Processes for the preparation of enantiomerically enriched r-fezolinetant

Abstract

The present invention relates to a process for the preparation of highly pure enantiomer of Fezolinetant, and in particular highly pure (R)-Fezolinetant. The invention also relates to chiral resolution by acid for the preparation of highly pure R-Fezolinetant. Additionally, the invention relates to a process for preparing intermediates useful for the preparation of (R)-Fezolinetant, their uses and a process for preparing (R)-Fezolinetant. In particular, the intermediate (R)-3-methylpiperazin-2-one is prepared by reductive amination with an imine reductase from Myxococcus stipitatus and ethylene diamine and ethyl pyruvate.

Description

PROCESSES FOR THE PREPARATION OF ENANTIOMERICALLY ENRICHED R-FEZOLINETANTFIELD OF THE DISCLOSURE

[0001] The present invention relates to a process for the preparation of highly pure enantiomer of Fezolinetant, and in particular highly pure (R)-Fezolinetant. The invention also relates to chiral resolution by acid for the preparation of highly pure R-Fezolinetant.Additionally, the invention relates to a process for preparing intermediates useful for the preparation of (R)-Fezolinetant, their uses and a process for preparing (R)-Fezolinetant.BACKGROUND OF THE DISCLOSURE

[0002] Fezolinetant which has the chemical name, (4-fluorophenyl)-[(8R)-8-methyl-3-(3- methyl-l,2,4-thiadiazol-5-yl)-6,8- dihydro-5H-[l,2,4]triazolo[4,3-a]pyrazin-7-yl]methanone is reported to be an antagonist of the GPCR known as the tachykinin NK3 receptor, and it is developed for the treatment of menopausal hot flashes (HF) and / or other menopausal symptoms such as night sweats and / or sleep and mood disturbances. Fezolinetant has recently been investigated for the treatment of polycystic ovary syndrome (PCOS), endometriosis, benign prostate hyperplasia, and uterine fibroids.

[0003] Several approaches for the enantioselective preparation of (R)-Fezolinetant have been described in the art. The process for racemic Fezolinetant is described in U.S. Patent No. 9,422,299 and ACS Med. Chem. Lett. 2015, 6, 736-740, which may then be subjected to chiral preparative HPLC for chiral separation. The use of chiral preparative HPLC for large scale production is not widely appreciated since it is time consuming and an expensive technique.

[0004] A novel, chiral synthesis of Fezolinetant is described in U.S. Patent No. 10,787,458. According to this approach the retention of stereochemistry with respect to the starting material is preserved, except to the extent that racemization occurs as a minor side reaction. However, no information for Fezolinetant chiral impurity is described (nominated compound 5 in US'458). Repetition of the described procedure resulted in incomplete reaction, degradation of starting material and about 3-4% of the undesired enantiomer (S)-Fezolinetant measured at the end of reaction.

[0005] Fractional crystallization of racemic mixture is common technique for chiral separating. In this technique, chiral substance (e.g. base or acid) is added and converts theenantiomer to a mixture of diastereomers that have different solubility and can be separated from a solution.

[0006] Resolution of racemic Fezolinetant, a chiral base, through the formation of diastereomeric salts requires adequate and suitable chiral acid. Furtheremore, since Fezolinetant is very weak base (pKa ~2), potential chiral strong acids that can be used for this separation are very limited. In addition, strong acids which are not chiral (e.g. HBr, p- toluenesulfonic acid and methanesulfonic acid), were found to be much less effective for this purification.

[0007] Furthermore, it was found that fraction crystallization of racemic Fezolinetant with chiral acid resulted with low yield since the desired enantiomer crystallized out together with racemic compound. In addition, the desired enantiomer left in the mother liquor is contaminated with 0.5-1.3 A% of (S)-Fezolinetant. Further crystallization from this mother liquor results with the same chiral purity or with minor purification as was in starting mother liquor. Therefore, there is a need for improved purification procedure with high efficiency and high yield.

[0008] U.S. Patent No. 10,787,458 discloses the following process for preparing Fezolinetant and related compounds:

[0009] Enantioselective synthesis of (R)-Fezolinetant can be achieved starting from chiral starting materials. For example, a chirally pure compound A (wherein R1is methyl) in the above reaction scheme from US 10,787,458, can be used as starting material for the preparation of (R)-Fezolinetant. The compound (R)-3-methylpiperazin-2-one may be prepared according to the process disclosed in CN111825677, in two steps starting from ethyl L-lactate or methyl L-lactate:

[0010] However, although the chiral ethyl L-lactate or methyl L-lactate are readily available, these starting materials are very expensive. Moreover, the process disclosed in CN111825677 involves chromatographic purification for both the intermediate and the (R)-3- methylpiperazin-2-one product. The use of chromatographic procedures for industrial scale syntheses is highly undesirable, due to the need to employ large volumes of solvents and energy-inefficient solvent removal step. This is particularly undesirable in the present case, because the chromatographic procedures are conducted at a very early stage of the synthesis.

[0011] CN108129404 discloses a different approach to obtain (R)-3-methylpiperazin-2-one:Nevertheless, the process also involves two steps and requires a chiral starting material. As for the above process, the starting materials for this process are very costly, and the process similarly involves chromatographic purification of the intermediate and the (R)-3- methylpiperazin-2-one product.

[0012] The above-discussed prior art processes for preparing (R)-3-methylpiperazin-2-one, a useful starting material in the enantioselective synthesis of Fezolinetant, are therefore not practical or economical for industrial scale syntheses. Accordingly, there exists a need for theprovision of an economical process for preparing (R)-3-methylpiperazin-2-one (herein also referred to as FZL-10) on an industrial scale.SUMMARY OF THE DISCLOSURE

[0013] In a first aspect of the disclosure, it has been surprisingly found that highly pure (R)- Fezolinetant can be obtained by preparing a solution of an enantiomerically enriched racemic mixture, removing solid racemic Fezolinetant, crystallization of the mother liquor to isolate (R)-Fezolinetant and purification with chiral acid.

[0014] Processes for production of highly pure (R)-Fezolinetant include preparation of enantiomerically enriched racemic mixture solution, crystallizing mother liquor enriched with (R)-Fezolinetant, and purifying the isolated R-Fezolinetant with chiral acid.

[0015] The process for producing highly pure (R)-Fezolinetant includes preparation of enantiomerically enriched racemic mixture solution of Fezolinetant, removing the solid racemic Fezolinetant, in embodiment by evaporation of the solvent or cooling, preferably cooling, crystallizing the enriched mother liquor by drying to isolate (R)-Fezolinetant and purifying the isolated (R)-Fezolinetant with chiral acid.

[0016] Highly pure (R)-Fezolinetant is obtained by preparation of an enantiomerically enriched racemic mixture solution, removing racemate Fezolinetant by crystallization to isolate the enriched mother liquor, crystallizing (R)-Fezolinetant from the enriched mother liquor, and purifying (R)-Fezolinetant with chiral acid.

[0017] In embodiments, highly pure (R)-Fezolinetant is obtained by preparing mother liquor enriched with (R)-Fezolinetant, removing racemic Fezolinetant, crystallizing the R- enantiomer from the enriched mother liquor and purifying (R)-Fezolinetant with chiral acid.

[0018] In embodiments, highly pure (R)-Fezolinetant is obtained by preparing an enantiomerically enriched racemic mixture with suitable solvent, optionally by heating, separating solid racemic Fezolinetant by evaporation or cooling, in embodiment cooling, isolating the enantiomerically enriched mother liquor, optionally the enriched mother liquor is purified with activated carbon, crystallizing and isolating (R)-Fezolinetant from the enriched mother liquor by drying, and purifying the isolated (R)-Fezolinetant with chiral acid.

[0019] In embodiments, 100% chiral purity (R)-Fezolinetant is obtained by purification process described herein.

[0020] In embodiments, enantiomerically enriched racemic mixture is obtained by adding suitable solvent to Fezolinetant, optionally by heating. Fezolinetant can be prepared by the process described as follows.

[0021] Fezolinetant in high yield and in short time can be obtained by reacting 3-methyl- l,2,4-thiadiazole-5-carbohydrazide with (R)-(4-fluorophenyl)(5-methoxy-6-methyl-3,6- dihydropyrazin-l(2H)-yl)m ethanone in anhydrous solvent, optionally with base, under heating. The resulting reaction mixture is (R)-Fezolinetant which typically contains: about 1 to about 6%, about 1% to about 5%, about 1% to about 4%, about 1% to about 3%, about 2% to about 3%, about 2% to about 4%, about 3% to about 4%, or about 1.2% to about 3%, about 1.5% to about 2%, or about 1.6% to about 1.8% of (S)-Fezolinetant.

[0022] Fezolinetant obtained by the procedure above can be purified by adding suitable solvent to produce an enantiomerically enriched racemic solution, isolating the enriched mother liquor by removing the precipitated racemic Fezolinetant by cooling, crystallization and separation of R-Fezolinetant from the mother liquor by drying and purification with chiral acid to obtain highly pure (R)-Fezolinetant. The suitable solvent can be added directly to the reaction mixture after completion of the reaction. Alternatively, the reaction product can be isolated by any suitable method, and a mixture an anhydrous solvent can be added to form a solution, to which the suitable solvent can be added to precipitate racemic Fezolinetant.

[0023] In embodiments, purification process according to this invention includes preparation of Fezolinetant in anhydrous solvent, optionally with base, isolating mother liquor enriched with (R)-Fezolinetant by suitable solvent, removing precipitated racemic Fezolinetant by cooling and crystallizing R-Fezolinetant from the mother liquor, and purification with chiral acid to obtain highly pure (R)-Fezolinetant.

[0024] The present disclosure is based on a surprising and unexpected discovery that a (S)- Fezolinetant in an enantiomerically enriched racemic mixture, can be removed as a racemic mixture with (R)-Fezolinetant, in the form of a solid. The mother liquor obtained after removal of the solid, is therefore further enriched with (R)-Fezolinetant. This first step therefore produces a solution which has a greater enantiomeric excess of (R)-Fezolinetant compared with the starting enantiomerically enriched racemic mixture (i.e. “an enantiomerically enriched racemic solution of Fezolinetant”). (R)-Fezolinetant having a reduced content of (S)-Fezolinetant may be obtained from this mother liquor. The resulting (R)-Fezolinetant may be further purified using a chiral acid, if required. The furtherpurification using a chiral acid proceeds more efficiently compared with the purification of racemic mixture of Fezolinetant (i.e. non-enantiomerically enriched) using a chiral acid.

[0025] A further aspect of the present disclosure provides a process for the preparation of (R)-3-methylpiperazin-2-one (FZL-10), which is useful as an intermediate for the enantioselective synthesis of Fezolinetant. The process involves a biocatalytic stereoselective reductive amination of an alkyl pyruvate with ethylene diamine. In particular, the process comprises enzymatic stereoselective reductive amination of an alkyl pyruvate with ethylene diamine. Preferably, the process is carried out in the presence of an imine reductase or reductive aminase enzyme, optionally with a co-factor regenerating system.

[0026] The process for preparing FZL-10 according to the present disclosure advantageously enables to production of chirally pure FZL-10 in only a single step from inexpensive, and non-chiral, starting materials. Particularly FZL-10 prepared according to the disclosed process advantageously has a very high enantiomeric purity. The disclosed process also avoids the use of preparative chiral chromatography purification. By way of contrast, prior art methods for preparing (R)-3-methylpiperazin-2-one involve at least two reaction steps, wherein each step involves the use of chiral chromatography to purify the products.

[0027] According to another aspect of the disclosure, FZL-10 prepared according to the disclosed process may be converted to (R)-Fezolinetant having a high enantiomeric purity and in high yield. The disclosure further comprises the use of the process for preparing FZL- 10 as described in any aspect or embodiment herein, for the preparation of (R)-Fezolinetant.

[0028] The present disclosure further comprises a process for preparing (R)-Fezolinetant comprising biocatalytic stereoselective reductive amination of an alkyl pyruvate with ethylene diamine, as described in any aspect or embodiment disclosed herein, to from FZL- 10, and converting the FZL-10 to (R)-Fezolinetant. The FZL-10 can be converted to (R)- Fezolinetant by any known process, such as by the process disclosed in US 10,787,458. Optionally the FZL-10 can be converted to (R)-Fezolinetant by the processes disclosed herein.

[0029] According to any aspect or embodiment of the disclosure, the (R)-Fezolinetant prepared from the FZL-10 obtained by the disclosed processes, may be subjected to further enantiomeric purification according to the enantiomeric purification processes described herein. The resulting purified (R)-Fezolinetant may optionally be subjected to an addiitonal chiral purification using by diastereomeric salt separation, or by selective salt precipitation from a suitable acid. The processes described herein may provide highly pure (R)-Fezolinetant having a very high enantiomeric purity (for example, about 99% to 100%, about 99.5% to 100%, about 99.7% to 100%, about 99.8% to 100%, about 99.9% to 100%, or about 100% of (R)-Fezolinetant (i.e. wt% or area% by chiral HPLC).BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 shows amino acid sequence listings of particularly useful enzymes that may be used in accordance with the biocatalytic processes disclosed herein.DETAILED DESCRIPTION OF THE DISCLOSURE

[0031] According to one aspect, highly pure (R)-Fezolinetant is obtained by preparation of an enantiomerically enriched racemic solution, removing precipitated racemic Fezolinetant, isolating R-Fezolinetant from the enriched mother liquor and purifying R-Fezolinetant with chiral acid.

[0032] In embodiments, highly pure (R)-Fezolinetant is produced by crystallization of (R)- Fezolinetant with chiral acid, preferably strong chiral acid.

[0033] In embodiments, highly pure (R)-Fezolinetant is obtained by diastereomeric salt crystallization of (R)-Fezolinetant obtained from enriched mother liquor.

[0034] The process for highly pure (R)- Fezolinetant comprises preparation of enantiomerically enriched racemic solution of Fezolinetant, removing the solid racemic Fezolinetant, in embodiment by evaporation of the solvent or cooling, preferably cooling, crystallizing and isolating R-Fezolinetant from mother liquor by drying and purifying (R)- Fezolinetant with chiral acid.

[0035] Process for highly pure (R)-Fezolinetant comprising preparation of enantiomerically enriched racemic solution with suitable solvent; separating from said solution solid racemic Fezolinetant by cooling and isolating the enriched mother liquor, optionally mother liquor is purified by activated carbon before crystallizing (R)-Fezolinetant, the isolated (R)- Fezolinetant is purified with chiral acid, preferably strong chiral acid.

[0036] Process for highly pure (R)-Fezolinetant comprising the preparation of enantiomerically enriched racemic Fezolinetant solution, from said solution separating the solid racemic Fezolinetant by cooling and filtration to isolate the enriched mother liquor comprising the desired enantiomer, optionally purification with activated carbon, crystallization of (R)-Fezolinetant from the enriched mother liquor by drying, optionally concentration under reduced pressure and drying and purifying the isolated (R)-Fezolinetant with chiral acid, preferably strong chiral acid.

[0037] In embodiments, purification procedure according to this invention includes preparation of Fezolinetant, adding suitable solvent to obtain enantiomerically enriched racemic solution, isolating enriched mother liquor by removing racemate Fezolinetant, crystallizing the isolated enriched mother liquor to obtain the (R)-Fezolinetant and purifying the isolated (R)-Fezolinetant with chiral acid to obtain highly pure (R)-Fezolinetant.

[0038] In embodiments, highly pure (R)-Fezolinetant is obtained by process comprising the preparation of enantiomerically enriched racemic solution by adding suitable solvent to Fezolinetant racemic mixture, cooling to isolate the enriched mother liquor with (R)- Fezolinetant by removing the solid racemic Fezolinetant, crystallizing (R)-Fezolinetant from the enriched mother liquor by drying, optionally concentration under reduced pressure and heating and purifying the isolated (R)-Fezolinetant with chiral acid, preferably strong chiral acid.

[0039] This invention describes processes for the preparation of highly pure (R)-Fezolinetant. The process includes preparation of enantiomerically enriched racemic solution by adding suitable solvent to racemic Fezolinetant mixture, isolating the enriched mother liquor by crystallizing racemate Fezolinetant, optionally enriched mother liquor is purified by activated carbon, crystallization from the enriched mother liquor by drying, optionally concentration under reduced pressure and drying to isolate (R)-Fezolinetant, and purifying the isolated (R)- Fezolinetant by chiral acid, preferably crystallization with strong chiral acid to isolate highly purified (R)-Fezolinetant.

[0040] In embodiments, 100% (R)-Fezolinetant is obtained when using the purification procedure according to this invention.

[0041] As defined herein, racemic Fezolinetant refers to a mixture of equal quantities of two enantiomers (S)-Fezolinetant : (R)-Fezolinetant = 50:50.

[0042] As defined herein, "highly pure (R)-Fezolinetant" refers to about 99.0-100%, (R)- Fezolinetant, in embodiment 99.5-100% (R)-Fezolinetant, in embodiment 100% (R)- Fezolinetant as measured by any known technique.

[0043] As defined herein, “enantiomerically enriched racemic solution” refers to a solution of Fezolinetant which is enriched in (R)-Fezolinetant. Particularly the solution of Fezolinetant is more enriched in (R)-Fezolinetant than the Fezolinetant starting material. In the process of any aspect or embodiment of the disclosure, enantiomerically enriched racemic solution is typically the product obtained after precipitating and removing racemic Fezolinetant from the Fezolinetant starting material, to form a mother liquor (i.e. the enantiomerically enrichedracemic solution); or removing racemic Fezolinetant from the Fezolinetant starting material, isolating the enantiomerically enriched Fezolinetant, and dissolving in an anhydrous solvent to form an enantiomerically enriched racemic solution. Preferably, according to any aspect or embodiment of the disclosure, the “enantiomerically enriched racemic solution” is the mother liquor obtained from removal of precipitated racemic Fezolinetant from the Fezolinetant starting material. According to any aspect or embodiment. Typically, the Fezolinetant starting material contains a major amount of (R)-Fezolinetant and a minor amount of (S)- Fezolinetant. According to any aspect or embodiment, the Fezolinetant starting material contains (R)-Fezolinetant, and: about 1 to about 6%, about 1% to about 5%, about 1% to about 4%, about 1% to about 3%, about 2% to about 3%, about 2% to about 4%, about 3% to about 4%, or about 1.2% to about 3%, about 1.5% to about 2%, or about 1.6% to about 1.8% of (S)-Fezolinetant. Such Fezolinetant starting material can be obtained by the processes described and exemplified herein, or by the process disclosed in US 10,787,458. For example, Fezolinetant can be obtained by reacting 3-methyl-l,2,4-thiadiazole-5- carbohydrazide with (R)-(4-fluorophenyl)(5-methoxy-6-methyl-3,6-dihydropyrazin-l(2H)- yl)methanone in an anhydrous solvent, optionally with base, under heating.

[0044] As defined herein, "enriched mother liquor" refers to solution enriched by the desired enantiomer, (R)-Fezolinetant. In embodiment enriched mother liquor is containing at least 95%, 96%, 97%, 98%, 99% or 99.5% of (R)-Fezolinetant. As discussed above, the “enriched mother liquor” corresponds to the “enantiomerically enriched racemic solution” which is obtained after precipitation and removal of solid racemic Fezolinetant from the Fezolinetant starting material. The term “enriched mother liquor” may also correspond to the “enantiomerically enriched racemic solution” obtained after precipitation and removal of solid racemic Fezolinetant from the Fezolinetant starting material, isolating the enantiomerically enriched Fezolinetant from the mother liquor, and redissolving the enantiomerically enriched Fezolinetant in an anhydrous solvent (as described in any aspect or embodiment of the disclosure), to form a solution. Preferably, according to any aspect or embodiment of the disclosure, “enriched mother liquor” refers to the mother liquor obtained after precipitation and removal of solid racemic Fezolinetant starting material as discussed above.

[0045] As defined herein, chiral acid is optically active acid to improve enantiomeric excess.

[0046] As defined herein, strong chiral acid refers to chiral acid having pKa lower than 0.

[0047] As defined herein, Fezolinetant Form 5 refers to Form 5 described in PCT application PCT / US2021 / 044436 (i.e. Inf 1 Publication No. WO 2022 / 031773).

[0048] A thing, e.g., a reaction mixture, may be characterized herein as being at, or allowed to come to "room temperature", often abbreviated "RT." This means that the temperature of the thing is close to, or the same as, that of the space, e.g., the room or fume hood, in which the thing is located. Typically, room temperature is from about 20 °C to about 30 °C, about 22 °C to about 27 °C, or about 25 °C.

[0049] A process or step may be referred to herein as being carried out "overnight." This refers to a time interval, e.g., for the process or step, that spans the time during the night, when that process or step may not be actively observed. This time interval is from about 8 to about 20 hours, about 10 to about 18 hours, or about 16 hours.

[0050] The amount of solvent employed in a chemical process, e.g., a reaction or crystallization, may be referred to herein as a number of "volumes" or "vol" or "V." For example, a material may be referred to as being suspended in 10 volumes (or 10 vol or 10V) of a solvent. In this context, this expression would be understood to mean milliliters of the solvent per gram of the material being suspended, such that suspending 5 grams of a material in 10 volumes of a solvent means that the solvent is used in an amount of 10 milliliters of the solvent per gram of the material that is being suspended or, in this example, 50 mL of the solvent. In another context, the term "v / v" may be used to indicate the number of volumes of a solvent that are added to a liquid mixture based on the volume of that mixture. For example, adding methyl tert-butyl ether (MTBE) (1.5 v / v) to a 100 ml reaction mixture would indicate that 150 mL of MTBE was added.

[0051] Unless otherwise indicated, reference to a concentration, such as wt% or vol % of a reagent, solvent, or other additive to a reaction mixture, relates to the percentage relative to the total, final reaction mixture.

[0052] As used herein, the term "reduced pressure" refers to a pressure of from about 10 mb ar to 50 mb ar.

[0053] As used herein and unless indicated otherwise, the term "ambient conditions" refer to atmospheric pressure and a temperature of about 22-24°C.

[0054] As used herein, the term “cofactor regeneration system” means an enzyme-substrate pair, mixed to the required constituents of the enzymatic reaction (including the cofactor). In the course of imine reductase-catalyzed oxidation, cofactor regeneration system has the role to oxidize the reduced form of the P-nicotinamide adenine dinucleotide phosphate cofactor(NADPH) to the oxidized form (NADP+), therefore allowing the use of catalytic amount of NADP+. It is generally understood that the cofactor regeneration system is named after the enzyme that is used for the oxidation of NADPH to NADP+. Typically, the name implicates the suitable coupled substrate. For example, the NOX cofactor regeneration system implicates the use of molecular oxygen (O2) as a substrate together with NOX enzyme, whereas the ADH or KRED cofactor regeneration systems implicate the use of a carbonyl compound as substrate. In case of imine reductase catalyzed reduction, cofactor regeneration system has the role to reduce the oxidized form of the P-nicotinamide adenine dinucleotide phosphate cofactor (NADP+) to the reduced form (NADPH), therefore allowing the use of catalytic amount of NADP+. GDH cofactor regeneration system implicates the use of glucose as a substrate together with GDH enzyme.

[0055] As used herein, references to an amino acid sequence have a specified percentage identity to a specified amino acid sequence refers to the degree of similarity between the two amino acid sequences. The percentage may be determined by comparing with the naked eye or using a bioinformatic algorithm. The latter enables calculation of the degree of homology by aligning sequences for comparison. The homology between the two amino acid sequences may be calculated as a percentage. The useful automated algorithms may be used in GAP, BESTFIT, FASTA, and TFASTA computer software modules of Wisconsin Genetics Software Package (Genetics Computer Group, Madison, Wis., USA). Other useful algorithms and homology determinations on alignment are already automated in software such as FASTP, BLAST, BLAST2, PSIBLAST, and CLUSTAL W. Preferably, CLUSTAL Omega (http s : / / www . uniprot . org / ali gn) was used for the sequence identity calculations.

[0056] In embodiments, a process for producing pure (R)-Fezolinetant includes:I. Preparing enantiomerically enriched racemic solutionII. Isolating enriched mother liquor by crystallizing solid racemic Fezolinetant by evaporation of the solvent or cooling, preferably by cooling. Optionally washing the solid racemic Fezolinetant and combining the filtrate with the enriched isolated mother liquor. Optionally purifying the enriched mother liquor with activated carbon.III. (R)-Fezolinetant is crystallized from the enriched mother liquor by drying,IV. The isolated (R)-Fezolinetant is purified by chiral acid, preferably crystallization with strong chiral acid to obtain highly pure (R)-Fezolinetant.

[0057] The enantiomerically enriched racemic solution in step I, is obtained by adding suitable solvent to Fezolinetant. The Fezolinetant may be obtained by reaction of 3-methyl- l,2,4-thiadiazole-5-carbohydrazide with (R)-(4-fluorophenyl)(5-methoxy-6-methyl-3,6- dihydropyrazin-l(2H)-yl)m ethanone in an anhydrous solvent, optionally with base, under heating as described above. The Fezolinetant may be obtained according to the process described in the prior art, for example in US 10,787,458. The Fezolinetant product from the reaction is primarily (R)-Fezolinetant, but typically contains: about 1 to about 6%, about 1% to about 5%, about 1% to about 4%, about 1% to about 3%, about 2% to about 3%, about 2% to about 4%, about 3% to about 4%, or about 1.2% to about 3%, about 1.5% to about 2%, or about 1.6% to about 1.8% of (S)-Fezolinetant. Typically, the Fezolinetant formed in the process is in solution with an anhydrous solvent. For example, the Fezolinetant may be in the reaction mixture as a solution in the anhydrous reaction solvent, or the Fezolinetant may be isolated from the reaction mixture, and dissolved in an anhydrous solvent to form a solution of Fezolinetant in the anhydrous solvent. The anhydrous solvent may be an anhydrous alcohol, particularly anhydrous methanol, anhydrous 2-propanol and anhydrous ethanol, preferably anhydrous methanol.

[0058] The suitable solvent which is added to the Fezolinetant can be selected from acetone, methyl ethyl ketone, tetrahydrofuran, 2-methyltetrahydrofuran and acetonitrile, in embodiments acetonitrile. In embodiments, the reaction mixture is kept at RT for about 1-3 hours, preferably for 1 hour.

[0059] In embodiments, the reaction mixture is cooled to 10 °C -0 °C, preferably 0 °C for about 1 - 16h, preferably for 3h to crystallize racemic Fezolinetant according to step II.

[0060] The addition of the suitable solvent surprisingly enables the precipitation of racemic Fezolinetant from the Fezolinetant solution. This process therefore removes (S)-Fezolinetant as a racemic mixture with (R)-Fezolinetant, as a solid, leaving a solution (i.e. enantiomerically enriched mother liquor) which is more enantiomerically enriched in (R)- Fezolinetant compared with the Fezolinetant obtained in the reaction step. Hence, this step enables (S)-Fezolinetant to be easily removed to produce (R)-Fezolinetant having a greater enantiomeric excess. The resulting (R)-Fezolinetant may be further purified using a chiral acid in order to achieve an even greater enantiomeric purity.

[0061] The solid racemic Fezolinetant is removed from the enriched mother liquor by any conventional method (e.g., centrifugation, filtration, or decantation), optionally washing theisolated solid with suitable solvent and combining the filtrate with the enriched mother liquor, optionally purifying the enriched mother liquor with activated carbon.

[0062] The isolated enriched mother liquor is purified by adding activated carbon to the solution and heating to 40-60 °C, preferably 55 °C for about 0.5-5 hours, preferably 1 hour, and filtration, preferably hot filtration at about 40-60 °C, optionally, washing the Celite (i.e. diatomaceous earth, diatomite, or kieselguhr) with the suitable solvent and combining the filtrate to the enriched mother liquor.

[0063] (R)-Fezolinetant is crystallized from the enriched mother liquor, according to stage III by drying. Optionally, the solution is concentrated under reduced pressure, optionally concentration to about 3 V / w and heated to 50-75 °C, preferably 60 °C for about 10 min-2 hours, preferably 30 min. (R)-Fezolinetant is isolated by cooling, preferably to 0 °C for about 1-4 hours, preferably for 2 hours and to obtain (R)-Fezolinetant (having about 1% of (S)- Fezolinetant).

[0064] According to any embodiment of the disclosed process, the (R)-Fezolinetant obtained from the mother liquor may be further purified with a chiral acid. The isolated (R)- Fezolinetant is purified with chiral acid according to step IV, preferably crystallization with strong chiral acid, in a mixture of solvents, optionally under heating and cooling to isolate the optically active (R)-Fezolinetant salt.

[0065] Potential strong chiral acid can be selected from ethanesulfonic acid, benzenesulfonic acid, tosic acid and (lS)-(+)-Camphor-10-sulfonic acid, preferably (lS)-(+)-Camphor-10- sulfonic acid.

[0066] In embodiment, (R)-Fezolinetant is purified with (lS)-(+)-Camphor-10-sulfonic acid.

[0067] Any suitable solvent or solvent combination can be used to selectively crystallise the (R)-Fezolinetant camsylate from the diastereomeric salt mixture. Acetonitrile combination with tert-butyl methyl ether (MTBE) ethyl acetate or acetone (1 : 1 ), methanol combination with isopropanol, ethyl acetate or MTBE (1 : 1), ethanol combination with ethyl acetate (1 : 1) and acetone combination with ethyl acetate (1 : 1) and without combination are preferred solvents for enantiomeric purification with the (lS)-(+)-Camphor-10-sulfonic acid.

[0068] In embodiments, (R)-Fezolinetant, (lS)-(+)-Camphor-10-sulfonic acid and acetonitrile / MTBE mixture (1 : 1) are heated to 40-60 °C, preferably 50 °C for about 1-5 h, preferably 3h under stirring, followed by cooling to 0 °C and drying at 40-60 °C to isolate (R)-Fezolinetant camsylate.

[0069] In embodiments, the isolated (R)-Fezolinetant camsylate is suspended in methanol and water (5 V / w, 1 V / w) at RT for about 30 min - 2 hours, preferably 1 h under stiring, optionaly with seeding and additional water is added (9 V / w) to isolate by precipitation and optionally drying to 40-60 °C, preferably 50 °C, to obtain highly pure (R)-Fezolinetant (chiral HPLC: 100A%).

[0070] About 0.001-0.01 w / w, preferably, 0.005 w / w seeding with Fezolinetant Form 5 is used.

[0071] The Fezolinetant that is used in the disclosed process to which the suitable solvent is added in order to precipitate racemic Fezolinetant as a solid, thereby providing an enantiomerically enriched racemic solution (i.e. a mother liquor which is more enriched in (R)-Fezolinetant compared with the starting Fezolinetant) may be obtained by any process suitable for preparing (R)-Fezolinetant, such as US 10,787,458. The Fezolinetant product from the reaction is primarily (R)-Fezolinetant, but typically contains: about 1 to about 6%, about 1% to about 5%, about 1% to about 4%, about 1% to about 3%, about 2% to about 3%, about 2% to about 4%, about 3% to about 4%, or about 1.2% to about 3%, about 1.5% to about 2%, or about 1.6% to about 1.8% of (S)-Fezolinetant. Typically, the Fezolinetant product is in solution with an anhydrous solvent. For example, the Fezolinetant may be in the reaction mixture as a solution in an anhydrous solvent, or the Fezolinetant may be isolated from the reaction mixture, and the enantiomerically enriched racemic solution in step I, is obtained by adding suitable solvent to Fezolinetant. The Fezolinetant can be prepared by heating 3 -methyl- 1, 2, 4-thiadiazole-5 -carbohydrazide and (R)-(4-fluorophenyl)(5-methoxy-6- methyl-3,6-dihydropyrazin-l(2H)-yl)methanone in anhydrous solvent and optionally base.

[0072] In embodiments, anhydrous solvent is selected from anhydrous alcohol, in embodiment anhydrous methanol, anhydrous 2-propanol and anhydrous ethanol, preferably anhydrous methanol. Preferably, according to any embodiment, the anhydrous solvent contains: about 1% or less, about 0.8% or less, about 0.5% or less, about 0.4% or less, about 0.3% or less, about 0.2% or less, about 0.1% or less, about 0.05% or less, or about 0% water, for example as measured by KF titration.

[0073] For the optional base, preferably an organic base selected from DIPEA (N,N- diisopropylethylamine), 2,6-di-tert-Butyl-pyridine and pyridine is used, preferably DIPEA.

[0074] In embodiments, heating to 50-70 °C, preferably to 58 °C for 5-24 hours, preferably for 22 hours until reaction completed to obtain Fezolinetant. The Fezolinetant is primarily (R)-Fezolinetant, but as discussed above, the product typically contains minor quantities of(S)-Fezolinetant. The disclosed purification process enables removal of further amounts of (S)-Fezolinetant in order to further improve the enantiomeric purity of the (R)-Fezolinetant. As discussed herein, the removal of (S)-Fezolinetant can be achieved by the addition of a suitable solvent to a solution of the (R)-Fezolinetant in an anhydrous solvent (preferably anhydrous 2-propanol, anhydrous ethanol or anhydrous methanol, preferably anhydrous methanol), thereby precipitating racemic Fezolinetant as a solid which can be readily removed, to provide a mother liquor which is more enantiomerically enriched in (R)- Fezolinetant compared with the Fezolinetant obtained in the reaction step.

[0075] In embodiments suitable solvents are selected from acetone, methyl ethyl ketone, tetrahydrofuran, 2-methyltetrahydrofuran and acetonitrile, in embodiment acetonitrile is added to the Fezolinetant (which is preferably obtained from the reaction described above, and which is primarily (R)-Fezolinetant typically containing minor amounts of (S)- Fezolinetant), which is preferably in solution in an anhydrous solvent (preferably anhydrous 2-propanol, anhydrous ethanol or anhydrous methanol, preferably anhydrous methanol). The Fezolinetant may be present in the anhydrous solvent in the reaction mixture after reaction completion, or alternatively, the Fezolinetant may be isolated from the reaction mixture and dissolved in the anhydrous solvent. According to any aspect or embodiment of the disclosed purification process for (R)-Fezolinetant, the ratio of the anhydrous solvent (preferably methanol, ethanol or 2-propanol, and particularly methanol) to the suitable solvent (preferably acetone, methyl ethyl ketone, tetrahydrofuran, 2-methyltetraydrofuran or acetonitrile, and particularly acetonitrile), is: about 1 : 1 to about 1 :4, about 1 : 1.2 to about 1 :3.5, about 1 : 1.4 to about 1:3, about 1 : 1.4 to about 1 :2.5, about 1 : 1.4 to about 1 :2.2, or about 1 : 1.5 to about 1 :2. Preferably, the ratio of the anhydrous solvent (preferably methanol) to suitable solvent (preferably acetonitrile) is: about 1 : 1.2 to about 1 :2.5, about 1 : 1.3 to about 1 :2.4, about 1 : 1.4 to about 1 :2.3, or about 1 : 1.5 to about 1 : 1.2, about 1 :2, or about 1 : 1.5. In embodiments, the ratio between the added acetonitrile to the dry methanol is 2: 1 to obtain enantiomerically enriched racemic solution enriched mother liquor and further isolating (R)- Fezolinetant and purification with chiral acid according to procedure described herein.

[0076] According to any aspect or embodiment of the disclosed purification process for (R)- Fezolinetant, the precipitated racemic Fezolinetant may be isolated and removed by filtration. The solution may be cooled prior to filtration, preferably to a temperature of: about -5 °C to about 15 °C, about -2 °C to about 10 °C, about -2 °C to about 5 °C, about -1 °C to about 2 °C, or about 0 °C.

[0077] According to any aspect or embodiment of the purification process, the isolated mother liquor following removal of the solid racemic Fezolinetant may be treated with activated carbon prior to isolation of the (R)-Fezolinetant. The treating may comprise combining the mother liquor with activated carbon and heating (typically to a temperature of: about 40 °C to about 70 °C, about 45 °C to about 65 °C, about 50 °C to about 60 °C, or about 55 °C). Owing to the removal of the (S)-Fezolinetant as a racemic mixture by precipitation using the suitable solvent, the resulting (R)-Fezolinetant isolated from the mother liquor is further enriched in (R)-Fezolinetant, and hence has a higher enantiomeric purity. The enantiomeric purity of the resulting (R)-Fezolinetant may be further increased by purification with a chiral acid as discussed herein.

[0078] A further aspect of the disclosure provides a process for the preparation of compound FZL-10:FZL-10 comprising enzymatic stereoselective reductive amination of an alkyl pyruvate with ethylene diamine. Any suitable alkyl pyruvate may be used in this process. For example, the alkyl pyruvate may be a Ci to Cs alkyl pyruvate, preferably a Ci to C3 alkyl pyruvate. Methyl pyruvate, ethyl pyruvate, propyl pyruvate or isopropyl pyruvate may be used. Particularly, the alkyl pyruvate may be ethyl pyruvate.

[0079] The process is preferably carried out in the presence of an imine reductase or reductive aminase enzyme, preferably an imine reductase enzyme. The process may be carried out in the presence of a cofactor regenerating system, optionally a cofactor regenerating system containing NADPH, NADP+or their mixture, as cofactor.

[0080] Suitable cofactor regenerating systems that may be used comprise a cofactor regenerating enzyme. The cofactor regenerating enzyme may be selected from: an alcohol dehydrogenase (ADH), a glucose dehydrogenase (GDH), or a ketoreductase (KRED).Optionally, the cofactor regenerating enzyme is selected from: an alcohol dehydrogenase, or a glucose dehydrogenase preferably in the presence of glucose. Particularly suitable cofactorregenerating enzymes are an alcohol dehydrogenase from Lactobacillus brevis (LbADH) or a glucose dehydrogenase in the presence of glucose.

[0081] According to any aspect or embodiment of the process, the cofactor regenerating system may comprise sodium salt of nicotinamide adenine dinucleotide phosphate (NADP+) and Lactobacillus brevis alcohol dehydrogenase (LbADH) cofactor regenerating enzyme. Alternatively, the cofactor regenerating system may comprise sodium salt of nicotinamide adenine dinucleotide phosphate (NADP+) and glucose dehydrogenase (GDH) cofactor regenerating enzyme.

[0082] Preferred enzymes for the stereoselective reductive amination of the alkyl pyruvate with ethylene diamine to form FZL-10 according to any aspect or embodiment of the present disclosure, include imine reductases or reductive aminase enzymes. Imine reductase enzymes are particularly preferred. According to any aspect or embodiment of the process, the imine reductase enzyme has an amino acid sequence corresponding to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 (Figure 1), which are commercially available from Prozomix Limited as PRO-IRED-028, PRO-IRED-042, PRO- IRED-048, PRO-IRED-128, PRO-IRED-364, and PRO-IRED-374, respectively. Also preferred is an imine reductase enzyme isolated from Myxococcus stipitatus (herein referred to as imine reductase T171, SEQ ID NO: 7, Figure 1), or imine reductase enzymes having an amino acid sequence that comprises at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identity to any of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.

[0083] Particularly preferred imine reductase enzymes have amino acid sequences corresponding to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 (Figure 1), which are commercially available from Prozomix Limited as PRO-IRED-028, PRO-IRED-042, PRO-IRED-048, PRO-IRED-128, PRO-IRED-364, and PRO-IRED-374, respectively. Imine reductases were explored by Prozomix, and were found in soil sample in the United Kingdom (Marshall et al. Nat. Chem. 2021 13, 140-148). Prozomix imine reductase termed as pIR, pIRED in the above cited literature refers to identical enzymes as the PRO-IRED coding if the respective number codes coincide (e.g. pIR128 and pIRED128 is identical with PRO-IRED-128). Imine reductases can be clustered to superfamilies (SFaml) according to Imine Reductase Engineering Database (Fademrech et al. Proteins 2016, 84 (5), 600-610; https: / / ired.biocatnet.de / ).

[0084] A particularly preferred imine reductase enzyme for use the biocatalytic process for preparing FZL-10 according to any aspect or embodiment disclosed herein, has the amino acid sequence corresponding to SEQ ID NO: 7 (Figure 1) (also referred to herein as T-171). This imine reductase enzyme is an NAD(P)-dependent oxidoreductase isolated from Myxococcus stipitatus (NCBI reference sequence WP_015347361.1, https: / / www. ncbi. nln nih. gov / protein / WP 015347361}.

[0085] Preferably, the enzyme for the stereoselective reductive amination according to any aspect or embodiment of the present disclosure is an imine reductase enzyme having an amino acid sequence that comprises at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identity to any of: SEQ ID NO: 3, SEQ ID NO: 5, , SEQ ID NO: 6, or SEQ ID NO: 7; or SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, or particularly SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 7; and more particularly SEQ ID NO: 7.

[0086] Alternatively, the enzyme for the stereoselective reductive amination according to any aspect or embodiment of the present disclosure is an imine reductase enzyme having an amino acid sequence that comprises at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identity to: at least 50, at least 100, at least 150, at least 200, or at least 250, contiguous amino acid residues of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, or particularly SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 7; and more particularly SEQ ID NO: 7.

[0087] Particularly, the enzyme for the stereoselective reductive amination according to any aspect or embodiment of the present disclosure is an imine reductase enzyme having an amino acid sequence corresponding to: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, or particularly SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 7; and more particularly SEQ ID NO: 7.

[0088] The biocatalytic process for preparing FZL-10 may be generally represented by the following scheme (which exemplifies a cofactor regenerating system containing NADPH, NADP+):FZL-10

[0089] According to any aspect or embodiment of the disclosure, the enzyme for the stereoselective reductive amination is purified or partially purified. The enzyme may be in a lyophilized or lysed state. The enzyme may be purified, and / or lyophilized.

[0090] The reductive amination reaction according to any aspect or embodiment of the disclosure may be carried out in the presence of an aqueous buffer, optionally wherein the buffer is a potassium phosphate, a (4-(2-hydroxyethyl)piperazine-l -ethane-sulfonic acid (HEPES) / HC1, or the buffer may be ethylene diamine.

[0091] According to any aspect or embodiment, the reductive amination reaction may be carried out in the presence of a cosolvent, optionally wherein the cosolvent is selected from: an ether, preferably a C4 to G> ether and more preferably, tetrahydrofuran, diisopropyl ether or methylcyclopentyl ether; an ester, preferably a C4 to Ce ether and more preferably isopropyl acetate; an aromatic hydrocarbon preferably a Ce to Cs aromatic hydrocarbon and more preferably toluene; and alcohol, preferably a C2 to C4 alcohol and more preferably isopropanol; a ketone, preferably a C3 to Ce ketone and more preferably acetone or methylethyl ketone; dimethylsulfoxide, dimethyl formamide, N-methylpyrrolidine, or acetonitrile. However, a cosolvent may not be required.

[0092] According to any aspect or embodiment, the reductive amination reaction may be carried out in the presence of a cosolvent, wherein the cosolvent is selected from: an ether, preferably a C4 to Ce ether and more preferably, tetrahydrofuran, diisopropyl ether or methylcyclopentyl ether; an ester, preferably a C4 to Ce ether and more preferably isopropyl acetate; an aromatic hydrocarbon preferably a Ce to Cs aromatic hydrocarbon and more preferably toluene; and alcohol, preferably a C2 to C4 alcohol and more preferably isopropanol; or dimethylsulfoxide. Particularly suitable cosolvents may be selected from: diisopropyl ether, methylcyclopentyl ether, isopropyl acetate, toluene, isopropanol, or dimethylsulfoxide.

[0093] According to any aspect or embodiment, the reductive amination reaction may be carried out in the presence of an aqueous buffer, and wherein the concentration of thecofactor NADP which is optionally added to the reaction, based on the aqueous phase, is in the ratio of ranges from 0.0 mM to 2 mM in particular from 0.2 mM to 0.25 mM.

[0094] According to any aspect or embodiment, the process comprises combining an alkyl pyruvate with an aqueous mixture comprising: ethylene diamine and an imine reductase or reductive aminase enzyme, and optionally an enzyme co-factor regenerating system.

[0095] According to any aspect or embodiment, the alkyl pyruvate is preferably combined with an aqueous mixture comprising: ethylene diamine, an imine reductase enzyme, a cofactor regenerating system comprising monosodium salt of nicotinamide adenine dinucleotide phosphate (NADP+), an alcohol dehydrogenase, and optionally a cosolvent. The pH of the aqueous mixture is preferably about 5 to about 9, about 7 to about 9, about 7.5 to about 8.8, about 7.8 to about 8.7, about 8.2 to about 8.7, about 8.4 to about 8.7, or about 8.0 to 8.5, or about 8.5. The pH of the aqueous mixture may be adjusted, if necessary, with a mineral acid, preferably hydrochloric acid.

[0096] According to any aspect or embodiment, the process preferably comprises adding the alkyl pyruvate to the aqueous mixture. Preferably, the alkyl pyruvate may be added gradually, portion wise or continuously (i.e. in small amounts) to the aqueous mixture, wherein the addition is preferably over a suitable period of time. Preferably, the addition takes place portion wise or continuously: over the first 15-40% of the total reaction time, or over the first 15-30% of the total reaction time, or over the first 15-25% of the total reaction time, or over the first 18-22% of the total reaction time, or the first about 20% of the total reaction time. According to any aspect or embodiment, the alkyl pyruvate is added portion wise or continuously to the aqueous mixture over a period of: about 30 minutes to about 10 hours, about 2 hours to about 8 hours, about 2 hours to about 6 hours, about 3 hours to about 6 hours, about 3 hours to about 5 hours, or about 4 hours.

[0097] According to any aspect or embodiment, the aqueous mixture is prepared by combining ethylene diamine, water and, if required, adjusting the pH to: about 7.5 to about 8.8, about 7.8 to about 8.7, about 8.2 to about 8.7, about 8.4 to about 8.7, or about 8.0 to 8.5, or about 8.5; adding the enzyme cofactor regenerating system, preferably comprising monosodium salt of nicotinamide adenine dinucleotide phosphate (NADP+) and either: an alcohol dehydrogenase or glucose dehydrogenase in the presence of glucose; adding the imine reductase or reductive aminase enzyme, and optionally adding a cosolvent.

[0098] According to any aspect or embodiment, the aqueous mixture is heated to a temperature of: about 20 °C to about 40 °C, about 22 °C to about 38 °C, about 25 °C to about38 °C, about 25 °C to about 36 °C, about 26 °C to about 35 °C, about 27 °C to about 33 °C, about 28 °C to about 32 °C, about 29 °C to about 31 °C, or about 25 °C to about 30 °C, prior to combining with the alkyl pyruvate.

[0099] According to any aspect or embodiment, the concentration of the total alkyl pyruvate added to the reaction mixture is preferably about 1.4 g / L to about 50 g / L, about 2.5 g / L to about 40 g / L, about 2.5 g / L to about 20 g / L, about 3 to about 15 g / L, about 4 g / 1 to about 12 g / L; or about 5 g / L to about 10 g / L.

[0100] According to any aspect or embodiment, the cosolvent, when present, may be used in the reaction mixture at a concentration (v / v%) of: about 0.2% to about 10%, about 0.5% to about 8%, about 0.5% to about 5%, about 0.8% to about 2%, or about 0.8% to about 1.5%, about 0.9% to about 1.2%, or about 1%.

[0101] According to any aspect or embodiment, the ethylene diamine may be used in the reaction mixture in an amount of: about 1.5 to about 8, about 1.8 to about 7, about 2 to about 6, or about 2.5 to about 5, mole equivalents relative to alkyl pyruvate.

[0102] According to any aspect or embodiment, the enzyme for the stereoselective reductive amination is preferably an imine reductase as described above. The enzyme is preferably present in the reaction mixture in a concentration (w / w%) of: about 1 to about 200, about 1 to about 150, about 1 to about 100, about 2 to about 50, about 3 to about 30, about 4 to about 28, or about 5 to about 25.

[0103] According to any aspect or embodiment, the enzyme regeneration cofactor, when used, is preferably sodium salt of nicotinamide adenine dinucleotide phosphate. The enzyme regeneration cofactor may preferably be present in the reaction mixture at a concentration of: 0.15 to about 3.5, about 0.2 to about 2.8, or about 0.25 to about 2.5 mM.

[0104] According to any aspect or embodiment, the cofactor regenerating enzyme is preferably an alcohol dehydrogenase preferably from Lactobacillus brevis, or a glucose dehydrogenase wherein glucose is present. Preferably, the cofactor regenerating enzyme is present in the reaction mixture at a concentration (w / w%) of: 0.25 to about 5, about 0.4 to about 3.5, about 0.6 to about 3, about 0.8 to about 2.5, or about 1 to about 2,

[0105] According to any aspect or embodiment, the cofactor regenerating enzyme is preferably an alcohol dehydrogenase preferably from Lactobacillus brevis, or a glucose dehydrogenase wherein glucose is present, wherein the cofactor regenerating enzyme is present in the reaction mixture at a concentration (w / w%) of: 0.25 to about 5, about 0.4 to about 3.5, about 0.6 to about 3, about 0.8 to about 2.5, or about 1 to about 2.

[0106] Preferably, according to any aspect or embodiment of the disclosure, the cofactor regenerating enzyme may ne glucose dehydrogenase and wherein glucose is present, wherein glucose is present in the reaction mixture in an amount of: about 1.0 to about 2.2 about 1.02 to about 2.1, or about 1.05 to about 2.0 mole equivalents relative to the alkyl pyruvate.

[0107] According to any aspect or embodiment, the alkyl pyruvate is a Ci to Cs alkyl pyruvate, preferably a Ci to C3 alkyl pyruvate. Particularly, the alkyl pyruvate may be methyl pyruvate, ethyl pyruvate, propyl pyruvate or isopropyl pyruvate, and preferably ethyl pyruvate.

[0108] According to any aspect or embodiment, the cofactor regenerating enzyme is preferably an alcohol dehydrogenase preferably from Lactobacillus brevis, or a glucose dehydrogenase and wherein glucose is present, wherein the cofactor regenerating enzyme is present in the reaction mixture at a concentration (w / w%) of: 0.25 to about 5, about 0.4 to about 3.5, about 0.6 to about 3, about 0.8 to about 2.5, or about 1 to about 2.

[0109] Preferably, according to any aspect or embodiment of the process, the pH of the reaction mixture is maintained at: about 5 to about 9, about 7 to about 9, about 7.5 to about 8.8, about 7.8 to about 8.7, about 8.2 to about 8.7, about 8.4 to about 8.7, or about 8.0 to 8.5, or about 8.5. The pH of the reaction mixture may be adjusted, if necessary, with an organic base, preferably ethylenediamine.

[0110] According to any aspect or embodiment, the reductive amination reaction may be carried out at: about 20 °C to about 40 °C, about 22 °C to about 38 °C, about 25 °C to about 38 °C, about 25 °C to about 36 °C, about 26 °C to about 35 °C, about 27 °C to about 33 °C, about 28 °C to about 32 °C, about 29 °C to about 31 °C, or wherein the reductive amination reaction is carried out at about 25 °C to about 30 °C. The reaction time may be: about 1 hour to about 72 hours, about 2 hours to about 48 hours, about 3 hours to about 38 hours, or about 4 hours to about 36 hours, about 6 hours to about 30 hours, about 10 hours to about 28 hours, about 12 hours to about 26 hours, or about 12 hours to about 24 hours.

[0111] According to any aspect or embodiment, the FZL-10 obtained by the disclosed process may be isolated. FZL-10 may be isolated from the disclosed processes according to any aspect or embodiment, by a process comprising: basifying the reaction mixture, concentrating the mixture, extracting the mixture with an organic solvent (preferably a C4 to Cs aliphatic alcohol) to form a solution of FZL-10 in the organic solvent, and crystallizing FZL-10 from the solution. In the basifying step, the reaction mixture may be adjusted to a pHof about 9.5 to about 12.5, about 9.5 to about 11, about 9.5 to about 10.5, about 9.8 to about 10.2, or about 10. Any suitable base can be used, preferably an inorganic based, particularly an alkali metal hydroxide, and more particularly sodium hydroxide or potassium hydroxide, and most preferably potassium hydroxide. Suitable organic solvents for the extraction include C4 to Cs aliphatic alcohols, optionally a C4 to Ce aliphatic alcohol, and preferably a C4 aliphatic alcohol, more preferably 1-butanol, isobutanol or sec-butanol, preferably 1-butanol. After the extraction step, sodium chloride may be added to the solution of FZL-10 in the organic solvent, and the mixture stirred, preferably for a period of: about 1 to about 24 hours, about 4 to about 18 hours, about 5 to about 15 hours, about 6 to about 10 hours, about 7 to about 9 hours, or about 8 hours. The liquid phase may be separated from the solid phase by e.g. filtration. The resulting solution may be concentrated by evaporation under reduced pressure. The evaporation may be carried out to reduce the solvent volume in order to facilitate or to initiate crystallization of the product. Crystallization may be initiated by the addition of seed crystals of FZL-10, optionally stirring the seeded mixture (preferably at a temperature of: about -5 °C to about 15 °C, about -2 °C to about 12 °C, about 0 °C to about 10 °C, about 2 °C to about 8 °C, about 4 °C to about 6 °C, or about 5 °C), and collecting the product by filtration. The seeded mixture may be stirred for an appropriate period of time, particularly: about 30 minutes to about 24 hours, about 1 hour to about 18 hours, about 2 hours to about 10 hours, about 3 hours to about 6 hours, or about 5 hours.

[0112] Following filtration, according to any aspect or embodiment, the FZL-10 may be slurried in a C4 to Cs aliphatic alcohol (optionally a C4 to Ce aliphatic alcohol, and preferably a C4 aliphatic alcohol, more preferably 1-butanol, isobutanol or sec-butanol, and particularly 1-butanol), preferably at a temperature of: about -5 °C to about 15 °C, about - 2 °C to about 12 °C, about 0 °C to about 10 °C, about 2 °C to about 8 °C, about 4 °C to about 6 °C, or about 5 °C. The FZL-10 product may be isolated, preferably by filtration, and optionally dried.

[0113] The process of any aspect or embodiment herein may further comprise converting the FZL-10 to (R)-Fezolinetant or a salt thereof. (R)-Fezolinetant may be prepared from the FZL-10 by a process comprising:(i) reacting the compound FZL-10 with 4-fluorobenzoyl chloride to form a compound of formula FZL-9:(ii) reacting FZL-9 with trimethyloxonium tetrafluoroborate to form a compound of formula FZL-11:FZL-11; and(iii) reacting the compound FZL-11 with a compound of formula FZL-3 :FZL-3 to form (R)-Fezolinetant:

[0114] For example, (R)-Fezolinetant may be prepared from the FZL-10 by a process according to the following scheme:

[0115] According to any aspect or embodiment, the resulting (R)-Fezolinetant may be subject to enantiomeric purification. The enantiomeric purification of the (R)-Fezolinetant may be carried out according to the procedures described in any aspect or embodiment of the enantiomeric purification process for (R)-Fezolinetant described herein. Particularly, the (R)- Fezolinetant prepared using FZL-10 prepared by the disclosed process, may be purified by a process comprising:(a) providing a solution of the (R)-Fezolinetant in a solvent, preferably wherein the solvent is an alcohol, and more preferably methanol, ethanol or 2-propanol, and particularly methanol;(b) combining the solution with a solvent selected from: acetone, methyl ethyl ketone, tetrahydrofuran, 2-methyltetrahydrofuran and acetonitrile, and particularly acetonitrile, preferably by adding the solvent to the solution to form a mixture;(c) filtering the mixture and collecting the filtrate;(d) optionally stirring the filtrate with activated carbon and removing the activated carbon (when used);(e) crystallizing purified (R)-Fezolinetant from the mixture;(f) isolating the purified (R)-Fezolinetant, preferably by filtration; and(g) optionally drying the (R)-Fezolinetant.

[0116] The solvent used in step (a) may be the anhydrous solvent described in the disclosed purification process discussed herein. According to any aspect or embodiment of the disclosure, the solvent in step (a) is preferably anhydrous methanol. The solvent used in step (b) may be the suitable solvent described in the disclosed purification process discussed herein. According to any aspect or embodiment of the disclosure, the solvent in step (b) is preferably acetonitrile. Step (b) preferably comprises stirring the mixture, preferably at room temperature, optionally for a period of about 0.5 to about 8 hours, about 0.5 to about 6 hours, about 0.5 to about 4 hours, or about 0.5 to about 3 hours, about 1 to about 3 hours, or about 1 hour. As discussed for the enantiomeric purification process for (R)-Fezolinetant disclosed herein, step (b), which preferably comprises addition of the solvent to the solution in step (a), effects the precipitation of minor amounts of (S)-Fezolinetant, that may be present from the synthetic process described. The (S)-Fezolinetant is precipitated as a racemic mixture with (R)-Fezolinetant as a solid. The filtration step (c) enables removal of the solid, thereby producing a filtrate which is further enriched in (R)-Fezolinetant. The (R)-Fezolinetant in the filtrate may be further purified in step (d), by stirring the filtrate with activated carbon. Preferably the stirring is at a temperature of about 40 °C to about 60 °C, about 50 °C to about 60 °C, or about 55 °C. Step (d) may be carried out for a suitable period of time to purify the (R)-Fezolinetant in the filtrate, preferably for a period of about 0.5 hours to about 5 hours, about 0.5 to about 3 hours, or about 1 hour. The activated carbon may be removed by filtration. Purified (R)-Fezolinetant may be isolated by cooling, preferably to a temperature of about -5 °C to about 5 °C, about -2 °C to about 2 °C, about -1 °C to about 1 °C, or about 0 °C, to crystallize (R)-Fezolinetant, or by concentrating the filtrate from step (c) or step (d) and optionally cooling, preferably to a temperature of about -5 °C to about 5 °C, about -2 °C to about 2 °C, about -1 °C to about 1 °C. Preferably, step (e) comprises concentrating the filtrate from step (c) or step (d), and cooling, preferably to a temperature of about -5 °C to about 5 °C, about -2 °C to about 2 °C, about -1 °C to about 1 °C, or about 0 °C.

[0117] Alternatively, or additionally, enantiomeric purification or further enantiomeric purification of the (R)-Fezolinetant, either from the synthetic procedure according to the disclosed process, or from the enantiomeric purification process described herein, may be carried out by diastereomeric salt separation using a chiral acid, preferably wherein the chiral acid is (lS)-(+)-camphor-10-sulfonic acid; or by selective crystallization using an acid selected from ethanesulfonic acid, benzenesulfonic acid and tosic acid. The purified (R)-Fezolinetant (lS)-(+)-camphor-10-sulfonic acid, (R)-Fezolinetant esylate, (R)-Fezolinetant besylate, or (R)-Fezolinetant tosylate, may be crystallized from a solvent combination selected from: acetonitrile / tert-butyl methyl ether, acetonitrile / ethyl acetate, acetonitrile / acetone, methanol / isopropanol, methanol / ethyl acetate, methanol / tert-butyl methyl ether, ethanol / ethyl acetate, acetone / ethyl acetate. The process may comprise heating (R)-Fezolinetant, and: (lS)-(+)-camphor-10-sulfonic acid, (R)-Fezolinetant esylate, (R)- Fezolinetant besylate, or (R)-Fezolinetant tosylate, in a solvent combination of: acetonitrile and tert-butyl methyl ether. The heating may be to a temperature of 40-60 °C, and more preferably 50 °C. The heating may be carried out for a period of about 1-5 hours, preferably about 3 hours. The resulting (R)-Fezolinetant camsylate, (R)-Fezolinetant esylate, (R)- Fezolinetant besylate, or (R)-Fezolinetant tosylate, which is selectively crystallized from the solution, may be isolated by cooling, preferably to: about -10 °C to about 10 °C, about -5 °C to about 5 °C, about -2 °C to about 2 °C, or about 0 °C. The purified (R)-Fezolinetant camsylate, (R)-Fezolinetant esylate, (R)-Fezolinetant besylate, or (R)-Fezolinetant tosylate may be dried. The resulting (R)-Fezolinetant camsylate, (R)-Fezolinetant esylate, (R)- Fezolinetant besylate, or (R)-Fezolinetant tosylate, may be suspended in a mixture of water and methanol, preferably at room temperature, and optionally seeded to precipitate (R)- Fezolinetant, which can be isolated, preferably by filtration; and optionally dried. Preferably the enantiomeric purification of the (R)-Fezolinetant or further enantiomeric purification of (R)-Fezolinetant is carried out by diastereomeric salt separation using a chiral acid, preferably wherein the chiral acid is (lS)-(+)-camphor-10-sulfonic acid.

[0118] The processes for preparing (R)-Fezolinetant according to any aspect or embodiment described herein may further comprise combining the (R)-Fezolinetant with at least one pharmaceutically acceptable acid, to form a pharmaceutically acceptable salt of (R)- Fezolinetant. The processes for preparing (R)-Fezolinetant or pharmaceutically acceptable salt thereof, according to any aspect or embodiment described herein may further comprise combining the (R)-Fezolinetant or pharmaceutically acceptable salt thereof, to form a pharmaceutical composition or formulation.

[0119] (R)-Fezolinetant or pharmaceutically acceptable salt of (R)-Fezolinetant prepared according to process of the disclosure herein, may be combined with at least one pharmaceutically acceptable excipient to form a pharmaceutical composition or formulation.

[0120] The present disclosure further includes the use of a process for preparing FZL- 10, in the synthesis of (R)-Fezolinetant or a salt thereof.

[0121] The present disclosure includes a process for preparing (R)-Fezolinetant, comprising preparing the compound FZL-10 by a process according to any aspect or embodiment described herein, and converting the FZL-10 to (R)-Fezolinetant or a pharmaceutically acceptable salt of (R)-Fezolinetant. Preferably, the FZL-10 is converted to (R)-Fezolinetant or a pharmaceutically acceptable salt of (R)-Fezolinetant by a process according to any aspect or embodiment of the process disclosed herein.EXAMPLES

[0122] Determination of chiral purity was performed with Column: Chiralpak IA 5.0pm C18 4.6x250 mm and mobile phase: 0.1% DEA in HexaneZEtOAc (1 : 1 v / v); flow rate: 1.0 mL / min; UV absorbance at 280 nm. Imine reductase enzymes were obtained from Prozomix, with the exception of T171 (SEQ ID NO: 7).Example 1. Preparation of (R)-(4-fluorophenyl)(8-methyl-3-(3-methyl-L2,4-thiadiazol-5-yl)-5,6-dihydro-rL2,41triazolo[4,3-a1pyrazin-7(8H)-yl)methanone

[0123] N,N-Diisopropylethylamine (31.32 mL, 179.806 mmol, 1.50 molEq), followed by 3-methyl-l,2,4-thiadiazole-5-carbohydrazide (21.80 grams, 137.851 mmol, 1.15 molEq), were added to a solution of (R)-(4-fluorophenyl)(5-methoxy-6-methyl-3,6-dihydropyrazin- l(2H)-yl)methanone (30.0 grams, 119.871 mmol) in anhydrous methanol (150 mL, 5.0 V / w). The resultant suspension was heated to 58 °C and kept at this temperature for about 20-22 hours until completion under stirring. Reaction mixture was gradually cooled down to 22 °C and acetonitrile (300 mL, 5.0 V / w) was added, over the course of 1 hour.

[0124] Resulted suspension was stirred at 22 °C for 1 hour, then cooled to 0 °C, continued stirring at 0 °C for 3 hours and then filtered. The filter cake was washed twice with acetonitrile (2 x 30 mL, 2 x 1 V / w). Obtained filtrate was heated to 55 °C, activated carbon(6.0 grams, 0.2 w / w) was added and the mixture is stirred for 1 hour prior to hot filtration over celite. Celite was washed twice with acetonitrile, heated to 50 °C, (2 x 30 mL, 2 x 1 V / w).

[0125] Obtained filtrate was concentrated under reduced pressure to a volume of 90 mL (3 V / w). Resulting suspension was stirred at 60 °C for 30 minutes, then gradually cooled down to 0 °C, stirred at 0 °C for 2 hours and filtered. Filter cake was washed twice with ethyl acetate (2 x 30 mL, 2 x 1 V / w) and dried under reduced pressure at 50 °C to afford (R)-(4- fluorophenyl)(8-methyl-3-(3-methyl-l,2,4-thiadiazol-5-yl)-5,6-dihydro-[l,2,4]triazolo[4,3- a]pyrazin-7(8H)-yl)m ethanone (27.0 grams, 63% yield) as white crystalline solid (HPLC purity 99.90 A%, chiral HPLC: 99.0 A%).Example 2, Preparation of (R)-(4-fluorophenyl)(8-methyl-3-(3-methyl-L2,4-thiadiazol-5-yl)-5,6-dihvdro-[L2,41triazolo[4,3-a1pyrazin-7(8H)-yl)methanone: ((!S,4R)-7,7-dimethyl-2- oxobicyclo[2.2.11heptan-l-yl)methanesulfonate

[0126] (lS)-(+)-10-Camphorsulfonic acid (camsylate acid, 778 mg, 3.348 mmol, 1.20 molEq) was added to a suspension of (R)-(4-fluorophenyl)(8-methyl-3-(3-methyl-l,2,4- thiadiazol-5-yl)-5,6-dihydro-[l,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methanone (1.0 g, 2.790 mmol) in a ACN / tert-butyl methyl ether (1 / 1) (8.2 mL, 8.2 V / w). Resulted mixture was heated to 50 °C and is stirred at given temperature for 3 hours.

[0127] Obtained solution was gradually cooled down to 0 °C, stirred at 0 °C for 1 hour and filtered. Filter cake is washed twice with tert-Butyl methyl ether (2 x 2 mL, 2 x 2 V / w) and dried under reduced pressure at 50 °C to afford (R)-(4-fluorophenyl)(8-methyl-3- (3-methyl-l,2,4-thiadiazol-5-yl)-5,6-dihydro-[l,2,4]triazolo[4,3-a]pyrazin-7(8H)- yl)methanone ((1 S,4R)-7,7-dimethyl-2-oxobicyclo[2.2. l]heptan-l-yl)m ethanesulfonate (1.4 grams, 85% yield) as white crystalline solid (HPLC purity 99.90 A%, chiral HPLC: 100 A%).Example 3, (R)-(4-fluorophenyl)(8-methyl-3-(3-methyl-L2,4-thiadiazol-5-yl)-5,6-dihvdro- rL2,41triazolo[4,3-a1pyrazin-7(8H)-yl)methanone

[0128] (R)-(4-fluorophenyl)(8-methyl-3-(3-methyl-l,2,4-thiadiazol-5-yl)-5,6- dihydro-[l,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methanone : ((1 S,4R)-7,7-dimethyl-2- oxobicyclo[2.2.1]heptan-l-yl)methanesulfonate (1.0 gram, 1.693 mmol) was suspended in a methanol (5.0 mL, 5.0 V / w). Water (1.0 mL, 1.0 V / w) was added at 22 °C, in a dropwise manner. Seed with Fezolinetant Form 5 (0.005 w / w) was introduced and suspension was stirred at given temperature for 1 hour. Additional water (9.0 mL, 9.0 V / w) was added at 22 °C, in a dropwise manner, and suspension was stirred for 2 hours.

[0129] Suspension was filtered, and washed twice with MeOH / H2O (1 / 2) (2 x 2 mL, 2 x 2 V / w) and dried under reduced pressure at 50 °C to afford (R)-(4-fluorophenyl)(8- methyl-3-(3-methyl-l,2,4-thiadiazol-5-yl)-5,6-dihydro-[l,2,4]triazolo[4,3-a]pyrazin-7(8H)- yl)methanone (560 mg, 93% yield) as white crystalline solid (HPLC purity 99.93 A%, chiral HPLC: 100 A%).Example 4, Preparation of FZL-10 by LbADH cofactor regeneration

[0130] Distilled water (9.3 liters) and ethylenediamine (137.7 g) were mixed under stirring in a glass reactor with 10 liters of working volume. The obtained solution was cooled to 25 °C, and the pH of the solution was slowly adjusted to 8.50 by adding diluted aqueous HC1 solution to it (0.35 liters of HC1 solution was used). Monosodium salt of nicotinamide adenine dinucleotide phosphate (NADP+, 1.531 g) was added to and dissolved into the solution and the pH was adjusted back to 8.50 with diluted HC1 solution. Alcohol dehydrogenase from Lactobacillus brevis, recombinantly produced by fermentation in Escherichia coli (LbADH, 2.13 g) and imine reductase (IRED) from Myxococcus stipitatus also recombinantly expressed (T-171, 5.33 g) were added and dissolved into the solution. Isopropanol (100 ml) was finally added, and the reaction volume was completed to 10 liters with distilled water. Before starting the reaction, the temperature of the mixture was set to 30 °C. Ethyl pyruvate (106.0 grams in total) was gradually added to the reaction mixture during a 4-hour timeframe, while the pH of the reaction mixture was kept constantly at pH = 8.5 by addition of ethylenediamine (in total 55.0 grams of ethylenediamine were added). The stirring and pH adjustment continued for 24 hours, when the conversion was analyzed by HPLC. Conversion of 97.6% was reached after a total reaction time of 24 hours.Isolation of FZL-10

[0131] The pH of the final reaction mixture was adjusted to 10.0 by addition of KOH solution (5.0 M) to the reaction mixture. The solvent content of the reaction mixture was partially evaporated under vacuum to a final volume of one-tenth of the initial volume. A large amount of ethylenediamine was evaporated during this step. Perlite (5.3 grams) and 1- butanol (1 liter) were added to the concentrate, kept under stirring for 30 minutes, thereafter filtered. The filtrate separated to aqueous (lower) and organic (upper) phases, the organic phase was separated and collected in a second reactor. The remaining aqueous phase was extracted four times with 4^1 liters of 1 -butanol, the butanolic extracts were united with the organic phase from the second reactor. Sodium chloride (5.3 grams) was added, and the mixture was stirred for 8 hours, thereafter the salt was let to settle, and the liquid phase was separated and concentrated to 4% of its initial volume by evaporation under vacuum. Perlite (5.3 grams) and ethyl acetate (1060 grams) were added to the concentrate, which was stirred for 30 minutes, thereafter filtered. Final concentration of the filtrate was performed by evaporation under vacuum, to 50% of the initial volume (corresponding to ~500 g / 1 of FZL- 10). The crystallization of the product was initiated by adding seed crystals of FZL-10 into the solution. The crystallization of FZL-10 was exothermic. The mixture was stirred at 5 °C for 5 hours, thereafter the suspension was filtered. The crystals were resuspended in 1- butanol precooled to 5 °C (21.2 grams), filtered and dried at 50 °C. FZL-10 was isolated in 115.3 g amount, corresponding to 55% yield (corrected by assay, relatively to the amount of ethyl pyruvate). The chromatographic purity of the obtained product was 99.9 area%, and its enantiomeric excess (ee) was 99.8.Example 5, Enzymatic process with LbADH cofactor regeneration

[0132] A I L reactor is charged with deionized water (750 ml) and ethylene diamine (5.52 g / 91.8 mmol [1 mol. eq]) to get a solution at 20-25 °C. The pH of the solution is adjusted to 8.0-8.5 by slow addition of dilute aqueous HC1 solution. Sodium salt of nicotinamide adenine dinucleotide phosphate (NADP+) (193 mg / 0.256 mmol) is charged to the reactor and stirred at 20-25 °C until dissolution. The pH of the solution is adjusted to 8.0-8.5. 535 mg T-171 imine reductase [I-RED] enzyme (SEQ ID NO: 7) is charged to the reactor and stirred at 20-30 °C until dissolution. 40 ml of 50 mM aqueous sodium chloride solution is charged to a second reactor. 107 mg Lactobacillus brevis alcohol dehydrogenase (LbADH) cofactor regenerating enzyme is dissolved in this solution. LbADH enzyme solution from the second reactor ischarged to the first reactor. The volume of the final bioconversion mixture is adjusted to 1 L with deionized water.

[0133] The enzymatic reaction is conducted at 30 °C. Ethyl pyruvate (10.65 g / 91.8 mmol) is added in portions over a period four hours to the stirred solution and the pH maintain in the range 8.0-8.5 by the addition of ethylene diamine. The reaction is monitored by HPLC. FZL-10 is isolated according to the procedure in Example 7.Example 6, Enzymatic process with GDH cofactor regeneration

[0134] A 10 L reactor is charged with deionized water (7500 ml) and ethylene diamine (54.1 g / 0.9 mol [1 mol. eq]) to get a solution at 20-25 °C. The pH of the solution is adjusted to 8.0-8.5 by slow addition of diluted aqueous HC1 solution. Sodium salt of nicotinamide adenine dinucleotide phosphate (NADP+) (1531 mg / 2 mmol) is charged to the reactor and stirred at 20-25 °C until dissolution. The pH of the solution is adjusted to 8.0-8.5. Glucose (186.5 g / 1.05 mol) is charged to the reactor and stirred at 20-25 °C until dissolution. Regenerating enzyme glucose dehydrogenase (GDH) 2.09 g is added to this solution and stirred at 20-25 °C until dissolution. Finally, 5.2 g T-171 I-RED enzyme is charged to the reactor and stirred at 20-25 °C until dissolution. The volume of the final bioconversion mixture is adjusted to 1 L with deionized water.

[0135] The enzymatic reaction is conducted at 30 °c. Ethyl pyruvate (104.5 g / 900 mmol) is added in portions over a period of four hours to the stirred solution and the pH maintain in the range 8.0-8.5 by the addition of ethylene diamine. The reaction is monitored by HPLC. The FZL-10 is isolated according to the procedure in Example 7.Example 7, Isolation of FZL-10 from enzymatic process

[0136] After reaction completion the product is isolated from the reaction mixture according to the following procedure:-the aqueous reaction mixture is concentrated until a concentration of FZL-10 no less than 100 g / L is reached. Evaporation is carried out in vacuum or reduced pressure, and the maximum bath temperature is 50 °C;-the residue is cooled to 20-30 °C and mixed with n-butanol (at least equal volume relative to the volume of the residue) to form a suspension. The enzyme residue is precipitated and filtered off from the mixture;-the solvent phase of the filtrate is separated, and the aqueous phase is re-extracted with n- butanol (preferably at least four times). The phases are separated, and the solvent phase is collected;-the water content of the combined solvent phase is salted out by the addition of solid sodium chloride. The phases are separated, and the solvent phase is collected;-after the evaporation of the solvent phase, the product is isolated as base or can be converted to a salt or a pharmaceutically acceptable salt. The product may be subjected to enantiomeric purification as described herein.

Claims

CLAIMS1. A process for the preparation of compound FZL-10:FZL-10 comprising enzymatic stereoselective reductive amination of an alkyl pyruvate with ethylene diamine.

2. The process according to Claim 1, which is carried out in the presence of an imine reductase or reductive aminase enzyme.

3. The process according to Claim 1 wherein the reaction is carried out in the presence of a cofactor regenerating system containing NADPH, NADP+or their mixture as cofactor.

4. The process according to Claim 3, wherein the cofactor regenerating system comprises a cofactor regenerating enzyme, optionally wherein the cofactor regenerating enzyme is selected from: an alcohol dehydrogenase (ADH), a glucose dehydrogenase (GDH), or a ketoreductase (KRED); preferably wherein the cofactor regenerating enzyme is selected from: an alcohol dehydrogenase, or a glucose dehydrogenase preferably in the presence of glucose; and particularly: an alcohol dehydrogenase from Lactobacillus brevis or a glucose dehydrogenase in the presence of glucose.

5. The process according to any of Claims 1, 2 or 3, wherein the cofactor regenerating system comprises sodium salt of nicotinamide adenine dinucleotide phosphate (NADP+) and Lactobacillus brevis alcohol dehydrogenase (Lb ADH) cofactor regenerating enzyme, or wherein the cofactor regenerating system comprises sodium salt of nicotinamide adenine dinucleotide phosphate (NADP+) and glucose dehydrogenase (GDH) cofactor regenerating enzyme.The process according to any of Claims 1, 2, 3, 4, or 5, wherein the enzyme for the stereoselective reductive amination has an amino acid sequence that comprises at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identity to any of: SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NON, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO: 7:preferably wherein the enzyme for the stereoselective reductive amination has an amino acid sequence that comprises at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identity to any of: SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:6, or SEQ ID NO: 7; or particularly SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, and more particularly SEQ ID NO: 7.

7. The process according to any of Claims 1, 2, 3, 4, 5, or 6, wherein the enzyme has an amino acid sequence that comprises at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identity to: at least 50, at least 100, at least 150, at least 200, or at least 250, contiguous amino acid residues of: SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO: 6, or SEQ ID NO:7, and particularly SEQ ID NO: 3, SEQ ID NO:5, SEQ ID NO: 6 and SEQ ID NO: 7, and more particularly SEQ ID NO: 7.

8. The process according to any of Claims 1, 2, 3, 4, 5, 6, or 7, wherein the enzyme for the stereoselective reductive amination has an amino acid sequence corresponding to: SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and particularly SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7; and more particularly SEQ ID NO: 7.

9. The process according to any of Claims 1, 2, 3, 4, 5, 6, 7, or 8, wherein the enzyme for the stereoselective reductive amination is purified or partially purified, or wherein the enzyme is in a lyophilized or lysed state, preferably wherein the enzyme is purified and / or lyophilized.

10. The process according to any of Claims 1, 2, 3, 4, 5, 6, 7, 8 or 9, wherein the reductive amination reaction is carried out in the presence of an aqueous buffer, optionally wherein the buffer is a potassium phosphate, a (4-(2-hydroxyethyl)piperazine-l -ethane- sulfonic acid (HEPES) / HC1, or ethylene diamine.

11. The process according to any of Claims 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, wherein the reductive amination reaction is carried out in the presence of a cosolvent, optionally wherein the cosolvent is selected from: an ether, preferably a C4 to Ce ether and more preferably, tetrahydrofuran, diisopropyl ether or methylcyclopentyl ether; an ester, preferably a C4 to Ce ether and more preferably isopropyl acetate; an aromatic hydrocarbon preferably a Ce to Cs aromatic hydrocarbon and more preferably toluene; and alcohol, preferably a C2 to C4 alcohol and more preferably isopropanol; a ketone, preferably a C3 to Ce ketone and more preferably acetone or methylethyl ketone; dimethylsulfoxide, dimethyl formamide, N-methylpyrrolidine, or acetonitrile.

12. The process according to any of Claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, wherein the reductive amination reaction is carried out in the presence of a cosolvent, wherein the cosolvent is selected from: an ether, preferably a C4 to Ce ether and more preferably, tetrahydrofuran, diisopropyl ether or methylcyclopentyl ether; an ester, preferably a C4 to Ce ether and more preferably isopropyl acetate; an aromatic hydrocarbon preferably a Ce to Cs aromatic hydrocarbon and more preferably toluene; and alcohol, preferably a C2 to C4 alcohol and more preferably isopropanol; or dimethylsulfoxide.

13. The process according to any of Claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, wherein the reductive amination reaction is carried out in the presence of a cosolvent, wherein the cosolvent is selected from: diisopropyl ether, methylcyclopentyl ether, isopropyl acetate, toluene, isopropanol, or dimethylsulfoxide.

14. The process according to any of Claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13, wherein the reductive amination reaction is carried out in the presence of an aqueous buffer, and wherein the concentration of the cofactor NADP added, based on the aqueous phase, is in the ratio of ranges from 0.0 mM to 2 mM, in particular from 0.2 mM to 0.25 mM.

15. The process according to any of Claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, wherein the process comprises combining an alkyl pyruvate with an aqueous mixture comprising: ethylene diamine and an imine reductase or reductive aminase enzyme, and optionally an enzyme co-factor regenerating system.

16. The process according to any of Claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, wherein the process comprises combining the alkyl pyruvate with an aqueous mixture comprising: ethylene diamine, an imine reductase enzyme, a cofactor regenerating system comprising monosodium salt of nicotinamide adenine dinucleotide phosphate (NADP+), an alcohol dehydrogenase, and optionally a cosolvent.

17. The process according to Claim 15 or Claim 16, wherein the pH of the aqueous mixture is: about 5 to about 9, about 7 to about 9, about 7.5 to about 8.8, about 7.8 to about 8.7, about 8.2 to about 8.7, about 8.4 to about 8.7, or about 8.0 to 8.5, or about 8.

518. The process according to Claim 17, wherein the pH of the aqueous mixture is adjusted if necessary, using a mineral acid, preferably hydrochloric acid.

19. The process according to any of Claims 15, 16, 17, of 18, wherein the alkyl pyruvate is added to the aqueous mixture.

20. The process according to any of Claims 15, 16, 17, 18, or 19, wherein the alkyl pyruvate is added portion wise or continuously to the aqueous mixture over a suitable period of time, preferably wherein the addition takes place portion wise or continuously: over the first 15-40% of the total reaction time, or over the first 15-30% of the total reaction time, or over the first 15-25% of the total reaction time, or over the first 18-22% of the total reaction time, or the first about 20% of the total reaction time.

21. The process according to any of Claims 15, 16, 17, 18, 19, or 20, wherein the alkyl pyruvate is added portion wise or continuously to the aqueous mixture over a period of: about 30 minutes to about 10 hours, about 2 hours to about 8 hours, about 2 hours to about 6 hours, about 3 hours to about 6 hours, about 3 hours to about 5 hours, or about 4 hours.

22. The process according to any of Claims 15, 16, 17, 18, 19, 20, or 21, wherein the aqueous mixture is prepared by combining ethylene diamine, water and, if required, adjusting the pH to: about 7.5 to about 8.8, about 7.8 to about 8.7, about 8.2 to about 8.7, about 8.4 to about 8.7, or about 8.0 to 8.5, or about 8.5; adding the enzyme cofactor regenerating system, preferably comprising monosodium salt of nicotinamide adenine dinucleotide phosphate (NADP+) and either an alcohol dehydrogenase or glucose dehydrogenase; adding the imine reductase or reductive aminase enzyme and optionally adding a cosolvent.

23. The process according to any of Claims 15, 16, 17, 18, 19, 20, 21, or 22, wherein the aqueous mixture is heated to a temperature of: about 20 °C to about 40 °C, about 22 °C to about 38 °C, about 25 °C to about 38 °C, about 25 °C to about 36 °C, about 26 °C to about 35 °C, about 27 °C to about 33 °C, about 28 °C to about 32 °C, about 29 °C to about 31 °C, or about 25 °C to about 30 °C, prior to combining with the alkyl pyruvate.

24. The process according to any of Claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23, wherein the concentration of alkyl pyruvate in the reaction is: about 1.4 g / L to about 50 g / L, about 2.5 g / L to about 40 g / L, about 2.5 g / L to about 20 g / L, about 3 to about 15 g / L, about 4 g / 1 to about 12 g / L; or about 5 g / L to about 10 g / L.

25. The process according to any of Claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24, wherein the cosolvent, when present is used in the reaction mixture at a concentration (v / v%) of: about 0.2% to about 10%, about 0.5% to about 8%, about 0.5% to about 5%, about 0.8% to about 2%, or about 0.8% to about 1.5%, about 0.9% to about 1.2%, or about 1%.

26. The process according to any of Claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, wherein the ethylene diamine is used in the reaction mixture in an amount of: about 1.5 to about 8, about 1.8 to about 7, about 2 to about 6, or about 2.5 to about 5, mole equivalents relative to alkyl pyruvate.27 The process according to any of Claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26, wherein the enzyme for the stereoselective reductive amination is preferably an imine reductase, wherein the enzyme is present in the reaction mixture in a concentration (w / w%) of: about 1 to about 200, about 1 to about 150, about 1 to about 100, about 2 to about 50, about 3 to about 30, about 4 to about 28, or about 5 to about 25.

28. The process according to any of Claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27, wherein the enzyme regeneration cofactor is preferably sodium salt of nicotinamide adenine dinucleotide phosphate, and wherein the enzyme regeneration cofactor is present in the reaction mixture at a concentration of: 0.15 to about 3.5, about 0.2 to about 2.8, or about 0.25 to about 2.5 mM.

29. The process according to any of Claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28, wherein the cofactor regenerating enzyme is preferably an alcohol dehydrogenase preferably from Lactobacillus brevis, or a glucose dehydrogenase wherein glucose is present, wherein the cofactor regenerating enzyme is present in the reaction mixture at a concentration (w / w%) of: 0.25 to about 5, about 0.4 to about 3.5, about 0.6 to about 3, about 0.8 to about 2.5, or about 1 to about 2,30. The process according to any of Claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29, wherein the cofactor regenerating enzyme is preferably an alcohol dehydrogenase preferably from Lactobacillus brevis, or a glucose dehydrogenase wherein glucose is present, wherein the cofactor regenerating enzyme is present in the reaction mixture at a concentration(w / w%) of: 0.25 to about 5, about 0.4 to about 3.5, about 0.6 to about 3, about 0.8 to about 2.5, or about 1 to about 2,31. The process according to any of Claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, wherein the cofactor regenerating enzyme is glucose dehydrogenase and wherein glucose is present, wherein glucose is present in the reaction mixture in an amount of: about 1.0 to about 2.2 about 1.02 to about 2.1, or about 1.05 to about 2.0 mole equivalents relative to the alkyl pyruvate.

32. The process according to any of Claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31, wherein the alkyl pyruvate is a Ci to Cs alkyl pyruvate, preferably a Ci to C3 alkyl pyruvate.

33. The process according to any of Claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32, wherein the alkyl pyruvate is methyl pyruvate, ethyl pyruvate, propyl pyruvate or isopropyl pyruvate, and preferably wherein the alkyl pyruvate is ethyl pyruvate.

34. The process according to any of Claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33, wherein the cofactor regenerating enzyme is preferably an alcohol dehydrogenase preferably from Lactobacillus brevis, or a glucose dehydrogenase and wherein glucose is present, wherein the cofactor regenerating enzyme is present in the reaction mixture at a concentration (w / w%) of: 0.25 to about 5, about 0.4 to about 3.5, about 0.6 to about 3, about 0.8 to about 2.5, or about 1 to about 2,35. The process according to any of Claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34, wherein the pH of the reaction mixture is maintained at: about 5 to about 9, about 7 to about 9, about 7.5 to about 8.8, about 7.8 to about 8.7, about 8.2 to about 8.7, about 8.4 to about 8.7, or about 8.0 to 8.5, or about 8.5.

36. The process according to Claim 35, wherein the pH of the reaction mixture is adjusted if necessary, with an organic base, preferably ethylenediamine.

37. The process according to any of Claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36, wherein the reductive amination reaction is carried out at: about 20 °C to about 40 °C, about 22 °C to about 38 °C, about 25 °C to about 38 °C, about 25 °C to about 36 °C, about 26 °C to about 35 °C, about 27 °C to about 33 °C, about 28 °C to about 32 °C, about 29 °C to about 31 °C, or wherein the reductive amination reaction is carried out at about 25 °C to about 30 °C.

38. The process according to any of Claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37, wherein the reaction time is: about 1 hour to about 72 hours, about 2 hours to about 48 hours, about 3 hours to about 38 hours, or about 4 hours to about 36 hours, about 6 hours to about 30 hours, about 10 hours to about 28 hours, about 12 hours to about 26 hours, or about 12 hours to about 24 hours.

39. The process according to any of Claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38, further comprising isolating FZL-10.

40. The process according to Claim 39, wherein FZL-10 is isolated by a process comprising: basifying the reaction mixture, concentrating the mixture, extracting the mixture with an organic solvent, preferably a C4 to Cs aliphatic alcohol, to form a solution of FZL-10 in the organic solvent, and crystallizing FZL-10 from the solution.

41. The process according to Claim 40, wherein the reaction mixture is adjusted to a pH of: about 9.5 to about 12.5, about 9.5 to about 11, about 9.5 to about 10.5, about 9.8 to about 10.2, or about 10.

42. The process according to Claim 40 or Claim 41, wherein the basifying is with an inorganic base, particularly an alkali metal hydroxide, and more particularly sodium hydroxide or potassium hydroxide, and most preferably potassium hydroxide.

43. The process according to any of Claims 40, 41 or 42, wherein the organic solvent is a C4 to Cs aliphatic alcohol, optionally a C4 to Ce aliphatic alcohol, and preferably a C4 aliphatic alcohol, more preferably 1 -butanol, isobutanol or sec-butanol, preferably 1- butanol.

44. The process according to any of Claims 40, 41, 42, or 43, wherein prior to the crystallizing step, sodium chloride is added to the solution of FZL-10 and the mixture stirred, preferably for a period of: about 1 to about 24 hours, about 4 to about 18 hours, about 5 to about 15 hours, about 6 to about 10 hours, about 7 to about 9 hours, or about 8 hours, followed by separating the liquid phase and concentrating.

45. The process according to any of Claims 40, 41, 42, 43, or 44, wherein the crystallization is initiated by the addition of seed crystals of FZL-10, optionally stirring the seeded mixture, and collecting the product by filtration.

46. The process according to Claim 45, wherein the seeded mixture is stirred at a temperature of: about -5 °C to about 15 °C, about -2 °C to about 12 °C, about 0 °C to about 10 °C, about 2 °C to about 8 °C, about 4 °C to about 6 °C, or about 5 °C; optionally wherein the seeded mixture is stirred for a period of: about 30 minutes to about 24 hours, about 1 hour to about 18 hours, about 2 hours to about 10 hours, about 3 hours to about 6 hours, or about 5 hours.

47. The process according to any of Claims 40, 41, 42, 43, 44, 45, or 46, further comprising suspending the isolated FZL-10 in a C4 to Cs aliphatic alcohol, a C4 to Ce aliphatic alcohol, and preferably a C4 aliphatic alcohol, more preferably 1 -butanol, isobutanol or sec-butanol, and preferably 1 -butanol; preferably at a temperature of: about -5 °C to about 15 °C, about -2 °C to about 12 °C, about 0 °C to about 10 °C, about 2 °C to about 8 °C, about 4 °C to about 6 °C, or about 5 °C, collecting FZL-10, preferably by filtration, and optionally drying.

48. The process according to any preceding claim, further comprising converting the FZL- 10 to (R)-Fezolinetant or a salt of (R)-Fezolinetant or.

49. The process according to Claim 48, wherein FZL-10 is converted to (R)-Fezolinetant by a process comprising:(i) reacting the compound FZL-10 with 4-fluorobenzoyl chloride to form a compound of formula FZL-9:(ii) reacting FZL-9 with trimethyloxonium tetrafluoroborate to form a compound of formula FZL-11:(iii) reacting the compound FZL-11 with a compound of formula FZL-3 :to form (R)-Fezolinetant:

50. The process according to Claim 48 or Claim 49, further comprising enantiomeric purification of the (R)-Fezolinetant.

51. The process according to Claim 50, wherein the (R)-Fezolinetant is purified by a process comprising:(a) forming a solution of the (R)-Fezolinetant in an anhydrous solvent, preferably wherein the solvent is an alcohol, and more preferably methanol, ethanol or 2- propanol, and particularly methanol;(b) combining the solution with a solvent selected from: acetone, methyl ethyl ketone, tetrahydrofuran, 2-methyltetrahydrofuran and acetonitrile, and particularly acetonitrile, preferably by adding the solvent to the solution to form a mixture;(c) filtering the mixture and collecting the filtrate;(d) optionally stirring the filtrate with activated carbon and removing the activated carbon; a(e) crystallizing purified (R)-Fezolinetant from the mixture;(f) isolating the purified (R)-Fezolinetant, preferably by filtration; and(g) optionally drying the (R)-Fezolinetant.

52. The process according to Claim 51, wherein the solvent in step (a) is anhydrous methanol, and the solvent in step (b) is acetonitrile.

53. The process according to Claim 51 or Claim 52, wherein step (b) comprises stirring the mixture, preferably at room temperature, optionally for a period of: about 0.5 to about 8 hours, about 0.5 to about 6 hours, about 0.5 to about 4 hours, or about 0.5 to about 3 hours, about 1 to about 3 hours, or about 1 hour.

54. The process according to any of Claims 51, 53 or 53, wherein step (d) comprises stirring the filtrate from step (c) with activated carbon at a temperature of: about 40 °C to about 60 °C, about 50 °C to about 60 °C , or about 55 °C, preferably for a period of: about 0.5 hours to about 5 hours, about 0.5 to about 3 hours, or about 1 hour.

55. The process according to any of Claims 51, 52, 53, or 54, wherein the activated carbon is removed in step (d) by filtration.

56. The process according to any of Claims 51, 52, 53, 54, or 55, wherein step (e) comprises by cooling the filtrate from step (d) or step (e), preferably to a temperature of: about -5 °C to about 5 °C, about -2 °C to about 2 °C, about -1 °C to about 1 °C, or about 0 °C.

57. The process according to any of Claims 51, 52, 53, 54, 55, or 56, wherein step (e) comprises concentrating the filtrate from step (d) or step (e)58. The process according to any of Claims 51, 52, 53, 54, 55, 56, or 57, wherein step (e) comprises concentrating the filtrate from step (c) or step (d), and cooling, preferably to a temperature of: about -5 °C to about 5 °C, about -2 °C to about 2 °C, about -1 °C to about 1 °C, or about 0 °C.

59. The process according to any of Claims 51, 52, 53, 54, 55, 56, 57, or 58, further comprising enantiomeric purification of the (R)-Fezolinetant: by diastereomeric salt separation using a chiral acid, preferably wherein the chiral acid is (lS)-(+)-camphor- 10-sulfonic acid; or by selective crystallization using an acid selected from ethanesulfonic acid, benzenesulfonic acid and tosic acid.

60. The process according to Claim 59, wherein purified (R)-Fezolinetant (lS)-(+)- camphor-10-sulfonic acid, (R)-Fezolinetant esylate, (R)-Fezolinetant besylate, or (R)- Fezolinetant tosylate, is crystallized from a solvent combination selected from: acetonitrile / tert-butyl methyl ether, acetonitrile / ethyl acetate, acetonitrile / acetone, methanol / isopropanol, methanol / ethyl acetate, methanol / tert-butyl methyl ether, ethanol / ethyl acetate, acetone / ethyl acetate.

61. The process according to Claim 59 or Claim 60, comprising heating (R)-Fezolinetant, (lS)-(+)-camphor-10-sulfonic acid, (R)-Fezolinetant esylate, (R)-Fezolinetant besylate, or (R)-Fezolinetant tosylate, in a solvent combination of: acetonitrile and tert-butyl methyl ether, preferably wherein the heating is to a temperature of 40-60 °C, more preferably 50 °C, optionally wherein the heating is for a period of about 1-5 hours, preferably about 3 hours.

62. The process according to Claim 61, wherein the (R)-Fezolinetant camsylate, (R)- Fezolinetant esylate, (R)-Fezolinetant besylate, or (R)-Fezolinetant tosylate, is isolated by cooling, preferably to: about -10 °C to about 10 °C, about -5 °C to about 5 °C, about -2 °C to about 2 °C, or about 0 °C; and optionally drying the purified (R)- Fezolinetant camsylate, (R)-Fezolinetant esylate, (R)-Fezolinetant besylate, or (R)- Fezolinetant tosylate.

63. The process according to Claim 62, wherein the (R)-Fezolinetant camsylate, (R)- Fezolinetant esylate, (R)-Fezolinetant besylate, or (R)-Fezolinetant tosylate, is suspended in a mixture of water and methanol, preferably at room temperature, and optionally seeding to precipitate (R)-Fezolinetant; optionally isolating the (R)- Fezolinetant, preferably by filtration; and optionally drying the (R)-Fezolinetant.

64. The process according to any of Claims 59, 60, 61, 62, or 63, wherein the enantiomeric purification of the (R)-Fezolinetant is by diastereomeric salt separation using a chiral acid, preferably wherein the chiral acid is (lS)-(+)-camphor-10-sulfonic acid.

65. The process according to any of Claims 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, or 64, which further comprises combining the (R)-Fezolinetant with at least one pharmaceutically acceptable acid to form a pharmaceutically acceptable salt of (R)-Fezolinetant.

66. The process according to any of Claims 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65, further comprising combining the (R)-Fezolinetant orpharmaceutically acceptable salt of (R)-Fezolinetant, with at least one pharmaceutically acceptable excipient to form a pharmaceutical composition or formulation.

67. Use of the process as defined in any of Claims 1 to 47 for the preparation of (R)- Fezolinetant or a salt thereof.

68. The process for preparing (R)-Fezolinetant, comprising preparing the compound FZL- 10 by the process according to any of Claims 1 to 47, and converting the FZL-10 to (R)-Fezolinetant or a pharmaceutically acceptable salt of (R)-Fezolinetant.

69. The process according to Claim 67, wherein FZL-10 is converted to (R)-Fezolinetant or a pharmaceutically acceptable salt of (R)-Fezolinetant by the process according to any of Claims 49 to 66.