Method for preparing high-purity allopregnanolone and its intermediates

Abstract

The present invention relates to an efficient and industrially applicable method for preparing and purifying allopregnanolone and its intermediates without the aid of column chromatography.

Description

Technical Field

[0001] The present invention belongs to the field of drug synthesis, and more particularly relates to an efficient and industrially applicable method for preparing and purifying allopregnanolone (isopregnanolone / allopregnanolone) and its useful intermediates. Background Art

[0002] Allopregnanolone [516-54-1], also known as 3α-hydroxy-5α-pregnane-20-one, 5α-pregnane-3α-ol-20-one or 3α,5α-tetrahydroprogesterone (3α,5α-THP) and brexanolone (USAN), is an endogenous inhibitory pregnane neurosteroid. Synthesized from progesterone in the brain, it is a potent positive allosteric modulator of the action of gamma-aminobutyric acid (GABA) at the GABA-A receptor. Allopregnanolone has actions similar to those of benzodiazepines at the GABA-A receptor. Allopregnanolone has the same effects as those of other positive allosteric modulators of GABA action, including anxiolytic, sedative, and anticonvulsant activity. The structure of allopregnanolone is depicted below.

[0003]

[0004] Brenolone injection for intravenous use (Brenolone IV) is a novel product being developed by Sage Therapeutics for the treatment of postpartum depression (PPD), a serious and potentially life-threatening condition for which there is currently no specifically indicated drug therapy. The FDA anticipates approval of Brenolone IV in March 2019.

[0005] Several methods for the synthesis of this compound have been disclosed in the prior art.

[0006] Purdy et al. (J. Med. Chem. 1990, 33, 1572-1581) describe the direct preparation of diastereomers of allopregnanolone from isopregnanolone, which differ structurally only in the orientation of the hydroxyl group at carbon 3 of the steroid A ring. 3 ) and diethyl azodicarboxylate (DEAD), using trifluoroacetic acid (TFA) to achieve inversion of the 3β-hydroxy group via the Mitsunobu reaction without isolating the intermediate trifluoroacetate (Scheme 1). The crude product was worked up and crystallized to remove 5% of the unreacted starting material (1), yielding allopregnanolone (2a) in 74% yield, mp (melting point) 168-170°C. The product was further purified by dry column chromatography on silica gel and crystallized from aqueous ethanol; yield 54%, mp (melting point) 174-176°C.

[0007]

[0008] Solution 1

[0009] WO 2009 / 108804 discloses in Example 6 a similar procedure for the preparation of allopregnanolone (14) from allopregnanolone (12) without isolation of the intermediate trifluoroacetate (Scheme 2). After purification by silica column, allopregnanolone was obtained in 92% overall yield.

[0010]

[0011] Solution 2

[0012] Processes involving the isolation of the intermediate mitsinyl ester have also been proposed, such as for example in WO 93 / 03732, WO 2012 / 127176 and CN 103396467 (Schemes 3, 4 and 5, respectively).

[0013] In Example 4 of WO 93 / 03732, allopregnanolone was reacted with acetic acid, triphenylphosphine (PPh 3 ) and diisopropyl azodicarboxylate (DIAD), and the product residue was purified by flash chromatography on silica gel to give 3α-acetoxy-5α-pregnane-20-one in 97.5% yield. The ester was hydrolyzed with perchloric acid and the reaction mixture was stirred for three days to give crude allopregnanolone as disclosed in Example 5.

[0014]

[0015] Solution 3

[0016] WO 2012 / 127176 discloses in Example 4 that allopregnanolone is reacted with benzoic acid, triphenylphosphine (PPh 3 ) and diisopropyl azodicarboxylate (DIAD) to give a crude residue which was subjected to purification by chromatography to give the corresponding benzoate (3) in 90% yield. Hydrolysis was carried out in Example 5 under harsh conditions (KOH, MeOH, at reflux for 2.5 h) followed by purification by chromatography to give allopregnanolone (4) in 80% yield (mp (melting point) 161.7-162.8° C.). The low melting point reflects that the product is not sufficiently purified.

[0017]

[0018] Solution 4

[0019] CN 103396467 discloses in Example 5 that by using o-anisylbenzoic acid together with triphenylphosphine (PPh 3) and diisopropyl azodicarboxylate (DIAD) were used to convert allopregnanolone. In this procedure, the crude intermediate ester was precipitated and hydrolyzed with sodium hydroxide in methanol at 60 °C for 2 h to obtain a white solid with an overall yield of 52% for two steps.

[0020]

[0021] Solution 5

[0022] Like any synthetic compound, allopregnanolone may contain extraneous compounds or impurities. These impurities may include unreacted starting materials, reaction byproducts, side reaction products, and / or degradation products. Impurities in allopregnanolone or any active pharmaceutical ingredient ("API") are undesirable and, in extreme cases, may even be harmful to patients treated with a dosage form of the API. Therefore, identifying impurities in APIs generated during the manufacturing process and reducing / eliminating the presence of impurities in the final product is critical to commercialization.

[0023] The inventors have thoroughly examined the processes disclosed in the cited documents and have found that they do not reproducibly produce allopregnanolone in a sufficiently pure state and / or require a chromatographic purification step. Purification by column chromatography is not suitable, or at least undesirable, for industrial-scale production of large quantities of API, requiring extremely large amounts of solvent, which makes the process expensive and disadvantageous from an environmental point of view.

[0024] There is therefore a need to develop a process suitable for producing highly pure allopregnanolone on a commercial scale. Summary of the invention

[0025] The present invention solves the above needs by providing a simple and industrially applicable process for preparing and purifying allopregnanolone and its intermediates. The process provided by the present invention allows obtaining highly pure allopregnanolone and its intermediates without the aid of column chromatography. The present invention also provides novel intermediates that can be used to prepare allopregnanolone.

[0026] After extensive research, the present inventors have identified two important impurities associated with the process reported in the prior art for preparing allopregnanolone from allopregnanolone by Mitsunobu reaction and subsequent hydrolysis. These undesirable impurities are 5α-pregn-2-en-20-one (also referred to herein as impurity I or elimination impurity) and 3α-hydroxy-5α,17α-pregnane-20-one (also referred to herein as impurity II or epimer impurity).

[0027] The present inventors have conducted extensive experiments with the aim of reducing / eliminating such impurities and obtaining allopregnanolone in a highly pure form. As a result of this research, the inventors have surprisingly discovered a new process based on the following key features: careful selection of the acid to be reacted with allopregnanolone, separation and purification of the intermediate ester by means of a specific non-chromatographic procedure, and careful selection of the final hydrolysis conditions.

[0028] Therefore, in a first aspect, the present invention relates to a process for preparing and purifying the 3-carboxylate of allopregnanolone, the process comprising:

[0029] - reacting allopregnanolone with a strong carboxylic acid (e.g., a carboxylic acid with a pKa ≤ 3) under Mitsunobu conditions;

[0030] - precipitating the 3-carboxylate of allopregnanolone in a solvent system comprising water and an organic solvent; and

[0031] - Recrystallizing the precipitate of the 3-carboxylate of allopregnanolone in a non-polar solvent.

[0032] In another aspect, the present invention relates to a method for preparing allopregnanolone, the method comprising:

[0033] - the 3-carboxylate of allopregnanolone obtained by the process defined above; and

[0034] - subjecting the 3-carboxylic acid ester of allopregnanolone thus obtained to hydrolysis under neutral conditions (e.g. with an alcohol, without the aid of any acid or base), mildly alkaline conditions (e.g. with a base, the conjugate acid of which has a pKa equal to or less than 11), or strongly alkaline conditions (e.g. with a base, the conjugate acid of which has a pKa equal to or greater than 12, for an amount of time and temperature suitable to maintain the level of 3α-hydroxy-5α,17α-pregnane-20-one at 0.5% or less.

[0035] Optionally, the method further comprises the step of purifying allopregnanolone, such as by recrystallization.

[0036] In another aspect, the present invention relates to allopregnanolone or a 3-carboxylate of allopregnanolone, said compound being obtainable according to a process as disclosed herein.

[0037] In another aspect, the present invention relates to 3-carboxylate esters of allopregnanolone at high purity levels, and more particularly, containing 5α-pregn-2-en-20-one in an amount of 0.5% or less.

[0038] In another aspect, the present invention relates to allopregnanolone at high purity levels, and more particularly, comprising 5α-pregn-2-en-20-one and 3α-hydroxy-5α,17α-pregnane-20-one in an amount of 0.15% or less in total.

[0039] In another aspect, the present invention relates to a 3-carboxylate of allopregnanolone selected from the group consisting of:

[0040] 2,6-Dinitrobenzoate,

[0041] 2,4-Dinitrobenzoate,

[0042] Chloroacetate,

[0043] Dichloroacetate,

[0044] Trichloroacetate,

[0045] Cyanoacetate,

[0046] Fluoroacetate,

[0047] difluoroacetate, and

[0048] o-Nitrobenzoate.

[0049] These aspects and preferred embodiments thereof are also further defined below in the detailed description and in the claims. DETAILED DESCRIPTION

[0050] The present inventors have developed a chromatography-free method for preparing highly pure allopregnanolone. The method of the present invention is simple, inexpensive, reproducible and well suited for industrial scale.

[0051] definition

[0052] As used herein, the term "about" means a slight variation of a specified value, preferably within 10% of a specified value. However, the term "about" may mean a higher tolerance for variation, depending on, for example, the experimental technique used. The technician understands the variation of the specified value and the variation is within the context of the present invention. Further, in order to provide a more concise description, some quantitative expressions given herein are not modified with the term "about". It should be understood that, whether or not the term "about" is explicitly used, each amount given herein is meant to refer to an actual given value, and is also meant to refer to an approximate value of such a given value that would be reasonably inferred by a person of ordinary skill in the art, including equivalents and approximate values ​​to such a given value due to experimental and / or measurement conditions.

[0053] "Room temperature" or its abbreviation "rt" herein means that the reaction or method is carried out without heating or cooling. Generally, room temperature can be understood as a temperature between about 15°C and about 30°C, or more specifically a temperature between about 20°C and about 25°C.

[0054] The term "organic solvent" includes, for example, cyclic and acyclic ethers (such as Et 2 O、iPr 2 O、tBu 2 O, 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, tert-butanol), sulfoxides (DMSO) and mixtures thereof.

[0055] The term "water-soluble solvent" refers to a solvent that can be completely mixed with water, i.e., mixed in all proportions, or partially mixed, i.e., mixed in some proportions. Water-soluble solvents include, for example, organic solvents of which 5 g or more can be dissolved in 100 g of water at a temperature of 25° C. In a specific embodiment, the water-soluble solvent is selected from organic solvents having a miscibility in water at 25° C. greater than 50% by weight.

[0056] As used herein, the term "non-polar solvent" refers to a solvent whose polarity is low enough to induce crystal formation of polar compounds such as 3-esters of allopregnanolone. The selection of suitable non-polar solvents is well within the knowledge of the skilled person in view of the present disclosure. More particularly, the term "non-polar solvent" refers to a solvent having a Log P>2. Non-polar solvents have a low dielectric constant, 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 2 O、iPr 2 O、tBu 2 O, MeOtBu, 1,4-dioxane, tetrahydrofuran, methyltetrahydrofuran).

[0057] The term "Mitsunobu conditions" refers to conditions suitable for converting allopregnanolone to the 3-carboxylate of allopregnanolone by reaction with a specific carboxylic acid. The reagent used in Mitsunobu conditions is preferably a phosphine (typically triphenylphosphine (PPh 3)), azodicarboxylic acid esters and optional tertiary amine additives. Examples of azodicarboxylic acid esters include, but are not limited to, diethyl azodicarboxylate (DEAD), diisopropyl azodicarboxylate (DIAD), di-tert-butyl azodicarboxylate, 1,1'-(azocarbonyl)dipiperidine, dibenzyl azodicarboxylate and / or the like. In some embodiments, reagents for Mitsunobu conditions can be selected so that reagents can 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, polystyrene-based diphenylphosphine and / or the like can be used to replace triphenylphosphine. In some embodiments, triphenylphosphine or its equivalent and / or azodicarboxylic acid esters can be anchored to a resin such as, for example, a polystyrene resin. In some embodiments, cyanomethylenetri-n-butylphosphorane can be used as a reagent for Mitsunobu conditions.

[0058] The term "neutral conditions" preferably refers to hydrolysis reactions (transesterification) for alcohols such as MeOH, EtOH without the addition of any acid or base.

[0059] The term "mildly basic conditions" preferably refers to hydrolysis reactions using a base whose conjugate acid has a pKa equal to or less than 11. Suitable bases include, but are not limited to, alkali metal and alkaline earth metal carbonates and bicarbonates, such as potassium carbonate, sodium carbonate, barium carbonate, cesium carbonate, potassium bicarbonate, and sodium bicarbonate.

[0060] The term "severely alkaline conditions" preferably refers to the hydrolysis reaction using a base whose conjugate acid has a pKa equal to or greater than 12, for a short time and / or at a low temperature to avoid the production of 3α-hydroxy-5α,17α-pregnane-20-one in an amount greater than 0.5%. The selection of a suitable time and temperature is well within the knowledge of the skilled person in view of the present disclosure. Suitable bases include, but are not limited to, alkali metals and alkaline earth metals C 1-6 Alkoxides and hydroxides such as potassium methoxide, sodium methoxide, potassium ethoxide, sodium ethoxide, lithium hydroxide, potassium hydroxide, sodium hydroxide, and calcium hydroxide.

[0061] Unless otherwise stated, references to purity are to be understood as referring to HPLC purity.

[0062] Preparation and purification of 3-ester of allopregnanolone from allopregnanolone

[0063] In reproducing the conditions disclosed in the prior art, the inventors have noted that the appropriate choice of carboxylic acid to be reacted with allopregnanolone is important to avoid / reduce the formation of impurities, especially during the hydrolysis following the Mitsunobu reaction. Keeping the impurity levels within acceptable limits allows for the purification of both the intermediate 3-carboxylic acid ester and allopregnanolone without reliance on column chromatography. It has also been discovered that a method is needed to separate and purify the intermediate ester to obtain a method suitable for large-scale production of highly pure allopregnanolone. If the intermediate ester is not purified from certain impurities, but is used in a crude form for the subsequent hydrolysis reaction, the result of effective purification of the final allopregnanolone is not feasible.

[0064] In particular, the inventors have found that the Mitsunobu reaction always produces significant amounts, typically about 7-17%, of 5α-pregn-2-en-20-one (also referred to herein as impurity I or the eliminated impurity):

[0065]

[0066] 1 H NMR (400MHz, CDCl3): δ5.57(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).

[0067] 13 C NMR (400MHz, CDCl3): δ209.7,125.9,125.8,63.9,56.8,54.0,44.2,41.5,3 9.9,39.2,35.7,34.7,31.8,31.6,30.3,28.7,24.5,22.9,21.0,13.5,11.8.

[0068] Elimination of impurities is therefore inherent to the Mitsunobu reaction. Attempts to prepare allopregnanolone without isolation and purification of the intermediate 3-ester have either failed or provided unsatisfactory results because the final product needs to be subjected to complex column chromatography, which does not result in an acceptable degree of purity.

[0069] The present invention provides a method for preparing and purifying 3-ester of allopregnanolone from allopregnanolone. In a preferred embodiment, the method comprises:

[0070] - treating allopregnanolone with an organic acid having a pKa of 3 or less under Mitsunobu conditions;

[0071] - precipitating the crude reaction mixture in a solvent system comprising a mixture of water and an organic solvent; and

[0072] - Recrystallize the precipitate from a non-polar solvent.

[0073] The selection of an organic acid with a low pKa is crucial, especially because it allows the subsequent complete hydrolysis of the 3-ester of allopregnanolone. For example, formic acid (pKa = 3.75), benzoic acid (pKa = 4.20), acetic acid (pKa 4.75), orthomethoxybenzoic acid (pKa = 4.09), 3-nitrobenzoic acid (pKa 3.46), 4-nitrobenzoic acid (pKa 3.43) have been found to be unsuitable. In an embodiment, the carboxylic acid with a pKa ≤ 3 is selected from the group consisting of monofluoroacetic acid, difluoroacetic acid and trifluoroacetic acid, monochloroacetic acid, dichloroacetic acid 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 specific embodiment, the carboxylic acid is chloroacetic acid or dinitrobenzoic acid. Typically, the amount of the carboxylic acid is about 1-4 eq, more particularly about 2-3 eq.

[0074] Suitable photo-delay conditions preferably include a phosphine such as PPh 3 , azodicarboxylic acid esters and optional tertiary amine additives. The phosphine can be selected from the group consisting of triphenylphosphine, dicyclohexylphenylphosphine, diethylphenylphosphine, tributylphosphine, diphenyl-2-pyridylphosphine, 4-(dimethylamino)phenyldiphenylphosphine, isopropyldiphenylphosphine, tri-tert-butylphosphine, tri-n-octylphosphine, tricyclohexylphosphine, polystyrene-based diphenylphosphine or a mixture thereof. The azodicarboxylic acid ester can be selected from the group consisting of diethyl azodicarboxylate (DEAD), diisopropyl azodicarboxylate (DIAD), di-tert-butyl azodicarboxylate, 1,1'-(azocarbonyl)dipiperidine, dibenzyl azodicarboxylate or a mixture thereof. According to a preferred embodiment, the reagent for the Mitsunobu reaction is or includes triphenylphosphine (PPh 3 ) and an azodicarboxylate selected from DEAD and DIAD. Suitable amounts of phosphine and azodicarboxylate are generally in the range of about 1-2 eq. In a specific embodiment, about 1.5 eq of phosphine and about 1.4 eq of azodicarboxylate are added. Preferably, the azodicarboxylate, typically a solution of azodicarboxylate, is slowly added or added dropwise to a reaction mixture comprising allopregnanolone, phosphine and an organic acid. In a specific embodiment, the Mitsunobu reaction is carried out in the presence of NaOBz (e.g., about 1-2 eq).

[0075] The Mitsunobu reaction is usually carried out in the presence of an organic solvent, which can be selected from the group consisting of cyclic and acyclic ethers (e.g., Et 2 O、iPr 2 O、tBu2 In some 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 a specific embodiment, the solvent is 1,4-dioxane or a mixture of 1,4-dioxane and tetrahydrofuran (THF).

[0076] In an embodiment, allopregnanolone is treated with an organic acid at a suitable temperature of about 0-45° C. (e.g., about 15-45° C.) for a time sufficient for ester formation, typically between about 1-24 hours and more particularly between about 2-12 hours or between about 4-8 hours. In an embodiment, the reaction mixture may be stirred for a sufficient time and at a suitable temperature to complete ester formation. The reaction may be followed by thin layer chromatography (TLC or HPLC or UPLC).

[0077] After completion, water is preferably added to the crude reaction mixture and the resulting suspension may be filtered to obtain a wet cake comprising the 3-carboxylic acid ester of allopregnanolone.

[0078] For solvents that are not miscible with water, it is necessary to remove the solvent, preferably by evaporation, and replace it with a solvent that is miscible with water (a water-soluble solvent), such as 1,4-dioxane, MeOH, EtOH, IPA, ACN, etc., and then add water.

[0079] The isolation of the 3-ester of allopregnanolone is carried out according to the invention by precipitation in a solvent system comprising water and an organic solvent. Such precipitation allows the reduction / elimination of polar and basic impurities, such as by-products derived from phosphines and azodicarboxylates.

[0080] Preferably, the organic solvent used to precipitate 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, and even more particularly, it is 1,4-dioxane.

[0081] Different ratios of water and organic solvent can be used. According to one embodiment, the ratio of water to organic solvent in the solvent system is about 0.1:1 to 1:0.1, more specifically 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.

[0082] In a more specific embodiment, the 3-ester of crude allopregnanolone may be conveniently precipitated with a solvent system comprising water and an organic solvent in a ratio of about 1:1, wherein the organic solvent is 1,4-dioxane, acetonitrile, or isopropanol.

[0083] Once precipitated, the 3-ester of allopregnanolone is preferably dried (eg, under reduced pressure).

[0084] Purification of the 3-ester of allopregnanolone is achieved using recrystallization in a non-polar solvent. Advantageously, recrystallization allows the elimination or reduction of the amount of 5α-pregn-2-ene-20-one (i.e., impurity I or elimination of the impurity) 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 that can be used to induce 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 2 O、iPr 2 O、tBu 2 O, MeOtBu, methyltetrahydrofuran). In a specific embodiment, the non-polar solvent is selected from the group consisting of hexane, cyclohexane, heptane, toluene, iPr 2 O, MeOtBu or mixtures thereof; more particularly, cyclohexane, heptane; and even more particularly, heptane.

[0085] Thus, the present invention provides a 3-carboxylate of allopregnanolone at a high purity level, including 5α-pregn-2-en-20-one in an amount of no greater than 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).

[0086] Hydrolysis of 3-ester of allopregnanolone to allopregnanolone

[0087] The inventors have discovered that hydrolysis of certain 3-esters of allopregnanolone (e.g., esters of acetate, formate, isobutyrate, benzoate, o-methoxybenzoate, or 3- or 4-nitrobenzoate) produces large amounts of 3α-hydroxy-5α,17α-pregnane-20-one (also referred to herein as impurity II or the epimeric impurity):

[0088]

[0089] 1 H NMR (400 MHz, CDCl 3 ): δ4.00(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).

[0090] 13 C NMR (400MHz, CDCl 3 ): δ212.9,66.5,61.5,53.6,50.4,45.9,39.1,36.2,36.0,35.8,35.5,32.9,32.3,32.2,29.0,28.6,25.9,24.4,21.1,20.8,11.2.

[0091] For purification suitable for commercial scale, it is convenient to keep the level of 3α-hydroxy-5α,17α-pregnane-20-one no higher than 0.5% during the hydrolysis reaction. This can be successfully achieved with 3-esters of allopregnanolone derived from carboxylic acids having low pka (e.g., pka of 3 or less), which are readily hydrolyzed under the conditions disclosed in the present invention. Furthermore, if obtained according to the process disclosed above, such 3-esters of allopregnanolone also contain low amounts of 5α-pregn-2-en-20-one (≤0.5%).

[0092] In the present invention, the hydrolysis is carried out under neutral conditions (e.g. in the presence of an alcohol without the addition of any acid or base), mildly alkaline conditions (e.g. in the presence of a base whose conjugate acid has a pKa ≤ 11), or strongly alkaline conditions (e.g. in the presence of a base whose conjugate acid has a pKa ≥ 12, for no extended time and / or not at high temperature).

[0093] According to a specific embodiment, the ester is hydrolyzed under neutral conditions using an alcohol selected from methanol, ethanol, propanol, isopropanol, sec-butyl alcohol and tert-butyl alcohol. More particularly, the alcohol is MeOH or EtOH. In an embodiment, the ester is stirred in alcohol for a sufficient time (e.g., about 1-24h or about 1-12h) and a suitable temperature (e.g., about 15-40°C or about 15-25°C) to complete the hydrolysis. The reaction can be followed by thin layer chromatography (TLC).

[0094] According to a specific embodiment, the ester is hydrolyzed under mild alkaline conditions using a base selected from alkali metal and alkaline earth metal carbonates and bicarbonates such as potassium carbonate, sodium carbonate, barium carbonate, cesium carbonate, potassium bicarbonate and sodium bicarbonate. More particularly, the base is potassium carbonate, sodium carbonate, potassium bicarbonate or sodium bicarbonate, and even more particularly, the base is potassium carbonate, sodium carbonate. The reaction can be carried out in the presence of alcohols such as methanol, ethanol, propanol, isopropanol, sec-butyl alcohol or tert-butyl alcohol as a solvent. In an embodiment, the alcohol is MeOH or EtOH. In an embodiment, the ester is preferably treated with any mentioned base in an amount of about 0.2-3eq. or about 0.5-2eq. at a suitable temperature of about 15-40°C or about 15-30°C for a sufficient time for hydrolysis, generally between about 1-4h. In an embodiment, the reaction mixture can be stirred to complete hydrolysis at a sufficient time (e.g., about 1-4h) and a suitable temperature (e.g., about 15-40°C or about 15-30°C).

[0095] According to a specific embodiment, under severe alkaline conditions, a catalyst selected from alkali metals and alkaline earth metals C 1-6 The alkali of alkoxide and hydroxide hydrolyzes the ester. More particularly, the alkali is potassium methoxide, sodium methoxide, potassium ethoxide, sodium ethoxide, lithium hydroxide, potassium hydroxide, sodium hydroxide and calcium hydroxide, and even more particularly, the alkali is sodium methoxide, potassium hydroxide or sodium hydroxide. The reaction can be carried out in the presence of alcohols such as methanol, ethanol, propanol, isopropanol, sec-butyl alcohol or tert-butyl alcohol as solvents. In an embodiment, the alcohol is MeOH or EtOH. The use of a strong base (equal to or greater than a pKa of 12) requires keeping the hydrolysis reaction for a short time and / or at low temperatures to prevent 3α-hydroxy-5α, 17α-pregnane-20-one from being produced in an amount greater than 0.5%. In an embodiment, hydrolysis is carried out under severe alkaline conditions for no more than about 2h, more particularly no more than about 1.5h and even more particularly no more than about 1h 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 a time sufficient for hydrolysis (e.g., no more than about 2 h, more particularly no more than about 1.5 h, and even more particularly no more than about 1 h). In an embodiment, the reaction mixture can be stirred for a sufficient time (e.g., about 0.5-2 h) and a suitable temperature (e.g., no higher than 40° C. or 30° C.) to complete the hydrolysis.

[0096] Advantageously, allopregnanolone is obtained in a high purity, including 3α-hydroxy-5α,17α-pregnane-20-one (also referred to herein as impurity II or an epimeric impurity) in an amount of no greater than 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).

[0097] The isolation of allopregnanolone can be carried out, for example, by precipitation in a solvent system comprising water and an organic solvent. Preferably, the organic solvent used to precipitate 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, tert-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, and even more particularly, it is methanol.

[0098] Different ratios of water and organic solvent can be used. According to one embodiment, the ratio of water to organic solvent in the solvent system is about 0.1:1 to 1:0.1, more specifically 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.

[0099] In a more specific embodiment, crude allopregnanolone may be conveniently precipitated with 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.

[0100] Once precipitated, the allopregnanolone is preferably dried (eg, under reduced pressure).

[0101] If desired, further purification of allopregnanolone can be achieved, for example, using recrystallization. Examples of solvents that can be used to induce 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 specific embodiment, the non-polar solvent is selected from MeOH, EtOH, IPA, acetone, ACN, and the like, and even more particularly, MeOH / water.

[0102] Advantageously, recrystallization allows to eliminate or reduce the level of impurities in order to obtain a high purity level of allopregnanolone, including 5α-pregn-2-en-20-one and 3α-hydroxy-5α,17α-pregnane-20-one in an amount of 0.15% or less in total. In a preferred variant of the invention, the amount of both impurities in the final allopregnanolone is at most 0.10%.

[0103] The present invention allows obtaining allopregnanolone with a high degree of purity, such as higher than 98%, higher than 99% and even higher than 99.5%, in compliance with the requirements usually imposed by good manufacturing practices (GMP) for pharmaceutical products. In a specific embodiment, the purity of allopregnanolone is ≥ 99.5% and the total content of impurities I and II is equal to or lower than 0.15%, preferably equal to or lower than 0.10%.

[0104] In a preferred embodiment of the present invention, allopregnanolone is prepared by a method comprising the following steps:

[0105] - reacting allopregnanolone with a strong carboxylic acid under Mitsunobu conditions;

[0106] - precipitating the 3-carboxylate of allopregnanolone in a solvent system comprising water and an organic solvent;

[0107] - recrystallizing the precipitate of the 3-carboxylate of allopregnanolone in a non-polar solvent;

[0108] - subjecting the 3-carboxylate of the allopregnanolone thus obtained to hydrolysis under neutral conditions, mildly alkaline conditions or strongly alkaline conditions to obtain allopregnanolone; and

[0109] - precipitation and recrystallization, thereby obtaining allopregnanolone having a total content of the total impurities I and II of 0.15% or less.

[0110] The present disclosure provides allopregnanolone having a total content of impurities I and II equal to or less than 0.15% for preparing pharmaceutical compositions. The present disclosure also covers the use of allopregnanolone having a total content of impurities I and II equal to or less than 0.15% for preparing pharmaceutical compositions. The present disclosure includes methods for preparing the above-mentioned pharmaceutical compositions. These methods include combining allopregnanolone having a total content of impurities I and II equal to or less than 0.15% with at least one pharmaceutically acceptable excipient. Allopregnanolone and pharmaceutical compositions of allopregnanolone of the present disclosure can be used as drugs, in particular, drugs for treating postpartum depression (PPD). The present disclosure also provides a method for treating postpartum depression (PPD), the method comprising administering a therapeutically effective amount of allopregnanolone of the present disclosure to a subject in need of treatment.

[0111] Allopregnanolone 3-ester

[0112] In another aspect, the present invention provides the following 3-esters of allopregnanolone:

[0113] Pregnane-20-one, 3-(2,6-dinitrobenzoyloxy)-, (3α,5α)-

[0114] Pregnane-20-one, 3-(2,4-dinitrobenzoyloxy)-, (3α,5α)-

[0115] Pregnane-20-one, 3-(chloroacetyloxy)-, (3α,5α)-

[0116] Pregnane-20-one, 3-(dichloroacetyloxy)-, (3α,5α)-

[0117] Pregnane-20-one, 3-(trichloroacetyloxy)-, (3α,5α)-

[0118] Pregnane-20-one, 3-(cyanoacetoxy)-, (3α,5α)-

[0119] Pregnane-20-one, 3-(fluoroacetyloxy)-, (3α,5α)-

[0120] Pregnane-20-one, 3-(difluoroacetyloxy)-, (3α,5α)-

[0121] Pregnane-20-one, 3-(2-nitrobenzoyloxy)-, (3α,5α)-

[0122] These esters can be prepared by reacting allopregnanolone with the corresponding carboxylic acid (ie, 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.

[0123] It should be understood that the scope of the present disclosure includes all possible combinations of the embodiments disclosed herein.

[0124] The following examples merely illustrate certain embodiments of the present invention and are not to be construed as limiting the same in any way.

[0125] Example

[0126] 1.-Isopregnanolone is converted to alpha-3-chloroacetate of brexanolone

[0127]

[0128] Mitsunobu: pregnanolone (36 g, 113.2 mmol), PPh 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 the addition, the reaction mass was stirred at 35 °C until the reaction was complete (4 h). Impurity I (5α-pregnane-2-ene-20-one) was eliminated: about 9-12%.

[0129] Workup and isolation: After cooling at 25°C, water (540 mL) was added and the resulting suspension was stirred for 30 min and then cooled to 20-25°C. The suspension was filtered and the wet cake was washed with 100 mL of a mixture of dioxane / water 1:1. 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-pregnane-2-ene-20-one) was eliminated: 3.5%.

[0130] Recrystallization: The dry cake obtained is suspended in heptane (15 ml / g) and heated at 75° C. until completely dissolved. The solution is then cooled at 0 / 10° C. and the precipitate is filtered off. The solid is 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 of impurity I (5α-pregn-2-en-20-one): 0.3%.

[0131] 1 H NMR (400 MHz, CDCl 3 ): δ5.07(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).

[0132] 13 C NMR (400MHz, CDCl 3 ): δ209.5,166.7,72.6,63.8,56.7,54.0,44.2,41.4,39.9,39.0,35.8, 35.4,32.8,32.7,31.8,31.5,28.2,26.0,24.4,22.8,20.8,13.5,11.4.

[0133] Isolation of 5α-pregn-2-en-20-one from the dioxane / water mother liquor obtained in the precipitation of the ester: The mother liquor was diluted with more water (approximately 3 liters), a suspension was formed and the solid was isolated by filtration. The filtrate was suspended in 400 ml of heptane, stirred at 40° C. during one hour and filtered again, and evaporation of the liquid also gave a solid which was chromatographed and characterized by NMR:

[0134] 1H NMR (400MHz, CDCl3): δ5.57(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).

[0135] 13 C NMR (400MHz, CDCl3): δ209.7,125.9,125.8,63.9,56.8,54.0,44.2,41.5,3 9.9,39.2,35.7,34.7,31.8,31.6,30.3,28.7,24.5,22.9,21.0,13.5,11.8.

[0136] The NMR signals matched the data found in the literature (Tetrahedron (60) 2004, 11851-11860).

[0137] 2.-α-3-Chloroacetate is hydrolyzed to brenoside.

[0138]

[0139] Hydrolysis: Chloroacetate-brenolone (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 h.

[0140] Post-treatment and separation: 150 ml of water was added and cooled at 0 / 5°C and the solid was filtered. The wet cake was then washed with 60 mL of a mixture of methanol / water 1:0.5. The product was dried under reduced pressure at 50°C to give 22 g of crude bryosone (yield: 93%; purity:>99%). Dimer impurity II (3α-hydroxy-5α,17α-pregnane-20-one): 0.23%.

[0141] Recrystallization: The obtained dry cake was suspended in MeOH (15 vol) and stirred at 30°C for 30 min, then water (7.5 vol) was added and the resulting suspension was cooled to 5-10°C and filtered, the wet cake was dried to obtain 20 g of pure bryosone as a white solid (purity:> 99.8%; DSC 176°C), epimeric impurity II (3α-hydroxy-5α, 17α-pregnane-20-one): 0.08%. No elimination of impurity I (5α-pregnane-2-ene-20-one) was detected.

[0142] 3.- Hydrolysis of α-3-chloroacetate containing 0.73% of 5-α-pregn-3-en-20-one (Comparative)

[0143] Hydrolysis: Chloroacetate-brenolone (5 g, 12.7 mmol, with 0.73% elimination of impurities 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%).

[0144] Workup: 150 ml of water was added and cooled at 0 / 5°C and the solid was filtered. The wet cake was then washed with 7.5 mL of a mixture of methanol / water 1:0.5. The product was dried under reduced pressure at 50°C to obtain 3.77 g of crude brexanolone (yield: 93%; purity:>98.93%). Impurity I (5α-pregnant-2-ene-20-one) was eliminated: 0.52%.

[0145] 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 to obtain a filtered suspension. The wet cake was dried to obtain 3.3 g of bryosone as a white solid. Purity>99.7%, elimination of impurity I (5-α-pregnant-3-ene-20-one): 0.21%.

[0146] Thus, starting from an ester comprising more than 0.5% of eliminated impurity I, it is not possible to reduce its amount to less than 0.15%.

[0147] 4.- Hydrolysis of α-3-chloroacetate containing 0.5% impurity 5α-pregn-2-en-20-one

[0148] The method disclosed in Example 3 was followed, but using as starting material the alpha-3-chloroacetate containing 0.5% of eliminated impurity I. 3.2 g of brexanolone were obtained with a purity of >98% and a content of eliminated impurity I of about 0.13%.

[0149] Thus, starting from an ester containing 0.5% of impurity I eliminated, it is possible to reduce this amount to less than 0.15%.

[0150] 5.-Hydrolyzed α-3-chloroacetate containing 0.5% of 3α-hydroxy-5α,17α-pregnane-20-one

[0151] Hydrolysis: Chloroacetate-brenosulone (5 g, 12.7 mmol, doped with 0.5% of diastereomeric impurities) 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%),

[0152] Post-treatment: 150 ml of water was added and cooled at 0 / 5°C and the solid was filtered. The wet cake was then washed with 7.5 mL of a mixture of methanol / water 1:0.5. The product was dried under reduced pressure at 50°C to obtain 3.82 g of crude bryosone (yield: 94%; purity:>99.43%). Dimeric impurity II (3α-hydroxy-5α,17α-pregnane-20-one): 0.24%. Impurity I (5α-pregnane-2-ene-20-one) was eliminated: 0.18%.

[0153] 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 to obtain a filtered suspension, and the wet cake was dried to obtain 3.3 g of brucenolide as a white solid. Purity: 99.81%. Dimer impurity II (3α-hydroxy-5α, 17α-pregnane-20-one): 0.1%. Elimination of impurity I (5α-pregnane-2-ene-20-one): 0.02%.

[0154] 6.-Conversion of allopregnanolone to alpha-3-chloroacetate of brexanolone

[0155]

[0156] Mitsunobu: pregnanolone (14 g, 44 mmol), PPh 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 the addition, the reaction mass was stirred at 35 °C until the reaction was complete (4 h). Impurity I (5α-pregnane-2-ene-20-one) was eliminated: about 8%.

[0157] After cooling at 25° C., the reaction mass (250 ml) was divided into 7 samples (35 ml each, containing about 2 g of ester final product). Each sample was subjected to evaporation, the residue was redissolved in 30 ml of different kinds of water-miscible solvents (MeOH, ACN, DMF, IPA, acetone, dioxane) and induced precipitation with the same volume of water.

[0158] 7.-Conversion of allopregnanolone to alpha-3-trifluoroacetate of brexanolone

[0159]

[0160] Mitsunobu: pregnanolone (1 g, 3.14 mmol), PPh 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 the addition, the reaction mass was stirred at 35 ° C during one hour, and then 1.2 eq of NaOBz was added as a solid, and the reaction mixture was stirred again at 35 ° C during 20 h (starting material was still 6%).

[0161] Workup and precipitation: After cooling at 25°C, water (10 mL) was added and the resulting suspension was stirred for 30 min, then cooled to 10-15°C. The suspension was filtered and the wet cake was washed with 2 mL of a mixture of dioxane / water 1:1. The product was dried under reduced pressure at 50°C to give 0.75 of the ester, with 14.6% impurity elimination by HPLC.

[0162] 8.-α-3-benzoyl ester of brexanolone (comparison)

[0163]

[0164] Mitsunobu: pregnanolone (1 g, 3.14 mmol), PPh 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 the addition, the reaction mass was stirred at 35 ° C for 5 hours (elimination of impurities of about 8%).

[0165] After workup and precipitation cooling at 25°C, water (10 mL) was added and the resulting suspension was stirred for 30 min, then cooled to 10-15°C. The suspension was filtered and the wet cake was washed with dioxane / water 1: 1. The product was dried under reduced pressure at 50°C to give 0.1 g of >96% ester (elimination of impurities 0.5%).

[0166] Hydrolysis.- 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, only 4% of brexanolone final product and 9% of diastereomeric impurity were present along with the ester starting material.

[0167] As can also be understood in this example and also from Example 12, the benzoyl ester of brexanolone does not provide brexanolone with acceptable purity levels.

[0168] 9.-Brenosolone from allopregnanolone in a "one-pot" process using chloroacetic acid (comparative).

[0169]

[0170] Triphenylphosphine (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 the starting material (K1) was consumed, NaHCO was added. 3 7% aqueous solution (5ml / g). The organic phase is subsequently washed with water (5ml / g) and evaporated under reduced pressure. Trace toluene is removed by further distillation with methanol. The ester residue is suspended in methanol (10ml / g) and potassium carbonate (0.17g / g) is added. The suspension is stirred at 30°C during 1h (K2). The product is then precipitated by adding water (5ml / g). The reaction mass is cooled at 5°C, filtered and washed with a mixture of methanol / water 1:0.5 (2ml / g) (TH1). The resulting wet cake is further washed with heptane (5ml / g) (TH2).

[0171]

[0172] As can be appreciated, the one-pot process resulted in elevated amounts of eliminated impurities (9%) that could not be separated from the brexanolone.

[0173] 10.- Purification of brenoside from allopregnanolone in a "one-pot" process using trifluoroacetic acid and by chromatography (contrast)

[0174]

[0175] Triphenylphosphine (1.2 g), trifluoroacetic 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. for 24 h. After consumption of the starting material, the reaction mixture was evaporated under reduced pressure to a residue.

[0176] 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 to obtain 0.69 g of brexanolone, purity: 97%.

[0177] As can be appreciated, the one-pot process results in brenosanolone having low purity levels.

[0178] 11.-Bryosanolone acetate using toluene and sodium benzoate as solvent (comparative)

[0179]

[0180] DIAD (0.46 g), acetic acid (0.15 ml) and pregnanolone (0.5 g) along with toluene (15 ml) were charged in a round bottom flask. The reaction mass was cooled at 0 °C and PPh 3 (0.6 g) was added followed by the addition of NaOBz (0.34 g). The resulting reaction mass was kept stirring at 25°C during 15 h. After consumption of the starting material, the reaction mixture was evaporated under reduced pressure to a residue (elimination of impurities content about 14%).

[0181] This example shows that the elimination of impurities is inherent to the Mitsunobu reaction.

[0182] 12.- Hydrolysis of benzoic acid esters, nitrobenzoic acid esters and 2,6-dinitrobenzoic acid esters:

[0183]

[0184]

[0185]

[0186] As can be seen in the table, both benzoate and 4-nitrobenzoate are not easily hydrolyzed, requiring very harsh conditions, which lead to the formation of diastereomers (which are difficult to purify). However, dinitrobenzoate hydrolyzes in only one hour without generating diastereomers.

[0187] 13.-Hydrolysis of α-3-acetate to obtain brexanolone. (Comparative)

[0188]

[0189] 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) was added and the reaction mass was stirred at 40 ° C for 4 h. HPLC control showed that the starting material disappeared. The solvent was concentrated to a volume of 5 mL under reduced pressure. The mixture was poured into water (87 mL) and stirred at room temperature for 1 hour.

[0190] The precipitate was then filtered and the wet cake was washed with water (10 mL) and dried under vacuum at 50 °C.

[0191] HPLC analysis of the solid showed the presence of 8.6% of the epimeric impurity (II).

[0192] 14.-3-(2,4-dinitrobenzoyl ester) of brexanolone

[0193]

[0194] Allopregnanolone (0.5 g, 1.57 mmol), PPh 3 (0.6g, 1.5eq) and 2,4-nitrobenzoic acid (0.5g, 2.5eq) were suspended in THF (10mL). The mixture was cooled at 0 / 5°C and a solution of DIAD (0.45mL, 1.4eq) in THF (5mL) was added dropwise. At the end of the addition, the reaction mass was stirred at room temperature for 5 hours. 1mL of water was added, the solvent was evaporated and the residue was purified by column chromatography. 0.24g of solid product was obtained (yield 32.7%).

[0195] 15.-Bryosanolone 3-(3,5-dinitrobenzoyl ester)

[0196]

[0197] Allopregnanolone (0.5 g, 1.57 mmol), PPh 3 (0.6g, 1.5eq) and 3,5-dinitrobenzoic acid (0.5g, 2.5eq) were suspended in THF (10mL). The mixture was cooled at 0 / 5°C and a solution of DIAD (0.45mL, 1.4eq) in THF (5mL) was added dropwise. At the end of the addition, the reaction mass was stirred at room temperature for 5 hours. 1mL of water was added, the solvent was evaporated and the residue was purified by column chromatography. 0.43g of solid product was obtained (yield 53.5%).

[0198] Using the same procedure, 2-nitrobenzoyl ester of bryosanolone and 2,6-dinitrobenzoyl ester of bryosanolone were prepared.

[0199] 16.-Bryonolide 3-dichloroacetate

[0200]

[0201] Allopregnanolone (5.0 g, 15.7 mmol), PPh 3 (6.0g, 1.5eq) and dichloroacetic acid (3.4mL) were suspended in dioxane (55mL). The mixture was cooled at 15°C and a solution of DIAD (4.5mL, 1.4eq) in dioxane (20mL) was added dropwise. At the end of the addition, the reaction mass was stirred at 35°C until completion. 75mL of water was added, the mixture was stirred for 0.5h, filtered out and washed with 10mL (1:1) water / dioxane and dried. 3.8g of white solid product (yield 56.4%) was obtained.

[0202] 17.-Bryonolide 3-trichloroacetate

[0203]

[0204] Allopregnanolone (5.0 g, 15.7 mmol), PPh 3 (6.0g, 1.5eq) and trichloroacetic acid (6.45g) were suspended in dioxane (55mL). The mixture was cooled at 15°C and a solution of DIAD (4.5mL, 1.4eq) in dioxane (20mL) was added dropwise. At the end of the addition, the reaction mass was stirred at 35°C until completion. 75mL of water was added, the two phases were separated and the aqueous layer was extracted with 50mL of DCM. The organic layer was concentrated to obtain a yellow oil.

Claims

1. A method for preparing allopregnanolone, the method comprising: - reacting allopregnanolone with a strong carboxylic acid having a pKa ≤ 3 under Mitsunobu conditions; - precipitating the 3-carboxylate of allopregnanolone 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; - recrystallizing the precipitate of the 3-carboxylate of allopregnanolone in a non-polar solvent selected from the group consisting of hexane, cyclohexane, heptane, toluene, iPr2O, MeOtBu or mixtures thereof; and - subjecting the 3-carboxylate of the allopregnanolone thus obtained to hydrolysis under neutral conditions, mildly alkaline conditions or strongly alkaline conditions.

2. The method according to claim 1, wherein: The strong carboxylic acid is selected from the group consisting of mono-, di- and trifluoroacetic acid, mono-, di- and trichloroacetic acid, cyanoacetic acid and o- and dinitrobenzoic acid.

3. The method according to claim 1, wherein: The 3-carboxylate of allopregnanolone is subjected to alcohol hydrolysis without the addition of any acid or base.

4. The method according to claim 1, wherein: The 3-carboxylate of allopregnanolone is subjected to hydrolysis with a base whose conjugate acid has a pKa≤11.

5. The method according to claim 4, wherein: The conjugate acid of the base has a pKa≤11, and the base is an alkali metal or alkaline earth metal carbonate or bicarbonate.

6. The method according to claim 1, wherein: The 3-carboxylate of allopregnanolone is subjected to hydrolysis with a base whose conjugate acid has a pKa > 12, at an amount and temperature suitable to maintain the level of 3α-hydroxy-5α,17α-pregnane-20-one at 0.5% or less.

7. The method according to claim 6, wherein: The conjugate acid of the base has a pKa ≥ 12, and the base is an alkali metal or alkaline earth metal C 1-6 Alkoxide or hydroxide.

8. The method according to claim 6, wherein: The hydrolysis is carried out for no more than 2 h and / or at a temperature of 15-40°C.

9. The method according to claim 1, comprising the steps of: - reacting allopregnanolone with a strong carboxylic acid having a pKa ≤ 3 under Mitsunobu conditions; - precipitating the 3-carboxylate of allopregnanolone in a solvent system comprising water and an organic solvent; - recrystallizing the precipitate of the 3-carboxylate of allopregnanolone in a non-polar solvent; - subjecting the 3-carboxylic acid ester of the allopregnanolone thus obtained to hydrolysis under neutral conditions, mildly alkaline conditions or strongly alkaline conditions to obtain allopregnanolone; as well as - precipitating and recrystallizing to obtain allopregnanolone having a total content of impurities I and II of 0.15% or less, wherein the impurity I is 5α-pregn-2-en-20-one and the impurity II is 3α-hydroxy-5α,17α-pregnane-20-one.

10. A 3-carboxylic acid ester of allopregnanolone having a carboxylic acid with a pKa of ≤ 3, said ester comprising 5α-pregn-2-en-20-one in an amount of 0.5% or less.

11. A 3-carboxylate of allopregnanolone selected from the group consisting of: Pregnane-20-one, 3-(2,6-dinitrobenzoyloxy)-, (3α,5α)- Pregnane-20-one, 3-(2,4-dinitrobenzoyloxy)-, (3α,5α)- Pregnane-20-one, 3-(chloroacetyloxy)-, (3α,5α)- Pregnane-20-one, 3-(dichloroacetyloxy)-, (3α,5α)- Pregnane-20-one, 3-(trichloroacetyloxy)-, (3α,5α)- Pregnane-20-one, 3-(fluoroacetyloxy)-, (3α,5α)- Pregnane-20-one, 3-(difluoroacetyloxy)-, (3α,5α)- Pregnane-20-one, 3-(2-nitrobenzoyloxy)-, (3α,5α)- Pregnane-20-one, 3-(cyanoacetoxy)-, (3α,5α)-.

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