Process for preparing high purity allopregnanolone and intermediates thereof

Abstract

To provide an efficient and industrially applicable method for preparing and purifying allopregnanolone and intermediates thereof without the aid of column chromatography.SOLUTION: A method of preparing and purifying a 3-carboxylic acid ester of allopregnanolone, comprising: reacting isoallopregnanolone with a strong carboxylic acid having a pKa of 3 or less under Mitsunobu conditions; precipitating the 3-carboxylic acid ester of allopregnanolone in a solvent system comprising water and an organic solvent; and recrystallizing the precipitate of the 3-carboxylic acid ester of allopregnanolone in a non-polar solvent. Also, a method of preparing allopregnanolone comprises obtaining a 3-carboxylic acid ester of allopregnanolone as described above; and hydrolyzing the obtained 3-carboxylic acid ester of allopregnanolone under neutral, weakly basic, or energetic basic conditions.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention is in the field of pharmaceutical synthesis, and more particularly relates to an efficient and industrially applicable method for preparing and purifying allopregnanolone, and intermediates useful therefor. [Background technology]

[0002] Allopregnanolone [516-54-1], also known as 3α-hydroxy-5α-pregnan-20-one, 5α-pregnan-3α-ol-20-one, or 3α,5α-tetrahydroprogesterone (3α,5α-THP) and brexanolone (USAN), is a pregnane neurosteroid with an endogenous inhibitory effect. Synthesized from progesterone in the brain, allopregnanolone is a potent positive allosteric modulator of γ-aminobutyric acid (GABA) action at GABA-A receptors. Allopregnanolone exhibits effects similar to other positive allosteric modulators of GABA action at GABA-A receptors, such as benzodiazepines, including anxiolytic, sedative, and anticonvulsant activities. The structure of allopregnanolone is shown below. [ka]

[0003] Brexanolone IV is a novel product developed by Sage Therapeutics to treat postpartum depression (PPD), a serious and potentially life-threatening condition for which no specifically indicated medications currently exist. Food and Drug Administration approval for brexanolone IV is expected in March 2019.

[0004] Several methods have been disclosed in the state of the art for the synthesis of this compound.

[0005] Purdy et al. (J. Med. Chem., 1990, Vol. 33, pp. 1572-1581) described the direct preparation of isoallopregnanolone, an epimer of allopregnanolone that differs structurally only in the orientation of the hydroxyl group at the 3-carbon of the steroid A ring. Inversion of the 3β-hydroxy group was achieved by a Mitsunobu reaction using trifluoroacetic acid (TFA) in the presence of triphenylphosphine (PPh3) and diethyl azodicarboxylate (DEAD) without isolation of a trifluoroacetate ester intermediate (Scheme 1). Workup and recrystallization of the crude product removed 5% of the unreacted starting material (1) to give allopregnanolone (2a) (74% yield, melting point (mp): 168-170 °C). This product was further purified by silica gel dry column chromatography and crystallized from aqueous ethanol (54% yield, mp: 174-176 °C). [ka]

[0006] WO 2009 / 108804 discloses a similar procedure for preparing allopregnanolone (14) from isoallopregnanolone (12) in Example 6 without isolating the trifluoroacetic acid ester intermediate (Scheme 2). After purification on a silica column, allopregnanolone was obtained (overall yield: 92%). [ka]

[0007] Methods involving isolation of Mitsunobu ester intermediates have also been proposed, for example, in WO 93 / 03732, WO 2012 / 127176, and CN 103396467 (Schemes 3, 4, and 5, respectively).

[0008] In Example 4 of WO 93 / 03732, isoallopregnanolone was reacted with acetic acid, triphenylphosphine (PPh3), and diisopropyl azodicarboxylate (DIAD), and the resulting residue was purified by silica gel flash chromatography to give 3α-acetoxy-5α-pregnan-20-one (yield: 97.5%). As disclosed in Example 5, this ester was hydrolyzed with perchloric acid, and the reaction mixture was stirred for 3 days to give crude allopregnanolone. [ka]

[0009] In Example 4, WO 2012 / 127176 discloses that isoallopregnanolone was reacted with benzoic acid, triphenylphosphine (PPh3), and diisopropyl azodicarboxylate (DIAD) to give a crude residue, which was purified by chromatography to give the corresponding benzoate ester (3) (yield: 90%). In Example 5, hydrolysis was carried out under harsh conditions (KOH, MeOH, 2.5 hours at reflux), followed by chromatographic purification to give allopregnanolone (4) (yield: 80%, mp: 161.7-162.8°C). The low melting point indicates that the product could not be sufficiently purified. [ka]

[0010] In Example 5 of Chinese Patent No. 103396467, isoallopregnanolone was converted to allopregnanolone using o-methoxybenzoic acid together with triphenylphosphine (PPh3) and diisopropyl azodicarboxylate (DIAD). In this procedure, the crude ester intermediate was precipitated and then hydrolyzed with sodium hydroxide in methanol at 60°C for 2 hours to give a white solid (overall yield for the two steps: 52%). [ka]

[0011] Like other synthetic compounds, allopregnanolone may contain extraneous compounds or impurities. These impurities may include unreacted starting materials, reaction by-products, side reaction products, and / or degradation products. In allopregnanolone, or any active pharmaceutical ingredient ("API"), impurities are undesirable and, in extreme cases, may even be harmful to patients receiving the API dosage form. Therefore, identifying API impurities generated during manufacturing and reducing / eliminating their presence in the final product is critical to commercialization.

[0012] After careful consideration of the methods disclosed in the above cited documents, the present inventors have found that these methods do not reproducibly yield allopregnanolone in a sufficiently pure state and / or require a chromatographic purification step, which is inappropriate, or at least undesirable, for industrial-scale production of large quantities of API, and which requires very large amounts of solvents, making the method expensive and environmentally unfriendly.

[0013] In light of the above, there is a need to develop a method suitable for producing high-purity allopregnanolone on a commercial scale. Summary of the Invention

[0014] The present invention addresses the above-mentioned needs by providing a simple and industrially applicable method for preparing and purifying allopregnanolone and its intermediates. The method provided in the present invention makes it possible to obtain highly pure allopregnanolone and its intermediates without the aid of column chromatography. The present invention also provides novel intermediates useful for the preparation of allopregnanolone.

[0015] Through extensive research, the present inventors have identified two important impurities associated with the current reported method for preparing allopregnanolone from isoallopregnanolone via the Mitsunobu reaction followed by hydrolysis: 5α-pregn-2-en-20-one (also referred to herein as impurity I or removed impurity) and 3α-hydroxy-5α,17α-pregnan-20-one (also referred to herein as impurity II or epimeric impurity).

[0016] The present inventors have carried out extensive experiments with the intention of reducing / removing such impurities and obtaining allopregnanolone in a highly purified form. As a result of this research, the present inventors have surprisingly discovered a new methodology based on the following key features: careful selection of the acid to be reacted with isoallopregnanolone, isolation and purification of the ester intermediate by a specific method other than chromatography, and careful selection of the final hydrolysis conditions.

[0017] Thus, in a first aspect, the present invention provides a method for preparing and purifying a 3-carboxylic acid ester of allopregnanolone, comprising: reacting isoallopregnanolone with a strong carboxylic acid (e.g., a carboxylic acid having a pKa of 3 or less) under Mitsunobu conditions; precipitating the 3-carboxylic acid ester of allopregnanolone in a solvent system comprising water and an organic solvent; and recrystallizing the precipitate of the 3-carboxylic acid ester of allopregnanolone in a non-polar solvent; The present invention covers a method including:

[0018] In another aspect, the present invention provides a method for preparing allopregnanolone, comprising the steps of: Obtaining the 3-carboxylic acid ester of allopregnanolone by the above method; and hydrolyzing the 3-carboxylic acid ester of allopregnanolone thus obtained under neutral conditions (e.g., hydrolysis with an alcohol without the aid of an acid or a base), under weakly basic conditions (e.g., hydrolysis with a base whose conjugate acid has a pKa of 11 or less), or under energetic basic conditions (e.g., hydrolysis with a base whose conjugate acid has a pKa of 12 or more for a time and at a temperature suitable to maintain the level of 3α-hydroxy-5α,17α-pregnan-20-one at 0.5% or less); The present invention covers a method including:

[0019] Optionally, the method further comprises a step of purifying the allopregnanolone, such as by recrystallization.

[0020] In another aspect, the present invention is directed to allopregnanolone or a 3-carboxylic acid ester of allopregnanolone, obtainable according to one of the methods disclosed herein.

[0021] In another aspect, the present invention is directed to a 3-carboxylic acid ester of allopregnanolone at a high purity level, more particularly one containing 0.5% or less of 5α-pregn-2-en-20-one.

[0022] In another aspect, the present invention is directed to high purity levels of allopregnanolone, more particularly, to those containing 5α-pregn-2-en-20-one and 3α-hydroxy-5α,17α-pregnan-20-one in total at 0.15% or less.

[0023] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: 2,6-dinitrobenzoic acid esters, 2,4-dinitrobenzoic acid esters, chloroacetic acid esters, dichloroacetic acid esters, trichloroacetic acid esters, cyanoacetate, fluoroacetic acid esters, difluoroacetic acid esters, and O-Nitrobenzoic acid esters The present invention relates to a 3-carboxylic acid ester of allopregnanolone selected from the group consisting of:

[0024] These aspects and preferred embodiments thereof are further defined in the detailed description and claims that follow. DETAILED DESCRIPTION OF THE INVENTION

[0025] The present inventors have developed a chromatography-free method for preparing highly pure allopregnanolone, which is simple, inexpensive, reproducible, and well suited for industrial scale.

[0026] definition As used herein, the term "about" refers to a slight variation of a particular value, preferably within 10% of the particular value. However, the term "about" may also refer to a greater tolerance for variation depending on the experimental techniques used, etc. Variations in the above-mentioned particular values ​​are understood by those skilled in the art and are within the scope of the present invention. Furthermore, for the sake of brevity, some quantitative expressions described herein are not modified by the term "about." Regardless of whether the term "about" is explicitly used, any quantity described herein is understood to refer to an actual given value, and also to an approximation to such a given value that can be reasonably estimated based on ordinary skill in the art, including equivalents and approximations due to the experimental and / or measurement conditions of such a given value.

[0027] "Room temperature" or its abbreviation "rt" as used herein means that a reaction or method is carried out without heating or cooling. Generally, room temperature may be understood as a temperature of about 15°C to about 30°C, or more specifically, about 20°C to about 25°C.

[0028] The term "organic solvent" includes, for example, cyclic and acyclic ethers (e.g., EtO, iPrO, tBuO, 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.

[0029] The term "water-soluble solvent" refers to a solvent that is miscible with water completely, i.e., in all proportions, or partially, i.e., in some proportions. Water-soluble solvents include, for example, those that contain 5 g or more of an organic solvent that is soluble in 100 g of water at a temperature of 25° C. In certain embodiments, the water-soluble solvent is selected from organic solvents that are miscible with more than 50% by weight of water at 25° C.

[0030] As used herein, the term "nonpolar solvent" refers to a solvent with sufficiently low polarity to induce crystal formation of a polar compound, such as a 3-ester of allopregnanolone. For the present disclosure, the selection of a suitable nonpolar solvent is well within the knowledge of one skilled in the art. More specifically, the term "nonpolar solvent" refers to a solvent with a Log P greater than 2. Nonpolar solvents have a low dielectric constant, e.g., less than 5. In embodiments, the nonpolar 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., EtO, iPrO, tBuO, MeOtBu, 1,4-dioxane, tetrahydrofuran, methyltetrahydrofuran).

[0031] The term "Mitsunobu conditions" refers to conditions suitable for converting isoallopregnanolone to a 3-carboxylic acid ester of allopregnanolone by reaction with a specific carboxylic acid. The reagents used in the Mitsunobu conditions are preferably a phosphine (typically triphenylphosphine (PPh3)), an azodicarboxylic acid ester, and an optional tertiary amine additive. Examples of azodicarboxylic acid esters include, but are not limited to, diethyl azodicarboxylate (DEAD), diisopropyl azodicarboxylate (DIAD), di-t-butyl azodicarboxylate, 1,1'-(azocarbonyl)dipiperidine, dibenzyl azodicarboxylate, and the like. In some embodiments, the reagents for the Mitsunobu conditions may be selected so that they can be reused 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, etc. may be used in place of triphenylphosphine. In some embodiments, triphenylphosphine, or its equivalent, and / or azodicarboxylic acid ester may be immobilized on a resin, such as, for example, a polystyrene resin. In some embodiments, cyanomethylenetri-n-butylphosphorane may be used as a reagent for Mitsunobu conditions.

[0032] The term "neutral conditions" preferably refers to the use of alcohols such as MeOH, EtOH, etc. in the hydrolysis reaction without the addition of an acid or base (transesterification).

[0033] The term "weakly basic conditions" preferably refers to the use in the hydrolysis reaction of a base whose conjugate acid has a pKa of 11 or less. Suitable bases include, but are not limited to, alkali and alkaline earth metal carbonates and bicarbonates, such as potassium carbonate, sodium carbonate, barium carbonate, cesium carbonate, potassium hydrogenocarbonate, and sodium hydrogenocarbonate.

[0034] The term "energetic basic conditions" preferably refers to the use of a base in the hydrolysis reaction, in which the conjugate acid has a pKa of 12 or greater, for a short time and / or at a low temperature to avoid the production of 3α-hydroxy-5α,17α-pregnan-20-one in an amount greater than 0.5%. For the present disclosure, the selection of a suitable time and temperature is well within the knowledge of those skilled in the art. Suitable bases include C bases of alkali metals and alkaline earth metals, such as potassium methoxide, sodium methoxide, potassium ethoxide, sodium ethoxide, lithium hydroxide, potassium hydroxide, sodium hydroxide, and calcium hydroxide. 1~6 These include, but are not limited to, alkoxides and hydroxides.

[0035] Unless otherwise specified, references to purity may be understood to mean HPLC purity.

[0036] Preparation and purification of 3-esters of allopregnanolone from isoallopregnanolone When reproducing the conditions disclosed in the prior art, the inventors noted that the appropriate selection of the carboxylic acid to be reacted with isoallopregnanolone is important to avoid / reduce the formation of impurities, particularly during hydrolysis after the Mitsunobu reaction. Maintaining impurity levels within acceptable limits makes it possible to purify both the 3-carboxylic acid ester intermediate and allopregnanolone without relying on column chromatography. It was also found that isolation and purification of the ester intermediate is necessary to make this method suitable for large-scale production of highly pure allopregnanolone. If the ester intermediate is used in a crude form in the subsequent hydrolysis reaction without being purified from certain impurities, effective purification of the final allopregnanolone would be impossible.

[0037] Specifically, the inventors have found that the Mitsunobu reaction always yields a significant amount, typically about 7-17%, of 5α-pregn-2-en-20-one (also referred to herein as Impurity I or the removed impurity). [ka]

[0038] 1 H NMR (400 MHz, CDCl3): 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).

[0039] 13 C NMR (400 MHz, CDCl3): 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.

[0040] The removal of impurities is inherent to the Mitsunobu reaction. Attempts to prepare allopregnanolone without isolating and purifying the 3-ester intermediate have either failed or been unsatisfactory because the final product requires complex column chromatography, which does not result in acceptable purity.

[0041] The present invention provides a method for preparing and purifying a 3-ester of allopregnanolone from isoallopregnanolone, and in a preferred embodiment, the method comprises: Reacting isoallopregnanolone with an organic acid having a pKa of 3 or less under Mitsunobu conditions; precipitating the crude reaction mixture into a solvent system comprising water and an organic solvent; and recrystallizing the precipitate in a non-polar solvent; Includes.

[0042] The selection of an organic acid with a low pKa is particularly important, as it allows for subsequent clean hydrolysis of the 3-ester of allopregnanolone. For example, formic acid (pKa = 3.75), benzoic acid (pKa = 4.20), acetic acid (pKa = 4.75), o-methoxybenzoic acid (pKa = 4.09), 3-nitrobenzoic acid (pKa = 3.46), and 4-nitrobenzoic acid (pKa = 3.43) have been found to be unsuitable. In one embodiment, the carboxylic acid having a pKa of 3 or less is selected from the group consisting of monofluoroacetic acid, difluoroacetic acid, trifluoroacetic acid, monochloroacetic acid, dichloroacetic acid, trichloroacetic acid, cyanoacetic acid, 2-nitrobenzoic acid, and dinitrobenzoic acid (e.g., 2,4-dinitrobenzoic acid, 2,6-dinitrobenzoic acid, and 3,5-dinitrobenzoic acid). In a more specific embodiment, the carboxylic acid is chloroacetic acid or dinitrobenzoic acid. Typically, the amount of carboxylic acid is about 1 to 4 eq, more specifically about 2 to 3 eq.

[0043] Suitable Mitsunobu conditions preferably include a phosphine such as PPh3, an azodicarboxylic acid ester, and an optional tertiary amine additive. The phosphine may be selected from the group consisting of, for example, triphenylphosphine, dicyclohexylphenylphosphine, diethylphenylphosphine, tributylphosphine, diphenyl-2-pyridylphosphine, 4-(dimethylamino)phenyldiphenylphosphine, isopropyldiphenylphosphine, tri-tert-butylphosphine, tri-n-octylphosphine, tricyclohexylphosphine, polystyryldiphenylphosphine, or a mixture thereof. The azodicarboxylic acid ester may be selected from the group consisting of, for example, diethyl azodicarboxylate (DEAD), diisopropyl azodicarboxylate (DIAD), di-t-butyl azodicarboxylate, 1,1'-(azocarbonyl)dipiperidine, dibenzyl azodicarboxylate, or a mixture thereof. In a preferred embodiment, the reagents used in the Mitsunobu reaction are or include triphenylphosphine (PPh3) and an azodicarboxylic acid ester selected from DEAD and DIAD. Suitable amounts of phosphine and azodicarboxylic acid ester typically range from about 1 to 2 eq. In certain embodiments, about 1.5 eq of phosphine and about 1.4 eq of azodicarboxylic acid ester are added. Preferably, the azodicarboxylic acid ester, typically an azodicarboxylic acid ester solution, is added slowly or dropwise to a reaction mixture containing isoallopregnanolone, a phosphine, and an organic acid. In certain embodiments, the Mitsunobu reaction is carried out in the presence of NaOBz (e.g., about 1 to 2 eq).

[0044] The Mitsunobu reaction is typically carried out in the presence of an organic solvent, which may be selected from the group consisting of cyclic and acyclic ethers (e.g., EtO, iPrO, tBuO, MeOtBu, 1,4-dioxane, tetrahydrofuran, methyltetrahydrofuran), halogenated solvents (e.g., dichloromethane, chloroform), and aromatic solvents (e.g., toluene, xylene), or a mixture thereof. In preferred embodiments, 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 certain embodiments, the solvent is 1,4-dioxane or a mixture of 1,4-dioxane and tetrahydrofuran (THF).

[0045] In one embodiment, isoallopregnanolone is treated with an organic acid at a suitable temperature of about 0 to 45°C (e.g., about 15 to 45°C) for a time sufficient to form an ester, typically about 1 to 24 hours, more specifically about 2 to 12 hours or about 4 to 8 hours. In one embodiment, the reaction mixture may be stirred at a suitable temperature for a time sufficient to complete ester formation. The reaction may be followed by thin layer chromatography (TLC, HPLC, or UPLC).

[0046] Upon completion, water may be added, preferably to the crude reaction mixture, and the resulting suspension may be filtered to obtain a wet filter cake containing the 3-carboxylic acid ester of allopregnanolone.

[0047] If a water-immiscible solvent is used, it will be necessary to remove, preferably evaporate, the solvent and replace it with a water-miscible solvent (a water-soluble solvent), such as 1,4-dioxane, MeOH, EtOH, IPA, ACN, etc., before adding water.

[0048] According to the present invention, the isolation of the 3-ester of allopregnanolone is carried out by precipitation in a solvent system comprising water and an organic solvent, which allows the reduction / removal of polar and basic impurities, such as phosphine-derived by-products and azodicarboxylic acid ester-derived by-products.

[0049] Preferably, the organic solvent used in 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 mixtures thereof; even more particularly, the water-soluble solvent is 1,4-dioxane, acetonitrile, or isopropanol; even more particularly, the water-soluble solvent is 1,4-dioxane.

[0050] Various ratios of water and organic solvent may be used. In one embodiment, the ratio of water to organic solvent in the solvent system ranges from about 0.1:1 to 1:0.1, and more particularly from 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.

[0051] In more particular embodiments, it may be advantageous to precipitate the crude allopregnanolone 3-ester using a solvent system comprising water and an organic solvent in about a 1:1 ratio, wherein the organic solvent is 1,4-dioxane, acetonitrile, or isopropanol.

[0052] After precipitation, the 3-ester of allopregnanolone is preferably dried (eg, dried under vacuum).

[0053] Purification of the 3-ester of allopregnanolone is achieved by recrystallization in a non-polar solvent. Recrystallization is advantageously performed to remove or reduce the amount of 5α-pregn-2-en-20-one (i.e., impurity I or removed impurities) to about 0.5% or less (e.g., about 0.4% or less, 0.3% or less, 0.2% or less, 0.1% or less). 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., EtO, iPrO, tBuO, MeOtBu, methyltetrahydrofuran). In certain embodiments, the non-polar solvent is selected from the group consisting of hexane, cyclohexane, heptane, toluene, iPrO, MeOtBu, or mixtures thereof; more particularly, selected from the group consisting of cyclohexane, heptane; and even more particularly, heptane.

[0054] That is, the present invention provides a highly pure 3-carboxylic acid ester of allopregnanolone containing 5α-pregn-2-en-20-one in an amount not exceeding 0.5% (e.g., about 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, 0.1% or less).

[0055] Hydrolysis of 3-esters of allopregnanolone to allopregnanolone The present inventors have discovered that hydrolysis of certain allopregnanolone 3-esters (e.g., acetate, formate, isobutyrate, benzoate, o-methoxybenzoate, 3-nitrobenzoate, or 4-nitrobenzoate) produces large amounts of 3α-hydroxy-5α,17α-pregnan-20-one (also referred to herein as impurity II or the epimeric impurity). [ka]

[0056] 1 H NMR (400 MHz, CDCl3): 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).

[0057] 13 C NMR (400 MHz, CDCl3): 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.

[0058] For purification suitable for commercial scale, it is advantageous to maintain the level of 3α-hydroxy-5α,17α-pregnan-20-one at 0.5% or less during the hydrolysis reaction. This may be achieved by using 3-esters of allopregnanolone obtained from carboxylic acids with low pKa (e.g., pKa of 3 or less), which readily hydrolyze under the conditions disclosed herein. Furthermore, when obtained according to the methods disclosed above, such 3-esters of allopregnanolone also contain only small amounts of 5α-pregn-2-en-20-one (0.5% or less).

[0059] In the present invention, hydrolysis is carried out under neutral conditions (e.g., in the presence of an alcohol without the addition of an acid or a base), under weakly basic conditions (e.g., in the presence of a base whose conjugate acid has a pKa of 11 or less), or under energetically basic conditions (e.g., in the presence of a base whose conjugate salt has a pKa of 12 or more, for a non-prolonged time and / or at a moderate temperature).

[0060] In certain embodiments, the ester is hydrolyzed under neutral conditions using an alcohol selected from methanol, ethanol, propanol, isopropanol, sec-butanol, and t-butanol. More particularly, the alcohol is MeOH or EtOH. In one embodiment, the ester is stirred in the alcohol for a sufficient time (e.g., about 1 to 24 hours, or about 1 to 12 hours) at a suitable temperature (e.g., about 15 to 40°C or about 15 to 25°C) to complete the hydrolysis. The reaction may be followed by thin layer chromatography (TLC).

[0061] In certain embodiments, the ester is hydrolyzed under mildly basic conditions using a base selected from carbonates and bicarbonates of alkali and alkaline earth metals, 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 or 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 a solvent. In one embodiment, the alcohol is MeOH or EtOH. In one embodiment, the ester is treated with any of the above bases, preferably in an amount of about 0.2 to 3 eq. or about 0.5 to 2 eq., at a suitable temperature of about 15 to 40° C. or about 15 to 30° C. for a sufficient time to hydrolyze, typically about 1 to 4 hours. In one embodiment, the ester may be stirred at a suitable temperature (e.g., about 15 to 40° C. or about 15 to 30° C.) for a sufficient time (e.g., about 1 to 4 hours) to complete the hydrolysis.

[0062] In certain embodiments, the ester is a C 1~6 The hydrolysis is carried out under energetic basic conditions using a base selected from 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; 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 a solvent. In one embodiment, the alcohol is MeOH or EtOH. The use of a strong base (pKa of 12 or higher) requires maintaining the hydrolysis reaction for a short time and / or at a low temperature to avoid the production of 3α-hydroxy-5α,17α-pregnan-20-one in an amount greater than 0.5%. In one embodiment, hydrolysis under energetic basic conditions is carried out for about 2 hours or less, more particularly about 1.5 hours or less, even more particularly about 1 hour or less, and / or at a temperature of about 15-40° C., more particularly about 15-30° C. In one embodiment, the ester is treated with any of the above 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 a time sufficient for hydrolysis (e.g., about 2 hours or less, more particularly about 1.5 hours or less, even more particularly about 1 hour or less). In one embodiment, the reaction mixture may be stirred for a sufficient time (e.g., about 0.5-2 hours) at a suitable temperature (e.g., 40° C. or less or 30° C. or less) to complete the hydrolysis.

[0063] Advantageously, allopregnanolone is obtained in a highly pure state, containing 3α-hydroxy-5α,17α-pregnan-20-one (also referred to herein as impurity II or the epimeric impurity) in an amount not exceeding 0.5% (e.g., about 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, 0.1% or less).

[0064] Isolation of allopregnanolone may be carried out, for example, by precipitation in a solvent system containing water and an organic solvent. Preferably, the organic solvent used to precipitate 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 mixtures thereof. Even more particularly, the water-soluble solvent is 1,4-dioxane, acetonitrile, or methanol. Even more particularly, the water-soluble solvent is methanol.

[0065] Various ratios of water and organic solvent may be used. In one embodiment, the ratio of water to organic solvent in the solvent system ranges from about 0.1:1 to 1:0.1, and more particularly from 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.

[0066] In more particular embodiments, it may be advantageous to precipitate the crude allopregnanolone using a solvent system comprising water and an organic solvent in a ratio of about 0.5:1, wherein the organic solvent is 1,4-dioxane, acetonitrile, or methanol.

[0067] After precipitation, the allopregnanolone is preferably dried (eg, dried under vacuum).

[0068] If desired, further purification of allopregnanolone can be achieved, for example, by 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 certain embodiments, the non-polar solvent is selected from MeOH, EtOH, IPA, acetone, ACN, etc., and more particularly, MeOH / water.

[0069] Advantageously, recrystallization makes it possible to remove or reduce the level of impurities in order to obtain highly pure allopregnanolone containing 5α-pregn-2-en-20-one and 3α-hydroxy-5α,17α-pregnan-20-one in a total amount of 0.15% or less. In a preferred variant of the invention, the amount of both impurities in the final allopregnanolone is at most 0.10%.

[0070] The present invention makes it possible to obtain allopregnanolone with a high purity, such as greater than 98%, greater than 99%, or even greater than 99.5%, which meets the requirements normally imposed by Good Manufacturing Practices (GMP). In certain embodiments, the purity of allopregnanolone is 99.5% or greater, and the total content of impurities I and II is 0.15% or less, preferably 0.10% or less.

[0071] In a preferred embodiment of the present invention, allopregnanolone is prepared by a method comprising the following steps: Reacting isoallopregnanolone with strong carboxylic acids under Mitsunobu conditions; precipitating the 3-carboxylic acid ester of allopregnanolone in a solvent system comprising water and an organic solvent; Recrystallizing the precipitate of allopregnanolone 3-carboxylic acid ester in a non-polar solvent; Hydrolyzing the 3-carboxylic acid ester of allopregnanolone thus obtained under neutral conditions, weakly basic conditions, or energetically basic conditions to obtain allopregnanolone; and By precipitating and recrystallizing, allopregnanolone having a total content of impurities I and II of 0.15% or less is obtained.

[0072] The present disclosure provides allopregnanolone used in the preparation of a pharmaceutical composition, the total content of impurities I and II of which is 0.15% or less. The present disclosure also encompasses the use of allopregnanolone, the total content of impurities I and II of which is 0.15% or less, to prepare a pharmaceutical composition. The present disclosure also includes a method for preparing the pharmaceutical composition. The method includes combining allopregnanolone, the total content of impurities I and II of which is 0.15% or less, with at least one pharmaceutically acceptable excipient. The allopregnanolone and pharmaceutical compositions of allopregnanolone of the present disclosure can be used as pharmaceuticals, particularly as pharmaceuticals for the treatment of postpartum depression (PPD). The present disclosure also provides a method for treating postpartum depression (PPD), comprising administering a therapeutically effective amount of the allopregnanolone of the present disclosure to a subject in need of treatment.

[0073] 3-ester of allopregnanolone In another embodiment, the present invention provides the following 3-esters of allopregnanolone: 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-(cyanoacetyloxy)-, (3α,5α)- Pregnan-20-one, 3-(fluoroacetyloxy)-, (3α,5α)- Pregnan-20-one, 3-(difluoroacetyloxy)-, (3α,5α)- Pregnan-20-one, 3-(2-nitrobenzoyloxy)-, (3α,5α)-

[0074] These esters may be prepared by reacting isoallopregnanolone with the corresponding carboxylic acid (i.e., 2,6-dinitrobenzoic acid, 2,4-dinitrobenzoic acid, chloroacetic acid, dichloroacetic acid, trichloroacetic acid, cyanoacetic acid, fluoroacetic acid, difluoroacetic acid, or 2-nitrobenzoic acid) under Mitsunobu conditions.

[0075] It is to be understood that all possible combinations of the embodiments disclosed herein are encompassed within the scope of the present disclosure.

[0076] The following examples are merely illustrative of certain embodiments of the present invention and are not intended to limit the invention in any way. [Example]

[0077] 1. Conversion of isoallopregnanolone to the α-3-chloroacetate ester of brexanolone [ka]

[0078] Mitsunobu: Isopregnanolone (36 g, 113.2 mmol), PPh3 (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 to 15 °C, and a solution of DIAD (32.4 mL, 1.4 eq) in dioxane (140 mL) was added dropwise. After the addition, the reaction mixture was stirred at 35 °C until the reaction was complete (4 h). Impurity I (5α-pregn-2-en-20-one): approximately 9-12%.

[0079] Workup and isolation: After cooling to 25°C, water (540 mL) was added, and the resulting suspension was stirred for 30 minutes, then cooled to 20-25°C. The suspension was filtered, and the wet cake was washed with 100 mL of a 1:1 dioxane / water mixture. The product was dried under reduced pressure at 50°C to give 32.45 g of a white solid (yield: 70%; purity: 96%). Impurity I (5α-pregn-2-en-20-one): 3.5%.

[0080] Recrystallization: The resulting dry cake was suspended in heptane (15 ml / g) and heated to 75°C until dissolution was complete. The solution was then cooled to 0 / 10°C, and the precipitate was filtered off. The solid was washed with fresh heptane (2 ml / g) and dried under reduced pressure at 50°C. Weight: 29.2 g; Yield: 90%; Purity: 99.15%. Removed impurity I (5α-pregn-2-en-20-one): 0.3%.

[0081] 1 H NMR (400 MHz, CDCl3): 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).

[0082] 13 C NMR (400 MHz, CDCl3): 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.

[0083] Isolation of 5α-pregn-2-en-20-one from the dioxane / water mother liquor obtained by ester precipitation: The mother liquor was diluted with more water (approximately 3 L) to form a suspension, and the solid was filtered off. The filtrate was suspended in 400 ml of heptane and stirred at 40° C. for 1 hour, then filtered again and evaporated to give a solid that was chromatographed and characterized by NMR.

[0084] 1 H NMR (400 MHz, CDCl3): 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).

[0085] 13 C NMR (400 MHz, CDCl3): 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.

[0086] The NMR signals were consistent with data found in the literature (Tetrahedron (Vol. 60) 2004, pp. 11851-11860).

[0087] 2. Hydrolysis of α-3-chloroacetate to brexanolone [ka]

[0088] 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 at 30° C. for 1 hour.

[0089] Workup and isolation: 150 ml of water was added, cooled to 0 / 5°C, and the solid was filtered off. The wet cake was then washed with 60 mL of a 1:0.5 methanol / water mixture. The product was dried under vacuum at 50°C to give 22 g of crude brexanolone (yield: 93%; purity: >99%). Epimeric impurity II (3α-hydroxy-5α,17α-pregnan-20-one): 0.23%.

[0090] Recrystallization: The resulting dried cake was suspended in MeOH (15 volumes) and stirred at 30°C for 30 minutes. Water (7.5 volumes) was then added, the resulting suspension was cooled to 5-10°C, filtered, and the wet cake was dried to give 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%. Removed impurity I (5α-pregn-2-en-20-one): not detected.

[0091] 3. Hydrolysis of α-3-chloroacetate containing 0.73% 5-α-pregn-3-en-20-one (Comparative Example)

[0092] Hydrolysis: Chloroacetate-brexanolone (5 g, 12.7 mmol, containing 0.73% of the 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 at 20-25 °C for 1 h (starting material content: 0.35%).

[0093] Workup: 150 ml of water was added, cooled to 0 / 5°C, and the solid was filtered off. The wet cake was then washed with 7.5 mL of a 1:0.5 methanol / water mixture. The product was dried under vacuum at 50°C to give 3.77 g of crude brexanolone (yield: 93%; purity: >98.93%). Impurity I (5α-pregn-2-en-20-one): 0.52%.

[0094] Recrystallization: The resulting dry cake was suspended in MeOH (25 volumes) and dissolved at 35-40°C. After the reaction mixture was cooled to 20-25°C, water (12.5 volumes) was slowly added to obtain a suspension, which was then filtered. The wet cake was dried to give 3.3 g of brexanolone as a white solid. Purity: >99.7%. Impurity I (5-α-pregn-3-en-20-one): 0.21%.

[0095] Thus, if one started with an ester containing more than 0.5% of the removed impurity I, it was not possible to reduce the amount below 0.15%.

[0096] 4. Hydrolysis of α-3-chloroacetate containing 0.5% 5α-pregn-2-en-20-one impurity

[0097] The method disclosed in Example 3 was followed, except that the starting material used was α-3-chloroacetic acid ester containing 0.5% of the removed impurity I. 3.2 g of brexanolone was obtained with a purity of greater than 98% and a content of approximately 0.13% of the removed impurity I.

[0098] Thus, when starting with an ester containing 0.5% of the removed impurity I, the amount could be reduced to less than 0.15%.

[0099] 5. Hydrolysis of α-3-chloroacetate containing 0.5% 3α-hydroxy-5α,17α-pregnan-20-one

[0100] Hydrolysis: Chloroacetate-brexanolone (5 g, 12.7 mmol, doped with 0.5% epimer impurity) was suspended in methanol (50 mL) and potassium carbonate (0.85 g, 0.5 eq) was added. The reaction mixture was stirred at 20-25 °C for 1 h (starting material content: 0.30%).

[0101] Workup: 150 ml of water was added, cooled to 0 / 5°C, and the solid was filtered off. The wet cake was then washed with 7.5 mL of a 1:0.5 methanol / water mixture. The product was dried under vacuum at 50°C to give 3.82 g of crude brexanolone (yield: 94%; purity: >99.43%). Epimeric impurity II (3α-hydroxy-5α,17α-pregnan-20-one): 0.24%. Removed impurity I (5α-pregn-2-en-20-one): 0.18%.

[0102] Recrystallization: The resulting dried cake was suspended in MeOH (25 volumes) and dissolved at 35-40°C. The reaction mixture was cooled to 20-25°C, and then water (12.5 volumes) was slowly added to obtain a suspension. The suspension was filtered, and the wet cake was dried to obtain 3.3 g of brexanolone as a white solid. Purity: 99.81%. Epimeric impurity II (3α-hydroxy-5α,17α-pregnan-20-one): 0.1%. Removed impurity I (5α-pregn-2-en-20-one): 0.02%.

[0103] 6. Conversion of isoallopregnanolone to the α-3-chloroacetate ester of brexanolone [ka]

[0104] Mitsunobu: Isopregnanolone (14 g, 44 mmol), PPh3 (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 to 15 °C, and a solution of DIAD (32.4 mL, 1.4 eq) in dioxane (70 mL) was added dropwise. After the addition, the reaction mixture was stirred at 35 °C until the reaction was complete (4 h). Removal of impurity I (5α-pregn-2-en-20-one): approximately 8%.

[0105] After cooling to 25°C, the reaction mixture (250 ml) was divided into seven samples (35 ml each, containing approximately 2 g of the final ester product). All samples were evaporated, and the residue was redissolved in 30 ml of different water-miscible solvents (MeOH, ACN, DMF, IPA, acetone, dioxane) and precipitated with an equal volume of water.

[0106] 7. Conversion of isoallopregnanolone to the α-3-trifluoroacetate ester of brexanolone [ka]

[0107] Mitsunobu: Isopregnanolone (1 g, 3.14 mmol), PPh3 (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 to 15 °C, and a solution of DIAD (0.9 mL, 1.4 eq) in dioxane (5 mL) was added dropwise. After the addition, the reaction mixture was stirred at 35 °C for 1 h. Then, 1.2 eq of NaOBz was added as a solid, and the reaction mixture was stirred again at 35 °C for 20 h (starting material still 6%).

[0108] Workup and precipitation: After cooling to 25°C, water (10 mL) was added, and the resulting suspension was stirred for 30 min before being cooled to 10-15°C. The suspension was filtered, and the wet cake was washed with 2 mL of a 1:1 dioxane / water mixture. The product was dried under vacuum at 50°C to give 0.75% of the ester, with 14.6% of the impurities removed by HPLC.

[0109] 8. α-3-benzoyl ester of brexanolone (comparative example) [ka]

[0110] Mitsunobu: Isopregnanolone (1 g, 3.14 mmol), PPh3 (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 to 15 °C, and a solution of DIAD (0.9 mL, 1.4 eq) in dioxane (5 mL) was added dropwise. After the addition, the reaction mixture was stirred at 35 °C for 5 h (removal of approximately 8% of the impurities).

[0111] Workup and Precipitation After cooling to 25°C, water (10 mL) was added and the resulting suspension was stirred for 30 minutes before being cooled to 10-15°C. The suspension was filtered and the wet cake was washed with 1:1 dioxane / water. The product was dried under vacuum at 50°C to give 0.1 g of the ester in >96% purity (0.5% impurities removed).

[0112] hydrolysis The above benzoyl ester was added to a solution of 70 volumes of MeOH and 12 eq of NaOH, and the reaction mixture was heated at 40°C for 30 hours. A sample was taken and analyzed by HPLC, which showed that only 4% of the brexanolone final product was present, and 9% of the epimeric impurity was present along with the ester starting material.

[0113] It can be seen in this example, and from Example 12, that acceptable purity of brexanolone cannot be obtained from the benzoyl ester of brexanolone. 9. Brexanolone from isopregnanolone in a "one-pot" method using chloroacetic acid (Comparative Example) [ka]

[0114] Triphenylphosphine (1.2 g / g), chloroacetic acid (0.75 g / g), and isopregnanolone were added to a round-bottom flask, followed by toluene (10 ml / g). The suspension was cooled to 15°C, and a solution of DIAD (0.95 ml / g) in toluene (5 ml / g) was added dropwise, maintaining the temperature below 25°C. The resulting yellow solution was heated at 35°C for 4 hours. After the starting material was consumed (K1), a 7% aqueous solution of NaHCO3 (5 ml / g) was added. The organic phase was then washed with water (5 ml / g) and evaporated under reduced pressure. 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 was stirred at 30°C for 1 hour (K2). The product was then precipitated by adding water (5 ml / g). The reaction product was cooled to 5°C, filtered, and washed with a 1:0.5 methanol / water mixture (2 ml / g) (TH1). The resulting wet cake was further washed with heptane (5 ml / g) (TH2). [Table 1]

[0115] It can be seen that the one-pot method removes an increased amount of impurities (9%) that cannot be separated from brexanolone.

[0116] 10. Brexanolone from isopregnanolone in a "one-pot" method using trifluoroacetic acid and purification by chromatography (Comparative Example) [ka]

[0117] Triphenylphosphine (1.2 g), trifluoroacetic acid (0.6 ml, 2.5 eq), NaOBz (0.9 g), and isopregnanolone (1 g) were added to a round-bottom flask, followed by THF (10 ml). The suspension was cooled to 15°C, and a solution of DIAD (0.95 ml) in THF (5 ml) was added dropwise, maintaining the temperature below 25°C. The resulting reaction mixture was stirred at 25°C for 24 hours. After the starting material was consumed, the reaction mixture was evaporated under reduced pressure to give a residue.

[0118] MeOH (20 ml) was added, the mixture was refluxed for 24 hours, the solvent was evaporated, and the residue was purified by column chromatography to give 0.69 g of brexanolone (purity: 97%).

[0119] It will be appreciated that the one-pot process will only yield brexanolone of low purity.

[0120] 11. Brexanolone acetate using toluene and sodium benzoate as solvent (Comparative Example) [ka]

[0121] DIAD (0.46 g), acetic acid (0.15 ml), isopregnanolone (0.5 g), and toluene (15 ml) were added to a round-bottom flask. The reaction mixture was cooled to 0°C, and PPh3 (0.6 g) was added, followed by NaOBz (0.34 g). The resulting reaction mixture was stirred at 25°C for 15 hours. After the starting material was consumed, the reaction mixture was evaporated under reduced pressure to obtain a residue (removed impurity content: approximately 14%).

[0122] This example demonstrates that the removed impurities are inherent to the Mitsunobu reaction.

[0123] 12. Hydrolysis of benzoates, nitrobenzoates, and 2,6-dinitrobenzoates: [ka] [Table 2]

[0124] As can be seen from the table, both the benzoate and 4-nitrobenzoate esters hydrolyze easily, requiring very energetic conditions that lead to the formation of epimers (which are difficult to purify), but the dinitrobenzoate ester hydrolyzes in just one hour without producing the epimer.

[0125] 13. Obtaining Brexanolone by Hydrolysis of α-3-Acetate Ester (Comparative Example) [ka]

[0126] In a round-bottom flask, 3-α-pregnanolone acetate (1 g) was dissolved in 15 mL of methanol. 0.26 g of sodium hydroxide (2.4 eq) was added, and the reaction mixture was stirred at 40°C for 4 hours. HPLC control showed the disappearance of the starting material. The solvent was concentrated under reduced pressure to a volume of 5 mL. The mixture was poured into water (87 mL) and stirred at room temperature for 1 hour.

[0127] The precipitate was then filtered off, and the wet cake was washed with water (10 mL) and dried under vacuum at 50°C.

[0128] HPLC analysis of this solid showed the presence of 8.6% of the epimeric impurity (II).

[0129] 14. 3-(2,4-dinitrobenzoyl ester) of brexanolone [ka]

[0130] Isopregnanolone (0.5 g, 1.57 mmol), PPh3 (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 to 0 / 5°C, and a solution of DIAD (0.45 mL, 1.4 eq) in THF (5 mL) was added dropwise. After the addition, the reaction mixture was stirred at room temperature for 5 hours. 1 mL of water was added, the solvent was evaporated, and the residue was purified by column chromatography. 0.24 g of solid product was obtained (yield: 32.7%).

[0131] 15. 3-(3,5-dinitrobenzoyl ester) of brexanolone [ka]

[0132] Isopregnanolone (0.5 g, 1.57 mmol), PPh3 (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 to 0 / 5°C, and a solution of DIAD (0.45 mL, 1.4 eq) in THF (5 mL) was added dropwise. After the addition, the reaction mixture was stirred at room temperature for 5 hours. 1 mL of water was added, the solvent was evaporated, and the residue was purified by column chromatography. 0.43 g of solid product was obtained (yield: 53.5%).

[0133] Using a similar procedure, the 2-nitrobenzoyl ester of brexanolone and the 2,6-dinitrobenzoyl ester of brexanolone were prepared.

[0134] 16. 3-Dichloroacetate of Brexanolone [ka]

[0135] Isopregnanolone (5.0 g, 15.7 mmol), PPh3 (6.0 g, 1.5 eq), and dichloroacetic acid (3.4 mL) were suspended in dioxane (55 mL). The mixture was cooled to 15 °C, and a solution of DIAD (4.5 mL, 1.4 eq) in dioxane (20 mL) was added dropwise. After the addition, the reaction mixture was stirred at 35 °C until completion. 75 mL of water was added, and the mixture was stirred for 0.5 h, filtered, washed with 10 mL (1:1) water / dioxane, and dried. 3.8 g of white solid product was obtained (yield: 56.4%).

[0136] 17. 3-Trichloroacetate of Brexanolone [ka]

[0137] Isopregnanolone (5.0 g, 15.7 mmol), PPh3 (6.0 g, 1.5 eq), and trichloroacetic acid (6.45 g) were suspended in dioxane (55 mL). The mixture was cooled to 15 °C, and a solution of DIAD (4.5 mL, 1.4 eq) in dioxane (20 mL) was added dropwise. After the addition, the reaction mixture was stirred at 35 °C until completion. 75 mL of water was added, the two phases were separated, and the aqueous layer was extracted with 50 mL of DCM. The organic layer was concentrated to give a yellow oil.

Claims

1. 1. A method for preparing and purifying a 3-carboxylic acid ester of allopregnanolone, comprising: reacting isoallopregnanolone with a strong carboxylic acid having a pKa of 3 or less under Mitsunobu conditions; precipitating the 3-carboxylic acid ester of allopregnanolone in a solvent system comprising water and an organic solvent; and recrystallizing the precipitate of the 3-carboxylic acid ester of allopregnanolone in a non-polar solvent; A method comprising:

2. 1. A method for preparing allopregnanolone, comprising: Obtaining a 3-carboxylic acid ester of allopregnanolone by the method defined in claim 1; and hydrolyzing the 3-carboxylic acid ester of allopregnanolone thus obtained under neutral conditions, weakly basic conditions, or energetically basic conditions; A method comprising:

3. 3. The method of claim 1, wherein the carboxylic acid is selected from the group consisting of monofluoroacetic acid, difluoroacetic acid, trifluoroacetic acid, monochloroacetic acid, dichloroacetic acid, trichloroacetic acid, cyanoacetic acid, orthonitrobenzoic acid, and dinitrobenzoic acid.

4. The method according to any one of claims 1 to 3, wherein the 3-carboxylic acid ester of allopregnanolone is precipitated in a solvent system comprising water and a water-soluble organic solvent.

5. the 3-carboxylic acid 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 a mixture thereof; and / or The precipitate of the 3-carboxylic acid ester of allopregnanolone can be extracted with hexane, cyclohexane, heptane, toluene, iPr ２ 5. The method of claim 1, wherein the product is recrystallized in a non-polar solvent selected from the group consisting of O, MeOtBu, or a mixture thereof.

6. 6. The method according to any one of claims 1 to 5, wherein the 3-carboxylic acid ester of allopregnanolone is hydrolyzed with an alcohol without the addition of an acid or a base.

7. 6. The method of any one of claims 1 to 5, wherein the 3-carboxylic acid ester of allopregnanolone is hydrolyzed with a base whose conjugate acid has a pKa of 11 or less, such as an alkali metal or alkaline earth metal carbonate or bicarbonate.

8. The 3-carboxylic acid ester of allopregnanolone is reacted with an alkali metal or alkaline earth metal C ester at a time and temperature suitable to maintain the level of 3α-hydroxy-5α,17α-pregnan-20-one at an amount of 0.5% or less. １～６ 6. The method of any one of claims 1 to 5, wherein the conjugate acid is hydrolyzed with a base having a pKa of 12 or greater, such as an alkoxide or hydroxide.

9. 9. The method of claim 8, wherein the hydrolysis is carried out for about 2 hours or less and / or at a temperature of about 15-40°C.

10. The following steps: Reacting isoallopregnanolone with a strong carboxylic acid under Mitsunobu conditions; precipitating the 3-carboxylic acid ester of allopregnanolone in a solvent system comprising water and an organic solvent; recrystallizing the precipitate of the 3-carboxylic acid ester of allopregnanolone in a non-polar solvent; The 3-carboxylic acid ester of allopregnanolone thus obtained is hydrolyzed under neutral conditions, weakly basic conditions, or energetically basic conditions to obtain allopregnanolone; and By precipitating and recrystallizing, allopregnanolone having a total content of impurities I and II of 0.15% or less can be obtained. The method according to any one of claims 1 to 9, comprising:

11. A 3-carboxylic acid ester of allopregnanolone having a 5α-pregn-2-en-20-one content of 0.5% or less.

12. Allopregnanolone having a total content of 5α-pregnan-2-en-20-one and 3α-hydroxy-5α,17α-pregnan-20-one of 0.15% or less.

13. 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)- Pregnan-20-one, 3-(cyanoacetyloxy)-, (3α,5α)- 3-carboxylic acid esters of allopregnanolone selected from:

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