Synthesis of pyrrolidine compounds

Abstract

Provided are methods for the preparation of certain substituted pyrrolidine compounds, forms of (2S,4R)-1-(2-aminoacetyl)-4-benzamidopyrrolidine-2-carboxylic acid hydrochloride, and methods for preparing and using these forms.

1 . A method for preparing a compound of formula (I) or a salt thereof: 
       
         
           
           
               
               
           
         
       
       wherein:
 one of R 1  and R 2  is hydrogen or C 1 -C 6  alkyl, and the other is —C(O)R 5 ;
 wherein R 5  is chosen from:
 optionally substituted C 6 -C 10  aryl; 
 optionally substituted heteroaryl including 5-10 atoms; 
 optionally substituted C 1 -C 20  alkyl; 
 optionally substituted C 1 -C 20  haloalkyl; 
 optionally substituted C 7 -C 12  aralkyl; 
 optionally substituted heteroaralkyl including 6-12 atoms; 
 optionally substituted C 3 -C 10  cycloalkyl; and 
 optionally substituted C 3 -C 10  cycloalkenyl; 
 
 
 W is C 1-6  alkylene; and 
 R 3  and R 4  may be the same or different and are each independently chosen from hydrogen, optionally substituted C 1 -C 6  alkyl, —C(O)OR 6 , and —C(O)R 9 ;
 wherein R 6  is optionally substituted C 1 -C 20  alkyl; and 
 wherein R 9  is chosen from
 hydrogen; 
 optionally substituted C 6 -C 10  aryl; 
 optionally substituted heteroaryl including 5-10 atoms; 
 optionally substituted C 1 -C 20  alkyl; 
 optionally substituted C 3 -C 10  cycloalkyl; and 
 optionally substituted heterocycloalkyl including 5-10 atoms; 
 
 
 
       the method comprising:
 converting a compound of formula (II) or a salt thereof: 
 
       
         
           
           
               
               
           
         
       
       wherein:
 R 7  is chosen from hydrogen and optionally substituted C 1 -C 6  alkyl; 
 
       to a compound of formula (III) or a salt thereof: 
       
         
           
           
               
               
           
         
       
       and, where R 7  in formula (III) is optionally substituted C 1 -C 6  alkyl,
 reacting the compound of formula (III) with a metal hydroxide or other suitable base to provide a compound of formula (I) or a salt thereof. 
 
     
     
         2 . The method of  claim 1 , wherein converting the compound of formula (II) to a compound of formula (III) comprises contacting the compound of formula (II) with a compound of formula (IV): 
       
         
           
           
               
               
           
         
       
     
     
         3 . The method of  claim 2 , wherein the compound of formula (II) is contacted with the carboxylic acid of formula (IV) in the presence of a coupling agent. 
     
     
         4 . The method of  claim 3 , wherein the coupling agent is a carbodiimide. 
     
     
         5 . The method of  claim 4 , wherein the coupling agent is N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride or dicyclohexylcarbodiimide. 
     
     
         6 . The method of  claim 2 , wherein the compound of formula (II) is contacted with the carboxylic acid of formula (IV) in the presence of a coupling agent and a hydroxylated moiety. 
     
     
         7 . The method of  claim 6 , wherein the hydroxylated moiety is N-hydroxybenzotriazole, 1-hydroxy-7-azabenzotriazole (HOAt), or pentafluorophenol. 
     
     
         8 . The method of  claim 2 , wherein the carboxylic acid of formula (IV) is converted to a mixed anhydride. 
     
     
         9 . The method of  claim 8 , wherein the compound of formula (IV) is Boc-Gly-OH and the mixed anhydride is formed by treatment of the compound of formula (IV) with ethyl chloroformate. 
     
     
         10 . The method of  claim 1 , wherein the metal hydroxide is a Group IA metal hydroxide. 
     
     
         11 . The method of  claim 10 , wherein the Group IA metal hydroxide is chosen from NaOH, KOH, and LiOH. 
     
     
         12 . The method of  claim 11 , wherein the Group IA metal hydroxide is NaOH. 
     
     
         13 . The method of  claim 1 , wherein the reaction of the compound of formula (III) with a metal hydroxide or other suitable base is conducted in the presence of at least one solvent. 
     
     
         14 . The method of  claim 13 , wherein said at least one solvent is a mixture of two or more solvents. 
     
     
         15 . The method of  claim 14 , wherein said at least one solvent is a mixture of water and a C 1 -C 3  alcohol. 
     
     
         16 . The method of  claim 15 , wherein the C 1 -C 3  alcohol is CH 3 OH. 
     
     
         17 . The method of  claim 1 , wherein the reaction of the compound of formula (III) with a metal hydroxide or other suitable base is conducted at a temperature of at most about 5° C. 
     
     
         18 . The method of  claim 17 , wherein the reaction of the compound of formula (III) with a metal hydroxide or other suitable base is conducted at a temperature of from about −10° C. to about 5° C. 
     
     
         19 . The method of  claim 18 , wherein the reaction of the compound of formula (III) with a metal hydroxide or other suitable base is conducted at a temperature of from about −5° C. to about 5° C. 
     
     
         20 . The method of  claim 19 , wherein the reaction of the compound of formula (III) with a metal hydroxide or other suitable base is conducted at a temperature of from about −5° C. to about 1° C. 
     
     
         21 . The method of  claim 1 , wherein the reaction of the compound of formula (III) with a metal hydroxide or other suitable base provides a compound of formula (I-A) or a salt thereof: 
       
         
           
           
               
               
           
         
       
       wherein R 3  is chosen from hydrogen and optionally substituted C 1 -C 6  alkyl. 
     
     
         22 . The method of  claim 21 , further comprising converting the compound of formula (I-A) or a salt thereof to a compound of formula (I-B) or a salt thereof: 
       
         
           
           
               
               
           
         
       
     
     
         23 . The method of  claim 21 , wherein R 3  is hydrogen, and R 6  is tert-butyl. 
     
     
         24 . The method of  claim 21 , wherein R 3  is hydrogen, and R 6  is benzyl. 
     
     
         25 . The method of  claim 22 , wherein converting the compound of formula (I-A) or a salt thereof to a compound of formula (I-B) or a salt thereof comprises contacting the compound of formula (I-A) with at least one acid. 
     
     
         26 . The method of  claim 25 , wherein the at least one acid is HCl. 
     
     
         27 . The method of  claim 1 , wherein the compound of formula (II) is prepared by a method comprising:
 contacting a compound of formula (V):   
       
         
           
           
               
               
           
         
       
       wherein:
 R 2  is chosen from hydrogen and optionally substituted C 1 -C 6  alkyl; 
 R 7  is chosen from hydrogen and optionally substituted C 1 -C 6  alkyl; and 
 R 8  is a nitrogen protecting group; 
 
       with an activated carboxylic acid to provide a compound of formula (VII) or a salt thereof: 
       
         
           
           
               
               
           
         
       
       and
 removing the protecting group from the compound of formula (VII). 
 
     
     
         28 . The method of  claim 27 , wherein the activated carboxylic acid is a compound of formula (VI):
   R 5 C(O)Cl  (VI).   
     
     
         29 . The method of  claim 28 , wherein removing the protecting group from the compound of formula (VII) provides the compound of formula (II) as an HCl salt. 
     
     
         30 . The method of  claim 27 , wherein R 8  is —C(O)OR a , in which R a  is chosen from:
 optionally substituted C 6 -C 10  aryl;   optionally substituted heteroaryl including 5-10 atoms;   optionally substituted C 1 -C 20  alkyl;   optionally substituted C 1 -C 20  haloalkyl;   optionally substituted C 7 -C 12  aralkyl;   optionally substituted heteroaralkyl including 6-12 atoms;   optionally substituted C 3 -C 10  cycloalkyl; and   optionally substituted C 3 -C 10  cycloalkenyl.   
     
     
         31 . The method of  claim 30  wherein R 8  is —C(O)O(tert-butyl) or —C(O)O(benzyl). 
     
     
         32 . The method of  claim 27 , wherein R 8  is an acid labile nitrogen protecting group and removing the protecting group from the compound of formula (VII) comprises contacting the compound of formula (VII) with at least one acid. 
     
     
         33 . The method of  claim 32  wherein the at least one acid is HCl or trifluoroacetic acid. 
     
     
         34 . The method of  claim 27  wherein R 8  in formula (VII) is a nitrogen protecting group that is susceptible to cleavage by hydrogenolysis and removing the protecting group from the compound of formula (VII) comprises subjecting the compound of formula (VII) to catalytic hydrogenation. 
     
     
         35 . The method of  claim 34  wherein the nitrogen protecting group is —C(O)O(benzyl). 
     
     
         36 . The method of  claim 34  wherein subjecting the compound of formula (VII) to catalytic hydrogenation comprises contacting the compound of formula (VII) with H 2  gas and a transition metal catalyst. 
     
     
         37 . The method of  claim 36  wherein the transition metal catalyst is palladium. 
     
     
         38 . The method of  claim 27 , wherein R 8  in formula (VII) is a nitrogen protecting group that is susceptible to cleavage by hydrogenolysis and removing the protecting group from the compound of formula (VII) comprises subjecting the compound of formula (VII) to transfer hydrogenation. 
     
     
         39 . The method of  claim 38  wherein subjecting the compound of formula (VII) to transfer hydrogenation comprises contacting the compound of formula (VII) with cyclohexene and a transition metal catalyst. 
     
     
         40 . The method of  claim 39  wherein the transition metal catalyst is palladium. 
     
     
         41 . The method of  claim 27  wherein a compound of formula (V), in which R 2  is hydrogen, is prepared by a method comprising
 contacting a compound of formula (VIII):   
       
         
           
           
               
               
           
         
       
       wherein R 7  is chosen from hydrogen and optionally substituted C 1 -C 6  alkyl, 
       with a compound of the formula (R b )(R c )NH, wherein R b  and R c  are not hydrogen to provide a compound of formula (IX): 
       
         
           
           
               
               
           
         
       
       and
 (ii) converting the compound of formula (IX) to a compound of formula (V), in which R 2  is hydrogen. 
 
     
     
         42 . The method of  claim 41  wherein the compound of the formula (R b )(R c )NH is chosen from phthalimide, O-tert-butyl carbamate, N-Boc ethyl oxamate, benzhydryl amine, trityl amine, lithium hexamethyldisilazane, triphenylsilyl amine, LiNH 2 , allylamine, and bis(allyl)amine. 
     
     
         43 . The method of  claim 27 , wherein R 2  is hydrogen, wherein a stereoisomer or an enriched stereoisomeric mixture of a compound of formula (V) is used and wherein said stereoisomer or enriched stereoisomeric mixture of a compound of formula (V) is prepared by a method comprising:
 contacting a compound of formula (X)   
       
         
           
           
               
               
           
         
         wherein
 R 7  is chosen from hydrogen and optionally substituted C 1 -C 6  alkyl; and 
 R 8  is hydrogen or a nitrogen protecting group 
 
         with a compound of formula R w CO 2 H wherein R w  is lower alkyl or lower haloalkyl; a compound of formula P(R d ) 3 , in which R d  at each occurrence is, independently, optionally substituted C 6 -C 10  aryl or optionally substituted heteroaryl including 5-10 atoms; and a di(C 1 -C 6  alkyl)azodicarboxylate to provide a compound of formula (XI): 
       
       
         
           
           
               
               
           
         
         contacting the compound of formula (XI) with a metal hydroxide to provide a compound of formula (XII) or a salt thereof: 
       
       
         
           
           
               
               
           
         
         contacting the compound of formula (XII) with a compound of formula (R b )(R c )NH (in which R b  and R c  are other than hydrogen) to provide a compound of formula (XIII): 
       
       
         
           
           
               
               
           
         
         and 
         converting the compound of formula (XIII) to a compound of formula (V), in which R 2  in formula (V) is hydrogen. 
       
     
     
         44 . The method of  claim 43  wherein R 8  is C(O)OR a  wherein R a  is chosen from
 optionally substituted C 6 -C 10  aryl;   optionally substituted heteroaryl including 5-10 atoms;   optionally substituted C 1 -C 20  alkyl;   optionally substituted C 1 -C 20  haloalkyl;   optionally substituted C 7 -C 12  aralkyl;   optionally substituted heteroaralkyl including 6-12 atoms;   optionally substituted C 3 -C 10  cycloalkyl; and   optionally substituted C 3 -C 10  cycloalkenyl.   
     
     
         45 . The method of  claim 43  wherein R 8  is —C(O)O(tert-butyl) or —C(O)O(benzyl). 
     
     
         46 . The method of  claim 45  wherein R 8  is —C(O)O(benzyl). 
     
     
         47 . The method of  claim 43  wherein the compound of formula P(R d ) 3  is triphenylphosphine. 
     
     
         48 . The method of  claim 43  wherein the di(C 1 -C 6  alkyl)azodicarboxylate is diisopropyl azodicarboxylate. 
     
     
         49 . The method of  claim 1 , wherein W is —CH 2 —. 
     
     
         50 . The method of  claim 1 , wherein R 5  is optionally substituted C 6 -C 10  aryl or optionally substituted heteroaryl including 5-10 atoms. 
     
     
         51 . The method of  claim 50 , wherein R 5  is optionally substituted C 6 -C 10  aryl. 
     
     
         52 . The method of  claim 51 , wherein R 5  is phenyl. 
     
     
         53 . The method of  claim 1 , wherein each of R 3  and R 4  is hydrogen. 
     
     
         54 . The method of  claim 1 , wherein R 7  is CH 3 . 
     
     
         55 . The method of  claim 1 , wherein the —NR 1 R 2  group and the —COOH group in formula (I) are trans with respect to one another. 
     
     
         56 . The method of  claim 55 , wherein the ring carbon attached to the —NR 1 R 2  group has the R configuration, and the ring carbon attached to the —COOH group has the S configuration. 
     
     
         57 . The method of  claim 1 , wherein the —NR 1 R 2  group and the —COOR 7  group in formula (II) are trans with respect to one another. 
     
     
         58 . The method of  claim 57 , wherein the ring carbon attached to the —NR 1 R 2  group has the R configuration, and the ring carbon attached to the —COOR 7  group has the S configuration. 
     
     
         59 . The method of  claim 1 , wherein the compound of formula (I) or a salt thereof is: 
       
         
           
           
               
               
           
         
         the compound of formula (II) or a salt thereof is: 
       
       
         
           
           
               
               
           
         
         the compound of formula (III) or a salt thereof is: 
       
       
         
           
           
               
               
           
         
       
     
     
         60 . The method of  claim 2 , wherein the compound of formula (I) or a salt thereof is: 
       
         
           
           
               
               
           
         
         the compound of formula (II) or a salt thereof is: 
       
       
         
           
           
               
               
           
         
         the compound of formula (III) or a salt thereof is: 
       
       
         
           
           
               
               
           
         
         the compound of formula (IV) or a salt thereof is Boc-Gly-OH. 
       
     
     
         61 . The method of  claim 59 , wherein the reaction of the compound of formula (III) with a metal hydroxide or other suitable base provides a compound of formula (I-A-1) or a salt thereof: 
       
         
           
           
               
               
           
         
       
     
     
         62 . The method of  claim 61 , further comprising converting the compound of formula (I-A-1) or a salt thereof to a compound of formula (I-B-1) or a salt thereof: 
       
         
           
           
               
               
           
         
       
     
     
         63 . The method of  claim 59 , wherein the compound of formula (II) is prepared by a method comprising:
 contacting a compound of formula (V-1):   
       
         
           
           
               
               
           
         
       
       with benzoyl chloride to provide a compound of formula (VII-1): 
       
         
           
           
               
               
           
         
       
       and
 removing —C(O)O(tert-butyl) from the compound of formula (VII-1). 
 
     
     
         64 . A composition comprising
 (1) a compound of formula (I) or a salt thereof   
       
         
           
           
               
               
           
         
         
           a compound of formula (III) or a salt thereof. 
         
       
       
         
           
           
               
               
           
         
       
       wherein
 one of R 1  and R 2  is hydrogen or C 1 -C 6  alkyl, and the other is —C(O)R 5 ;
 wherein R 5  is chosen from:
 optionally substituted C 6 -C 10  aryl; 
 optionally substituted heteroaryl including 5-10 atoms; 
 optionally substituted C 1 -C 20  alkyl; 
 optionally substituted C 1 -C 20  haloalkyl; 
 optionally substituted C 7 -C 12  aralkyl; 
 optionally substituted heteroaralkyl including 6-12 atoms; 
 optionally substituted C 3 -C 10  cycloalkyl; and 
 optionally substituted C 3 -C 10  cycloalkenyl; 
 
 
 W is C 1-6  alkylene; 
 R 3  and R 4  may be the same or different and are each independently chosen from hydrogen, optionally substituted C 1 -C 6  alkyl, —C(O)OR 6 , and —C(O)R 9 ;
 wherein R 6  is optionally substituted C 1 -C 20  alkyl; and 
 wherein R 9  is chosen from
 hydrogen; 
 optionally substituted C 6 -C 10  aryl; 
 optionally substituted heteroaryl including 5-10 atoms; 
 optionally substituted C 1 -C 20  alkyl; 
 optionally substituted C 3 -C 10  cycloalkyl; and 
 optionally substituted heterocycloalkyl including 5-10 atoms; and 
 
 
 R 7  is chosen from hydrogen and optionally substituted C 1 -C 6  alkyl; and 
 (2) a detectable amount of one or more compounds selected from: 
 N-((7R,8aS)-1,4-dioxooctahydropyrrolo[1,2-a]pyrazin-7-yl)benzamide or a salt thereof, i.e., a compound of formula 
 
       
         
           
           
               
               
           
         
         or a salt thereof; 
         triphenylphosphine; 
         triphenylphosphine oxide; 
         a hydrazine dicarboxylate; 
         triethylamine; 
         benzotriazole; 
         (2S,4R)-4-benzamido-1-(2-(tert-butylamino)acetyl)pyrrolidine-2-carboxylic acid or a salt thereof, i.e., a compound of formula: 
       
       
         
           
           
               
               
           
         
         or a salt thereof; 
         and a compound of formula (II) or a salt thereof: 
       
       
         
           
           
               
               
           
         
       
     
     
         65 . The composition of  claim 64  comprising greater than about 99% of a compound of formula (I). 
     
     
         66 . The composition of  claim 65  comprising greater than about 99.9% of a compound of formula (I). 
     
     
         67 . The composition of  claim 64 , wherein the compound of formula (I) is (2S,4R)-1-(2-aminoacetyl)-4-benzamidopyrrolidine-2-carboxylic acid or a salt thereof. 
     
     
         68 . The composition of  claim 67  wherein the compound of formula (I) is (2S,4R)-1-(2-aminoacetyl)-4-benzamidopyrrolidine-2-carboxylic acid hydrochloride. 
     
     
         69 . The composition of  claim 64 , wherein the compound of formula (I) is (2S,4R)-1-(2-aminoacetyl)-4-benzamidopyrrolidine-2-carboxylic acid hydrochloride monohydrate. 
     
     
         70 . The composition of  claim 67  wherein R 1  is H, R 2  is —(CO)Ph, and R 7  is methyl. 
     
     
         71 . A pharmaceutical formulation comprising a composition of  claim 64  and one or more pharmaceutically acceptable carriers. 
     
     
         72 . A method for treating a condition selected from cardiovascular disease; osteoporosis; inflammation of airway epithelium; disorders of alveolar tissue; bladder incontinence; impaired hearing; endothelial lesions; Type I or Type II diabetes; diabetic retinopathy; diabetic neuropathy; atherosclerosis; CNS related conditions; seizures; ischemia; dental tissue disorders; kidney diseases; anaemia; leukopenia; thrombocytopenia; pancytopenia; superficial wounds; deep wounds resulting from trauma; bone fractures; erectile dysfunction; urinary bladder incontinence; neuropathic pain; subchronic and chronic inflammation; cancer; failure of bone marrow; stem cell transplantation; conditions arising during transplantation of cells and tissues; conditions arising during medical procedures; conditions caused by an excess of reactive oxygen species, free radicals or nitric oxide; diseases or disorders of pregnancy; female infertility; and stroke, comprising administering to a patient a therapeutically effective amount of the composition of  claim 64 . 
     
     
         73 . A compound that is (2S,4R)-1-(2-aminoacetyl)-4-benzamidopyrrolidine-2-carboxylic acid hydrochloride. 
     
     
         74 . A compound that is (2S,4R)-1-(2-aminoacetyl)-4-benzamidopyrrolidine-2-carboxylic acid hydrochloride monohydrate. 
     
     
         75 . The compound of  claim 74 , having a desolvation onset temperature of about 104° C. 
     
     
         76 . The compound of  claim 74 , having two endothermic transitions with onset temperatures of about 104° C. and 168° C. 
     
     
         77 . The compound of  claim 74 , having powder x-ray diffraction peaks at about 12.5° 2θ and 19.0° 2θ. 
     
     
         78 . The compound of  claim 77 , further having powder x-ray diffraction peaks at about 17.1° 2θ, 19.7° 2θ, 20.5° 2θ, 21.8° 2θ, 23.3° 2θ, and 23.4° 2θ. 
     
     
         79 . The compound of  claim 78 , further having powder x-ray diffraction peaks at about 5.4° 2θ, 20.7° 2θ, 21.2° 2θ, 23.1° 2θ, 26.4° 2θ, and 27.4° 2θ. 
     
     
         80 . The compound of  claim 74 , having an X-ray powder diffraction pattern substantially as in  FIG. 2 . 
     
     
         81 . A method for preparing (2S,4R)-1-(2-aminoacetyl)-4-benzamidopyrrolidine-2-carboxylic acid hydrochloride monohydrate, comprising providing a solution of (2S,4R)-1-(2-aminoacetyl)-4-benzamidopyrrolidine-2-carboxylic acid hydrochloride in a crystallization medium, wherein the crystallization medium comprises at least one water-miscible organic solvent and water, and maintaining the solution for a time and under conditions suitable for forming the (2S,4R)-1-(2-aminoacetyl)-4-benzamidopyrrolidine-2-carboxylic acid hydrochloride monohydrate. 
     
     
         82 . The method of  claim 81 , wherein the at least one water-miscible organic solvent comprises at least one solvent selected from water-miscible alcohols, water-miscible ketones, and water-miscible ethers. 
     
     
         83 . The method of  claim 82 , wherein the at least one water-miscible organic solvent is a water-miscible C 1 -C 4  alcohol. 
     
     
         84 . The method of  claim 83 , wherein the alcohol is isopropanol. 
     
     
         85 . The method of  claim 82 , wherein the at least one water-miscible organic solvent is a water-miscible C 1 -C 6  ketone. 
     
     
         86 . The method of  claim 85 , wherein the ketone is acetone. 
     
     
         87 . The method of  claim 82 , wherein the at least one water-miscible organic solvent ether is a water-miscible C 1 -C 6  ether. 
     
     
         88 . The method of  claim 87 , wherein the ether is t-butyl methyl ether. 
     
     
         89 . The method of  claim 82 , wherein the at least one water-miscible organic solvent comprises a mixture of isopropanol, acetone, and t-butyl methyl ether. 
     
     
         90 . The method of  claim 81 , wherein the (2S,4R)-1-(2-aminoacetyl)-4-benzamidopyrrolidine-2-carboxylic acid hydrochloride monohydrate has powder x-ray diffraction peaks at about 12.5° 2θ and 19.0° 2θ. 
     
     
         91 . The method of  claim 90 , wherein the (2S,4R)-1-(2-aminoacetyl)-4-benzamidopyrrolidine-2-carboxylic acid hydrochloride monohydrate has powder x-ray diffraction peaks at about 17.1° 2θ, 19.7° 2θ, 20.5° 2θ, 21.8° 2θ, 23.3° 2θ, and 23.4° 2θ. 
     
     
         92 . The method of  claim 91 , wherein the (2S,4R)-1-(2-aminoacetyl)-4-benzamidopyrrolidine-2-carboxylic acid hydrochloride monohydrate has powder x-ray diffraction peaks at about 5.4° 2θ, 20.7° 2θ, 21.2° 2θ, 23.1° 2θ, 26.4° 2θ, and 27.4° 2θ. 
     
     
         93 . The method of  claim 81 , wherein the (2S,4R)-1-(2-aminoacetyl)-4-benzamidopyrrolidine-2-carboxylic acid hydrochloride monohydrate has an X-ray powder diffraction pattern as substantially shown by  FIG. 2 . 
     
     
         94 . The method of  claim 81 , wherein the conditions suitable for forming the (2S,4R)-1-(2-aminoacetyl)-4-benzamidopyrrolidine-2-carboxylic acid hydrochloride monohydrate comprise maintaining the solution at room temperature 
     
     
         95 . The method of  claim 81 , wherein the conditions suitable for forming the (2S,4R)-1-(2-aminoacetyl)-4-benzamidopyrrolidine-2-carboxylic acid hydrochloride monohydrate comprise evaporation of at least some of the crystallization medium. 
     
     
         96 . The method of  claim 81 , wherein the crystallization medium comprises up to 5% by volume of water. 
     
     
         97 . The method of  claim 81 , further comprising drying the (2S,4R)-1-(2-aminoacetyl)-4-benzamidopyrrolidine-2-carboxylic acid hydrochloride monohydrate at elevated temperature and under vacuum. 
     
     
         98 . A pharmaceutical formulation comprising one or more pharmaceutically acceptable carriers and a compound of  claim 73 . 
     
     
         99 . A method for treating a condition selected from cardiovascular disease; osteoporosis; inflammation of airway epithelium; disorders of alveolar tissue; bladder incontinence; impaired hearing; endothelial lesions; Type I or Type II diabetes; diabetic retinopathy; diabetic neuropathy; atherosclerosis; CNS related conditions; seizures; ischemia; dental tissue disorders; kidney diseases; anaemia; leukopenia; thrombocytopenia; pancytopenia; superficial wounds; deep wounds resulting from trauma; bone fractures; erectile dysfunction; urinary bladder incontinence; neuropathic pain; subchronic and chronic inflammation; cancer; failure of bone marrow; stem cell transplantation; conditions arising during transplantation of cells and tissues; conditions arising during medical procedures; conditions caused by an excess of reactive oxygen species, free radicals or nitric oxide; diseases or disorders of pregnancy; female infertility; and stroke, comprising administering to a patient a therapeutically effective amount of the compound of  claim 73 . 
     
     
         100 .- 101 . (canceled)