Process for preparing 3α-hydroxy-5α-pregnane-20-one (brenolone)

Abstract

The present invention relates to a novel process for synthesizing 3α-hydroxy-5α-pregnane-20-one, which is generally known as bryosone, wherein the corresponding cyclic 20-ketal compound or cyclic 20-ketalthiolate compound of formula (IV) is deprotected using iodine in an organic solvent.

Description

[0001] describe Field of the Invention

[0002] The present invention relates to the field of synthesis processes for active ingredients for pharmaceutical use, and in particular to a process for the industrial-scale preparation of 3α-hydroxy-5α-pregnane-20-one, an active ingredient that can be used to treat postpartum depression. The compound is also known by the name of allopregnanolone or the USAN name brexanolone (the USAN name brexanolone will be used for the rest of the text) and has the following formula:

[0003] Prior art

[0004] Brenolone was first described in patent GB 442,319 assigned to Schering-Kahlbaum Aktiengesellschaft in 1934. This patent reports a brief experimental description of the preparation of the compound in question starting from 3-hydroxy-bisnorcholenic acid, which, however, is of historical academic value only and has no practical use.

[0005] A more recent synthesis is described in the article "Synthesis of the allylic gonadal steroids, 3α-hydroxy-4-pregnen-20-one and 3α-hydroxy-4-androsten-17-one, and of 3α-hydroxy-5α-pregnan-20-one", JP Wiebe et al., Steroids (1985) Vol. 45, No. 1, pp. 39-51. Figure 1 The synthesis is reported in the scheme in ; in this synthetic route, 55 mg of brenosone (compound 10 in the figure) was obtained after chromatographic purification by reacting 150 mg of compound 9 with potassium tri-sec-butylborohydride (K selectride).

[0006] The article "Synthesis, metabolism, and pharmacological activity of 3α-hydroxysteroids which potentiate GABA-receptor-mediated chloride ion uptake in ratcerebral cortical synaptoneurosomes", RH Purdy et al., Journal of Medicinal Chemistry, 1990, Vol. 33 (6), 1572-1581, describes another synthetic route to brexanolone, which is described with reference to Scheme 1 on page 1573. In this synthesis, a brexanolone sample (intermediate 2a) (melting point = 174° C.-176° C.) was obtained from the corresponding 3β isomer (intermediate 1) in a final yield of 54% after crystallization and chromatographic purification.

[0007] Patent EP 2 688 902 B1 describes a further synthesis of this compound. In this patent (Example 5), a sample of brucetolone is obtained from the corresponding benzoate (Intermediate 3, Scheme 1 on page 8) with a final yield of 80% after chromatographic purification. However, the melting point of 161.7°C-162.8°C is significantly lower than the melting point described in the cited article by RHPurdy et al. This data, coupled with the fact that the European text does not contain purity information, casts doubt on the quality of the product obtained. After experimental examinations conducted by the inventors to evaluate the content of this patent, it seems possible that the product obtained in this patent is brucetolone containing 16% of diastereomers at position 17, which has a melting range (DSC) between 163.8°C<T<165.6°C.

[0008] In the course of their experimental activities, the inventors have consistently observed epimerization at position 17 when brexanolone or one of its pregnane structure synthetic intermediates with an unprotected carbonyl function is exposed to acidic or basic pH, capable of generating an enolate intermediate or an enol in the course of the reaction.

[0009]

[0010] The formation of the double bond generates an intermediate with a planar structure between positions 17 and 20, which evolves in the reaction regenerating the single bond, wherein the steric configuration is both β (major) and α (minor), but quantitatively relevant for the purpose of the desired quality of the drug product. Despite the research activities carried out by the inventors, it has proven difficult, if not impossible, to determine the pH limits at which these undesirable reactions occur, since the reactions can occur in organic or aqueous organic solvents, at a variety of concentrations and temperatures and with different acids or bases.

[0011] In the course of their experimental activities, the inventors also attempted to protect the carbonyl group at position 20 as a ketal using ethylene glycol, but all subsequent hydrolysis tests resulted in epimerization at position 17. The conditions tested for ketal deprotection were: 1 M HCl in methanol at 25 °C; 10 mol% p-toluenesulfonic acid (PTSA) in acetone at 55 °C; 5 mol% PTSA in acetone at 25 °C; 5 mol% PTSA in acetone at 0 °C; 1 mol% PTSA in acetone at 25 °C; 5 mol% pyridinium p-toluenesulfonate (PPTS) in acetone at 25 °C; anhydrous FeCl in an acetone / dichloromethane mixture at 25 °C. 3 .

[0012] Eliminating or at least limiting this undesirable chemical reaction (epimerization) is relevant both to process yield and to the quality of brexanolone.

[0013] The article "Improved syntheses of aromatase inhibitors and neuroactivesteroids efficient oxidations and reductions at key positions for bioactivity", A. S. Campos Neves et al., Tetrahedron, 55 (1999) pp. 3255-64 describes the epimerization of steroids similar to brexanolone at position 17 in an acid environment (Scheme 1 on page 3257); this undesirable reaction involves the formation of 12% of by-products, which are eliminated by crystallization with a yield of 60%, obtaining an intermediate with a purity of 95%-97%, which is still far from pharmaceutical quality.

[0014] The object of the present invention is to provide a synthetic route for the preparation of brexanolone which is industrially applicable and allows obtaining a product of pharmaceutical quality, overcoming the criticalities associated with the processes described in the prior art. SUMMARY OF THE INVENTION

[0016] This object is achieved by the present invention, which relates to a method for preparing bryosperone, which comprises deprotecting a cyclic ketal or a cyclic thioketal of the general formula (IV) using iodine in an organic solvent according to the following reaction scheme:

[0017]

[0018] wherein in the case of ketals, X=O (oxygen), and in the case of thioketals, X=S (sulfur), and R is selected from ethylene (—CH 2 -CH 2 -), propylene (-CH 2 -CH 2 -CH 2 -) and 2,2-dimethylpropylene (-CH 2 -C(CH 3 ) 2 -CH 2 -) group.

[0019] The brenosone obtained by means of this preparation process has an amount of the epimer at position 17 lower than 0.15%, as determined by HPLC analysis.

[0020] In a preferred embodiment, the present invention relates to a process for synthesizing brexanolone, the process comprising the following steps:

[0021] 1) catalytic hydrogenation of the double bonds at positions 5 and 6 of pregnenolone to obtain the corresponding saturated steroid of formula (I), wherein the hydrogen atom at position 5 of the steroid skeleton is arranged in a spatial arrangement α:

[0022]

[0023] 2) Reversing the spatial orientation of the 3-hydroxyl group of the compound of formula (I), which is protected as a benzoate at the end of the reaction, to obtain a compound of formula (II):

[0024]

[0025] 3) Protecting the carbonyl group at position 20 of compound (II) as ketal or thioketal to obtain a compound of formula (III), wherein X=O or X=S, and R is selected from ethylene (-CH 2 -CH 2 -), propylene (-CH 2 -CH 2 -CH 2 -) and 2,2-dimethylpropylene (-CH 2 -C(CH 3 ) 2 -CH 2 -) group:

[0026]

[0027] 4) hydrolyzing the benzoate of compound (III) to obtain a compound of general formula (IV) in which X and R have the meanings reported above:

[0028]

[0029] 5) Deprotecting the 20-position of the compound of general formula (IV) to obtain brenosulone:

[0030]

[0031] In a second aspect of the present invention, the present invention relates to the following intermediate synthetic compounds:

[0032]

[0033] That is, a compound of formula (III) wherein R is ethylene both in the case where X=S and in the case where X=O; and a compound of formula (IV) wherein R is ethylene in the case where X=S. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 : NMR spectrum of the brexanolone-epimer mixture obtained in the preparation described in Example 6.

[0036] Figure 2 : NMR spectrum of the brexanolone-epimer mixture obtained in the preparation described in Example 7.

[0037] Figure 3 : NMR spectrum of diastereomer-free brexanolone. An extension from 2.3 ppm to 3.2 ppm is reported.

[0038] Figure 4 : DSC thermogram of pure brexanolone obtained according to the procedure of Example 5. DETAILED DESCRIPTION OF THE INVENTION

[0040] In the present description and claims, if the name of a compound is inconsistent with the structural formula reported for it, the latter must be assumed to be correct.

[0041] In a first aspect of the present invention, the object of the present invention is a process for preparing bryosole, which comprises deprotecting a cyclic ketal or a cyclic thioketal of the general formula (IV) using iodine in an organic solvent according to the following reaction scheme:

[0042]

[0043] wherein in the case of ketals, X=O (oxygen), and in the case of thioketals, X=S (sulfur), and R is selected from ethylene (—CH 2 -CH 2 -), propylene (-CH 2 -CH 2 -CH 2 -) and 2,2-dimethylpropylene (-CH 2 -C(CH 3 ) 2 -CH 2 -) group.

[0044] In a preferred embodiment of the present invention, R is an ethylene group -CH 2 -CH 2 -.

[0045] Depending on whether the compound of formula (IV) is a ketal or a thioketal, the reaction conditions are different.

[0046] In the case where X═O, the ketal must be hydrolyzed in a neutral environment, avoiding the presence of acids or bases. Preferably, for the deprotection of the ketal, iodine is used in an amount between 1 and 10% by weight relative to the amount of compound (IV).

[0047] As reaction solvent it is possible to use anhydrous acetone or a mixture of dichloromethane and acetone operating in the absence of water. Operating with anhydrous acetone or with a mixture of dichloromethane and acetone in the absence of water is decisive for the outcome of the reaction.

[0048] The reaction temperature is between -5°C and the reflux temperature of the reaction mixture.

[0049] The reaction time is between 5 minutes and 90 minutes. Preferably, it is operated within the range between 10 minutes and 45 minutes.

[0050] In the case of X = S, the thioketal is reacted with iodine in an organic solvent at a temperature between -30°C and 20°C in the presence of a solid inorganic base such as sodium carbonate, lithium carbonate, potassium carbonate, calcium carbonate, sodium bicarbonate, lithium bicarbonate and potassium bicarbonate; the preferred base is sodium bicarbonate. As organic solvent, dichloromethane (DCM), methanol or preferably a mixture thereof can be used.

[0051] The reaction time is between 2 hours and 36 hours and is related to the amount of iodine used in the reaction and the reaction temperature. Preferably, operation is carried out in a temperature range between -25°C and -5°C for a period between 1 hour and 18 hours.

[0052] The inventors have observed experimentally that, by means of this synthetic process, it is possible to obtain brenosone with an amount of diastereomers at position 17 lower than 0.15% (value determined by HPLC analysis), which corresponds to the maximum amount allowed by the ICH guidelines published by the European Medicines Agency (EMA) for the presence of identified impurities in active ingredients and intermediates (API) of the pharmaceutical industry, without having conducted pharmacological studies that would allow the presence of said impurities in higher amounts. This result is possible by operating under the conditions of the invention, without resorting to chromatographic purification or to specific purification techniques applied to the brenosone produced.

[0053] In particular, in the case of X = S, the brexanolone obtained according to the process of the invention is substantially free of its diastereomer at position 17 and therefore does not require a specific purification treatment to eliminate this impurity. In the case of X = O, a minimum amount of less than 0.5% of the diastereomer at position 17 is observed in the crude product, which is easily reduced to a value of less than 0.1% by crystallization.

[0054] In a preferred embodiment, the present invention relates to a complete process for the industrial scale synthesis of brexanolone comprising steps 1 to 5 reported above.

[0055] The reactions of step 1) from pregnenolone to the compound of formula (I), step 2) from the compound of formula (I) to the compound of formula (II), and step 4) from the compound of general formula (III) to the compound of general formula (IV) can be performed according to the instructions available in the literature, such as those reported in patent EP 2 688 902 B1; therefore, these steps are described in outline below.

[0056] In step 1), intermediate (I) is produced starting from pregnenolone:

[0057]

[0058] The starting pregnenolone is a commercially available compound. Methods for performing this step are known in the art.

[0059] For the purposes of the present invention, preferred conditions are to use 5% palladium on carbon (Pd / C) as the hydrogenation catalyst. The amount used is about 5% of the weight of the pregnenolone to be hydrogenated. The hydrogenation pressure is between 2 bar and 5 bar. The reaction solvent is tetrahydrofuran (THF). The hydrogenation temperature is between 35° C. and 45° C. The hydrogenation time is variable between 3 hours and 7 hours, and the reaction is terminated when the hydrogen consumption stops.

[0060] In step 2), the spatial orientation of the 3-hydroxyl group of intermediate (I) is reversed, and at the end of the reaction, the hydroxyl group is protected as a benzoate (intermediate (II)):

[0061]

[0062] This step can also be performed according to methods known in the art.

[0063] In the case of the present invention, this step is preferably carried out by preparing a benzoic acid ester of the 3-hydroxyl group by reacting the intermediate (I) with benzoic acid in tetrahydrofuran (THF) as a solvent, and allowing the system to react with triphenylphosphine and diisopropyl azodicarboxylate (DIAD) at 15°C to 25°C for 14 hours to 20 hours.

[0064] The intermediate (II) thus obtained is then purified by refluxing it in an alcohol, preferably methanol, to obtain a quality suitable for the further synthesis.

[0065] In step 3), the carbonyl group at position 20 of intermediate (II) is protected as ketal or thioketal to obtain an intermediate of general formula (III), wherein X=O or X=S, and R is selected from ethylene (-CH 2 -CH 2 -), propylene (-CH 2 -CH 2 -CH 2 -) and 2,2-dimethylpropylene (-CH 2 -C(CH 3 ) 2 -CH 2 -) group:

[0066]

[0067] Ketal (X = O) can be obtained by refluxing a mixture of intermediate (II) with toluene, ethylene glycol, propylene glycol or 2,2-dimethylpropylene glycol, triethyl orthoformate and p-toluenesulfonic acid. In this case, the diastereomer 17α is formed in an amount between 5% and 10%, which is eliminated by crystallizing the crude intermediate (III) with an organic solvent such as, for example, ethyl acetate, isopropyl acetate or 2-propanol.

[0068] In the selected boron trifluoride etherate (BF 3 ·Et 2 O), zinc iodide (ZnI 2 ), titanium tetrachloride (TiCl 4 ), tin dichloride (SnCl 2 ), magnesium iodide (MgI 2 ), lithium perchlorate (LiClO 4), aluminum trifluoromethanesulfonate (also known as aluminum triflate, Al(OTf) 3 ), lithium tetrafluoroborate (LiBF 4 ) and cobalt dichloride (CoCl 2 ) in the presence of a catalyst selected from acetic acid, dichloromethane (CH 2 Cl 2 ), ether, acetonitrile, tetrahydrofuran (THF) and 1,2-dichloroethane and for a period of time between 8 hours and 36 hours, it is possible to obtain thioketal. Preferred reaction conditions envisage the use of acetic acid as solvent, the use of BF 3 Etherates (BF 3 ·Et 2 O) as a catalyst at a temperature of 30±5°C for a period of 24 hours to 28 hours.

[0069] In step 4), the benzoic acid ester of the intermediate of general formula (III) is hydrolyzed to obtain the intermediate of general formula (IV):

[0070]

[0071] This step is executable according to the instructions available in the prior art.

[0072] In the present case, this step is preferably carried out by refluxing intermediate (III) in methanol in the presence of a strong base such as sodium hydroxide for a period of 20h-28h and monitoring the progress of the reaction, for example by TLC.

[0073] Finally, step 5) of the process corresponds to the deprotection method of the ketal or thioketal according to the first aspect of the invention described in detail above.

[0074] The present invention will be further illustrated by the following examples.

[0075] Instruments, methods and experimental conditions

[0076] NMR:

[0077] NMR spectrometer JEOL 400 YH (400 MHz); JEOL Delta software v5.1.1;

[0078] Spectra recorded in deuterated solvents: Chloroform-d, D 99.8%, containing 0.1% (v / v) tetramethylsilane (TMS) as internal standard; Chloroform-d, “100%”, D 99.96%, containing 0.03% (v / v) TMS; CD 3 OD; and DMSO-d 6 .

[0079] MS

[0080] Instrument: DSQ-trace Thermofisher;

[0081] Sample introduction - direct exposure probe (dep);

[0082] Chemical ionization (CI) of methane;

[0083] Source temperature: 200°C.

[0084] DSC

[0085] Instrument: Perkin Elmer model Diamond;

[0086] Perkin Elmer standard aluminum capsule and cap, code 02190041;

[0087] Scanning speed: 10℃ / min;

[0088] Temperature range: from -20°C to 220°C.

[0089] HPLC

[0090] For pregnenolone, intermediate (I) and brexanolone

[0091] Agilent chromatography system model 1260 Infinity; UV detector model 1260 DAD VL; column Advanced Chromatography Technologies ACE 3 C18-PFP 150×3 mm, 3 μm; mobile phase A: water, mobile phase B: acetonitrile, isocratic method 50 / 50, wavelength 200 nm, flow rate 0.5 ml / min, injection volume 5 μl, run time 25 min, temperature 25 °C;

[0092] For intermediate (II), intermediate (III) and intermediate (IV)

[0093] Agilent chromatography system model 1260 Infinity; UV detector model 1260 DAD VL; column: SupelcoAscentis C8150×4.6mm, 5μm; mobile phase A: water, mobile phase B: methanol; 85 / 15 isocratic method, wavelength 216nm, flow rate 1ml / min, injection volume 5μl, running time 45min, temperature 25°C.

[0094] LC / Ms / Ms System

[0095] Agilent chromatography system model 1100 with UV DAD detector connected to API 2000 mass spectrometer by Applied Biosystem.

[0096] TLC

[0097] MERCK: TLC silica gel 60F 254 Aluminum sheet 20×20cm, code 1.0554.0001.

[0098] HPTLC

[0099] MERCK: HPTLC silica gel 60 F with concentration zone 10×2.5 cm 254 , code 1.13727.0001.

[0100] TLC detector

[0101] Acid solution of cerium phosphomolybdate. Preparation: 25 g of phosphomolybdic acid hydrate (Aldrich), 10 g of cerium (IV) sulfate hydrate (Aldrich) and 600 ml of water are stirred until dissolved with 60 ml of 95%-98% sulfuric acid (Aldrich 258105); make it up to a final volume of 1000 ml with water; impregnate a plate with the solution and then heat until blue coloration occurs.

[0102] TLC and HPTLC Eluents

[0103] The conditions for the TLC examination are reported in the Experimental Procedures.

[0104] Specifications for the experimental description of the examples:

[0105] Unless otherwise indicated, water used in the experimental descriptions should be understood as pure water.

[0106] Unless otherwise indicated, organic solvents used in the experimental descriptions are understood to be of "technical grade".

[0107] Unless otherwise indicated, reagents and catalysts used in the experimental descriptions are understood to be of commercial quality.

[0108] Example 1

[0109] This example relates to step 1) of the process of the invention.

[0110]

[0111] 10 g of pregnenolone were dissolved in 300 ml of THF at 20±5° C. and the solution was loaded into the hydrogenation reactor. 0.50 g of 5% Pd / C were added. Two vacuum / hydrogen cycles were performed and finally hydrogen was loaded up to a pressure of 4 bar.

[0112] The reagent mixture was heated to 40 ± 5 °C and maintained under stirring until the end of hydrogen consumption was observed (approximately 5 hours). 1 H-NMR examination noted the complete disappearance of the starting pregnenolone.

[0113] The reagent mixture is cooled to 20 ± 5 ℃, and filtered on diatomaceous earth, washed with 50ml of THF. Remove THF with a rotary evaporator and heat until 40 ℃-45 ℃. Add methyl ethyl ketone (MEK, 50ml), and the mixture is distilled with a rotary evaporator, heated until 45 ℃ twice. Load 30ml of MEK, and the mixture is stirred under reflux for 10 minutes (no dissolution is noted). The mixture is cooled to 0 ℃, and kept under stirring for 1h, after which it is filtered and washed with cold MEK. The solid is dried in a 40 ℃ baking oven for 4h, obtaining 9.2g of a white solid (intermediate (I)).

[0114] Pregnenolone Analysis:

[0115] 1 H-NMR, CDCl 3 : 5.36-5.35 (1H, m, H-6); 3.57-3.49 (1H, m, H-3); 2.54 (1H, t, J = 9Hz, H-17); 2.34-2.15 (2H, m); 2.13 (3H , s, H-21); 2.07-1.97 (2H, m); 1.89-1.82 (2H, m); 1.73-1.41 (10H, m); 1.29-1.06 (3H, m); 1.01 (3H, s, CH 3 ); 0.63(3H, s, CH 3 );

[0116] Mass spectrum: 316 (M + ).

[0117] Analysis of intermediate (I):

[0118] 1 H-NMR, CDCl 3 :3.64-3.56(1H,m,H-3);2.52(1H,t,J=9Hz,H-17);2.2-2.1(1H,m);2.11(3H,s,H-2 1); 2.00 (1H, dt, J = 3-11.4Hz); 1.84-1.53 ​​(8H, m); 1.50-0.84 (11H, m); 0.80 (3H, s, CH 3 ); 0.68 (1H, dt, J = 4-12Hz); 0.60 (3H, s, CH 3 );

[0119] Mass spectrum: 318 (M + ).

[0120] Example 2

[0121] This example relates to step 2) of the process of the invention.

[0122]

[0123] At 20 ℃-25 ℃ under nitrogen atmosphere, 15.8g of the intermediate (I) obtained in the previous example was loaded into a flask. 390ml of THF was added, and the mixture was stirred until dissolved. 9.1g of benzoic acid and 19.4g of triphenylphosphine were added. In the reaction mixture, 15.3ml of 94% DIAD was added dropwise over 30 minutes, keeping the temperature below 30 ℃. The mixture was stirred at 20±5 ℃ for 16 hours, and a TLC check was performed to observe the complete disappearance of the starting reagent. The TLC check was performed under the following conditions:

[0124] - Start: In CH 2 Cl 2 middle;

[0125] - Sample: reaction mixture in ethyl acetate. The organic phase is deposited;

[0126] - Eluent: toluene / isopropyl acetate 8 / 2; Plate: HPTLC glass;

[0127] -Detector: UV / cerium phosphomolybdate.

[0128] THF was distilled in a rotary evaporator under vacuum at 45±5° C. The residue obtained was dissolved with 250 ml of ethyl acetate at 20±5° C. and 200 ml of NaHCO were added. 3 saturated aqueous solution. Stirring was continued at 20 ± 5 °C for 10 minutes and then the phases were separated. The organic phase was treated with 200 ml of NaHCO 3saturated aqueous solution, and the phases are separated. The aqueous phase is recombined and re-extracted with ethyl acetate. The organic phase is recombined and washed with 130 ml of an aqueous solution saturated with NaCl. The phases are separated, and the organic phase is washed with 100 ml of water. The phases are separated, and the organic phase is distilled at 45 ± 5 ° C under reduced pressure using a rotary evaporator. 150 ml of heptane is added, and the mixture is distilled at 40 ± 5 ° C under reduced pressure using a rotary evaporator until all solvents are removed, and then for another 30 minutes. 62.0 g of an almost white solid is obtained. The solid is suspended in 120 ml of MeOH at 20 ± 5 ° C, and the suspension is heated at reflux for 10 minutes; no complete dissolution is observed. The system is allowed to cool naturally to 20 ± 5 ° C, and then cooled to 0 ± 5 ° C with an ice bath, at which temperature it is kept under stirring for 1 hour. The mixture is filtered on a Buchner funnel by washing with 40 ml of MeOH pre-cooled at 0 ± 5 ° C. The solid was dried in an oven at 45±5°C under vacuum for 16 hours to obtain 19.7 g of a white solid (Intermediate (II)).

[0129] Analysis of intermediate (II):

[0130] 1 H-NMR, CDCl 3 : 8.07 (2H, dd, J = 1.5-8.6 Hz, aromatic H); 7.59-7.53 (1H, m, aromatic H); 7.46 (2H, t, J = 7.8 Hz, aromatic H); 5.29 (1H, m, H-3); 2.54 (1H, t, J = 9 Hz, H-17); 2.20-0.80 (22H, m); 2.12 (3H, s, H-21); 0.85 (3H, s, CH 3 ); 0.62(3H, s, CH 3 );

[0131] Mass spectrum: 423 (M + +1).

[0132] Example 3

[0133] This example involves the case where X = S and R = ethylene -CH 2 -CH 2 -step 3 of the process of the present invention).

[0134]

[0135] In a reaction flask under nitrogen, 17 g of the intermediate (II) obtained in the previous example and then 85 ml of glacial acetic acid were charged: no complete dissolution was noted. 3.7 ml of 1,2-ethanedithiol were added. 3.7 ml of boron trifluoride-ether complex (BF 3 ·Et 2 O). Another 3.8 ml of ethanedithiol was added in batches, the system was kept at 30±5° C. under stirring for a total of 26 h, and the progress of the reaction was checked by TLC (constant intermediate (II) spot). The TLC check was carried out under the following conditions:

[0136] - Starting: in THF;

[0137] -Sample: NaOH 10% + toluene;

[0138] - Eluent: heptane / isopropyl acetate 8:2; Plate: silica gel;

[0139] -Detector: UV / cerium phosphomolybdate.

[0140] After the reaction is complete, the mixture is cooled to 20 ° C. A 10 wt / % NaOH solution cooled to 0 ° C is prepared separately by diluting 226 g of 30 wt / % NaOH solution with 452 g of water. In the case of no more than 10 ° C, the reaction mixture is poured into a cold soda solution (the formation of a precipitate is observed). The reaction flask is washed with water (150 ml), and the water is added to the alkaline mixture containing the product. The suspension thus obtained is stirred for 1 h at a temperature of ≤ 20 ° C. The solid is filtered on a Buchner funnel and washed with water (500 ml) until a neutral pH is reached. The solid on the filter is dissolved with dichloromethane (400 ml), washed with 1.5% sodium hypochlorite solution, and then washed with water. By removing the solvent by evaporation under reduced pressure at 45 ° C, 22.6 g of a yellow solid (crude intermediate (III)) is obtained. 20 g of this intermediate is loaded into a flask under nitrogen. 60 ml of MEK is added, and the mixture is heated under reflux (complete dissolution is not observed). The system was allowed to cool naturally to 25°C, and then cooled to 0°C for 1 hour. The mixture was filtered on a Buchner funnel, and the solid was washed with cold MEK. The solid was dried under reduced pressure at 45°C for 2 hours to obtain 10.3 g of a white solid (Intermediate (III)).

[0141] Analysis of intermediate (III):

[0142] 1 H-NMR, CDCl 3: 8.07 (2H, dd, J = 1.4-8 Hz, aromatic H); 7.56 (1H, dt, J = 1.4-7.3 aromatic H); 7.46 (2H, t, J = 7.5 Hz, aromatic H); 5.30-5.28 (1H, m, H-3); 3.40-3.13 (4H, m, SCH 2 CH 2 S); 2.12-0.89 (23H, m); 1.87 (3H, s, H-21); 0.84 (3H, s, CH 3 ); 0.81(3H, s, CH 3 );

[0143] 13 C-NMR, CDCl 3 :165.92 (C=O); 132.70 (aromatic CH); 131.14 (aromatic C); 129.53 (2 aromatic CH); 128.32 (2 aromatic CH); 71.41 (C, SCS); 70.70 (CH, C-3); 60.76 (CH); 56.41 (CH); 54.14 (CH); 44.27 (C); 41.41 (CH 2 ); 40.39(CH); 40.06(CH 2 ); 37.29 (CH 2 ); 35.86(C); 35.61(CH 3 , C-21); 35.10(CH); 33.17(CH 2 ); 32.97 (CH 2 ); 31.74 (CH 2 ); 28.32 (CH 2 ); 26.98 (CH 2 ); 26.29 (CH 2 ); 23.94 (CH 2 ); 20.73 (CH 2 ); 13.36 (CH 3 ); 11.41(CH 3 );

[0144] Mass spectrum: 498 (M + ).

[0145] Example 4

[0146] This example involves the case where X = S and R = ethylene -CH 2 -CH 2 -step 4 of the process of the present invention).

[0147]

[0148] Under a stream of nitrogen, 10.1 g of the intermediate (III) obtained in the previous example was loaded into a flask. 250 ml of methanol was added; no complete dissolution was noted. 20.3 g of NaOH was added, and the mixture was kept under stirring and heated under reflux for 25 hours. A TLC check was performed and completion of the reaction was observed. The TLC check was performed under the following conditions:

[0149] - Initial: in dichloromethane;

[0150] -Reaction: in dichloromethane;

[0151] - Eluent: heptane / isopropyl acetate 8:2; Plate: silica gel;

[0152] -Detector: UV / cerium phosphomolybdate.

[0153] The reaction mixture was cooled to 25°C and 300 ml of water was added. Methanol was distilled under reduced pressure using a rotary evaporator. Formation of a precipitate was observed, 350 ml of dichloromethane was added and the system was kept at 35°C under stirring for 10 minutes, noting complete dissolution of the solid. The phases were separated and the aqueous phase was re-extracted at 35°C with 100 ml of dichloromethane. The organic phases were recombined and washed with water (3 times x 300 ml) until a neutral pH was reached. The solvent was removed by evaporation under reduced pressure to obtain 8.3 g of a white solid (intermediate (IV)).

[0154] Analysis of intermediate (IV):

[0155] 1 H-NMR, CDCl 3 :4.04(1H,m,H-3);3.4-3.1(4H,m,SCH 2 CH 2 S); 2.11-0.86 (23H, m); 1.86 (3H, s, H-21); 0.80 (3H, s, CH 3 ); 0.78 (3H, s, CH 3 );

[0156] 13 C-NMR, CDCl 3 : 71.43 (C, SCS); 66.58 (CH, C-3); 60.70 (CH); 56.43 (CH); 54.11 (CH); 44.25 (C); 41.39 (SCH 2 ); 40.08(CH 2 ); 39.07 (CH); 37.29 (SCH2 ); 36.04(C); 35.84(CH 2 ); 35.58 (CH 3 , C-21); 35.12(CH); 32.12(CH 2 ); 31.81(CH 2 ); 28.98 (CH 2 ); 28.48 (CH 2 ); 26.97 (CH 2 ); 23.93 (CH 2 ); 20.66 (CH 2 ); 13.35(CH 3 ); 11.18 (CH 3 );

[0157] Mass spectrum: 394 (M + ).

[0158] Example 5

[0159] This example relates to step 5) of the process according to the invention starting from the thioketal.

[0160]

[0161] Under nitrogen, 8 g of intermediate (IV) obtained in the previous example was loaded into a flask. Dichloromethane (56 ml) and methanol (88 ml) were added; no complete dissolution was observed. The system was cooled to -20 °C and NaHCO was added. 3 (15.3 g). Add I 2 (15.5 g), and the system was kept at -20 °C under stirring for 4 h, and NaHCO was added in batches 3 (4.95 g) and I 2 (5.15 g), and the reaction progress was checked by TLC (complete reaction). TLC check was carried out under the following conditions:

[0162] - Starting: in THF;

[0163] -Reaction: In Na 2 S 2 O 3 The mixture was added to an aqueous solution, extracted with dichloromethane, and the organic phase was seeded;

[0164] - Eluent: heptane / isopropyl acetate 1:1; Plate: silica gel;

[0165] -Detector: UV / cerium phosphomolybdate.

[0166] By adding Na2 S 2 O 3 ·5H 2 The reaction was quenched with a solution of 1H 2 O (45.3 g) in water (90 ml), keeping the temperature below -15°C (an exotherm was noted, but no problems with freezing of the existing water were observed). The system was warmed to 20°C and the suspension was filtered on a decalite panel, washing the solid on the filter with dichloromethane preheated to 35°C. The phases were separated and the aqueous phase was extracted with dichloromethane; the organic phases were recombined and washed with water.

[0167] The solvent was removed by evaporation under reduced pressure using a rotary evaporator to obtain 6.2 g of a yellow powder (crude brexanolone), which was examined in HPLC; the HPLC chromatogram of the crude brexanolone thus obtained showed that the content of the diastereomer was undetectable.

[0168] 4.5 g of crude bryosanolone were purified using a chromatography column (120 g of silica gel) eluting first with dichloromethane and then with acetone to obtain 4.1 g of product which was crystallized from dichloromethane / methyl tert-butyl ether to obtain 3.9 g of pure bryosanolone. Figure 4 The thermogram obtained from the product using the DSC technique is shown (1.158 mg of sample; test conditions indicated above); for clarity of presentation, only the portion of the thermogram above 120° C. is shown. The figure shows that the sample melts between 174° C. and 176° C., which is the melting range of the pure product reported in the article by RH Purdy et al. mentioned above.

[0169] Analysis of Brenolone:

[0170] 1 H-NMR, CDCl 3 : 4.05 (1H, m, H-3); 2.53 (1H, t, J = 9Hz, H-17); 2.20-2.10 (1H, m); 2.11 (3H, s, H-21); 2.00 (1H, dt, J = 3.2-11.9Hz); 1.85-0.75 (20H, m); 0.78 (3H, s, CH 3 ); 0.60(3H, s, CH 3 );

[0171] Mass spectrum: 318 (M + ).

[0172] Example 6

[0173] This example involves the epimerization of brexanolone in an alkaline environment.

[0174]

[0175] 50 mg of bryosone obtained after the preparation of Example 5 was dissolved in 1.25 ml of methanol and refluxed with 38 mg of NaOH for 16 h. The reaction mixture was cooled, the methanol was evaporated under reduced pressure, the solid was taken up with water and dichloromethane, the phases were separated, and the organic phase was washed until a neutral pH was reached. The dichloromethane was evaporated to obtain a crude product, which was purified by a chromatographic column (heptane / isopropyl acetate 6:4) to obtain 22 mg of product. By NMR analysis, the content of the diastereomer 17α was determined based on the signal ratio of the 17-position proton.

[0176] Figure 1 The NMR spectrum is shown to be extended from 0 ppm to 4.3 ppm; the 17-position proton signals are at 2.53 ppm and 2.79 ppm, based on which the amounts of brexanolone and diastereomers were calculated. The diastereomer content is 19%.

[0177] For intermediate (I), epimerization was also tested under similar conditions, with comparable results obtained in terms of epimer formation.

[0178] H-NMR,CDCl 3 : 4.05 (1H, m, H-3); 4.03 (1H, m, H-3 isomer 17α); 2.79 (dd, J = 2.7-8.2 Hz, H-17 isomer 17α); 2.53 (1H, t, J = 9 Hz, H-17); 2.120 (s, H-21 isomer 17α); 2.115 (3H, s, H-21); 0.904 (s, CH 3 Isomer 17α); 0.781 (3H, s, CH3); 0.764 (s, CH 3 Isomer 17α); 0.603 (3H, s, CH 3 ).

[0179] Example 7

[0180] This example involves the epimerization of brexanolone in an acid environment.

[0181] 50 mg of bryosperone obtained after the preparation of Example 5 was dissolved in 1.2 ml of THF, 0.5 ml of 2M HCl was added thereto, and the mixture was heated under reflux for 16 h. The reaction mixture was cooled, THF was evaporated under vacuum, the solid was dissolved with water and dichloromethane, the phases were separated, and the organic phase was washed until a neutral pH was reached. Dichloromethane was evaporated under reduced pressure to obtain 42 mg of a white solid.

[0182] Figure 2shows an expansion of the NMR spectrum from 2.5 ppm to 4.3 ppm; ref. Figure 1 , the signals associated with brexanolone and the epimers are the same as indicated in Example 6. The signal ratios show that the product has an 18% 17α epimer content.

[0183] Example 8

[0184] This example involves the case where X=O and R=ethylene-CH 2 -CH 2 -step 3 of the process of the present invention).

[0185]

[0186] Under nitrogen flow, 10 g of the intermediate (II) obtained as described in Example 2 was dissolved in 150 ml of toluene. Ethylene glycol (26.5 ml), triethyl orthoformate (25.2 ml) and p-toluenesulfonic acid (0.36 g) were added. The mixture was warmed to reflux temperature (105° C.-110° C.) and kept under stirring for 2 hours. A TLC check was performed to confirm the completion of the reaction (disappearance of the starting compound); the TLC check was performed under the following conditions:

[0187] - Start: In CH 2 Cl 2 middle;

[0188] -Sample: In NaHCO 3 + reaction mixture in toluene;

[0189] - Eluent: toluene / isopropyl acetate 97 / 3; Plate: HPTLC;

[0190] -Detector: UV / cerium phosphomolybdate.

[0191] The reaction mixture was cooled to 10°C and poured into 4% NaHCO pre-cooled at T < 5°C. 3 The system was kept under stirring for 10 minutes, the phases were separated, and the aqueous phase was re-extracted with 20 ml of toluene. The organic phases were recombined and washed with water. The solvent was removed under vacuum to obtain 13.4 g of crude intermediate (III) (white solid).

[0192] 5.2 g of the crude intermediate (III) was suspended in 15.6 ml of ethyl acetate and refluxed for 5 minutes. The suspension was cooled by keeping it at 0 ° C for 1 h and then filtered on a Buchner funnel by washing with ethyl acetate. The procedure was repeated twice more and, after drying to constant weight (T = 50 ° C and reduced pressure), 4.1 g of pure intermediate (III) (white solid) was obtained.

[0193] Analysis of intermediate (III):

[0194] 1 H-NMR, CDCl 3 : 8.07 (2H, dd, J = 1.4-8.5 Hz, aromatic H); 7.56 (1H, t, J = 7.3 aromatic H); 7.46 (2H, t, J = 7.5 Hz, aromatic H); 5.29-5.27 (1H, m, H-3); 4.01-3.85 (4H, m, OCH 2 CH 2 O); 2.04 (1H, dt, J = 3-11.9, H-17); 1.88-0.76 (22H, m); 1.302 (3H, s, H-21); 0.847 (3H, s, CH 3 ); 0.768 (3H, s, CH 3 );

[0195] 13 C-NMR, CDCl 3 :165.92 (C=O); 132.69 (aromatic CH); 131.15 (aromatic C); 129.53 (2 aromatic CH); 128.32 (2 aromatic CH); 111.97 (C, OCO); 70.72 (CH, C-3); 65.25 (OCH 2 ); 63.17(OCH 2 ); 58.37(CH); 56.39(CH); 54.33(CH); 42.02(C); 40.44(CH); 39.63(CH 2 );35.90(C);34.95(CH);33.19(CH 2 ); 33.00(CH); 31.83(CH 2 ); 28.37 (CH 2 ); 26.30(CH 2 ); 24.59 (CH 3 , C-21); 23.69 (CH 2 ); 22.88 (CH 2 ); 20.62(CH 2 ); 13.05(CH 3 ); 11.43 (CH 3 );

[0196] Mass spectrum: 466 (M + ).

[0197] Analysis of the 17α isomer of intermediate (III):

[0198] 1 H-NMR: The 17α isomer has the following characteristic signals: 1.323 (3H, s, H-21); 0.837 (3H, s, CH 3 ); 0.813 (3H, s, CH 3 ).

[0199] Example 9

[0200] This example involves the case where X=O and R=ethylene-CH 2 -CH 2 -step 4 of the process of the present invention).

[0201]

[0202] 3.2 g of the ketal benzoate obtained as described in Example 8 was suspended in 80 ml of methanol, and then 6.8 g of NaOH was added. The reaction mixture was continued at reflux (65° C.) for 16 h without noticing any dissolution. A TLC check was performed to verify the completion of the reaction (disappearance of the starting product). The TLC check was performed under the following conditions:

[0203] - Start: In CH 2 Cl 2 middle;

[0204] - Sample: reaction mixture in THF;

[0205] - Eluent: toluene / isopropyl acetate 6 / 4; Plate: TLC;

[0206] -Detector: UV / cerium phosphomolybdate.

[0207] The reaction mixture was cooled to 25°C and 100 ml of water was added. The solvent was removed under vacuum at 45°C. The reaction mixture was extracted with DCM and the organic phase was then washed with water until a neutral pH was reached. The organic fraction was concentrated under reduced pressure to obtain 2.6 g of intermediate (IV) as a white solid of suitable quality for continued synthesis.

[0208] Analysis of intermediate (IV):

[0209] 1 H-NMR, CDCl 3 :4.04(1H,m,H-3);4.04-3.83(4H,m,OCH 2 CH 2O); 2.015 (1H, dt, J = 3.2-11.9, H-17); 1.82-0.72 (23H, m); 1.293 (3H, s, H-21); 0.780 (3H, s, CH 3 ); 0.751(3H, s, CH 3 );

[0210] 13 C-NMR, CDCl 3 : 112.00 (C, OCO); 66.59 (CH, C-3); 65.23 (OCH 2 ); 63.19(OCH 2 );58.31(CH);56.41(CH);54.31(CH);42.01(C);39.66(CH 2 );39.14(CH);36.10(C);35.87(CH 2 ); 34.97 (CH); 32.16 (CH 2 ); 31.91(CH 2 ); 29.00(CH 2 ); 28.55(CH 2 ); 24.58 (CH 3 , C-21); 23.70(CH 2 ); 22.88 (CH 2 ); 20.56 (CH 2 ); 13.07(CH 3 ); 11.20(CH 3 );

[0211] Mass spectrum: 362 (M + ).

[0212] Analysis of the 17α isomer of intermediate (IV):

[0213] The 17α isomer has the following characteristic signals: 1.300 (3H, s, H-21); 0.792 (3H, s, CH 3 ); 0.772(3H, s, CH 3 ).

[0214] Example 10

[0215] This example relates to step 5) of the process of the invention starting from the ketal.

[0216]

[0217] 1.5 g of the intermediate (IV) obtained according to the procedure of Example 9 was placed under stirring with 22.5 ml of anhydrous acetone and 105 mg of iodine. The mixture was placed under reflux (T = 58 ° C.) and after stirring under reflux for 10 minutes, a clear solution was obtained. TLC inspection confirmed the completion of the reaction. TLC inspection was carried out under the following conditions:

[0218] - Start: In CH 2 Cl 2 middle;

[0219] - Sample: reaction mixture diluted in acetone;

[0220] - Eluent: toluene / isopropyl acetate 6 / 4; Plate: TLC;

[0221] -Detector: UV / cerium phosphomolybdate.

[0222] Acetone was removed by distillation under reduced pressure, and the residue was dissolved with DCM (50 ml). The organic phase was first washed with 5% aqueous sodium thiosulfate solution, and then washed with water. The organic phase was then dried and concentrated under reduced pressure to obtain 1.25 g of crude brexanolone.

[0223] HPLC analysis of the crude brexanolone thus obtained, without any further purification, showed that the product contained the 17α isomer in an amount equal to 0.29%.

[0224] 1.0 g of the previously obtained sample was completely dissolved under reflux with 7 ml of isopropyl acetate, cooled to 0° C. for 1 h and finally filtered. The solid was dried at 50° C. to obtain 0.9 g of pure bryosanolone which, analyzed by HPLC, contained the 17α isomer in an amount equal to 0.10%.

[0225] Example 11 (Comparative)

[0226] This example involves deprotection of the ketal to form bryosone under conditions different from those of the present invention.

[0227] Under nitrogen, 0.1 g of intermediate (IV) obtained as described in Example 9 was charged into a flask. DCM (1.1 ml) and methanol (0.7 ml) were added; no complete dissolution was observed. NaHCO 3 (0.096 g). Then add I 2(0.112 g), and the mixture was kept at 25 ° C under stirring for 20 h, then the mixture was gradually warmed to 40 ° C in 6 h, and the progress of the reaction was checked by TLC. The mixture was cooled to 0 ° C, and the reaction was quenched by adding a solution of sodium thiosulfate (0.25 g) in water (5 ml) dropwise on the reaction mixture, keeping the temperature below 10 ° C. The mixture was warmed to 25 ° C, and the suspension was filtered on a diatomaceous earth plate, and the solid on the filter was washed with DCM at 35 ° C. The phases were separated and the aqueous phase was extracted with DCM. The organic phases were recombined and washed with water. The solvent was removed by evaporation under reduced pressure with a rotary evaporator to obtain 0.09 g of brucenolide, whose diastereomer content was equal to 0.7%.

[0228] Example 12

[0229] 40 mg of the intermediate (IV) obtained according to the procedure of Example 9 was mixed with 0.8 ml of CH 2 Cl 2 Place under stirring together with 0.08ml of acetone. Add 0.7mg of iodine. The mixture is stirred at 25°C and the reaction is confirmed to be complete (disappearance of the starting compound) by TLC inspection after 10 minutes. The mixture is kept under stirring at 25°C for another 18 hours to test the stability of the product. The formation of degradation products is not noted after TLC inspection. The organic phase is first washed with 5% sodium thiosulfate aqueous solution and then washed with water. The organic phase is concentrated under reduced pressure drying to obtain 36mg of brucerolon.

[0230] HPLC analysis of the brexanolone thus obtained, without any further purification, showed that the product contained the 17α epimer in an amount equal to 0.04%.

[0231] Embodiment 13

[0232] 40 mg of the intermediate (IV) obtained according to the procedure of Example 9 was mixed with 0.8 ml of CH 2 Cl 2 Place under stirring together with 0.081 ml of acetone. Add 2.8 mg of iodine. The mixture is stirred at 0 ° C, and after 15 minutes, the reaction is confirmed to be complete (disappearance of the starting compound) by TLC inspection. The organic phase is first washed with 5% sodium thiosulfate aqueous solution, and then washed with water. The organic phase is dried and concentrated under reduced pressure to obtain 35 mg of brucenolide.

[0233] HPLC analysis of the brexanolone thus obtained, without any further purification, showed that the product contained the 17α epimer in an amount equal to 0.09%.

Claims

1. A process for the synthesis of 3α-hydroxy-5α-pregnane-20-one, i.e., brexanolone, having an amount of diastereomer at position 17 less than 0.15%, said process comprising the following steps: 1) catalytic hydrogenation of the double bonds at positions 5 and 6 of pregnenolone to obtain the corresponding saturated steroid of formula (I), wherein the hydrogen atom at position 5 of the steroid skeleton is arranged in a spatial arrangement α: 2) Reversing the spatial orientation of the 3-hydroxyl group of the compound of formula (I), which is protected as a benzoate at the end of the reaction, to obtain a compound of formula (II): 3) Protecting the carbonyl group at position 20 of compound (II) as ketal or thioketal to obtain a compound of general formula (III), wherein X=O or X=S, and R is a group selected from ethylene (-CH2-CH2-), propylene (-CH2-CH2-CH2-) and 2,2-dimethylpropylene (-CH2-C(CH3)2-CH2-): 4) hydrolyzing the benzoate of compound (III) to obtain a compound of general formula (IV) in which X and R have the meanings reported in step 3): 5) Using iodine in an organic solvent, deprotect the 20-position of the compound of general formula (IV) to obtain brenolon:

2. The process according to claim 1, wherein step 1) is carried out using 5% palladium on carbon (Pd / C) as hydrogenation catalyst in an amount of 5% relative to the weight of pregnenolone, at a hydrogen pressure between 2 and 5 bars, in tetrahydrofuran (THF) as solvent, at a temperature between 35° C. and 45° C., and for a period of between 3 and 7 hours.

3. The process according to any one of claims 1 and 2, wherein step 2) is carried out by reacting compound (I) with benzoic acid in tetrahydrofuran (THF) as solvent and allowing to react with triphenylphosphine and diisopropyl azodicarboxylate (DIAD) at a temperature between 15°C and 25°C for a time between 14 hours and 20 hours.

4. The process according to claim 1, wherein in step 3), the carbonyl group at position 20 of compound (II) is protected as ketal, and compound (II) is reacted with a mixture of toluene, a diol selected from ethylene glycol, propylene glycol and 2,2-dimethylpropylene glycol, triethyl orthoformate and p-toluenesulfonic acid under reflux.

5. The process according to claim 1, wherein in step 3), the carbonyl group at position 20 of compound (II) is protected as thioketal, and compound (II) is reacted with ethanedithiol or propylenedithiol in a solvent selected from acetic acid, dichloromethane (CH2Cl2), diethyl ether, acetonitrile, tetrahydrofuran (THF) and 1,2-dichloroethane in the presence of a catalyst selected from boron trifluoride etherate, zinc iodide (ZnI2), titanium tetrachloride (TiCl4), tin dichloride (SnCl2), magnesium iodide (MgI2), lithium perchlorate (LiClO4), aluminum trifluoromethanesulfonate (Al(OTf)3), lithium tetrafluoroborate (LiBF4) and cobalt dichloride (CoCl2) at a temperature in the range of between 15°C and 45°C and for a period of time between 8 hours and 36 hours.

6. The process according to claim 1, wherein step 4) is carried out by refluxing compound (III) in methanol in the presence of sodium hydroxide for a period of time between 20 hours and 28 hours.

7. A compound of the general formula (III), wherein X=O or X=S, and R is a group selected from ethylene (-CH2-CH2-), propylene (-CH2-CH2-CH2-) and 2,2-dimethylpropylene (-CH2-C(CH3)2-CH2-):

8. The compound according to claim 7, wherein R is ethylene and X is sulfur:

9. The compound according to claim 7, wherein R is ethylene and X is oxygen:

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