Process and intermediates for preparing nematrivor

Abstract

The present invention relates to intermediates and efficient processes for the preparation of nemategravir (compounds of formula I) and intermediates useful in the preparation of nemategravir. # imgabs0 #

Description

[0001] The present invention relates to an intermediate and an efficient method for preparing Nematevir and an intermediate useful in preparing Nematevir. Nematevir is an antiviral compound with effective inhibitory activity against coronavirus 3CL protease and is a product approved for the treatment of COVID-19 The active ingredient in. Namatevir and methods for its preparation have been disclosed in PCT International Patent Application WO 2021 / 250648 and US Patent Application Publication 2022 / 0062232A1 and US Patent No. 11,351,149. SUMMARY OF THE INVENTION

[0002] The present invention provides intermediates used in the preparation of Nematevir and synthetic methods for preparing intermediates (described in reaction scheme 1), and it contains several method modifications compared to the previously disclosed methods. The product of step 1 of reaction scheme 1 is (1R, 2S, 5S) -6,6- dimethyl -3- azabicyclo [3.1.0] hexane -2- sodium formate, which is a new salt form (sodium replaces lithium) compared to the previous method, and step 2 and step 3 reaction conditions are modified. Additional solid form characterization data of several raw materials and intermediates used in the method are provided. Provided are methods for preparing and isolating (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-trifluoroacetylamino)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamidomethylethylketone (MEK) solvate and characterization data of the compound.

[0003] Reaction Scheme 1

[0004]

[0005] BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Figure 1 : Effect of the relationship between initial concentration of API (PF-07321332, MTBE solvate) in isopropyl acetate (mL / g), heptane addition time (hours), seed loading (% wt PF-07321332 Form 1 / wt PF-07321332 MTBE solvate), and seed size (microns) on the PF-07321332 Form 1 particle size distribution (PSD's) in final D[v,0.5] counts in microns.

[0007] Figure 2: Correlation between seed size and final particle size distribution after crystallization of PF-07321332 Form 1, using 0.75 wt% seed loading (blue circles) and 0.5 wt% seed loading (orange circles) in bench-scale (100 mL) experiments.

[0008] Figure 3 : Particle size distributions (PSDs) obtained for PF-07321332 Form 1 showed D[v,0.5] in microns greater than 50 batches at 1 location.

[0009] Figure 4 : Particle size distributions (PSDs) obtained for PF-07321332 Form 1 showed D[v,0.5] in microns greater than 50 batches at 1 location.

[0010] Figure 5 :PXRD pattern of (S)-3,3-dimethyl-2-(2,2,2-trifluoroacetylamino)butyric acid, i.e., compound V

[0011] Figure 6 :(1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid methyl ester hydrochloride; PXRD pattern of compound VII

[0012] Figure 7 :PXRD spectrum of sodium (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate, the initial form of compound VI

[0013] Figure 8 :PXRD pattern of sodium (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate Form B

[0014] Fig. 9 :PXRD pattern of substance A of sodium (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate

[0015] Fig.10 :PXRD pattern of (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetylamino)butyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid form 1

[0016] Fig.11 :PXRD pattern of the "new form" of (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetylamino)butyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid

[0017] Fig.12 :PXRD pattern of (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl)propanamide hydrochloride form 1

[0018] Fig.13 :PXRD pattern of (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl)propionamide hydrochloride form 2

[0019] Fig.14 :PXRD pattern 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-trifluoroacetylamino)butyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamidomethylethylketone solvate

[0020] Fig.15 :PXRD pattern of PF-07321332 isopropyl acetate solvate DETAILED DESCRIPTION OF THE INVENTION

[0022] The following embodiments EMB-1 to EMB-32 are representative embodiments of the present invention, which should be construed in a non-limiting manner.

[0023] 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-trifluoroacetylamino)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide, methyl ethyl ketone solvate.

[0024] EMB-2 is a method 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-trifluoroacetylamino)butyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide, i.e., compound II

[0025]

[0026] The method of reacting a compound of formula IV with a compound of formula III comprises steps (a) to (d):

[0027] (a) combining (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetylamino)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid, i.e., compound IV, and methyl ethyl ketone, followed by the addition of 2-hydroxypyridine N-oxide and triethylamine to provide a first mixture S3-M1;

[0028] (b) combining (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl)propionamide hydrochloride, i.e., compound III, 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride, and methyl ethyl ketone to provide a second mixture S3-M2;

[0029] (c) combining the first mixture S3-M1 from step (a) with the second mixture S3-M2 from step (b) to provide a third mixture S3-M3; and

[0030] (d) Stirring the third mixture S3-M3 from step (c) to provide 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-trifluoroacetylamino)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide, i.e., compound II.

[0031] EMB-3 is the method 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-trifluoroacetylamino)butyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid, i.e., compound IV, 2 L of methyl ethyl ketone per kg of compound IV, 0.9 equivalent of 2-hydroxypyridine N-oxide and 2.50 equivalents of triethylamine.

[0032] 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 and stirred at about 25 °C for about 30 minutes and then warmed to about 50 °C.

[0033] EMB-5 is a method of any one of EMB-2 to EMB-4, wherein in step (b) the second mixture S3-M2 contains 1.05 equivalents of (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl)propanamide hydrochloride, i.e., 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.

[0034] EMB-6 is the method of any one of EMB-2 to EMB-5, wherein in step (b) the second mixture S3-M2 is prepared at about 25°C and stirred at about 25°C for 30 minutes and then warmed to about 50°C.

[0035] EMB-7 is the method 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 combined with the second mixture S3-M2 from step (b) at about 50°C to provide a third mixture S3-M3 while maintaining the temperature of the third mixture S3-M3 at about 50°C.

[0036] EMB-8 is the method of any one of EMB-2 to EMB-7, wherein in step (d) the third mixture S3-M2 from step (c) is stirred at about 50°C for at least 6 hours.

[0037] EMB-9 is a method 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-trifluoroacetylamino)butyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide, i.e., compound II

[0038]

[0039] The method of reacting a compound of formula IV with a compound of formula III comprises steps (a) to (d):

[0040] (a) combining (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetylamino)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid, Compound IV (1.0 equivalent) and methyl ethyl ketone (2 L / kg Compound IV) at about 25°C, followed by the addition of 2-hydroxypyridine N-oxide (0.90 equivalent) and triethylamine (2.50 equivalent) to provide a first mixture S3-M1, which was stirred for about 30 minutes and then warmed to about 50°C;

[0041] (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 / kg Compound IV) to provide a second mixture S3-M2, which was stirred for about 30 minutes and then warmed to about 50° C.;

[0042] (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 provide a third mixture S3-M3; and

[0043] (d) stirring the third mixture S3-M3 from step (c) at about 50° C. for at least 6 hours to provide 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-trifluoroacetylamino)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide, i.e., compound II.

[0044] EMB-10 is the process of EMB-9, wherein the amount of acylurea impurities IMP-S3-3, i.e., (2S,4S)-4-(2-aminoethyl)-5-oxopyrrolidine-2-carboxamide, and IMP-S3-4, i.e., (1R,2S,5S)-N-(2-((3S,5S)-5-carbamoyl-2-oxopyrrolidine-3-yl)ethyl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetylamino)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide formed does not exceed 10%.

[0045] EMB-11 is the method of EMB-9, wherein the amount of acylurea impurities IMP-S3-3, i.e. (2S,4S)-4-(2-aminoethyl)-5-oxopyrrolidine-2-carboxamide, and IMP-S3-4, i.e. (1R,2S,5S)-N-(2-((3S,5S)-5-carbamoyl-2-oxopyrrolidin-3-yl)ethyl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetylamino)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide formed does not exceed 5%.

[0046] EMB-12 is the method of any one of EMB-9 to EMB-11, wherein the amount of the rearranged impurity IMP-S3-3, i.e., (2S,4S)-4-(2-aminoethyl)-5-oxopyrrolidine-2-carboxamide, formed does not exceed 2%.

[0047] EMB-13 is the method of any one of EMB-9 to EMB-11, wherein the amount of the rearranged impurity IMP-S3-4 formed, i.e., (1R, 2S, 5S)-N-(2-((3S, 5S)-5-carbamoyl-2-oxopyrrolidin-3-yl)ethyl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetylamino)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide, does not exceed 2%.

[0048] EMB-14 is a method for preparing (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetylamino)butyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid, i.e., compound IV

[0049]

[0050] The process of reacting a compound of formula V with a compound of formula VI comprises steps (a) to (c):

[0051] (a) combining (S)-3,3-dimethyl-2-(2,2,2-trifluoroacetylamino)butanoic acid (Compound V), methanesulfonyl chloride, and isopropyl acetate to provide a first mixture S2-M1;

[0052] (b) adding triethylamine to the first mixture S2-M1 to provide a second mixture S2-M2;

[0053] (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 provide a third mixture S2-M3; and

[0054] (d) The third mixture S2-M3 is stirred to provide (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetylamino)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid, ie, Compound IV.

[0055] EMB-15 is the method of EMB-14, wherein in step (a) the first mixture S2-M1 contains 1.2 equivalents of (S)-3,3-dimethyl-2-(2,2,2-trifluoroacetylamino)butyric acid, i.e., compound V, 1.1 equivalents of methanesulfonyl chloride and 20 mL of isopropyl acetate per g of compound V.

[0056] EMB-16 is the method 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 provide a second mixture S2-M2.

[0057] EMB-17 is the method of any one of EMB-14 to EMB-16, wherein in step (c), 1.0 equivalent of sodium (1R, 2S, 5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate, i.e., compound VI, is added to the second mixture S2-M2 to provide a third mixture S2-M3, which is stirred for about 4 hours.

[0058] EMB-18 is the method of EMB-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.

[0059] EMB-19 is the method of EMB-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 original volume, providing organic layers S2-M4.

[0060] EMB-20 is the method of EMB-19, wherein the organic layer S2-M4 is heated to 60°C and 1 volume of heptane is added thereto, and then the resulting mixture is cooled to 10°C, stirred for 3 hours and the resulting solid is collected by filtration, washed with 1:1 isopropyl acetate / heptane and dried to provide (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetylamino)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid, i.e., IV.

[0061] EMB-21 is a method 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, i.e., Compound I

[0062]

[0063] The method comprises steps (a) to (d):

[0064] (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-carboxamidomethyl tert-butyl ether, i.e., Compound I', in isopropyl acetate, wherein the concentration of Compound I' in isopropyl acetate is from about 7 mL to about 9 mL of isopropyl acetate per 1 gram of Compound I';

[0065] (b) seeding the solution with 0.5 wt % to 0.75 wt % 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

[0066] (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 heptane per gram of compound I'; and

[0067] (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.

[0068] EMB-22 is the process 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'.

[0069] EMB-23 is the method 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 w%.

[0070] EMB-24 is the method of any one of EMB-21 to EMB-23, wherein the amount of heptane added in step (c) is about 12 mL / gram of compound I'.

[0071] EMB-25 is the process of any of EMB-21 to EMB-24, wherein heptane is added over a period of about 10 hours.

[0072] EMB-26 is the method of any one of EMB-21 to 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.

[0073] EMB-27 is the method 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.

[0074] EMB-28 is the method 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.

[0075] EMB-29 is the method 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 approximately 15 microns.

[0076] 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.

[0077] EMB-31 is the compound of EMB-30, wherein the compound is crystalline.

[0078] EMB-32 is a compound of EMB-31, which is Fig.15 PXRD characterization as described in .

[0079] Preparation of raw materials and intermediates

[0080] Synthetic routes for preparing intermediate compounds of Formula 1 are provided in Reaction Schemes A and B and synthetic routes for preparing compounds of Formula 2 are provided in Reaction Schemes C, D and E.

[0081] The compounds of Formula 1 can be prepared by the following methods described below and in Reaction Schemes A and B. For the compounds of Formula 1, it is understood that in R 1 In the case where is methyl and the compound is in the form of its hydrochloride salt, the compound is (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid methyl ester hydrochloride, which is also referred to as Compound VII in Reaction Scheme 1.

[0082] Reaction Scheme A

[0083]

[0084] In reaction scheme A, variable R 1 , R 4 and R 5 represents an alkyl group, including but not limited to methyl, ethyl and isopropyl. The variable Ar 1 represents an aryl group, including but not limited to 2-methoxyphenyl. 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, 4 th ed.; John Wiley & Sons, Inc., 2006.

[0085] Compounds of formula 1 can be prepared from cyclopropanation of A2 followed by deprotection. Compound A2 can be prepared from A3 or A4 by converting the hydroxyl group of compound A3 or A4 into an activated group using a reaction using reagents including but not limited to toluenesulfonyl chloride, methanesulfonyl chloride, trifluoromethanesulfonic anhydride and sodium iodide, followed by elimination with a base to provide compound A2. 1 The group can be installed before or after this step by methods well known to those skilled in the art (ie, using R 1 A2 can also be prepared by reduction of pyrrole A5 or decarboxylation of A6. A2 can also be prepared by intermolecular cyclization of A7, and the reaction conditions include but are not limited to metal-catalyzed α-vinylation.

[0086] Formula 1 compounds can also be prepared from the functionalization of A8 via conversion, including but not limited to amide reduction to imines followed by cyanation and esterification. These conversions may require one or more chemical or enzyme catalysts. A8 can be formed via intramolecular cyclopropanation of A9 or A10, cyclopropanation by A11, or metal-catalyzed hydroxylation CH functionalization by A12, and A12 can be prepared from A13. Formula 1 compounds can be prepared from A14 oxidized to carboxylic acid followed by esterification. Conversion may require a metal catalyst. Formula 1 compounds can be prepared via the functionalization of A15 via conversion, including but not limited to metalation with an organometallic reagent, followed by carboxylation and borylation, followed by carboxylation. A15 can be prepared from reactions, including but not limited to coupling A16 with an amine source, reduction and cyclization of A17, cyclopropanation of A18, or reduction of A22. A22 can be synthesized as follows: 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 can be formed from the reaction of A23 or A28 with A24, A25 or A33 in the presence of dialkyl sulfide or the cyclopropanation of A26 or A29 with a cyclopropanation agent including but not limited to A30. A27 can also be prepared by reacting A31 with A32 in the presence of a base.

[0087] Reaction Scheme B

[0088]

[0089] In reaction scheme B, variable 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. 4 , R 7 and R 8 represents amine protecting groups well known to those skilled in the art.

[0090] The compound of formula 1 can be prepared by cyclization of B2 and B3 with an ammonia source in the presence of one or more chemical or enzyme catalysts and a reducing agent. If necessary, the resulting product can be subjected to another reduction reaction. B2 can be prepared by coupling of B4 and B5.

[0091] Formula 1 compounds can be prepared from the cyclization of B6 under reducing conditions in the presence of one or more chemical or enzyme catalysts. B6 can be prepared from B7 under conditions well known to those skilled in the art. B8 can be treated with a chlorinating agent in the presence of a base to form a formula 1 compound. B8 can also be prepared from B9 via B10 by conversion including but not limited to ozonolysis.

[0092] The compound of formula 1 can be prepared from the intramolecular cyclopropanation of B11, B12, B13 or B14. The cyclopropanation reaction may require one or more chemical and / or enzyme catalysts and the resulting product may need to be reduced to form the compound of formula 1. B12 can be prepared from B11 and B14 can be prepared from B13. B16 can be oxidized and olefinated to form B15, which can then be converted to B13 under conditions well known to those skilled in the art. The compound of formula 1 can also be prepared from the intramolecular carbon-carbon bonding of B17 under reducing conditions and B17 can be formed from the functionalization of B18 (i.e., the introduction of the group OSO by double bond rearrangement). 2 R 10 , Cl, Br, etc.). B20 and B21 can react in the presence of a base to form B19, which can then be reduced to a compound of Formula 1 in the presence of one or more chemical and / or enzyme catalysts. B22 or B24 can react with an azomethine ylide derived from B23 or B25 to form a compound of Formula 1 or A2, and A2 can then be cyclopropanated to form a compound of Formula 1.

[0093] The compounds of formula 2 can be synthesized by the following methods described in Schemes C, D and E.

[0094] Reaction Scheme C

[0095]

[0096] In scenario C, variable 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. 2 , R 6 and R 7 represents amine protecting groups well known to those skilled in the art.

[0097] Formula 2 compounds can be prepared by reductive amination of the C2 ketone moiety and reduction conversion of the enone moiety to compound C2. This conversion may require one or more chemical or enzyme catalysts. Compound C2 can be prepared from, including but not limited to, the following reactions: Wittig reaction of compound C3 and compound C4 or aldol reaction of compound C3 and compound C5 or C6, followed by decarboxylation under conditions well known to those skilled in the art if necessary. Compound C12 can react with C33 in the presence of a base to form C34. C34 can undergo stereoselective hydrolysis of esters (desymmetrization, where R 1 The resulting acid moiety can be reacted with a nitrogen source to produce an amide, which can then be reacted under Curtius, Lossen, or Hofmann rearrangement conditions to produce compound 2.

[0098] Formula 2 compound can also be prepared by the reductive amination of compound C7 and the conversion can require one or more chemical or enzyme catalysts.Compound C7 can be prepared from the alkylation of compound C9 and compound C10 or the alkylation of compound C9 and compound C11, followed by oxidation of the resulting product C8.The conversion can require one or more chemical or enzyme catalysts.Compound C7 can also be prepared from the reaction of compound C12 and compound C13 in the presence of thiazolium salts or the reaction of compound C14 and compound C15 in the presence of catalysts such as tertiary amines and phosphines.Compound C7 can also be prepared from the coupling of C27 and C28 to form C26 or C29, followed by rearrangement via C25 or C30.

[0099] Formula 2 compounds can also be prepared by converting compound C31 through reductive amination of the ketone moiety of C31 and reduction of the enone moiety. This conversion may require one or more chemical or enzyme catalysts. Compound C31 can be prepared from C16 in the presence of ammonia or from C17 in the presence of an acid or base. Compounds C16 and C17 can be prepared from the reaction of compound C18 with C19 or C20, respectively, under conditions well known to those skilled in the art. Compound C18 can be prepared from compound C32 in the presence of reagents including but not limited to acetic anhydride. Compound C31 can also be prepared from compound C21 under conditions involving an acid or base. Compound C21 can be prepared from the alkylation of C23 and C24, followed by reaction with C20 and decarboxylation.

[0100] Reaction Scheme D

[0101]

[0102] In Reaction Scheme D, variable R 1 , R 3 , R 10 , R 13 , R 14 , R15 and R 16 represents an alkyl or aryl group, including but not limited to methyl, ethyl, isopropyl and tolyl. 2 , R 4 , R 5 , R 7 , R 8 , R 9 and R 11 represents amine protecting groups well known to those skilled in the art.

[0103] Formula 2 compounds can be prepared by the reaction of compound D8 with matching compounds including but not limited to compounds D2, D3, D4, D5, D6 and D7. The conversion may require components including but not limited to chemical or enzyme catalysts and acids or bases. It will be appreciated by those skilled in the art that the resulting products from these conversions may require further conversion. For example, the resulting products from the alkylation of D8 and D7 may require reductive amination in the presence of a chemical catalyst or enzyme.

[0104] The compounds of formula 2 can be prepared by reducing compound D10 in the presence of one or more enzyme catalysts or one or more chemical catalysts, followed by deprotection if necessary. Compound D10 can be prepared by reacting 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 can also be prepared by reacting D13 or D14 with D2, D3 or D7 in the presence of one or more metals or organic catalysts, followed by additional transformations such as aminolysis, reductive amination, and / or deprotection.

[0105] Formula 2 compounds can be prepared by reduction of compound D15. This conversion may require one or more chemical or enzyme catalysts. D15 can be prepared as follows: D16 is reacted with D2, D3, D4, D5, D6 or D7 in the presence of one or more chemical catalysts, one or more enzymes and / or one or more bases, followed by additional conversions such as reductive amination, and / or deprotection. Formula 2 compounds can be prepared by decarboxylation of compound D17 in the presence or absence of a catalyst. Compound D17 can then be prepared as follows: D18 is reacted with D2, D3, D4, D5, D6 or D7 in the presence of one or more chemical catalysts, one or more enzymes and / or one or more bases, followed by additional conversions such as reductive amination, and / or deprotection.

[0106] The compound of formula 2 can be prepared by reduction of compound D19, D20, D21 or D22. The conversion may require one or more chemical or enzyme catalysts. D19 and D20 can be prepared as follows: D16 is reacted with D23 or D24 in the presence of an acid or a base. D21 and D22 can be prepared as follows: D8 is reacted with D23 or D24 in the presence of an acid or a base. Alternatively, D8 or D16 can be reacted with D28 in the presence of a base and then reacted with R 1 OH alcoholysis to produce D19, D20, D21 or D22.

[0107] The compound of formula 2 can be prepared by reduction of compound D25. This conversion may require one or more chemical or enzyme catalysts. Compound D25 can be prepared as follows: reacting D11 with D26 in the presence of a metal catalyst or reacting D12 with D26 in the presence of a metal catalyst, followed by aminolysis.

[0108] Compounds of Formula 2 can be prepared by reacting compound D27 with compound D2, D3, D4, D5, D6 or D7 in the presence of a reducing agent including but not limited to zinc and manganese and a metal catalyst, followed by additional transformations including but not limited to deprotection and reductive amination.

[0109] Reaction Scheme E

[0110]

[0111] In Scheme E, R 1 , R 2 , R 5 and R 16 The group is an alkyl or aryl group, including but not limited to methyl, ethyl, isopropyl and tolyl. 3 , R 4 , R 6 , R 7 , R 8 , R 9 , R 12 , R 13 , R 14 , R 15 and R 17 The protecting groups are well known to those skilled in the art. 1

[0112] The N-protected glutamate E2 can be reacted with a base, and the resulting anion can be reacted with reagents including, but not limited to, E3, E4, E5, E6, E7, E8, E9, and E10. The resulting product can then be converted to 2 using methods well known to those skilled in the art.

[0113] The N-protected glutamate E20 can react with a base, and the resulting product can react with reagents 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 can undergo reductive amination in the presence of a chemical or enzyme catalyst such as a transaminase. The resulting product can then be converted to 2 using methods well known to those skilled in the art.

[0114] E2 can be converted to E23 via amidation. E23 can then be reacted with a base to produce compound 2.

[0115] Compound 2 can be prepared by rearrangement and deprotection (if necessary) of E24. E24 can be prepared by cyanide addition to E25 followed by olefin and nitrile reduction or reduction and deprotection of E26. E25 or E26 can be prepared from E27 under conditions well known to those skilled in the art.

[0116] E28 or E29 can be converted into compound 2 under conditions well known to those skilled in the art. For example, E28 can be subjected to olefin reduction conditions, followed by saponification of ester, asymmetric decarboxylation of nitrile and reduction to produce E30 in the presence of chemical or enzyme catalysts such as but not limited to alkene reductase, which can be carried out in the presence of chemical or enzyme catalysts. Alternatively, E29 can be subjected to reduction of alkene and cyano group in the presence of chemical and enzyme catalysts to produce E30. After deprotection, intramolecular cyclization of E30 (if necessary) can then produce compound 2.

[0117] Compound 2 can be converted to compound E11 by methods including, but not limited to, halogenation with N-halosuccinimide and enzyme halogenation. Compound E11 can then react with reagents including, but not limited to, E12 and KCN. The resulting product can then be converted to 2 using methods well known to those skilled in the art.

[0118] Compound E15 or E16 can be reacted with a reagent such as E13 or E14 in the presence of a base and / or a chiral catalyst to form 1.

[0119] 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 performed in a stereoselective manner; in addition, the conversion from E21 to 1 can be performed via enzymatic resolution or dynamic kinetic asymmetric transformation.

[0120] Compound 2 can be prepared by reaction of E17 and E18 followed by deprotection if necessary. E17 can be prepared from E19 by methods well known to those skilled in the art.

[0121] Method for preparing Namatevir

[0122] It was found in step 2 of reaction scheme 1 that impurities such as (S)-N,N-diethyl-3,3-dimethyl-2-(2,2,2-trifluoroacetylamino)butyramide (denoted as diethylamide impurity, IMP-S2-1), (1R,2S,5S)-3-((R)-3,3-dimethyl-2-(2,2,2-trifluoroacetylamino)butyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (denoted as diisomeric impurities, IMP-S2-2) and 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]hexane-3-yl)-3,3-dimethyl-1-oxobutan-2-yl)-2,2,2-trifluoroacetamide (denoted as the bisamide impurity, IMP-S2-3) (see reaction scheme below). Advantageously, the present invention minimizes the formation of these undesirable impurities. The formation mechanism of (S)-N,N-diethyl-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanamide IMP-S2-1 is not clear, but does not involve the presence of diethylamine in the triethylamine (TEA) used in the reaction. In the case of adding methanesulfonyl chloride to a mixture of (S)-3,3-dimethyl-2-(2,2,2-trifluoroacetylamino)butyric acid, i.e., compound V, in the presence of triethylamine, or in the case of using a greater excess of triethylamine (3.0 equivalents vs. 2.5 equivalents of TEA) in the reaction, diethylamide impurity formation was observed. In both cases, the amount of diethylamide impurity and bisamide impurity was minimized by adding methanesulfonyl chloride to the compound of formula V in isopropyl acetate before adding triethylamine. The use of methanesulfonyl chloride and TEA was also found to be advantageous because minimal epimerization occurred when those reagents were used in the amidation reaction. The present invention therefore provides significant advantages because the claimed step 2 method 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 minimizes the amount of epimerized product IMP-S2-2 formed by controlling the amount of triethylamine base used.

[0123] Reaction Scheme 1 Step 2 with Possible Impurities

[0124]

[0125] Impurities that can be formed in the step 2 reaction:

[0126]

[0127] It was discovered in the process of step 3 of reaction scheme 1 that under certain reaction conditions acylurea impurities as well as rearrangement related impurities can be formed. The structures of the acylurea impurities that can be formed in step 3 are described 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-trifluoroacetylamino)butyryl)-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-trifluoroacetylamino)butyryl)-N-((3-(dimethylamino)propyl)carbamoyl)-N-ethyl-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide. It was found that under certain reaction conditions, (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl)acrylamide can undergo a rearrangement reaction to form a rearranged impurity IMP-S3-3, which can further react under the step 3 amidation reaction conditions to form an additional impurity designated as 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-trifluoroacetylamino)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide. It is desirable to minimize the amount of IMP-S3-4 formed to avoid further carryover of this impurity and subsequent reaction under the reaction conditions of Step 4 in which the amide moiety on the lactam ring of IMP-S3-4 can 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-trifluoroacetylamino)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide.

[0128] Step 3 reaction, wherein possible impurities formed in step 3 and step 4 of reaction scheme 1

[0129]

[0130] Acyl urea impurity formed in step 3

[0131]

[0132] The products formed in steps 3 and 4 and the rearrangement byproducts from step 3

[0133]

[0134] In step 3, it was found that 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, had an effect on the amount of acylurea impurities IMP-S3-1 and IMP-S3-2, as shown in column 4 of the table below: Step 3 reaction conditions. In the case of using unrecrystallized (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl)propanamide hydrochloride, Compound III at 50° C. in a two-reactor setup, no more than 5% of acylurea impurities IMP-S3-1 and IMP-S3-2 were formed, no more than 2% of rearranged impurity IMP-S3-3 was formed, with favorable reaction kinetics (less than 3% of the initial compound IV remained after ~2 hours reaction time). In the case of using recrystallized (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl)propanamide hydrochloride, compound III, at 50° C. in a two-reactor setup, no more than 10% of the acylurea impurities IMP-S3-1 and IMP-S3-2 are formed, and no more than 2% of the rearranged impurity IMP-S3-3 is formed, with favorable reaction kinetics (less than 3% of the initial compound IV remains after ~6 hours reaction time). The two-reactor process provides favorable reaction kinetics when carried out at 50° C. while maintaining relatively low levels of acylurea and rearranged impurities that can be formed. The disadvantage of the initial process conditions (column 2 of the table below) is the less than desirable reaction kinetics (16 hours or 60-100 hours reaction time, depending on the type of compound III used), while the one-reactor process when carried out at 50° C. causes a large amount of rearranged impurity formation (see column 3 of the table, no less than 50% or no less than 40% or rearranged impurity formation).

[0135] Table: Step 3 reaction conditions

[0136]

[0137] Step 5: Namatevir API crystallization method with optimized particle size control

[0138] Crystallization of PF-07321332 Anhydrous Form 1 (Compound I) begins with dissolving PF-07321332 MTBE solvate (Compound I') in isopropyl acetate solvent, followed by seeding with anhydrous Form 1 solid and adding heptane as an antisolvent. It is this step that is important in controlling particle size and polymorphic form. Therefore, crystallization and isolation of PF-07321332 is an important process for controlling the physical properties of the API for optimal drug product performance.

[0139] The initial concentration of API (PF-07321332MTBE solvate, Compound I') in isopropyl acetate, the time of heptane addition, the seed size and the seed amount had a detectable effect on the final particle size distribution. Statistical analysis was performed using Design Expert. The results of the analysis were graphically summarized using main effect plots and contour plots to facilitate the visualization of statistically significant factor effects and allow evaluation of their practical importance. Figure 1 Shown is the effect of the relationship between the initial concentration of the API in isopropyl acetate (mL / g), heptane addition time (hours), seed loading (% wt Compound I (Form I) / wt Compound I' (MTBE solvate)), and seed size (microns) on the particle size distributions (PSD's) of the final D[v,0.5] counts in microns.

[0140] Crystallization method has been carried out in a robust and consistent manner on a small to large scale. In addition, the target particle size is achieved via direct crystallization, without the grinding operation after crystallization. The method shows size-dependent growth, which depends on the size of the seed material. In order to minimize the dependence on the seed size, the seed loading and heptane addition duration are adjusted. The seed loading is optimized between 0.2 wt % and 1.5 wt %, and its target value is 0.75 wt % in all scales. The heptane addition duration is adjusted to 10 hours, and the range is 6 hours to 15 hours. These changes help to achieve the desired final particle size, independent of the seed particle size added. The method relies on primary nucleation and secondary nucleation rather than size-dependent crystal growth. The data obtained from different scales show a consistent particle size supply with minimal variation. The method wherein the seed loading is about 0.75 wt % and heptane is added in about 10 hours provides a consistent preparation of Namatevir, wherein the particle size distribution is well controlled. Figure 2 Figure 2 shows the correlation between seed size and final particle size distribution after API crystallization using 0.75 wt% seed loading (blue circles) and 0.5 wt% seed loading (orange circles) in a lab-scale (100 mL) experiment. Figure 3 and Figure 4The batch history data show that the process delivers particle size distributions in a robust and consistent manner, showing in each case greater than 50 batches of PSDs obtained with respect to D[v,0.5]. Individual batches were generally found to have particle size distributions ranging from about 12 microns to about 18 microns, from about 14 microns to about 16 microns, with the majority having a D[v,0.5] of about 15 microns.

[0141] Experimental Procedure

[0142] The synthesis of various compounds of the present invention is described below. Additional compounds within the scope of the present invention can be prepared using the methods described in these examples, either alone or in combination with techniques generally known in the art. All starting materials in these preparations and examples are commercially available or can be prepared by methods known in the art or as described herein.

[0143] All reactions were performed under nitrogen or argon atmosphere with continuous stirring unless otherwise stated. When appropriate, reaction equipment was dried with a heat gun under dynamic vacuum, and anhydrous solvents (Sure-Seal from Aldrich Chemical Company, Milwaukee, Wisconsin) were used. TM products or DriSolv from EMD Chemicals, Gibbstown, NJ TM product). In some cases, commercial solvents are passed through the filling 5-Molecular sieve column until the following QC standards for water are obtained: a) <100ppm for dichloromethane, toluene, N,N-dimethylformamide and tetrahydrofuran; b) <180ppm for methanol, ethanol, 1,4-dioxane and diisopropylamine. For very sensitive reactions, the solvent is further treated with sodium metal, calcium hydride or molecular sieves and distilled just before use. Other commercial solvents and reagents are used without further purification. For syntheses that cite procedures in other embodiments or methods, the reaction conditions (reaction time and temperature) may vary. The product is generally dried under vacuum and then carried to further reactions or.

[0144] The progress of the reaction can be monitored by thin layer chromatography (TLC), liquid chromatography-mass spectrometry (LCMS), high performance liquid chromatography (HPLC) and / or gas chromatography-mass spectrometry (GCMS) analysis. TLC can be performed on pre-coated silica gel plates using a fluorescent indicator (254 nm excitation wavelength) and under UV light and / or with I 2 、KMnO 4 、CoCl 2, phosphomolybdic acid and / or ammonium molybdate cerium dye visualization.LCMS data can be obtained on Agilent 1100 series equipment, using Leap Technologies automatic sampler, Gemini C18 column, acetonitrile / water gradient, and trifluoroacetic acid, formic acid or ammonium hydroxide regulator.Column eluent is analyzed by Waters ZQ mass spectrometer, and scans from 100 to 1200Da with positive and negative ion modes.Other similar equipment can also be used.HPLC data are generally also obtained on Agilent 1100 series equipment, using indicated column, acetonitrile / water gradient, and trifluoroacetic acid or ammonium hydroxide regulator.GCMS data are obtained with Hewlett Packard 6890 furnace, using HP 6890 syringe, HP-1 column (12m x 0.2mm x 0.33μm), and helium carrier gas.Sample can be analyzed on HP 5973 mass selective detector, and scans from 50 to 550Da with electron ionization. Purification is performed by medium performance liquid chromatography (MPLC) using an Isco CombiFlash Companion, AnaLogix IntelliFlash 280, Biotage SP1, or Biotage IsoleraOne device and pre-packed Isco RediSep or Biotage Snap silica cartridges. Chiral purification is performed by chiral supercritical fluid chromatography (SFC), typically using Berger or Thar devices; columns such as ChiralPAK-AD, -AS, -IC, Chiralcel-OD or -OJ columns; and CO with methanol, ethanol, 2-propanol or acetonitrile. 2 mixtures, either alone or modified with trifluoroacetic acid or propan-2-amine. UV detection can be used to trigger fraction collection. For syntheses citing procedures in other examples or methods, purification may vary: generally, the solvents and solvent ratios used for the eluent / gradient are selected to provide the appropriate R f s or retention time.

[0145] Mass spectrometry data are reported from LCMS analyses. Mass spectrometry (MS) was performed using atmospheric pressure chemical ionization (APCI), electrospray ionization (ESI), electron impact ionization (EI), or electron scattering ionization (ES) sources. Proton NMR spectroscopy ( 1 H NMR) chemical shifts are given in ppm 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) and are referenced to the residual peak of the deuterated solvent (chloroform, 7.26 ppm; CD 2 HOD, 3.31ppm; acetonitrile-d 2, 1.94ppm; dimethyl sulfoxide-d 5 , 2.50 ppm; DHO, 4.79 ppm). Peak shapes are described as follows: s, singlet; d, doublet; dd, double doublet; ddd, double doublet; dt, double triplet; t, triplet; q, quartet; qd, double quartet; quin, quintet; m, multiplet; br s, broad singlet; app, distinct. Analytical SFC data were generally obtained on a Berger analytical instrument as described above. Optical rotation data were obtained on a PerkinElmer Model 343 polarimeter using a 1 dm cell. Microanalyses were performed by Quantitative Technologies Inc. and were within 0.4% of the calculated values.

[0146] Unless otherwise stated, chemical reactions were performed at room temperature (about 23 degrees Celsius). Unless otherwise stated, all reactants were obtained commercially and used without further purification or were prepared by methods known in the literature.

[0147] The terms "concentrate", "evaporate" and "concentrate in vacuo" refer to the removal of solvent on a rotary evaporator under reduced pressure with a bath temperature of less than 60°C or at a specified temperature. The abbreviations "min" and "h" stand for "minute" and "hour", respectively. The term "TLC" refers to thin layer chromatography, "room temperature or ambient temperature" means a temperature of 18 to 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; "CO" means temperature. 2 " is carbon dioxide; "eq." or "equiv." is equivalent; "DMSO-d 6 " is hexadeuterated dimethyl sulfoxide; "g" is gram; "HCl" is hydrogen chloride; "HOPO" is 2-hydroxypyridine-N-oxide; "HRMS" is high resolution mass spectrometry; "Hz" is hertz; "iPrOAc" is isopropyl acetate; "K" is Kelvin; "kg" is kilogram; "L" is liter; "M" is molar or molar concentration; "mbar" is millibar; "MEK" is methyl ethyl ketone; "MeOH" is methanol; "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; "NaHCO 3 "It is sodium bicarbonate;" 2 SO 4" is sodium sulfate; "PXRD" is powder X-ray diffraction; and "THF" is tetrahydrofuran.

[0148] Hydrogenations can be performed in a Parr shaker under pressurized hydrogen or in a Thales-nano H-Cube flow hydrogenation apparatus under full hydrogen at a flow rate of 1-2 mL / min at the specified temperature or as otherwise specified.

[0149] HPLC, UPLC, LCMS, GCMS and SFC retention times were measured using the methods described in the procedures.

[0150] The optical rotation of an enantiomer can be measured with a polarimeter. According to its observed optical rotation data (or its specific optical rotation data), the enantiomer with clockwise rotation is designated as the (+)-enantiomer and the enantiomer with counterclockwise rotation is designated as the (-)-enantiomer. Racemic compounds are indicated by not depicting or describing stereochemistry or by the presence of (+ / -) adjacent to the structure; in this latter case, the indicated stereochemistry represents only one of the two enantiomers constituting the racemic mixture.

[0151] The compounds and intermediates described below are named using the naming conventions provided with ACD / ChemSketch 2019.1.1 file version C05H41 build 110712 (Advanced Chemistry Development, Inc., Toronto, Ontario, Canada) or using ChemDraw 18.0 ( Informatics; perkinelmer.com). The naming conventions provided with ACD / ChemSketch 2019.1.1 or ChemDraw 18.0 are well known to those skilled in the art, and it is believed that the naming conventions provided with ACD / ChemSketch 2019.1.1 generally conform to the recommended organic chemical nomenclature of IUPAC (International Union of Pure and Applied Chemistry) and CAS Index rules.

[0152] General Methods for Solid Form Characterization

[0153] Solid form data for certain starting materials, intermediates and products were obtained using the powder X-ray diffraction method described below.

[0154] Powder X-ray Diffraction:

[0155] Powder X-ray diffraction analysis was performed with a Bruker AXSD4 Endeavor diffractometer equipped with a Cu radiation source. The divergence slit was set at 0.6 mm, while the secondary optics used a variable slit. The diffracted radiation was detected by a PSD-Lynx Eye detector. The X-ray tube voltage and amperage were set to 40 kV and 40 mA, respectively. Data were collected in a Theta-2Theta goniometer at Cu wavelengths from 3.0 to 40.0 degrees 2-Theta, using a step size of 0.020 degrees and a step time of 0.3 seconds. Samples were prepared as follows: they were placed in a silicon low background sample holder and rotated during collection.

[0156] Powder X-ray diffraction analysis was performed with a Bruker AXSD8 Advance diffractometer equipped with a Cu radiation source. The diffracted radiation was detected by a LYNXEYE_EX detector with a motorized slit. Both the primary and secondary were equipped with 2.5 soller slits. The X-ray tube voltage and amperage were set to 40 kV and 40 mA, respectively. Data were collected in a Theta-Theta goniometer with a locked coupled scan at Cu K-α (average) wavelength from 3.0 to 40.0 degrees 2-Theta, using an increment of 0.02 degrees and a scan speed of 0.5 seconds per step. The sample was prepared as follows: placed in a silicon low background sample holder.

[0157] Data were collected using Bruker DIFFRAC Plus software and analyzed by EVA diffract plus software in both instruments. The PXRD data files were not processed before peak finding. Peaks selected with a threshold of 1 were used for preliminary peak attribution using the peak finding algorithm in the EVA software. To ensure validity, adjustments were made manually; the output of automated attribution was visually inspected and the peak position was adjusted according to the peak maximum. Peaks with a relative intensity ≥ 3% were generally selected. In general, unresolved or noise-consistent peaks were not selected. Typical errors associated with peak positions from PXRD described in the USP are up to + / -0.2°2-Theta (USP-941). Unless otherwise stated, the variance of each peak reported in the PXRD peak table is + / -0.2°2-θ.

[0158] Preparation of raw materials

[0159] Preparation of (1R, 2S, 5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid methyl ester hydrochloride; Compound VII

[0160] Reaction Scheme SM-1

[0161]

[0162] The title compound was prepared according to the following procedure as described above in Reaction Scheme SM-1.

[0163] CoBr 2 (0.05-0.15 equivalents), (1E,1'E)-1,1'-(pyridine-2,6-ylidene)bis(N-(2-(tert-butyl)phenyl)ethane-1-imine) or (1E,1'E)-1,1'-(pyridine-2,6-ylidene)bis(N-(2-isopropylphenyl)ethane-1-imine) (0.05-0.15 equivalents, i.e., ligand), and tetrahydrofuran (10 vol) were added to the reactor. Zn (2.25-2.5 equivalents) was added. I 2 (0.25 eq.) / tetrahydrofuran (1-2 vol). A purple solution was obtained. (S)-2,5-dihydro-1H-pyrrole-1,2-dicarboxylic acid 1-(tert-butyl) 2-methyl ester (1 eq., Compound VII") was added as a pure oil. 2,2-dichloropropane or 2,2-dibromopropane (1.5-2.0 eq.) / tetrahydrofuran (1-3 vol) was slowly added and stirred until the reaction was complete (Compound VII' was formed). The reaction mixture was filtered through Celite. Methyl tert-butyl ether (MTBE), HCl and water were used during this operation. 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 washed with HCl (3 eq.) / methanol or with tetrahydrofuran and H Cl gas (3 equivalents) was used for treatment. After the reaction was completed, (1R, 2S, 5S)-6,6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxylic acid methyl ester hydrochloride was separated as a solid from MTBE and THF. The typical yield is 50-80%. For example, 3.00g of (S)-2,5-dihydro-1H-pyrrole-1,2-dicarboxylic acid 1-(tert-butyl) 2-methyl ester was converted into 1.98g of (1R, 2S, 5S)-6,6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxylic acid methyl ester hydrochloride (80% yield). Product characterization data are consistent with previous reports: Oruganti, S. et al. Tetrahedron, 2017, 73, 4285.

[0164] The product (1R, 2S, 5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid methyl ester hydrochloride; the PXRD of compound VII is Figure 6 Available in.

[0165] Preparation of (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl)propanoic acid methyl ester p-toluenesulfonate The title compound was prepared as described in Scheme SM-2 and described below.

[0166] Reaction Scheme SM-2

[0167]

[0168] Add (tert-butyloxycarbonyl)-L-glutamic acid dimethyl ester (50.2g, 1.00 equivalent) and tetrahydrofuran (753mL, 15mL / g) to the reactor. The solution is cooled to -78°C in a dry ice / acetone bath. A solution of bis(trimethylsilyl)amide lithium (1M) in THF (371mL 2.1 equivalents) is precooled and added via a cannula. A solution of bromoacetonitrile (13.5mL, 1.07 equivalents) in THF (70mL, 1.4mL / g) is precooled and added to the reactor. After the addition is complete, the mixture is stirred for at least 45 minutes, keeping the temperature below -70°C. A solution of methanol (20mL, 0.4mL / g) in tetrahydrofuran (25mL, 0.5mL / g) is added to the reaction mixture. Acetic acid (40.5mL, 4 equivalents) / tetrahydrofuran (100mL, 2mL / g) is added to the reaction mixture via an addition funnel. The mixture was warmed to -20 ° C in 30 minutes. Sodium chloride (12 mass %) / water (251 mL, 5 mL / g) was added to the mixture. The layers were separated. The organic layer was concentrated to 2-3 mL / g. Toluene (1000 L, 20 mL / g) was added to the residue. The mixture was concentrated to ~20 mL / g and then distilled at a constant volume, maintaining 20 mL / g. Diatomaceous earth (10 g, 0.2 g / g) was added to the solution and the slurry was stirred for 2 h. The slurry was filtered on a diatomaceous earth bed (10 g, 0.2 g / g). The filter cake was washed with toluene (1 mL / g). The filtrate was concentrated to ~2-3 mL / g. Methanol (250 mL, 5 mL / g) was added. Constant volume distillation was performed and replaced with methanol (650 mL, 13 mL / g). The resulting (2S,4R)-dimethyl 2-((tert-butoxycarbonyl)amino)-4-(cyanomethyl)pentanedioate (44.5 g in solution, 80% in situ yield) was carried forward as a light amber solution

[0169] The reactor was charged with Raney Nickel 2400 (6.05 g, 100 wt%). A solution of (2S,4R)-dimethyl 2-((tert-butoxycarbonyl)amino)-4-(cyanomethyl)pentanediolate in methanol (6 g input, 40.83 g solution) was added, followed by additional methanol (120 mL, 20 mL / g amount) and ammonia (7N in MeOH, 2.7 mL, 1.0 equiv). The reactor was heated with N 2 Purge three times, then rinse with H 2Purge three times. The reactor was pressurized to 5 bar and heated to 24 ° C and stirred for 24 hours. The reactor was rinsed and sampled to confirm the completion of the reaction (i.e., the uncyclized intermediate (2S, 4S)-2-(2-aminoethyl)-4-((tert-butoxycarbonyl)amino)pentanedicarboxylic acid dimethyl ester was converted to the cyclized intermediate (S)-2-((tert-butoxycarbonyl)amino)-3-((S)-2-oxopyrrolidin-3-yl)propionic acid methyl ester). The reaction was filtered and the catalyst was washed twice with methanol (5mL / g, 30mL). In preparation for the subsequent steps, the filtrate was concentrated to ~2-3mL / g in vacuo. Isopropanol (20mL / g, 125mL) was added and concentrated to remove residual MeOH and water to a target total volume of 7mL / g (6mL / g IPA). p-Toluenesulfonic acid (pTsOH) monohydrate (1.5 equivalents) was added to the solution. The mixture was diluted with isopropanol (20 mL / g) and the solution was reconcentrated to remove water. MTBE (4 mL / g) was added to the reactor. The slurry was warmed to 50 ° C and kept overnight. The mixture was cooled to 10 ° C in 2 hours. The mixture was kept 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 (S)-2-amino-3-((S)-2-oxopyrrolidine-3-yl) propionic acid methyl ester p-toluenesulfonate was separated as a white crystalline solid (86% yield, 2 steps).

[0170] 1 H NMR (600 MHz, DMSO-d 6 , 298K): δ8.54 (s, 2H), 7.98 (s, 1H), 7.95 (s, 1H), 7.65 (s, 1H), 7.49 (d, J = 8.0Hz, 2H), 7.12 (d, J = 8.0Hz, 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-d 6 , 298K): δ178.2, 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 + )C 8 H 15 N 2 O 3 + Calculated: 187.1077, Measured: 187.1077

[0171] Alternative preparation of methyl (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl)propanoate p-toluenesulfonate

[0172] The title compound was prepared as depicted in Reaction Scheme SM-3 and as described below.

[0173] Reaction Scheme SM-3

[0174]

[0175] Step 1: Preparation of (S,Z)-4-((dimethylamino)methylene)-5-oxopyrrolidine-1,2-dicarboxylic acid 1-(tert-butyl) 2-methyl ester

[0176] (S)-5-oxopyrrolidine-1,2-dicarboxylic acid 1-(tert-butyl) 2-methyl ester (10.00 g) was added to toluene (60 mL) with stirring at 20°C to provide a clear colorless solution. To this solution was added tert-butoxybis(dimethylamino)methane (Brederecks reagent) (8.60 g, 10.2 mL, 1.2 eq.) in one portion and the resulting reaction mixture was stirred at a temperature of 95-100°C for 16 hours. The reaction was checked for completion by UPLC (2.7% of the starting material remained) and the temperature was then adjusted to 80°C. 40 mL of n-heptane was added to provide a yellow solution and the mixture was cooled to 60°C, at which time product seeds (if any) could be added. The mixture was cooled to 5°C within 2 hours and the resulting slurry was stirred at 5°C for 2 hours. 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 provide (S,Z)-1-(tert-butyl)2-methyl 4-((dimethylamino)methylene)-5-oxopyrrolidine-1,2-dicarboxylate as a white to off-white solid (10.3 g, 84% yield). 1 HNMR (400MHz, 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). >97% purity, 1 H NMR

[0177] Step 2: Preparation of (S,E)-4-(cyanomethylene)-5-oxopyrrolidine-1,2-dicarboxylic acid 1-(tert-butyl) 2-methyl ester

[0178] To a 1-neck 250mL round-bottom flask equipped with a toxic gas scrubber and under an inert atmosphere, (S, Z)-4-((dimethylamino)methylene)-5-oxopyrrolidine-1,2-dicarboxylic acid 1-(tert-butyl) 2-methyl ester (5.00g, 1.00 equivalent) and acetic acid (30.0mL) were added. The mixture was stirred at 20°C to form a solution, to which potassium cyanide (1.20g, 1.10 equivalents) was added. The reaction mixture was stirred at 20°C for 24 hours to obtain a dark yellow solution. Water (210mL, 7 volumes) was slowly added to the dark yellow solution within 25 minutes to precipitate the product. The mixture was then stirred for 5 minutes and subsequently filtered on a sinter funnel. The resulting white solid was washed with water (3x30mL) and dried with an air stream under vacuum on a sinter funnel. The resulting (S,E)-1-(tert-butyl)2-methyl 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). >95% purity, 1 H NMR. The material was used in the subsequent step without further purification.

[0179] 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)

[0180] Methanol (200 ml, 20 ml / g) was stirred in a reactor at 5°C and acetyl chloride (2.79 mL, 1.1 eq) was added thereto, which was added dropwise to control the exotherm produced to produce anhydrous HCl (and 1.1 eq methyl acetate byproduct). The solution was then warmed to 20°C. (S,E)-4-(cyanomethylene)-5-oxopyrrolidine-1,2-dicarboxylic acid 1-(tert-butyl) 2-methyl ester (10.0 g, 1.0 eq, limiting reagent) as a solid was added in one batch to the HCl methanol solution to provide a clear, colorless solution. To this solution was added 5% Pd / C A503023-5 type (2.0 g). The reactor was purged three times with nitrogen, then three times with hydrogen, then pressurized to 50 psi with hydrogen and the mixture was stirred at 600 rpm for 16 hours. The reactor was rinsed and sampled to confirm the reaction was complete. The reaction was incomplete (>3% residual nitrile intermediate), so hydrogenation was performed 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 (2x 10mL, 2x 1mL / g). The resulting methanol filtrate containing (2S, 4S)-4-(2-aminoethyl)-5-oxopyrrolidine-1,2-dicarboxylic acid 1-(tert-butyl) 2-methyl ester hydrochloride was used as is in subsequent steps.

[0181] 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)

[0182] Methanol (200 ml) was stirred at 5°C and acetyl chloride (2.79 mL, 1.1 eq) was added dropwise to control the exotherm produced to produce anhydrous HCl (and 1.1 eq methyl acetate byproduct). In a separate hydrogenation vessel, 2-propanol (50 mL) was stirred at 20°C and to it was added (S,E)-4-(cyanomethylene)-5-oxopyrrolidine-1,2-dicarboxylic acid 1-(tert-butyl) 2-methyl ester (10.0 g, 1.0 eq, limiting reagent) to produce a slurry. 5% Pd / C A503023-5 type (2.0 g) was added as a solid to the slurry. The reactor was purged three times with nitrogen, then purged three times with hydrogen, then pressurized to 50 psi with hydrogen and stirred at 600 rpm for 2 hours. The reactor was purged with nitrogen, then the HCl methanol solution was added. The reactor was purged three times with nitrogen, then purged three times with hydrogen, then pressurized to 50 psi with hydrogen and stirred at 600 rpm for 3 days. The reactor was rinsed and sampled to confirm that the reaction was complete (>3% residual nitrile intermediate), then filtered through Arbocel to remove the catalyst, and the filter was washed with methanol (2x 10 mL). The resulting methanol / 2-propanol filtrate containing (2S, 4S)-4-(2-aminoethyl)-5-oxopyrrolidine-1,2-dicarboxylic acid 1-(tert-butyl) 2-methyl ester hydrochloride was used as is for subsequent steps.

[0183] Step 4: Preparation of methyl (S)-2-((tert-butyloxycarbonyl)amino)-3-((S)-2-oxopyrrolidin-3-yl)propanoate (as a solution in isopropanol)

[0184] The methanol solution of (2S,4S)-4-(2-aminoethyl)-5-oxopyrrolidine-1,2-dicarboxylic acid 1-(tert-butyl)2-methyl ester hydrochloride from step 3 above was stirred at 25°C, and 1 M NaHCO was slowly added thereto. 3 Aqueous solution (89.2 mL, 2.5 eq.) was used to control the generated CO 2The resulting subdivided suspension was stirred at 25°C for 18 hours or until the reaction was confirmed to be complete by liquid chromatography (no more than 0.5% area residual starting material remaining). The reaction was then quenched by adding citric acid (4.11 g, 0.60 equiv.) / water (30 mL) in portions and tested by pH paper to confirm that the pH was 5-7. The mixture was then concentrated in vacuo (90-100 mbar) to a volume of approximately 100-120 mL. Ethyl acetate (100 mL) was added thereto, the mixture was stirred at 25°C for 10 minutes, and then the organic and aqueous layers were separated. Ethyl acetate (100 mL) was added to the aqueous layer, stirred at 25°C for 10 minutes, and then the layers were separated. The organic layers were combined and concentrated in vacuo (200 mbar) to a final volume of approximately 30 mL. 2-Propanol (200 mL) was added thereto and the solution was 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 was checked by Karl Fischer analysis to confirm that the water content was less than 1% by weight and then used as is in the subsequent step.

[0185] Step 5: Preparation of (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl)propionic acid methyl ester p-toluenesulfonate

[0186] The isopropanol solution of (S)-methyl 2-((tert-butoxycarbonyl)amino)-3-((S)-2-oxopyrrolidin-3-yl)propanoate from step 4 was stirred at 20°C. Isopropanol (200 mL) and p-toluenesulfonic acid monohydrate (10.33 g, 1.5 eq) were added to a separate container. The p-TSA solution was concentrated to a final volume of approximately 70 mL in vacuo (90-100 mbar) to remove water. The concentrated p-TSA solution was then added to the (S)-methyl 2-((tert-butoxycarbonyl)amino)-3-((S)-2-oxopyrrolidin-3-yl)propanoate solution, followed by an in-line rinse of 2-propanol (20 mL). The solution was distilled to a final volume of approximately 70 mL (7 mL / g) under vacuum (90-100 mbar) with a cannula temperature of 40°C to remove additional water. The sample solution is used for Karl-Fischer analysis (no more than 1% by weight water). If>1%wt water, then add additional 2-propanol as needed and repeat the reduced pressure distillation in step 5 until the target water content is achieved. The reaction mixture is heated to 50°C at atmospheric pressure and stirred for 12-18 hours until the reaction is completed (liquid chromatography does not observe raw materials). The desired product crystallizes during this maintenance period. After the reaction is completed, tert-butyl methyl ether (50mL) is added in a batch. The resulting slurry is cooled from 50°C to 10°C within 2 hours. The slurry is stirred at 10°C for 1 hour and then filtered under vacuum. The crystallization container is rinsed with tert-butyl methyl ether (40mL) and transferred to the filter as a filter cake washing. The product filter cake is pumped to dryness under vacuum to remove liquid, and then the product is dried under vacuum at 40°C. The desired (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl) propionic acid methyl ester p-toluenesulfonate is separated, which is a white crystalline solid.

[0187] The yield of steps 3-5 of the above process using MeOH / AcCl hydrogenation was 44% (20% overall, steps 1-5), wherein the product had 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% as determined by Q-NMR. The yield of steps 3-5 of the process using isopropanol 2-stage hydrogenation was 63% (30% overall, steps 1-5), wherein the product had 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% as determined by Q-NMR.

[0188] Preparation of [(1R)-7,7-dimethyl-2-oxo-norbornane-1-yl]methanesulfonic acid (2S)-2-amino-3-[(3S)-2-oxopyrrolidin-3-yl]propionic acid methyl ester salt

[0189] The title compound was prepared as depicted in Reaction Scheme SM-4 and as described below.

[0190] Reaction Scheme SM-4

[0191]

[0192] A 2M NaOH (4.8 mL, 4.8 mmol) aqueous solution was added to a mixture of (1S,2S)-1-[(2,3-dimethylcycloprop-2-ene-1-ylidene)amino]indan-2-ol hydrochloride (557 mg, 0.96 mmol) in MTBE (6 mL) at 20°C and stirred for 10 minutes. The organic layer was collected and the aqueous layer was washed with MTBE (4 mL). 2 SO 4 The combined organic fractions were filtered on the upper column, and the filtrate was directly added to a mixture of 2-(diphenylmethyleneamino)acetic acid methyl ester (97.0%, 5.00g, 19.1mmol) and 3-methylene-2-oxo-pyrrolidine-1-carboxylic acid tert-butyl ester (4.15g, 21.1mmol) in MTBE (10mL). The mixture was stirred at 35°C for 3.5 hours, and then (-)-CSA (9.10g, 38.4mmol) was added in batches at 35°C. The mixture was heated to 65°C for 18 hours, then diluted with acetone (70mL). The mixture was refluxed for 20 minutes and diluted with MTBE (130mL). The mixture was refluxed for 4 hours, then cooled to 20°C and filtered. The solid was washed with a 2:1 mixture (3x 30mL) of MTBE and acetone. The solid was collected and dried under high vacuum to provide the title compound [(1R)-7,7-dimethyl-2-oxo-norbornan-1-yl]methanesulfonic acid (2S)-2-amino-3-[(3S)-2-oxopyrrolidin-3-yl]propanoic acid methyl ester salt; (5.79 g, 69%, >25:1 dr) as a solid. MS (ESI) [M+H-CSA] + 187.1. 1 H NMR (500 MHz, methanol-d 4)δ4.24(dd, J=9.7, 3.6Hz, 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=14.8Hz, 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: Four interchangeable protons are not seen. 13 C NMR (126 MHz, methanol-d 4 )δ218.28, 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.

[0193] Preparation of (S)-tert-butyl 3-((S)-2-((diphenylmethylene)amino)-3-methoxy-3-oxopropyl)-2-oxopyrrolidine-1-carboxylate;

[0194] The title compound was prepared as depicted in Reaction Scheme SM-5 and as described below.

[0195] Reaction Scheme SM-5

[0196]

[0197] It should be noted that all solvents used in this procedure were purchased as anhydrous grade and degassed by bubbling with nitrogen for about 30 minutes. 2 The reaction was carried out in a glove box. 4 ]PF 6A 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 plate, followed by a solution of (R)-FeSulPhos in THF (0.020 M, 25 μL, 0.5 μmol, 1 mol%). 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. Then, 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 added, and the vial was sealed and stirred at 500 rpm for 24 hours at -10°C to -4°C. 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 (S)-tert-butyl 3-((S)-2-((diphenylmethylene)amino)-3-methoxy-3-oxopropyl)-2-oxopyrrolidine-1-carboxylate was formed in 74% assay yield (rt 6.17 minutes, m / z+ve=451). The reference time was compared to an independently synthesized sample of (S)-tert-butyl 3-((S)-2-((diphenylmethylene)amino)-3-methoxy-3-oxopropyl)-2-oxopyrrolidine-1-carboxylate to identify the product.

[0198] SFC method: (Chiracel OX-H 250mm x 4.6mm x 5um (P / N: 63325), mobile phase A: CO 2 , mobile phase B: isopropanol + 0.2% 7N ammonia / methanol, 3 mL min -1 , 40°C, UV detection at 210 nm. 0-1 min 5% B, 1-9 min 5-60% B, 9-9.5 min 60% B, 9.5-10 min 60-5% B.

[0199] Using the above procedure, the resulting (S)-3-((S)-2-((diphenylmethylene)amino)-3-methoxy-3-oxopropyl)-2-oxopyrrolidine-1-carboxylic acid tert-butyl ester can be converted to [(1R)-7,7-dimethyl-2-oxo-norbornan-1-yl]methanesulfonic acid (2S)-2-amino-3-[(3S)-2-oxopyrrolidin-3-yl]propanoic acid methyl ester salt

[0200] Preparation of (S)-3,3-dimethyl-2-(2,2,2-trifluoroacetylamino)butyric acid, i.e., compound V

[0201] The title compound was prepared as depicted in Reaction Scheme SM-6 and as described below.

[0202] Reaction Scheme SM-6

[0203]

[0204] A methanol solution of (S)-2-amino-3,3-dimethylbutyric acid is prepared and sodium methoxide is added. Ethyl trifluoroacetate is added and the mixture is stirred until the reaction is complete. The reaction is diluted with ethyl acetate and washed with brine. Heptane is added to the organic layer containing (S)-3,3-dimethyl-2-(2,2,2-trifluoroacetylamino)butyric acid, and the solution is concentrated to 10 mL / g at 50°C. This is repeated three times to remove as much ethyl acetate as possible. After the second distillation, seeds of (S)-3,3-dimethyl-2-(2,2,2-trifluoroacetylamino)butyric acid may be introduced. After the final distillation, the mixture is cooled to 20°C and particles are formed, which are then filtered and washed with heptane. The solid is dried at 40°C. Crystal data is obtained and provided in the table below, and the PXRD pattern of the substance is as shown in Figure 5 supply.

[0205] Table - Crystal data and structural refinement of (S)-3,3-dimethyl-2-(2,2,2-trifluoroacetylamino)butyric acid, i.e., compound V:

[0206]

[0207] Figure 5 A PXRD pattern is provided for (S)-3,3-dimethyl-2-(2,2,2-trifluoroacetylamino)butanoic acid, which is characterized by the PXRD peaks in the table below.

[0208] Table - PXRD peaks of (S)-3,3-dimethyl-2-(2,2,2-trifluoroacetylamino)butyric acid:

[0209] Example

[0210] Step 1: Preparation of sodium (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate

[0211]

[0212] Combine (1R, 2S, 5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid methyl ester hydrochloride (Compound VII, 40 g, 195 mmol, 1.0 equiv)), tetrahydrofuran (80 mL, 2 mL / g Compound VII) and water (40 mL, 1 mL / g Compound VII) and stir at 25°C. Add triethylamine (40.7 mL, 292 mmol, 1.5 equiv) and stir the mixture for 30 minutes. The pH of the stirred mixture should be no less than 8.5. Stop stirring and allow the phases to separate. Remove the aqueous phase to provide an organic solution of (1R, 2S, 5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid methyl ester. In a separate container, sodium hydroxide (8.16 g, 204 mmol, 1.05 equiv), tetrahydrofuran (360 mL, 9 mL / g Compound VII) and water (40 mL, 1 mL / g Compound VII) were combined and heated to 40°C with stirring. An organic solution of (1R, 2S, 5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid methyl ester was added to the mixture over no less than 15 minutes, and the resulting mixture was stirred at 40°C for 4 hours. The reaction was judged complete by UPLC analysis of a sample (the goal was to have no more than 4% (1R, 2S, 5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid methyl ester remaining, and stirring was continued if the reaction was not complete). Upon completion, the mixture was cooled to 20°C and stirred for no less than 2 hours. The solid was collected by filtration, rinsed with 96:4 THF / water (80 mL), and dried in a vacuum oven at 70 °C to provide sodium (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate, Compound VI.

[0213] 1 H NMR (600 MHz, CD 3 OD-d 4 , 298K): δ3.37 (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).

[0214] 13 C NMR (150 MHz, CD 3 OD-d 4 , 298K): δ181.6, 64.3, 47.2, 37.7, 31.6, 27.1, 20.9, 14.2.

[0215] HRMS: (ESI + )C 8 H 14 O2 N + Calculated: 156.1019, measured: 156.1020 (mass deviation + 0.83ppm)

[0216] Several solid forms of sodium (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate, Compound VI, have been isolated and characterized by PXRD.

[0217] Figure 7 A PXRD pattern of the starting form of (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate is provided and is characterized by the peaks in the table below.

[0218] Table - PXRD peaks (2-theta degrees) of the initial form of sodium (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate:

[0219] Angle [2θ] Relative Strength Angle [2θ] Relative Strength 5.5 19 25.8 11 5.9 9 26.0 8 6.6 59 26.7 4 10.7 2 28.1 3 16.0 9 28.9 6 16.3 6 29.8 4 16.7 10 30.3 8 17.0 52 30.8 8 17.3 42 32.0 3 17.7 15 32.5 5 18.5 7 33.8 7 19.0 15 34.6 4 19.3 100 35.8 6 21.3 45 36.6 8 23.2 12 36.9 7 23.5 17 37.6 3 24.2 20

[0220] Figure 8 A PXRD pattern designated Form B of sodium (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate is provided and is characterized by the peaks in the table below.

[0221] Table - PXRD Peaks of Sodium (1R,2S,5S)-6,6-Dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate Form B:

[0222]

[0223] Fig. 9 A PXRD pattern is provided for a form of sodium (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate designated Material A, which is characterized by the PXRD peaks in the table below.

[0224] Table - PXRD peaks of sodium (1R, 2S, 5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate, i.e. substance A:

[0225]

[0226] Step 2: Preparation of (1R, 2S, 5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetylamino)butyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid, i.e., compound IV

[0227]

[0228] (S)-3,3-dimethyl-2-(2,2,2-trifluoroacetylamino)butyric acid, Compound V (38.9 g, 169 mmol, 1.2 equiv), methanesulfonyl chloride (17.8 g, 155 mmol, 1.1 equiv) and isopropyl acetate (500 mL, 20 mL / g Compound V) were combined and stirred at 20°C. Triethylamine (49.0 mL, 423 mmol, 2.5 equiv) was added 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 equiv) was added, and the mixture was stirred for 4 hours. A sample of the reaction mixture was obtained and analyzed for reaction completion (no more than 3% VI, UPLC). If the reaction was not completed, additional triethylamine may be added. The reaction mixture was quenched by adding aqueous citric acid (74 g citric acid monohydrate, 353 mmol, 2.5 eq. in 150 mL water) and the mixture was heated to 40°C. The mixture was stirred for at least 10 minutes and then the layers were allowed to clarify. The aqueous phase was removed and the organic phase was washed with 2 portions of water (125 mL each). The organic phase was cooled to 10-15°C and concentrated by vacuum distillation (-100 mbar, gradually warming to a maximum jacket temperature of 60°C) to a volume of approximately 192 mL. The mixture was analyzed for water content (Karl-Fischer); if the water was greater than 3 wt%, the vacuum distillation was repeated with additional isopropyl acetate. The solution was heated to 60°C and heptane (192 mL) was added. The mixture was stirred and cooled to 10°C at a rate of ~-12°C / hour. The slurry was stirred at 10°C for 3 hours. The solid was collected by filtration and rinsed with 1:1 iPrOAc / heptane (100 mL). The solid was dried in a vacuum oven at 50 °C to provide (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetylamino)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid, Compound IV.

[0229] 1 H NMR (600 MHz, DMSO-d 6 , 298K): δ12.72 (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).

[0230] 13 C NMR (150 MHz, DMSO-d 6, 298K): d 172.3, 167.6, 156.9( 2 J CF =37Hz), 115.8( 2 J CF =288Hz), 59.2, 58.1, 47.2, 34.7, 29.7, 26.7, 26.2, 25.7, 18.8, 12.1.

[0231] Note: 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 the solution. Only the major resonance (Z rotamer, 92%) is listed here.

[0232] HRMS: (ESI + )C 16 H 24 F 3 N 2 O 4 + Calculated: 365.1610, measured: 365.1684 (mass deviation + 0.5ppm)

[0233] Preparation of crystalline (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetylamino)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid Form 1 by seeded crystallization from aqueous isopropanol

[0234] Step 1:

[0235] In a 100 mL reactor, (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetylamino)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) were added and stirred at 500 rpm for 30 minutes to dissolve.

[0236] Step 2: Add in reverse

[0237] In a separate 100 mL reactor were added water (75 mL, 4163.2 mmol) and isopropanol (3.75 mL, 49.0 mmol) (95-05 v / v%), then (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetylamino)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (150 mg, 0.4116 mmol) was added to act as a seed and the mixture was heated to 50°C at a rate of 5°C / min and stirred for 30 minutes. A solution of (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetylamino)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid in isopropanol (from step 1 above) was added over a period of 6 hours. The resulting mixture was stirred at 50°C for 1 hour and then cooled to 10°C at a rate of -0.1°C / min and stirred overnight. The resulting slurry was then filtered and the solids were washed with a mixture of isopropanol (5 mL, 65.39 mmol) and water (15 mL, 832.64 mmol). The resulting solid was dried at 60° C. under vacuum overnight to provide crystalline (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetylamino)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.

[0238] Table - Crystal data and structure refinement of (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetylamino)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid Form 1:

[0239]

[0240] Fig.10 A PXRD pattern is provided for (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetylamino)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.

[0241] Table - PXRD Peaks of (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetylamino)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid Form 1:

[0242]

[0243] Fig.11 The PXRD pattern of (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetylamino)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid "New Form" is described, which is characterized by the PXRD peaks in the table below.

[0244] Table - PXRD peaks (2-theta degrees) of (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetylamino)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid "New Form":

[0245] Angle [2-Theta] Relative Strength Angle [2-Theta] Relative Strength Angle [2-Theta] Relative Strength 10.2 3 25.7 11 33.7 1 10.8 100 26.1 3 34.1 1 12.6 22 26.8 2 34.6 4 13.3 21 27.1 2 35.1 3 13.8 88 27.4 7 35.5 1 16.3 59 27.8 7 36.3 4 17.1 39 28.0 3 36.9 6 18.7 5 28.2 3 38.2 6 18.9 2 29.0 2 38.8 2 19.5 20 29.5 4 39.3 1 19.9 18 29.8 2 39.6 2 20.3 2 30.1 2 39.8 2 20.6 26 30.6 7 40.2 2 20.8 63 30.9 3 42.2 3 21.7 6 31.2 3 44.2 2 22.2 20 32.0 8 44.7 3 23.4 8 32.7 4 47.0 2 23.7 18 32.9 4 47.3 2 24.5 7 33.1 5 25.4 9 33.5 1

[0246] Step 3: Comparative method 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-trifluoroacetylamino)butyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide, i.e., compound II

[0247] *EDC = 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide

[0248] (1R, 2S, 5S) -3- ((S) -3, 3- dimethyl -2- (2, 2, 2- trifluoroacetylamino) butyryl) -6, 6- dimethyl -3- azabicyclo [3.1.0] hexane -2- carboxylic acid, Compound IV (1.0 kg, 2.74 mol, 1.0 equiv) and methyl ethyl ketone (5.0 L, 5 L / kg Compound IV) were combined and stirred at 25 ° C. 2-Hydroxypyridine N-oxide (0.228 kg, 2.05 mol, 0.75 equiv) and triethylamine (0.416 kg, 4.11 mol, 1.50 equiv) were added and the resulting slurry was stirred for 30 minutes. (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl)propionamide hydrochloride, i.e., compound III (0.597 kg, 2.87 mol, 1.05 equiv.) and 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDC, 0.682 kg, 3.56 mol, 1.30 equiv.) were added and stirred for 16 hours. The reaction was sampled to determine completion (the goal was to have no more than 1.0% of compound IV remaining). If the reaction was not complete, the mixture was stirred for an additional time. NaCl aqueous solution (3.0 L, 14 wt % saline solution) was added to quench the reaction and stirred for 30 minutes. Stirring was stopped and the layers were allowed to clarify. The lower aqueous phase was removed and the organic phase was washed with a second portion of NaCl aqueous solution (3.0 L, 14 wt % saline solution) according to the same protocol. The organic phase was then concentrated by distillation at reduced pressure at 0.3 bar, while additional isopropyl acetate (13 L) was added to maintain a constant volume of ~6 L / kg, and the distillation was terminated at 7 L / kg. The sample was analyzed for water content (Karl-Fischer), with a target of no more than 0.2 wt % 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-trifluoroacetylamino)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide, Compound II, was used in Step 4 without further purification.

[0249] (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-trifluoroacetylamino)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamidomethylethylketone (MEK) solvate II Preparation and Isolation of MEK Solvate

[0250] Separate the sample of title MEK solvate by following procedure.Carry out step 3 reaction with 150g scale as described above and allow stirring for 3 days.Gained slurry is combined with NaCl (325mL, 14 wt % aqueous solution), warmed to 48 ℃ and stirred, causing two transparent phases.Remove water phase, wash organic phase with the NaCl aqueous solution (400mL, 14 wt % solution) of second part.Concentrate organic phase under partial vacuum at 45 ℃, add extra MEK.It is repeated until water content is reduced to 6% and volume is~4mL / g raw material.Mixture is cooled to 15 ℃ gradually and keeps 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-trifluoroacetylamino)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamidomethylethylketone solvate.

[0251] 1 H NMR (600 MHz, DMSO-d 6, 298K): δ 9.40 (d, J = 8.6 Hz, 1H), 8.76 (d, J = 9.2 Hz, minor), 8.48 (d, J = 7.2 Hz, minor), 8.28 (d, J = 8.9 Hz, 1H), 7.65 (s, minor), 7.55 (s, 1H), 7.45-7.39 (m, minor), 7.35-7.26 (m, 1H), 7.06-6.99 (m, 1H), 6.92 (s, 4.49 (s, minor), 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, minor), 4.09 (ddd, J = 11.1, 7.2, 3.8 Hz, minor), 3.89 (dd, J = 10.3, 5.5 Hz, 1H), 3.67 (d, J = 10.4 Hz, 1H), 3.58 (d d, J = 12.4, 5.6 Hz, minor), 3.54 (dd, minor), 3.40 (d, J = 12.5 Hz, minor), 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, minor), 1.94 (ddd, J = 13.5, 12.0, 3.6 Hz, 1H), 1.73 (dt, J = 12.4, 9.1 Hz, minor), 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, minor), 1.02 (s, 3H), 0.98 (s, 9H), 0.91 (t, J = 7.3 Hz, 3H), 0.84 (s, 3H).

[0252] 13 C NMR (150 MHz, DMSO-d 6, 298K): δ 208.83, 178.92 (minor), 178.62, 173.74 (minor), 173.51, 170.63, 170.52 (minor), 167.94 (minor), 167.21, 156.87 (q, J = 36.9 Hz), 155.52 (d, J = 36.7 Hz-minor), 118.96-112.77 (m), 60.61 (minor), 60.25, 58.11, 57.67 (minor), 51.78 (minor), 50.32, 47.70, 47.50 (minor ), 45.51(minor), 42.09(minor), 40.06, 39.29, 37.88(minor), 37.32, 36.48(minor), 36.16, 35.85, 34.67, 34.55, 34.09, 33.58(minor), 30.57, 29.35, 27.65(minor), 27.39, 27.10, 26.40, 26.28, 25.87, 24.86(minor), 19.05(minor), 18.58, 13.24(minor), 12.38, 8.51(minor), 7.66.

[0253] Note: 1 H and 13 In the C NMR spectrum, two sets of resonances are observed due to the presence of both E and Z amide bond rotamers in solution. Where relevant, the lower rotamer signal is designated as "minor".

[0254] HRMS: (ESI + )C 23 H 35 F 3 N 5 O 5 + Calculated: 518.2585, measured: 518.2582 (mass deviation -0.46ppm)

[0255] Alternative Step 3: Optimized Method 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-trifluoroacetylamino)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide

[0256]

[0257] *EDC = 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide

[0258] In reactor A, (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetylamino)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid, Compound IV (1.0 kg, 2.74 mol, 1.0 equiv) and methyl ethyl ketone (2.0 L, 2 L / kg Compound IV) were combined and stirred at 25° C. 2-Hydroxypyridine N-oxide (0.274 kg, 2.47 mol, 0.90 equiv) and triethylamine (0.694 kg, 6.86 mol, 2.50 equiv) were added, and the resulting slurry was stirred for 30 minutes and then warmed to 50° C. In reactor B, (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl)propionamide hydrochloride, i.e., compound III (0.597 kg, 2.87 mol, 1.05 equivalents) and 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDC, 0.682 kg, 3.56 mol, 1.30 equivalents) were combined with methyl ethyl ketone (3.0 L, 3 L / kg compound IV), the resulting slurry was stirred for 30 minutes and then warmed to 50°C. The current solution from reactor A was then transferred into reactor B while maintaining the temperature in reactor B at 50°C and continued stirring for at least 6 hours. The reaction was sampled to determine completion (the goal was to have no more than 3% of compound IV unreacted). If the reaction was not complete, the mixture was stirred for an additional time. The reaction was quenched at 50°C by adding an aqueous NaCl solution (3.0 L, 14 wt % brine solution, 3.0 L / kg compound IV) and kept stirring for 30 minutes. Stirring was stopped and the layers were allowed to settle. The lower aqueous phase was removed and the organic phase was washed with a second portion of aqueous NaCl solution (3.0 L, 14 wt % brine solution) according to the same protocol. The cooled organic phase was then concentrated by distillation under reduced pressure at 0.3 bar while adding additional isopropyl acetate (18 L, 18 L / kg Compound IV) to maintain a constant volume of ~6 L / kg, ending the distillation at 8 L / kg. The samples were analyzed for water content (Karl-Fischer), with a goal of no more than 0.2 wt % 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-trifluoroacetylamino)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide was used in step 4 without further purification.

[0259] The (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl)propanamide hydrochloride, Compound III, used in the aforementioned reaction may exist in some solid form.

[0260] Fig.12A PXRD pattern is provided for (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl)propanamide hydrochloride salt Form 1, which is characterized by the PXRD peaks in the table below.

[0261] Table - PXRD Peaks of (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl)propanamide hydrochloride salt Form 1:

[0262] Angle (2Theta degrees) Relative Strength Angle (2Theta degrees) Relative Strength 9.97 3 29.24 13 11.67 1 30.98 6 14.17 1 31.78 2 16.08 1 32.32 23 16.35 1 32.79 10 17.10 14 33.10 1 17.27 3 33.50 6 18.23 24 33.70 4 19.21 4 33.90 3 20.83 20 35.27 3 22.20 58 36.20 3 22.97 12 36.42 6 23.35 34 36.75 6 23.79 2 36.95 7 24.62 3 37.83 3 25.10 100 38.58 2 26.85 11 39.44 7 28.39 14 39.75 1

[0263] Fig.13 A PXRD pattern is provided for (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl)propanamide hydrochloride salt Form 2, which is characterized by the PXRD peaks in the table below.

[0264] Table - PXRD Peaks of (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl)propanamide hydrochloride salt Form 2:

[0265]

[0266] Fig.14 Provided is a PXRD pattern 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-trifluoroacetylamino)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamidomethylethylketone (MEK) solvate, which is characterized by the PXRD peaks in the table below.

[0267] Table - PXRD peaks 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-trifluoroacetylamino)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamidomethylethylketone solvate:

[0268]

[0269] Table - Crystal data and structure refinement 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-trifluoroacetylamino)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamidomethylethylketone solvate:

[0270]

[0271] 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-carboxamidomethyl tert-butyl ether solvate, I'

[0272]

[0273] A 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-trifluoroacetylamino)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide, Compound II, prepared above in Step 4 in isopropyl acetate (assuming quantitative conversion, 2.74 mol, 1.0 equiv) was combined with N-methylmorpholine (1.11 kg, 10.4 mol, 4.0 equiv) and stirred at 20°C. Trifluoroacetic anhydride (1.15 kg, 5.20 mol, 2.0 equiv) was added over 60 minutes, maintaining the reaction temperature at no more than 25°C. The resulting mixture was stirred for 1 hour. A sample was analyzed to determine the reaction was complete (no more than 0.5% of Compound II remained). If the reaction is not complete, keep stirring for another 60 minutes, and add additional trifluoroacetic anhydride if necessary. Add water (3.0L, 3.0L / kg compound IV of the previous step) to quench the reaction, keep stirring for 30 minutes, then stop and let each layer clarify. Remove the aqueous phase and wash the organic phase with a second 3.0L portion of water. The organic phase is then concentrated to a volume of 3.5L (3.5L / kg compound IV of the previous step) by vacuum distillation (0.1 bar). Add isopropyl acetate (5.0L, 5.0L / kg compound IV of the previous step) and concentrate the solution to a volume of 3.5L (3.5L / kg compound IV of the previous step) by vacuum distillation. Stir the solution at 50°C and add methyl tert-butyl ether (MTBE) within 60 minutes. If product crystallization does not occur during this addition, PF-07321332MTBE solvate seeds (10g, 1.0 wt %, based on compound IV of the previous step) may be added. Additional portions of MTBE (6.0 L, 6.0 L / kg of compound IV from the previous step) were added over 3 hours. The slurry was 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 was collected by filtration, rinsed with 2.0 L / kg of a 80:20 MTBE:iPrOAc solution of compound IV from the previous step, and dried at no more than 50°C.

[0274] 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, i.e., isopropyl acetate solvate, was obtained according to the following procedure.

[0275] PF-07328615 (in methyl ethyl ketone) (7.87g, 15.2mmol) is added to the 50mL reactor equipped with a magnetic stirrer. Some solids have been crystallized from the PF-07328615 solution. Isopropyl acetate (for distillation) (72mL, 614.04mmol) is added thereto while constant volume distillation is carried out, Tj-Tr is set to 15°C, Tj max is set to 70°C, and vacuum is set to 300 mbar (actual vacuum is 295-305 mbar). It is diluted to 7.5mL / g (42mL) with isopropyl acetate. Cool to 20°C (Tj), stir speed is set to 350rpm and then 1-methylmorpholine (6.7mL, 61mmol) is added in 5 minutes, then trifluoroacetic anhydride (4.28mL, 30.4mmol) is added via a syringe pump in 1 hour and stirred for one hour after the addition is completed. 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), Tj-Tr was set to 15°C, Tj max was set to 70°C, and the vacuum was set to 100 mbar (the actual vacuum was 100-105 mbar). Isopropyl acetate (28 mL, 238.79 mmol) was added thereto and the mixture was distilled again to a concentration of 3.5 mL / g. Solid crystallized when the volume reached ~30 mL. 1 mL of the solution was filtered (producing 71 mg of wet solid) and analyzed by PXRD (see Fig.15 ).

[0276] Fig.15 A PXRD pattern 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-carboxamideacetic acid isopropyl ester solvate is provided, which is characterized by the following PXRD peaks.

[0277] Table - PXRD peaks 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 acetate isopropyl solvate

[0278]

Claims

1. 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-trifluoroacetylamino)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide, methyl ethyl ketone solvate.

2. A method 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-trifluoroacetylamino)butyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide, i.e., compound II The method of reacting a compound of formula IV with a compound of formula III comprises steps (a) to (d): (a) combining (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetylamino)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid, i.e., compound IV, and methyl ethyl ketone, followed by the addition of 2-hydroxypyridine N-oxide and triethylamine to provide a first mixture S3-M1; (b) combining (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl)propionamide hydrochloride, i.e., compound III, 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride, and methyl ethyl ketone to provide 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 provide a third mixture S3-M3; and (d) Stirring the third mixture S3-M3 from step (c) to provide 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-trifluoroacetylamino)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide, i.e., compound II.

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-trifluoroacetylamino)butyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid, i.e., compound IV, 2 L of methyl ethyl ketone per kg of compound IV, 0.9 equivalent of 2-hydroxypyridine N-oxide and 2.50 equivalents of triethylamine.

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

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)propionamide hydrochloride, i.e., 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. 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 and stirred at about 25°C for 30 minutes and then warmed to about 50°C.

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 combined with the second mixture S3-M2 from step (b) at about 50°C to provide a third mixture S3-M3 while maintaining the temperature of the third mixture S3-M3 at about 50°C.

8. The process 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. A method 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-trifluoroacetylamino)butyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide, i.e., compound II The method of reacting a compound of formula IV with a compound of formula III comprises steps (a) to (d): (a) combining (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetylamino)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid, Compound IV (1.0 equivalent) and methyl ethyl ketone (2 L / kg Compound IV) at about 25°C, followed by the addition of 2-hydroxypyridine N-oxide (0.90 equivalent) and triethylamine (2.50 equivalent) to provide a first mixture S3-M1, which was 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 / kg Compound IV) to provide a second mixture S3-M2, which was 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 provide 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 provide 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-trifluoroacetylamino)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide, i.e., compound II.

10. The process of claim 9, wherein the amount of acyl urea impurities IMP-S3-3, i.e. (2S,4S)-4-(2-aminoethyl)-5-oxopyrrolidine-2-carboxamide and IMP-S3-4, i.e. (1R,2S,5S)-N-(2-((3S,5S)-5-carbamoyl-2-oxopyrrolidin-3-yl)ethyl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetylamino)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide formed does not exceed 10%.

11. The process of claim 9, wherein the amount of acyl urea impurities IMP-S3-3, i.e. (2S,4S)-4-(2-aminoethyl)-5-oxopyrrolidine-2-carboxamide and IMP-S3-4, i.e. (1R,2S,5S)-N-(2-((3S,5S)-5-carbamoyl-2-oxopyrrolidin-3-yl)ethyl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetylamino)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide formed does not exceed 5%.

12. The process of any one of claims 9 to 11, wherein the amount of rearranged impurity IMP-S3-3 (2S, 4S)-4-(2-aminoethyl)-5-oxopyrrolidine-2-carboxamide formed does not exceed 2%.

13. The process of any one of claims 9 to 11, wherein the amount of rearranged impurity IMP-S3-4, i.e., (1R,2S,5S)-N-(2-((3S,5S)-5-carbamoyl-2-oxopyrrolidin-3-yl)ethyl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetylamino)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide, formed is not more than 2%.

14. A method for preparing (1R, 2S, 5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetylamino)butyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid, i.e., compound IV The process of reacting a compound of formula V with a compound of formula VI comprises steps (a) to (d): (a) combining (S)-3,3-dimethyl-2-(2,2,2-trifluoroacetylamino)butanoic acid (Compound V), methanesulfonyl chloride, and isopropyl acetate to provide a first mixture S2-M1; (b) adding triethylamine to the first mixture S2-M1 to provide 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 provide a third mixture S2-M3; and (d) Stirring the third mixture S2-M3 provides (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetylamino)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid, ie, Compound IV.

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

16. The process 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 provide the second mixture S2-M2.

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

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. 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 about 40% of its initial volume to provide organic layers S2-M4.

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

21. A method 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, i.e., Compound I The method comprises 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-carboxamidomethyl tert-butyl ether, i.e., Compound I', in isopropyl acetate, wherein the concentration of Compound I' in isopropyl acetate is from about 7 mL to about 9 mL of isopropyl acetate per 1 gram of Compound I'; (b) seeding the solution with 0.5 wt % to 0.75 wt % 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 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.

22. The process 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. The process 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 w%.

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

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

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. 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. 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. 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. 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. The compound of claim 30, wherein the compound is crystalline.

32. The compound of claim 31 characterized by the following PXRD pattern:

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