PREPARATION PROCESS OF THE EPOXYKETONE PEPTIDE IMMUNOPROTEASOME INHIBITOR, AND ITS PRECURSORS

Abstract

The present methods are described for preparing [(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-(morpholin-4-yl)acetamido] propanamido]propanamide (compound “G”) of formula (1) and its precursors (compound “E” and compound “F”) of formula (2) and (3) respectively.

Description

Descriptive Report of the Patent of Invention About PREPARATION PROCESS OF THE EPOXYKETONE PEPTIDE IMMUNOPROTEASOME INHIBITOR, AND ITS PRECURSORS 232001 JJB / jcand Requested by: KEZAR LIFE SCIENCES, located at 300 Utah Ave. Suite 105, South San Francisco. California EU OF AMERICA PREPARATION PROCESS OF THE EPOXYKETONE PEPTIDE IMMUNOPROTEASOME INHIBITOR, AND ITS PRECURSORS BACKGROUND TECHNICAL FIELD The description refers to methods and processes for the preparation of (2S,3F?)- / V-[(2S)-3(cyclopent-1-en-1-yl)-1-[(2 / ?)-2-methyloxiran-2-yl]-1-oxopropan-2-yl]-3-hydroxy-3-(4-methoxyphenyl)-2-[(2S)-2-[2-(morpholin-4-yl)acetamido]propanamido]propanamide, and its precursors. DESCRIPTION OF RELATED TECHNOLOGY The compound, (2S,3R)-A / -[(2S)-3-(cyclopent-1-en-1-yl)-1-[(2 / ?)-2-methyloxiran-2-yl]-1oxopropan-2-yl]-3-hydroxy-3-(4-methoxyphenyl)-2-[(2S)-2-[2-(morpholin-4-1)acetamido]propanamido]propanamide (“compound G”), is useful as an immunoproteasome inhibitor: O HO' OMe (G) In eukaryotes, protein degradation is predominantly mediated through the ubiquitin pathway, in which proteins targeted for destruction are linked to the 76-amino-acid polypeptide ubiquitin. Once targeted, ubiquitinated proteins then serve as substrates for the 26S proteasome, a multicatalytic protease that cleaves proteins into short peptides through the action of its three main proteolytic activities. Although it has a general function in intracellular protein turnover, 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-κB activation, antigen processing, and proinflammatory signal transduction. The 20S proteasome is a cylindrical, 700 kDa multicatalytic protease complex composed of 28 subunits arranged in four rings. 232001 L· In yeasts and other eukaryotes, seven different β-subunits form the outer rings, and seven different β-subunits comprise the inner rings. The α-subunits serve as binding sites for the 19S (PA700) and 1IS (PA28) regulatory complexes, as well as a physical barrier to the internal proteolytic chamber formed by the two β-subunit rings. Consequently, in vivo, the proteasome is considered to exist as a 26S particle (the 26S proteasome). In vivo experiments have shown that inhibition of the 20S form of the proteasome can be readily correlated with inhibition of the 26S proteasome. Cleavage of amino-terminal prosequences of the β-subunits during particle formation exposes the amino-terminal threonine residues, which serve as catalytic nucleophiles.The subunits responsible for catalytic activity in proteasomes thus possess an amino-terminal nucleophilic residue, and these subunits belong to the family of N-terminal nucleophilic hydrolases (where the N-terminal nucleophilic residue is, for example, Cys, Ser, Thr, and other nucleophilic residues). This family includes, for example, penicillin G acylase (PGA), penicillin V acylase (PVA), glutamine PRPP amidotransferase (GAT), and bacterial glycosylparaginase. In addition to the ubiquitously expressed β subunits, higher vertebrates also possess three interferon-γ-inducible β subunits (LMP7, LMP2, and MECL1), which replace their normal counterparts, B5, B1, and B7, respectively, thereby altering the catalytic activities of the proteasome.Through the use of different peptide substrates, three main proteolytic activities have been defined for the 20S eukaryotic proteasome: chymotrypsin-like (CT-L) activity, which cleaves after large hydrophobic residues; trypsin-like (TL) activity, which cleaves after basic residues; and peptidylglutamyl peptide hydrolysis (PGPH) activity, which cleaves after acidic residues. Two other, 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 conferred by different catalytic sites, since inhibitors, point mutations in the β subunits, and the exchange of interferon-adducting β subunits alter these activities to varying degrees. The publication PCT N.WO2014 / 152134, which is incorporated herein by reference, describes epoxy tripeptide immunoproteasome inhibitors, such as Compound G, and methods for their small-scale synthesis. However, large-scale synthesis of epoxy tripeptide immunoproteasome inhibitors, such as Compound G, is required for commercial development. SUMMARY OF THE INVENTION In one aspect, the description provides a method of preparation (2S,3 / ?)- / V-[(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-(morpholin-4-yl)acetamido]propanamido]propanamide (compound “G”) which includes: (a) mix a tertiary amine base and a suspension of: (i) (2S,3 / ?)-3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-(2-morpholinoacetamido)propanamido)propanoic acid (compound Έ”): u HO' OMe(E), and (i) (S)-3-(cyclopent-1-en-1-yl)-1-((F?)-2-methyloxiran-2-yl)-1-oxopropan2-amino salt (compound “F”): Γ) X \<° _h3n^Y^ x 0 (F), where X' is a counterion; in an aprotic solvent to form a mixture; and (b) mixing a coupling agent and the mixture from step (a) to form compound G; where the temperature of each mixing stage is maintained at -20 °C 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 tosylate, naphthalenesulfonate, or 4-nitrobenzenesulfonate. For example, X' is tosylate. In several embodiments, the aprotic solvent is selected from the group consisting of acetonitrile (“AON”), dichloromethane (“DCM”), tetrahydrofuran (“THF”), dimethylacetamide (“DMAc”), ethyl acetate (“EtOAc”), isopropyl acetate (“IpOAc”), dimethylformamide (“DMF”), and combinations thereof. For example, the aprotic solvent may be DCM. In some cases, the tertiary amine base is selected from the group consisting of β-,β-diisopropylethylamine (“DIPEA”), triethylamine (“TEA”), β-methylmorpholine (“NMM”), 2,2,6,6-tetramethylpiperidine (“TMP”), 2,4,6-trimethylpyridine (“collidine”), and combinations thereof. For example, the tertiary amine base may include DIPEA. In several cases, the molar ratio of the tertiary amine base to compound E ranges from 1:1 to 4:1. In some embodiments, the coupling agent comprises a carbodiimide reagent, a phosphonium reagent, a uronium reagent, an iminium reagent, an imidazolium reagent, an organophosphorus reagent, an acid chloride reagent, a chloroformate reagent, or a pyridinium reagent. In several embodiments, the uronium reagent is selected from the group 1[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium hexafluorophosphate (“HATII”), O-(Benzotriazol-1-yl)-tetramethyluronium hexafluorophosphate (“HBTII”), and combinations thereof. For example, the uronium reagent may be HATU. In some cases, the molar ratio of the coupling agent to compound E is 1:1. The coupling reagent further comprises a coupling additive. In some embodiments, the coupling additive is selected from the group consisting of a benzotriazole, a dicarboximide, a succinimide, and combinations thereof.In various embodiments, the coupling additive is selected from the group consisting of N-hydroxysuccinimide (“HOSu”), M-hydroxy-5-norbomene-2,3-dicarboximide (“HONB”), 1-hydroxybenzotriazole (“HOBt”), 6-chloro-1-hydroxybenzotriazole (“CI-HOBt”), 1-hydroxy-7-azabenzotriazole (“HOAt’j”), and combinations thereof. In several cases, the temperature of each mixing stage is maintained between -15 °C and 25 °C. In some cases, the mixing of stage (a) comprises stirring the mixture for up to 10 minutes. In several embodiments, the mixing of stage (b) comprises stirring for up to two hours. In some embodiments, compound G is washed with one or more of the following: water, monobasic potassium phosphate, sodium bicarbonate, and sodium sulfate. In several embodiments, compound E is prepared by mixing a reducing agent and (2S,3R)-3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-(2-i morpholinoacetamido)propanamido)benzyl propanoate (compound “D”) ? I II I H ? or A H0 il I ^^OMe (D), 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; 4,4-di-tert-butylbiphenyllithium (“Li DTBBP”), and combinations thereof. In some embodiments, the mixing of the reducing agent and compound D takes place under a nitrogen atmosphere. The mixing of the reducing agent and compound D can take place for up to 4 hours. Furthermore, the mixing can take place at a temperature in the range of 10 °C to 20 °C. In several cases, the preparation of compound E further includes one or more of the following: filtration of compound E through diatomaceous earth; washing of compound E; and crystallization of compound E with THF and water. Another aspect of the description provides a method for preparing the salt (S)-3(cyclopent-1-en-1-yl)-1-((F?)-2-methyloxiran-2-yl)-1-oxopropan-2-aminium (compound “F”) which includes: (a) mixing trifluoroacetic acid (“TFA”) and fer-butyl-((S)-3-(cyclopent-1-en-1 -yl)-1 ((F?)-2-methyloxiran-2-yl)-1-oxopropan-2-yl)carbamate (compound “H”): AAH 0 (H), in an aprotic solvent at a temperature in the range of -5 °C to 5 °C to form a mixture; (b) concentrating the mixture; and (c) mixing an acid and the concentrated mixture from step (b) at a temperature in the range of -5 °C to 5 °C to form compound F, where X' is a conjugate base of the acid. In some embodiments, the acid is selected from the group consisting of toluenesulfonic acid, trifluoromethanesulfonic acid, acetic acid, trifluoroacetic acid, naphthalenesulfonic acid, 4-nitrobenzenesulfonic acid, sulfonic acid, methylsulfonic acid, benzenesulfonic acid, nitric acid, HF, HCl, HBr, and combinations thereof. For example, the acid may be selected from the group consisting of toluenesulfonic acid, naphthalenesulfonic acid, 4-nitrobenzenesulfonic acid, and combinations thereof. In some cases, the molar ratio of the acid to compound H is 1:1. In several cases, the molar ratio of TFA to compound H is 8:1. In various embodiments, the aprotic solvent in step (a) is selected from the group consisting of acetonitrile (“AON”), dichloromethane (“DCM”), tetrahydrofuran (“THF”), dimethylacetamide (“DMAc”), methyl tert-butyl ether (“MTBE”), isopropyl ether (“IRE”), and combinations thereof. For example, the aprotic solvent may include DCM.In some cases, the temperature in step (a), step (c), or both is 0 °C. In several cases, the mixture in step (b) is concentrated at a temperature in the range of 15 °C to 25 °C. In several embodiments, the mixing in step (a) comprises stirring for 2 hours. In some cases, the mixing in step (c) comprises stirring for 10 to 12 hours. In some embodiments, the concentrated mixture in step (b) is also washed with a polar aprotic solvent at a temperature in the range of 15 °C to 25 °C. Suitable polar aprotic solvates include diethyl ether, tetrahydrofuran (“THF”), acetonitrile (“ACN”), methyl tert-butyl ether (“MBTE”), isopropyl ether (“IPE”), and combinations thereof. For example, the polar aprotic solvent may include MBTE. In some cases, the method also includes one or more of the following steps: filtration of compound F, washing of compound F with a polar aprotic solvent, and drying of compound F.The polar aprotic solvent for washing compound F can be selected from the group consisting of diethyl ether, tetrahydrofuran (“THF”), acetonitrile (“ACN”), methyl tert-butyl ether (“MBTE”), isopropyl ether (“IPE”), and combinations thereof. Yet another aspect of the description provides a method of preparation of (2S,3R)-3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-(2-morpholinoacetamido)propanamido)propanoate benzyl (compound “D”) OMe (D); which includes: (a) mix a tertiary amine base and a suspension of: salt of (2S,3F?)-1 -(benzyloxy)-3-hydroxy-3-(4-methoxyphenyl)-1 -oxopropan-2-aminium (compound “B”): MeO OH OBn x. h3n o (B), where X' is a counterion; and (i) (2-morpholinoacetyl)-L-alanine (compound “C”): EITHER OH u O (C), in an aprotic solvent to form a mixture; and (b) mixing the coupling agent and the mixture from step (a) to form compound D; where the temperature of each mixing stage is maintained at -5 °C to 5 °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. For example, X' may be chloride. In some cases, the aprotic solvent is selected from the group consisting of acetonitrile (“AON”), dichloromethane (“DCM”), tetrahydrofuran (“THF”), dimethylacetamide (“DMAc”), and combinations thereof. For example, the aprotic solvent may include ACN. In various embodiments, the tertiary amine base is selected from the group consisting of / V, / V-diisopropylethylamine (“DIPEA”), triethylamine (“TEA”), N-methylmorpholine (“NMM”), 2,2,6,6-tetramethylpiperidine (“TMP”), 2,4,6-trimethylpyridine (“collidine”), and combinations thereof. For example, the tertiary amine base may include DIPEA.In several cases, the coupling agent comprises a carbodiimide reagent, a phosphonium reagent, a uronium reagent, an iminium reagent, an imidazolium reagent, an organophosphorus reagent, an acid chloride reagent, a chloroformate reagent, or a pyridinium reagent. In some embodiments, the uronium reagent is selected from the group 1[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium hexafluorophosphate (“HATU”), O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (“HBTU”), and combinations thereof. For example, the uronium reagent may include HATU. In some embodiments, the molar ratio of the coupling agent to compound B is 1:1. In several embodiments, the coupling reagent further includes a coupling additive. The coupling additive can be selected from the group consisting of a benzotriazole, a dicarboximide, a succinimide, and combinations thereof.For example, the coupling reagent may be selected from the group consisting of β-hydroxysuccinimide (“HOSu”), α-hydroxy-5-norbornene-2,3-dicarboximide (“HONB”), 1-hydroxybenzotriazole (“HOBt”), 6-chloro-1-hydroxybenzotriazole (“CI-HOBt”), 1-hydroxy-7-azabenzotriazole (“HOAt”), and combinations thereof. In some cases, the temperature of each mixing step is maintained between -5 °C and 5 °C. In some embodiments, the mixing of step (b) comprises mixing portions of the coupling agent with the mixture from step (a) for 30 minutes. In several cases, the mixing of step (b) comprises stirring for 2 hours. In some embodiments, the method further includes washing compound D with one or more of the following: water, isopropyl acetate, monobasic potassium phosphate, sodium bicarbonate, sodium sulfate, and THF. Compound B can be prepared by mixing (i) an acid and (ii) benzyl (2S,3R)-2((tert-butoxycarbonyl)amino)-3-hydroxy-3-(4-methoxyphenyl)propanoate (compound “A”): MeO OH r, .... OBn BocHN or (A) in a polar aprotic solvent, to form compound B. In some embodiments, the acid is selected from the group consisting of p-toluenesulfonic acid, trifluoromethanesulfonic acid, acetic acid, trifluoroacetic acid, naphthalenesulfonic acid, 4-nitrobenzenesulfonic acid, sulfonic acid, methylsulfonic acid, nitric acid, HF, HCl, HBr, and combinations thereof. For example, the acid may include trifluoroacetic acid or HCl. In some embodiments, the polar aprotic solvent is selected from the group consisting of ethyl acetate, N-methylpyrrolidone (“NMP”), tetrahydrofuran (“THF”), acetone, dimethylformamide (“DMF”), acetonitrile (“ACN”), dimethyl sulfoxide (“DMSO”), dichloromethane (“DCM”), and combinations thereof. For example, the polar aprotic solvent may include ethyl acetate, DCM, or combinations thereof. In some cases, the mixing step includes stirring at a temperature ranging from 15 °C to 25 °C.The method may also include filtration of compound B, drying of compound B, or both. Another aspect of the description provides a crystalline form of the chloride salt of (2S,3R)-1-(benzyloxy)-3-hydroxy-3-(4-methoxyphenyl)-1-oxopropan-2-ammonium (compound “B9 Cl”) MeO OH OBn cr h3n O (B-CI), characterized by a powder X-ray diffraction pattern comprising peaks at 4.6, 9.2, 13.8, 18.5, and 32.9 ± 0.2° 2Θ using Cu Ka radiation. Yet another aspect of the description provides a crystalline form of (2S,3R)3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-(2-morpholinoacetamido)propanamido)propanoic acid (compound Έ”): characterized by a powder X-ray diffraction pattern comprising 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. Yet another aspect of the description provides a crystalline salt form of (S)-3(cyclopent-1-en-1-yl)-1-((R)-2-methyloxiran-2-yl)-1-oxopropan-2-aminium (compound “F”): where X' is tosylate, characterized by a powder X-ray diffraction pattern comprising peaks at 6.8, 7.1, 7.4, 14.2, 14.8, 17.0, 17.5, 17.8, 18.5, 18.7, 20.1, 20.3, 23.0, 23.6, 24.5, 29.3, and 31.2 ± 0.2° 2Θ using Cu Ka radiation. Other aspects and advantages will become apparent to those skilled in the art upon review of the following detailed description. Although the methods described herein are capable of various embodiments, the description hereafter will include specific embodiments, with the understanding that the description is illustrative and is not intended to limit the invention to the specific embodiments described herein. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 represents the characteristic differential scanning calorimetry (“DSC”) curve for the salt (2S,3R)-1-(benzyloxy)-3-hydroxy-3-(4-methoxyphenyl)-1-oxopropan-2-aminium (compound “B”). FIG. 2 represents the characteristic DSC thermogram of (2S,3R)-3-hydroxy-3(4-methoxyphenyl)-2-((S)-2-(2-morpholinoacetamido)propanamido)propanoic acid (compound FIG. 3 represents the characteristic thermogravimetric analysis (“TGA”) data for compound E. FIG. 4 represents the characteristic X-ray diffraction pattern (“XRPD”) for compound E. FIG. 5 represents the characteristic DSC thermogram for the tosylate salt of (S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methyloxiran-2-yl)-1-oxopropan-2-amino salt (compound F"). FIG. 6 represents the characteristic thermogravimetric analysis (“TGA”) data for the tosylate salt of compound F. FIG. 7 represents the characteristic DSC thermogram of the naphthalenesulfonic acid salt of compound F. FIG. 8 represents the characteristic thermogravimetric analysis (“TGA”) data of the naphthalenesulfonic acid salt of compound F. FIG. 9 represents the characteristic XRPD pattern for the tosylate salt of compound F. FIG. 10 represents a single-crystal X-ray diffraction (“XRD”) of the tosylate salt of compound F. DETAILED DESCRIPTION Described herein is a process for the preparation of (2S,3R)-A / -[(2S)-3(cyclopent-1 -en-1 -i I)-1 -[(2 / ?)-2-methyloxiran-2-yl]-1 -oxopropan-2-yl]-3-hydroxy-3-(4methoxyphenyl)-2-[(2S)-2-[2-(morpholin-4-yl)acetamido]propanamido]propanamide (compound “G”): O HO' OMe (G) and its precursors, and in some cases, the process is for the large-scale preparation of compound G. The overall scheme for the preparation of compound G is shown below in Scheme 1. Scheme 1. (F) 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 can be identified by their chemical structure and / or their chemical name. When the chemical structure and the chemical name conflict, the chemical structure determines the identity of the compound. Unless otherwise stated, the terms and abbreviations used in this descriptive memorandum include the normal and customary meaning for those of the mid-level trade. Since the contribution of this description is not limited to the specific embodiments or aspects described herein, it provides a person skilled in the art with additional embodiments, including changes and modifications to suit various uses and conditions. For example, the changes and modifications to materials, synthesis methods, or procedures described herein will be obvious to a person of average skill. When ranges are used herein for physical properties, such as molecular weight, or chemical properties, such as chemical formulas, they are understood to include all combinations and subcombinations of specific ranges and embodiments. Preparation of Compound G In one aspect, a method for preparing compound G is provided herein. Compound G can be prepared in two steps—step (a) and step (b). In step (a), a mixture is formed by mixing together a tertiary amine base and a suspension that includes: (i) (2S,3R)-3-hydroxy-3-(4-methoxyphenyl)-2((S)-2-(2-morpholinoacetamido)propanamido)propanoic acid (compound “E”): (i) (S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methyloxiran-2-yl)-1-oxopropan-2-amino salt (compound “F”): where X' is a counterion, in an aprotic solvent to form a mixture. In step (b), the mixture from step (a) and a coupling agent are mixed together at a temperature in the range of approximately -20 °C to approximately 25 °C to form compound G. The counterion (Xj) 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 can be tosylate, naphthalenesulfonate, or 4-nitrobenzenesulfonate. For example, X' can be tosylate. The aprotic solvent can be any aprotic solvent (or solvent mixture) in which the nucleophilic acyl substitution reaction between compounds E and F can proceed. Suitable aprotic solvents include acetonitrile (“AON”), dichloromethane (“DCM”), tetrahydrofuran (“THF”), dimethylacetamide (“DMAc”), ethyl acetate (“EtOAc”), isopropyl acetate (“iPrOAc”), dimethylformamide (“DMF”), and combinations thereof. In several embodiments, the aprotic solvent is selected from the group consisting of AON, THF, DMF, and DCM. For example, the aprotic solvent may include DCM. Compound E and compound F may be present in a molar ratio of approximately 0.8:1 to 1.3:1. In some embodiments, compounds E and F are present in a ratio of approximately 0.9:1 to 1.1:1. For example, the molar ratio of compounds E and F may be approximately 1:1, or in the range of 1:1.11 to 1:1.15. The tertiary amine base can be any tertiary amine base that can promote or catalyze the nucleophilic acyl substitution reaction between compounds E and F. Suitable tertiary amine bases may include, for example, diisopropylethylamine (“DIPEA”), triethylamine (“TEA”), dimethylmorpholine (“NMM”), 2,2,6,6-tetramethylpiperidine (“TMP”), 2,4,6-trimethylpyridine (“collidine”), and combinations thereof. For example, the tertiary amine base may include DIPEA. The tertiary amine base may be present in a molar ratio to compound E ranging from approximately 1:1 to approximately 4:1. In some embodiments, the tertiary amine base and Compound E are present in a ratio of approximately 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 approximately 3.5:1 or 3.9:1. The coupling agent may include, for example, a carbodiimide reagent, a phosphonium reagent, a uronium reagent, an iminium 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). Carbodiimide may include, for example, 1,3-dichlorohexylcarbodiimide (“DCC”), 1,3-diisopropylcarbodiimide (“DIC”), 1-ethyl-3-(3-dlmethylaminopropyl)carbodiimide (“EDC”), and / or isopropylcarbodiimide (“CIC”), and combinations thereof. The phosphonium agent may include, for example, (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (“BOP”) or benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (“PyBOP”), and combinations thereof.The uronium agent may include, for example, 1[Bis(dimethylamino)methylene]-1 / 7-1,2,3-triazolo[4,5-]pyridinium 3-oxide hexafluorophosphate (“HATU”), O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (“HBTU”), and combinations thereof. 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 coupling agent may be present in a molar ratio to the compound in the range of approximately 0.8:1 to approximately 1:5. In some embodiments, the coupling agent and Compound E are present in a ratio of approximately 0.9:1 to 1.1:1.For example, the ratio of the coupling agent to compound E can be approximately 1:1 or 1.11:1. The coupling reaction can be carried out in the presence of a coupling additive. Coupling additives are known in the art, and any suitable additive for the formation of compound G can be used. Suitable coupling additives include, for example, benzotriazoles, dicarboximides, and succinimides. In some embodiments, the coupling additive is selected from the group consisting of β-hydroxysuccinimide (“HOSu”), β-hydroxy-5-norbomene-2,3-dicarboximide (“HONB”), 1-hydroxybenzotriazole (“HOBt”), 6-chloro-1-hydroxybenzotriazole (“CI-HOBt”), 1-hydroxy-7-azabenzotriazole (“HOAt”), and combinations thereof. For example, the coupling additive may include HOBt. The temperature of each mixing stage is maintained within a range of approximately -20 °C to approximately 25 °C. In some embodiments, the temperature of each mixing stage is maintained within a range of approximately -15 °C to approximately 25 °C. In some cases, the temperature of each mixing stage is maintained within a range of approximately -5 °C to approximately 15 °C. For example, the temperature of each mixing stage can be maintained within a range of approximately -5 °C to approximately 5 °C. The temperature of each mixing stage can be the same or different. In step (a) of the preparation of compound G, mixing can occur for a period of up to approximately 30 minutes (e.g., up to approximately 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 minutes). In some embodiments, mixing in step (a) can occur for up to approximately 10 minutes. In some cases, mixing in step (a) can occur for at least approximately 30 seconds or at least approximately 1 minute (e.g., at least approximately 2, 3, 4, 5, 6, 7, 8, or 9 minutes). For example, the mixing of step (a) can occur for approximately 30 seconds to approximately 30 minutes, or for approximately 1 minute to approximately 20 minutes, or for approximately 2 minutes to approximately 15 minutes, or for approximately 5 minutes to approximately 10 minutes. In step (b) of the preparation of compound G, mixing can occur for a period of up to approximately 3 hours (e.g., up to approximately 1, 1.5, 2, 2.5, or 3 hours). In some embodiments, mixing in step (b) can occur for up to approximately 2 hours. In some cases, mixing in step (b) can occur for at least approximately 30 minutes, or at least approximately 1 hour, or at least approximately 1.5 hours. For example, mixing in step (b) can occur for approximately 30 minutes to approximately 3 hours, or for approximately 30 minutes to approximately 2.5 hours, or for approximately 1 hour to approximately 2 hours. In step (b), the coupling reaction can occur under a nitrogen atmosphere. In some cases, the coupling reaction does not occur under a nitrogen atmosphere. After step (b), compound G can be washed with one or more solvents. The washing temperature can optionally range from approximately 0°C to approximately 25°C, or from approximately 15°C to approximately 25°C. Suitable washing solvents include, for example, water, potassium monobasic phosphate, sodium bicarbonate, sodium sulfate, and combinations thereof. In some embodiments, water is added to compound G after step (b), and the resulting two-phase mixture is separated into an aqueous layer and an organic layer before washing. In several cases, compound G can be washed with water, potassium monobasic phosphate, 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 approximately 10 minutes to form a mixture, and (b) mixing the mixture from step (a) with approximately 1 molar equivalent of HATU for up to approximately two hours under a nitrogen atmosphere, where the temperature for each stage is in the range of approximately -20 °C to approximately 25 °C, or approximately -20 °C to 0 °C. The resulting mixture can be inactivated with water to produce a two-phase mixture. The organic layer can then be separated, washed with water, then with monobasic potassium phosphate, followed by sodium bicarbonate and sodium sulfate (sequently). Preparation of Compound E Compound E can be prepared by mixing a reducing agent and (2S,3R)-3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-(2-morpholinoacetamido)propanamido)benzyl propanoate (compound “D”) The reducing agent can be any suitable agent capable of removing the benzyl group from 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; 4,4'-di-tert-butylbiphenyllithium (“Li DTBBP”), and combinations thereof. For example, the reducing agent can be H₂ in the presence of Pd / C. The mixture of the reducing agent and compound D is produced in any solvent capable of allowing the reduction reaction to occur. For example, the solvent may include THF, methanol, or a combination of these. In some embodiments, compound D is supplied under a nitrogen atmosphere prior to exposure to a hydrogen atmosphere. In several embodiments, the hydrogen atmosphere is set at approximately 15 psi. The mixing of the reducing agent and Compound D can occur for a period of at least 30 minutes and up to approximately 5 hours (e.g., up to approximately 2, 2.5, 3, 3.5, 4, or 4.5 hours). In some embodiments, the mixing can occur for up to approximately 4 hours. In some cases, the mixing of the reducing agent and Compound D can occur for at least approximately 30 minutes, or at least approximately 1 hour (e.g., at least approximately 1.5, 2, 2.5, 3, or 3.5 hours). For example, the mixing can occur for approximately 30 minutes to approximately 5 hours, or approximately 1 hour to approximately 4 hours, or approximately 2 hours to approximately 4 hours. The temperature of the mixture is maintained in a range of approximately 10 °C to approximately 20 °C. In some embodiments, the temperature is maintained at approximately 17 °C. In some cases, after mixing is complete, compound E is filtered, such as through diatomaceous earth (i.e., diatomite). The resulting filtrate can be φ subsequently wash with a suitable solvent (e.g., water, methanol, water and their combinations). 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 powder diffraction (XRPD) pattern shown in Figures 2, 3, and 4, respectively. For example, crystallization of Compound E can be achieved in THF and water by heating Compound E to a temperature in the range of approximately 50 °C to approximately 70 °C, or approximately 60 °C to approximately 70 °C, or approximately 55 °C to approximately 65 °C, and then cooling to approximately 0 °C. Accordingly, another aspect of the present description is a crystalline form of compound E, which is characterized by an XRPD pattern comprising 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° 28 using Cu Ka radiation, as shown in Figure 4. For example, Compound E can be prepared by mixing a reducing agent, such as H₂, in the presence of Pd / C, and Compound D under a nitrogen atmosphere at 10°C to 20°C for a period of at least 30 minutes up to 4 hours. Compound E can be filtered through diatomaceous earth, and the resulting filter cake can be washed (e.g., with water, methanol, and / or THF). Compound E can be crystallized by heating to approximately 60°C to 70°C, adjusting the temperature to approximately 55°C to 65°C and adding THF to the mixture, heating the mixture again to 60°C to 70°C, adding water to the heated mixture, cooling the mixture again to 55°C to 65°C, adding a seed crystal to the mixture, and stirring the seeded mixture for approximately two hours at 0°C. Filtration, washing, and drying of the cooled mixture produces crystallized compound E. Preparation of Compound F In another aspect, a method for preparing compound F is provided herein. ° (F), where X is a counterion. Compound F can be prepared in three stages—stages (a), (b), and (c). In stage i (a), a mixture is formed by mixing together an aprotic solvent, trifluoroacetic acid (“TFA”), and tert-butyl-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methyloxiran-2-yl)1-oxopropan-2-yl)carbamate (compound H): at a temperature in the range of approximately -5 °C to approximately 5 °C. In step (b), the mixture from step (a) is concentrated. In step (c), the concentrated mixture from step (b) is mixed with an acid at a temperature in the range of approximately -5 °C to 5 °C to form compound F. The acid can be any acid capable of forming a salt with the ammonium group of compound F. Suitable acids include, for example, p-toluensulfonic acid, trifluoromethanesulfonic acid, acetic acid, trifluoroacetic acid, naphthalenesulfonic acid, 4-nitrobenzenesulfonic acid, sulfonic acid, methylsulfonic acid, benzenesulfonic acid, nitric acid, HF, HCl, HBr, and combinations thereof. In some embodiments, the acid is selected from the group consisting of p-toluensulfonic acid, naphthalenesulfonic acid, 4-nitrobenzenesulfonic acid, and combinations thereof. For example, the acid may include p-toluensulfonic acid. The aprotic solvent in step (a) can be any aprotic solvent (or mixture of solvents) in which the reaction can proceed. Suitable aprotic solvents may include acetonitrile (“AON”), dichloromethane (“DCM”), tetrahydrofuran (“THF”), dimethylacetamide (“DMAc”), methyl tert-butyl ether (“MTBE”), isopropyl ether (“IPE”), and combinations thereof. For example, the aprotic solvent may include DCM. The trifluoroacetic acid in step (a) may be present in a molar ratio to Compound H in the range of approximately 15:1 to 5:1. In some embodiments, the trifluoroacetic acid and Compound H are present in a ratio of approximately 10:1 to 7.5:1. For example, the molar ratio of trifluoroacetic acid and Compound H may be approximately 8:1. In some embodiments, the deprotection reaction of step (a) takes place under a nitrogen atmosphere. The temperature of the mixture in step (a), step (c), or step (a) and step (c) is maintained in a range of approximately -5 °C to approximately 5 °C, or to approximately 0 °C. In some embodiments, the mixture is concentrated in step (b) at a temperature in a range of approximately 15 °C to approximately 25 °C. In some cases, the mixing of step (a) can occur for a period of at least 30 minutes up to approximately 3 hours (for example, up to approximately 1, 1.5, 2, 2.5, or 3 hours). In some embodiments, the mixing of step (a) can occur for a period of up to approximately 2 hours. In some cases, the mixing of step (a) can occur for at least approximately 30 minutes, or at least approximately 1 hour, or at least approximately 1.5 hours. For example, the mixing of step (a) can occur for approximately 30 minutes to approximately 3 hours, or approximately 30 minutes to approximately 2.5 hours, or approximately 1 hour to approximately 2 hours. In several cases, the mixing in step (c) can occur over a period of at least 5 hours up to approximately 12 hours (e.g., up to approximately 7, 8, 9, 10, or 11 hours). In some embodiments, the mixing in step (c) can occur over a period of up to approximately 10 to 12 hours. In some cases, the mixing in step (c) can occur for at least approximately 5 hours (e.g., at least approximately 6, 7, 8, 9, or 10 hours). For example, the mixing in step (c) can occur for approximately 5 hours to approximately 12 hours, or approximately 10 hours to approximately 12 hours. In some cases, the concentrated mixture from step (b) can be rinsed with a polar aprotic solvent. Suitable polar aprotic solvates include, for example, diethyl ether, tetrahydrofuran (“THF”), acetonitrile (“ACN”), methyl tert-butyl ether (“MBTE”), isopropyl ether (“IPE”), and combinations thereof. For example, the polar aprotic solvent could be MBTE. After step (c), compound F can optionally be filtered at a temperature in a range of approximately -5 °C to approximately 5 °C, washed with one or more polar aprotic solvents (e.g., diethyl ether, tetrahydrofuran (“THF”), acetonitrile (“ACN”), methyl tert-butyl ether (“MBTE”), isopropyl ether (“IPE”), and combinations thereof), and / or dried. Compound F can be crystallized to form a polymorph, characterized by the differential scanning calorimetry (“DSC”) thermogram, thermogravimetric analysis (“TGA”) data, and powder X-ray diffraction (“XRPD”) pattern depicted in Figures 5, 6, 7, 8, and 9. Accordingly, another aspect of the present description is a crystalline form of Compound F, such as the tosylate salt of Compound F, which is characterized by an XRPD pattern comprising peaks at 6.8, 7.1, 7.4, 14.2, 14.8, 17.0, 17.5, 17.8, 18.5, 18.7, 20.1, 20.3, 23.0, 23.6, 24.5, 29.3, and 31.2 ± 0.2° using Cu Ka radiation, as shown in Figure 9. The tosylate form of compound F can also be characterized by a single-crystal X-ray diffraction (XRD) structure, as described in the Examples section below. The crystal, as depicted in Figure 10, has a unit cell dimension of a = 13.264 (3) A, α = 90°, b = 5.6920 (11) A, b = 109.410 (4)°, c = 13.416 (3) A, γ = 90° and belongs to space group P21. The Flack parameter is 0.03 (0.08 su). Crystallizations using other acids such as 2-naphthalenesulfonic, methanesulfonic, benzenesulfonic, phosphoric, and sulfuric acids did not provide X-ray quality crystals in the following solvents: toluene, diethyl ether, MTBE, 1,4-dioxane, ethyl acetate, acetone, acetonitrile, butanol, isopropanol, and hexane / ethyl acetate (1:1 ratio). For example, compound F can be prepared by (a) mixing an aprotic solvent (e.g., DCM), TFA, and compound H in a molar ratio of 8:1 at approximately 0 °C under a nitrogen atmosphere for up to 2 hours, (b) concentrating the mixture at approximately 15 °C to 25 °C, and (c) mixing the concentrated mixture with an acid (e.g., p-toluenesulfonic acid) at approximately 0 °C for 10 to 12 hours. The resulting compound F can be filtered at approximately 0 °C, washed with a polar aprotic solvent (e.g., MBTE), and dried under vacuum. Preparation of Compound D In another aspect, a method for preparing compound D is provided herein. Compound D can be prepared in two steps—step (a) and step (b). In step (a), a mixture is prepared by mixing together a tertiary amine base and a suspension of compound B and Compound C in an aprotic solvent: (i) (2S,3R)-1-(benzyloxy)-3-hydroxy-3-(4-methoxyphenyl)-1-oxopropan-2-aminium salt (compound “B”): MeO OH x. H3N 0Bn ° (B), where X' is a counterion, and (i) (2-morpholinoacetyl)-L-alanine (compound “C”): In step (b), the mixture from step (a) and a coupling agent are mixed together at a temperature in a range of approximately -5 °C to approximately 5 °C to form compound D. The counterion (X) can be any anion capable of forming an ionic bond with the ammonium group of compound B. 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 chloride. The aprotic solvent can be any aprotic solvent (or solvent mixture) in which the nucleophilic acyl substitution reaction between compounds B and C proceeds. Suitable aprotic solvents may include 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 include ACN. Compound B and Compound C may be present in a molar ratio of approximately 0.65:1 to 1.1:1. In some embodiments, Compounds B and C are present in a ratio of approximately 0.75:1 to 1:1. For example, the molar ratio of Compounds B and C may be approximately 0.8:1. The tertiary amine base can be any tertiary amine base that can promote or catalyze the nucleophilic acyl substitution reaction between compounds B and C. Suitable tertiary amine bases may include, for example, A / ,A / -diisopropylethylamine (“DIPEA”), triethylamine (“TEA”), V-methylmorpholine (“NMM”), 2,2,6,6-tetramethylpiperidine (“TMP”), 2,4,6-trimethylpyridine (“collidine”), or combinations thereof. For example, the tertiary amine base may include DIPEA. The tertiary amine base may be present in a molar ratio to compound B ranging from approximately 1:1 to approximately 3.5:1. For example, the molar ratio of the tertiary amine base to compound B may be approximately 3.5:1.The coupling agent may include, for example, a carbodiimide reagent, a phosphonium reagent, a uranium reagent, an iminium reagent, an imidazolium reagent, an organophosphorus reagent, an acid chloride reagent, a chloroformate reagent, a pyridinium reagent, or combinations thereof, as previously described for the preparation of compound G. Examples of carbodiimide reagent, phosphonium reagent, uranium reagent, iminium reagent, imidazolium reagent, organophosphorus reagent, acid chloride reagent, chloroformate reagent, or pyridinium reagent are described above for the preparation of compound G. In some embodiments, the uranium agent may include HATU, HBTU, and combinations thereof. For example, the uranium agent may be HATU. The coupling agent may be present in a molar ratio to compound B ranging from approximately 1:1 to approximately 1:3.In some embodiments, the coupling agent and Compound B are present in a ratio of approximately 1:1 to 1:2. For example, the ratio of the coupling agent to Compound B may be approximately 1:1.5. The coupling reaction can be carried out in the presence of a coupling additive. Examples of quantities of these coupling additives are described for the preparation of compound G. The temperature of each mixing stage is maintained within a range of approximately -5 °C to approximately 5 °C. In some embodiments, the temperature of each mixing stage is maintained at approximately 0 °C. The temperature of each mixing stage may be the same or different. In step (b) of the preparation of compound D, the mixture may include portions of mixing the coupling agent with the mixture from step (a) for a period of at least 1 minute up to approximately 30 minutes (e.g., up to approximately 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, the coupling agent portions can be added to the mixture of step (a) over a period of at least approximately 1 minute (e.g., at least approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 minutes). For example, the coupling agent portions can be added to the mixture of step (a) over a period of approximately 1 minute to approximately 30 minutes, or approximately 10 minutes to approximately 30 minutes, or approximately 20 minutes to approximately 30 minutes.The mixing in step (b) may also include stirring the mixture for up to approximately 3 hours (e.g., up to approximately 1, 1.5, 2, 2.5, or 3 hours). In some embodiments, stirring may occur for up to approximately 2 hours. In some cases, stirring may occur for at least approximately 30 minutes, or at least approximately 1 hour, or at least approximately 1.5 hours. For example, stirring may occur for approximately 30 minutes to approximately 3 hours, or approximately 30 minutes to approximately 2.5 hours, or approximately 1 hour to approximately 2 hours. After step (b), compound D can be inactivated and / or washed with one or more solvents at a temperature in the range of approximately 15 °C to approximately 25 °C. Suitable solvents for inactivation and / or washing include, for example, water, isopropyl acetate, potassium monobasic phosphate, sodium bicarbonate, sodium sulfate, THF, and combinations thereof. For example, compound D can be prepared by (a) mixing compound B and compound (C) together (1:1 molar ratio) and a tertiary amine base (e.g., DIPEA) in ACN, and (b) mixing the mixture from step (a) with approximately 1 molar equivalent of HATU in portions over a period of approximately 30 minutes, and then stirring the mixture for a period of up to approximately 2 hours, where the temperature for each stage is approximately 0 °C. The resulting mixture from step (b) can be inactivated with, for example, sodium bicarbonate to form a two-phase mixture. The organic phase can then be separated and washed with sodium bicarbonate, monobasic potassium phosphate, and / or sodium sulfate. Preparation of Compound B Compound B can be prepared by mixing (i) an acid and (ii) (2S,3R)-2((tert-butoxycarbonyl)amino)-3-hydroxy-3-(4-methoxyphenyl)propanoate benzyl (compound “A’j: MeO OH _ ,,,, OBn BocHN 0 (A) in an aprotic solvent. The acid can be any acid capable of deprotecting the amino group in compound A. Suitable acids include, for example, p-toluenesulfonic acid, trifluoromethanesulfonic acid, acetic acid, trifluoroacetic acid, naphthalenesulfonic acid, 4-nitrobenzenesulfonic acid, sulfonic acid, methylsulfonic acid, nitric acid, HF, HCl, HBr, and combinations thereof. In some embodiments, the acid includes trifluoroacetic acid or HCl. The aprotic solvent can be any solvent in which the deprotection reaction can occur. Suitable solvents include ethyl acetate, methylpyrrolidone (NMP), tetrahydrofuran (THF), acetone, dimethylformamide (DMF), acetonitrile (ACN), dimethyl sulfoxide (DMSO), dichloromethane (DCM), and combinations thereof. For example, the solvent might include ethyl acetate, DCM, or a combination of these. In some embodiments, the temperature of the mixture during the mixing stage is maintained in a range of approximately 15 °C to approximately 25 °C, or to approximately 20 °C. In some cases, after the mixing is complete, compound B is filtered and dried under vacuum to form a crystalline polymorph, which is characterized by the DSC thermogram depicted in Figure 1. Therefore, another aspect of the present description is a crystalline form of compound B, which is characterized by an XRPD pattern comprising peaks at 4.6, 9.2, 13.8, 18.5 and 32.9 ± 0.2o 2Θ using Cu Ka radiation. For example, compound B can be prepared by mixing an acid (e.g., HCl) and compound A together at 20 °C, filtering and drying the resulting compound B. EXAMPLES The following examples are provided for illustration and are not deemed to limit the scope of the invention. General synthesis outline Compound G can be prepared according to Scheme 1, shown above. Example 1: Large-scale preparation of the HCI salt of the (2S,3R)-1-(benzyloxy)3-hydroxy-3-(4-methoxyphenyl)-1-oxopropan-2-aminium salt (Compound “B”): 0 (B), Ethyl acetate (58.5 kg) at 20 °C was charged with HCl gas (6.8 kg). Benzyl (2S,3R)-2-((tert-butoxycarbonyl)amino)-3-hydroxy-3-(4-methoxyphenyl)propanoate (compound “A”) was dissolved in this solution: MeO OH OBn BocHN θ (A) (5 kg, 12.5 mol, predissolved in 32.5 g of ethyl acetate). The suspension was stirred at 20 °C and, after completion as determined by HPLC, filtered and dried under vacuum at 45 °C to provide a crystalline polymorph of compound B (3.85 kg) as the HCl salt. LC / MS (LRMS(MH) m / z: 302). HPLC purity 97.9%. The characteristic DSC curve is shown in Figure 1. Example 2: Small-scale synthesis of the TFA salt of the (2S,3R)-1-(benzyloxy)3-hydroxy-3-(4-methoxyphenyl)-1-oxopropan-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 min, then diluted with DCM (100 mL). Saturated NaHCO3 solution (aqueous, 100 mL) was added, and the two layers separated. The aqueous layer was extracted with DCM (2 x 100 mL), and the combined organic layers were dried in anhydrous sodium sulfate and then concentrated to give crude compound B (5.0 g, 84% yield) as the TFA salt. LC / MS (LRMS(MH) m / z: 302. Example 3: Large-scale preparation of (2S,3R)-3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2 (2-morpholinoacetamido)propanamido)propanoate (Compound “D”) To compound B (3.8 kg) and (2-morpholinoacetyl)-L-alanine (compound “C”): (2.5 kg) at 20 °C, acetonitrile (30.4 kg) was added. The temperature was adjusted to 0 °C and A / , / Vdiisopropylethylamine (“DIPEA”) (3.19 kg) was added, followed by 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-]pyridinium 3-oxide hexafluorophosphate (“HATU”) (5.22 kg) in portions over 30 min. The reaction mixture was stirred for 2 h at 0 °C, then inactivated with 3.5% NaHCO3 (aqueous, 46 kg) and stirred for 30 min. After settling for 1 h at 20 °C, solid NaHCO3 was added and the mixture was stirred for 30 min, then allowed to settle again at 20 °C for 1 h. The aqueous layer was diluted with water (30.6 kg), extracted with isopropyl acetate (“iPrOAc’j” (23.4 kg), and the organic layers were combined. The organic layers were extracted with iPrOAc (3 x 27 kg), washed with 3.5% NaHCO3 (aqueous, 30 kg), KH2PO4 (aqueous, 3 x 65 kg), water (15 kg), 7% NaHCO3 (aqueous, 2 x 61 kg), and 5% Na2SO4 (aqueous, 3 x 55 kg).The solution was concentrated to 18 L then extracted with tetrahydrofuran (“THF”) (4 x 22.8 L) to provide the product (5.04 kg, 90% yield, 97.9% purity by HPLC) as a solution in THF (34.5% by weight, 14.6 kg total). Similar results were obtained using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCI (“EDC”) (1,1 equiv.) instead of HATU as the coupling reagent. 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 flash column chromatography on silica gel (petroleum ether / EtOAc = 2:1 to 1:2) to give Compound D (5.8 g, 78% yield) as a colorless solid. LC / MS (LRMS(MH) m / z: 500). Example 5: Large-scale preparation of (2S,3R)-3-hydroxy-3-(4-methoxypheniD-2((S)-2-(2-morpholine-acetamido)propanamido)propanoic acid (compound “E”): To a solution of (2S,3R)-3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-(2-morpholinoacetamido)propanamido)benzyl propanoate (compound “D”) (5.04 kg as a 34.5 wt% solution in THF), THF (3.25 kg) was added, followed by methanol (7.0 kg). A nitrogen atmosphere was established inside the reaction vessel, and Pd / C (10%, 473 g) was added under nitrogen protection. THF (500 g) and methanol (1 kg) were added to wash the reaction vessel, and a hydrogen atmosphere (15 psi) was established. The reaction 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, extracted with THF (4 x 45 kg), and heated to 60–70 °C. After 2 h, the temperature was adjusted to 50–60 °C and THF (30 kg) was added. The mixture was heated again to 60–70 °C for 2 h. Water (370 kg) at 60–70 °C was added to this solution, and 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 h. The suspension was concentrated twice to 5–6 volumes and stirred for 2 h at 0 °C. The mixture was filtered using THF (10 kg) for washing. The wet cake was dried to yield a crystalline polymorph of compound E (3.54 kg, 97.6% purity). Characteristic DSC, TGA, and XRPD data are shown in Figures 2–4. Example 6: Small-scale synthesis of Compound E

Claims

1. A method for preparing (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-(morpholin-4-yl)acetamido]propanamido]propanamide (compound “G”) (FORMULA 1) characterized in that it comprises: (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”): (FORMULA 2) and (ii) salt (S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methyloxiran-2-yl)-1-oxopropan-2-aminium (compound “F”): (FORMULA 3) where X- is a counterion; in an aprotic solvent to form a mixture and (b) mixing a coupling agent and the mixture of step (a) to form compound G; wherein the temperature of each mixing step is maintained at -20°C to 25°C. 19 Claims follow