Synthesis of (2S,5R)-5-(2-chlorophenyl)-1-(2'-methoxy-[1,1'-biphenyl]-4- carbonyl)pyrrolidine-2-carboxylic acid

Abstract

The present invention relates to a process for the preparation of (2S,5R)-5-(2-chlorophenyl)-1-(2'-methoxy-[1,1'-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid (1), comprising the preparation of (2S,5R)-methyl 5-(2-chlorophenyl)pyrrolidine-2-carboxylate hydrochloride (6.HCl) as a synthetic intermediate:

Description

Technical Field

[0001] This invention relates to the field of chemical synthesis and provides a method for preparing (2S,5R)-5-(2-chlorophenyl)-1-(2′-methoxy-[1,1′-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid (1), comprising preparing (2S,5R)-5-(2-chlorophenyl)pyrrolidine-2-carboxylic acid methyl ester hydrochloride (6.HCl) as a synthetic intermediate:

[0002] Background of the Invention

[0004] The pyrrolidine carboxylic acid derivatives disclosed in WO2011 / 073376 can be used to treat inflammatory diseases, as reported in WO2015 / 078949. In particular, the applicant demonstrated that (2S,5R)-5-(2-chlorophenyl)-l-(2′-methoxy-[1,1′-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, hereinafter referred to as compound (1), is particularly advantageous for the treatment of inflammatory diseases.

[0005] In WO2011 / 073376 and WO2015 / 078949, the synthesis of compound (1) was reported according to the following synthetic route:

[0006]

[0007] Grignard reagents were prepared from 1-bromo-2-chlorobenzene (2) in the presence of isopropyl magnesium chloride and lithium chloride. This Grignard reagent was added to N-Boc-L-pyroglutamic acid methyl ester (3) to provide intermediate (4a). Post-treatment of the reaction product included basic treatment and separation of intermediate (4a) by rapid chromatography. Under acidic conditions, one-pot deprotection of Boc and formation of a cyclic imine yielded a cyclic imine intermediate (5), which was reduced with a borohydride reagent to give a mixture of pyrrolidine ester epiomers (6 & 6′). The epiomers were separated by rapid chromatography, with the pyrrolidine ester intermediate (6) isolated, after two rapid chromatography steps, resulting in a diastereomeric excess of up to 97%. The pyrrolidine ester intermediate (6) was then acylated with an acyl chloride (7) using a standard amide coupling procedure. The reaction mixture was purified by rapid chromatography; subsequently, the epiomer of interest was saponified with lithium hydroxide to give the desired carboxylic acid compound (1).

[0008] This synthetic route is robust in obtaining good diastereomeric excess (de%) and enantiomeric excess (ee%). However, the synthetic route for compound (1) is not suitable for scale-up production. In fact, multiple purification steps are required by rapid silica gel chromatography, one of which is particularly difficult to separate the epimers (6) and (6′), which is not suitable for large-scale production.

[0009] To overcome the aforementioned drawbacks and improve the overall yield, the applicant provides a scalable process for this invention, comprising the preparation of a novel synthetic intermediate, (2S,5R)-5-(2-chlorophenyl)pyrrolidine-2-carboxylic acid methyl ester hydrochloride (6.HCl):

[0010]

[0011] As detailed in the Experimental Section, it was also demonstrated that the sodium salt of compound (1), hereinafter referred to as compound (l.Na), surprisingly resulted in enhanced properties compared to the acidic form of compound (1) and other salts. In particular, the sodium salt (l.Na) exhibited better water solubility and improved stability compared to the acidic form of (1). Therefore, the present invention also relates to the sodium salt (l.Na) and its preparation process. Summary of the Invention

[0012] Therefore, the present invention relates to a method for preparing (2S,5R)-5-(2-chlorophenyl)pyrrolidine-2-carboxylic acid methyl ester hydrochloride (6.HCl).

[0013]

[0014] The method includes the steps of forming a hydrochloride salt and separating the epimers, including crystallizing a mixture of epimers (6) and (6′) in the presence of hydrochloric acid in a solvent selected from alcohol solvents, isopropyl acetate, and mixtures thereof:

[0015]

[0016] The epimerization ratio (6):(6′) is at least 4:1.

[0017] In one embodiment, the method of the present invention further includes a subsequent recrystallization step in a solvent selected from alcohol solvents, isopropyl acetate, and mixtures thereof.

[0018] In one embodiment, the alcohol solvent used in the method of the present invention is selected from isopropanol, methanol, and mixtures thereof.

[0019] In one embodiment, a mixture of epiomers (6) and (6') with an epimeric ratio (6):(6') of at least 4:1 is obtained by reducing compound (5) with sodium triacetoxyborohydride in the presence of an acid in a solvent selected from dichloromethane, acetonitrile, isopropyl acetate, and mixtures thereof, wherein the solvent is preferably dichloromethane.

[0020]

[0021] In one embodiment, the acid is selected from acetic acid and trifluoroacetic acid; preferably, the acid is acetic acid.

[0022] In one embodiment, compound (5) is obtained by: a) subjecting compounds (2) and (3) to a Knochel-Grignard reaction in the presence of isopropyl magnesium chloride and lithium chloride:

[0023]

[0024] Quenching with an aqueous solution containing acid yields a mixture of compounds (4a) and (4b):

[0025]

[0026] and,

[0027] b) The mixture of compounds (4a) and (4b) obtained in step a) is contacted with an acid, preferably trifluoroacetic acid or hydrochloric acid, in a solvent selected from dichloromethane, methanol, isopropanol and mixtures thereof, preferably dichloromethane, at a temperature of 0°C to 25°C to obtain compound (5).

[0028] The present invention further relates to a method for preparing (2S,5R)-5-(2-chlorophenyl)-1-(2′-methoxy-[1,1′-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid (1):

[0029]

[0030] include:

[0031] a) According to the method of the present invention, compound (6.HCl) is obtained;

[0032] b) Formation of methyl ester (9) from the compound (6.HCl) obtained in step a) via the following steps:

[0033]

[0034] b1) In the presence of potassium carbonate, compound (7) is used to acetylate compound (6.HCl) in a mixture of toluene and water as solvent.

[0035]

[0036] Alternatively, b2) in the presence of a base and an acid activator, compound (6.HCl) is coupled to compound (8).

[0037]

[0038] c) In the presence of sodium hydroxide and tetrabutylammonium bromide (TBAB), methyl ester (9) was saponified in a mixture of toluene and water as solvent to give an amorphous acid compound (1).

[0039] This invention also relates to a method for preparing sodium (1.Na) of (2S,5R)-5-(2-chlorophenyl)-1-(2′-methoxy-[1,1′-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylate:

[0040]

[0041] include:

[0042] a) According to the method of the present invention, acid compound (1) is obtained; and

[0043] b) Sodium salt formation step, comprising contacting the acid compound (1) obtained from step a) with sodium hydroxide in methanol as a solvent to obtain an amorphous sodium salt (l.Na).

[0044] In one embodiment, the method for preparing (l.Na) further includes an optional step c) of precipitation in the presence of isopropanol.

[0045] The present invention also provides (2S,5R)-5-(2-chlorophenyl)pyrrolidine-2-carboxylic acid methyl ester hydrochloride (6.HCl):

[0046]

[0047] This invention also relates to (2S)-5-(2-chlorophenyl)-5-hydroxypyrrolidine-1,2-dicarboxylic acid-1-tert-butyl ester 2-methyl ester (4b):

[0048]

[0049] Another object of the present invention is sodium (2S,5R)-5-(2-chlorophenyl)-1-(2′-methoxy-[1,1′-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylate (1.Na):

[0050]

[0051] The present invention further provides a pharmaceutical composition comprising sodium (2S,5R)-5-(2-chlorophenyl)-1-(2′-methoxy-[1,1′-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylate (1.Na) and at least one pharmaceutically acceptable carrier. In one embodiment, sodium (2S,5R)-5-(2-chlorophenyl)-1-(2′-methoxy-[1,1′-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylate (1.Na) is used as a medicament. In one embodiment, sodium (2S,5R)-5-(2-chlorophenyl)-1-(2′-methoxy-[1,1′-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylate (1.Na) is used for the treatment and / or prevention of inflammation.

[0052] definition

[0053] In this invention, the following terms have the following meanings:

[0054] An "alcohol solvent" is a substance that dissolves a solute (a chemically distinct liquid, solid, or gas) to form a solution. It is an alcohol, an organic compound containing at least one hydroxyl functional group (-OH) bonded to a carbon atom. Examples of alcohol solvents include methanol, ethanol, and isopropanol.

[0055] - An "epomer" refers to one of a pair of diastereomers. These two epimers have at least one stereoisomeric center with opposite configurations. All other stereoisomeric centers in the molecule are identical in each epimer. Epimerization refers to the conversion of one epimer into another.

[0056] "Administration" or its variations (e.g., "dosage") means the provision of an active agent or active ingredient, alone or as part of a pharmaceutically acceptable composition, to a patient for the treatment or prevention of a condition, symptom, or disease.

[0057] - "Patient" refers to a mammal, more preferably a human, who is waiting to receive or is receiving medical care, or is / will be the subject of a medical procedure.

[0058] "Pharmaceutical acceptable" refers to components in a pharmaceutical composition that are compatible with each other and harmless to the recipient.

[0059] - A "pharmaceutically acceptable carrier" is a substance that, when administered to animals (preferably humans), will not produce adverse reactions, allergic reactions, or other adverse effects. It includes any and all inactive substances, such as solvents, solubilizers, antioxidants, surfactants, stabilizers, emulsifiers, buffers, pH adjusters, preservatives (or freshness preservers), antimicrobial and antifungal agents, isotonic agents, granulators or binders, lubricants, disintegrants, glidants, diluents or fillers, adsorbents, dispersants, suspending agents, coating agents, bulking agents, gelatin (for soft and hard capsules), release agents, absorption delay agents, sweeteners, flavorings, etc. For human administration, the formulation should meet the general safety and purity standards required by regulatory agencies (FDA office or EMA, etc.).

[0060] - "Prevention", "prevention" and "avoidance", as used herein, refer to methods of delaying or preventing the onset of a condition or disease and / or its accompanying symptoms, preventing a patient from developing a condition or disease, or reducing the risk of a patient developing a condition or disease.

[0061] - A “therapeutic effective dose” refers to the amount or dosage of an active ingredient intended to: (1) delay or prevent the onset of a disease (e.g., an inflammatory disease) in a subject without causing significant negative or adverse side effects; (2) reduce the severity or incidence of a disease (e.g., an inflammatory disease); (3) slow or stop the progression, aggravation, or worsening of one or more symptoms of a disease (e.g., an inflammatory disease) affecting a subject; (4) improve the symptoms of a disease (e.g., an inflammatory disease) affecting a subject; or (5) cure a disease (e.g., an inflammatory disease) affecting a subject. A therapeutic effective dose may be administered before the onset of a disease (e.g., an inflammatory disease) for a preventative or preventative effect. Alternatively, a therapeutic effective dose may be administered after the onset of a disease (e.g., an inflammatory disease) for a therapeutic effect.

[0062] "Treatment" or "treatment" refers to therapeutic treatment; wherein the aim is to prevent or alleviate a target pathological condition or disease (e.g., an inflammatory disease). A disease, infection, or condition can be considered successfully "treated" if, after receiving treatment according to the invention, the subject or mammal exhibits one or more of the following: an observable and / or measurable reduction or disappearance of one or more symptoms associated with the specific disease or condition (e.g., an inflammatory disease); a reduction in morbidity and mortality; and / or an improvement in quality of life. The parameters described above for assessing successful treatment and improvement of a disease are readily measurable using routine procedures familiar to physicians.

[0063] - "Subject" refers to a mammal, preferably a human. In one embodiment, the subject is diagnosed with a disease, such as an inflammatory disease. In one embodiment, the subject is a patient, preferably a human patient, who is awaiting or receiving medical care, or has been / is / will be a subject of a medical procedure, or is under surveillance for the development or progression of a disease (e.g., an inflammatory disease). In one embodiment, the subject is a human patient receiving treatment and / or under surveillance for the development or progression of a disease (e.g., an inflammatory disease). In one embodiment, the subject is male. In another embodiment, the subject is female. In one embodiment, the subject is an adult. In another embodiment, the subject is a child.

[0064] Detailed description

[0065] Preparation process of acid compound (1)

[0066] Therefore, the present invention relates to a novel method for preparing acid compounds (1) that can be scaled up.

[0067] Preparation of hydrochloride intermediate (6.HCl)

[0068] In particular, a novel synthetic intermediate, (2S,5R)-5-(2-chlorophenyl)pyrrolidine-2-carboxylic acid methyl ester hydrochloride (6.HCl), is provided instead of the corresponding pyrrolidine ester (6). In practice, separating the epimers (6) and (6′) to isolate intermediate (6) requires purification by rapid chromatography in the previously disclosed process, a step that is difficult to scale up for production.

[0069] In contrast, in the method according to the invention, the mixture of epiomers (6) and (6′) is separated by crystallization of the hydrochloride to obtain hydrochloride (6.HCl), which has excellent enantiomeric and diastereomeric excess. Separating the hydrochloride (6.HCl) by crystallization avoids the need for rapid chromatographic purification to separate the epiomer (6).

[0070] Therefore, the present invention provides a method for preparing (2S,5R)-5-(2-chlorophenyl)pyrrolidine-2-carboxylic acid methyl ester hydrochloride (6.HCl):

[0071]

[0072] The method includes the steps of forming a hydrochloride salt and separating the epimers, including crystallizing a mixture of epimers (6) and (6′) in the presence of hydrochloric acid in a solvent selected from alcohol solvents, isopropyl acetate, and mixtures thereof:

[0073]

[0074] The differential isomer ratio (6):(6′) is at least 4:1.

[0075] In one embodiment, the preparation of (6.HCl) further includes a subsequent recrystallization step in a solvent selected from alcohol solvents, isopropyl acetate, and mixtures thereof.

[0076] In one embodiment, the alcohol solvent used for the crystallization and / or recrystallization steps is selected from isopropanol, methanol, and mixtures thereof.

[0077] In one embodiment, the solvent used for the recrystallization step is the same as the solvent used for the crystallization step.

[0078] In one embodiment, the solvent used for the crystallization and / or recrystallization steps is isopropanol. In another embodiment, the solvent used for the crystallization and / or recrystallization steps is a mixture of isopropanol and isopropyl acetate. In yet another embodiment, the solvent used for the crystallization and / or recrystallization steps is a mixture of methanol and isopropyl acetate.

[0079] In one embodiment, the duration of the crystallization step is from 0.5 hours to 24 hours, preferably at least 1 hour.

[0080] In one embodiment, the temperature range for the crystallization step is 0°C to room temperature (about 25°C), preferably 0°C to 15°C, and more preferably 0°C to 5°C.

[0081] During the formation and crystallization of hydrochloride, a corresponding hydrochloride epimer (6′HCl) is also formed:

[0082]

[0083] In one embodiment, this preparation process separates the hydrochloride (6.HCl) at a diastereomeric ratio of at least 94:6, preferably at least 98:2, and more preferably greater than 99:1 (6.HCl):(6′HCl). According to one embodiment, the diastereomeric ratio can be expressed as a percentage of HPLC area, a mass percentage, or a molar percentage. The ratio expressed as a mass percentage and the ratio expressed as a molar percentage are the same. In one embodiment, the ratios expressed as a percentage of HPLC area, a mass percentage, and a molar percentage are the same.

[0084] Preparation of a mixture of epimers (6) and (6′)

[0085] The mixture of epimers (6) and (6′) should exhibit an epimer ratio of at least 4:1 (6):(6′) so that the hydrochloride (6.HCl) can be separated with high purity.

[0086] In one embodiment, a mixture of epiomers (6) and (6') wherein the epiomer ratio (6):(6') is at least 4:1 is obtained by reducing compound (5) with sodium triacetoxyborohydride in the presence of an acid in a solvent selected from dichloromethane, acetonitrile, isopropyl acetate, and mixtures thereof.

[0087]

[0088] In one embodiment, the acid is selected from acetic acid and trifluoroacetic acid; preferably, the acid is acetic acid.

[0089] In one embodiment, the solvent is preferably dichloromethane, more preferably anhydrous dichloromethane.

[0090] In one embodiment, a solution containing compound (5), an acid, and a reaction solvent is gradually added to a solution containing sodium triacetoxyborohydride and a reaction solvent.

[0091] In one implementation, the reduction is carried out in a temperature range of 0°C to 5°C.

[0092] In one embodiment, the reaction is stopped before (5) complete reduction. Preferably, the reduction reaction is stopped after a period of 3 to 20 hours, more preferably 9 to 20 hours, and even more preferably 12 to 18 hours. This has the advantage of avoiding the formation of excessive amounts of the following acetylation byproduct (N-Ac-6):

[0093]

[0094] In one embodiment, the reaction is terminated by adding a saturated sodium carbonate solution.

[0095] Preparation of cyclic imine intermediate (5)

[0096] In one embodiment, compound (5) is obtained by:

[0097] a) Compounds (2) and (3) undergo a Knochel-Grignard reaction in the presence of isopropyl magnesium chloride and lithium chloride:

[0098]

[0099] A mixture of compounds (4a) and (4b) was obtained:

[0100]

[0101] and,

[0102] b) Dissolve the mixture of compounds (4a) and (4b) obtained in step a) in an acid in a solvent selected from dichloromethane, methanol, isopropanol, and mixtures thereof.

[0103] Compound (5) was obtained by contacting the sample at temperatures ranging from 0°C to 25°C.

[0104] In one embodiment, compound (5) is obtained as follows:

[0105] a) Compounds (2) and (3) undergo a Knochel-Grignard reaction in the presence of isopropyl magnesium chloride and lithium chloride:

[0106]

[0107] Quenching with an aqueous solution containing acid yields a mixture of compounds (4a) and (4b):

[0108]

[0109] and,

[0110] b) The mixture of compounds (4a) and (4b) obtained in step a) is contacted with an acid in a solvent selected from dichloromethane, methanol, isopropanol and mixtures thereof at a temperature of 0°C to 25°C to obtain compound (5).

[0111] In one embodiment, in step a), a Grignard reagent is first prepared from 1-bromo-2-chlorobenzene (2) in the presence of isopropyl magnesium chloride and lithium chloride. The Grignard reagent is then added to N-Boc-L-pyroglutamic acid methyl ester (3) to provide intermediates (4a) and (4b).

[0112] In one embodiment, step a) is carried out in THF, preferably anhydrous THF, as a solvent.

[0113] In one embodiment, step a) is carried out at a temperature of 0°C to 25°C. Preferably, the formation of the Grignard reagent is carried out in a temperature range of 15°C to 25°C, followed by mixing of the reactants in a temperature range of 0-5°C. In one embodiment, the formation of intermediates (4a) and (4b) from the Grignard reagent is carried out in a temperature range of 0°C to 5°C.

[0114] In one implementation, the reaction in step a) is quenched by adding an aqueous solution.

[0115] Using the related terminology of "quenching," it refers to the action of deactivating any unreacted reagent in a chemical reaction. In the Knochel-Grignard reaction of the method of this invention, the unreacted Grignard reagent is deactivated by the addition of an aqueous solution.

[0116] In one embodiment, the reaction in step a) is quenched by acid treatment, specifically by adding an aqueous solution containing an acid. In one embodiment, the aqueous solution containing an acid used to quench the reaction is selected from aqueous solutions of citric acid or acetic acid. Preferably, an aqueous solution of citric acid is used to quench the reaction. In one embodiment, quenching the reaction by adding an aqueous solution containing an acid does not necessarily result in an acidic pH in the reaction mixture. In one embodiment, quenching by acid treatment can set the pH of the reaction mixture from an initial pH of 12 to 4.

[0117] In one implementation, the reaction in step a) is not quenched by alkali treatment.

[0118] In one implementation, intermediates (4a) and (4b) were not separated, let alone purified by rapid chromatography.

[0119] In one embodiment, the acid used in step b) is trifluoroacetic acid (TFA) or hydrochloric acid; trifluoroacetic acid is preferred.

[0120] In one embodiment, the solvent used in step b) is preferably dichloromethane, more preferably anhydrous dichloromethane. In one embodiment, in step b), when TFA is used as the acid, the solvent is dichloromethane, preferably anhydrous dichloromethane. In another embodiment, the solvent used in step b) is preferably methanol or isopropanol. In one embodiment, in step b), when hydrochloric acid is used as the acid, the solvent is methanol or isopropanol.

[0121] In one embodiment, step b) is carried out for a period of 1 to 50 hours, preferably 15 to 50 hours; more preferably 20 to 45 hours. In one embodiment, the acid used in step b) is TFA, and the reaction is carried out for a period of 15 to 50 hours; preferably 18 to 48 hours; 18 to 23 hours or 24 to 44 hours.

[0122] Preparation of compound (1)

[0123] Therefore, the present invention also relates to a method for preparing the amorphous form of (2S,5R)-5-(2-chlorophenyl)-1-(2′-methoxy-[1,1′-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid (1):

[0124]

[0125] include:

[0126] a) A method for preparing hydrochloride (6.HCl) according to the present invention;

[0127] b) Formation of methyl ester (9) from the compound (6.HCl) obtained in step a) via the following steps:

[0128]

[0129] b1) In the presence of potassium carbonate, compound (7) is used to acetylate compound (6.HCl) in a mixture of toluene and water as solvent.

[0130]

[0131] Alternatively, b2) in the presence of a base and an acid activator, compound (6.HCl) is coupled to compound (8).

[0132]

[0133] c) In the presence of sodium hydroxide and tetrabutylammonium bromide (TBAB), methyl ester (9) was saponified in a mixture of toluene and water as solvent to give an amorphous acid compound (1).

[0134] Therefore, the present invention also relates to a method for preparing the amorphous form of (2S,5R)-5-(2-chlorophenyl)-1-(2′-methoxy-[1,1′-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid (1):

[0135]

[0136] include:

[0137] a) A method for preparing hydrochloride (6.HCl) according to the present invention;

[0138] b)b1) In the presence of potassium carbonate, compound (7) is used to acetylate compound (6.HCl) in a mixture of toluene and water as solvent.

[0139] and

[0140] c) In the presence of sodium hydroxide and tetrabutylammonium bromide (TBAB), methyl ester (9) was saponified in a mixture of toluene and water as solvent to give an amorphous acid compound (1).

[0141] In one embodiment, acyl chloride (7) is obtained from the corresponding acid (8) by reacting with SOCl2 in the presence of catalytic dimethylformamide (DFM), preferably in toluene as a solvent:

[0142]

[0143] Therefore, the present invention also relates to a method for preparing (2S,5R)-5-(2-chlorophenyl)-1-(2′-methoxy-[1,1′-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid (1), comprising:

[0144] a) A method for preparing hydrochloride (6.HCl) according to the present invention;

[0145] b)b2) In the presence of a base and an acid activator, compound (8) is used to couple compound (6.HCl).

[0146]

[0147] and

[0148] c) In the presence of sodium hydroxide and tetrabutylammonium bromide (TBAB), methyl ester (9) was saponified in a mixture of toluene and water as solvent to give an amorphous acid compound (1).

[0149] In step b2), the acid activator may be selected from known acid-amine coupling activators, such as propylphosphonic anhydride (T3P), N,N′-dicyclohexylcarbodiimide (DCC), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDCI-HCl), or 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU). In one embodiment, the acid activator is selected from T3P, DCC, EDCI-HCl, and HATU, with T3P being preferred.

[0150] In step b2), the base is, for example, triethylamine (TEA), diisopropylethylamine (DIEA), or N-methylmorpholine (NMM).

[0151] In one embodiment, step b2) is carried out in a solvent selected from dimethylformamide (DMF), dichloromethane (DCM), isopropyl acetate (iPrOAC), and acetonitrile (CAN).

[0152] In one implementation, step b2) is performed in a temperature range from room temperature (about 25°C) to 60°C.

[0153] In one implementation, step b2) lasts for 1 to 20 hours.

[0154] In one embodiment, the method for preparing compound (1) further includes a subsequent step (d) of precipitation in the presence of cyclohexane. According to one embodiment, at the end of saponification in step (c), toluene may be partially evaporated to concentrate the reaction mixture, and then cyclohexane may be added at a toluene:cyclohexane volume ratio of 1:8 to 1:10.

[0155] In one embodiment, step b) of the method for preparing compound (1) does not involve purification by rapid chromatography.

[0156] In one embodiment, the method for preparing compound (1) is shown in the figure below:

[0157]

[0158] This preparation method can provide an amorphous acid compound (1) in a total yield of at least 10 mol%, preferably at least 15 mol%, more preferably at least 30 mol%. In one embodiment, the total yield of the amorphous acid compound (1) is provided to be from 12 mol% to 34 mol%.

[0159] Synthesis of intermediates

[0160] As described above, the use of the hydrochloride intermediate (6.HCl) is essential for providing the scalable process of this invention, particularly because the intermediate (6.HCl) is separated in high purity, avoiding the need to perform rapid chromatography to separate the diastereomer (6), while exhibiting excellent enantiomeric excess. In one embodiment, the hydrochloride intermediate (6.HCl) is obtained with a diastereomer excess (de) of at least 97.5%, typically about 99.6% de. In one embodiment, the hydrochloride intermediate (6.HCl) is obtained with an enantiomeric excess (ee) of about 99.9%, or even more than 99.9% ee.

[0161] Therefore, the present invention also relates to (2S,5R)-5-(2-chlorophenyl)pyrrolidine-2-carboxylic acid methyl ester hydrochloride (6.HCl):

[0162]

[0163] The use of intermediate (4b) in conjunction with the previously disclosed intermediate (4a) enables the method of the present invention to obtain intermediate (5) with an improved yield compared to the previously reported method. Intermediate (4b) was never obtained in the previously reported method. In the method of the present invention, the acquisition of (4b) is made possible by acid treatment for quenching the Knochel-Grignard reaction carried out with reactants (2) and (3).

[0164] Therefore, the present invention also relates to (2S)-5-(2-chlorophenyl)-5-hydroxypyrrolidine-1,2-dicarboxylic acid-1-tert-butyl ester 2-methyl ester (4b):

[0165]

[0166] Sodium salt of compound (1)

[0167] As detailed in the experimental section, the sodium salt of compound (1), namely compound (l.Na), was demonstrated to have surprisingly enhanced properties compared to the acid form of compound (1) and other salts. In particular, the sodium salt (l.Na) exhibits better water solubility and improved stability.

[0168] Therefore, the present invention also relates to sodium (1.Na)-5-(2-chlorophenyl)-1-(2′-methoxy-[1,1′-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylate.

[0169]

[0170] In one embodiment, (2S,5R)-5-(2-chlorophenyl)-1-(2′-methoxy-[1,1′-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid sodium salt (1.Na) is amorphous.

[0171] This invention also relates to a method for preparing sodium (1.Na) of (2S,5R)-5-(2-chlorophenyl)-1-(2′-methoxy-[1,1′-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylate.

[0172]

[0173] include:

[0174] a) The method for preparing acid compound (1) according to the present invention as described above;

[0175] b) Sodium salt formation step, comprising contacting the acid compound (1) obtained in step a) with sodium hydroxide in methanol as a solvent to produce an amorphous sodium salt (l.Na).

[0176] In one embodiment, the method for producing the sodium salt (l.Na) further includes an optional step c) of precipitation in the presence of isopropanol. In one embodiment, step c) is carried out by solvent exchange distillation.

[0177] This preparation method can provide a sodium salt (l.Na) with a total yield of at least 10 mol%, preferably at least 15 mol%, more preferably at least 30 mol%. In one embodiment, the method of the present invention provides a sodium salt (l.Na) with a total yield of 10 to 33 mol%. This preparation method can also provide a sodium salt (l.Na) wherein the enantiomeric and diastereomeric excess exceeds 97.6%, preferably exceeding 99.5%.

[0178] As in its corresponding acidic form, the sodium salt (l.Na) is particularly suitable for treating inflammatory diseases. Therefore, the present invention further provides a pharmaceutical composition comprising sodium (2S,5R)-5-(2-chlorophenyl)-1-(2′-methoxy-[1,1′-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylate (l.Na) and at least one pharmaceutically acceptable carrier.

[0179] The present invention also relates to sodium (2S,5R)-5-(2-chlorophenyl)-1-(2′-methoxy-[1,T-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylate (l.Na) as a pharmaceutical.

[0180] In particular, the present invention relates to sodium (2S,5R)-5-(2-chlorophenyl)-1-(2′-methoxy-[1,1′-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylate (1.Na) for the treatment and / or prevention of inflammation.

[0181] In one implementation, the compound (l.Na) is used to delay the onset of inflammatory diseases in patients.

[0182] In one embodiment, the compound (l.Na) is used to treat and / or prevent diseases selected from: rheumatoid arthritis; inflammatory bowel disease (IBD), including but not limited to Crohn's disease and ulcerative colitis; colitis; collagenous colitis; lymphocytic colitis; immune-related enterocolitis (including adverse events in response to cancer treatment with checkpoint inhibitors such as CTLA-4, PD-1, and PD-L1 inhibitors); cystitis; celiac disease; irritable bowel syndrome; intestinal flora imbalance, including antibiotic-induced flora imbalance leading to bacterial infections (e.g., Clostridium difficile infection, pneumococcal infection, etc.); Paget's disease; osteoporosis; multiple myeloma; uveitis; acute and chronic myeloid leukemia; pancreatic β-cell destruction; rheumatoid spondylitis; osteoarthritis; gouty arthritis and other arthritis conditions; gout; adult respiratory distress syndrome (ARDS); chronic inflammatory lung disease; silicosis; pulmonary sarcoidosis; psoriasis. Rhinitis; Allergic reactions; Contact dermatitis; Pancreatitis; Allergies; Hepatitis, including hepatitis B virus infection; Asthma; Muscle atrophy; Cachexia, such as cachexia secondary to infection or malignancy, cachexia secondary to acquired immunodeficiency syndrome; Reiter's syndrome; Type I diabetes; Bone resorption disease; Graft-versus-host disease; Ischemia-reperfusion injury; Traumatic brain injury; Multiple sclerosis; Cerebral malaria; Sepsis; Septic shock; Toxic shock syndrome Endotoxic shock; Gram-negative sepsis; fever and myalgia caused by infections such as influenza; heartburn; anorexia-inducing intestinal hormones (e.g., PYY, GLP-1), such as inflammatory conditions observed in overeating-related diseases, obesity, type 2 diabetes, etc.; microbiome-related neurological and mood disorders, including autism spectrum disorders, schizophrenia, depression, major depressive disorder, and neurodegenerative diseases characterized by neuroinflammation, including Alzheimer's disease and Parkinson's disease.

[0183] In one embodiment, the compound (l.Na) is used to treat and / or prevent diseases selected from: inflammatory bowel disease (IBD), including but not limited to Crohn's disease and ulcerative colitis; colitis; collagenous colitis; lymphocytic colitis; immune-related enterocolitis (including adverse events in response to cancer treatment with checkpoint inhibitors such as CTLA-4, PD-1, and PD-L1 inhibitors); cystitis; celiac disease; irritable bowel syndrome; and intestinal flora imbalance, including antibiotic-induced flora imbalance leading to bacterial infections (e.g., Clostridium difficile infection, pneumococcal infection, etc.).

[0184] The present invention also relates to a method for treating and / or preventing inflammation in patients with such need, comprising administering to said patient a therapeutically effective amount of sodium (2S,5R)-5-(2-chlorophenyl)-1-(2′-methoxy-[1,1′-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylate (1.Na).

[0185] In one embodiment, the present invention provides a method for delaying the onset of an inflammatory disease in a patient in need, comprising administering to the patient a therapeutically effective amount of sodium (2S,5R)-5-(2-chlorophenyl)-1-(2′-methoxy-[1,1′-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylate (1.Na).

[0186] In one embodiment, the present invention provides a method for treating and / or preventing, in patients with this need, diseases selected from: rheumatoid arthritis; inflammatory bowel disease (IBD), including but not limited to Crohn's disease and ulcerative colitis; colitis; collagenous colitis; lymphocytic colitis; immune-related enterocolitis (including adverse events in response to cancer treatment with checkpoint inhibitors such as CTLA-4, PD-1, and PD-L1 inhibitors); cystitis; celiac disease; irritable bowel syndrome; and gut microbiota dysbiosis, including those leading to... Antibiotic-induced dysbiosis of bacteria (e.g., Clostridium difficile infection, pneumococcal infection, etc.); Paget's disease; osteoporosis; multiple myeloma; uveitis; acute and chronic myeloid leukemia; pancreatic β-cell destruction; rheumatoid spondylitis; osteoarthritis; gouty arthritis and other arthritis conditions; gout; adult respiratory distress syndrome (ARDS); chronic inflammatory lung disease; silicosis; pulmonary sarcoidosis; psoriasis; rhinitis; allergic reactions; contact dermatitis; pancreatitis; allergies; hepatitis, including hepatitis B virus infection. Infections; asthma; muscle atrophy; cachexia, such as cachexia secondary to infection or malignancy, cachexia secondary to acquired immunodeficiency syndrome; Reiter's syndrome; type 1 diabetes; bone resorption disease; graft-versus-host disease; ischemia-reperfusion injury; traumatic brain injury; multiple sclerosis; cerebral malaria; sepsis; septic shock; toxic shock syndrome; endotoxin shock; Gram-negative sepsis; fever and myalgia caused by infections such as influenza; heartburn; anorexia-inducing intestinal hormones (e.g., PYY, GLP-1), for example... Inflammatory conditions observed in overeating-related diseases, obesity, type 2 diabetes, etc.; microbiome-related neurological and mood disorders, including autism spectrum disorders, schizophrenia, depression, major depressive disorder, and neurodegenerative diseases characterized by neuroinflammation, including Alzheimer's disease and Parkinson's disease; the method includes administering a therapeutically effective amount of sodium (2S,5R)-5-(2-chlorophenyl)-1-(2′-methoxy-[1,1′-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylate (1.Na) to the patient.

[0187] In one embodiment, the present invention provides a method for treating and / or preventing, in a patient with this need, diseases selected from: inflammatory bowel disease (IBD), including but not limited to Crohn's disease and ulcerative colitis; colitis; collagenous colitis; lymphocytic colitis; immune-associated enterocolitis (including adverse events as a response to cancer treatment with checkpoint inhibitors such as CTLA-4, PD-1, PD-L1 inhibitors); cystitis; celiac disease; irritable bowel syndrome; intestinal flora imbalance, including antibiotic-induced flora imbalance leading to bacterial infection (e.g., Clostridium difficile infection, pneumococcal infection, etc.); the method comprising administering to said patient a therapeutically effective amount of sodium (2S,5R)-5-(2-chlorophenyl)-1-(2′-methoxy-[1,1′-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylate (1.Na).

[0188] The present invention also relates to the use of sodium (2S,5R)-5-(2-chlorophenyl)-1-(2′-methoxy-[1,1′-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylate (1.Na) in the preparation of medicaments for treating and / or preventing inflammation.

[0189] In one embodiment, the present invention provides the use of compound (l.Na) in the preparation of a medicament for delaying the onset of inflammatory diseases in patients.

[0190] In one embodiment, the present invention provides the use of compound (l.Na) in the preparation of a medicament for treating and / or preventing diseases selected from: rheumatoid arthritis; inflammatory bowel disease (IBD), including but not limited to Crohn's disease and ulcerative colitis; colitis; collagenous colitis; lymphocytic colitis; immune-related enterocolitis (including adverse events in response to cancer treatment with checkpoint inhibitors such as CTLA-4, PD-1, PD-L1 inhibitors); cystitis; celiac disease; irritable bowel syndrome; intestinal flora imbalance, including antibiotic-induced flora imbalance leading to bacterial infections (e.g., Clostridium difficile infection, pneumococcal infection, etc.); Paget's disease; osteoporosis; multiple myeloma; uveitis; acute and chronic myeloid leukemia; pancreatic β-cell destruction; rheumatoid spondylitis; osteoarthritis; gouty arthritis and other arthritis conditions; gout; adult respiratory distress syndrome (ARDS); chronic inflammatory lung disease; silicosis; lung disease. Sarcoidosis; psoriasis; rhinitis; allergic reactions; contact dermatitis; pancreatitis; allergies; hepatitis, including hepatitis B virus infection; asthma; muscle atrophy; cachexia, such as cachexia secondary to infection or malignancy, cachexia secondary to acquired immunodeficiency syndrome; Reiter's syndrome; type 1 diabetes mellitus; bone resorption disease; graft-versus-host disease; ischemia-reperfusion injury; traumatic brain injury; multiple sclerosis; cerebral malaria; sepsis; septic shock; toxic shock Syndrome; endotoxin shock; Gram-negative sepsis; fever and myalgia caused by infections such as influenza; heartburn; anorexia-inducing intestinal hormones (e.g., PYY, GLP-1), such as inflammatory conditions observed in overeating-related diseases, obesity, type 2 diabetes, etc.; microbiome-related neurological and mood disorders, including autism spectrum disorders, schizophrenia, depression, major depressive disorder, and neurodegenerative diseases characterized by neuroinflammation, including Alzheimer's disease and Parkinson's disease.

[0191] In one embodiment, the present invention provides the use of compound (l.Na) in the preparation of a medicament for treating and / or preventing diseases selected from: inflammatory bowel disease (IBD), including but not limited to Crohn's disease and ulcerative colitis; colitis; collagenous colitis; lymphocytic colitis; immune-related enterocolitis (including adverse events as a response to cancer treatment with checkpoint inhibitors such as CTLA-4, PD-1, PD-L1 inhibitors); cystitis; celiac disease; irritable bowel syndrome; intestinal flora imbalance, including antibiotic-induced flora imbalance leading to bacterial infections (e.g., Clostridium difficile infection, pneumococcal infection, etc.).

[0192] Typically, sodium salts (l.Na) can be formulated into pharmaceutical preparations comprising (l.Na) and at least one pharmaceutically acceptable carrier, diluent, excipient and / or adjuvant, and optionally one or more other pharmaceutically active compounds.

[0193] By way of non-limiting examples, such formulations may be suitable for oral administration, parenteral administration (e.g., by intravenous, intramuscular, or subcutaneous injection or intravenous infusion), topical administration (including ocular administration), inhalation administration, administration via skin patches, implants, suppositories, etc. Such suitable forms of administration, depending on the route of administration, may be solid, semi-solid, or liquid, and the methods and carriers, diluents, and excipients used in their preparation are clear to those skilled in the art; see the latest edition of Remington's Pharmaceutical Sciences.

[0194] Some preferred but non-limiting examples of such pharmaceutical preparations include tablets, pills, powders, lozenges, sachets, capsules, elixirs, suspensions, emulsions, solutions, syrups, aerosols, ointments, creams, lotions, soft and hard gelatin capsules, suppositories, drops, sterile injections for asabolus administration and / or for continuous administration, and sterile packaged powders (usually reconstituted before use), which may be formulated with carriers, excipients, and diluents that are inherently suitable for the preparation, such as lactose, dextran, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, astragalus gum, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, polyethylene glycol, cellulose, (sterile) water, methylcellulose, methyl- and propyl hydroxybenzoates, talc, magnesium stearate, edible oils, vegetable oils, and mineral oils, or suitable mixtures thereof. The formulation may optionally contain other substances commonly used in pharmaceutical preparations, such as lubricants, wetting agents, emulsifiers and suspending agents, dispersants, disintegrants, swelling agents, fillers, preservatives, sweeteners, flavoring agents, flow regulators, releasing agents, etc. The composition may also be formulated to provide a rapid, sustained, or delayed release of the active compound contained therein.

[0195] The pharmaceutical formulations of the present invention are preferably unit dosage forms and can be suitably packaged in, for example, boxes, blister packs, vials, bottles, pouches, ampoules, or any other suitable single- or multi-dose reservoirs or containers (which may be suitably labeled); optionally including one or more instruction sheets containing product information and / or instructions for use. Typically, such unit dosage forms contain 0.05 to 1000 mg, usually 1 to 500 mg, of a sodium salt (L.Na), for example, about 10, 25, 50, 100, 200, 300, or 400 mg per unit dosage form.

[0196] Generally, depending on the condition to be prevented or treated and the route of administration, sodium salts (l.Na) are typically administered at doses of 0.01 to 100 mg per kilogram of patient body weight per day, more often 0.1 to 50 mg, such as 1 to 25 mg, for example, about 0.5, 1, 5, 10, 15, 20 or 25 mg. They can be administered as a single daily dose, or divided into one or more daily doses, or essentially continuously (e.g., using intravenous infusion). Example

[0197] The invention is further illustrated by the following examples. The reaction schemes described in the Examples section illustrate different possible methods by way of example.

[0198] Materials and Methods

[0199] All reported temperatures are expressed in degrees Celsius (°C); unless otherwise stated, all reactions were carried out at room temperature (rt). The term "room temperature" as used herein refers to a temperature between 10°C and 30°C, preferably 20 ± 5°C.

[0200] Reactants: Unless otherwise stated, solvents, reagents and starting materials were purchased from commercial suppliers and used as is.

[0201] Use the following abbreviations:

[0202] a%: HPLC area percentage,

[0203] AcOH: Acetic acid

[0204] cc: concentrate

[0205] DMF: Dimethylformamide,

[0206] dr: diastereomeric proportion,

[0207] ee: enantiomer excess

[0208] eq: equivalent

[0209] g: gram

[0210] GC: Gas chromatography

[0211] HPLC: High Performance Liquid Chromatography

[0212] IPA: Isopropyl alcohol

[0213] iPrOAc: Isopropyl acetate

[0214] L: Lift,

[0215] MeCN: Acetonitrile

[0216] MeOH: Methanol

[0217] M: mol / L

[0218] mL: milliliters

[0219] mol: mole

[0220] mmol: millimole,

[0221] min / mins: minutes

[0222] MTBE: Methyl tert-butyl ether.

[0223] sat: saturated

[0224] T3P: Propylphosphonic anhydride;

[0225] TEA: Triethylamine;

[0226] TFA: Trifluoroacetic acid

[0227] THF: Tetrahydrofuran

[0228] vol: volume.

[0229] All compounds disclosed in this application were obtained using ChemDraw Ultra purchased from CambridgeSoft (Cambridge, MA, USA). name.

[0230] All compounds disclosed in this application were obtained using ChemDraw Ultra from Cambridge Soft (Cambridge, MA, USA). name.

[0231] General procedure: The general synthesis route is shown in the figure below:

[0232]

[0233] analyze

[0234] Reversed-phase HPLC method

[0235] -For phases 1-2

[0236] Chromatographic column: Kinetex EVO C18 100*2.1mm*2.6μm

[0237] Eluent: A: H2O 0.1% TFAB: ACN

[0238] Flow rate: 0.5 ml / min

[0239] Temperature: 25℃

[0240] Gradient: A:B 98:2 (1 min) - 9 - 10:90 (3 min) - 0.1 - 98:2 (9.9 min)

[0241] Detection: 210nm

[0242] Retention time: (3) 8.6 min; (4a) 10.7 min; (4b) 10.9 min; (5) 8.0 min.

[0243] -For stages 3-4

[0244] Chromatographic column: Poroshell 120 EC-C18 150x4.6mmx2.7μm

[0245] Eluent: A: 20 mmol ammonium acetate in water; B: MeCN

[0246] Temperature: 25℃

[0247] Flow rate: 0.5 ml / min

[0248] Gradient: A:B 95:5(2min)-12min-10:90(3min)-0.1min-95:5(7.9min)

[0249] Detection: 210nm

[0250] Retention time: (5) 12.7 min; (N-Ac-6) 13.1 min; (6) 14.0 min; (6′) 14.4 min; (5) 8.0 min

[0251] -For stages 5-7

[0252] Chromatographic column: Kinetex EVO C18 100*2.1mm*2.6μm

[0253] Eluent: A: H2O 0.1% TFA B: ACN

[0254] Temperature: 25℃

[0255] Flow rate: 0.5 ml / min

[0256] Gradient: A:B 95:5 (0 min) -10 -45:55 (5 min) -4 -10:90 (3 min) -0.1 -95:5 (7.9 min)

[0257] Detection: 210nm

[0258] The sample was dissolved in a mixture of MeCN:water:(TFA) = 50:50:(0.1%).

[0259] Retention times: (8) 11.7 min; (1) 13.6 min; (1.Na) 13.6 min; (9) 14.7 min;

[0260] Chiral HPLC (normal phase)

[0261] -For stages 3-4

[0262] Column: Lux i-Cellulose-5 100x4.6mmx5μm

[0263] Eluent: A: n-Hexane:IPA:DEA = 80:20:0.1

[0264] Flow rate: 0.5 ml / min

[0265] Temperature: 25℃

[0266] Detection: 230nm

[0267] Gradient: A: 100 (25 min)

[0268] The sample was dissolved in an elution buffer of ~1 mg / mL.

[0269] Retention time: (6) 7.1 min; (6), (6′), (6) enantiomers, and mixture of (6′) enantiomers: 7.1 min, 5.8 min, 8.0 min and 4.7 min, respectively.

[0270] -For stages 6-7

[0271] Column: Lux i-Amylose-1 100x4.6mmx5μm

[0272] Eluent: A: n-Hexane:IPA:TFA = 80:20:0.1

[0273] Flow rate: 0.5 ml / min

[0274] Temperature: 37℃

[0275] Gradient: A: 100 (30 min)

[0276] Detection: 280nm

[0277] The sample is dissolved in the eluent at a concentration of approximately 0.5 mg / mL.

[0278] Retention times: (1′”) 9.8 min; (1″) 11.5 min; (1′) 17.1 min and (1 / l.Na) 22.4 min

[0279] Structure of acid diastereomers

[0280]

[0281] Example 1: Preparation of compound (1) and its sodium salt in batches of 25g

[0282] Phase 1: Synthesis of (4a) and (4b): Methyl (S)-2-((tert-butoxycarbonyl)amino)-5-(2-chlorophenyl)-5-oxovalerate and 2-methyl (2S)-5-(2-chlorophenyl)-5-hydroxypyrrolidine-1,2-dicarboxylic acid-1-tert-butyl ester

[0283] Under a nitrogen atmosphere, 39.35 g (205.5 mmol, 1.0 eq) of 1-bromo-2-chlorobenzene (2) in 34.0 mL of THF was added dropwise over 10 min to 192 mL (211.2 mmol, 1.02 eq) of iPrMgCl·LiCl in THF (commercially available, 1.1 M in THF) and 32 mL of stirred THF. The mixture was warmed to room temperature for 30 min and then stirred at 15–25 °C for 1 h. The mixture was then recooled to 0 °C and 50.0 g (50.0 mmol, 1.0 eq) of Boc-L-pyroglutamic acid methyl ester in 110 mL of THF was added dropwise over 100 min at 0 °C–5 °C. The resulting mixture was stirred at 0 °C until complete (1 h). The reaction was monitored by HPLC. After completion, the temperature was maintained below 5°C, and 190 mL of a 10% by weight citric acid solution was added to the cooled reaction mixture (pH 3.5). The mixture was then warmed to 20-25°C, producing two clear layers. THF was stripped at 200-250 mbar in a rotary evaporator at 40°C for 1 hour. The remaining aqueous phase with yellow oil was extracted with 2 × 75 mL CH₂Cl₂. The combined organic phase (272 g) had a water content of 0.55 w / w%. Approximately 100 g of solvent was evaporated from this solution. 100 g of CH₂Cl₂ was added to the resulting concentrate and removed under vacuum (500 mbar, 40°C), and the process was repeated. In total, 3 x 100 g of solvent was removed from the product, resulting in a water content of <0.01 w / w%. To determine the content of the resulting solution (151 g), 1.315 g (1 mL) was evaporated to give 603 mg of oil. Calculated yield: 94% (69.2 g); HPLC purity: 59.6a% (4b) and 28.9a% (4a).

[0284] Phase 2: Synthesis of (5): (S)-5-(2-chlorophenyl)-3,4-dihydro-2H-pyrrole-2-carboxylic acid methyl ester

[0285] Under a nitrogen atmosphere, 90.0 mL (133.2 g, 1168 mmol, 6.0 eq) of TFA and 245 mL of anhydrous dichloromethane were added to a 500 mL three-necked round-bottom flask. The solution was cooled between 0 and 10 °C, and then, while maintaining this temperature, 69.2 g (194 mmol, 1.0 eq) of a crude mixture of (4a) and (4b) dissolved in 61 mL (81 g) of CH2Cl2 (HPLC: 59 a% of (4b)) (as a 151 g solution from the Grignard reaction) was added after 30 min. The resulting yellow mixture was warmed to 20–25 °C and stirred overnight at ambient temperature, monitored by HPLC. The mixture gradually darkened. After completion (stirring for 24 h), 274 mL of saturated Na2CO3 solution (prepared from 56.5 g, 533 mmol Na2CO3 and 257 g deionized water) was added at 20–25 °C (without increasing the temperature). The pH changed to 7–8. The organic phase was separated; the aqueous phase was extracted with 50 mL of CH2Cl2. The combined organic phases were evaporated (at 40 °C, under vacuum) to give 38.46 g of a purple oil (5) (yield 83%). HPLC purity: 94.0 a%.

[0286] Phase 3: Synthesis of mixtures (6) & (6′): Methyl 2-(2S,5R)-5-(2-chlorophenyl)pyrrolidine-2-carboxylate and methyl 2-(2S,5S)-5-(2-chlorophenyl)pyrrolidine-2-carboxylate

[0287] All reactions were carried out under an inert atmosphere.

[0288] Preparation of mixture "A"-NaB(OAc)3H. 6.23 g (164.6 mmol, 1.25 eq) of NaBH4 and 157 mL of anhydrous CH2Cl2 were added to a glass flask. The suspension was cooled to 0 °C, and at this temperature, 28.25 mL (29.66 g, 493.8 mmol, 3.75 eq) of AcOH was added. A large amount of gas was observed to escape. The reaction mixture was heated to 20 °C and stirred for 15 min, then cooled back to 0–5 °C.

[0289] Preparation of solution “B” - solution (5). 30.1 mL (31.6 g, 526.8 mmol, 4.0 eq) of AcOH was added to 31.3 g (131.7 mmol, 1.0 eq) of (5) (isolated) in 31 mL of anhydrous CH2Cl2 at a cooled (0-5 °C) temperature.

[0290] The temperature was maintained between 0 and 5 °C. Solution “B” (1.0 eq (5) and 4.0 eq AcOH in CH2Cl2) was added to mixture “A” (1.25 eq NaB(OAc)3H in CH2Cl2) for 45 min. At the same time, 125 mL of anhydrous CH2Cl2 was added to mixture “B” to keep it stirable. The resulting brown solution was stirred at 0-5 °C for 24 h and monitored by HPLC. The temperature was kept below 10 °C. 200 mL of saturated Na2CO3 solution was added to the reaction mixture and the pH was set to 4 to 7. (A large amount of gas escaped). The resulting layers were separated, and the aqueous layer was extracted with 50 mL of CH2Cl2. The combined organic layers were vacuum evaporated on a rotary evaporator at 40 °C. Yield: 98.3% (31.05 g (6) & (6′) mixture, 129.5 mmol), brown oil. HPLC purity: 73.8a% of (6) (dr: 84 / 16).

[0291] Phase 4: Crystals of methyl 2-(2S,5R)-5-(2-chlorophenyl)pyrrolidine-2-carboxylic acid hydrochloride (6.HCl)

[0292] Under an inert atmosphere, 27.5 g (114.7 mmol) of (6) and (6′) (HPLC 73.8a% of (6); dr: 84 / 16) were dissolved in 25 mL (1 vol) of IPA. This solution (keeping the temperature below 15 °C) was added to a mixture of 13.15 g of IPA containing HCl (HCl content: 4.99 g, 137 mmol, 1.19 eq) and 3 mL of IPA. Addition was stopped when half was reached, as the mixture became too thick. 28 mL of iPrOAc was added for dilution, and then addition was continued. The mixture was cooled to 0–5 °C, stirred for 20 min, and then filtered. The filtered solid was washed with 4 x 14 mL of cold iPrOAc and dried under vacuum at 30 °C. The resulting white solid (19.3 g) was confirmed by HPLC to have 94a%. Therefore, the salt (19.3 g) was purified by slurry purification with 58 mL of hot IPA. Cool to 0-5°C, stir for 30 min, filter, wash with 2 x 10 mL of cold IPA, and dry under vacuum at 30°C. Yield: 51.5% (16.3 g) white solid (6.HCl). HPLC purity: 99.9a%; chiral HPLC purity: 99.9a%.

[0293] Phase 5: Synthesis of methyl ester (9): (2S,5R)-5-(2-chlorophenyl)-1-(2′-methoxy-[1,1′-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid methyl ester

[0294] Solution "A": Under an inert atmosphere, 27.47 g (120.4 mmol, 1.0 eq) of 2′-methoxy-[1,1′-biphenyl]-4-carboxylic acid (8) was suspended in 137 mL of anhydrous toluene, and then 425 μL (0.05 eq) of DMF was added. 9.6 mL (156.4 mmol, 1.3 eq) of SOCl2 was added at 20–25 °C. The resulting suspension was heated to 50 °C and stirred at this temperature for 24 hours to form 2′-methoxy-[1,1′-biphenyl]-4-carbonyl chloride (7). The suspension turned into a yellow solution. Monitoring: High performance liquid chromatography (HPLC)

[0295] Mixture "B": 26.7 g (96.79 mmol, 0.9 eq) (6·HCl) was suspended in 81 mL of toluene under an inert atmosphere. 80.27 g (581 mmol, 5.4 eq) of K₂CO₃ dissolved in 72 mL of water was added to the suspension at 10 °C.

[0296] After stirring for 20 min, solution "A" was added to mixture "B", maintaining the temperature between 0-10℃. The reaction was heated to 20-25℃ and stirred overnight. The layers separated; the aqueous phase was extracted with 20 mL of toluene, and the combined organic phases were washed with 20 mL of saturated NaCl solution. Yield: 95% (41.38 g) (9). HPLC purity: 97 a% at 254 nm. (Content: 186 g toluene solution)

[0297] Phase 6: Synthesis of compound (1): (2S,5R)-5-(2-chlorophenyl)-1-(2′-methoxy-[1,1′-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid

[0298] Under an inert atmosphere, 9.3 g (232.57 mmol, 4.0 eq) of NaOH was dissolved in 65 mL of water, and 185 mg (0.58 mmol, 0.01 eq) of TBAB was added to a solution of 26.16 g (58.14 mmol, 1.0 eq, 91.23 g solution) in toluene (9) at 20–25 °C. The mixture was then stirred at 20–25 °C for 18 hours and monitored by HPLC (sample preparation: 20 μL of the reaction mixture was added to 100 μL of toluene and 200 μL of 2M HCl, the organic phase was separated and evaporated).

[0299] Post-treatment: 23 mL (268 mmol) ccHCl was added to the mixture to adjust the pH to 1. The layers were separated, the aqueous phase was extracted with 26 mL toluene, and the combined organic phases were washed with 2 x 30 mL saturated sodium chloride solution. The organic phase was dehydrated using a Dean-Stark water separator (130 °C oil temperature). The concentrated toluene solution (1 volume ratio (1) content) was added to 8 volumes of cyclohexane, and the mixture was stirred at 20-25 °C for 4 hours and filtered. Yield: 88% (22.2 g) amorphous (1). HPLC purity: 95.9 a%. Chiral HPLC purity: 97.4 a%. Water content: 0.14 w / w%.

[0300] Phase 7: Sodium salt formation (l.Na): (2S,5R)-5-(2-chlorophenyl)-1-(2′-methoxy-[1,1′-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid sodium salt

[0301] Procedure A: Under an inert atmosphere, 25 g (57.35 mmol, 1.0 eq) of compound (1) was dissolved in 75 mL of anhydrous MeOH. The solution was cooled to 20–25 °C, and then 13.1 mL (containing 3.09 g, 57.3 mmol, 1.0 eq) of NaOMe in MeOH solution (25 wt%) was added (temperature increased to 32 °C). After stirring at ambient temperature for 1.5 hours, the solution was evaporated under vacuum at 40 °C to give 25.6 g of white amorphous solid. Yield: 97% (25.6 g) (l. Na). HPLC purity: 96.6 a% (at 210 nm). Chiral HPLC purity: 99.5 a%.

[0302] Procedure B (Solvent Exchange): Under an inert atmosphere, 10 g (22.94 mmol, 1.0 eq) of compound (1) was dissolved in 30 mL of anhydrous MeOH. The solution was cooled to 20–25 °C, and then 5.25 mL of a MeOH solution (25 wt%) containing 1.24 g, 22.94 mmol, 1.0 eq) of NaOMe was added (temperature increased to 32 °C). After stirring at ambient temperature for 1.5 hours, 240 mL of IPA (MeOH:IPA = 1:8) was added, and the resulting suspension was subjected to solvent exchange (T). 油 =95℃, at T 油 (At 50°C, it becomes a clear solution). The concentrate (8 volumes, becomes cloudy) was cooled to 20-25°C and then filtered. Yield: 92% (9.7 g) (l. Na). HPLC purity: 95.2% (at 210 nm). Chiral HPLC purity: 97.5%.

[0303] Example 2: Prepare 400g batches of hydrochloride (6·HCl)

[0304] Phase 1: Synthesis of (4a) and (4b): Methyl (S)-2-((tert-butoxycarbonyl)amino)-5-(2-chlorophenyl)-5-oxovalerate and 2-methyl (2S)-5-(2-chlorophenyl)-5-hydroxypyrrolidine-1,2-dicarboxylic acid-1-tert-butyl ester

[0305] Under a nitrogen atmosphere, 5100 mL (5.610 mol, 1.05 eq) of iPrMgCl·LiCl in THF (commercially available, 1.1 M THF solution) and 820 mL of anhydrous THF were added to a 20 L glass reactor. The mixture was cooled to 0–5 °C, and then, while maintaining the temperature below 10 °C, 1043.6 g (5.451 mmol, 1.02 eq) of 1-bromo-2-chlorobenzene dissolved in 910 mL of anhydrous THF was added dropwise to the iPrMgCl·LiCl solution over 45 min (2). The mixture was warmed to room temperature over 30 min, and then stirred at 15–25 °C for 1 h. The reaction was monitored by GC (sample preparation: 200 μL of the reaction mixture was added to a mixture of 500 μL of saturated NaCl and 500 μL of THF, the organic phase was separated and used as is). After completion, the mixture was recooled to 0-5°C for 30 min, and a solution of 1.30 kg (5.344 mmol, 1.0 eq) of Boc-L-pyroglutamic acid methyl ester (3) in 2880 mL of anhydrous THF was added dropwise over 90 min at 0°C-5°C. The resulting mixture was stirred at 0°C until completion (1-2 h). The reaction was monitored by HPLC. After completion, while keeping the temperature below 15°C, 5470 mL of 10% citric acid solution (from a mixture of 600 g citric acid monohydrate and 5.4 L deionized water) was added to the cooled reaction mixture to set the pH from 12 to 4, and then the mixture was warmed to 20-25°C to produce two transparent layers. THF (~8.3 L) was stripped in 150-250 mbar vacuum at 35-40°C (internal temperature, thermostat temperature 50°C) for 4 h. The remaining aqueous phase containing the yellow oil was cooled to 20–25 °C and then extracted with 2 × 2.4 L of CH₂Cl₂. The combined organic phase (~6 L) had a water content of 0.74 w / w%. To reduce the water content, it was removed by 5 × 1.5 L of CH₂Cl₂ (replaced with fresh CH₂Cl₂ for each 1.5 L of CH₂Cl₂ distilled off) for 5 hours at 30–35 °C and 550–600 mbar. The water content became 0.2 w / w%. To determine the content of the obtained 4.61 L (5650 g) solution, 51 mL (63 g) was evaporated to give 18.9 g of yellow oil. The yield was calculated to be 89% (1693.4 g, 4.75 mol) as a solution of 4.61 L (5650 g) in CH₂Cl₂. The HPLC purity of the solution was 39.6a% (4b); 15.9a% (4a).

[0306] Phase 2: Synthesis of (5): (S)-5-(2-chlorophenyl)-3,4-dihydro-2H-pyrrole-2-carboxylic acid methyl ester

[0307] Under a nitrogen atmosphere, 5.2 L of anhydrous CH2Cl2 and 2268 mL (3356 g, 29.43 mmol, 6.0 eq) of TFA were added to a 20 L glass reactor. The solution was cooled to 0–10 °C and then kept at this temperature for 70 min. 4.2 L (5556 g, containing 1745 g, 4.91 mol, 1.0 eq) of a crude mixture of (4a) and (4b) was added (as a CH2Cl2 solution from the Grignard reaction (stage 1)). The resulting yellow mixture was warmed to 20–25 °C (for 40 min) and stirred at ambient temperature (for 21 h), monitored by HPLC. The mixture gradually darkened. After completion (stirring for 21 h), 6.6 L of saturated Na2CO3 solution (from a mixture of 1540 g Na2CO3 and 7.0 L of deionized water) was added at 20–25 °C (without increasing the temperature) to set the pH from 1 to 7.5–8.0 (for 90 min). The organic phase was separated, and the aqueous phase was extracted with 1.0 L of CH2Cl2. The combined organic phase (10.1 L) had a water content of 0.22 w / w%. This organic solution was concentrated to 3.9 L and combined with 1.0 L of anhydrous CH2Cl2 for rinsing the reactor to remove product residue. The yield was determined by evaporating a small sample (35.8 g, containing 6.9 g of product) under vacuum at 40 °C. Calculated yield: 89% (1041 g) (5). HPLC purity: 66.7 a%.

[0308] Phase 3: Synthesis of mixtures (6) & (6′): Methyl 2-(2S,5R)-5-(2-chlorophenyl)pyrrolidine-2-carboxylate and methyl 2-(2S,5S)-5-(2-chlorophenyl)pyrrolidine-2-carboxylate

[0309] All reactions were carried out under an inert atmosphere.

[0310] Preparation of mixture "A"-NaB(OAc)3H. 5.17 L of anhydrous CH2Cl2 and 165.7 g (4.38 mmol, 1.25 eq) of NaBH4 were charged into a 20 L glass reactor. The white suspension was cooled to 0–5 °C, and 789 g (751 mL, 14.02 mol, 3.75 eq) of AcOH was added at this temperature after 30 min. A large amount of gas was observed to escape. The reaction was heated to 20 °C and stirred for 10 h, and the resulting white slurry was cooled to 0–5 °C after 1 h.

[0311] Preparation of solution “B” – solution (5). 918 g (3.86 mol, corrected to 3.5 mmol (80%, based on HPLC), 1.0 eq) of CH2Cl2 (2.0 L (2502 g)) and 3800 mL of anhydrous CH2Cl2 were placed in a 10 L glass flask. The mixture was cooled to 0–5 °C and 841.7 g (802 mL, 14.01 mol, 4.0 eq) of AcOH was added.

[0312] The temperature was maintained between 0 and 5 °C. Solution “B” (1.0 eq (5) and 4.0 eq AcOH in CH2Cl2) was added to mixture “A” (1.25 eq NaB(OAc)3H in CH2Cl2) over 1.5 hours. The resulting brownish-purple solution was stirred at 0-5 °C and monitored by HPLC. After stirring at 0-5 °C for 13 hours, the amount of starting material was less than 3a%, allowing the reaction to be post-processed. The pH of the mixture was ~4. The (6) & (6′) reaction mixture (12.0 L) was removed from the reactor and kept at 10 °C. 5.0 L of saturated Na2CO3 was added to a 20 L glass reactor. The temperature was kept below 15 °C (over 2 hours), and 12.0 L of the (6) & (6′) reaction mixture solution was added to the saturated Na2CO3 solution. A large amount of gas and foam was observed to escape. The pH after addition was 7. To set the pH to 7-8, another 400 mL of saturated Na2CO3 solution was required. After setting the pH, the mixture was stirred for 20 min. The two layers were separated, and the aqueous phase was extracted with 1 L of CH2Cl2. The combined organic layers (~10.5 L) were evaporated under vacuum (600 mbar) at 40 °C (thermostat: 50 °C, internal temperature 30-33 °C). Yield: 91% (834.8 g crude (6) & (6′) in 4713 g CH2Cl2 solution). HPLC purity: 74% of (6) (dr88 / 12).

[0313] Phase 4: Crystals of (6.HCl)-(2S,5R)-5-(2-chlorophenyl)pyrrolidine-2-carboxylate methyl carboxylate hydrochloride

[0314] Under an inert atmosphere, 4.1 kg of CH2Cl2 solution (containing 1720 g (6) & (6′)) was exchanged for 2-propanol. The reaction temperature was 15-40 °C, and the pH was 100-600 mbar. After 4 hours, 3822 mL of solution was obtained. Next, 1950 mL of IPA (cooled to below 5 °C) and 850 mL of HCl (822 g, containing 287 g, 7.89 mol, 1.1 eq HCl) in an IPA solution were loaded into a 20 L glass reactor. The mixture was cooled to 0-5 °C, and after 1 hour, 3.822 kg (containing 1.719 kg, 7.17 mol, 1.0 eq) of (6) & (6′) in IPA was added to the mixture. The resulting solution was heated to reflux (72 °C, after 1 hour), stirred for 10 min, and cooled back to 20-25 °C (after 2 hours), and then 1.72 L of iPrOAc was added. The mixture was cooled to -5°C over 2.5 hours. Seeded crystals were then formed at 60°C and 34°C. After stirring overnight at -5°C, the sample was collected: approximately 10 mL of the mixture contained 220 mg of precipitate, meaning a total salt content of 192 g. The reaction mixture (suspension) was stirred again at -10°C for 20 hours, but the sample was expected to contain only 271 g of salt. To improve the yield, the solvent was removed: 5160 mL of solvent was removed at 30°C and 50–100 mbar. 1750 mL (1 volume) of iPrOAc was added to the residue, and the suspension was cooled to -10°C and stirred for 17 hours. The sample yield was predicted to be 900 g of wet material. The sample was filtered through a G3 glass filter and washed with 2 x 1 L IPA:iPrOAc = 1:1 and 2 x 0.5 L iPrOAc. Wet mass: 822 g, dried overnight at 20–30°C and 200–300 mbar. Yield: 21% (480 g), obtained as a pink solid (6.HCl) after prolonged drying. HPLC purity: 96.1 a%, chiral HPLC: ee: 99.9%, dr: 96.3 / 3.5.

[0315] Recrystallization of (6.HCl): 1500 mL of IPA was added to a magnetically stirred 2 L glass flask and heated to 67 °C. At this temperature, 478.6 g of (6.HCl) was added over 50 min, and the mixture was heated to reflux. After reaching 80 °C (solution), the mixture was allowed to cool back to 20–25 °C over 80 min. The resulting suspension was filtered, washed with 200 mL of IPA, and dried under vacuum at 30 °C. Dried product: 398 g, white solid (6.HCl). HPLC purity: 99.5a% (dr: 99.5a%). Chiral HPLC purity: 98.9a% (dr: 98.9 / 1.1).

[0316] Example 3: Prepare 350g batches of hydrochloride (6·HCl).

[0317] Phase 4: Crystals of methyl 2-(2S,5R)-5-(2-chlorophenyl)pyrrolidine-2-carboxylic acid hydrochloride (6.HCl)

[0318] Under an inert atmosphere, a solution of (6&6′) (1174 g crude (6&6′) in 3404 g CH2Cl2; HPLC: 64.9a% (6)) was distilled under vacuum at 40 °C (200-400 mbar); solvent exchange from CH2Cl2 to iPrOAc was performed to obtain 1378 g (containing 1174 g 6&6′) of solution (solvent iPrOAc). Next, 996 g (904 mL, containing 199.2 g, 5.457 mol, 1.1 eq HCl) of iPrOAc solution and 290 mL of anhydrous MeOH were added to a 5 L glass reactor equipped with a mechanical stirrer. The mixture was cooled to 0-5 °C, and after 1 hour, 1378 g (containing 1174 g, 4.897 mol, 1.0 eq HCl) of crude (6&6′) in iPrOAc was added to the mixture. (Final solvent ratio: MeOH:iPrOAc = 1:4). The resulting solution was seeded and crystallized to form a suspension. The suspension was stirred overnight (18 hours) at 0–5 °C and then filtered. Filtered through a G3 glass filter and washed with 3 x 470 mL of cold MeOH:iPrOAc = 1:4 to obtain 458 g of wet material. Drying at 30 °C and 200–300 mbar for 2 hours gave 392.7 g of white solid (6.HCl). HPLC purity: 96.0a% (dr: 98.4 / 1.6). Chiral HPLC purity: 98.0a% (dr: 98.4 / 1.6).

[0319] Recrystallization of (6.HCl): 785 mL of a (2 Vol) MeOH:iPrOAc = 1:4 mixture was added to a 5 L glass flask under mechanical stirring at 20–25 °C, followed by the addition of 392.7 g of (6.HCl). The mixture was heated to reflux for 30 min. After reaching 80 °C (solution), the mixture was stirred at this temperature for 10–15 min, then cooled to 0 °C. After stirring at 0 °C for 30 min, the suspension was filtered and washed with 2 x 225 mL of cold MeOH:iPrOAc = 1:4. The resulting wet substance, 398 g, was dried overnight under vacuum (200–300 mbar) at 30 °C to give 341.4 g of a white solid (6.HCl). HPLC purity: 97.8a% (dr: 98.9 / 1.1). Chiral HPLC purity: 98.7a% (dr: 98.8 / 1.2a%).

[0320] Example 4:Synthesis of Methyl Ester (9) – Alternative Coupling Stage 5′

[0321] Phase 5′: Synthesis of methyl ester (9): (2S,5R)-5-(2-chlorophenyl)-1-(2′-methoxy-[1,1′-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid methyl ester

[0322] Mixture “A”: 4.34 g (15.71 mmol, 1.0 eq) (6·HCl) was suspended in 22 mL (5V) of anhydrous iPrOAc under an inert atmosphere. 3.3 mL (23.57 mmol, 1.5 eq) of TEA (water content: 0.05%) was added to the suspension, and the resulting white suspension (mixture “A”) was stirred at room temperature for 1 hour.

[0323] Solution "B": In a 100 mL round-bottom flask under an inert atmosphere, 3.59 g (15.71 mmol, 1.0 eq) (8) was suspended in 22 mL (5V) of anhydrous iPrOAc. 4.4 mL (31.42 mmol, 2.0 eq) of TEA (water content: 0.05%) was added to the suspension. 20.00 g of 50 wt% T3P / EtOAc solution (31.42 mmol, 2.0 eq, 10.00 g T3P) was added dropwise to the obtained suspension over 2 min, maintaining the temperature between 20-25 °C. The resulting milky white solution "B" was stirred at room temperature for 1 hour.

[0324] After stirring for 1 hour, solution "B" was added to mixture "A" at room temperature, and then stirred overnight under a nitrogen atmosphere and monitored by HPLC. The reaction mixture was then quenched with 90 mL of water, maintaining the temperature below 20 °C. After phase separation, the organic phase was washed with 80 mL of water (pH = 1-2) and 2 × 40 mL of 5% Na₂CO₃ solution (pH = 10).

[0325] Add 15 mL of toluene to the obtained iPrOAc solution, and then concentrate it under vacuum at 40 °C. Add 14 mL of toluene to the concentrate, and then concentrate it again. After concentrating to 12.0 g, the iPrOAc content of the resulting solution is 1.5%. Considering that the yield of the coupling reaction is 100%, the toluene solution of (9) is further used for stage 6 hydrolysis without separation.

[0326] Example 5: Properties of sodium salts (l.Na)

[0327] 5.1. Solubility in water

[0328] Objective: Compound (1) is poorly soluble in water in its acidic form. Solubility determination was performed to determine whether salts of compound (1) have an improving property in this respect.

[0329] Methods: The solubility in water was investigated as described below. 150 mg of the study materials (1), (l.Na), and (l.K) were transferred to a glass bottle and 1.0 mL of deionized water was added. The mixture was circulated using vortexing and sonication over 5 min. The pH was then checked; the pH was adjusted to 9.0–10.0 by accurately adding 2 M NaOH aqueous solution to (1) and (l.Na) or accurately adding KOH aqueous solution to (l.K). Several cycles of vortexing and sonication were then applied for 30 min. The pH was adjusted to the range of 7.2–7.6 by carefully adding NaOH, KOH, or HCl aqueous solution. The vortexing and sonication cycles and pH adjustments were repeated until the pH stabilized within the range of 7.2–7.6. The suspension was then centrifuged at 3000 rpm and the supernatant was filtered through a membrane filter. The resulting filtrate was diluted 1000-fold with DMSO and analyzed by HPLC against a standard solution of known concentration. The obtained data are shown in the table below.

[0330] Results: Comparison of solubility data of sodium salt (l.Na) with free acid (l.Na) and potassium salt (l.K):

[0331]

[0332] The solubility of (l.Na) in water was confirmed to be ~130 mg / mL.

[0333] These results indicate that the solubility of sodium salt (l.Na) in water is surprisingly increased compared to that of free acid (1) and potassium salt (lK). Specifically, the solubility of sodium salt (l.Na) increased by 2 times compared to free acid (1) and by 1.5 times compared to potassium salt (lK). This improved solubility in water is beneficial for increasing dissolution rate and bioavailability, which is particularly important for high-concentration aqueous formulations, such as those used in animal maximum tolerated dose studies.

[0334] 5.2. Stability

[0335] Objective: To conduct forced degradation studies to determine the stability of the acid form (1) and the sodium salt (1.Na).

[0336] method:

[0337] For photolysis stability, an OSRAM DULUXTWIST 23W 1600 1m 6500K cold daylight lamp was used, positioned 20 cm away from the sample. A 1 mg / mL sample was prepared in an ACN / water (3:7 v / v) solution and irradiated with light in a capped glass bottle placed on a reflective surface at ambient temperature (approximately 22°C).

[0338] For thermal stability, the powder was placed in a sealed glass bottle and exposed to high temperatures in a constant-temperature oven at 115°C.

[0339] Samples were analyzed by RP-HPLC and chiral HPLC at T0, 1 week, and 3 weeks. For RP-HPLC analysis, samples were analyzed directly from the solution used for stability studies. For chiral HPLC, aliquots were evaporated in Genevac at 40°C and redissolved in the mobile phase.

[0340] result

[0341]

[0342] Compared to the free acid (1), the sodium salt (l.Na) exhibited better photolytic and thermal stability after 3 weeks. Specifically, in the photolytic stability assay, after 3 weeks, the purity of the acid form (1) decreased by 23.6%, while the purity of the sodium salt (l.Na) decreased by only 2.3%. In the thermal stability assay, after 3 weeks, the purity of the acid form (1) decreased by 28.1%, while the purity of the sodium salt (l.Na) decreased by only 0.4%.

Claims

1. A method for preparing (2S,5R)-5-(2-chlorophenyl)pyrrolidine-2-carboxylic acid methyl ester hydrochloride (6.HCl), The method includes the steps of forming a hydrochloride salt and separating the epimers, including crystallizing a mixture of epimers (6) and (6') in the presence of hydrochloric acid in a solvent selected from isopropanol, isopropyl acetate, a mixture of isopropanol and isopropyl acetate, and a mixture of methanol and isopropyl acetate. The epimerization ratio (6):(6') is at least 4:

1.

2. The method of claim 1, further comprising a subsequent recrystallization step in a solvent selected from isopropanol, isopropyl acetate, a mixture of isopropanol and isopropyl acetate, and a mixture of methanol and isopropyl acetate thereof.

3. The method according to claim 1, wherein the compound (5) is reduced in the presence of an acid by sodium triacetoxyborohydride in a solvent selected from dichloromethane, acetonitrile, isopropyl acetate, and mixtures thereof to obtain a mixture of epimers (6) and (6') wherein the epimer ratio (6):(6') is at least 4:

1. 。 4. The method according to claim 3, wherein the solvent is dichloromethane.

5. The method according to claim 3, wherein the acid is selected from acetic acid and trifluoroacetic acid.

6. The method according to claim 3, wherein the acid is acetic acid.

7. The method according to claim 3, wherein compound (5) is obtained by: a) Compounds (2) and (3) undergo a Knochel-Grignard reaction in the presence of isopropyl magnesium chloride and lithium chloride: Quenching with an aqueous solution containing acid yields a mixture of compounds (4a) and (4b): and, b) The mixture of compounds (4a) and (4b) obtained in step a) is contacted with an acid in a solvent selected from dichloromethane, methanol, isopropanol and mixtures thereof at a temperature of 0°C to 25°C to obtain compound (5).

8. The method according to claim 7, wherein the acid is trifluoroacetic acid or hydrochloric acid.

9. The method according to claim 7, wherein the solvent is dichloromethane.

10. A method for preparing (2S,5R)-5-(2-chlorophenyl)-l-(2'-methoxy-[1,1'-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid (1): include: a) The method according to any one of claims 1 to 9 yields compound (6.HCl); b) Formation of methyl ester (9) from the compound (6.HCl) obtained in step a) via the following steps: b1) In the presence of potassium carbonate, compound (6.HCl) is acetylated with compound (7) in a mixture of toluene and water as solvent. ， Or b2) In the presence of a base and an acid activator, the compound (6.HCl) is coupled to the compound (8). ； c) In the presence of sodium hydroxide and tetrabutylammonium bromide (TBAB), methyl ester (9) was saponified in a mixture of toluene and water as solvent to give an amorphous acid compound (1).

11. A method for preparing sodium (1.Na) of (2S,5R)-5-(2-chlorophenyl)-1-(2'-methoxy-[1,1'-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylate: include: a) The method according to claim 10 yields acid compound (1); and b) The sodium salt formation step includes contacting the acid compound (1) obtained from step a) with sodium hydroxide in methanol as a solvent to obtain an amorphous sodium salt (l.Na).

12. The method of claim 11, further comprising optional step c) of precipitation in the presence of isopropanol.

13. (2S,5R)-5-(2-chlorophenyl)pyrrolidine-2-carboxylic acid methyl ester hydrochloride (6.HCl): 。 14. (2S)-5-(2-chlorophenyl)-5-hydroxypyrrolidine-1,2-dicarboxylic acid-1-tert-butyl ester 2-methyl ester (4b): 。

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