EA041535B1 - METHOD FOR PRODUCING EPOXYKETONE PEPTIDE IMMUNOPROTEASOME INHIBITOR - Google Patents

Description

State of the art

The field of technology.

Description refers to the method and process for obtaining (2S,3R)-N-[(2S)-3-(cyclopent-1-en-1-yl)-1[(2R)-2-methyloxiran-2-yl]-1 -oxopropan-2-yl]-3-hydroxy-3-(4-methoxyphenyl)-2-[(2S)-2-[2-(morpholin4-yl)acetamido]propanamido]propanamide.

Description of the prior art.

Compound (2S,3R)-N-[(2S)-3-(cyclopent-1-en-1-yl)-1-[(2R)-2-methyloxiran-2-yl]-1-oxopropan-2yl] -3-Hydroxy-3-(4-methoxyphenyl)-2-[(2S)-2-[2-(morpholin-4-yl)acetamido]propanamido]propanamide (Compound G) is useful as an immunoproteasome inhibitor.

In eukaryotes, protein degradation is predominantly mediated by the ubiquitin metabolic pathway, in which proteins destined for degradation are ligated to the 76 amino acid polypeptide ubiquitin. Once tagged, ubiquitinated proteins then serve as substrates for the 26S proteasome, a multicatalytic protease that cleaves proteins into short peptides through three major proteolytic activities. Despite the general function of intracellular protein metabolism, proteasome-mediated degradation also plays a key role in many processes, such as major histocompatibility complex (MHC) class I antigen presentation, apoptosis, cell growth regulation, NF-kB activation, antigen processing, and transduction of pro-inflammatory signals.

The 20S proteasome is a cylindrical multicatalytic complex of 700 kDa proteases, consisting of 28 subunits organized into four rings. In yeast and other eukaryotes, 7 different α subunits form the outer rings and 7 different β subunits make up the inner rings. The α-subunits serve as binding sites for the regulatory complexes 19S (PA700) and 1 IS (PA28), as well as a physical barrier to the internal proteolytic chamber formed by two rings of β-subunits. Thus, the proteasome is thought to exist in vivo as a 26S particle (the 26S proteasome). In vivo experiments have shown that inhibition of the 20S proteasome form can be easily correlated with inhibition of the 26S proteasome. Cleavage of the amino-terminal sequences of the β-subunits during particle formation acts on the amino-terminal threonine residues, which serve as catalytic nucleophiles. Thus, the subunits responsible for catalytic activity in proteasomes have an amino-terminal nucleophilic residue and these subunits belong to the family of N-terminal nucleophilic (Ntn) hydrolases (wherein the nucleophilic N-terminal residue is, for example, Cys, Ser, Thr and other nucleophilic fragments). This family includes, for example, penicillin G acylase (PGA), penicillin V acylase (PVA), glutamine PRPP amidotransferase (GAT), and bacterial glycosylasparaginase. In addition to the ubiquitously expressed β-subunits, higher vertebrates also have three interferon-γ-induced β-subunits (LMP7, LMP2, and MECL1) that replace their normal counterparts, B5, B1, and B7, respectively, altering the catalytic activity of the proteasome. Through the use of various peptide substrates for the eukaryotic 20S proteasome, three major proteolytic activities have been identified: chymotrypsin-like activity (CT-L), which cleaves after large hydrophobic residues; trypsin-like activity (TL), which cleaves after basic residues; and peptidylglutamylpeptide hydrolyzing activity (PGPH), which cleaves after acid residues. Two additional less characterized activities have also been attributed to the proteasome: BrAAP activity, which cleaves after branched chain amino acids; and SNAAP activity, which cleaves after small neutral amino acids. The main proteolytic activities of the proteasome appear to be mediated by various catalytic sites, starting with inhibitors, point mutations in the β-subunits and exchange of interferon-γ-induced β-subunits alter these activities to varying degrees.

PCT Publication No. WO 2014/152134, which is incorporated herein by reference, describes tripeptide epoxy immunoproteasome inhibitors such as Compound G and methods for their small scale synthesis. However, large scale synthesis of tripeptide epoxy immunoproteasome inhibitors such as Compound G is required for commercial development.

The essence of the invention

In one aspect, the invention provides a process for the preparation of (2S,3R)-N-[(2S)-3-(cyclopent-1-en1-yl)-1-[(2R)-2-methyloxiran-2-yl]-1 -oxopropan-2-yl]-3-hydroxy-3-(4-methoxyphenyl)-2-[(2S)-2-[2(morpholin-4-yl)acetamido]propanamido]propanamide (compound G)

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comprising (a) mixing a tertiary amine base and a suspension of (i) (2S,3R)-3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-(2-morpholinoacetamido)-propanamido)propanoic acid (compound E)

(ii) ^)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methyloxiran-2-yl)-1-oxopropan-2-aminium salt (compound F)

where X ^- represents a counterion;

in an aprotic solvent to form a mixture; and (b) mixing the condensing agent and the mixture of step (a) to form compound G;

wherein the temperature of each mixing step is maintained at -20 to 25°C.

In some embodiments, X ^- is selected from the group consisting of tosylate, triflate, acetate, naphthalenesulfonate, 4-nitrobenzenesulfonate, sulfate, methyl sulfate, nitrate, fluoride, chloride, bromide, and combinations thereof. In some cases X ^- is a tosylate, naphthalenesulfonate or 4-nitrobenzenesulfonate. For example, X ^- is a tosylate.

In various embodiments, the aprotic solvent is selected from the group consisting of acetonitrile (ACN), dichloromethane (DCM), tetrahydrofuran (THF), dimethylacetamide (DMAc), ethyl acetate (EtOAc), isopropyl acetate (iPrOAc), dimethylformamide (DMF), and combinations thereof. For example, the aprotic solvent may be DCM.

In some instances, the tertiary amine base is selected from the group consisting of N,Ndiisopropylethylamine (DIPEA), triethylamine (TEA), N-methylmorpholine (NMM), 2,2,6,6tetramethylpiperidine (TMP), 2,4,6-trimethylpyridine ( collidine) and combinations thereof. For example, a tertiary amine base may include DIPEA. In various cases, the molar ratio of tertiary amine base to compound E ranges from 1:1 to 4:1.

In some embodiments, the condensing agent includes a carbodiimide reagent, a phosphonium reagent, an uronium reagent, an immonium reagent, an imidazolium reagent, an organophosphorus reagent, an acid chloride reagent, a chloroformate reagent, or a pyridinium reagent. In various embodiments, the uronium reagent is selected from the group 1-[bis(dimethylamino)methylene]-1H1,2,3-triazol[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), )-N,N,N',N'tetramethyluronium hexafluorophosphate (HBTU) and combinations thereof. For example, the uronium reagent may be HATU. In some cases, the molar ratio of the condensing agent to compound E is 1 to 1. The condensing agent further comprises a condensing agent. In some embodiments, the condensing agent is selected from the group consisting of benzotriazole, dicarboximide, succinimide, and combinations thereof. In various embodiments, the condensing agent is selected from the group consisting of N-hydroxysuccinimide (HOSu), N-hydroxy-5-norbornene-2,3-dicarboximide (HONB), 1-hydroxybenzotriazole (HOBt), 6-chloro-1hydroxybenzotriazole (Cl -HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), and combinations thereof.

In various cases, the temperature of each mixing stage is maintained at -15 to 25°C. In some cases, the mixing in step (a) includes stirring the mixture for up to 10 minutes. In various embodiments, mixing in step (b) includes stirring for up to 2 hours. In some embodiments, Compound G is washed with one or more of the following: water, potassium phosphate monobasic, sodium bicarbonate, and sodium sulfate.

In various embodiments, compound E is prepared by mixing a reducing agent and benzyl (2S,3R)-3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-(2-morpholinoacetamido)propanamido)propanoate (compound D )

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to form compound E. In some cases, the reducing agent is selected from the group consisting of H2, Pd/C; H2, Pd(OH)2/C; Li; Na; lithium-4,4'-di-tert-butylbiphenyl (Li DTBBP) and combinations thereof. In some embodiments, the mixing of the reducing agent and compound D occurs under a nitrogen atmosphere. Mixing of the reducing agent and compound D can take place up to 4 hours. In addition, mixing can take place at a temperature in the range from 10 to 20°C. In various cases, obtaining compound E further includes one or more of the following steps: filtering compound E through diatomaceous earth; washing compound E; and crystallization of compound E with THF and water.

Additional aspects and advantages will be apparent to those skilled in the art from a review of the following detailed description. While the methods described herein are capable of being implemented in various forms, the description hereinafter includes specific embodiments with the understanding that the description is illustrative and does not limit the invention to the specific embodiments described herein.

Detailed description

This document describes a process for the preparation of (2S,3R)-N-[(2S)-3-(cyclopent-1-en-1-yl)-1-[(2R)-2methyloxiran-2-yl]-1-oxopropane -2-yl]-3-hydroxy-3-(4-methoxyphenyl)-2-[(2S)-2-[2-(morpholin-4yl)acetamido]propanamido]propanamide (compound G)

and in some cases the method is designed for large-scale preparation of compound G. The general scheme for obtaining compound G is shown in Scheme 1 below.

(G)

Scheme 1

The optical purity of compound G is controlled during synthesis by the quality of the starting materials and the specific reagents used for the transformations.

The compounds described herein may be identified either by their chemical structure and/or chemical name in this document. When a chemical structure and a chemical name conflict, the chemical structure is decisive for the identification of the compound.

Unless otherwise indicated, terms and abbreviations used in this specification include

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Since the contribution of the present description is not limited to the specific implementation options or aspects described in this document, the description provides a person skilled in the art with additional implementation options, including changes and modifications to adapt to different applications and conditions. For example, changes and modifications to materials, synthetic methods, or techniques described herein will be apparent to those skilled in the art.

The ranges used herein for physical properties such as molecular weight or chemical properties such as chemical formulas, all combinations and subcombinations of the ranges, and specific embodiments therein, are intended to be included.

Preparation of Compound G.

In one aspect, provided herein is a process for preparing Compound G. Compound G can be prepared in two steps: step (a) and step (b). In step (a), a mixture is formed by mixing a tertiary amine base and a suspension which includes (i) (2S,3R)-3-hydroxy-3-(4-methoxyphenyl)-2-(S)-2(2-morpholino -acetamido)propanamudo)propanoic acid (compound E)

(ii) (S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methyloxiran-2-yl)-1-oxopropan-2-aminium salt (compound F)

^X ° (F), where X ^- is a counterion in an aprotic solvent to form a mixture.

In step (b), the mixture from step (a) and the condensing agent are mixed together at a temperature in the range of about -20 to about 25° C. to form compound G.

The counterion (X ^- ) can be any anion capable of forming an ionic bond with the ammonium group of compound F. In some embodiments, X ^- is selected from the group consisting of tosylate, triflate, acetate, naphthalenesulfonate, 4-nitrobenzenesulfonate, sulfate, methyl sulfate, nitrate, fluoride, chloride, bromide and combinations thereof. In some cases X ^- may be tosylate, naphthalenesulfonate or 4-nitrobenzenesulfonate. For example, X ^- may be tosylate.

The aprotic solvent can be any aprotic solvent (or mixture of solvents) in which the nucleophilic acyl substitution reaction between compounds E and F can take place. Suitable aprotic solvents include acetonitrile (ACN), dichloromethane (DCM), tetrahydrofuran (THF), dimethylacetamide (DMAc ), ethyl acetate (EtOAc), isopropyl acetate (iPrOAc), dimethylformamide (DMF), and combinations thereof. In various embodiments, the aprotic solvent is selected from the group consisting of ACN, THF, DMF, and DCM. For example, an aprotic solvent may include DCM.

Compound E and compound F may be present in a molar ratio of 0.8:1 to 1.3:1. In some embodiments, Compounds E and F are present in a ratio of about 0.9:1 to 1.1:1. For example, the molar ratio of compounds E and F may be about 1:1, or in the range of 1:1.11 to 1:1.15.

The tertiary amine base may be any tertiary amine base that can activate or catalyze the nucleophilic acyl substitution reaction between compounds E and F. Suitable tertiary amine bases may include, for example, N,N-diisopropylethylamine (DIPEA), triethylamine (TEA), N -methylmorpholine (NMM), 2,2,6,6tetramethylpiperidine (TMP), 2,4,6-trimethylpyridine (collidine) and combinations thereof. For example, a tertiary amine base may include DIPEA. The tertiary amine base may be present in a molar ratio to compound E ranging from about 1:1 to about 4:1. In some embodiments, the tertiary amine base and Compound E are present in a ratio of about 2.5:1 to 4:1, or 2.5:1 to 3.5:1. For example, the ratio of tertiary amine base to compound E may be about 3.5:1 or 3.9:1.

The condensing agent may include, for example, a carbodiimide reagent, a phosphonium reagent, a uronium reagent, an immonium reagent, an imidazolium reagent, an organophosphorus reagent, an acid chloride reagent, a chloroformate reagent, a pyridinium reagent, or combinations thereof. See, for example, Han & Kim, Tetrahedron Report, 60:2447-2467 (2004); Montalbetti and Falque, Tetrahedron, 61:10827-10852 (2005). The carbodiimide may include, for example, N,N'-dicyclohexylcarbodiimide (DCC),

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1,3-diisopropylcarbodiimide (DIC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), or isopropylcarbodiimide (CIC) and combinations thereof. The phosphonium agent may include, for example, (benzotriazol1-yl-oxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP) or benzotriazol-1-yloxytripyrrolidinephosphonium hexafluorophosphate (PyBOP) and combinations thereof. The uronium agent may include, for example, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazol[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), O-(benzotriazol-1-yl) -N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) and combinations thereof. For example, a ronium agent might include HATU. The imidazolium agent may include, for example, 1,1'-carbonyldiimidazole (CDI).

The acid chloride agent includes, for example, pivaloyl chloride, 2,4,6-trimethylbenzoyl chloride, and combinations thereof. The chloroformate agent may include, for example, ethyl chloroformate, isobutyl chloroformate, and combinations thereof. The condensing agent may be present in a molar ratio to compound E ranging from about 0.8:1 to about 1:5. In some embodiments, the condensing agent and Compound E are present in a ratio of 0.9:1 to 1.1:1. For example, the ratio of condensing agent to compound E may be about 1:1 or 1.11:1.

The condensation reaction can be carried out in the presence of a condensing additive. Condensing additives are known in the art, and any suitable additive can be used to form Compound G. Suitable condensing additives include, for example, benzotriazoles, dicarboximides, and succinimides. In some embodiments, the condensing additives are selected from the group consisting of N-hydroxysuccinimide (HOSu), N-hydroxy-5-norbornene-2,3-dicarboximide (HONB), 1-hydroxybenzotriazole (HOBt), 6-chloro-1-hydroxybenzotriazole (Cl -HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), and combinations thereof. For example, the condensing agent may include HOBt.

The temperature of each stage of mixing is maintained in the range from about -20°C to about 25°C. In some embodiments, the temperature of each mixing stage is maintained in the range of about -15°C to about 25°C. In some cases, the temperature of each stage of mixing is maintained in the range from about -5°C to about 15°C. For example, the temperature of each mixing stage may be maintained in the range of about -5°C to about 5. The temperature of each mixing stage may be the same or different.

In step (a) of preparing compound G, mixing may occur for up to about 30 minutes (e.g., up to about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 16, 18 , 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 minutes). In some embodiments, mixing in step (a) may occur for about 10 minutes. In some instances, mixing in step (a) may occur for at least about 30 seconds, or at least about 1 minute (e.g., at least about 2, 3, 4, 5, 6, 7, 8, or 9 minutes) . For example, mixing in step (a) may occur for about 30 seconds to about 30 minutes, or about 1 minute to about 20 minutes, or about 2 minutes to about 15 minutes, or about 5 minutes to about 10 minutes. .

In step (b) of preparing Compound G, mixing may occur for up to about 3 hours (eg, up to about 1, 1.5, 2, 2.5, or 3 hours). In some embodiments, the mixing in step (b) may occur for about 2 hours. In some cases, the mixing in step (b) may occur for at least about 30 minutes, or at least about 1 hour, or at least about 1.5 hours. For example, the mixing in step (b) may occur for about 30 minutes to about 3 hours, or from about 30 minutes to about 2.5 hours, or from about 1 hour to about 2 hours.

In step (b), the coupling reaction may take place under a nitrogen atmosphere. In some cases, the condensation reaction does not take place under a nitrogen atmosphere.

After step (b) compound G can be washed with one or more solvents. The temperature during washing may optionally range from about 0°C to about 25°C, or from about 15°C to about 25°C. Suitable washing solvents include, for example, water, potassium phosphate monobasic, sodium bicarbonate, sodium sulfate, and combinations thereof. In some embodiments, water is added to compound G after step (b) and the resulting biphasic mixture is separated into an aqueous layer and an organic layer before washing. In various cases, Compound G can be washed with water, potassium phosphate monobasic, sodium bicarbonate and sodium sulfate.

For example, compound G can be prepared by (a) mixing compound E and compound F (1:1 molar ratio) in DCM for up to about 10 minutes to form a mixture, and (b) mixing the mixture from step (a) and about 1 molar equivalent of HATU for up to about 2 hours under nitrogen, where the temperature of each stage is in the range from about -20°C to about 25°C or from about -20 to 0°C. The resulting mixture can be quenched with water to obtain a two-phase mixture. The organic layer can be separated, washed with water, then with monobasic potassium phosphate, then with sodium bicarbonate and sodium sulfate (in succession).

Preparation of compound E.

Compound E can be prepared by mixing a reducing agent and benzyl (2S,3R)-3-hydroxy3-(4-methoxyphenyl)-2-((S)-2-(2-morpholinoacetamido)propanamido)propanoate (compound D)

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The reducing agent may be any suitable agent capable of removing the benzyl group in compound D to form the carboxylic acid of compound E. Suitable reducing agents include, for example, H2 in the presence of Pd/C or Pd(OH)2/C; Li; Na; lithium-4,4'-di-tert-butylbiphenyl (Li DTBBP) and combinations thereof. For example, the reducing agent may be H2 in the presence of Pd/C.

The mixing of the reducing agent and compound D can take place in any solvent capable of allowing the reduction reaction to proceed. For example, the solvent may include THF, methanol, or a combination thereof.

In some embodiments, Compound D is placed under a nitrogen atmosphere prior to being exposed to a hydrogen atmosphere. In various embodiments, the hydrogen atmosphere is set at about 15 psi.

Mixing of the reducing agent and Compound D may occur over a period of time of at least 30 minutes to about 5 hours (eg, up to about 2, 2.5, 3, 3.5, 4, or 4.5 hours). In some embodiments, mixing may occur for about 4 hours. 2, or 2.5, or 3, or 3.5 hours). For example, mixing may occur for about 30 minutes to about 5 hours, or about 1 hour to about 4 hours, or about 2 hours to about 4 hours.

The mixing temperature is maintained in the range from about 10°C to about 20°C. In some embodiments, the temperature is maintained at about 17°C.

In some cases, after mixing is complete, Compound E is filtered, for example, through diatomaceous earth (ie, diatomaceous earth). The resulting filtrate may be subsequently washed with a suitable solvent (eg, water, methanol, water, and combinations thereof).

Compound E (with or without washing) can be crystallized to form a polymorph characterized by the differential scanning calorimetry (DSC) thermogram, thermogravimetric analysis (TGA) data, and X-ray diffraction (XRPD) pattern depicted in FIG. 2, 3 and 4, respectively. For example, compound E can crystallize in THF and water when compound E is heated to a temperature in the range of about 50°C to about 70°C, or about 60°C to about 70°C, or about 55°C to about 65°C and then cooling to a temperature of about 0°C. Thus, another aspect of the present disclosure is the crystalline form of compound E, which is characterized by an x-ray pattern including peaks at 6.2, 8.5, 9.7, 12.7, 13.7, 16.0, 16.9, 17, 2, 18.4, 18.9, 19.2, 19.7, 22.5, 24.7, 25.4, 28.7 and 29.7 ± 0.2° 2θ using Cu Ka radiation as shown in FIG. 4.

For example, compound E can be prepared by mixing together a reducing agent such as H2 in the presence of Pd/C and compound D under a nitrogen atmosphere, at a temperature of 10 to 20°C, for a period of time of at least 30 minutes to 4 hours. Compound E can be filtered through diatomaceous earth and the resulting filter cake can be washed (eg with water, methanol and/or THF). Compound E can be crystallized by heating it to a temperature of about 60 to 70°C, bringing the temperature to a temperature of about 55 to 65°C and adding THF to the mixture, heating the mixture to a temperature of 60 to 70°C, adding water to the heated mixture while cooling the mixture to a temperature of 55 to 65°C, adding a seed crystal to the mixture and stirring the seeded mixture for about 2 hours at 0°C. Filtration, washing and drying the cooled mixture leads to the crystallization of compound E.

Examples

The following examples are provided for illustration and are not intended to limit the scope of the invention.

General synthesis scheme.

Compound G can be prepared according to Scheme 1 shown above.

Example 1 Large scale preparation of the HCl salt of (2S,3R)-1-(benzyloxy)-3-hydroxy-3-(4methoxyphenyl)-1-oxopropane-2-aminium salt (compound B).

Oh (W)

Ethyl acetate (58.5 kg) at 20° C. was saturated with gaseous HCl (6.8 kg). Benzyl(2S,3R)-2((tert-butoxycarbonyl)amino)-3-hydroxy-3-(4-methoxyphenyl)propanoate (compound A) was dissolved in this solution

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(5 kg, 12.5 mol, previously dissolved in 32.5 kg of ethyl acetate). The suspension was stirred at 20° C. and after completion as determined by HPLC, filtered and dried in vacuo at 45° C. to give the crystalline polymorph of Compound B (3.85 kg) as the HCl salt. LC/MS (HPMS(MH) m/z: 302). HPLC purity 97.9%. The DSC characteristic curve is shown in FIG. 1.

Example 2 Small scale synthesis of TFA salt (2S,3R)-1-(benzyloxy)-3-hydroxy-3-(4methoxyphenyl)-1-oxopropane-2-aminium salt (compound B).

Trifluoroacetic acid (TFA) (20 ml) was added to a solution of compound A (7.0 g, 17.4 mmol) in dichloromethane (DCM) (50 ml) at 0°C. The mixture was stirred for 30 minutes, then diluted with DCM (100 ml). Saturated NaHCO ₃ (aqueous, 100 ml) was added and the two layers were separated. The aqueous layer was extracted with DCM (2x100 ml) and the combined organic layers were dried over anhydrous sodium sulfate, then concentrated to give crude Compound B (5.0 g, 84% yield) as the TFA salt. LC/MS (HPMS(MH) m/z: 302.

Example 3 Large scale preparation of (2S,3R)-3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-(2morpholinoacetamido)propanamido)propanoate (compound D).

To compound B (3.8 kg) and (2-morpholinoacetyl)-L-alanine (compound C)

(2.5 kg) at 20°C was added acetonitrile (30.4 kg). The temperature was brought to 0°C and N,N-diisopropylethylamine (DIPEA) (3.19 kg) was added followed by 1-[bis(dimethylamino)methylene]-1H-1,2,3triazole[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) (5.22 kg) in batches over 30 minutes. The reaction mixture was stirred for 2 h at 0° C., then quenched with 3.5% NaHCO ₃ (aqueous, 46 kg) and stirred for 30 min. After 1 h at 20°C, solid NaHCO ₃ was added and the mixture was stirred for 30 min, then held at 20°C again for 1 h. The aqueous layer was diluted with water (30.6 kg), extracted with isopropyl acetate (iPrOAc) (23.4 kg) and the organic layers were combined. The organic layers were displaced by iPrOAc (3x27 kg), washed with 3.5% NaHCO ₃ (aqueous, 30 kg), KH2PO4 (aqueous, 3x65 kg), water (15 kg), 7% NaHCO ₃ (aqueous, 2x61 kg) and 5% Na ₂ SO ₄ (water, 3x55 kg). The solution was concentrated to 18 L, then displaced with tetrahydrofuran (THF) (4 x 22.8 L) to give the product (5.04 kg, 90% yield, 97.9% HPLC purity) as a solution in THF (34.5 wt. %, total 14.6 kg).

Similar results were obtained using 1-ethyl-3-(3dimethylaminopropyl)carbodiimide HCl (EDC) (1.1 eq.) instead of HATU as the condensing agent.

Example 4 Small Scale Synthesis of Compound D

The reagents HATU (6.79 g, 17.9 mmol) and DIPEA (9.63 ml, 59.2 mmol) were added to a solution of Compound B (TFA salt, 5.0 g, 14.8 mmol) and Compound C (3 .36 g, 15.9 mmol) in dimethylformamide (DMF) (100 ml) at 0°C. The reaction mixture was allowed to warm to room temperature and stirred for 1 h. The mixture was concentrated and the residue was purified by silica gel flash column chromatography (petroleum ether/EtOAc=2:1 to 1:2) to give compound D (5.8 g, 78% yield) as a colorless solid. LC/MS (HPMS(MH) m/z: 500.

Example 5 Large scale preparation of (2S,3R)-3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-(2morpholinoacetamido)propanamido)propanoic acid (compound E).

benzyl(2S,3R)-3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-(2 morpholinoacetamido)propanamido)propanoate solution (compound D) (5.04 kg as 34.5 wt.% solution in

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THF) and THF (3.25 kg) was added followed by methanol (7.0 kg). The interior of the reaction vessel was vented to N ₂ and Pd/C (10%, 473 g) was added under nitrogen. Added THF (500 g) and methanol (1 kg) to wash the reaction vessel and create an atmosphere of H ₂ (15 psi). The reaction mixture was stirred for 4 h at 17°C, then filtered through diatomaceous earth. The wet cake was washed with methanol (30 kg), concentrated to 3-4 volumes, displaced THF (4x45 kg) and heated to 60-70°C. After 2 hours the temperature was brought to 50-60° C. and THF (30 kg) was added. The mixture was again heated to 60-70°C for 2 hours. To this solution was added water (370 kg) at 60-70°C, then the mixture was cooled to 55-65°C. Seed crystals (18.0 g) were added and the mixture was stirred at 55-65°C for 1 hour. Twice the suspension was concentrated to 5-6 volumes and stirred for 2 hours at 0°C. The mixture was filtered using THF (10 kg) for washing. The wet cake was dried to give the crystalline polymorph of Compound E (3.54 kg, 97.6% purity). Characteristic data for DSC, TGA and XRPD are shown in FIG. 2-4.

Example 6 Small Scale Synthesis of Compound E

To a solution of compound D (5.8 g, 11.6 mol) in THF (120 ml) was added Pd/C (1.5 g, 10%). The mixture was stirred under H ₂ (1 atm) at room temperature overnight, then filtered through a pad of celite. The filtrate was concentrated under reduced pressure and the residue was washed with EtOAc (20 ml) to give compound E (4.8 g, ~100% yield) as a colorless solid.

Example 7 Large scale preparation of (S)-3-(cyclopent-1-en-1-yl)-1-((R)-2methyloxiran-2-yl)-1-oxopropan-2-aminium salt tosylate salt (compound F ).

D) X \<° _H ₃ N^γ^ ^{X +} ° (F)

To a solution of tert-butyl ((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methyloxiran-2-yl)-1-oxopropan-2yl)carbamate (compound H ).

(134 g) in DCM (402 ml) at 0°C was added TFA (414.3 g, 8 eq.) at a rate maintaining the internal temperature at -5-5°C. The reaction mixture was stirred for 2 hours at this temperature under N ₂ atmosphere. The dark mixture was then concentrated to remove DCM and TFA at 15-25°C. The solution was displaced with methyl tert-butyl ether (MBTE) (5x2 L). HPLC analysis showed that 2.72 eq. TFA and MTBE (804 ml) was added at 15-25°C. To this solution was added p-toluenesulfonic acid (PTSA) (83.6 g) at 0°C and the mixture was stirred at this temperature for 10-12 h. ml) and the filter cake was dried in vacuo at 15-25° C. for 16-18 hours to give Compound F (126 g, 99.4% purity) as the tosylate salt. Characteristic curves for DSC and TGA are shown in FIG. 5 and 6 and the XRPD characteristic curve is shown in FIG. 9.

Example 8 Small Scale Synthesis of the Naphthalenesulfonate Salt of Compound F

DCM (8 ml) was added to compound H (2 g) and the mixture was cooled to 5°C. TFA (8 ml) was added at a rate that kept the internal temperature below 10°C. The mixture was then stirred at ambient temperature for 30 minutes, then concentrated in vacuo. Toluene (3x5 ml) was added to remove excess TFA. EtOAc (4 ml) was added to the TFA salt followed by 2-naphthalenesulfonic acid (6.78 mmol, 1.41 g, dissolved in 10 ml EtOAc). The mixture was stirred at room temperature and a colorless solid precipitated over 5 minutes. The mixture was stirred for another 15 min, then filtered using EtOAc (10 ml) for washing. The solid was placed under vacuum for 16 hours to give the naphthalenesulfonate salt as a colorless solid (1.62 g, 72% yield). Characteristic curves for DSC and TGA are shown in FIG. 7 and 8.

A similar procedure was used to prepare 4-nitrobenzenesulfonic acid of compound F.

Example 9 Large Scale Production of (2S,3R)-N-[(2S)-3-(cyclopent-1-en-1-yl)-1-[(2R)-2methyloxiran-2-yl]-1-oxopropan- 2 -yl]-3-hydroxy-3-(4-methoxyphenyl)-2-[(2S)-2-[2-(morpholin-4yl)acetamido]propanamido]propanamide (compound G).

- 8 041535

DCM (1.46 kg) was added to compound E (110.0 g) and compound F (tosylate salt, 110.0 g) and the suspension was cooled from -15 to 5°C. DIPEA (122.1 g) was added at a rate maintaining an internal temperature of -15 to -5°C. The mixture was then stirred for 10 minutes and HATU (114.4 g) was added to this solution at -15 to -5°C under nitrogen. The mixture was stirred for 2 h at -15 to -5° C. and Compound F (3.91 g) was added. After 10 minutes, the internal temperature was brought to 5-15° C., then water (1100 g) was added. The solution was stirred for 30-60 min and held for 30-60 min. The organic layer was separated and washed with water (1100 g) for 30-60 minutes and the mixture was kept for 30-60 minutes. The organic phases were combined and the temperature was raised to 15-25°C. The solution was concentrated to 2-4 volumes in vacuo (<45°C). Added iPrOAc (957 g) and the solution was concentrated to 2-4 volumes in vacuum (<45°C). The solution was washed with 5% KH2PO4 (aq. 1100 g), 1% KH2PO4 (aq. 2x1100 g), 7% NaHCO ₃ (aq. 1100 g) and 5% Na ₂ SO ₄ (aq. 1100 g). The product was obtained as a 10.53 wt% solution in iPrOAc (89.8%).

Example 10 Small-Scale Production of Compound G

HATU (5.35 g, 14.1 mmol) and DIPEA (9.55 ml, 58.7 mmol) were added to a solution of compound E (4.8 g, 11.7 mmol) and compound F (TFA salt, 3, 46 g, 11.7 mmol) in DMF (90 ml) at 0°C. The reaction mixture was allowed to warm to room temperature and stirred for 30 minutes. The mixture was concentrated and the residue was purified by silica gel flash column chromatography (petroleum ether/EtOAc=2:1 to EtOAc) to give Compound G (4.8 g, 70% yield, 95.2% purity by HPLC) as a colorless solid substances. LC/MS (HPMS(MH) m/z: 587).

The above description is provided for clarity of understanding only, and is not intended to be undue restrictive, as various modifications may be understood by those skilled in the art within the scope of the present invention.

In this description and in the claims that follow, unless the context otherwise requires, it is to be understood that the word contains and variants such as contain and containing are intended to include the specified integer or step or group of integers or steps, but not the exclusion of any another integer or stage or group of integers or stages.

In this specification, when compositions are described as containing components or materials, it is contemplated that said compositions may also consist essentially of, or consist of, any combination of said components or materials, unless otherwise indicated. Similarly, if the methods are described as including certain steps, it is contemplated that said methods may also consist essentially of, or consist of, any combination of said steps, unless otherwise indicated. The invention illustratively described herein can be properly practiced in the absence of any element or step that is not described in this document.

The practical implementation of the method described in this document, and its individual stages can be implemented manually and/or using automatic devices provided by electronic equipment. While the methods are described with reference to specific implementations, those skilled in the art will recognize that other methods of implementing the actions associated with said methods may be used. For example, the order of the various steps may be changed without departing from the scope or spirit of said method, unless otherwise described. In addition, some of the individual steps may be combined, omitted, or further subdivided into further steps.

All patents, publications and references cited in this document are incorporated herein by reference in their entirety. In the event of any conflict between the present disclosure and the incorporated patents, publications, and references, the present disclosure shall govern.

Claims (15)

CLAIM 1. Method for obtaining (2S,3R)-N-[(2S)-3-(cyclonent-1-en-1-yl)-1-[(2R)-2-methyloxiran-2-yl]-1oxopropan-2 -yl]-3-hydroxy-3-(4-methoxyphenyl)-2-[(2S)-2-[2-(morpholin-4yl)acetamido]propanamido]propanamide (compound G) - 9 041535 comprising (a) mixing a tertiary amine base and a suspension of (i) (2S,3R)-3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-(2-morpholinoacetamido)propanamido)propanoic acid ( compound E) About 9 I H? ⁿ SI ^ΗΟ II Ί ^^OMe (e) _and (ii) (S)-3-(cyclopent-1-en-1-yl)-1 -((R)-2-methyloxiran-2-yl)-1 -oxopropane-2-aminium salt (compound F) D 4.0 _H ₃ N^γ^ ^{x +} O(E)^ where X- is the counterion, in an aprotic solvent to form a mixture; and (b) mixing the condensing agent and the mixture of step (a) to form compound G, the temperature of each mixing step being maintained at -20 to 25°C. 2. The method of claim 1 wherein X- is selected from the group consisting of tosylate, triflate, acetate, naphthalenesulfonate, 4-nitrobenzenesulfonate, sulfate, methyl sulfate, nitrate, fluoride, chloride, bromide, and combinations thereof. 3. The method according to claim 1 or 2, characterized in that the aprotic solvent is selected from the group consisting of acetonitrile (ACN), dichloromethane (DCM), tetrahydrofuran (THF), dimethylacetamide (DMAc), ethyl acetate (EtOAc), isopropyl acetate (iPrOAc ), dimethylformamide (DMF), and combinations thereof. 4. Process according to any one of claims 1 to 3, characterized in that the tertiary amine base is selected from the group consisting of N,N-diisopropylethylamine (DIPEA), triethylamine (TEA), N-methylmorpholine (NMM), 2,2, 6,6-tetramethylpiperidine (TMP), 2,4,6-trimethylpyridine (collidine) and combinations thereof. 5. Process according to any one of claims 1 to 4, characterized in that the molar ratio of tertiary amine base to compound E is in the range from 1:1 to 4:1. 6. The method according to any one of claims 1 to 5, characterized in that the condensing agent comprises a carbodiimide reagent, a phosphonium reagent, an uronium reagent, an immonium reagent, an imidazolium reagent, an organophosphorus reagent, an acid chloride reagent, a chloroformate reagent, or a pyridinium reagent. 7. The method according to any one of claims 1 to 6, characterized in that the molar ratio of the condensing agent to the compound E is 1 to 1. 8. The method according to any one of claims 1 to 7, characterized in that the condensing agent further comprises a condensing additive. 9. The method of claim 8, wherein the condensing agent is selected from the group consisting of benzotriazole, dicarboximide, succinimide, and combinations thereof. 10. Process according to any one of claims 1 to 9, characterized in that the temperature of each mixing step is maintained at -15 to 25°C. 11. Method according to any one of claims 1 to 10, characterized in that the mixing in step (a) comprises stirring the mixture for up to 10 minutes. 12. The method according to any one of claims 1 to 11, characterized in that the mixing in step (b) includes stirring for up to 2 hours. 13. The method according to any one of claims 1 to 12, further comprising washing the compound G with one or more of the following components: water, potassium phosphate monobasic, sodium bicarbonate and sodium sulfate. 14. The method according to any one of claims 1 to 13, characterized in that the compound E is obtained by mixing the reducing agent and benzyl-(2S,3R)-3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2 -(2- - 10 041535 morpholinoacetamido)propanamido)propanoate (compound D) to form E. 15. The method according to claim 14, characterized in that the reducing agent is selected from the group consisting of H2,