Processes and intermediates useful for preparing nilamtrervir

Abstract

The present invention relates to nilmatrervir (a compound of formula I) [Formula 1] This application is directed to intermediates and efficient methods for preparing TIFF2025525405000046.tif4952, as well as intermediates useful in the preparation of nilmatrervir. [Figure 1] TIFF2025525405000047.tif65139

Description

[Technical Field]

[0001] The present invention is directed to intermediates and efficient processes for preparing nilmatrellvir, as well as intermediates useful in the preparation of nilmatrellvir. Nilmatrellvir is an antiviral compound with potent inhibitory activity against coronavirus 3CL protease and is the active ingredient in the product Paxlovid®, which is approved for use in the treatment of COVID-19. Nilmatrellvir and processes for its preparation are disclosed in PCT International Patent Application Publication No. WO 2021 / 250648, U.S. Patent Application Publication No. 2022 / 0062232 A1, and U.S. Patent No. 11,351,149. [Background technology]

[0002] Summary of the Invention [Problem to be solved by the invention]

[0003] [Means for solving the problem]

[0004] The present invention provides intermediates used in the preparation of nilmatrervir and synthetic methods for preparing the intermediates, as depicted in Reaction Scheme 1, which contain several process modifications compared to previously disclosed methods. The product of Step 1 in Reaction Scheme 1 is sodium (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate, which is a new salt form (sodium instead of lithium) compared to previous methods, and the reaction conditions in Steps 2 and 3 have been modified. Additional solid form characterization data is provided for several starting materials and intermediates used in the process. A process for the preparation and isolation of (1R,2S,5S)-N-((S)-1-amino-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide, methyl ethyl ketone (MEK) solvate is presented along with characterization data for the compound.

[0005] [ka]

[0006] [ka] [Brief explanation of the drawings]

[0007] [Figure 1] FIG. 1 shows the relationship between initial concentration (mL / g) of API (PF-07321332, MTBE solvate) in isopropyl acetate, heptane addition time (in hours), seed loading (% wt of PF-07321332, Form 1 per weight of MTBE solvate), and seed size (in microns) at final D[v,0.5] count in microns of particle size distribution (PSD) of PF-07321332, Form 1. [Figure 2] FIG. 1 shows the correlation between seed size and final particle size distribution after crystallization of PF-07321332, Form 1, at 0.75 wt% seed loading (blue circles) and 0.5 wt% seed loading (orange circles) in lab-scale (100 mL) experiments. [Figure 3] FIG. 1 is a diagram of the particle size distribution (PSD) obtained for PF-07321332, Form 1, showing D[v,0.5] in microns for over 50 batches at one location. [Figure 4] FIG. 1 is a diagram of the particle size distribution (PSD) obtained for PF-07321332, Form 1, showing D[v,0.5] in microns for over 50 batches at one location. [Figure 5] FIG. 1 shows the PXRD pattern for (S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoic acid (Compound V). [Figure 6] FIG. 1 shows the PXRD pattern for methyl (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate, hydrochloride salt (Compound VII). [Figure 7] FIG. 1 shows the PXRD pattern of sodium (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate (Compound VI), initial form. [Figure 8] FIG. 1 shows the PXRD pattern of sodium (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate, Form B. [Figure 9] FIG. 1 shows the PXRD pattern of sodium (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate, Material A. [Figure 10] FIG. 1 shows the PXRD pattern of (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid, Form 1. [Figure 11]FIG. 1 shows the PXRD pattern of (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid, a new form. [Figure 12] FIG. 1 shows the PXRD pattern for (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl)propanamide hydrochloride, Form 1. [Figure 13] FIG. 1 shows the PXRD pattern for (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl)propanamide hydrochloride, Form 2. [Figure 14] FIG. 1 shows the PXRD pattern for (1R,2S,5S)—N—((S)-1-amino-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide, methyl ethyl ketone solvate. [Figure 15] FIG. 1 shows the PXRD pattern for PF-07321332 isopropyl acetate solvate. DETAILED DESCRIPTION OF THE INVENTION

[0008] The following embodiments EMB-1 to EMB-32 are representative embodiments of the present invention and should not be construed as limiting.

[0009] EMB-1 is the compound (1R,2S,5S)-N-((S)-1-amino-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide, methyl ethyl ketone solvate.

[0010] EMB-2 is a process for preparing (1R,2S,5S)—N-((S)-1-amino-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Compound II), comprising:

[0011] [ka] Steps (a) to (d): (a) combining (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (compound IV) and methyl ethyl ketone, followed by the addition of 2-hydroxypyridine N-oxide and triethylamine to obtain a first mixture, S3-M1; (b) combining (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl)propanamide hydrochloride (compound III), 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride, and methyl ethyl ketone to obtain a second mixture, S3-M2; (c) combining the first mixture S3-M1 from step (a) with the second mixture S3-M2 from step (b) to obtain a third mixture S3-M3; and (d) stirring the third mixture S3-M3 from step (c) to obtain compound (1R,2S,5S)—N—((S)-1-amino-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (compound II); reacting a compound of formula IV with a compound of formula III,

[0012] EMB-3 is the process of EMB-2, wherein in step (a), the first mixture, S3-M1, contains 1.0 equivalent of (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (Compound IV), 2 L of methyl ethyl ketone per kg of Compound IV, 0.9 equivalents of 2-hydroxypyridine N-oxide, and 2.50 equivalents of triethylamine.

[0013] EMB-4 is the method of EMB-2 or EMB-3, wherein in step (a), the first mixture, S3-M1, is prepared at about 25°C, stirred at about 25°C for about 30 minutes, and then warmed to about 50°C.

[0014] EMB-5 is any one of the processes EMB-2 to EMB-4, wherein in step (b), the second mixture, S3-M2, comprises 1.05 equivalents of (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl)propanamide hydrochloride (Compound III), 1.30 equivalents of 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride, and 3 L of methyl ethyl ketone per kg of Compound IV.

[0015] EMB-6 is any one of the methods EMB-2 to EMB-5, wherein in step (b), the second mixture, S3-M2, is prepared at about 25°C, stirred at about 25°C for 30 minutes, and then warmed to about 50°C.

[0016] EMB-7 is the process of any one of EMB-2 to EMB-6, wherein in step (c), the first mixture, S3-M1, from step (a), is at about 50°C and is combined with a second mixture, S3-M2, from step (b), which is at about 50°C, to obtain a third mixture, S3-M3, while maintaining the temperature of the third mixture, S3-M3, at about 50°C.

[0017] EMB-8 is the process of any one of EMB-2 to EMB-7, wherein in step (d) the third mixture from step (c), S3-M2, is stirred at about 50° C. for at least 6 hours.

[0018] EMB-9 is a process for preparing (1R,2S,5S)—N-((S)-1-amino-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Compound II), comprising:

[0019] [ka] Steps (a) to (d): (a) combining (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (Compound IV, 1.0 equivalent) and methyl ethyl ketone (2 L per kg of Compound IV) at about 25° C., followed by the addition of 2-hydroxypyridine N-oxide (0.90 equivalents) and triethylamine (2.50 equivalents) to obtain a first mixture, S3-M1, which is stirred for about 30 minutes and then warmed to about 50° C.; (b) combining (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl)propanamide hydrochloride (Compound III, 1.05 equivalents), 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (1.30 equivalents), and methyl ethyl ketone (3 L per kg of Compound IV) to obtain a second mixture, S3-M2, which is stirred for about 30 minutes and then warmed to about 50° C.; (c) combining the first mixture, S3-M1, from step (a) with the second mixture, S3-M2, from step (b) while maintaining the temperature at about 50° C. to obtain a third mixture, S3-M3; and (d) stirring the third mixture, S3-M3, from step (c) at about 50° C. for at least 6 hours to obtain compound (1R,2S,5S)—N—((S)-1-amino-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (compound II); reacting a compound of formula IV with a compound of formula III,

[0020] EMB-10 is the process of EMB-9, wherein the amount of acylurea impurities IMP-S3-3, (2S,4S)-4-(2-aminoethyl)-5-oxopyrrolidine-2-carboxamide, and IMP-S3-4, (1R,2S,5S)—N-(2-((3S,5S)-5-carbamoyl-2-oxopyrrolidin-3-yl)ethyl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide formed is 10% or less.

[0021] EMB-11 is the process of EMB-9, in which the amount of acylurea impurities IMP-S3-3, (2S,4S)-4-(2-aminoethyl)-5-oxopyrrolidine-2-carboxamide, and IMP-S3-4, (1R,2S,5S)—N-(2-((3S,5S)-5-carbamoyl-2-oxopyrrolidin-3-yl)ethyl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide formed is 5% or less.

[0022] EMB-12 is the process of any one of EMB-9 through EMB-11, wherein the amount of rearrangement impurity IMP-S3-3, (2S,4S)-4-(2-aminoethyl)-5-oxopyrrolidine-2-carboxamide formed is 2% or less.

[0023] EMB-13 is the process of any one of EMB-9 to EMB-11, wherein the amount of rearrangement impurity IMP-S3-4, (1R,2S,5S)—N-(2-((3S,5S)-5-carbamoyl-2-oxopyrrolidin-3-yl)ethyl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide, formed is 2% or less.

[0024] EMB-14 is a process for preparing (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (compound IV), comprising:

[0025] [ka] Steps (a) to (c): (a) combining (S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoic acid (Compound V), methanesulfonyl chloride, and isopropyl acetate to obtain a first mixture, S2-M1; (b) adding triethylamine to the first mixture, S2-M1, to obtain a second mixture, S2-M2; (c) adding sodium (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate (compound VI) to the second mixture, S2-M2, to obtain a third mixture, S2-M3; and (d) stirring the third mixture, S2-M3, to obtain (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (compound IV) reacting a compound of formula V with a compound of formula VI,

[0026] EMB-15 is the method of EMB-14, wherein the first mixture, S2-M1, in step (a) contains 1.2 equivalents of (S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoic acid (Compound V), 1.1 equivalents of methanesulfonyl chloride, and 20 mL of isopropyl acetate per gram of Compound V.

[0027] EMB-16 is the process of EMB-14 or EMB-15, wherein in step (b) 2.5 equivalents of triethylamine are added to the first mixture, S2-M1, at about 20° C., at a rate such that the temperature does not exceed 25° C. to obtain a second mixture, S2-M2.

[0028] EMB-17 is the process of any one of EMB-14 to EMB-16, wherein in step (c), 1.0 equivalent of (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate sodium salt (Compound VI) is added to the second mixture, S2-M2, to obtain a third mixture, S2-M3, which is stirred for about 4 hours.

[0029] EMB-18 is the method of EMB-17, in which 2.5 equivalents of aqueous citric acid are added to the third mixture, S2-M3, and the resulting mixture is stirred at about 40° C. for at least 10 minutes.

[0030] EMB-19 is a method of EMB-18 in which the organic and aqueous layers of the resulting mixture are allowed to settle, and the organic isopropyl acetate layer is separated from the aqueous layer, washed with water, and concentrated to approximately 40% of its initial volume to yield an organic layer, S2-M4.

[0031] EMB-20 is the method of EMB-19 in which the organic layer S2-M4 is heated to 60° C. and one volume of heptane is added thereto, then the resulting mixture is cooled to 10° C. and stirred for 3 hours, and the resulting solid is collected by filtration, washed with 1:1 isopropyl acetate / heptane, and dried to give (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (IV).

[0032] EMB-21 is a process for preparing (1R,2S,5S)—N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (Form 1, Compound I), comprising:

[0033] [ka] Steps (a) to (d): (a) dissolving (1R,2S,5S)—N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide, methyl tert-butyl ether (Compound I′) in isopropyl acetate, wherein the concentration of Compound I′ in the isopropyl acetate is from about 7 mL to about 9 mL of isopropyl acetate per gram of Compound I′; (b) seeding the solution with 0.5% to 0.75% by weight of (1R,2S,5S)—N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide, Form 1; (c) adding heptane to the mixture from step (b) over a period of 6 to 15 hours, wherein the amount of heptane added is about 10 mL to about 14 mL of heptane per gram of Compound I'; and (d) isolating the obtained (1R,2S,5S)—N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide, Form 1 The method includes:

[0034] EMB-22 is the method of EMB-22, wherein the amount of isopropyl acetate used in step (a) is about 8 mL of isopropyl acetate per gram of Compound I'.

[0035] EMB-23 is the process of EMB-21 or EMB-22, wherein the amount of (1R,2S,5S)—N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide, Form 1 used to seed the solution in step (b) is about 0.75 wt %.

[0036] EMB-24 is the process of any one of EMB-21 to EMB-23, wherein the amount of heptane added in step (c) is about 12 mL per gram of Compound I'.

[0037] EMB-25 is any one of methods EMB-21 through EMB-24, in which heptane is added over about 10 hours.

[0038] EMB-26 is the process of any one of EMB-21 through EMB-25, wherein (1R,2S,5S)—N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide, Form 1, is isolated by filtration.

[0039] EMB-27 is the process of any one of EMB-21 to EMB-26, wherein the (1R,2S,5S)—N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide, Form 1 isolated in step (d) has a particle size distribution with a D[v,0.5] count of about 12 microns to about 18 microns.

[0040] EMB-28 is the process of any one of EMB-21 to EMB-27, wherein the (1R,2S,5S)—N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide, Form 1 isolated in step (d) has a particle size distribution with a D[v,0.5] count of about 14 microns to about 16 microns.

[0041] EMB-29 is the process of any one of EMB-21 to EMB-28, wherein the (1R,2S,5S)—N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide, Form 1 isolated in step (d) has a particle size distribution with a D[v,0.5] count of about 15 microns.

[0042] EMB-30 is the compound (1R,2S,5S)—N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide, isopropyl acetate solvate.

[0043] EMB-31 is a crystalline compound of EMB-30.

[0044] EMB-32 is a compound of EMB-31 characterized by the PXRD pattern as depicted in FIG.

[0045] Preparation of Starting Materials and Intermediates Synthetic routes for the preparation of intermediate compounds of Formula 1 are provided in Reaction Schemes A and B, and synthetic routes for the preparation of compounds of Formula 2 are provided in Reaction Schemes C, D, and E.

[0046] Compounds of Formula 1 may be prepared by the following methods as described below and depicted in Reaction Schemes A and B. For compounds of Formula 1, R 1 is methyl and the compound is in the form of its hydrochloride salt, the compound is methyl (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate, hydrochloride, which is also referred to as Compound VII in Reaction Scheme 1.

[0047] [ka]

[0048] In Reaction Scheme A, the variable group R 1 , R 4 and R 5 represents an alkyl group, including but not limited to methyl, ethyl, and isopropyl groups. 1 represents an aryl group, including but not limited to a 2-methoxyphenyl group.2 and R 3 represents an amine protecting group well known to those skilled in the art (see, for example, Wuts, PGM; Greene, TW Greene's Protective Groups in Organic Synthesis, 4th ed.; John Wiley & Sons, Inc., 2006).

[0049] Compounds of formula 1 may be prepared by cyclopropanation of A2, followed by deprotection. Compound A2 may be prepared from A3 or A4 by conversion of the hydroxyl group of compound A3 or A4 to an activated group by reaction with reagents including, but not limited to, tosyl chloride, mesyl chloride, triflic anhydride, and sodium iodide, followed by elimination with a base to give compound A2. R 1 The group is preferably substituted prior to this step (i.e., by replacing the hydrogen of the carboxylic acid group in A3 or A4 with R 1 A2 may be prepared by reduction of pyrrole A5 or decarboxylation of A6. A2 may be prepared by intermolecular cyclization of A7 under conditions including, but not limited to, metal-catalyzed alpha vinylation.

[0050] Compounds of Formula 1 may be prepared by functionalization of A8 via transformations including, but not limited to, reduction of the amide to an imine, followed by cyanation and esterification. These transformations may require chemical or enzymatic catalysis. A8 may be formed via intramolecular cyclopropanation of A9 or A10, by cyclopropanation of A11, or by metal-catalyzed carbonylative C—H functionalization of A12, which may be prepared from A13. Compounds of Formula 1 may be prepared by oxidation of A14 to a carboxylic acid, followed by esterification. The transformations may require metal catalysis. Compounds of Formula 1 may be prepared via functionalization of A15 via transformations including, but not limited to, metallation with an organometallic reagent, followed by carboxylation and boronation, followed by carboxylation. A15 may be prepared by reactions including, but not limited to, coupling of A16 with an amine source, reduction and cyclization of A17, cyclopropanation of A18, or reduction of A22. A22 can be synthesized by cyclopropanation and aminolysis of A19, aminolysis and cyclization of A20, aminolysis, cyclization and decarboxylation of A27, or oxidative ring contraction of A21. A20 or A27 may be formed by reaction of A23 or A28 with A24, A25, or A33 in the presence of a dialkyl sulfide, or by cyclopropanation of A26 or A29 with a cyclopropanation reagent, including but not limited to A30. A27 may also be prepared by the reaction between A31 and A32 in the presence of a base.

[0051] [ka]

[0052] In Reaction Scheme B, the variable group R 1 , R 2 , R 3 , R 5 , R 6 , R 9 and R 10 represents an alkyl or aryl group, including, but not limited to, methyl, ethyl, isopropyl, and tolyl groups.4 , R 7 and R 8 represents an amine protecting group, which is well known to those skilled in the art.

[0053] The compound of formula 1 can be prepared by cyclization of B2 and B3 with an ammonia source in the presence of a chemical or enzymatic catalyst and a reducing agent. The resulting product can be subjected to another reduction reaction, if necessary. B2 can be prepared by coupling B4 and B5.

[0054] Compounds of formula 1 may be prepared by cyclization of B6 under reducing conditions in the presence of a chemical or enzymatic catalyst. B6 may be prepared from B7 under conditions well known to those skilled in the art. B8 may be treated with a chlorinating agent in the presence of a base to form compounds of formula 1. B8 may be prepared from B9 via B10 by transformations including, but not limited to, ozonolysis.

[0055] Compounds of Formula 1 may be prepared by intramolecular cyclopropanation of B11, B12, B13, or B14. This cyclopropanation reaction may require chemical and / or enzymatic catalysis, and it may be necessary to reduce the resulting product to form the compound of Formula 1. B12 may be prepared from B11, and B14 may be prepared from B13. B16 may undergo oxidation and olefination to form B15, which may then be converted to B13 under conditions well known to those skilled in the art. Compounds of Formula 1 may also be prepared by intramolecular carbon-carbon bond formation of B17 under reducing conditions, where B17 is functionalized (i.e., double bond rearrangement to form the group OSOR) of B18. 10 , Cl, Br, etc.) B20 and B21 can react in the presence of a base to form B19, which can then be reduced in the presence of a chemical and / or enzymatic catalyst to form a compound of formula 1. B22 or B24 can react with an azomethine ylide derived from B23 or B25 to form a compound of either formula 1 or A2, which can then be cyclopropanated to form a compound of formula 1.

[0056] Compounds of formula 2 may be synthesized by the following methods as described in Schemes C, D, and E.

[0057] [ka]

[0058] In Scheme C, the variable group R 1 , R 4 , R 5 and R 8 represents an alkyl or aryl group, including, but not limited to, methyl, ethyl, isopropyl, and tolyl groups. 2 , R 6 and R 7 represents an amine protecting group, which is well known to those skilled in the art.

[0059] Compounds of formula 2 may be prepared by conversion of compound C2 by reductive amination of the ketone moiety and reduction of the enone moiety. This conversion may require chemical or enzymatic catalysis. Compound C2 may be prepared by reactions including, but not limited to, the Wittig reaction of compound C3 with compound C4 or the aldol reaction of compound C3 with compounds C5 or C6, followed by decarboxylation, if necessary, under conditions well known to those skilled in the art. Compound C12 can react with C33 in the presence of a base to form C34. C34 can be obtained by stereoselective hydrolysis of an ester (R 1 where one of the is now hydrogen). The resulting acid moiety can be reacted with a nitrogen source to prepare an amide, which can then be reacted under Curtius, Rossen, or Hoffmann rearrangement conditions to yield compound 2.

[0060] The compound of formula 2 may be prepared by reductive amination of compound C7, which may require chemical or enzymatic catalysis. Compound C7 may be prepared by alkylation of compound C9 with compound C10 or compound C9 with compound C11, followed by oxidation of the resulting product C8. This may require chemical or enzymatic catalysis. Compound C7 may be prepared by reacting compound C12 with compound C13 in the presence of a thiazolium salt or by reacting compound C14 with compound C15 in the presence of a catalyst such as a tertiary amine and a phosphine. Compound C7 may be prepared by coupling C27 and C28 to form C26 or C29, followed by rearrangement via C25 or C30.

[0061] The compound of formula 2 may be prepared by converting compound C31 by reductive amination of the ketone moiety and reduction of the enone moiety. This conversion may require chemical or enzymatic catalysis. Compound C31 may be prepared from C16 in the presence of ammonia or from C17 in the presence of acid or base. Compounds C16 and C17 may be prepared by reacting compound C18 with C19 or C20, respectively, under conditions well known to those skilled in the art. Compound C18 may be prepared from compound C32 in the presence of a reagent, including but not limited to, acetic anhydride. Compound C31 may be prepared from compound C21 under acid or base conditions. Compound C21 may be prepared by alkylation of C23 with C24, followed by reaction with C20 and decarboxylation.

[0062] [ka]

[0063] In Reaction Scheme D, the variable group R 1 , R 3 , R 10 , R 13 , R 14 , R 15 and R 16represents an alkyl or aryl group, including, but not limited to, methyl, ethyl, isopropyl, and tolyl groups. 2 , R 4 , R 5 , R 7 , R 8 , R 9 and R 11 represents an amine protecting group, which is well known to those skilled in the art.

[0064] Compounds of formula 2 may be prepared by the reaction of compound D8 with other compounds, including, but not limited to, compounds D2, D3, D4, D5, D6, and D7. The transformation may require components, including, but not limited to, chemical or enzymatic catalysts and acids or bases. Those skilled in the art will recognize that the products obtained by these transformations may require further transformations. For example, the product obtained by alkylation of D8 with D7 may require reductive amination in the presence of a chemical catalyst or enzyme.

[0065] Compounds of formula 2 may be prepared by reduction of compound D10 in the presence of an enzyme or chemical catalyst, followed by deprotection if necessary. Compound D10 may be prepared by reaction of compound D11 or D12 with D2, D3, or D7 in the presence of a metal catalyst and zinc or other metal as a stoichiometric reagent, followed by additional transformations such as aminolysis, reductive amination, and / or deprotection. Compound D10 may be prepared by reaction of D13 or D14 with D2, D3, or D7 in the presence of a metal or organic catalyst, followed by additional transformations such as aminolysis, reductive amination, and / or deprotection.

[0066] The compound of formula 2 may be prepared by reduction of compound D15. This transformation may require chemical or enzymatic catalysis. D15 may be prepared by reaction of D16 with D2, D3, D4, D5, D6, or D7 in the presence of a chemical catalyst, enzyme, and / or base, followed by additional transformations such as reductive amination and / or deprotection. The compound of formula 2 may be prepared by decarboxylation of compound D17 in the presence or absence of a catalyst. Compound D17 may then be prepared by reaction of D18 with D2, D3, D4, D5, D6, or D7 in the presence of a chemical catalyst, enzyme, and / or base, followed by additional transformations such as reductive amination and / or deprotection.

[0067] Compounds of formula 2 may be prepared by reduction of compounds D19, D20, D21, or D22. This conversion may require chemical or enzymatic catalysis. D19 and D20 may be prepared by reaction of D16 with D23 or D24 in the presence of an acid or base. D21 and D22 may be prepared by reaction of D8 with D23 or D24 in the presence of an acid or base. Alternatively, D8 or D16 may be reacted with D28 in the presence of a base, followed by reaction with R 1 Alcoholysis with OH can produce D19, D20, D21 or D22.

[0068] The compound of formula 2 may be prepared by reduction of compound D25. This conversion may require chemical or enzymatic catalysis. Compound D25 may be prepared by reaction of D11 with D26 in the presence of a metal catalyst or reaction of D12 with D26 in the presence of a metal catalyst, followed by aminolysis.

[0069] Compounds of formula 2 may be prepared by reaction of compound D27 with compounds D2, D3, D4, D5, D6 or D7 in the presence of a reducing agent and metal catalyst, including but not limited to zinc and manganese, followed by deprotection and further transformations, including but not limited to reductive amination.

[0070] [ka]

[0071] In Scheme E, R 1 , R 2 , R 5 and R 16 The R group is an alkyl or aryl group, including, but not limited to, methyl, ethyl, isopropyl, and tolyl groups. 3 , R 4 , R 6 , R 7 , R 8 , R 9 , R 12 , R 13 , R 14 , R 15 and R 17 The groups are protecting groups well known to those skilled in the art. 1 .

[0072] The N-protected glutamate ester E2 may be reacted with a base and the resulting anion may be reacted with a reagent including, but not limited to, E3, E4, E5, E6, E7, E8, E9, and E10. The resulting product may then be converted to 2 by using methods well known to those skilled in the art.

[0073] The N-protected glutamic acid ester E20 may be reacted with a base, and the resulting product may be reacted with a reagent including, but not limited to, E3, E4, E5, E6, E7, E8, E9, and E10. The alkylation may or may not be stereoselective. The resulting product may be subjected to reductive amination in the presence of a chemical or enzymatic catalyst, such as a transaminase. The resulting product may then be converted to 2 by using methods well known to those skilled in the art.

[0074] E2 can be converted to E23 via amidation, which can then be reacted with a base to yield compound 2.

[0075] Compound 2 may be prepared by rearrangement and deprotection (if necessary) of E24. E24 may be prepared by cyanide addition to E25 followed by alkene and nitrile reduction or reduction and deprotection of E26. E25 or E26 may be prepared from E27 under conditions well known to those skilled in the art.

[0076] E28 or E29 can be converted to compound 2 under conditions well known to those skilled in the art. For example, E28 can be subjected to olefin reduction conditions in the presence of a chemical or enzymatic catalyst, such as, but not limited to, an enereductase, followed by ester saponification, asymmetric decarboxylation, and nitrile reduction to produce E30, which can be carried out in the presence of a chemical or enzymatic catalyst. Alternatively, E29 can be subjected to reduction of the olefin and cyano group in the presence of a chemical and enzymatic catalyst to produce E30. Compound 2 can then be produced by intramolecular cyclization of E30, after deprotection if necessary.

[0077] Compound 2 can be converted to compound E11 by methods including, but not limited to, halogenation with N-halosuccinimides and enzymatic halogenation. Compound E11 can then be reacted with reagents including, but not limited to, E12 and KCN. The resulting product can then be converted to 2 by using methods well known to those skilled in the art.

[0078] Compounds E15 or E16 can be reacted with a reagent such as E13 or E14 to form 1 in the presence of a base and / or a chiral catalyst.

[0079] Compound E22 can be subjected to Strecker reaction conditions well known to those skilled in the art to prepare E21, which can then be converted to 1 under conditions well known to those skilled in the art. The synthesis of E21 can be carried out in a stereoselective manner, or the conversion of E21 to 1 can be carried out via enzymatic resolution or dynamic kinetic asymmetric transformation.

[0080] Compound 2 may be prepared by reaction of E17 and E18, followed by deprotection if necessary. E17 may be prepared from E19 via methods well known to those skilled in the art.

[0081] Process for the preparation of nilmatrervir In Step 2 of Reaction Scheme 1, under certain reaction conditions, (S)-N,N-diethyl-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanamide (diethylamide impurity, denoted as IMP-S2-1), (1R,2S,5S)-3-((R)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (epimeric impurity, denoted as IMP-S2-2) It was found that impurities such as N-((S)-1-((1R,2S,5S)-2-((1R,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-3-carbonyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexan-3-yl)-3,3-dimethyl-1-oxobutan-2-yl)-2,2,2-trifluoroacetamide (bisamide impurity, designated as IMP-S2-3) were formed (see reaction scheme below). Advantageously, the present invention minimizes the formation of these undesirable impurities. The mechanism of formation of (S)-N,N-diethyl-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanamide, IMP-S2-1, is unknown but is independent of the presence of diethylamine in the triethylamine (TEA) used in the reaction. The diethylamide impurity was observed to form when methanesulfonyl chloride was added to a mixture of (S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoic acid (Compound V) in the presence of triethylamine, or when a larger excess of triethylamine (3.0 equivalents to 2.5 equivalents of TEA) was used in the reaction. In both cases, the amount of diethylamide and bisamide impurities was minimized by adding methanesulfonyl chloride to the compound of Formula V in isopropyl acetate before the addition of triethylamine. The use of methanesulfonyl chloride and TEA was also found to be advantageous due to the minimal epimerization that occurs when using these reagents in the amidation reaction.Thus, the present invention provides significant advantages in that the claimed Step 2 process minimizes the formation of undesirable diethylamide and bisamide impurities, IMP-S2-1 and IMP-S2-3, by controlling the order of addition of methanesulfonyl chloride to the reaction mixture, and also minimizes the amount of epimerized product, IMP-S2-2, formed by controlling the amount of triethylamine base used.

[0082] Reaction of Potential Impurities with Step 2 of Scheme 1

[0083] [ka] It has been found that in Step 3 of the Reaction Scheme 1 process, an acylurea impurity and a rearrangement-related impurity can form under certain reaction conditions. The structures of the acylurea impurities that can form in Step 3 are depicted below as IMP-S3-1 and IMP-S3-2. IMP-S3-1 is (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-N-(3-(dimethylamino)propyl)-N-(ethylcarbamoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide, and IMP-S3-2 is (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-N-((3-(dimethylamino)propyl)carbamoyl)-N-ethyl-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide. It has been found that under certain reaction conditions, (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl)propenamide can undergo a rearrangement reaction to form the rearrangement impurity IMP-S3-3, which can further react under the amidation reaction conditions of Step 3 to form an additional impurity designated IMP-S3-4. IMP-S3-3 is (2S,4S)-4-(2-aminoethyl)-5-oxopyrrolidine-2-carboxamide, and IMP-S3-4 is (1R,2S,5S)-N-(2-((3S,5S)-5-carbamoyl-2-oxopyrrolidin-3-yl)ethyl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide.Minimizing the amount of IMP-S3-4 formed is desirable to avoid further carryover of this impurity and subsequent reaction under the reaction conditions of Step 4, in which the amide moiety in the lactam ring of IMP-S3-4 may be converted to a nitrile moiety to form an impurity designated IMP-S4-1, which is (1R,2S,5S)—N-(2-((3S,5S)-5-cyano-2-oxopyrrolidin-3-yl)ethyl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide.

[0084] Reaction of Step 3 with potential impurities formed in Steps 3 and 4 of Reaction Scheme 1

[0085] [ka]

[0086] [ka] In Step 3, the use of unrecrystallized (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl)propanamide hydrochloride (Compound III) or recrystallized (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl)propanamide hydrochloride (Compound III) was found to affect the amount of acylurea impurities IMP-S3-1 and IMP-S3-2, as shown in column 4 of the Table below: Reaction Conditions for Step 3. When unrecrystallized (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl)propanamide hydrochloride (Compound III) was used in two reactors set at 50° C., 5% or less of the acylurea impurities IMP-S3-1 and IMP-S3-2 were formed, and 2% or less of the rearrangement impurity IMP-S3-3 was formed with favorable reaction kinetics (less than 3% of the starting compound IV remained after about 2 hours of reaction time). When recrystallized (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl)propanamide hydrochloride (Compound III) was used in two reactors set at 50° C., 10% or less of the acylurea impurities IMP-S3-1 and IMP-S3-2 were formed, and 2% or less of the rearrangement impurity IMP-S3-3 was formed with favorable reaction kinetics (less than 3% of the starting compound IV remained after about 6 hours of reaction time). The two-reactor process, when run at 50° C., provides favorable reaction kinetics while maintaining relatively low levels of acylurea and rearrangement impurities that may form. The initial process conditions (row 2 in the table below) suffered from less-than-desirable reaction kinetics (16 hours or 60-100 hours of reaction time, depending on the type of compound III used), while the one-reactor process, when run at 50° C., resulted in the formation of large amounts of rearrangement impurity (see row 3 in the table; either 50% or more, or 40% or more, of the rearrangement impurity formed).

[0087] [Table 1-1]

[0088] [Table 1-2]

[0089] Step 5: Nilmatrervir API crystallization process with optimal particle size control Crystallization of PF-07321332 anhydrous Form 1, Compound I, begins with a solution of PF-07321332 MTBE solvate (Compound I') in isopropyl acetate solvent, followed by seeding with anhydrous Form 1 solid and the addition of heptane as an antisolvent. This step is critical for controlling particle size and polymorphic form. Therefore, crystallization and isolation of PF-07321332 is a key process for controlling API physical properties for optimal drug product performance.

[0090] The initial concentration of API (PF-07321332 MTBE solvate (Compound I')) in isopropyl acetate, heptane addition time, seed size, and seed amount had a detectable effect on the final particle size distribution. Statistical analysis was performed using Design Expert. Main effect plots and contour plots were used to graphically summarize the analytical results to facilitate visualization of statistically significant factor effects and allow assessment of their practical importance. Figure 1 shows the relationship between the initial concentration of API in isopropyl acetate (mL / g), heptane addition time (in hours), seed loading (% wt of Compound I, Form I per weight of Compound I', MTBE solvate), and seed size (in microns) at the final D[v,0.5] count in microns of the particle size distribution (PSD).

[0091] The crystallization process was performed in a robust and consistent manner from small to large scales. Furthermore, the target particle size was achieved through direct crystallization without the need for post-crystallization milling. The process exhibits size-dependent growth, which depends on the size of the seed material. To minimize the dependency on seed size, the seed loading and duration of heptane addition were adjusted. The seed loading was optimized between 0.2 wt% and 1.5 wt%, with a target value of 0.75 wt% across all scales. The duration of heptane addition was adjusted to 10 hours, ranging from 6 to 15 hours. These changes helped achieve the desired final particle size regardless of the seed size input. The process relies on primary and secondary nucleation instead of size-dependent crystal growth. Data obtained from different scales demonstrate consistent particle size delivery with minimal variability. A process with a seed loading of approximately 0.75 wt% and heptane addition over approximately 10 hours provides consistent production of nilmatrervir with well-controlled particle size distribution. Figure 2 shows the correlation between seed size and final particle size distribution after crystallization of API at 0.75 wt% seed loading (blue circles) and 0.5 wt% seed loading (orange circles) in laboratory-scale (100 mL) experiments. Batch history data shown in Figures 3 and 4 from two different locations demonstrate that the process delivers particle size distributions in a robust and consistent manner, and in each case shows the PSD obtained for D[v,0.5] of over 50 batches. The particle size distributions for individual batches were generally found to have D[v,0.5] in the ranges of about 12 microns to about 18 microns, about 14 microns to about 16 microns, with most having a D[v,0.5] of about 15 microns.

[0092] Experimental procedure The following illustrates the synthesis of various compounds of the present invention. Additional compounds within the scope of the present invention may be prepared using the methods illustrated in these examples, either alone or in combination with techniques generally known in the art. All starting materials in these preparations and examples are either commercially available or can be prepared by methods known in the art or as described herein.

[0093] All reactions were carried out under a nitrogen or argon atmosphere with continuous stirring unless otherwise noted. Where appropriate, reaction apparatus was dried using a heat gun under dynamic vacuum, and anhydrous solvents (Sure-Seal™ products from Aldrich Chemical Company, Milwaukee, Wisconsin, or DriSolv™ products from EMD Chemicals, Gibbstown, NJ) were used. In some cases, commercially available solvents were passed through a column packed with 4 Å molecular sieves until the following water QC criteria were reached: a) less than 100 ppm for dichloromethane, toluene, N,N-dimethylformamide, and tetrahydrofuran; b) less than 180 ppm for methanol, ethanol, 1,4-dioxane, and diisopropylamine. For highly sensitive reactions, solvents were further treated with sodium metal, calcium hydride, or molecular sieves and distilled immediately before use. Other commercially available solvents and reagents were used without further purification. For syntheses referencing procedures in other examples or methods, reaction conditions (reaction times and temperatures) may vary. The product was typically dried under vacuum before being carried on to further reactions.

[0094] Reaction progress can be monitored using thin-layer chromatography (TLC), liquid chromatography-mass spectrometry (LCMS), high-performance liquid chromatography (HPLC), and / or gas chromatography-mass spectrometry (GCMS). TLC is performed on precoated silica gel plates using a fluorescent indicator (254 nm excitation wavelength) and can be visualized under UV light and / or using I2, KMnO4, CoCl2, phosphomolybdic acid, and / or ceric ammonium molybdate stains. LCMS data can be acquired on an Agilent 1100 series instrument using a Leap Technologies autosampler, a Gemini C18 column, an acetonitrile / water gradient, and either trifluoroacetic acid, formic acid, or ammonium hydroxide modifiers. Column eluates are analyzed using a Waters ZQ mass spectrometer scanning from 100 to 1200 Da in both positive and negative ion modes. Other similar instruments can also be used. HPLC data were generally acquired on an Agilent 1100 series instrument using the indicated column, an acetonitrile / water gradient, and either trifluoroacetic acid or ammonium hydroxide modifier. GCMS data were acquired using an HP6890 injector, an HP-1 column (12 m × 0.2 mm × 0.33 μm), and a Hewlett Packard 6890 oven with helium carrier gas. Samples can be analyzed with an HP5973 mass-selective detector scanning from 50 to 550 Da using electron ionization. Purification was performed by medium-pressure liquid chromatography (MPLC) using an Isco Combiflash Companion, an AnaLogix IntelliFlash 280, a Biotage SP1, or a Biotage Isolera One instrument and prepacked Isco ReadySep or Biotage Snap Silica cartridges.Chiral purification was performed by chiral supercritical fluid chromatography (SFC), typically using a Berger or Thar instrument; columns such as ChiralPak-AD, -AS, -IC, Chiralcel-OD, or -OJ columns; and CO mixtures with methanol, ethanol, 2-propanol, or acetonitrile, alone or modified with trifluoroacetic acid or propan-2-amine. UV detection can be used to induce fraction collection. In syntheses referencing procedures in other examples or methods, purification may vary, and typically the solvents and solvent ratios used in the eluents / gradients are adjusted to the appropriate R. f or selected to provide retention times.

[0095] Mass spectrometry data are reported via LCMS analysis. Mass spectrometry (MS) is performed via atmospheric pressure chemical ionization (APCI), electrospray ionization (ESI), electron impact ionization (EI) or electron scattering ionization (ES) sources. Proton nuclear magnetic resonance spectroscopy ( 1 H NMR (H NMR) chemical shifts are reported in parts per million downfield from tetramethylsilane and were recorded on 300, 400, 500, or 600 MHz Varian, Bruker, or Jeol spectrometers. Chemical shifts are expressed in parts per million (ppm, δ) referenced to deuterated solvent residual peaks (chloroform, 7.26 ppm; CD2HOD, 3.31 ppm; acetonitrile-d2, 1.94 ppm; dimethyl sulfoxide-d5, 2.50 ppm; DHO, 4.79 ppm). Peak shapes are described as follows: s, singlet; d, doublet; dd, doublet of doublets; ddd, doublet of doublets of doublets; dt, doublet of triplets; t, triplet; q, quartet; qd, quartet of doublets; quin, quintet; m, multiplet; brs, broad singlet; app, apparent. Analytical SFC data were generally acquired on a Berger analytical instrument as described above. Optical rotation data were acquired on a PerkinElmer Model 343 polarimeter using a 1 dm cell. Microanalysis was performed by Quantitative Technologies Inc. and was within 0.4% of the calculated values.

[0096] Unless otherwise noted, chemical reactions were carried out at room temperature (approximately 23 degrees Celsius). Unless otherwise noted, all reactants were obtained commercially and used without further purification, or were prepared using methods known in the literature.

[0097] The terms "concentrated," "evaporated," and "concentrated in vacuo" refer to removal of solvent under reduced pressure on a rotary evaporator with a bath temperature of less than 60° C., or at the temperature as specified. The abbreviations "min" and "h" stand for "minutes" and "hours," respectively. The term "TLC" refers to thin layer chromatography, "room or ambient temperature" means a temperature between 18 and 25° C., "GCMS" refers to gas chromatography-mass spectrometry, "LCMS" refers to liquid chromatography-mass spectrometry, "UPLC" refers to ultra-performance liquid chromatography, "HPLC" refers to high-performance liquid chromatography, and "SFC" refers to supercritical fluid chromatography. Other abbreviations used include: "°C" is degrees Celsius, "CO2" is carbon dioxide, "eq." or "equiv." is equivalent, "DMSO-d6" is hexadeuterodimethylsulfoxide, "g" is grams, "HCl" is hydrogen chloride, "HOPO" is 2-hydroxypyridine-N-oxide, "HRMS" is high resolution mass spectroscopy, "Hz" is hertz, "iPrOAc" is isopropyl acetate, "K" is kelvin, "kg" is kilogram, "L" is liter, "M" is mole or molar concentration, "mbar" is millibar, and "MEK" is "MeOH" is methyl ethyl ketone, "MHz" is megahertz, "mg" is milligram, "μg" is microgram, "min" is minute, "mL" is milliliter, "μL" is microliter, "mm" is millimeter, "mmol" is millimole, "μmol" is micromole, "MTBE" is methyl tert-butyl ether, "NaCl" is sodium chloride, "NaHCO3" is sodium bicarbonate, "Na2SO4" is sodium sulfate, "PXRD" is powder X-ray diffraction, and "THF" is tetrahydrofuran.

[0098] Hydrogenation may be carried out in a Parr shaker under pressurized hydrogen gas or in a Thales-nano H cube flow hydrogenator with full hydrogen and a flow rate between 1-2 mL / min at the specified temperature, or as otherwise specified.

[0099] HPLC, UPLC, LCMS, GCMS and SFC retention times are measured using the methods noted in the procedures.

[0100] The optical rotation of an enantiomer can be measured using a polarimeter. According to its observed rotation data (or its specific rotation data), the enantiomer with clockwise rotation is designated the (+)-enantiomer, and the enantiomer with counterclockwise rotation is designated the (-)-enantiomer. A racemate is designated either by the absence of a drawn or written stereochemistry or by the presence of (+ / -) adjacent to the structure; in this latter case, the designated stereochemistry represents only one of the two enantiomers that make up the racemic mixture.

[0101] The compounds and intermediates described below were named using the naming conventions provided by ACD / ChemSketch 2019.1.1, file version C05H41, build 110712 (Advanced Chemistry Development, Inc., Toronto, Ontario, Canada) or using the structure naming feature in ChemDraw 18.0 (PerkinElmer® Informatics; perkinelmer.com). The naming conventions provided by ACD / ChemSketch 2019.1.1 or ChemDraw 18.0 are well known by those skilled in the art, and the naming conventions provided by ACD / ChemSketch 2019.1.1 are generally believed to conform to the IUPAC (International Union of Pure and Applied Chemistry) recommendations for the nomenclature of organic compounds and the CAS indexing rules.

[0102] General methods for solid form characterization Solid state morphology data was obtained for certain starting materials, intermediates and products using the following powder X-ray diffraction method.

[0103] Powder X-ray diffraction: Powder X-ray diffraction analysis was performed using a Bruker AXS D4 Endeavor diffractometer equipped with a Cu radiation source. The divergence slit was set to 0.6 mm, while a variable slit was used for the secondary optics. Diffracted radiation was detected by a PSD-LynxEye detector. The X-ray tube voltage and amperage were set to 40 kV and 40 mA, respectively. Data were collected on a theta-2theta goniometer at Cu wavelengths from 3.0 to 40.0 degrees 2theta using a 0.020 degree step width and a 0.3 second step time. Samples were prepared by placing them in a silicon low-background sample holder and rotating during collection.

[0104] Powder X-ray diffraction analysis was performed using a Bruker AXS D8 Advance diffractometer equipped with a Cu radiation source. Diffracted radiation was detected by a Lynx Eye EX detector with motorized slits. Both the primary and secondary beams were equipped with 2.5 Soller slits. The X-ray tube voltage and amperage were set at 40 kV and 40 mA, respectively. Data were collected on a theta-theta goniometer using a locked-couple scan of the Cu K-alpha (mean) wavelength from 3.0 to 40.0 degrees 2-theta in 0.02-degree increments, with a scan speed of 0.5 seconds per step. Samples were prepared by placing them in a silicon low-background sample holder.

[0105] Data were collected on both instruments using Bruker DIFFRAC Plus software, and analysis was performed using EVA Diffract Plus software. PXRD data files were not processed prior to peak searching. Preliminary peak assignments were made using peaks selected at a threshold of 1 using the peak search algorithm in the EVA software. To ensure validity, adjustments were made manually, the output of the automatic assignment was visually checked, and peak positions were adjusted to the peak maximum. Peaks with a relative intensity of 3% or greater were generally selected. Peaks that were unresolved or consistent with noise were typically not selected. Typical errors associated with peak positions from PXRD are listed in the USP as a maximum of + / - 0.2° 2-theta (USP-941). Unless otherwise noted, the variance for each peak reported in the PXRD peak table is + / - 0.2° 2-theta.

[0106] Preparation of starting materials Preparation of methyl (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate, hydrochloride (Compound VII)

[0107] [ka] The title compound is prepared according to the following procedures, as depicted above in Reaction Scheme SM-1.

[0108] CoBr2 (0.05-0.15 equivalents), (1E,1'E)-1,1'-(pyridine-2,6-diyl)bis(N-(2-(tert-butyl)phenyl)ethan-1-imine) or (1E,1'E)-1,1'-(pyridine-2,6-diyl)bis(N-(2-isopropylphenyl)ethan-1-imine) (0.05-0.15 equivalents, i.e., ligand), and tetrahydrofuran (10 volumes) were charged to a reactor. Zn (2.25-2.5 equivalents) was charged. I2 (0.25 equivalents) in tetrahydrofuran (1-2 volumes) was charged. A purple solution was obtained. 1-(tert-butyl) 2-methyl (S)-2,5-dihydro-1H-pyrrole-1,2-dicarboxylate (1 equivalent, compound VII") was charged as neat oil. 2,2-Dichloropropane or 2,2-dibromopropane (1.5-2.0 equivalents) in tetrahydrofuran (1-3 volumes) was added slowly and stirred until the reaction was complete (formation of compound VII'). The reaction mixture was filtered through Celite. Methyl tert-butyl ether (MTBE), HCl, and water were used during this procedure. The organic phase was washed with HCl, water, and dried over magnesium sulfate or sodium sulfate. The solution was concentrated to 1-2 volumes. The resulting solution was purified by HCl in methanol (3 equivalents) or tetrahydrofuran and HCl gas ( The reaction was treated with 3 equivalents of methyl (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate, hydrochloride, was isolated as a solid from MTBE and THF upon completion. Typical yields range from 50 to 80%. For example, 3.00 g of 1-(tert-butyl) 2-methyl (S)-2,5-dihydro-1H-pyrrole-1,2-dicarboxylate was converted to 1.98 g of methyl (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate, hydrochloride (80% yield). Product characterization data was consistent with previously reported data: Oruganti, S. et al., Tetrahedron, 2017, 73, 4285.

[0109] The PXRD for the product methyl (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate, hydrochloride salt (Compound VII) is provided in FIG.

[0110] Preparation of methyl (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl)propanoate para-toluenesulfonate The title compound is prepared as depicted in Reaction Scheme SM-2 and as described below.

[0111] [ka]

[0112] The reactor was charged with dimethyl(tert-butoxycarbonyl)-L-glutamate (50.2 g, 1.00 equiv.) and tetrahydrofuran (753 mL, 15 mL / g). The solution was cooled to -78°C in a dry ice / acetone bath. A pre-cooled solution of lithium bis(trimethylsilyl)amide (1 M) in THF (371 mL, 2.1 equiv.) was added via cannula. A pre-cooled solution of bromoacetonitrile (13.5 mL, 1.07 equiv.) in THF (70 mL, 1.4 mL / g) was charged to the reactor. After the addition was complete, the mixture was stirred for at least 45 minutes while maintaining the temperature below -70°C. The reaction mixture was charged with a solution of methanol (20 mL, 0.4 mL / g) in tetrahydrofuran (25 mL, 0.5 mL / g). To the reaction mixture, acetic acid (40.5 mL, 4 equiv.) in tetrahydrofuran (100 mL, 2 mL / g) was charged via addition funnel. The mixture was warmed to -20°C over 30 minutes. Sodium chloride (12% by weight) in water (251 mL, 5 mL / g) was charged to the mixture. The layers were separated. The organic layer was concentrated to 2-3 mL / g. To the residue, toluene (1000 L, 20 mL / g) was charged. The mixture was concentrated to approximately 20 mL / g, then a constant volume distillation was performed while maintaining the concentration at 20 mL / g. Diatomaceous earth (10 g, 0.2 g / g) was charged to the solution, and the slurry was stirred for 2 hours. The slurry was filtered through a bed of diatomaceous earth (10 g, 0.2 g / g). The filter cake was washed with toluene (1 mL / g). The filtrate was concentrated to approximately 2-3 mL / g. Methanol (250 mL, 5 mL / g) was charged. Methanol (650 mL, 13 mL / g) was replaced and a constant volume distillation was performed. The resulting dimethyl (2S,4R)-2-((tert-butoxycarbonyl)amino)-4-(cyanomethyl)pentanedioate was carried over as a light amber solution (44.5 g in solution, 80% in situ yield).

[0113] The reactor was charged with Raney Nickel 2400 (6.05 g, 100 wt%). A solution of dimethyl (2S,4R)-2-((tert-butoxycarbonyl)amino)-4-(cyanomethyl)pentanedioate in methanol (6 g charge, 40.83 g solution) was charged, followed by additional methanol (120 mL, 20 mL / g portion) and ammonia (7N in MeOH, 2.7 mL, 1.0 equiv). The reactor was purged three times with N2 and then three times with H2. The reactor was pressurized to 5 bar, heated to 24°C, and stirred for 24 hours. The reactor was purged and sampled to confirm completion of the reaction (i.e., the uncyclized intermediate dimethyl (2S,4S)-2-(2-aminoethyl)-4-((tert-butoxycarbonyl)amino)pentanedioate was converted to the cyclized intermediate methyl (S)-2-((tert-butoxycarbonyl)amino)-3-((S)-2-oxopyrrolidin-3-yl)propanoate). The reaction was filtered, and the catalyst was washed twice with methanol (5 mL / g, 30 mL). The filtrate was concentrated in vacuo to approximately 2-3 mL / g in preparation for the next step. Isopropanol (20 mL / g, 125 mL) was charged and concentrated to remove residual MeOH and water, targeting a total volume of 7 mL / g (6 mL / g IPA). The solution was charged with para-toluenesulfonic acid (pTsOH) monohydrate (1.5 equiv.). The mixture was diluted with isopropanol (20 mL / g), and the solution was reconcentrated to remove water. MTBE (4 mL / g) was charged to the reactor. The slurry was warmed to 50°C and held overnight. The mixture was cooled to 10°C over 2 hours. The mixture was held for 2 hours and then filtered. The filter cake was washed with isopropanol, and the product was dried in a vacuum oven at 45-50°C. 5.86 g of methyl (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl)propanoate para-toluenesulfonate was isolated as a white crystalline solid (86% yield over two steps). 1 H NMR (600 MHz, DMSO-d6, 298K): δ 8.54 (s, 2H), 7.98 (s, 1H), 7.95 (s, 1H), 7.65 (s, 1H), 7.49 (d, J = 8.0 Hz, 2H), 7.12 (d, J = 8.0 Hz, 2H), 4.32 (m, 1H), 4.21 (m, 1H), 3.75 (s, 3H), 3.19 (m, 2H), 2.56 (m, 1H), 2.55 (m, 1H), 2.29 (s, 3H), 2.26 (m, 1H), 2.01 (m, 1H), 1.89 (m, 1H), 1.66 (m, 1H). 13 C NMR (150 MHz, DMSO-d6, 298K): δ 178.2, 169.7, 145.5, 137.6, 128.0, 125.4, 52.8, 51.2, 39.8, 38.3, 31.6, 27.6, 20.7. HRMS: (ESI + ) C8H 15 N2O3 + Calculated value: 187.1077, Measured value: 187.1077

[0114] Alternative preparation of methyl (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl)propanoate para-toluenesulfonate The title compound is prepared as depicted in Reaction Scheme SM-3 and as described below.

[0115] [ka]

[0116] Step 1: Preparation of 1-(tert-butyl) 2-methyl (S,Z)-4-((dimethylamino)methylene)-5-oxopyrrolidine-1,2-dicarboxylate 1-(tert-butyl) 2-methyl (S)-5-oxopyrrolidine-1,2-dicarboxylate (10.00 g) was added to toluene (60 mL) with stirring at 20 °C to give a clear, colorless solution. To this solution, tert-butoxybis(dimethylamino)methane (Bredereck's reagent) (8.60 g, 10.2 mL, 1.2 equiv.) was added in one portion, and the resulting reaction mixture was stirred at 95–100 °C for 16 h. The reaction was checked for completion (2.7% starting material remained) by UPLC, and then the temperature was adjusted to 80 °C. 40 mL of n-heptane was added to give a yellow solution, and the mixture was cooled to 60 °C; at this point, seed crystals of the product, if available, could be added. The mixture was cooled to 5 °C over 2 h, and the resulting slurry was stirred at 5 °C for 2 h. The mixture was filtered, and the resulting filter cake was washed with toluene (10 mL). The resulting solid was dried under vacuum at 40° C. overnight to give 1-(tert-butyl) 2-methyl(S,Z)-4-((dimethylamino)methylene)-5-oxopyrrolidine-1,2-dicarboxylate as a white to off-white solid (10.3 g, 84% yield). 1 H NMR (400 MHz, dimethyl sulfoxide-d6) δ 6.97 (t, 1H), 4.50 (dd, 1H), 3.69 (s, 3H), 3.22 (m, 1H), 2.99 (s, 6H), 2.81 (m, 1H), 1.39 (s, 9H). 1 >97% purity by H NMR

[0117] Step 2: Preparation of 1-(tert-butyl) 2-methyl (S,E)-4-(cyanomethylene)-5-oxopyrrolidine-1,2-dicarboxylate To a 250 mL single-neck round-bottom flask under an inert atmosphere equipped with a toxic gas scrubber was added 1-(tert-butyl)2-methyl(S,Z)-4-((dimethylamino)methylene)-5-oxopyrrolidine-1,2-dicarboxylate (5.00 g, 1.00 equiv.) and acetic acid (30.0 mL). The mixture was stirred at 20° C. to form a solution, to which potassium cyanide (1.20 g, 1.10 equiv.) was added. The reaction mixture was stirred at 20° C. for 24 hours to give a dark yellow solution. Water (210 mL, 7 volumes) was added slowly over 25 minutes to precipitate the product. The mixture was then stirred for 5 minutes before being filtered through a sintered funnel. The resulting white solid was washed with water (3×30 mL) and dried on a sintered funnel using an air stream under vacuum. The resulting 1-(tert-butyl) 2-methyl (S,E)-4-(cyanomethylene)-5-oxopyrrolidine-1,2-dicarboxylate was obtained as a white solid (2.47 g, 51% yield). 1 H NMR (396 MHz, chloroform-d) δ 6.34 (t, J = 3.0 Hz, 1H), 4.74 (dd, J = 9.7, 3.0 Hz, 1H), 3.81 (s, 3H), 3.29 (qd, J = 9.9, 3.5 Hz, 1H), 3.01 (dt, J = 19.6, 2.7 Hz, 1H), 1.52 (s, 9H). 1 >95% pure by H NMR. This material was used in the next step without further purification.

[0118] Step 3: Preparation of 1-(tert-butyl) 2-methyl (2S,4S)-4-(2-aminoethyl)-5-oxopyrrolidine-1,2-dicarboxylate hydrochloride (as a solution in methanol) Methanol (200 ml, 20 ml / g) was stirred in a reactor at 5°C, to which acetyl chloride (2.79 mL, 1.1 equivalents) was added dropwise to control the resulting exotherm, producing anhydrous HCl (and 1.1 equivalents of methyl acetate by-product). The solution was then warmed to 20°C. 1-(tert-butyl)2-methyl(S,E)-4-(cyanomethylene)-5-oxopyrrolidine-1,2-dicarboxylate (10.0 g, 1.0 equivalents, limiting reagent) was added as a solid to the methanolic HCl solution in one portion to give a clear, colorless solution. To this solution was charged 5% Pd / C-form A503023-5 (2.0 g). The reactor was purged three times with nitrogen, then three times with hydrogen, and then pressurized to 50 psi with hydrogen, and the mixture was stirred at 600 rpm for 16 hours. The reactor was purged and sampled to confirm reaction completion. The reaction was incomplete (>3% residual nitrile intermediate), so it was hydrogenated for an additional 16 hours to reduce the nitrile intermediate content from 20% to 3%. The reaction mixture was filtered through Arbocel to remove the catalyst, and the filter was washed with methanol (2 × 10 mL, 2 × 1 mL / g). The resulting methanol filtrate, containing 1-(tert-butyl) 2-methyl(2S,4S)-4-(2-aminoethyl)-5-oxopyrrolidine-1,2-dicarboxylate hydrochloride, was used directly in the next step.

[0119] Alternative Step 3: Preparation of 1-(tert-butyl) 2-methyl (2S,4S)-4-(2-aminoethyl)-5-oxopyrrolidine-1,2-dicarboxylate hydrochloride (as a solution in methanol / isopropanol) Methanol (200 ml) was stirred at 5° C., and acetyl chloride (2.79 mL, 1.1 equivalents) was added dropwise to control the resulting exotherm, producing anhydrous HCl (and 1.1 equivalents of methyl acetate by-product). In a separate hydrogenation vessel, 2-propanol (50 mL) was stirred at 20° C., and 1-(tert-butyl) 2-methyl(S,E)-4-(cyanomethylene)-5-oxopyrrolidine-1,2-dicarboxylate (10.0 g, 1.0 equivalents, limiting reagent) was added to produce a slurry. 5% Pd / C-form A503023-5 (2.0 g) was charged to the slurry as a solid. The reactor was purged with nitrogen three times, then with hydrogen three times, then pressurized to 50 psi with hydrogen, and stirred at 600 rpm for 2 hours. The reactor was purged with nitrogen, and then the methanolic HCl solution was added. The reactor was purged three times with nitrogen, then three times with hydrogen, then pressurized to 50 psi with hydrogen and stirred at 600 rpm for 3 days. The reactor was purged and sampled to confirm reaction completion (>3% residual nitrile intermediate), then filtered through Arbocel to remove the catalyst, and the filter was washed with methanol (2 × 10 mL). The resulting methanol / 2-propanol filtrate, containing 1-(tert-butyl) 2-methyl(2S,4S)-4-(2-aminoethyl)-5-oxopyrrolidine-1,2-dicarboxylate hydrochloride, was used directly in the next step.

[0120] Step 4: Preparation of methyl (S)-2-((tert-butoxycarbonyl)amino)-3-((S)-2-oxopyrrolidin-3-yl)propanoate (as a solution in isopropanol) The methanolic solution of 1-(tert-butyl) 2-methyl(2S,4S)-4-(2-aminoethyl)-5-oxopyrrolidine-1,2-dicarboxylate hydrochloride from Step 3 is stirred at 25 °C, and 1 M aqueous NaHCO (89.2 mL, 2.5 equiv.) is slowly added to control the rate of CO off-gassing. The resulting fine suspension is stirred at 25 °C for 18 h or until the reaction is complete (less than 0.5 area % of the starting material remains) as determined by liquid chromatography. The reaction is then quenched by the portionwise addition of citric acid (4.11 g, 0.60 equiv.) in water (30 mL) and tested with pH indicator paper to ensure the pH is between 5 and 7. The mixture is then concentrated under vacuum (90-100 mbar) to a volume of approximately 100-120 mL. To this, ethyl acetate (100 mL) is added, and the mixture is stirred at 25° C. for 10 minutes, and then the organic and aqueous layers are separated. Ethyl acetate (100 mL) is added to the aqueous layer, and the mixture is stirred at 25° C. for 10 minutes, and then the layers are separated. The organic layers are combined and concentrated in vacuo (200 mbar) to a final volume of approximately 30 mL. To this, 2-propanol (200 mL) is added, and the solution is concentrated in vacuo (100 mbar) to a final volume of approximately 70 mL. The isopropanol solution of methyl (S)-2-((tert-butoxycarbonyl)amino)-3-((S)-2-oxopyrrolidin-3-yl)propanoate is checked by Karl Fischer analysis to confirm that the water content is less than 1% by weight, and then used directly in the next step.

[0121] Step 5: Preparation of methyl (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl)propanoate para-toluenesulfonate The isopropanol solution of methyl (S)-2-((tert-butoxycarbonyl)amino)-3-((S)-2-oxopyrrolidin-3-yl)propanoate from step 4 is stirred at 20 °C. In a separate vessel, isopropanol (200 mL) and p-toluenesulfonic acid monohydrate (10.33 g, 1.5 equiv.) are added. The p-TSA solution is concentrated in vacuo (90-100 mbar) to a final volume of approximately 70 mL to remove water. The concentrated p-TSA solution is then added to the methyl (S)-2-((tert-butoxycarbonyl)amino)-3-((S)-2-oxopyrrolidin-3-yl)propanoate solution, followed by a line rinse of 2-propanol (20 mL). The solution is distilled under vacuum (90-100 mbar) at a jacket temperature of 40°C to a final volume of approximately 70 mL (7 mL / g) to remove additional water. The solution is sampled for Karl Fischer analysis (≤1 wt% water). If water is greater than 1% wt, additional 2-propanol is added as needed, and vacuum distillation is repeated until the target water content is achieved in step 5. The reaction mixture is heated to 50°C at atmospheric pressure and stirred for 12-18 hours until completion is achieved (no starting material is observed by liquid chromatography). The desired product crystallizes during this hold. Upon completion of the reaction, tert-butyl methyl ether (50 mL) is added in one portion. The resulting slurry is cooled from 50°C to 10°C over 2 hours. The slurry is stirred at 10°C for 1 hour, then filtered under vacuum. The crystallization vessel is rinsed with tert-butyl methyl ether (40 mL) and transferred to the filter as a cake wash. The product cake is removed and dried under vacuum to deliquor, and the product is then dried under vacuum at 40° C. The desired methyl (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl)propanoate para-toluenesulfonate is isolated as a white crystalline solid.

[0122] The yield for steps 3-5 from the above process using MeOH / AcCl hydrogenation is 44% (20% overall yield for steps 1-5), with the product having 98.7% achiral purity, 0.58% RRT 0.194, 0.28% RRT 1.783; 1% diastereomer 1, 0.68% diastereomer 2, approximately 1% enantiomer, and 98.7% wt by Q-NMR. The yield for steps 3-5 from the process using isopropanol two-step hydrogenation is 63% (30% overall yield for steps 1-5), with the product having 98.7% achiral purity, 0.65% RRT 0.194, 0.24% RRT 1.784; 1% diastereomer 1, 0.40% diastereomer 2, approximately 1% enantiomer, and 99.2% wt by Q-NMR.

[0123] Preparation of [(1R)-7,7-dimethyl-2-oxo-norbornan-1-yl]methanesulfonate methyl (2S)-2-amino-3-[(3S)-2-oxopyrrolidin-3-yl]propanoate The title compound is prepared as depicted in Reaction Scheme SM-4 and as described below.

[0124] [ka]

[0125] A 2 M aqueous solution of NaOH (4.8 mL, 4.8 mmol) was added to a mixture of (1S,2S)-1-[(2,3-dimethylcycloprop-2-en-1-ylidene)amino]indan-2-ol hydrochloride (557 mg, 0.96 mmol) in MTBE (6 mL) at 20 °C and stirred for 10 min. The organic layer was collected, and the aqueous layer was rinsed with MTBE (4 mL). The combined organic fractions were filtered over NaSO, and the filtrate was added directly to a mixture of methyl 2-(benzhydrylideneamino)acetate (97.0%, 5.00 g, 19.1 mmol) and tert-butyl 3-methylene-2-oxo-pyrrolidine-1-carboxylate (4.15 g, 21.1 mmol) in MTBE (10 mL). The mixture was stirred at 35° C. for 3.5 hours, and then (−)-CSA (9.10 g, 38.4 mmol) was added in one portion at 35° C. The mixture was heated to 65° C. for 18 hours and then diluted with acetone (70 mL). The mixture was refluxed for 20 minutes and diluted with MTBE (130 mL). The mixture was refluxed for 4 hours, then cooled to 20° C. and filtered. The solid was washed with a 2:1 mixture of MTBE and acetone (3×30 mL). The solid was collected and dried under high vacuum to give the title compound [(1R)-7,7-dimethyl-2-oxo-norbornan-1-yl]methanesulfonate methyl (2S)-2-amino-3-[(3S)-2-oxopyrrolidin-3-yl]propanoate (5.79 g, 69%, dr >25:1) as a solid. MS (ESI) [M+H-CSA] + 187.1. 1 H NMR (500 MHz, methanol-d4) δ 4.24 (dd, J = 9.7, 3.6 Hz, 1H), 3.85 (s, 3H), 3.42 - 3.35 (m, 2H), 3.33 - 3.26 (m, 1H), 2.85 - 2.77 (m, 1H), 2.76 (d, J = 14.8 Hz, 1H), 2.70 - 2.61 (m, 1H), 2.47 - 2.37 (m, 1H), 2.38 - 2.29 (m, 1H), 2.23 (ddd, J = 15.1, 4.8, 3.7 Hz, 1H), 2.08 - 1.98 (m, 3H), 1.93 - 1.81 (m, 2H), 1.61 (ddd, J = 13.7, 9.3, 4.3 Hz, 1H), 1.41 (ddd, J = 11.9, 9.5, 4.2 Hz, 1H), 1.13 (s, 3H), 0.86 (s, 3H). Note: The four exchangeable protons are invisible. 13 C NMR (126 MHz, methanol-d4) δ 218.28, 181.61, 170.69, 59.57, 53.81, 53.79, 48.17, 44.05, 43.61, 42.04, 41.65, 32.96, 29.50, 27.79, 25.73, 20.43, 20.12.

[0126] Preparation of tert-butyl (S)-3-((S)-2-((diphenylmethylene)amino)-3-methoxy-3-oxopropyl)-2-oxopyrrolidine-1-carboxylate; The title compound is prepared as depicted in Reaction Scheme SM-5 and as described below.

[0127] [ka]

[0128] Note that all solvents used in this procedure were purchased as anhydrous grade and degassed by bubbling with nitrogen for approximately 30 minutes. The reaction was carried out inside an N2-filled glovebox. A solution of [Cu(MeCN)4]PF6 in THF (0.020 M, 25 μL, 0.5 μmol, 1 mol%), followed by a solution of (R)-FeSulPhos in THF (0.020 M, 25 μL, 0.5 μmol, 1 mol%), was dispensed into a 1 mL vial with a stir disk. The mixture was stirred at 25 °C for 23 hours, after which the THF was evaporated. A solution of diisopropylamine in THF (1.0 M, 25 μL, 25 μmol, 0.5 equiv.) was added, and the vial was then cooled to -10 °C while stirring at 500 rpm. A solution of methyl 2-((diphenylmethylene)amino)acetate (12.5 mg, 49 μmol, 1.0 equiv.) and tert-butyl 3-methylene-2-oxopyrrolidine-1-carboxylate (10.7 mg, 54 μmol, 1.1 equiv.) in isopropanol (170 μL) was then added, and the vial was sealed and stirred at 500 rpm between −10 and −4°C for 24 h. A 25 μL aliquot of the reaction mixture was then removed and diluted with 1 mL of MeCN. This sample was then analyzed via chiral SFC, which showed that the desired compound, tert-butyl (S)-3-((S)-2-((diphenylmethylene)amino)-3-methoxy-3-oxopropyl)-2-oxopyrrolidine-1-carboxylate, was formed in 74% assay yield (rt 6.17 min, m / z +ve = 451). The reference time was compared to an independently synthesized sample of tert-butyl (S)-3-((S)-2-((diphenylmethylene)amino)-3-methoxy-3-oxopropyl)-2-oxopyrrolidine-1-carboxylate to confirm the identity of the product.

[0129] SFC method: (Chiralcel OX-H 250mm x 4.6mm x 5um (P / N: 63325), Mobile phase A: CO2, Mobile phase B: 0.2% 7N ammonia in isopropyl alcohol + methanol, 3mL / min, 40℃, UV detection at 210nm. 0-1min 5% B, 1-9min 5-60% B, 9-9.5min 60% B, 9.5-10min 60-5% B).

[0130] Using the procedure described above, the resulting tert-butyl (S)-3-((S)-2-((diphenylmethylene)amino)-3-methoxy-3-oxopropyl)-2-oxopyrrolidine-1-carboxylate can be converted to methyl (2S)-2-amino-3-[(3S)-2-oxopyrrolidin-3-yl]propanoate [(1R)-7,7-dimethyl-2-oxo-norbornan-1-yl]methanesulfonate.

[0131] Preparation of (S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoic acid (Compound V) The title compound is prepared as depicted in Reaction Scheme SM-6 and as described below.

[0132] [ka]

[0133] A solution of (S)-2-amino-3,3-dimethylbutanoic acid was prepared in methanol and sodium methoxide was added. Ethyl trifluoroacetate was added, and the mixture was stirred until the reaction was complete. The reaction was diluted with ethyl acetate and washed with brine. Heptane was added to the organic layer containing (S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoic acid, and the solution was concentrated to 10 mL / g at 50 °C. This was repeated three times to remove as much ethyl acetate as possible. After the second distillation, seed crystals of (S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoic acid could be introduced. After the final distillation, the mixture was cooled to 20 °C, granulated, then filtered and washed with heptane. The solid was dried at 40 °C. Crystallographic data was obtained and provided in the table below, and the PXRD pattern for this material is provided as Figure 5.

[0134] [Table 2]

[0135] FIG. 5 provides the PXRD pattern for (S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoic acid, which is characterized by the PXRD peaks in the table below.

[0136] [Table 3] [Example]

[0137] Step 1: Preparation of sodium (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate

[0138] [ka] Methyl (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate, hydrochloride (Compound VII, 40 g, 195 mmol, 1.0 equiv.)), tetrahydrofuran (80 mL, 2 mL per g of Compound VII), and water (40 mL, 1 mL per g of Compound VII) are combined and stirred at 25°C. Triethylamine (40.7 mL, 292 mmol, 1.5 equiv.) is added, and the mixture is stirred for 30 minutes. The pH of the stirred mixture should not be below 8.5. Stirring is stopped, and the phases are allowed to separate. The aqueous phase is removed to obtain an organic solution of methyl (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate. In a separate vessel, sodium hydroxide (8.16 g, 204 mmol, 1.05 equiv.), tetrahydrofuran (360 mL, 9 mL per gram of Compound VII), and water (40 mL, 1 mL per gram of Compound VII) are combined and heated to 40° C. with stirring. To this mixture is added the organic solution of methyl (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate over 15 minutes, and the resulting mixture is stirred at 40° C. for 4 hours. A sample is analyzed by UPLC for reaction completion (targeting no more than 4% methyl (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate remaining; continue stirring if reaction is not complete). Upon completion, the mixture is cooled to 20° C. and stirred for 2 hours or more. The solid is collected by filtration, rinsed with 96:4 THF / water (80 mL), and dried in a vacuum oven at 70° C. to give sodium (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate (compound VI). 1 H NMR (600 MHz, CD3OD-d4, 298K): δ 3.37 (d, 1H), 3.31 (dd, 1H), 2.76 (dd, 1H), 1.62 (dd, 1H), 1.36 (m, 1H), 1.06 (s, 3H), 1.03 (s, 3H). 13 C NMR (150 MHz, CD3OD-d4, 298K): δ 181.6, 64.3, 47.2, 37.7, 31.6, 27.1, 20.9, 14.2. HRMS: (ESI + ) C8H 14 O2N + Calculated value: 156.1019, Measured value: 156.1020 (mass deviation +0.83 ppm)

[0139] Multiple solid forms of sodium (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate (Compound VI) were isolated and characterized by PXRD.

[0140] FIG. 7 provides the PXRD pattern of (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate, the primary form of which is characterized by the peaks in the table below.

[0141] [Table 4]

[0142] FIG. 8 provides the PXRD pattern for sodium (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate, designated as Form B, which is characterized by the peaks in the table below.

[0143] [Table 5]

[0144] FIG. 9 provides the PXRD pattern for a form of sodium (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate designated as Material A, which is characterized by the PXRD peaks in the table below.

[0145] [Table 6]

[0146] Step 2: Preparation of (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (compound IV)

[0147] [ka] (S)-3,3-Dimethyl-2-(2,2,2-trifluoroacetamido)butanoic acid (Compound V, 38.9 g, 169 mmol, 1.2 eq.), methanesulfonyl chloride (17.8 g, 155 mmol, 1.1 eq.), and isopropyl acetate (500 mL, 20 mL per g of Compound V) were combined and stirred at 20 °C. Triethylamine (49.0 mL, 423 mmol, 2.5 eq.) was charged at a rate such that the reaction temperature did not exceed 25 °C, and the resulting mixture was stirred for 1 hour. Sodium (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate (Compound VI, 25.0 g, 141 mmol, 1.0 eq.) was charged, and the mixture was stirred for 4 hours. A sample of the reaction mixture was taken and analyzed for reaction completion (VI by UPLC less than 3%). If the reaction is not complete, additional triethylamine may be added. The reaction mixture is quenched by the addition of aqueous citric acid (74 g citric acid monohydrate in 150 mL water, 353 mmol, 2.5 equiv.) and the mixture is heated to 40°C. The mixture is stirred for at least 10 minutes, and then the layers are allowed to settle. The aqueous phase is removed and the organic phase is washed with two portions of water (125 mL each). The organic phase is cooled to 10-15°C and concentrated to a volume of approximately 192 mL by vacuum distillation (-100 mbar, gradually warming to a maximum jacket temperature of 60°C). The mixture is analyzed for water content (Karl Fischer), and if there is more than 3 wt% water, the vacuum distillation is repeated with additional isopropyl acetate. The solution is heated to 60°C and heptane (192 mL) is added. The mixture is stirred and cooled to 10°C at a rate of approximately -12°C / hr. The slurry is stirred at 10°C for 3 hours. The solid is collected by filtration and rinsed with 1:1 iPrOAc / heptane (100 mL). The solid is dried in a vacuum oven at 50° C. to give (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (Compound IV). 1 H NMR (600 MHz, DMSO-d6, 298K): δ 12.72 (s, 1H), 9.42 (d, 1H), 4.44 (d, 1H), 4.15 (s, 1H), 3.85 (dd, 1H), 3.72 (d, 1H), 1.53 (dd, 1H), 1.41 (d, 1H), 1.01 (s, 3H), 1.00 (s, 9H), 0.82 (s, 3H). 13 C NMR (150 MHz, DMSO-d6, 298K): d 172.3, 167.6, 156.9 ( 2 J CF = 37 Hz), 115.8 ( 2 J CF = 288 Hz), 59.2, 58.1, 47.2, 34.7, 29.7, 26.7, 26.2, 25.7, 18.8, 12.1.

[0148] Notes: 1 H and 13 In the C NMR spectrum, two sets of resonances were observed due to the presence of both E and Z amide bond rotamers in solution. Only the major resonance (Z rotamer, 92%) is listed here. HRMS: (ESI + ) C 16 H 24 F3N2O4 + Calculated value: 365.1610, Measured value: 365.1684 (mass deviation +0.5 ppm)

[0149] Preparation of crystalline (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid, Form 1, prepared by a seeded crystallization process from aqueous isopropanol. Step 1: To a 100 mL reactor, (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (5.08 g, 13.9 mmol) and isopropanol (21.25 mL, 277.9 mmol) are added and stirred at 500 rpm for 30 minutes to dissolve.

[0150] Step 2: Reverse addition To a separate 100 mL reactor, water (75 mL, 4163.2 mmol) and isopropanol (3.75 mL, 49.0 mmol) (95-05 v / v%) are added, followed by seeding with (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (150 mg, 0.4116 mmol), and the mixture is heated to 50° C. at a rate of 5° C. / min and stirred for 30 min. A solution of (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (from step 1 above) in isopropanol is added over 6 hours. The resulting mixture is stirred at 50°C for 1 hour, and then the mixture is cooled to 10°C at a rate of -0.1°C / min and stirred overnight. The resulting slurry is then filtered, and the solid is washed with a mixture of isopropanol (5 mL, 65.39 mmol) and water (15 mL, 832.64 mmol). The resulting solid was dried under vacuum at 60° C. overnight to give crystalline (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid, Form 1. The solid was characterized by powder X-ray diffraction (PXRD) according to the methods described herein.

[0151] [Table 7]

[0152] FIG. 10 provides the PXRD pattern for (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid, Form 1, which is characterized by the PXRD peaks in the table below.

[0153] [Table 8]

[0154] FIG. 11 depicts the PXRD pattern for (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid, a new form, which is characterized by the PXRD peaks in the table below.

[0155] [Table 9]

[0156] Step 3: Comparative process for the preparation of (1R,2S,5S)-N-((S)-1-amino-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Compound II)

[0157] [ka] (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (Compound IV, 1.0 kg, 2.74 mol, 1.0 equivalent) and methyl ethyl ketone (5.0 L, 5 L per kg of Compound IV) are combined and stirred at 25° C. 2-Hydroxypyridine N-oxide (0.228 kg, 2.05 mol, 0.75 equivalent) and triethylamine (0.416 kg, 4.11 mol, 1.50 equivalent) are added, and the resulting slurry is stirred for 30 minutes. (S)-2-Amino-3-((S)-2-oxopyrrolidin-3-yl)propanamide hydrochloride (Compound III, 0.597 kg, 2.87 mol, 1.05 eq) and 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDC, 0.682 kg, 3.56 mol, 1.30 eq) are charged and stirring is maintained for 16 hours. The reaction is sampled for completion (targeting no more than 1.0% Compound IV remaining). If the reaction is not complete, the mixture is stirred for an additional hour. The reaction is quenched by the addition of aqueous NaCl (3.0 L of a 14 wt % brine solution) and stirring is maintained for 30 minutes. Stirring is stopped and the layers are allowed to settle. The lower aqueous phase is removed and the organic phase is washed with a second portion of aqueous NaCl (3.0 L of a 14 wt % brine solution) following the same protocol. The organic phase is then concentrated by vacuum distillation at 0.3 bar while adding additional isopropyl acetate (13 L) to maintain a constant volume of approximately 6 L / kg, and the distillation is terminated at 7 L / kg. Samples are analyzed for water content (Karl Fischer), with a target of 0.2 wt% or less. The resulting organic solution of (1R,2S,5S)-N-((S)-1-amino-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide, compound II, is used in Step 4 without further purification.

[0158] Preparation and isolation of (1R,2S,5S)-N-((S)-1-amino-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide, methyl ethyl ketone (MEK) solvate (II MEK solvate) A sample of the title MEK solvate was isolated by the following procedure. The reaction in Step 3 was carried out as described above on a 150 g scale and allowed to stir for 3 days. The resulting slurry was combined with aqueous NaCl (325 mL of a 14 wt % solution), warmed to 48° C., and stirred, resulting in two clear phases. The aqueous phase was removed, and the organic phase was washed with a second portion of aqueous NaCl (400 mL of a 14 wt % solution). The organic phase was concentrated under partial vacuum at 45° C., and additional MEK was added. This was repeated until the water content was reduced to 6%, and the volume was approximately 4 mL / g of starting material. The mixture was gradually cooled to 15° C. and held for 3 hours. The resulting solid was collected by filtration, rinsed with MEK, and dried in a vacuum oven to provide 132 g of (1R,2S,5S)—N-((S)-1-amino-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide, methyl ethyl ketone solvate. 1 H NMR (600 MHz, DMSO-d6, 298K): δ 9.40 (d, J = 8.6 Hz, 1H), 8.76 (d, J = 9.2 Hz, secondary), 8.48 (d, J = 7.2 Hz, secondary), 8.28 (d, J = 8.9 Hz, 1H), 7.65 (s, secondary), 7.55 (s, 1H), 7.45 - 7.39 (m, secondary), 7.35 - 7.26 (m, 1H), 7.06 - 6.99 (m, 1H), 6.92 (s, d), 4.49 (s, d), 4.43 (d, J = 8.6 Hz, 1H), 4.33 - 4.28 (m, 1H), 4.28 (s, 1H), 4.21 (d, J = 9.2 Hz, secondary), 4.09 (ddd, J = 11.1, 7.2, 3.8 Hz, secondary), 3.89 (dd, J = 10.3, 5.5 Hz, 1H), 3.67 (d, J = 10.4 Hz, 1H), 3.58 (dd, J = 12.4, 5.6 Hz, secondary), 3.54 (dd, secondary), 3.40 (d, J = 12.5 Hz, secondary), 3.16 - 3.10 (m, 1H), 3.03 (td, J = 9.4, 7.1 Hz, 1H), 2.46 - 2.36 (m, 3H), 2.18 - 2.10 (m, 1H), 2.07 (s, 3H), 2.02 (m, s), 1.94 (ddd, J = 13.5, 12.0, 3.6 Hz, 1H), 1.73 (dt, J = 12.4, 9.1 Hz, vice), 1.69 - 1.59 (m, 1H), 1.54 - 1.46 (m, 2H), 1.38 (d, J = 7.6 Hz, 1H), 1.19 (t, J = 7.3 Hz, vice), 1.02 (s, 3H), 0.98 (s, 9H), 0.91 (t, J = 7.3 Hz, 3H), 0.84 (s, 3H). 13 C NMR (150 MHz, DMSO-d6, 298K): δ 208.83, 178.92 (minor), 178.62, 173.74 (minor), 173.51, 170.63, 170.52 (Deputy), 167.94 (Deputy), 167.21, 156.87 (q, J = 36.9 Hz), 155.52 (d, J = 36.7 Hz-sub), 118.96 - 112.77 (m), 60.61 (secondary), 60.25, 58.11, 57.67 (secondary), 51.78 (secondary), 50.32, 47.70, 47.50 (secondary), 45.51 (secondary), 42.09 (secondary), 40.06, 39.29, 37.88 (secondary), 37.32, 36.48 (secondary), 36.16, 35.85, 34.67, 34.55, 34.09, 33.58 (secondary), 30.57, 29.35, 27.65 (secondary), 27.39, 27.10, 26.40, 26.28, 25.87, 24.86 (secondary), 19.05 (secondary), 18.58, 13.24 (secondary), 12.38, 8.51 (secondary), 7.66.

[0159] Notes: 1 H and 13 In the C NMR spectrum, two sets of resonances were observed due to the presence of both E and Z amide bond rotamers in solution. Where relevant, the less abundant rotamer signals are designated as "minor." HRMS: (ESI + ) C 23 H 35 F3N5O5 + Calculated value: 518.2585, Measured value: 518.2582 (mass deviation -0.46 ppm)

[0160] Alternative Step 3: Optimized process for the preparation of (1R,2S,5S)-N-((S)-1-amino-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide

[0161] [ka] In reactor A, (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (Compound IV, 1.0 kg, 2.74 mol, 1.0 equivalent) and methyl ethyl ketone (2.0 L, 2 L per kg of Compound IV) are combined and stirred at 25° C. 2-Hydroxypyridine N-oxide (0.274 kg, 2.47 mol, 0.90 equivalent) and triethylamine (0.694 kg, 6.86 mol, 2.50 equivalents) are added, and the resulting slurry is stirred for 30 minutes and then warmed to 50° C. In reactor B, (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl)propanamide hydrochloride (compound III, 0.597 kg, 2.87 mol, 1.05 eq.) and 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDC, 0.682 kg, 3.56 mol, 1.30 eq.) are combined with methyl ethyl ketone (3.0 L, 3 L per kg of compound IV), and the resulting slurry is stirred for 30 minutes and then warmed to 50° C. The current solution from reactor A is then transferred to reactor B, while maintaining the temperature in reactor B at 50° C., and stirring is continued for at least 6 hours. The reaction is sampled for completion (the goal is for no more than 3% of compound IV to remain unreacted). If the reaction is not complete, the mixture is stirred for an additional period of time. The reaction is quenched at 50°C by the addition of aqueous NaCl (3.0 L of a 14 wt% brine solution, 3.0 L per kg of Compound IV) and stirring is maintained for 30 minutes. Stirring is stopped and the layers are allowed to settle. The lower aqueous phase is removed and the organic phase is washed with a second portion of aqueous NaCl (3.0 L of a 14 wt% brine solution) following the same protocol. The organic phase is cooled and then concentrated by vacuum distillation at 0.3 bar while adding additional isopropyl acetate (18 L, 18 L per kg of Compound IV) to maintain a constant volume of approximately 6 L / kg, and the distillation is stopped at 8 L / kg. Samples are analyzed for water content (Karl Fischer) with a target of 0.2 wt% or less water.The resulting organic solution of (1R,2S,5S)—N-((S)-1-amino-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide is used in Step 4 without further purification.

[0162] The (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl)propanamide hydrochloride (compound III) used in the previous reaction can exist in certain solid forms.

[0163] FIG. 12 provides the PXRD pattern for (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl)propanamide hydrochloride, Form 1, which is characterized by the PXRD peaks in the table below.

[0164] [Table 10]

[0165] FIG. 13 provides the PXRD pattern for (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl)propanamide hydrochloride, Form 2, which is characterized by the PXRD peaks in the table below.

[0166] [Table 11]

[0167] FIG. 14 provides the PXRD pattern for (1R,2S,5S)—N—((S)-1-amino-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide, methyl ethyl ketone (MEK) solvate, which is characterized by the PXRD peaks in the table below.

[0168] [Table 12]

[0169] Table - Crystallographic data and structure refinement for (1R,2S,5S)-N-((S)-1-amino-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide, methyl ethyl ketone solvate: Identification code E222 Empirical formula C27 H42 F3 N5 O6 Formula weight 589.65 Temperature 273.15K Wavelength 1.54178Å Crystal system Orthorhombic system Space group P212121 Unit cell dimensions a=9.4471(3)Å α=90°. b=9.6463(3)Å β=90°. c = 35.4946(12)Å γ = 90°. Volume 3234.61(18)Å 3 Z4 Density (calculated value) 1.211 Mg / m 3 Absorption coefficient 0.821mm -1 F(000) 1256 Crystal size: 0.202 x 0.174 x 0.124 mm 3 Theta range for data collection: 4.750 to 79.276°. Exponent range: -11<=h<=8, -12<=k<=11, -45<=l<=43 Collected Reflections 20328 Independent reflection 6587 [R(int)=0.0534] Perfection to Theta = 67.679° 99.6% No absorption correction Maximum and minimum transmittance: 0.7543 and 0.5564 Refinement method F 2 Full matrix least squares for Data / Limits / Parameters 6587 / 5 / 397 F 2 Goodness of fit to 1.016 Final R index [I>2 sigma (I)] R1 = 0.0718, wR2 = 0.2067 R-index (all data) R1=0.0844, wR2=0.2218 Absolute structural parameter 0.14(16) Extinction coefficient n / a Maximum diffraction peak and vacancy 0.487 and -0.220 e.Å -3

[0170] Step 4: Preparation of (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide, methyl t-butyl ether solvate (I')

[0171] [ka] The (1R,2S,5S)—N-((S)-1-amino-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Compound II) prepared above in Step 4 (quantitative conversion expected, 2.74 mol, 1.0 equivalent) is combined with N-methylmorpholine (1.11 kg, 10.4 mol, 4.0 equivalents) and stirred at 20° C. Trifluoroacetic anhydride (1.15 kg, 5.20 mol, 2.0 equivalents) is added over 60 minutes while maintaining the reaction temperature below 25° C. The resulting mixture is stirred for 1 hour. Analyze a sample for reaction completion (no more than 0.5% compound II remaining). If the reaction is not complete, maintain stirring for another 60 minutes, charging additional trifluoroacetic anhydride if necessary. Quench the reaction by adding water (3.0 L, 3.0 L per kg of compound IV from the previous step), maintain stirring for 30 minutes, then stop and allow the layers to settle. Remove the aqueous phase, and wash the organic phase with a second 3.0 L portion of water. The organic phase is then concentrated by vacuum distillation (0.1 bar) to a volume of 3.5 L (3.5 L per kg of compound IV from the previous step). Add isopropyl acetate (5.0 L, 5.0 L per kg of compound IV from the previous step), and concentrate the solution by vacuum distillation to a volume of 3.5 L (3.5 L per kg of compound IV from the previous step). Stir the solution at 50° C., and add methyl t-butyl ether (MTBE) over 60 minutes. If no product crystallization occurs during this addition, PF-07321332 MTBE solvate seed crystals (10 g, 1.0 wt % based on compound IV from the previous step) can be added. An additional portion of MTBE (6.0 L, 6.0 L per kg of compound IV from the previous step) is added over 3 hours. The slurry is stirred at 50° C. for 1 hour, cooled to 20° C. at a rate of 0.1 K / min, and stirred at 20° C. for 2 hours.The solid is collected by filtration, rinsed with 2.0 L of 80:20 MTBE:iPrOAc solution per kg of Compound IV from the previous step, and dried at ≤50°C.

[0172] A form of (1R,2S,5S)—N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide, isopropyl acetate solvate was obtained according to the following procedure.

[0173] A 50 mL reactor equipped with a magnetic stirrer was charged with PF-07328615 in methyl ethyl ketone (7.87 g, 15.2 mmol). Some solid crystallized from the PF-07328615 solution. To this was added isopropyl acetate (for distillation) (72 mL, 614.04 mmol) while performing a constant volume distillation with Tj-Tr set at 15 °C, Tj max set at 70 °C, and vacuum set at 300 mbar (actual vacuum is 295-305 mbar). Dilute with isopropyl acetate to 7.5 mL / g (42 mL). After cooling to 20°C (Tj), the stirring speed was set to 350 rpm, and then 1-methylmorpholine (6.7 mL, 61 mmol) was added over 5 min, followed by trifluoroacetic anhydride (4.28 mL, 30.4 mmol) over 1 h via syringe pump. After the addition was complete, the mixture was stirred for 1 h. The reaction mixture was quenched with water (17 mL, 943.66 mmol), transferred to a separatory funnel, and the aqueous layer was removed. The organic layer was returned to the reactor and distilled to a concentration of 3.5 mL / g (20 mL) with Tj-Tr set to 15°C, Tj max set to 70°C, and vacuum set to 100 mbar (actual vacuum was 100-105 mbar). To this was added isopropyl acetate (28 mL, 238.79 mmol), and the mixture was distilled again to a concentration of 3.5 mL / g. A solid crystallized when the volume reached approximately 30 mL. 1 mL of the solution is filtered (yield 71 mg wet solid) and analyzed by PXRD (see Figure 15).

[0174] FIG. 15 provides the PXRD pattern for (1R,2S,5S)—N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide, isopropyl acetate solvate, which is characterized by the PXRD peaks in the following reports:

[0175] [Table 13]

Claims

1. The compound (1R,2S,5S)-N-((S)-1-amino-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide, methyl ethyl ketone solvate.

2. 1. A process for preparing (1R,2S,5S)-N-((S)-1-amino-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (compound II), comprising: 【Chemical 1】 Steps (a) to (d): (a) combining (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (compound IV) and methyl ethyl ketone, followed by the addition of 2-hydroxypyridine N-oxide and triethylamine to obtain a first mixture, S3-M1; (b) combining (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl)propanamide hydrochloride (compound III), 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride, and methyl ethyl ketone to obtain a second mixture, S3-M2; (c) combining the first mixture S3-M1 from step (a) with the second mixture S3-M2 from step (b) to obtain a third mixture S3-M3; and (d) stirring the third mixture S3-M3 from step (c) to obtain compound (1R,2S,5S)—N—((S)-1-amino-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (compound II). A process for reacting a compound of formula IV with a compound of formula III, comprising:

3. 3. The method of claim 2, wherein in step (a), the first mixture, S3-M1, comprises 1.0 equivalent of (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (Compound IV), 2 L of methyl ethyl ketone per kg of Compound IV, 0.9 equivalents of 2-hydroxypyridine N-oxide, and 2.50 equivalents of triethylamine.

4. 4. The method of claim 2 or 3, wherein in step (a), the first mixture, S3-M1, is prepared at about 25°C, stirred at about 25°C for about 30 minutes, and then warmed to about 50°C.

5. 5. The method of any one of claims 2 to 4, wherein in step (b), the second mixture, S3-M2, comprises 1.05 equivalents of (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl)propanamide hydrochloride (compound III), 1.30 equivalents of 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride, and 3 L of methyl ethyl ketone per kg of compound IV.

6. 6. The method of any one of claims 2 to 5, wherein in step (b), the second mixture, S3-M2, is prepared at about 25°C, stirred at about 25°C for 30 minutes, and then warmed to about 50°C.

7. 7. The method of any one of claims 2 to 6, wherein in step (c), the first mixture, S3-M1, from step (a) is at about 50°C and is combined with the second mixture, S3-M2, from step (b) that is at about 50°C to obtain a third mixture, S3-M3, while maintaining the temperature of the third mixture, S3-M3, at about 50°C.

8. 8. The method of any one of claims 2 to 7, wherein in step (d), the third mixture, S3-M2, from step (c) is stirred at about 50°C for at least 6 hours.

9. 1. A process for preparing (1R,2S,5S)-N-((S)-1-amino-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (compound II), comprising: 【Chemistry 2】 Steps (a) to (d): (a) combining (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (Compound IV, 1.0 equivalent) and methyl ethyl ketone (2 L per kg of Compound IV) at about 25° C., followed by the addition of 2-hydroxypyridine N-oxide (0.90 equivalents) and triethylamine (2.50 equivalents) to provide a first mixture, S3-M1, which is stirred for about 30 minutes and then warmed to about 50° C.; (b) combining (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl)propanamide hydrochloride (Compound III, 1.05 equivalents), 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (1.30 equivalents), and methyl ethyl ketone (3 L per kg of Compound IV) to obtain a second mixture, S3-M2, which is stirred for about 30 minutes and then warmed to about 50° C.; (c) combining the first mixture, S3-M1, from step (a) with the second mixture, S3-M2, from step (b) while maintaining the temperature at about 50° C. to obtain a third mixture, S3-M3; and (d) stirring the third mixture, S3-M3, from step (c) at about 50° C. for at least 6 hours to obtain compound (1R,2S,5S)—N—((S)-1-amino-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (compound II). A process for reacting a compound of formula IV with a compound of formula III, comprising:

10. 10. The process of claim 9, wherein the amount of acylurea impurities IMP-S3-3, (2S,4S)-4-(2-aminoethyl)-5-oxopyrrolidine-2-carboxamide, and IMP-S3-4, (1R,2S,5S)—N-(2-((3S,5S)-5-carbamoyl-2-oxopyrrolidin-3-yl)ethyl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide formed is 10% or less.

11. 10. The process of claim 9, wherein the amount of acylurea impurities IMP-S3-3, (2S,4S)-4-(2-aminoethyl)-5-oxopyrrolidine-2-carboxamide, and IMP-S3-4, (1R,2S,5S)—N-(2-((3S,5S)-5-carbamoyl-2-oxopyrrolidin-3-yl)ethyl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide formed is 5% or less.

12. 12. The process of any one of claims 9 to 11, wherein the amount of rearrangement impurity IMP-S3-3, (2S,4S)-4-(2-aminoethyl)-5-oxopyrrolidine-2-carboxamide formed is 2% or less.

13. 12. The process of any one of claims 9 to 11, wherein the amount of rearrangement impurity IMP-S3-4, (1R,2S,5S)-N-(2-((3S,5S)-5-carbamoyl-2-oxopyrrolidin-3-yl)ethyl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide formed is 2% or less.

14. 1. A process for preparing (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (compound IV), comprising: 【Chemistry 3】 Steps (a) to (d): (a) combining (S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoic acid (Compound V), methanesulfonyl chloride, and isopropyl acetate to obtain a first mixture, S2-M1; (b) adding triethylamine to the first mixture, S2-M1, to obtain a second mixture, S2-M2; (c) adding sodium (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate (Compound VI) to the second mixture, S2-M2, to obtain a third mixture, S2-M3; and (d) stirring the third mixture, S2-M3, to obtain (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (compound IV) reacting a compound of formula V with a compound of formula VI.

15. 15. The method of claim 14, wherein the first mixture, S2-M1, in step (a) comprises 1.2 equivalents of (S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoic acid (Compound V), 1.1 equivalents of methanesulfonyl chloride, and 20 mL of isopropyl acetate per gram of Compound V.

16. 16. The method of claim 14 or 15, wherein in step (b), 2.5 equivalents of triethylamine are added to the first mixture, S2-M1, at about 20° C., at a rate such that the temperature does not exceed 25° C. to obtain a second mixture, S2-M2.

17. 17. The method of any one of claims 14 to 16, wherein in step (c), 1.0 equivalent of (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate sodium (compound VI) is added to the second mixture, S2-M2, to obtain a third mixture, S2-M3, which is stirred for about 4 hours.

18. 18. The method of claim 17, wherein 2.5 equivalents of aqueous citric acid is added to the third mixture, S2-M3, and the resulting mixture is stirred at about 40° C. for at least 10 minutes.

19. 20. The method of claim 18, wherein the organic and aqueous layers of the resulting mixture are allowed to settle, and the organic isopropyl acetate layer is separated from the aqueous layer, washed with water, and concentrated to approximately 40% of its initial volume to provide an organic layer, S2-M4.

20. 20. The method of claim 19, wherein the organic layer S2-M4 is heated to 60° C. and one volume of heptane is added thereto, then the resulting mixture is cooled to 10° C. and stirred for 3 hours, and the resulting solid is collected by filtration, washed with 1:1 isopropyl acetate / heptane, and dried to give (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (IV).

21. 1. A process for preparing (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (Form 1, Compound I), comprising: 【Chemistry 4】 Steps (a) to (d): (a) dissolving (1R,2S,5S)—N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide, methyl tert-butyl ether (Compound I′) in isopropyl acetate, wherein the concentration of Compound I′ in the isopropyl acetate is from about 7 mL to about 9 mL of isopropyl acetate per gram of Compound I′; (b) seeding the solution with 0.5% to 0.75% by weight of (1R,2S,5S)—N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide, Form 1; (c) adding heptane to the mixture from step (b) over a period of 6 to 15 hours, wherein the amount of heptane added is from about 10 mL to about 14 mL of heptane per gram of Compound I'; and (d) isolating the resulting (1R,2S,5S)—N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide, Form 1 A method comprising:

22. 22. The method of claim 21, wherein the amount of isopropyl acetate used in step (a) is about 8 mL of isopropyl acetate per gram of Compound I'.

23. 23. The method of claim 21 or 22, wherein the amount of (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide, Form 1 used to seed the solution in step (b) is about 0.75 wt %.

24. 24. The method of any one of claims 21 to 23, wherein the amount of heptane added in step (c) is about 12 mL per gram of compound I'.

25. 25. The method of any one of claims 21 to 24, wherein the heptane is added over a period of about 10 hours.

26. 26. The method of any one of claims 21 to 25, wherein (1R,2S,5S)—N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide, Form 1, is isolated by filtration.

27. 27. The method of any one of claims 21 to 26, wherein the (1R,2S,5S)—N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide, Form 1 isolated in step (d) has a particle size distribution with a D[v,0.5] count of about 12 microns to about 18 microns.

28. 28. The method of any one of claims 21 to 27, wherein the (1R,2S,5S)—N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide, Form 1 isolated in step (d) has a particle size distribution with a D[v,0.5] count of about 14 microns to about 16 microns.

29. 29. The method of any one of claims 21 to 28, wherein the (1R,2S,5S)—N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide, Form 1 isolated in step (d) has a particle size distribution with a D[v,0.5] count of about 15 microns.

30. The compound (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide, isopropyl acetate solvate.

31. 31. The compound of claim 30, which is crystalline.

32. PXRD pattern 【Chemistry 5】 32. The compound of claim 31 , characterized by:

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