboxylic ester of allopregnanolone is precipitated in a solvent system comprising water and a water-soluble organic solvent.

18. The process according to claim 14, wherein the 3-carboxylic ester of allopregnanolone is precipitated in a solvent system comprising water and a water-soluble organic solvent selected from 1,4-dioxane, acetone, acetonitrile, DMF, methanol, ethanol, isopropanol or their mixtures, and/or the precipitate of the 3-carboxylic ester of allopregnanolone is recrystallized in a non-polar solvent selected from group consisting of hexane, cyclohexane, heptane, toluene, iPr.sub.2O, MeOtBu or their mixtures.

19. The process according to claim 14, wherein the 3-carboxylic ester of allopregnanolone is subjected to hydrolysis with an alcohol without adding any acid or base.

20. The process according to claim 14, wherein the 3-carboxylic ester of allopregnanolone is subjected to hydrolysis with a base whose conjugate acid has a pKa≤11.

21. The process according to claim 20, wherein the base whose conjugate acid has a pKa≤11 is an alkali or alkaline earth carbonate or bicarbonate.

22. The process according to claim 14, wherein the 3-carboxylic ester of allopregnanolone is subjected to hydrolysis with a base whose conjugate acid has a pKa≥12 for a time and at a temperature suitable to keep the level of 3α-hydroxy-5α,17α-pregnan-20-one in an amount of 0.5% or less.

23. The process according to claim 22, wherein the base whose conjugate acid has a pKa≥12 is an alkali or alkaline earth C.sub.1-6 alkoxide or hydroxide.

24. The process according to claim 22, wherein the hydrolysis is carried out for not more than about 2 h and/or at a temperature of about 15-40° C.

25. The process according to claim 14 which comprises the following steps: reacting isoallopregnanolone with a strong carboxylic acid under Mitsunobu conditions; precipitating the 3-carboxylic ester of allopregnanolone in a solvent system comprising water and an organic solvent; recrystallizing the precipitate of the 3-carboxylic ester of allopregnanolone in a non-polar solvent; subjecting the 3-carboxylic ester of allopregnanolone thus obtained to hydrolysis under neutral conditions, mild basic conditions or energetic basic conditions to afford allopregnanolone; and precipitating and recrystallizing thereby obtaining allopregnanolone with a total content of impurities I and II, in total, of 0.15% or below.

26. A 3-carboxylic ester of allopregnanolone containing an amount of 5α-pregn-2-en-20-one of 0.5% or less.

27. Allopregnanolone containing an amount of 5α-pregn-2-en-20-one and 3α-hydroxy-5α,17α-pregnan-20-one, in total, of 0.15% or less.

28. A 3-carboxylic ester of allopregnanolone selected from: Pregnan-20-one, 3-(2,6-dinitrobenzoyloxy)-, (3α,5α)-, Pregnan-20-one, 3-(2,4-dinitrobenzoyloxy)-, (3α,5α)-, Pregnan-20-one, 3-(chloroacetyloxy)-, (3α,5α)-, Pregnan-20-one, 3-(dichloroacetyloxy)-, (3α,5α)-, Pregnan-20-one, 3-(trichloroacetyloxy)-, (3α,5α)-, Pregnan-20-one, 3-(fluoroacetyloxy)-, (3α,5α)-, Pregnan-20-one, 3-(difluoroacetyloxy)-, (3α,5α)-, Pregnan-20-one, 3-(2-nitrobenzoyloxy)-, (3α,5α)-, or Pregnan-20-one, 3-(cyanoacetyloxy)-, (3α,5α)-.

29. The process according to claim 15, wherein the 3-carboxylic ester of allopregnanolone is subjected to hydrolysis with an alcohol without adding any acid or base.

30. The process according to claim 15, wherein the 3-carboxylic ester of allopregnanolone is subjected to hydrolysis with a base whose conjugate acid has a pKa≤11.

31. The process according to claim 15, wherein the 3-carboxylic ester of allopregnanolone is subjected to hydrolysis with a base whose conjugate acid has a pKa≥12 for a time and at a temperature suitable to keep the level of 3α-hydroxy-5α,17α-pregnan-20-one in an amount of 0.5% or less.

32. The process according to claim 31, wherein the hydrolysis is carried out for not more than about 2 h and/or at a temperature of about 15-40° C.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0039] The present inventors have developed a chromatography-free process for the preparation of highly pure allopregnanolone. The process of the present invention is simple, inexpensive, reproducible and is well suited for industrial scale.

Definitions

[0040] As used herein, the term “about” means a slight variation of the value specified, preferably within 10 percent of the value specified. Nevertheless, the term “about” can mean a higher tolerance of variation depending on for instance the experimental technique used. Said variations of a specified value are understood by the skilled person and are within the context of the present invention. Further, to provide a more concise description, some of the quantitative expressions given herein are not qualified with the term “about”. It is understood that, whether the term “about” is used explicitly or not, every quantity given herein is meant to refer to the actual given value, and it is also meant to refer to the approximation to such given value that would reasonably be inferred based on the ordinary skill in the art, including equivalents and approximations due to the experimental and/or measurement conditions for such given value.

[0041] By “room temperature” or its abbreviation “rt” is meant herein that the reactions or processes are performed without heating or cooling. Generally, by room temperature may be understood as a temperature between about 15° C. and about 30° C., or more particularly between about 20° C. and about 25° C.

[0042] The term “organic solvent” includes for example cyclic and acyclic ethers (e.g. Et.sub.2O, iPr.sub.2O, tBu.sub.2O, MeOtBu, 1,4-dioxane, tetrahydrofuran, methyltetrahydrofuran), hydrocarbon solvents (e.g. pentane, hexane, cyclohexane, heptane), halogenated solvents (e.g. dichloromethane, chloroform), aromatic solvents (e.g. toluene, xylene), ketones (e.g. acetone, butanone, pentanone, methyl ethyl ketone, ethyl isopropyl ketone), esters (e.g. EtOAc, iPrOAc), nitriles (e.g. acetonitrile, benzonitrile, propionitrile), amides (e.g. DMF, DMA, HMPA), alcohols (e.g. methanol, ethanol, propanol, isopropanol, sec-butanol, t-butanol), sulfoxides (DMSO) and mixtures thereof.

[0043] The term “water soluble solvent” refers to solvents capable mixing with water fully i.e. in all proportions, or partly i.e. in some proportions. Water soluble solvents include for instance organic solvents of which 5 g or more is soluble in 100 g of water at a temperature of 25° C. In a particular embodiment, the water soluble solvent is selected from an organic solvent having a miscibility in water greater than 50% by weight at 25° C.

[0044] As used herein, the term “non-polar solvent” refers to a solvent of sufficiently low polarity to induce crystal formation of a polar compound such as a 3-ester of allopregnanolone. Having regard to the present disclosure, the selection of suitable non-polar solvents is well within the knowledge of the skilled artisan. More particularly, the term “non-polar solvent” refers to a solvent having Log P>2. Non polar solvents have low dielectric constants, such as <5. In an embodiment, the non-polar solvent is selected from the group consisting of hydrocarbon solvents (e.g. pentane, hexane, cyclohexane, heptane), aromatic solvents (e.g. toluene, xylene) and cyclic and acyclic ethers (e.g. Et.sub.2O, iPr.sub.2O, tBu.sub.2O, MeOtBu, 1,4-dioxane, tetrahydrofuran, methyltetrahydrofuran).

[0045] The term “Mitsunobu conditions” refers to conditions suitable for performing the conversion of isoallopregnanolone into a 3-carboxylic ester of allopregnanolone by reaction with particular carboxylic acids. The reagents used for Mitsunobu conditions are preferably a phosphine (typically triphenyl phosphine (PPh.sub.3)), an azodicarboxylate, and an optional tertiary amine additive. Examples of azodicarboxylate include, but are not limited to, diethyl azodicarboxylate (DEAD), diisopropyl azodicarboxylate (DIAD), di-t-butyl azodicarboxylate, 1,1′-(azocarbonyl)dipiperidine, dibenzyl azodicarboxylate, and/or the like. In some embodiments, the reagents for Mitsunobu conditions may be selected such that the reagents may be recycled or recovered after the reaction is complete. In some embodiments, one or more of dicyclohexylphenylphosphine, diethylphenylphosphine, tributylphosphine, diphenyl-2-pyridylphosphine, 4-(dimethylamino)phenyldiphenylphosphine, isopropyldiphenylphosphine, tri-tert-butylphosphine, tri-n-octylphosphine, tricyclohexylphosphine, polystyryldiphenylphosphine and/or the like may be used instead of triphenyl phosphine. In some embodiments, the triphenyl phosphine, or an equivalent thereof and/or the azodicarboxylate may be anchored to a resin such as, for example, as polystyrene resin. In some embodiments, cyanomethylenetri-n-butylphosphorane may be used as a reagent for Mitsunobu conditions.

[0046] The term “neutral conditions” preferably refers to use in the hydrolysis reaction of an alcohol such as MeOH, EtOH without adding any acid or base (transesterification).

[0047] The term “mild basic conditions” preferably refers to the use in the hydrolysis reaction of a base whose conjugate acid has a pKa equal or below 11. Suitable bases include, but are not limited to, alkali and alkaline earth carbonates and bicarbonates such as potassium carbonate, sodium carbonate, barium carbonate, cesium carbonate, potassium hydrogenocarbonate, and sodium hydrogenocarbonate.

[0048] The term “energetic basic conditions” preferably refers to the use in the hydrolysis reaction of a base whose conjugate acid has a pKa equal or above 12 for short times and/or at low temperatures so as to avoid that 3α-hydroxy-5α,17α-pregnan-20-one is generated in an amount higher than 0.5%. Having regard to the present disclosure, the selection of suitable times and temperatures is well within the knowledge of the skilled artisan. Suitable bases include, but are not limited to, alkali and alkaline earth C.sub.1-6 alkoxides and hydroxides such as potassium methoxide, sodium methoxide, potassium ethoxide, sodium ethoxide, lithium hydroxide, potassium hydroxide, sodium hydroxide, and calcium hydroxide.

[0049] Unless otherwise stated, references to purity may be understood as HPLC purity.

Preparation and Purification of 3-Esters of Allopregnanolone from Isoallopregnanolone

[0050] In reproducing the conditions disclosed in the prior art, the present inventors noted that the adequate selection of the carboxylic acid to be reacted with isoallopregnanolone is important to avoid/reduce the formation of impurities, especially during the hydrolysis subsequent to the Mitsunobu reaction. Keeping the level of impurities within acceptable limits makes possible to purify both the intermediate 3-carboxylic ester and allopregnanolone without relying on column chromatography. It has been also found that isolation and purification of the intermediate ester is required in order to get a process suitable for large scale production of highly pure allopregnanolone. If the intermediate ester is not purified from certain impurities, but used in crude form for the subsequent hydrolysis reaction, then an effective purification of the final allopregnanolone turns out to be infeasible.

[0051] Specifically, the inventors have found that the Mitsunobu reaction always brings about a substantial amount, normally about 7-17%, of 5α-pregn-2-en-20-one (herein also referred to as impurity I or elimination impurity):

##STR00007##

[0052] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 5.57 (2H), 2.50 (1H), 2.10-2.18 (1H), 2.09 (3H), 1.81-2.0 (3H), 1.51-1.71 (5H), 1.07-1.43 (9H), 0.83-0.94 (1H), 0.75-0.76 (1H), 0.73 (3H), 0.59 (3H).

[0053] .sup.13C NMR (400 MHz, CDCl.sub.3): δ 209.7, 125.9, 125.8, 63.9, 56.8, 54.0, 44.2, 41.5, 39.9, 39.2, 35.7, 34.7, 31.8, 31.6, 30.3, 28.7, 24.5, 22.9, 21.0, 13.5, 11.8.

[0054] The elimination impurity is thus intrinsic to the Mitsunobu reaction. Attempts to prepare allopregnanolone without isolating and purifying the intermediate 3-ester failed or afforded unsatisfactory results due to the need of subjecting the final product to a complex column chromatography that does not lead to an acceptable degree of purity.

[0055] The present invention provides a method for the preparation and purification of 3-esters of allopregnanolone from isoallopregnanolone, said method comprising in a preferred embodiment: [0056] treating isoallopregnanolone with an organic acid having a pka of 3 or less under Mitsunobu conditions; [0057] precipitating the crude reaction mixture in a solvent system comprising a mixture of water and an organic solvent; and [0058] recrystallizing the precipitate in a non-polar solvent.

[0059] The election of an organic acid having a low pka is crucial, especially because it makes possible to subsequently conduct a clean hydrolysis of the 3-ester of allopregnanolone. For instance, it has been found that formic acid (pka=3.75), benzoic acid (pKa=4.20), acetic acid (pKa 4.75), orthometoxybenzoic acid (pKa=4.09), 3-nitrobenzoic acid (pKa 3.46), 4-nitrobenzoic acid (pKa 3.43) are unsuitable. In an embodiment, the carboxylic acid having a pka 3 is selected from the group consisting of mono-, di-, and trifluoroacetic acid, mono-, di-, and trichloroacetic acid, cyanoacetic acid, 2-nitrobenzoic acid and dinitrobenzoic acid (e.g. 2,4-, 2,6- and 3,5-dinitrobenzoic acid). In a more particular embodiment, the carboxylic acid is chloroacetic acid or dinitrobenzoic acid. Typically, the amount of carboxylic acid is about 1-4 eq, more particularly about 2-3 eq.

[0060] Suitable Mitsunobu conditions preferably comprise a phosphine such as PPh.sub.3, an azodicarboxylate, and an optional tertiary amine additive. The phosphine may be selected for instance from the group consisting of triphenylphosphine, dicyclohexylphenylphosphine, diethylphenylphosphine, tributylphosphine, diphenyl-2-pyridylphosphine, 4-(dimethylamino)phenyldiphenylphosphine, isopropyldiphenylphosphine, tri-tert-butylphosphine, tri-n-octylphosphine, tricyclohexylphosphine, polystyryldiphenylphosphine or a mixture thereof. The azodicarboxylate may be selected for instance from the group consisting of diethyl azodicarboxylate (DEAD), diisopropyl azodicarboxylate (DIAD), di-t-butyl azodicarboxylate, 1,1′-(azocarbonyl)dipiperidine, dibenzyl azodicarboxylate or a mixture thereof. According to a preferred embodiment, the reagents used for the Mitsunobu reaction are or comprise triphenyl phosphine (PPh.sub.3) and azodicarboxylate selected from DEAD and DIAD. Suitable amounts of phosphine and azodicarboxylate normally range about 1-2 eq. In a particular embodiment, about 1.5 eq phosphine and about 1.4 eq azodicarboxylate are added. Preferably, the azodicarboxylate, normally a solution of azodicarboxylate, is added slowly or dropwise to the reaction mixture comprising isoallopregnanolone, phosphine and organic acid. In a particular embodiment, the Mitsunobu reaction is carried out in the presence of NaOBz (e.g. about 1-2 eq).

[0061] The Mitsunobu reaction is normally carried out in the presence of an organic solvent which may be selected for instance from the group consisting of cyclic and acyclic ethers (e.g. Et.sub.2O, iPr.sub.2O, tBu.sub.2O, MeOtBu, 1,4-dioxane, tetrahydrofuran, methyltetrahydrofuran), halogenated solvents (e.g. dichloromethane, chloroform), and aromatic solvents (e.g. toluene, xylene) or a mixture thereof. In a preferred embodiment, the solvent is selected from tetrahydrofuran (THF), 1,4-dioxane, toluene and dichloromethane or a mixture thereof; even more preferably, the solvent is tetrahydrofuran (THF), 1,4-dioxane, or toluene. In particular embodiments, the solvent is 1,4-dioxane or a mixture of 1,4-dioxane and tetrahydrofuran (THF).

[0062] In an embodiment, isoallopregnanolone is treated with the organic acid at a suitable temperature of about 0-45° C. (e.g. about 15-45° C.) for time sufficient for ester formation, normally between about 1-24 h and more particularly between about 2-12 h or about 4-8 h. In an embodiment, the reaction mixture may be stirred for sufficient time and at suitable temperature for the completion of ester formation. The reaction may be followed by thin layer chromatography (TLC or HPLC or UPLC).

[0063] After completion, water is preferably added to the crude reaction mixture and the resulting suspension may be filtered to obtain a wet cake containing a 3-carboxylic ester of allopregnanolone.

[0064] With solvents not miscible with water, it is necessary to remove the solvent, preferably by evaporation, and replace it with a solvent miscible with water (water-soluble solvent), such as 1,4-dioxane, MeOH, EtOH, IPA, ACN, etc. before adding the water.

[0065] Isolation of the 3-ester of allopregnanolone is carried out according to the present invention by precipitation in a solvent system comprising water and an organic solvent. Such a precipitation allows reducing/eliminating polar and basic impurities such as byproducts derived from the phosphine and the azodicarboxylate.

[0066] Preferably, the organic solvent used for precipitating the crude 3-ester is a water-soluble solvent such as cyclic and acyclic ethers (e.g. 1,4-dioxane), ketones (e.g. acetone, methyl ethyl ketone), nitriles (e.g. acetonitrile, propionitrile), amides (e.g. DMF, DMA, HMPA), alcohols (e.g. methanol, ethanol, propanol, isopropanol), and mixtures thereof. More particularly, the water-soluble solvent is selected from 1,4-dioxane, acetone, acetonitrile, DMF, methanol, ethanol, isopropanol or their mixtures, even more particularly the water-soluble solvent is 1,4-dioxane, acetonitrile or isopropanol, and even still more particularly, it is 1,4-dioxane.

[0067] Different proportions of water and organic solvent may be used. According to one embodiment, the ratio water to organic solvent within the solvent system ranges from about 0.1:1 to 1:0.1, more particularly about 0.2:1 to 1:0.2, 0.3:1 to 1:0.3, 0.4:1 to 1:0.4, 0.5:1 to 1:0.5, 0.6:1 to 1:0.6, 0.7:1 to 1:0.7, 0.8:1 to 1:0.8, 0.9:1 to 1:0.9, and about 1:1.

[0068] In more specific embodiment, the crude 3-ester of allopregnanolone may be conveniently precipitated with a solvent system comprising water and an organic solvent at a ratio about 1:1, wherein the organic solvent is 1,4-dioxane, acetonitrile or isopropanol.

[0069] Once precipitated, the 3-ester of allopregnanolone is preferably dried (e.g. under reduced pressure).

[0070] Purification of the 3-ester of allopregnanolone is effected using recrystallization in a non-polar solvent. Advantageously, the recrystallization allows eliminating or reducing the amount of 5α-pregn-2-en-20-one (i.e. impurity I or elimination impurity) to about 0.5% or below (e.g. about 0.4% or below, 0.3% or below, 0.2% or below, 0.1% or below). Examples of non-polar solvents useful for inducing crystal formation include, but are not limited to, hydrocarbon solvents (e.g. pentane, hexane, cyclohexane, heptane), aromatic solvents (e.g. toluene, xylene) and cyclic and acyclic ethers (e.g. Et.sub.2O, iPr.sub.2O, tBu.sub.2O, MeOtBu, methyltetrahydrofuran). In a particular embodiment, the non-polar solvent is selected from the group consisting of hexane, cyclohexane, heptane, toluene, iPr.sub.2O, MeOtBu or their mixtures; more particularly, cyclohexane, heptane; and even more particularly, heptane.

[0071] Therefore, the invention provides 3-carboxylic esters of allopregnanolone of high purity level, comprising an amount of 5α-pregn-2-en-20-one not higher than 0.5% (e.g. about 0.5% or below, 0.4% or below, 0.3% or below, 0.2% or below, 0.1% or below).

Hydrolysis of 3-Esters of Allopregnanolone to Allopregnanolone

[0072] The inventors have found that the hydrolysis of certain 3-esters of allopregnanolone (e.g. the esters of acetic, formic, isobutyric, benzoic, orto methoxy benzoic, or 3- or 4-nitrobenzoic acid) generates large amounts of 3α-hydroxy-5α,17α-pregnan-20-one (herein also referred to as impurity II or epimeric impurity):

##STR00008##

[0073] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 4.00 (1H), 2.77 (1H), 2.10 (3H), 1.84-1.94 (1H), 1.53-1.79 (6H), 1.33-1.5 (6H), 1.07-1.33 (8H), 0.94-1.04 (1H), 0.89 (3H), 0.75 (3H), 0.67-0.63 (1H).

[0074] .sup.13C NMR (400 MHz, CDCl.sub.3): δ 212.9, 66.5, 61.5, 53.6, 50.4, 45.9, 39.1, 36.2, 36.0, 35.8, 35.5, 32.9, 32.3, 32.2, 29.0, 28.6, 25.9, 24.4, 21.1, 20.8, 11.2.

[0075] In the interest of a purification suitable at commercial scale, it is convenient to keep the level of 3α-hydroxy-5α,17α-pregnan-20-one not higher than 0.5% during the hydrolysis reaction. This may be successfully achieved with 3-esters of allopregnanolone derived from carboxylic acids having a low pka (e.g. pka of 3 or less), which readily hydrolyze in the conditions disclosed in the present invention. Moreover, if obtained according to the process disclosed hereinabove, such 3-esters of allopregnanolone also contain low amounts of 5α-pregn-2-en-20-one 0.5%).

[0076] In the present invention, the hydrolysis is carried out under neutral conditions (e.g. in the presence of an alcohol without adding any acid or base), mild basic conditions (e.g. in the presence of a base whose conjugate acid has a pKa≤11) or energetic basic conditions (e.g. in the presence of a base whose conjugate acid has a pKa≥12 for unprolonged times and/or at not high temperatures).

[0077] According to a particular embodiment, the ester is hydrolyzed under neutral conditions employing an alcohol selected from methanol, ethanol, propanol, isopropanol, sec-butanol, and t-butanol. More particularly, the alcohol is MeOH or EtOH. In an embodiment, the ester is stirred in alcohol for sufficient time (e.g. about 1-24 h, or about 1-12 h) and at suitable temperature (e.g. about 15-40° C. or about 15-25° C.) for the completion of the hydrolysis. The reaction may be followed by thin layer chromatography (TLC).

[0078] According to a particular embodiment, the ester is hydrolyzed under mild basic conditions employing a base selected from alkali and alkaline earth carbonates and bicarbonates such as potassium carbonate, sodium carbonate, barium carbonate, cesium carbonate, potassium hydrogenocarbonate, and sodium hydrogenocarbonate. More particularly, the base is potassium carbonate, sodium carbonate, potassium hydrogenocarbonate, or sodium hydrogenocarbonate, and even more particularly, the base is potassium carbonate, sodium carbonate. This reaction may be carried out in the presence of an alcohol such as methanol, ethanol, propanol, isopropanol, sec-butanol, or t-butanol as solvent. In an embodiment, the alcohol is MeOH or EtOH. In an embodiment, the ester is treated with any of the mentioned bases, preferably in an amount of about 0.2-3 eq. or about 0.5-2 eq., at a suitable temperature of about 15-40° C. or about 15-30° C. for time sufficient for hydrolysis, normally between about 1-4 h. In an embodiment, the reaction mixture may be stirred for sufficient time (e.g. about 1-4 h) and at suitable temperature (e.g. about 15-40° C. or about 15-30° C.) for the completion of the hydrolysis.

[0079] According to a particular embodiment, the ester is hydrolyzed under energetic basic conditions employing a base selected from alkali and alkaline earth C.sub.1-6 alkoxides and hydroxides. More particularly, the base is potassium methoxide, sodium methoxide, potassium ethoxide, sodium ethoxide, lithium hydroxide, potassium hydroxide, sodium hydroxide, and calcium hydroxide, and even more particularly, the base is sodium methoxide, potassium hydroxide, or sodium hydroxide. This reaction may be carried out in the presence of an alcohol such as methanol, ethanol, propanol, isopropanol, sec-butanol, or t-butanol as solvent. In an embodiment, the alcohol is MeOH or EtOH The use of strong bases (pKa equal or above 12) requires keeping the hydrolysis reaction for short times and/or at low temperatures so as to prevent that 3α-hydroxy-5α,17α-pregnan-20-one is generated in an amount higher than 0.5%. In an embodiment, the hydrolysis under energetic basic conditions is carried out for not more than about 2 h, more particularly not more than about 1.5 h and even more particularly not more than about 1 h and/or at a temperature of about 15-40° C., more particularly about 15-30° C. In an embodiment, the ester is treated with any of the mentioned bases, preferably in an amount of about 5-15 eq. or about 10 eq, at a suitable temperature of about 15-40° C. or about 15-30° C. for time sufficient for hydrolysis (e.g. not more than about 2 h, more particularly not more than about 1.5 h and even more particularly not more than about 1 h). In an embodiment, the reaction mixture may be stirred for sufficient time (e.g. about 0.5-2 h) and at suitable temperature (e.g. not higher than 40° C. or 30° C.) for the completion of the hydrolysis.

[0080] Advantageously, allopregnanolone is obtained in high purity, comprising an amount of 3α-hydroxy-5α,17α-pregnan-20-one (herein also referred to as impurity II or epimeric impurity) not higher than 0.5% (e.g. about 0.5% or below, 0.4% or below, 0.3% or below, 0.2% or below, 0.1% or below).

[0081] Isolation of allopregnanolone may be carried out for instance by precipitation in a solvent system comprising water and an organic solvent. Preferably, the organic solvent used for precipitating the crude allopregnanolone is a water-soluble solvent such as cyclic and acyclic ethers (e.g. 1,4-dioxane), ketones (e.g. acetone, methyl ethyl ketone), nitriles (e.g. acetonitrile, propionitrile), amides (e.g. DMF, DMA, HMPA), alcohols (e.g. methanol, ethanol, propanol, isopropanol, sec-butanol, t-butanol), and mixtures thereof. More particularly, the water-soluble solvent is selected from 1,4-dioxane, acetone, acetonitrile, DMF, methanol, ethanol, isopropanol or their mixtures, even more particularly the water-soluble solvent is 1,4-dioxane, acetonitrile or methanol, and even still more particularly, it is methanol.

[0082] Different proportions of water and organic solvent may be used. According to one embodiment, the ratio water to organic solvent within the solvent system ranges from about 0.1:1 to 1:0.1, more particularly about 0.2:1 to 1:0.2, 0.3:1 to 1:0.3, 0.4:1 to 1:0.4, 0.5:1 to 1:0.5, 0.6:1 to 1:0.6, 0.7:1 to 1:0.7, 0.8:1 to 1:0.8, 0.9:1 to 1:0.9, and about 1:1.

[0083] In more specific embodiment, crude allopregnanolone may be conveniently precipitated with a solvent system comprising water and an organic solvent at a ratio about 0.5:1, wherein the organic solvent is 1,4-dioxane, acetonitrile or methanol.

[0084] Once precipitated, allopregnanolone is preferably dried (e.g. under reduced pressure).

[0085] If desired, further purification of allopregnanolone may be effected for instance using recrystallization. Examples of solvents useful for inducing crystal formation include, but are not limited to hydrocarbon solvents (e.g. pentane, hexane, cyclohexane, heptane), aromatic solvents (e.g. toluene, xylene), ketones (e.g. acetone, methyl ethyl ketone), esters (e.g. EtOAc, iPrOAc), nitriles (e.g. acetonitrile, propionitrile), alcohols (e.g. methanol, ethanol, propanol, isopropanol), and mixtures thereof. In a particular embodiment, the non-polar solvent is selected from MeOH, EtOH, IPA, acetone, ACN etc, and even more particularly, MeOH/water.

[0086] Advantageously, the recrystallization allows eliminating or reducing the level of impurities so as to obtain allopregnanolone of high purity level, comprising an amount of 5α-pregn-2-en-20-one and 3α-hydroxy-5α,17α-pregnan-20-one, in total, of 0.15% or less. In preferred variants of the invention, the amount of said both impurities in the final allopregnanolone is at most 0.10%.

[0087] The present invention allows obtaining allopregnanolone with a high degree of purity such as above 98%, above 99% and even above 99.5%, complying with the requirements normally imposed by Good Manufacturing Practices (GMP) for Pharmaceutical Products. In a particular embodiment, the purity of allopregnanolone is ≥99.5% and the total content of impurities I and II is equal or below 0.15%, preferably equal or below 0.10%.

[0088] In a preferred embodiment of the present invention, allopregnanolone is prepared by a process comprising the following steps: [0089] reacting isoallopregnanolone with a strong carboxylic acid under Mitsunobu conditions; [0090] precipitating the 3-carboxylic ester of allopregnanolone in a solvent system comprising water and an organic solvent; [0091] recrystallizing the precipitate of the 3-carboxylic ester of allopregnanolone in a non-polar solvent; [0092] subjecting the 3-carboxylic ester of allopregnanolone thus obtained to hydrolysis under neutral conditions, mild basic conditions or energetic basic conditions to afford allopregnanolone; and [0093] precipitating and recrystallizing thereby obtaining allopregnanolone with a total content of impurities I and II, in total, of 0.15% or below.

[0094] The present disclosure provides allopregnanolone with a total content of impurities I and II equal or below 0.15% for use in the preparation of pharmaceutical compositions. The present disclosure also encompasses the use of allopregnanolone with a total content of impurities I and II equal or below 0.15% for the preparation of pharmaceutical compositions. The present disclosure comprises processes for preparing the above mentioned pharmaceutical compositions. The processes comprise combining allopregnanolone with a total content of impurities I and II equal or below 0.15% with at least one pharmaceutically acceptable excipient. Allopregnanolone and the pharmaceutical compositions of allopregnanolone of the present disclosure can be used as medicaments, particularly for the treatment of postpartum depression (PPD). The present disclosure also provides methods of treating of postpartum depression (PPD) comprising administering a therapeutically effective amount of allopregnanolone of the present disclosure to a subject in need of the treatment.

3-Esters of Allopregnanolone

[0095] In another aspect, the present invention provides the following 3-esters of allopregnanolone: [0096] Pregnan-20-one, 3-(2,6-dinitrobenzoyloxy)-, (3α,5α)- [0097] Pregnan-20-one, 3-(2,4-dinitrobenzoyloxy)-, (3α,5α)- [0098] Pregnan-20-one, 3-(chloroacetyloxy)-, (3α,5α)- [0099] Pregnan-20-one, 3-(dichloroacetyloxy)-, (3α,5α)- [0100] Pregnan-20-one, 3-(trichloroacetyloxy)-, (3α,5α)- [0101] Pregnan-20-one, 3-(cyanoacetyloxy)-, (3α,5α)- [0102] Pregnan-20-one, 3-(fluoroacetyloxy)-, (3α,5α)- [0103] Pregnan-20-one, 3-(difluoroacetyloxy)-, (3α,5α)- [0104] Pregnan-20-one, 3-(2-nitrobenzoyloxy)-, (3α,5α)-

[0105] These esters may be prepared by reacting isoallopregnanolone with the corresponding carboxylic acid (i.e. 2,6-dinitrobenzoic, 2,4-dinitrobenzoic, chloroacetic, dichloroacetic, trichloroacetic, cyanoacetic, fluoroacetic, difluoroacetic, or 2-nitrobenzoic acid) under Mitsunobu conditions.

[0106] It should be understood that the scope of the present disclosure includes all the possible combinations of embodiments disclosed herein.

[0107] The following examples are merely illustrative of certain embodiments of the invention and cannot be considered as restricting it in any way.

EXAMPLES

1.—Conversion of Isoallopregnanolone into α-3-Chloroacetate Ester of Brexanolone

[0108] ##STR00009##

[0109] Mitsunobu: Isopregnanolone (36 g, 113.2 mmol), PPh.sub.3 (43.2 g, 1.5 eq) and chloroacetic acid (27 g, 2.5 eq) were suspended in 1,4-dioxane (400 mL). The mixture was cooled at 15° C. and a solution of DIAD (32.4 mL, 1.4 eq) in dioxane (140 mL) was added dropwise. At the end of addition the reaction mass was stirred at 35° C. until the reaction was finished (4 h). Elimination Impurity I (5α-pregn-2-en-20-one): about 9-12%.

[0110] Work-up and isolation: After cooling down at 25° C., water (540 mL) was added and the resulting suspension was stirred 30 min and then cooled to 20-25° C. The suspension was filtered and the wet cake washed with 100 mL of a mixture dioxane/water 1:1. The product was dried at 50° C. under reduced pressure giving place to 32.45 g of a white solid (yield: 70%; purity: 96%). Elimination Impurity I (5α-pregn-2-en-20-one): 3.5%.

[0111] Recrystallization: The obtained dry cake was suspended in heptane (15 ml/g) and heated at 75° C. till complete dissolution. The solution was then cooled at 0/10° C. and the precipitated was filtered off. The solid was washed with fresh heptane (2 ml/g), and dried at 50° C. under reduced pressure. Weight: 29.2 g; Yield: 90%; Purity: 99.15%. Elimination Impurity I (5α-pregn-2-en-20-one): 0.3%.

[0112] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 5.07 (1H), 4.02 (2H), 2.40 (1H), 2.09-2.15 (1H), 2.07 (3H), 1.95-1.99 (1H), 1.72-1.95 (1H), 1.54-1.68 (4H), 1.44-1.51 (4H), 1.28-1.42 (3H), 1.08-1.26 (6H), 0.85-0.96 (1H), 0.72-0.81 (4H), 0.56 (3H).

[0113] .sup.13C NMR (400 MHz, CDCl.sub.3): δ 209.5, 166.7, 72.6, 63.8, 56.7, 54.0, 44.2, 41.4, 39.9, 39.0, 35.8, 35.4, 32.8, 32.7, 31.8, 31.5, 28.2, 26.0, 24.4, 22.8, 20.8, 13.5, 11.4.

[0114] Isolation of 5α-pregn-2-en-20-one from the dioxane/water mother liqueurs obtained in the ester precipitation: The mother liqueurs were diluted with more water (around 3 Liters), a suspension was formed and the solid was isolated by filtration. The filtrate was suspended in 400 ml of Heptane, stirred during one hour at 40° C. and filtered again, the liquid was evaporated to get also a solid that was submitted to chromatography and characterized by NMR:

[0115] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 5.57 (2H), 2.50 (1H), 2.10-2.18 (1H), 2.09 (3H), 1.81-2.0 (3H), 1.51-1.71 (5H), 1.07-1.43 (9H), 0.83-0.94 (1H), 0.75-0.76 (1H), 0.73 (3H), 0.59 (3H).

[0116] .sup.13C NMR (400 MHz, CDCl.sub.3): δ 209.7, 125.9, 125.8, 63.9, 56.8, 54.0, 44.2, 41.5, 39.9, 39.2, 35.7, 34.7, 31.8, 31.6, 30.3, 28.7, 24.5, 22.9, 21.0, 13.5, 11.8.

[0117] NMR signal match with the data found in literature (Tetrahedron (60) 2004, 11851-11860).

2.—Hydrolysis of α-3-Chloroacetate Ester to Brexanolone

[0118] ##STR00010##

[0119] Hydrolysis: Chloroacetate-Brexanolone (29.2 g, 74.11 mmol) was suspended in methanol (300 mL) and potassium carbonate (5 g, 0.5 eq) was added. The reaction mixture was stirred for 1 h at 30° C.

[0120] Work-up and isolation: 150 ml of water were added and cooled down at 0/5° C. and the solid filtered. The wet cake was then washed with 60 mL of a mixture methanol/water 1:0.5. The product was dried at 50° C. under reduced pressure, affording 22 g of crude Brexanolone (yield: 93%; purity: >99%). Epimeric Impurity II (3α-hydroxy-5α,17α-pregnan-20-one): 0.23%.

[0121] Recrystallization: The obtained dry cake was suspended in MeOH (15 vol) and stirred during 30 min at 30° C., then water (7.5 vol) was added and the resulting suspension was cooled to 5-10° C. and filtered, the wet cake was dried, giving place to 20 g of pure brexanolone as a white solid (purity: >99.8%; DSC 176° C.), Epimeric Impurity II (3α-hydroxy-5α,17α-pregnan-20-one): 0.08%. Elimination Impurity I (5α-pregn-2-en-20-one) not detected.

3.—Hydrolysis of α-3-Chloroacetate Ester Containing 0.73% of the 5-α-Pregna-3-En-20-One (Comparative)

[0122] Hydrolysis: chloroacetate-brexanolone (5 g, 12.7 mmol with a 0.73% of elimination impurity by HPLC) was suspended in methanol (50 mL) and potassium carbonate (0.85 g, 0.5 eq) was added. The reaction mixture was stirred for 1 h at 20-25° C. (starting material content: 0.35%).

[0123] Work-up: 150 ml of water were added and cooled down at 0/5° C. and the solid filtered. The wet cake was then washed with 7.5 mL of a mixture methanol/water 1:0.5. The product was dried at 50° C. under reduced pressure to obtain 3.77 g of crude brexanolone (yield: 93%; purity: >98.93%). Elimination impurity I (5α-pregn-2-en-20-one): 0.52%.

[0124] Recrystallization: The obtained dry cake was suspended in MeOH (25 vol) and dissolved at 35-40° C. The reaction mixture was cooled to 20-25° C., and then water (12.5 vol) was slowly added giving place to a suspension which was filtered. The wet cake was dried, resulting in 3.3 g of brexanolone as a white solid. Purity>99.7%, Elimination impurity I (5-α-pregna-3-en-20-one): 0.21%.

[0125] Thus starting from an ester containing more than 0.5% of elimination impurity I it was not possible to lower the amount of the same below 0.15%.

4.—Hydrolisis of α-3-Chloroacetate Ester Containing 0.5% of Impurity 5α-Pregn-2-En-20-One

[0126] The process disclosed in example 3 was followed but using α-3-chloroacetate ester containing 0.5% of elimination impurity I as starting material. It was obtained 3.2 g brexanolone with a purity of >98% and a content of elimination impurity I of about 0.13%.

[0127] Thus starting from an ester containing 0.5% of elimination impurity I it was possible to lower the amount of the same below 0.15%.

5.—Hydrolysis of α-3-Chloroacetate Ester Containing 0.5% of 3α-Hydroxy-5α,17α-Pregnan-20-One

[0128] Hydrolysis: Chloroacetate-Brexanolone (5 g, 12.7 mmol doped with 0.5% of the epimeric impurity) was suspended in methanol (50 mL) and potassium carbonate (0.85 g, 0.5 eq) was added. The reaction mixture was stirred for 1 h at 20-25° C. (starting material content: 0.30%),

[0129] Work-up: 150 ml of water were added and cooled down at 0/5° C. and the solid filtered. The wet cake was then washed with 7.5 mL of a mixture methanol/water 1:0.5. The product was dried at 50° C. under reduced pressure to obtain 3.82 g of crude brexanolone (yield: 94%; purity: >99.43%). Epimeric impurity II (3α-hydroxy-5α,17α-pregnan-20-one): 0.24%. Elimination impurity I (5α-pregn-2-en-20-one): 0.18%.

[0130] Recrystallization: The obtained dry cake was suspended in MeOH (25 vol) and dissolved at 35-40° C., the reaction mixture was cooled to 20-25° C., then water (12.5 vol) was slowly added giving place to a suspension which was filtered, the wet cake was dried, resulting in 3.3 g of Brexanolone as a white solid. Purity: 99.81%. Epimeric impurity II (3α-hydroxy-5α,17α-pregnan-20-one): 0.1%. Elimination impurity I (5α-pregn-2-en-20-one): 0.02%.

6.—Conversion of Isoallopregnanolone into α-3-Chloroacetate Ester of Brexanolone

[0131] ##STR00011##

[0132] Mitsunobu: Isopregnanolone (14 g, 44 mmol), PPh.sub.3 (16.8 g, 1.5 eq) and chloroacetic acid (10.5 g, 2.5 eq) were suspended in THF (140 mL). The mixture was cooled at 15° C. and a solution of DIAD (32.4 mL, 1.4 eq) in dioxane (70 mL) was added dropwise. At the end of addition the reaction mass was stirred at 35° C. until the reaction has finished (4 h). Elimination Impurity I (5α-pregn-2-en-20-one): about 8%.

[0133] After cooling down at 25° C., the reaction mass (250 ml), was divided into 7 samples (35 ml each, containing about 2 g of the ester final product). Every sample was subjected to evaporation, the residue was redissolved again in 30 ml of different kind of solvents miscible with water (MeOH, ACN, DMF, IPA, Acetone, Dioxane) and induced precipitation with the same volume of water.

7.—Conversion of Isoallopregnanolone into α-3-Trifluororoacetate Ester of Brexanolone

[0134] ##STR00012##

[0135] Mitsunobu: Isopregnanolone (1 g, 3.14 mmol), PPh.sub.3 (1.2 g, 1.5 eq) and trifluoroacetic acid (0.6 ml, d=1.489, 2.5 eq) were suspended in 1,4-dioxane (10 mL). The mixture was cooled at 15° C. and a solution of DIAD (0.9 mL, 1.4 eq) in dioxane (5 mL) was added dropwise. At the end of addition the reaction mass was stirred at 35° C. during one hour and then 1.2 eq of NaOBz were added as a solid and the reaction mixture was stirred again at 35° C. during 20 h (starting material still 6%).

[0136] Work-up and precipitation: After cooling down at 25° C., water (10 mL) was added and the resulting suspension was stirred 30 min then cooled to 10-15° C. The suspension was filtered and the wet cake washed with 2 mL of a mixture dioxane/water 1:1. The product was dried at 50° C. under reduced pressure giving place to 0.75 of the ester, elimination impurity amount 14.6% by HPLC.

8.—α-3-Benzoyl Ester of Brexanolone (Comparative)

[0137] ##STR00013##

[0138] Mitsunobu: Isopregnanolone (1 g, 3.14 mmol), PPh.sub.3 (1.2 g, 1.5 eq) and benzoic acid (0.6 g, 2.5 eq) were suspended in 1,4-dioxane (10 mL). The mixture was cooled at 15° C. and a solution of DIAD (0.9 mL, 1.4 eq) in dioxane (5 mL) was added dropwise. At the end of addition the reaction mass was stirred at 35° C. during 5 hours (elimination impurity amount about 8%).

[0139] Work-up and precipitation After cooling down at 25° C., water (10 mL) was added and the resulting suspension was stirred 30 min then cooled to 10-15° C. The suspension was filtered and the wet cake washed with dioxane/water 1:1. The product was dried at 50° C. under reduced pressure giving place to 0.1 g of the ester of >96% (elimination impurity 0.5%).

[0140] Hydrolisis.—The benzoyl ester was added to a solution of 70 vol of MeOH and 12 eq of NaOH, the reaction mixture was warmed at 40° C. for 30 h, a sample was taken and analyzed by HPLC, there was only 4% of the brexanolone final product and 9% of the Epimeric impurity together with the ester starting material.

[0141] As may also be appreciated in this example and also from example 12, the benzoyl ester of brexanolone cannot afford brexanolone with an acceptable level of purity.

9.—Brexanolone from Isopregnanolone in a “One-Pot” Process Using Chloroacetic Acid (Comparative)

[0142] ##STR00014##

[0143] Triphenyl phosphine (1.2 g/g), chloroacetic acid (0.75 g/g) and isopregnanolone were loaded in a round bottom flask, followed by toluene (10 ml/g). The suspension was cooled at 15° C. and a solution of DIAD (0.95 ml/g) in toluene (5 ml/g) was added dropwise, keeping the temperature below 25° C. The resulting yellow solution was heated at 35° C. during 4 h. After consumption of starting material (K1), a solution of NaHCO.sub.3 7% aq (5 ml/g) was added. The organic phase was later washed with water (5 ml/g) and evaporated to reduced pressure. The traces of toluene were removed by further distillation with methanol. The ester residue was suspended in methanol (10 ml/g) and potassium carbonate (0.17 g/g) was added. The suspension stirred at 30° C. during 1 h (K2). The product was then precipitated by addition of water (5 ml/g). The reaction mass was cooled at 5° C., filtered and washed with a mixture methanol/water 1:0.5 (2 ml/g) (TH1). The resulting wet cake was further washed with heptane (5 ml/g) (TH2).

TABLE-US-00001 5α-Pregn-2-en-20-one Sample (HPLC %) UV factor K1 16 0.5 K2 14 0.7 TH1 14 0.7 TH2 9 0.7

[0144] As may be appreciated, the one-pot process leads to an elevated amount of elimination impurity (9%) which cannot be separated from brexanolone.

10.—Brexanolone from Isopregnanolone in a “One-Pot” Process Using Trifluoroacetic Acid and Purifying by Chromatography (Comparative)

[0145] ##STR00015##

[0146] Triphenyl phosphine (1.2 g), trifluoro acetic acid (0.6 ml, 2.5 eq), NaOBz (0.9 g) and isopregnanolone (1 g) were loaded in a round bottom flask, followed by THF (10 ml). The suspension was cooled at 15° C. and a solution of DIAD (0.95 ml) in THF (5 ml) was added dropwise, keeping the temperature below 25° C. The resulting reaction mass was kept stirring at 25° C. during 24 h. After consumption of starting material the reaction mixture was evaporated to residue under reduced pressure.

[0147] MeOH (20 ml) was added and the mixture was refluxed for 24 h, the solvent was evaporated and the residue was purified by column chromatography column to obtain 0.69 g of Brexanolone, purity: 97%.

[0148] As may be appreciated, the one-pot process leads to brexanolone with a low level of purity.

11.—Brexanolone Acetate Using Toluene as Solvent and Sodium Benzoate (Comparative)

[0149] ##STR00016##

[0150] DIAD (0.46 g), acetic acid (0.15 ml), and isopregnanolone (0.5 g) and toluene (15 ml) were loaded in a round bottom flask. The reaction mass was cooled at 0° C. and PPh.sub.3 (0.6 g) followed by NaOBz (0.34 g) was added. The resulting reaction mass was kept stirring at 25° C. during 15 h. After consumption of starting material the reaction mixture was evaporated to residue under reduced pressure (elimination impurity content around 14%).

[0151] This example shows that elimination impurity is intrinsic to the Mitsunobu reaction.

12.—Benzoate, Nitrobenzoate, 2,6-Dinitrobenzoate Ester Hydrolysis

[0152] ##STR00017##

TABLE-US-00002 Ester/FP/ Ester Base/solvent Time/T.sup.a epimer Comments 1)Benzoate. NaOH (12  30 h/40° C. 87%/4%/9% No significant pKa benzoic eq)/MeOH hydrolysis even with acid: 4.2 (70 vol) strong base but a lot of epimer was already achieved. 2)Benzoate KOH (12 eq)/  30 h/40° C. 87%/4%/9% Same comments with pKa benzoic MeOH (70 KOH acid: 4.2 vol) 3) Benzoate MeONa (10   6 h/30° C. 17%/71%/14% Hydrolysis partially pKa benzoic eq)/MeOH performed but with acid: 4.2 (50 vol) significant amount of epimer. 4) Benzoate SO4H2 (10 0.5 h/RT  0%/92%/8% Hydrolysis completed pKa benzoic eq)/MeOH but with significant acid: 4.2 (25 vol) amount of epimer. 5) 4-nitro- NaOH(10 eq)/ 4.5 h/30° C. 67%/31%/2% Hydrolysis only benzoate MeOH (40 partially performed pKa 4-NO2- vol) benzoic acid: 3.4 6) 4-nitro- NaOH(10 eq)/  15 h/30° C.  0%/85%/14% Hydrolysis completed benzoate MeOH (40 but with significant pKa 4-NO2- vol) amount of epimer. benzoic acid: 3.4 7) 2,6- NaOH(10 eq)/   1 h/30° C. 0%/100%/0% Hydrolysis completed dinitrobenzoate MeOH (40 no epimer was pKa vol) detected. dinitrobenzoic acid: 1.14

[0153] As can be seen in the table, neither the benzoic ester nor the 4-nitrobenzoic ester hydrolyze easily, requiring very energetic conditions that cause the formation of the epimer (difficult to purify). The dinitrobenzoic ester, however, hydrolyzes in only one hour without giving the epimer.

13.—Hydrolysis of Alpha-3-Acetate Ester to Achieve Brexanolone. (Comparative)

[0154] ##STR00018##

[0155] In a round bottom flask, 3-alpha acetate-pregnanolone (1 g), was dissolved in 15 mL of methanol. 0.26 g of sodium hydroxide (2.4 eq) were added, and the reaction mass was stirred at 40° C. for 4 h. HPLC control showed disappearance of starting material. The solvent was concentrated under reduced pressure till a volume of 5 mL. The mixture was poured into water (87 mL), and stirred at room temperature for 1 hour.

[0156] The precipitate was then filtered and the wet cake was washed with water (10 mL) and dried under vacuum at 50° C.

[0157] An analysis by HPLC of the solid revealed the presence of 8.6% of the epimeric impurity (II).

14.—3-(2,4-Dinitrobenzoyl Ester) of Brexanolone

[0158] ##STR00019##

[0159] Isopregnanolone (0.5 g, 1.57 mmol), PPh.sub.3 (0.6 g, 1.5 eq) and 2,4-nitrobenzoic acid (0.5 g, 2.5 eq) were suspended in THF (10 mL). The mixture was cooled at 0/5° C. and a solution of DIAD (0.45 mL, 1.4 eq) in THF (5 mL) was added dropwise. At the end of addition, the reaction mass was stirred at room temperature during 5 hours. Water 1 mL was added, the solvent was evaporated and the residue was purified by column chromatography. 0.24 g of solid product were obtained (yield 32.7%).

15.—3-(3,5-Dinitrobenzoyl Ester) of Brexanolone

[0160] ##STR00020##

[0161] Isopregnanolone (0.5 g, 1.57 mmol), PPh.sub.3 (0.6 g, 1.5 eq) and 3,5-dinitrobenzoic acid (0.5 g, 2.5 eq) were suspended in THF (10 mL). The mixture was cooled at 0/5° C. and a solution of DIAD (0.45 mL, 1.4 eq) in THF (5 mL) was added dropwise. At the end of addition, the reaction mass was stirred at room temperature during 5 hours. Water 1 mL was added, the solvent was evaporated and the residue was purified by column chromatography. 0.43 g of solid product were obtained (yield 53.5%).

[0162] With the same procedure, 2-nitrobenzoyl ester of Brexanolone and 2,6-dinitrobenzoyl ester of Brexanolone were prepared.

16.—3-Dichloroacetic Ester of Brexanolone

[0163] ##STR00021##

[0164] Isopregnanolone (5.0 g, 15.7 mmol), PPh.sub.3 (6.0 g, 1.5 eq) and dichloroacetic acid (3.4 mL) were suspended in Dioxane (55 mL). The mixture was cooled at 15° C. and a solution of DIAD (4.5 mL, 1.4 eq) in Dioxane (20 mL) was added dropwise. At the end of addition, the reaction mass was stirred at 35° C. until completion. Water 75 mL were added, the mixture was stirred for 0.5 h, filtered off and washed with 10 mL (1:1) water/Dioxane and dried. 3.8 g of white solid product were obtained (yield 56.4%).

17.—3-Trichloroacetic Ester of Brexanolone

[0165] ##STR00022##

[0166] Isopregnanolone (5.0 g, 15.7 mmol), PPh.sub.3 (6.0 g, 1.5 eq) and trichloroacetic acid (6.45 g) were suspended in Dioxane (55 mL). The mixture was cooled at 15° C. and a solution of DIAD (4.5 mL, 1.4 eq) in Dioxane (20 mL) was added dropwise. At the end of addition, the reaction mass was stirred at 35° C. until completion. Water 75 mL were added, the two phases were separated and the aqueous layer was extracted with 50 mL DCM. The organic layer was concentrated to obtain a yellow oil.