CYANOPYRROLIDINES AS USP30 INHIBITORS AND FIBROSIS TREATMENT

Abstract

The present invention relates to novel treatments of fibrotic diseases, including, inter alia, fibrosis of the lung, liver and kidney, and to substituted-cyanopyrrolidines of formula (I) having activity as inhibitors of ubiquitin specific peptidase 30 (USP30), and compositions containing said inhibitors, for use in said treatments (I).

##STR00001##

Claims

1. A method of treatment of fibrosis in a mammal, comprising administering to said mammal a therapeutically effective amount of a compound of formula (I): ##STR00032## a tautomer thereof, or a pharmaceutically acceptable salt of said compound or tautomer, wherein: m is 0 to 5; n is 0 to 5; p is 0 to 3; ring A is a monocyclic heteroaryl ring containing 1, 2 or 3 heteroatoms, each independently selected from N, O and S; each R.sup.1 is independently selected from halo, cyano, hydroxy, (C.sub.1-C.sub.6)alkyl, (C.sub.1-C.sub.6)alkoxy, halo(C.sub.1-C.sub.6)alkyl, halo(C.sub.1-C.sub.6)alkoxy, and (C.sub.1-C.sub.6)alkoxy(C.sub.1-C.sub.6)alkyl; R.sup.1a and R.sup.1b, are each independently selected from hydrogen, halo, cyano, hydroxy, (C.sub.1-C.sub.6)alkyl, (C.sub.1-C.sub.6)alkoxy, halo(C.sub.1-C.sub.6)alkyl, halo(C.sub.1-C.sub.6)alkoxy, and (C.sub.1-C.sub.6)alkoxy(C.sub.1-C.sub.6)alkyl; R.sup.2 is selected from hydrogen, (C.sub.1-C.sub.6)alkyl, and (C.sub.1-C.sub.6)alkoxy(C.sub.1-C.sub.6)alkyl; each R.sup.3 is independently selected from hydrogen, halo, cyano, hydroxy, (C.sub.1-C.sub.6)alkyl, (C.sub.1-C.sub.6)alkoxy, (C.sub.1-C.sub.6)alkoxy(C.sub.1-C.sub.6)alkyl, halo(C.sub.1-C.sub.6)alkyl, halo(C.sub.1-C.sub.6)alkoxy, NH(C.sub.1-C.sub.6)alkyl, N((C.sub.1-C.sub.6)alkyl).sub.2, C(O)NH(C.sub.1-C.sub.6)alkyl, C(O)N((C.sub.1-C.sub.6)alkyl).sub.2, NHC(O)(C.sub.1-C.sub.6)alkyl, N((C.sub.1-C.sub.6)alkyl)C(O)(C.sub.1-C.sub.6)alkyl), C(O)(C.sub.1-C.sub.6)alkyl, C(O)O(C.sub.1-C.sub.6)alkyl, CO.sub.2H, CONH.sub.2, SO.sub.2NH(C.sub.1-C.sub.6)alkyl, and SO.sub.2N((C.sub.1-C.sub.6)alkyl).sub.2; each R.sup.4 is independently selected from halo, cyano, hydroxy, (C.sub.1-C.sub.6)alkyl, (C.sub.1-C.sub.6)alkoxy, (C.sub.1-C.sub.6)alkoxy(C.sub.1-C.sub.6)alkyl, halo(C.sub.1-C.sub.6)alkyl, halo(C.sub.1-C.sub.6)alkoxy, NH(C.sub.1-C.sub.6)alkyl, N((C.sub.1-C.sub.6)alkyl).sub.2, C(O)NH(C.sub.1-C.sub.6)alkyl, C(O)N((C.sub.1-C.sub.6)alkyl).sub.2, NHC(O)(C.sub.1-C.sub.6)alkyl, N((C.sub.1-C.sub.6)alkyl)C(O)(C.sub.1-C.sub.6)alkyl), C(O)(C.sub.1-C.sub.6)alkyl, C(O)O(C.sub.1-C.sub.6)alkyl, CO.sub.2H, CONH.sub.2, SO.sub.2NH(C.sub.1-C.sub.6)alkyl, and SO.sub.2N((C.sub.1-C.sub.6)alkyl).sub.2; and R.sup.5 and R.sup.6 are each independently selected from hydrogen, cyano, and (C.sub.1-C.sub.6)alkyl; or R.sup.5 and R.sup.6 together form a 3 to 6 membered cycloalkyl ring.

2. The method according to claim 1, wherein m is 0 or 1.

3. The method according to claim 1, wherein n is 0, 1 or 2.

4. The method according to claim 1, wherein p is 0 or 1.

5. The method according to claim 1, wherein ring A is selected from pyrrolyl, furanyl, thiophenyl, pyrazolyl, imidazolyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, triazolyl, oxadiazolyl, and thiadiazolyl.

6. The method according to claim 1, wherein R.sup.1a is selected from chloro, fluoro, methyl, ethyl, and methoxy.

7. The method according to claim 1, wherein R.sup.1a is selected from hydrogen, chloro, fluoro, cyano, hydroxy, methyl, ethyl, methoxy, and methoxymethyl.

8. The method according to claim 1, wherein R.sup.1b is selected from hydrogen, chloro, fluoro, cyano, hydroxy, methyl, ethyl, methoxy, and methoxymethyl.

9. The method according to claim 1, wherein: (i) m is 0, R.sup.1a is hydrogen, and R.sup.1b is methyl; (ii) m is 1, R.sup.1 is methyl, and R.sup.1a and R.sup.1b are each hydrogen; or (iii) m is 0, and R.sup.1a and R.sup.1b are each hydrogen.

10. The method according to claim 1, wherein R.sup.2 is selected from hydrogen and methyl.

11. The method according to claim 1, wherein each R.sup.3 is independently selected from hydrogen, chloro, fluoro, cyano, hydroxy, methyl, ethyl, propyl, methoxy, ethoxy, and methoxymethyl.

12. The method according to claim 1, wherein each R.sup.4 is independently selected from chloro, fluoro, cyano, hydroxy, methyl, ethyl, propyl, methoxy, ethoxy, methoxymethyl, CF.sub.3, and OCF.sub.3.

13. The method according to claim 1, wherein R.sup.5 and R.sup.6 are each independently selected from hydrogen, cyano and methyl.

14. The method according to claim 1, wherein the compound of formula (I) is selected from: 1-(3-cyanophenyl)-N-((1-cyanopyrrolidin-3-yl)methyl)-1H-1,2,4-triazole-3-carboxamide; (R)-1-(3-cyanophenyl)-N-((1-cyanopyrrolidin-3-yl)methyl)-1H-1,2,4-triazole-3-carboxamide; (S)-1-(3-cyanophenyl)-N-((1-cyanopyrrolidin-3-yl)methyl)-1H-1,2,4-triazole-3-carboxamide; 1-(3-cyanophenyl)-N-((1-cyanopyrrolidin-3-yl)methyl)-N-methyl-1H-1,2,4-triazole-3-carboxamide; (R)-1-(3-cyanophenyl)-N-((1-cyanopyrrolidin-3-yl)methyl)-N-methyl-1H-1,2,4-triazole-3-carboxamide; (S)-1-(3-cyanophenyl)-N-((1-cyanopyrrolidin-3-yl)methyl)-N-methyl-1H-1,2,4-triazole-3-carboxamide; N-((1-cyanopyrrolidin-3-yl)methyl)-1-phenyl-1H-1,2,4-triazole-3-carboxamide; (R)-N-((1-cyanopyrrolidin-3-yl)methyl)-1-phenyl-1H-1,2,4-triazole-3-carboxamide; (S)-N-((1-cyanopyrrolidin-3-yl)methyl)-1-phenyl-1H-1,2,4-triazole-3-carboxamide; N-(((2S,3S)-1-cyano-2-methylpyrrolidin-3-yl)methyl)-1-(3-cyanophenyl)-1H-1,2,4-triazole-3-carboxamide; N-(((2S,3R)-1-cyano-2-methylpyrrolidin-3-yl)methyl)-1-(3-cyanophenyl)-1H-1,2,4-triazole-3-carboxamide; N-(((2R)-1-cyano-2-methylpyrrolidin-3-yl)methyl)-1-(3-cyanophenyl)-1H-1,2,4-triazole-3-carboxamide; N-(((2R,3R)-1-cyano-2-methylpyrrolidin-3-yl)methyl)-1-(3-cyanophenyl)-1H-1,2,4-triazole-3-carboxamide; N-(((2R,3S)-1-cyano-2-methylpyrrolidin-3-yl)methyl)-1-(3-cyanophenyl)-1H-1,2,4-triazole-3-carboxamide; N-(((2S)-1-cyano-2-methylpyrrolidin-3-yl)methyl)-1-(3-cyanophenyl)-1H-1,2,4-triazole-3-carboxamide; 1-(3-cyanophenyl)-N-((1-cyanopyrrolidin-3-yl)methyl)-1H-1,2,3-triazole-4-carboxamide; (R)-1-(3-cyanophenyl)-N-((1-cyanopyrrolidin-3-yl)methyl)-1H-1,2,3-triazole-4-carboxamide; (S)-1-(3-cyanophenyl)-N-((1-cyanopyrrolidin-3-yl)methyl)-1H-1,2,3-triazole-4-carboxamide; 1-(3-chlorophenyl)-N-((1-cyanopyrrolidin-3-yl)methyl)-1H-1,2,3-triazole-4-carboxamide; (R)-1-(3-chlorophenyl)-N-((1-cyanopyrrolidin-3-yl)methyl)-1H-1,2,3-triazole-4-carboxamide; (S)-1-(3-chlorophenyl)-N-((1-cyanopyrrolidin-3-yl)methyl)-1H-1,2,3-triazole-4-carboxamide; 2-(3-chlorophenyl)-N-((1-cyanopyrrolidin-3-yl)methyl)-2H-1,2,3-triazole-4-carboxamide; (R)-2-(3-chlorophenyl)-N-((1-cyanopyrrolidin-3-yl)methyl)-2H-1,2,3-triazole-4-carboxamide; (S)-2-(3-chlorophenyl)-N-((1-cyanopyrrolidin-3-yl)methyl)-2H-1,2,3-triazole-4-carboxamide; (R)-3-(((5-phenylthiazol-2-yl)amino)methyl)pyrrolidine-1-carbonitrile; (S)-3-(((5-phenylthiazol-2-yl)amino)methyl)pyrrolidine-1-carbonitrile; N-((1-cyanopyrrolidin-3-yl)methyl)-2-phenyloxazole-5-carboxamide; N-((1-cyanopyrrolidin-3-yl)methyl)-3-phenylisoxazole-5-carboxamide; N-((1-cyanopyrrolidin-3-yl)methyl)-5-phenyl-1H-pyrazole-3-carboxamide; N-((1-cyanopyrrolidin-3-yl)methyl)-3-(o-tolyl)-1H-pyrazole-5-carboxamide; N-((1-cyanopyrrolidin-3-yl)methyl)-2-phenylthiazole-4-carboxamide; N-((1-cyanopyrrolidin-3-yl)methyl)-3-(2-fluorophenyl)-1H-pyrazole-5-carboxamide; N-((1-cyanopyrrolidin-3-yl)methyl)-5-phenyloxazole-2-carboxamide; (R)-3-(3-chlorophenyl)-N-((1-cyanopyrrolidin-3-yl)methyl)isoxazole-5-carboxamide; (R)-N-((1-cyanopyrrolidin-3-yl)methyl)-5-phenylisoxazole-3-carboxamide; (R)-N-((1-cyanopyrrolidin-3-yl)methyl)-5-phenylthiazole-2-carboxamide; (R)-N-((1-cyanopyrrolidin-3-yl)methyl)-4-phenylthiazole-2-carboxamide; (R)-N-((1-cyanopyrrolidin-3-yl)methyl)-1-phenyl-1H-pyrazole-3-carboxamide; (R)-N-((1-cyanopyrrolidin-3-yl)methyl)-2-phenyl-1H-imidazole-5-carboxamide; (R)-3-(2-chlorophenyl)-N-((1-cyanopyrrolidin-3-yl)methyl)isoxazole-5-carboxamide; (R)-3-(4-chlorophenyl)-N-((1-cyanopyrrolidin-3-yl)methyl)isoxazole-5-carboxamide; (R)-5-(3-cyanophenyl)-N-((1-cyanopyrrolidin-3-yl)methyl)-1,3,4-oxadiazole-2-carboxamide; (S)-5-(3-cyanophenyl)-N-((1-cyanopyrrolidin-3-yl)methyl)-1,3,4-oxadiazole-2-carboxamide; (R)-N-((1-cyanopyrrolidin-3-yl)methyl)-1-phenyl-1H-imidazole-4-carboxamide; (R)-1-(3-cyanophenyl)-N-((1-cyanopyrrolidin-3-yl)methyl)-1H-imidazole-4-carboxamide; (R)-1-(4-cyanophenyl)-N-((1-cyanopyrrolidin-3-yl)methyl)-1H-imidazole-4-carboxamide; (R)-N-((1-cyanopyrrolidin-3-yl)methyl)-1-(2-methoxyphenyl)-1H-imidazole-4-carboxamide; and (R)-N-((1-cyanopyrrolidin-3-yl)methyl)-1-(3-methoxyphenyl)-1H-imidazole-4-carboxamide; a tautomer thereof, or a pharmaceutically acceptable salt of said compound or tautomer.

15-18. (canceled)

19. method according to claim 1, wherein said compound is administered orally.

20. A method of treatment of fibrosis in a mammal, comprising administering to said mammal a therapeutically effective amount of a USP30 inhibitor, or a pharmaceutically acceptable composition comprising said inhibitor, wherein the mammal is suffering from fibrosis.

21. The method according to claim 20, wherein said compound is administered orally.

22-23. (canceled)

24. The method according to any one of claims 19 to 21, wherein the fibrosis is selected from fibrosis or a fibrotic disorder associated with the accumulation of extracellular matrix constituents that occurs following trauma, inflammation, tissue repair, immunological reactions, cellular hyperplasia, and neoplasia.

25. The method according to claim 24, wherein the fibrosis is selected from fibrosis or a fibrotic disorder associated with major organ diseases, fibroproliferative disorders, and scarring associated with trauma.

26. The method according to claim 25, wherein the fibrosis is selected from fibrosis or a fibrotic disorder associated with interstitial lung disease, liver cirrhosis, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, kidney disease, heart or vascular disease, diseases of the eye, systemic and local scleroderma, keloids, hypertrophic scars, atherosclerosis, restenosis, Dupuytren's contracture, surgical complications, chemotherapeutics drug-induced fibrosis, radiation-induced fibrosis, accidental injury and burns, retroperitoneal fibrosis, and peritoneal fibrosis/peritoneal scarring.

27. The method according to claim 26, wherein the fibrosis associated with interstitial lung disease is selected from sarcoidosis, silicosis, drug reactions, infections, collagen vascular diseases, rheumatoid arthritis, systemic sclerosis, scleroderma, pulmonary fibrosis, idiopathic pulmonary fibrosis, usual interstitial pneumonitis, interstitial lung disease, cryptogenic fibrosing alveolitis, bronchiolitis obliterans, and bronchiectasis.

28. The method according to claim 26, wherein the fibrosis associated with liver cirrhosis is selected from cirrhosis associated with viral hepatitis, schistosomiasis and chronic alcoholism.

Description

EXAMPLE 1

(R)-1-(3-cyanophenyl)-N-((1-cyanopyrrolidin-3-yl)methyl)-1H-1,2,4-triazole-3-carboxamide

[0287] ##STR00014##

[0288] Step a. To a stirred solution of 3-iodobenzonitrile (0.500 g, 2.18 mmol) and methyl 1H-1,2,4-triazole-3-carboxylate (CAS Number 4928-88-5; 0.277 g, 2.18 mmol) in DMSO (6 mL) was added L-proline (0.050 g, 0.44 mmol), Cu(I)I (0.083 g, 0.44 mmol) and K.sub.2CO.sub.3 (0.602 g, 4.34 mmol) at rt. The solution was heated at 80 C. for 18 h. The reaction mixture was cooled to rt and poured into ice cold water (100 mL) and extracted with EtOAc (450 mL). Combined organic extracts were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography (compound eluted in 35% EtOAc in hexane) yielding methyl 1-(3-cyanophenyl)-1H-1,2,4-triazole-3-carboxylate (0.105 g, 0.46 mmol). LCMS: Method H, 1.517 min, MS:ES+229.19.

[0289] Step b. To a stirred solution of methyl 1-(3-cyanophenyl)-1H-1,2,4-triazole-3-carboxylate (0.100 g, 0.44 mmol) and tert-butyl (R)-3-(aminomethyl)pyrrolidine-1-carboxylate (CAS Number 199174-29-3; 0.106 g, 0.53 mmol) in THF (4 mL) was added a solution of 1,5,7-triazabicyclo[4.4.0]dec-5-ene (CAS Number 5807-14-7; 0.092 g, 0.66 mmol) in THF (1 mL) at 0 C. The resulting mixture was stirred at rt for 18 h. The reaction mixture was poured into water (50 mL) and extracted with EtOAc (250 mL). Combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography (compound eluted in 5% MeOH in DCM) yielding tert-butyl (R)-3-((1-(3-cyanophenyl)-1H-1,2,4-triazole-3-carboxamido)methyl)-pyrrolidine-1-carboxylate. (0.083 g, 0.21 mmol), LCMS: Method H, 1.789 min, MS:ES+[M100] 297.26.

[0290] Step c. To a stirred solution of tert-butyl (R)-3-((1-(3-cyanophenyl)-1H-1,2,4-triazole-3-carboxamido)-methyl)pyrrolidine-1-carboxylate (0.075 g, 0.189 mmol) in DCM (4 mL) was added TFA (0.37 mL) at 0 C. and stirred for 1.5 h. Volatiles were distilled off under reduced pressure and the residue azeotropically distilled with DCM (350 mL) and dried under high vacuum yielding (S)-1-(3 -cyanophenyl)-N-(pyrrolidin-3-ylmethyl)-1H-1,2,4-triazole-3-carboxamide TFA salt, 0.110 g, crude. LCMS: Method H, 1.331 min, MS:ES+297.26.

[0291] Step d. To a stirred solution of (S)-1-(3-cyanophenyl)-N-(pyrrolidin-3-ylmethyl)-1H-1,2,4-triazole-3-carboxamide TFA salt (0.100 g, crude from previous step) in THF (5 mL) was added K.sub.2CO.sub.3 (0.100 g, 0.73 mmol) at 0 C. After stirring at 0 C. for 15 min, cyanogen bromide (0.026 g, 0.24 mmol) was added and stirring continued for a further 1 h. The resulting mixture was poured into water (50 mL) and extracted with EtOAc (250 mL). Combined organic extracts were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography (compound eluted in 95% EtOAc in hexane) yielding (R)-1-(3-cyanophenyl)-N-((1-cyanopyrrolidin-3-yl)methyl)-1H-1,2,4-triazole-3-carboxamide (0.030 g, 0.09 mmol). LCMS: Method J, 2.767 min, MS:ES320.2; .sup.1H NMR (400 MHz, DMSO-d6) ppm: 9.49 (s, 1 H), 8.93-8.96 (t, J=6.0 Hz, 1 H), 8.44 (s, 1 H), 8.23-8.25 (m, 1 H), 7.93-7.95 (d, J=7.6 Hz 1 H), 7.78-7.82 (m, 1 H), 3.34-3.42 (m, 3 H), 3.14-3.29 (m, 4 H), 1.89-1.94 (m, 1 H), 1.63-1.68 (m, 1 H).

EXAMPLE 2

(S)-1-(3-cyanophenyl)-N((1-cyanopyrrolidin-3-yl)methyl)-N-methyl-1H-1,2,4-triazole-3-carboxamide

[0292] ##STR00015##

[0293] Step a. To a stirred solution of tert-butyl (R)-3-((1-(3-cyanophenyl)-1H-1,2,4-triazole-3-carboxamido)-methyl)pyrrolidine-1-carboxylate (described in Example 1, steps a and b; 0.100 g, 0.25 mmol) in DMF (2 mL) was added NaH (60% in mineral oil; 0.008 g, 0.30 mmol) portion-wise at 0 C. After stirring for 15 min, methyl iodide (0.035 g, 0.25 mmol) was added and the resulting reaction mixture was stirred at rt for 1 h. The reaction was poured into ice water (50 mL) and extracted with EtOAc (250 mL). The combined organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography (eluted in 2% MeOH in DCM) yielding tert-butyl (R)-3-((1-(3-cyanophenyl)-N-methyl-1H-1,2,4-triazole-3-carboxamido)methyl)-pyrrolidine-1-carboxylate (0.095 g, 0.23 mmol). LCMS: Method H, 1.755 min, MS:ES+411.4.

[0294] Step b. A stirred solution of tert-butyl (R)-3-((1-(3-cyanophenyl)-N-methyl-1H-1,2,4-triazole-3-carboxamido)methyl)pyrrolidine-1-carboxylate (0.090 g, 0.22 mmol) in DCM (5 mL) was added TFA (0.45 mL) at 0 C. The reaction mixture was stirred at 0 C. to rt for 1.5 h. The resulting reaction mixture was concentrated under reduced pressure. The obtained residue was azeotropically distilled with DCM (350 mL) and dried under high vacuum yielding (S)-1-(3-cyanophenyl)-N-methyl-N-(pyrrolidin-3-ylmethyl)-1H-1,2,4-triazole-3-carboxamide TFA salt, 0.110 g, crude. LCMS: Method H, 1.325 min, MS:ES+311.28.

[0295] Step c. To a stirred solution of (S)-1-(3-cyanophenyl)-N-methyl-N-(pyrrolidin-3-ylmethyl)-1H-1,2,4-triazole-3-carboxamide TFA salt (0.100 g, crude from previous step) in THF (5 mL) was added K.sub.2CO.sub.3 (0.097 g, 0.71 mmol) at 0 C. After stirring for 10 min, cyanogen bromide (0.024 g, 0.24 mmol) was added and stirring continued for 1 h at rt. The resulting mixture was poured into water (50 mL) and extracted with EtOAc (250 mL). The combined organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography (compound eluted in 2% MeOH in DCM) yielding (S)-1-(3-cyanophenyl)-N-((1-cyanopyrrolidin-3-yl)methyl)-N-methyl-1H-1,2,4-triazole-3-carboxamide (0.028 g, 0.08 mmol). LCMS: Method J, 2.723 min, MS:ES+336.15; .sup.1H NMR (400 MHz, DMSO-d6) ppm: 9.48 (s, 1 H), 8.42-8.43 (d, J=4.0 Hz, 1H), 8.19-8.23 (m, 1H), 7.92-7.94 (d, .1=8.0 Hz, 1 H), 7.77-7.81 (t, J=8 Hz 1 H), 3.44-3.58 (m, 3 H), 3.36-3.41 (m, 1 H), 3.23-3.27 (m, 1 H), 2.99-3.10 (m, 4 H), 2.59-2.65 (m, 1 H), 1.86-1.89 (m, 1 H), 1.64-1.69 (m, 1 H).

EXAMPLE 3

[0296] N-((1-cyanopyrrolidin-3-yl)methyl)-1-phenyl-1H-1,2,4-triazole-3-carboxamide

##STR00016##

[0297] Prepared using analogous procedures to those described herein.

EXAMPLE 4

N-(((2R,3R)-1-cyano-2-methylpyrrolidin-3-yl)methyl)-1-(3-cyanophenyl)-1H-1,2,4-triazole-3-carboxamide and N-(((2R,3S)-1-cyano-2-methylpyrrolidin-3-yl)methyl)-1-(3-cyanophenyl)-1H-1,2,4-triazole-3-carboxamide

[0298] ##STR00017##

[0299] Step a. To a solution of methyl acetoacetate (200 g, 1724.13 mmol) and 1,2-dibromoethane (179.2 mL, 2068.95 mmol) in acetone (2000 mL) was added K.sub.2CO.sub.3 (356.8 g, 2586.19 mmol) at rt. The reaction mixture was heated at 70 C. for 24 h. The resulting mixture was cooled to rt, filtered through celite and washed with acetone (2100 mL). The filtrate was concentrated under reduced pressure to give a crude oil which was purified by column chromatography (4% EtOAc in hexane) to yield methyl 1-acetylcyclopropane-1-carboxylate (100 g, 704.22 mmol). LCMS: Method C, 1.47 min, MS: ES+143.14.

[0300] Step b. A solution of methyl 1-acetylcyclopropane-1-carboxylate (82.0 g, 577.46 mmol) and (R)-1-phenylethan-1-amine (69.87 g, 577.46 mmol) in toluene (820 mL) was charged in a dean stark glass assembly. The reaction mixture was heated to 130 C. for 24 h (water release was collected in dean stark assembly). The reaction mixture was cooled to rt and concentrated under reduced pressure. The resulting residue was purified by column chromatography (1.5% EtOAc in hexane) to yield methyl (R)-2-methyl-1-(1-phenylethyl)-4,5-dihydro-1H-pyrrole-3-carboxylate (51.0 g, 208.16 mmol). LCMS: Method C, 1.409 min, MS:ES+246.40.

[0301] Step c. To NaBH.sub.4 (35.5 g, 936.73 mmol) was carefully added acetic acid (765 mL, 15 vol) dropwise at 0 C. and stirred for 45 min at 0 C. A solution of methyl (R)-2-methyl-1-(1-phenylethyl)-4,5-dihydro-1H-pyrrole-3-carboxylate (51.0 g, 208.16 mmol) in acetonitrile (765 mL) was then added at 0 C. and the reaction mixture was stirred for 4 h at rt. The resulting mixture was diluted with water (4000 mL) and basified with solid Na.sub.2CO.sub.3. The aqueous layer was extracted with EtOAc (23000 mL). The combined organic phase was collected, dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The resulting residue was dissolved in hexane and insoluble solid was removed by filtration. The filtrate was evaporated under reduced pressure to yield a mixture of diastereomers (51.0 g, 208.16 mmol). LCMS: Method D-NH.sub.3, 28.96 min (92%, major diasteromer) and 29.114 min (7%, minor diastereomer), MS:ES+248.2; The diastereomeric mixture was dissolved in n-hexane (500 mL) to form a clear solution which was stirred at 78 C. for 2 h. The resulting precipitate was removed by filtration and wash with cold hexane to yield methyl (2R,3R)-2-methyl-1-((R)-1-phenylethyl)pyrrolidine-3-carboxylate (28 g, 113.36 mmol). LCMS: Method D-NH.sub.3, 28.819 min, Method D-FA: 12.098 min, MS:ES+248.2; .sup.1H NMR (400 MHz, DMSO-d.sub.6) ppm: 7.29-7.35 (m, 4H), 7.22-7.24 (m, 1 H), 3.60 (s, 3 H), 3.55-3.58 (m, 1 H), 3.34-3.37 (m, 1H) 3.06-3.09 (m, 1 H), 2.58-2.61 (m, 1 H), 2.43-2.47 (m, 1 H), 1.92-1.99 (m, 1 H), 1.80-1.83 (m, 1 H), 1.27 (d, J=6.71 Hz, 3 H), 0.71 (d, J=6.40 Hz, 3 H).

[0302] Step d.

##STR00018##

[0303] Methyl (2R,3R)-2-methyl-1-((R)-1-phenylethyl)pyrrolidine-3-carboxylate (26.0 g, 105.263 mmol) was dissolved in DBU (47.99 g, 315.723 mmol) and heated at 100 C. for 24 h in seal tube. The reaction mixture was poured into water (800 mL) and extracted ethyl acetate with (3700 mL). The combined organic layer was washed with water (100 mL), dried over Na.sub.2SO.sub.4 and evaporated under reduced pressure to give crude product which was purified by column chromatography (eluted at 30% ethyl acetate in hexane) to yield mixture of methyl (2R,3R)-2-methyl-1-((R)-1-phenylethyl)pyrrolidine-3-carboxylate and methyl (2R, 3S)-2-methyl-1-((R)-1-phenylethyl)pyrrolidine-3-carboxylate (22 g, 89.068 mmol). LCMS: Method D-FA, 11.94 min (21.72%) & 12.68 min (77.01%), MS:ES+248.4; .sup.1H NMR (400 MHz, CDCl.sub.3) ppm: 7.25-7.40 (m, 5H), 3.85-3.87 (m, 1H), 3.74 (s, 3H), 3.00-3.30 (m, 1H), 2.56-2.81 (m, 3H), 1.93-2.08 (m, 2H), 1.41 (m, 3H), 1.04 (dd, J=6.4 Hz, 3H).

[0304] Step e.

##STR00019##

[0305] To solution of methyl (2R,3S)-2-methyl-1-((R)-1-phenylethyl) pyrrolidine-3-carboxylate and methyl (2R,3R)-2-methyl-1-((R)-1-phenylethyl)pyrrolidine-3-carboxylate (10 g, 40.485 mmol) in methanol (100 mL) was added 1 g (10% Pd/C, 50% moisture). The reaction mixture was stirred at rt under hydrogen (40 barr) for 24 h. The resulting mixture was filtered through celit, washed with methanol (50 mL) and the filtrate was concentrated under reduced pressure to yield mixture of methyl (2R,3S)-2-methylpyrrolidine-3-carboxylate and methyl (2R,3R)-2-methylpyrrolidine-3-carboxylate (5.3 g, 37.062 mmol). LCMS: Method C, 0.31 min, MS:ES+144.1; .sup.1H NMR (400 MHz, CDCl.sub.3) ppm: 3.74 (s, 3H), 3.40-3.43 (m, 1H), 3.10-3.20 (m, 2H), 2.53-2.63 (m, 1H), 2.13-2.19 (m, 2H), 2.04-2.08 (m, 1H), 1.37 (dd, J=6.4 Hz, 3H).

[0306] Step f.

##STR00020##

[0307] To solution of methyl (2R,3S)-2-methylpyrrolidine-3-carboxylate and methyl (2R,3R)-2-methylpyrrolidine-3-carboxylate (5.3 g, 37.062 mmol) in THF (53 mL) were added 4-dimethylaminopyridine (0.53 g, 0.1 w/w) and di-tert-butyl dicarbonate (9.69 g, 44.449 mmol) at 0 C. under N.sub.2. The reaction mixture was allowed to warm to rt and stirred for a further 16 h. The resulting mixture was diluted with water (200 mL) and extracted with ethyl acetate (3200 mL).The combined organic layer was washed with water (200 mL), dried over Na.sub.2SO.sub.4 and evaporated under reduced pressure to give a crude product which was purified by column chromatography (eluted at 10% ethyl acetate in hexane) to yield mixture of 1-(tert-butyl) 3-methyl (2R, 3S)-2-methylpyrrolidine-1,3-dicarboxylate and 1-(tert-butyl) 3-methyl (2R, 3R)-2-methylpyrrolidine-1,3-dicarboxylate (8.0 g, 32.92 mmol). LCMS: Method C, 1.665 min, MS:ES+(56) 188; .sup.1H NMR (400 MHz, CDCl.sub.3) ppm: 4.00-4.30 (m, 1H), 3.74 (s, 3H), 3.52-3.56 (m, 1H), 3.35-3.39 (m, 1H), 2.71-2.72 (m, 1H), 2.11-2.16 (m, 2H), 1.50 (s, 9H), 1.32 (dd, J=5.6 Hz, 3H).

[0308] Step g.

##STR00021##

[0309] To a solution of 1-(tert-butyl) 3-methyl (2R,3S)-2-methylpyrrolidine-1,3-dicarboxylate and 1-(tert-butyl)-3-methyl (2R,3R)-2-methylpyrrolidine-1,3-dicarboxylate (8.0 g, 32.921 mmol) in THF: Water (5:1) (12 mL) was added lithium hydroxide monohydrate (2.07 g, 49.381 mmol) at 0 C. The resulting reaction mixture was warmed to rt and stirred for 6 h. The mixture was poured into water (300 mL) and washed with ethyl acetate (3300 mL). The aqueous layer was acidified with 1M HCl solution (200 mL) and extracted with ethyl acetate (3300 mL). The combined organic layer was dried over Na.sub.2SO.sub.4 and evaporated under reduced pressure to yield mixture of (2R, 3R)-1-(tert-butoxycarbonyl)-2-methylpyrrolidine-3-carboxylic acid and (2R,3S)-(tert-butoxycarbonyl)-2-methylpyrrolidine-3-carboxylic acid (6.5 g, 28.384 mmol). LCMS: Method D-AA, 8.770 min (18.63 mmol) and 9.105 min (81.37 min), MS:ES+(56) 156.25; .sup.1H NMR (400 MHz, DMSO-d.sub.6) ppm: 12.47 (s, 1H), 3.8-4.1 (m, 1H), 3.25-3.37 (m, 2H), 2.60-2.75(m, 1H), 2.05-2.12 (m, 1H), 1.89-1.97 (m, 1H), 1.40 (s, 9H), 1.18 (dd, J=5.6 Hz, 3H).

[0310] Step h.

##STR00022##

[0311] To a solution of (2R, 3R)-1-(tert-butoxycarbonyl)-2-methylpyrrolidine-3-carboxylic acid and (2R,3S)-1-(tert-butoxycarbonyl)-2-methylpyrrolidine-3-carboxylic acid (5.5 g, 24.017 mmol) in THF (50 mL) were added EDC.HCl (5.50 g, 28.795 mmol) and 1-hydroxybenzotriazole hydrate (3.67 g, 23.986 mmol) at 0 C. under N.sub.2. Triethylamine (16.16 mL, 120.087 mmol) and NH.sub.4Cl (6.42 g, 120.000 mmol) were added to the reaction mixture and stirred for 16 h at rt. The resulting reaction mixture was poured into saturated NaHCO.sub.3 solution (400 mL) and extracted with ethyl acetate (3400 mL). The combined organic layer was dried over Na.sub.2SO.sub.4 and evaporated under reduced pressure to give crude product which was purified by column chromatography (2% MeOH in DCM) to yield tert-butyl (2R,3R)-3-carbamoyl-2-methylpyrrolidine-1-carboxylate and tert-butyl (2R,3S)-3-carbamoyl-2-methylpyrrolidine-1-carboxylate (4.8 g, 21.056 mmol). LCMS: Method C, 1.380 min, MS:ES+(56) 173.2; .sup.1H NMR (400 MHz, CDCl.sub.3) ppm: 5.57 (br, 2H), 3.95-4.12 (m, 1H), 3.42-3.63 (m, 2H), 2.42-2.61(m, 1H), 2.09-2.11 (m, 2H), 1.47 (s, 9H), 1.30 (dd, J=5.6 Hz, 3H).

[0312] Step i.

##STR00023##

[0313] To a solution of tert-butyl (2R, 3R)-3-carbamoyl-2-methylpyrrolidine-1-carboxylate and tert-butyl (2R,3S)-3-carbamoyl-2-methylpyrrolidine-1-carboxylate (4.8 g, 21.052 mmol) in THF (20 mL) was added BH.sub.3.DMS (2M in THF) (15.78 mL, 31.57 mmol) drop wise at 0 C. under N.sub.2. The reaction mixture was allowed to warm to rt and stirred for 16 h at 70 C. The mixture was then cooled to rt and methanol (50 mL) added. The mixture was further heated at 80 C. for 2 h then cooled to rt, acidified with 1N HCl solution (240 mL) and extracted with ethyl acetate (3400 mL). The aqueous layer was basified with sodium bicarbonate solution (50 mL) and extracted with ethyl acetate (3300 mL) concentrated under reduced pressure to yield tert-butyl (2R,3R)-3-(amino methyl)-2-methylpyrrolidine-1-carboxylate and tert-butyl (2R,3S)-3-(amino methyl)-2-methylpyrrolidine-1-carboxylate (0.137 g, 0.640 mmol). LCMS: Method C, 1.322 min, MS:ES-56+159.31; .sup.1H NMR (400 MHz, DMSO-d.sub.6) ppm: 3.28-3.48 (m, 3H), 3.15-3.26 (m, 1H), 2.41-2.44 (m, 1H), 1.65-2.05 (m, 2H), 1.41-1.60 (m, 3H), 1.34 (s, 9H), 0.95 (dd, J=6.4 Hz, 3H).

[0314] Step j.

##STR00024##

[0315] A stirred solution of methyl 1H-1,2,4-triazole-3-carboxylate (1.66 g, 13.1004 mmol) and 3-iodobenzonitrile (2 g, 8.733 mmol) in DMSO (10 mL) was added K.sub.2CO.sub.3 (3.6 g, 26.200 mmol) at rt. The reaction mixture was purged with N.sub.2 for 15 min. L-proline (0.2 g, 1.746 mmol) and CuI (0.33 g, 1.746 mmol) were added at rt and the mixture was heated to 85 C. for 16 h. The mixture was then poured into water (200 mL) and extracted ethyl acetate (2100 mL). The aqueous layer was acidified with 1M HCl solution (150 mL) and extracted with ethyl acetate (4100 mL). The combined organic layer was washed with water (100 mL), dried over Na.sub.2SO.sub.4 and evaporated under reduced pressure to yield 1-(3-cyanophenyl)-1H-1,2,4-triazole-3-carboxylic acid (0.522 g, 63.11 mmol). LCMS: Method C, 1.29 min, MS:ES+215.23; .sup.1H NMR (400 MHz, DMSO-d.sub.6) ppm: 12.8 (br, 1H), 7.35 (t, J=8 Hz, 1H), 7.02 (d, J=7.6 Hz, 1H), 6.83 (s, 1H), 6.77-6.80 (dd, J=8.4Hz, 2H).

[0316] Step k.

##STR00025##

[0317] To a solution of 1-(3-cyanophenyl)-1H-1,2,4-triazole-3-carboxylic acid (0.156 g, 0.728 mmol) in THF (7 mL) were added HATU (0.319 g, 0.841 mmol) and DIPEA (0.232 g, 1.682 mmol) 0 C. under N.sub.2. The reaction mixture was stirred for 30 min and added tert-butyl (2R, 3R)-3-(amino methyl)-2-methylpyrrolidine-1-carboxylate and tert-butyl (2R, 3S)-3-(amino methyl)-2-methylpyrrolidine-1-carboxylate (0.120 g, 0.560 mmol). The reaction mixture was become clear yellow solution and stirred at rt for 16 h. To resulting reaction mixture was added water (100 mL) and extracted with ethyl acetate (325 mL). The combined organic layer was washed with saturated NaHCO.sub.3 solution (100 mL), dried over Na.sub.2SO.sub.4 and evaporated under reduced pressure to give crude product which was purified by Combi-flash chromatography (eluted at 0.2% MeOH in DCM) to yield tert-butyl (2R, 3R)-3-((1-(3-cyanophenyl)-1H-1,2,4-triazole-3-carboxamido)methyl)-2-methylpyrrolidine-1-carboxylate and tert-butyl (2R, 3S)-3-((1-(3-cyanophenyl)-1H-1,2,4-triazole-3-carboxamido) methyl)-2-methylpyrrolidine-1-carboxylate (0.106 g, 0.258 mmol). LCMS: Method C, 1.627 min, MS:ES+(100) 311.48.

[0318] Step l.

##STR00026##

[0319] To a solution of tert-butyl (2R, 3S)-3-((1-(3-cyanophenyl)-1H-1,2,4-triazole-3-carboxamido) methyl)-2-methylpyrrolidine-1-carboxylate and tert-butyl (2R, 3R)-3-((1-(3-cyanophenyl)-1H-1,2,4-triazole-3-carboxamido) methyl)-2-methylpyrrolidine-1-carboxylate (0.103 g, 0.251 mmol) in DCM (5 mL) was added TFA (0.2 mL, 2.512 mmol) 0 C. under N.sub.2 atmosphere. The reaction mixture was allowed to warm to rt and stirred for 2 h then concentrated under reduced pressure to yield 1-(3-cyanophenyl)-N-(((2R, 3R)-2-methylpyrrolidin-3-yl) methyl)-1H-1,2,4-triazole-3-carboxamide trifluoroacetate and 1-(3-cyanophenyl)-N-(((2R, 3S)-2-methylpyrrolidin-3-yl) methyl)-1H-1,2,4-triazole-3-carboxamide trifluoroacetate (0.140 g, 0.267 mmol). LCMS: Method C, 1.286 min, MS: ES+311.5.

[0320] Step m.

##STR00027##

[0321] To a solution of 1-(3-cyanophenyl)-N-(((2R, 3S)-2-methylpyrrolidin-3-yl) methyl)-1H-1,2,4-triazole-3-carboxamide trifluoroacetate and 1-(3-cyanophenyl)-N-(((2R, 3R)-2-methylpyrrolidin-3-yl) methyl)-1H-1,2,4-triazole-3-carboxamide trifluoroacetate (0.139 g, 0.327 mmol) in THF (5 mL) was added K.sub.2CO.sub.3 (0.090 g, 0.654 mmol) and the mixture was stirred for 10 min. CNBr (0.034 g, 0.392 mmol) was added at rt and reaction mixture was stirred at rt for 30 min. To resulting reaction mixture was added water (50 mL) and the mixture extracted with ethyl acetate (350 mL). The combined organic layer was washed with water (50 mL), dried over Na.sub.2SO.sub.4 and evaporated under reduced pressure to give crude product. The crude product was purified by Combi-flash chromatography (eluted at 0.4% MeOH in DCM) to yield N-(((2R, 3R)-1-cyano-2-methylpyrrolidin-3-yl) methyl)-1-(3-cyanophenyl)-1H-1,2,4-triazole-3-carboxamide (0.052g, 0.155 mmol) which was further purified by prep TLC (3% MeOH in DCM) and then prep HPLC (MeOH:ACN using 0.1% ammonia) then triturated with n-pentene (23 mL) to yield N-(((2R,3S)-1-cyano-2-methylpyrrolidin-3-yl)methyl)-1-(3-cyanophenyl)-1H-1,2,4-triazole-3-carboxamide and N-(((2R,3R)-1-cyano-2-methylpyrrolidin-3-yl)methyl)-1-(3-cyanophenyl)-1H-1,2,4-triazole-3-carboxamide (0.0028 g, 0.0083 mmol); LCMS: Method M, 17.82 (25.34%),18.29 min (68.88%), MS:ES+336; MS:ES+336.2.

[0322] The diastereoisomers may be separable by standard methods.

EXAMPLE 5

(R)-1-(3-cyanophenyl)-N-((1-cyanopyrrolidin-3-yl)methyl)-1H-1,2,3-triazole-4-carboxamide

[0323] ##STR00028##

[0324] Step a. To a stirred solution of sodium azide (0.424 g, 6.535 mmol) and CuSO.sub.4 (0.086 g, 0.544 mmol) in MeOH (8 mL) was added (3-cyanophenyl)boronic acid (0.8 g, 5.445 mmol) (CAS number 150255-96-2, available from Combi-Blocks) at rt. The reaction mixture was stirred at rt for 16 h. Ethyl propionate (0.587 g, 5.99 mmol) and a solution of sodium L-(+)-ascorbate (0.539 g, 0.272 mmol) in water (2 mL) was added to the reaction mixture which was stirred for a further 1 h at rt. The resulting reaction mixture was filtered, washed with water (35 mL) and the solid was dried under reduced pressure to yield ethyl 1-(3-cyanophenyl)-1H-1,2,3-triazole-4-carboxylate (0.25 g, 1.033 mmol). LCMS: Method C, 1.579 min; MS:ES+243.4.

[0325] Step b. To a stirred solution of ethyl 1-(3-cyanophenyl)-1H-1,2,3-triazole-4-carboxylate (0.2 g, 0.826 mmol) and tert-butyl (R)-3-(aminomethyppyrrolidine-1-carboxylate (0.165 g, 0.826 mmol) (CAS number 199174-29-3, available from Synthonix) in THF (5 mL) was added a solution of TBD (0.23 g, 1.653 mmol) in THF (1 mL) dropwise at 0 C. The reaction mixture was heated at 80 C. for 16 h. The resulting reaction mixture was diluted with water (40 mL) and extracted with EtOAc (340 mL). The combined organic phase was dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. Crude material was purified by flash column chromatography (69% EtOAc in hexane) to yield tert-butyl (R)-3-((1-(3-cyanophenyl)-1H-1,2,3-triazole-4-carboxamido)-methyl)-pyrrolidine-1-carboxylate (0.13 g, 0.328 mmol). LCMS: Method C, 1.659 min; MS:ES+397.48.

[0326] Step c. To a stirred solution of tert-butyl (R)-3-((1-(3-cyanophenyl)-1H-1,2,3-triazole-4-carboxamido)-methyl)-pyrrolidine-1-carboxylate (0.12 g, 0.303 mmol) in DCM (3 mL) was added TFA (1.2 mL, 10 volumes) dropwise at 0 C. and stirred at rt for 2 h. The resulting reaction mixture was concentrated under reduced pressure and the residue was further concentrated from DCM (35 mL) to yield (S)-1-(3-cyanophenyl)-N-(pyrrolidin-3-ylmethyl)-1H-1,2,3-triazole-4-carboxamide trifluoroacetate (0.14 g, 0.341 mmol). LCMS: Method C, 1.249 min; MS:ES+297.43.

[0327] Step d. To a stirred solution of (S)-1-(3-cyanophenyl)-N-(pyrrolidin-3-ylmethyl)-1H-1,2,3-triazole-4-carboxamide trifluoroacetate (0.13 g, 0.317 mmol) in THF (4 mL) was added K.sub.2CO.sub.3 (0.131 g, 0.951 mmol) at 0 C. and was stirred for 10 min. Cyanogen bromide (0.04 g, 0.380 mmol) was added portionwise at 0 C. and stirred at rt for 1.5 h. The resulting reaction mixture was diluted with water (30 mL) extracted with EtOAc (330 mL). The combined organic phase was dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. Crude material was purified by flash column chromatography (72% EtOAc in hexane) to yield (R)-1-(3-cyanophenyl)-N-((1-cyanopyrrolidin-3-yl) methyl)-1H-1,2,3-triazole-4-carboxamide (0.12 g, 0.374 mmol). LCMS: Method H, 2.97 min, MS: ES320.2; .sup.1H NMR (400 MHz, CDCl.sub.3) ppm: 8.61 (s, 1 H), 8.18 (s, 1 H), 8.05 (m, 1 H), 7.86-7.75 (m, 2 H), 7.41-7.36 (m, 1 H), 3.65-3.47 (m, 4 H), 3.31-3.27 (m, 1 H), 2.71-2.64 (m, 1H), 2.22-2.12 (m, 1 H), 1.87 -1.78 (m, 1 H). Chiral HPLC: 100% purity, retention time: 5.67 min

[0328] CHIRAL SFC Analytical Method for Title Compound:

[0329] Chiral compound was analysed on Waters SFC Investigator and PDA (Photodiode array) detector. The column was used Chiralcel OX-H 250*4.6 mm, 5 micron, column flow was 4.0 mL/min and ABPR (automated back-pressure regulator) was set to 100 bar. Mobile phase were used (A) liquid carbon dioxide (Liq. CO2) and (B) 0.1% diethylamine in propan-2-ol:acetonitrile (50:50).

[0330] The UV spectra were recorded at 246 nm Lambda max. Gradient ratio was, as described below.

TABLE-US-00009 % B Start % B End Time duration (min) 5 50 5 50 50 5

EXAMPLE 6

(R)-1-(3-chlorophenyl)-N-((1-cyanopyrrolidin-3-yl)methyl)-1H-1,2,3-triazole-4-carboxamide

[0331] ##STR00029##

[0332] Step a. To a stirred solution of 1-(3-chlorophenyl)-1H-1,2,3-triazole-4-carboxylic acid (0.15 g, 0.671 mmol) (CAS Number 944901-58-0, available from Synthonix) and tert-butyl (R)-3-(aminomethyl)pyrrolidine-1-carboxylate (0.134 g, 0.671 mmol) (CAS number 199174-29-3, available from Astatech) in pyridine (5 mL) was added POCl.sub.3 (0.307 g, 0.19 mL, 2.013 mmol) dropwise at 0 C. and stirred at 0 C. for 40 min. The resulting reaction mixture was poured into cold water (30 mL) and extracted with EtOAc (330 mL). The combined organic phase was dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography (70% EtOAc in hexane) to yield tert-butyl-(R)-3-((1-(3-chlorophenyl)-1H-1,2,3-triazole-4-carboxamido)-methyl)-pyrrolidine-1-carboxylate (0.235 g, 0.580 mmol). LCMS: Method C, 1.734 min; MS:ES+406.49.

[0333] Step b. To a stirred solution of tert-butyl (R)-3-((1-(3-chlorophenyl)-1H-1,2,3-triazole-4-carboxamido)-methyl)-pyrrolidine-1-carboxylate (0.23 g, 0.568 mmol) in DCM (5 mL) was added TFA (2.3 mL, 10 volumes) dropwise at 0 C. and stirred at rt for 1.5 h. The resulting reaction mixture was concentrated under reduced pressure and crude was further concentrated from DCM (35 mL) to yield (S)-1-(3-chlorophenyl)-N-(pyrrolidin-3-ylmethyl)-1H-1,2,3-triazole-4-carboxamide trifluoroacetate (0.35 g, 0.835 mmol). LCMS: Method C, 1.341 min, MS:ES+306.36.

[0334] Step c. To a stirred solution of (S)-1-(3-chlorophenyl)-N-(pyrrolidin-3-ylmethyl)-1H-1,2,3-triazole-4-carboxamide trifluoroacetate (0.34 g, 0.811 mmol) in THF (7 mL) was added K.sub.2CO.sub.3 (0.335 g, 2.434 mmol) at 0 C. and stirred at 0 C. for 10 min. Cyanogen bromide (0.103 g, 0.974 mmol) was added portion wise to the reaction mixture. The reaction mixture was stirred at rt for 1.5h. The resulting reaction mixture was diluted with water (50 mL) extracted with EtOAc (350 mL). The combined organic phase was dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography (80% EtOAc in hexane) to yield (R)-1-(3-chlorophenyl)-N-((1-cyanopyrrolidin-3-yl)-methyl)-1H-1,2,3-triazole-4-carboxamide (0.13 g, 0.394 mmol). LCMS: Method H, 3.59 min; MS:ES+331.10; .sup.1H NMR (400 MHz, DMSO-d6) ppm: 9.38 (s, 1 H), 8.97-8.94 (m, 1H), 8.14(s, 1H), 8.01-7.99 (d, J=8.0 Hz, 1 H), 7.69-7.61 (m, 2 H), 3.48-3.19 (m, 7 H), 2.00-1.92 (m, 1 H), 1.75-1.66 (m, 1 H). Chiral HPLC (method as previously shown): 100% purity, retention time: 4.42 min

EXAMPLE 7

(R)-2-(3-chlorophenyl)-N-((1-cyanopyrrolidin-3-yl)methyl)-2H-1,2,3-triazole-4-carboxamide

[0335] ##STR00030##

[0336] Step a. To a stirred solution of 2-(3-chlorophenyl)-2H-1,2,3-triazole-4-carboxylic acid (0.15 g, 0.671 mmol) (CAS Number 90839-69-3, available from Enamine) and tert-butyl (R)-3-(aminomethyl)pyrrolidine-1-carboxylate (0.134 g, 0.671 mmol) (CAS number 199174-29-3, available from Astatech) in pyridine (3 mL) was added POCl.sub.3 (0.307 g, 0.19 mL, 2.013 mmol) dropwise at 0 C. and the mixture was stirred for 30 min. The resulting reaction mixture was poured in to water (100 mL) and extracted with EtOAc (350 mL). The combined organic phase was washed with water (100 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The crude material was purified by flash column chromatography (42% EtOAc in hexane) to yield tert-butyl (R)-3-((2-(3 -chlorophenyl)-2H-1,2,3-triazole-4-carboxamido)-methyl)-pyrrolidine-1-carboxylate (0.175 g, 0.432 mmol). LCMS: Method C, 2.014 min; MS:ES+350.38 (M56).

[0337] Step b. To a stirred solution of tert-butyl (R)-3-((2-(3-chlorophenyl)-2H-1,2,3-triazole-4-carboxamido)-methyl)-pyrrolidine-1-carboxylate (0.17 g, 0.419 mmol) in DCM (10 mL) was added TFA (0.478 g, 0.3 mL, 4.197 mmol) dropwise at 0 C. The reaction mixture was stirred at rt for 2 h. The resulting reaction mixture was concentrated under reduced pressure and crude was further concentrated from DCM (35 mL) to yield (S)-2-(3-chlorophenyl)-N-(pyrrolidin-3-ylmethyl)-2H-1,2,3-triazole-4-carboxamide trifluoroacetate (0.339 g, 0.809 mmol). LCMS: Method C, 1.374 min; MS:ES+306.31.

[0338] Step c. A solution of (S)-2-(3-chlorophenyl)-N-(pyrrolidin-3-ylmethyl)-2H-1,2,3-triazole-4-carboxamide trifluoroacetate (0.335 g, 0.799 mmol) in THF (10 mL) was added K.sub.2CO.sub.3 (0.22 g, 1.599 mmol) and the mixture was stirred for 10 min at 0 C. Cyanogen bromide (0.102 g, 0.959 mmol) was added portionwise in to the reaction mixture and stirred at rt for 45 min. The resulting reaction mixture was diluted with water (50 mL) extracted with EtOAc (325 mL). The combined organic phase was washed with water (50 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography (45% EtOAc in hexane) and by trituration with n-hexane (215 mL) to yield (R)-2-(3-chlorophenyl)-N-((1-cyanopyrrolidin-3-yl)-methyl)-2H-1,2,3-triazole-4-carboxamide (0.094 g, 0.284 mmol). LCMS: Method J, 3.99 min, MS: ES+331.4; .sup.1H NMR (400 MHz, CDCl.sub.3) ppm: 8.31 (s, 1 H), 8.17 (s, 1H), 8.05-8.03 (m, 1H), 7.52-7.44 (m, 2 H), 7.08-6.99 (m, 1 H), 3.65-3.47 (m, 5 H), 3.32-3.27 (m, 1 H), 2.73-2.66 (m, 1 H), 2.20-2.13 (m, 1 H), 1.88-1.79 (m, 1 H). Chiral HPLC: 100% purity, retention time: 14.05 min. Chiral compound was analysed on Agillent 1200 series HPLC and PDA detector. The column was used Chiralcel OJ-H 250*4.6 mm, 5 micron, column flow was 1.0 mL/min. Mobile phase were used (A) 0.1% diethylamine in hexane and (B) 0.1% diethylamine in propan-2-ol:methanol (50:50). The UV spectra were recorded at 272 nm Lambda max. Gradient ratio was, as described below.

TABLE-US-00010 Time % A % B 0.01 90 10 5 85 15 10 65 35 15 45 55 20 35 65 25 35 65 25.01 90 10 30 90 10

EXAMPLE 8

(R)-N-((1-cyanopyrrolidin-3-yl)methyl)-1-phenyl-1H-1,2,4-triazole-3-carboxamide

[0339] ##STR00031##

[0340] May be prepared using analogous procedures to those described herein.

[0341] Biological Activity of Compounds of the Invention

[0342] Abbreviations:

[0343] TAMRA carboxytetramethylrhodamine

[0344] PCR polymerase chain reaction

[0345] PBS phosphate buffered saline

[0346] EDTA ethylenediaminetetraacetic acid

[0347] Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol

[0348] NP-40 Nonidet P-40, octylphenoxypolyethoxyethanol

[0349] BSA bovine serum albumin

[0350] PNS peripheral nervous system

[0351] BH3 Bcl-2 homology domain 3

[0352] PTEN phosphatase and tensin homologue

[0353] USP30 Biochemical IC50 Assay

[0354] Dilution plates were prepared at 21 times the final concentration (2100 M for a final concentration of 100 M) in 50% DMSO in a 96-well polypropylene V-bottom plate (Greiner #651201). A typical 8-point dilution series would be 100, 30, 10, 3, 1, 0.3, 0.1, 0.03 M final. Reactions were performed in duplicate in black 384 well plates (small volume, Greiner 784076) in a final reaction volume of 21 l. Either 1 l of 50% DMSO or diluted compound was added to the plate. USP30 was diluted in reaction buffer (40 mM Tris, pH 7.5, 0.005% Tween 20, 0.5 mg/ml BSA, 5 mM beta-mercaptoethanol) to the equivalent of 0.05 l/well and 10 l of diluted USP30 was added to the compound. Enzyme and compound were incubated for 30 min at room temp. Reactions were initiated by the addition of 50 nM of TAMRA labelled peptide linked to ubiquitin via an iso-peptide bond as fluorescence polarisation substrate. Reactions were read immediately after addition of substrate and following a 2-hour incubation at room temperature. Readings were performed on a Pherastar Plus (BMG Labtech). Excitation 540 nm; Emission 590 nm.

[0355] Activity of Exemplary Compounds in USP30 biochemical IC50 Assay

[0356] Ranges:

[0357] A*<0.01 M;

[0358] 0.01<A<0.1 M;

[0359] 0.1<B<1 M; 1<C<10 M;

[0360] D>10 M;

TABLE-US-00011 Example IC50 1 A* 2 A 3 B 4 A 5 B 6 B 7 A*

TABLE-US-00012 Examples of WO 2017/103614 Example IC50 9 A 10 B 20 B 21 B 22 B 25 B 26 B 31 B 38 B 55 A 58 B 61 B 62 B 63 B 64 B 66 B 67 B 68 A* 69 A 70 C 71 A 72 B 73 B 74 B

[0361] Compounds of the invention may be tested for efficacy in representative disease models, including in vivo models.

[0362] MT#1: (R)-5-(3-cyanophenyl)-N-((1-cyanopyrrolidin-3-yl)methyl)-1,3,4-oxadiazole-2-carboxamide (WO 2017/103614: Ex. 68)

[0363] Representative models and experimental results for MT#1:

[0364] Bleomycin-Induced Lung Fibrosis Model

[0365] Bleomycin historically has been used in the treatment of lymphoma, squamous cell carcinomas, germ cell tumors and malignant pleural effusion, where it is injected intrapleurally. Bleomycin acts by causing single and double-strand DNA breaks in tumor cells and thereby interrupting the cell cycle. However, a concomitant overproduction of reactive oxygen species can lead to an inflammatory response causing pulmonary damage and subsequent fibrosis. Pulmonary side effects in patients are more likely in patients that smoke, are of older age, or have pre-existing pulmonary conditions. Dose dependent lung toxicity develops in approximately 10% of patients receiving bleomycin, and is clinically associated with cough, dyspnea, fever, cyanosis, and deterioration of lung function parameters. Within weeks to months this response might progress to pulmonary fibrosis in approximately 1% of patients.

[0366] Bleomycin as an agent to induce experimental lung fibrosis was first described in dogs later in mice, hamsters, and rats. It causes an initial elevation of pro-inflammatory cytokines, followed by increased expression of pro-fibrotic markers (transforming growth factor-1, fibronectin, procollagen-1), with a peak around day 14. Fibrosis tends to dominate pathology from day 9 onwards. Bleomycin-induced lung fibrosis is currently the mainstay of preclinical in vivo modelling of Idiopathic Pulmonary Fibrosis in the pharmaceutical industry. Current standard of care drugs such as pirfenidone and nintedanib have been tested in this preclinical lung fibrosis model.

[0367] In this study, Male C57B/L6 mice between ages of 6 to 8 weeks were used for a dose ranging study with MT#1. Treatment group animals (n=10/group) received 1.5 U/kg of clinical bleomycin via oropharyngeal route on day 0 under inhalant anaesthesia. Animals were monitored for tolerance of the bleomycin administration procedure and subsequently bodyweights of all mice were recorded at least 3 times per week during the study. Dosing with MT#1 or positive control (pirfenidone) commenced on day 7, with final dosing administered 2 to 4 hours prior to harvest on day 21 post bleomycin. All treatments were administered orally. MT#1 was supplied as a nanosuspension prepared at 100 mg/ml in hydroxymethylpropylcellulose/PVP/SDS (0.5%/0.5%/0.1%).

[0368] The Following Table Summarizes Treatment Groups:

TABLE-US-00013 Test article Number of Treatment Treatment daily dosing animals per In life Group Bleomycin (days 7 to 21) volume group mortality 1 0 U/kg None 10 0% 2 1.5 U/kg Vehicle BID 200 l 10 0% 3 1.5 U/kg 50 mg/kg MT#1 200 l 10 0% nanosuspension BID 4 1.5 U/kg 15 mg/kg MT#1 200 l 10 10% nanosuspension BID 5 1.5 U/kg 5 mg/kg MT#1 200 l 10 10% nanosuspension BID 6 1.5 U/kg 100 mg/kg 200 l 10 0% pirfenidone BID 7 1.5 U/kg 15 mg/kg MT#1 400 l 10 10% nanosuspension BID + 100 mg/kg Pirfenidone BID

[0369] At day 21, lungs were harvested from each animal and weighed, followed by fixation in 10% neutral buffered formalin (NBF) for histopathological analysis. The lung samples were processed and embedded with all lobes from each mouse in one paraffin block. Coronal sections through the four major lobes were stained with Masson's Trichrome. For each animal, consecutive lung fields were examined at 200magnification in a raster pattern using a 20objective lens and a 10ocular lens. A modified Ashcroft score (Hubner et al., 2008) was recorded for each field. The Fibrotic Index was calculated as the sum of the modified Ashcroft field scores divided by the number of fields examined.

[0370] FIG. 1. Effects of MT#1 treatment on Ashcroft Score in Bleomycin-induced lung fibrosis.

[0371] Treatment with MT#1 resulted in a dose dependent reduction in fibrosis as assessed by the modified Ashcroft score (collagen deposition, septae thickening). A statistically significant effect compared to vehicle was observed at 50 mg/kg BID (Statistics=one-way ANOVA+Tukey post-testing; *p<0.05, **p<0.01).

[0372] Unilateral Ureteral Obstructive Kidney Disease Model (UUO)

[0373] Unilateral ureteral obstruction (UUO) causes renal injury characterized by tubular cell injury, interstitial inflammation and fibrosis. It serves as a model both of irreversible acute kidney injury and as a model of events taking place during human chronic kidney disease. Being a unilateral disease, it is not useful to study changes in global kidney function, but has the advantage of a low mortality and the availability of an internal control (the non-obstructed kidney). Experimental UUO has illustrated the molecular mechanisms of apoptosis, inflammation and fibrosis, all three key processes in kidney injury of any cause, thus providing information beyond obstruction. The UUO model is commonly used to study drugs for potential therapeutic benefit in kidney fibrosis, including TGF-beta receptor inhibitors (galunisertib; LY2157299) and anti-TGF-beta antibodies (fresolumimab).

[0374] In this study, adult C57B16 female mice at 7 to 8 weeks of age with average weight of around 18 to 20 g were used. To complete the ureteral obstruction, the abdominal cavity was exposed via a midline incision and the left ureter was ligated at two points with 4-0 silk and dissected. Successful unilateral ureteral obstruction was later confirmed by observation of dilation of the renal pelvis due to hydronephrosis.

[0375] The Following Table Summarizes Treatment Groups:

TABLE-US-00014 Test article Number Adminis- of com- tration Number pleted route (PO- of animals Treat- oral, IP- animals In life excluded ment Treatment intraperi- per mortal- from Group Surgery (days 0 to 10) toneal) group ity analysis 1 Sham None 3 0% 0 2 UUO Vehicle BID PO 10 20% 0 3 UUO 5 mg/kg MT#1 PO 10 10% 1 nanosuspension BID 4 UUO 15 mg/kg MT#1 PO 10 0% 0 nanosuspension BID 5 UUO 50 mg/kg MT#1 PO 10 10% 0 nanosuspension BID 6 UUO 20 mg/kg IN- IP 10 0% 0 1130 QD

[0376] At harvest, mice were anesthetised and both kidneys were harvested; the contralateral kidney serves as control for the UUO kidney. The kidneys were dissected along median line into two parts, of which one side was fixed in formaldehyde, for paraffin embedding and the other was snap frozen for protein or RNA isolation.

[0377] Formalin fixed tissue were embedded in paraffin blocks and cut into 5 m sections. All damaged kidneys, 5 contralateral kidneys from each group and 3 sham kidneys were analysed (in total n=80 kidney samples). Picrosirius Red and -SMA stain was performed to evaluate fibrotic changes and myofibroblast activation. Analysis was performed by a pathology trained technical staff member, without knowledge of the animal group assignment. High magnification images were taken of each slide an analysed for the surface area covered by the stain.

[0378] FIG. 2. Effects of MT#1 treatment in UUO model.

[0379] Treatment with MT#1 resulted in a dose dependent decrease in collagen deposition as measured by picrosirius red staining. Statistically significant decreases in collagen were observed at 15 and 50 mg/kg BID MT#1.

[0380] Diet-Induced Model of Non-Alcoholic Fatty Liver Disease (NAFLD)/Non-Alcoholic Steatohepatitis (NASH)

[0381] Mice were fed Amylin-diet (D09100301, Research Diets, US; 40% fat (18% trans-fat), 40% carbohydrate (20% fructose) and 2% cholesterol) for 37 weeks. Liver biopies were harvested and mice were randomization into treatment groups based on liver Colla1 quantification. Three weeks following biopsy surgery, study animals were dosed orally (P.O.) with either; Vehicle (0.5% HPMC/0.5% PVP/0.1% SDS nanosuspension), MT#1 @ 15 mg/kg QD or MT#1 @ 15 mg/kg QD staining or with Elafibranor @ 30 mg/kg QD (positive control) for eight (8) weeks. A control group of age- and sex-matched mice fed lean chow diet ad libitum were also administered vehicle (P.O) over the same 8 (eight) week period. Following the full dosing schedule, mice were sacrificed, whole livers resected and weighed, fixed in paraformaldehyde before being paraffin embedded. H&E stained slides were then prepared and the average % fractional area of lipid vacuoles (steatosis) determined for each section (@ 20objective) using Visiomorph software (Visophram, Denmark). The average % fractional area was multiplied by the total liver weight to determine the total liver lipid content per animal. Values expressed as mean of n=9-12+SEM. Dunnett's test one-factor linear model. **: P<0.01, ***: P<0.001 compared to DIO-NASH Vehicle.

[0382] Effects of MT/41 treatment in NAFLD/NASH model.

[0383] FIG. 3. MT#1 administration resulted in a dose-dependent reduction in total liver lipid content in NASH animals, an effect that was statistically significant in mice administered MT#115 mg/kg BID (P.O.).

[0384] FIG. 4. Neither MT#1 or Elafibranor (positive control) significantly improved fibrotic stage scores in the same diet-induced Nonalcoholic Fatty Liver Disease (NAFLD)/Nonalcoholic Steatohepatitis (NASH).

[0385] Summary of fibrosis stage scores of pre- and post-study liver biopsies harvested from the diet-induced NAFLD/NASH study. Fibrosis stage scores are determined by assessment of picosirius red stained liver sections for relative appearance and location of collagen staining, where; 0=No staining, 1=Perisinusoidal or periportal, 2=Perisinusoidal and periportal and 4=Bridging collagen staining observed. For each group of animals with higher (worsening), same or lower (improvement) in score at post-compared to pre-study is indicated by the height of the bar. One-sided Fisher's exact test with Bonferroni correction. No differences at significance level 0.05.

[0386] Despite eliciting a statistically significant reduction in liver steatosis, MT#1 did not significantly improve fibrotic stage scores in treated animals. However, in this particular study, Elafibranor, whilst significantly reducing liver steatosis also failed to statistically improve fibrosis scoring in post-biopsy samples. Hence, it is possible that a longer period of compound administration may have been required to see a robust effect on fibrosis in this particular preclinical model of NAFLD/NASH.

[0387] Ischemia-Induced Acute Kidney Injury Model (AKI)

[0388] Severe blood loss during major operations, sepsis and cardio-thoracic surgeries are common causes for acute kidney injury (AKI). The incidence of AKI after lung transplantation is 50 to 60% and leads to increased morbidity and mortality of those patients. Similar AKI rates are present in other types of solid organ transplantation (i.e. liver, heart). Underlying cellular mechanisms include rapid complement activation, generation of oxygen free radicals, up regulation of cell adhesion molecules and inflammatory cell infiltration. Long term consequences are progressive interstitial fibrosis and chronic kidney disease. AKI can be induced by bilateral renal pedical clamping resulting in ischemia reperfusion injury (IRI) resulting in severe loss of renal function tubular damage and inflammation.

[0389] 12 to 15-week old male C57B1/6N mice or CD-1 mice (Charles River, Germany) are used for the study. Under anaesthesia, a midline incision is performed and both renal pedicles are clipped with a microaneurysm clip for 30 min. Without treatment mice show an increase of creatinine within 24 h of 3 to 6 fold and mortality can reach 30 to 40% with the first 4 days after surgery.