Sulfonamide-substituted cyanopyrrolidines with activity as DUB inhibitors

Abstract

The present invention relates to a class of sulfonamide-substituted cyanopyrrolidines of Formula (Ia) and (Ib) with activity as inhibitors of deubiquitilating enzymes, in particular, ubiquitin C-terminal hydrolase L1 (UCHL1) and ubiquitin C-terminal hydrolase 30 or ubiquitin specific peptidase 30 (USP30), having utility in a variety of therapeutic areas including cancer and conditions involving mitochondrial dysfunction: (Formulae (Ia), (Ib)). ##STR00001##

Claims

1. A compound of formula (Ia): ##STR00063## or a tautomer thereof, or a pharmaceutically acceptable salt of said compound or tautomer, wherein: m is 1 to 4; n is 0 or 1; one R.sup.1 group and NR.sup.2 are situated on adjacent ring atoms, and said R.sup.1 together with R.sup.2 forms a pyrrolidine ring; each other R.sup.1 group 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; L.sup.1 is selected from a covalent bond, (C.sub.1-C.sub.4)alkylene, and (C.sub.2-C.sub.4)alkenylene; L.sup.2 is selected from a covalent bond, (C.sub.1-C.sub.4)alkylene, (C.sub.2-C.sub.4)alkenylene, and (C.sub.0-C.sub.3)alkylene-X—(C.sub.0-C.sub.3)alkylene; X is selected from O, S, SO, SO.sub.2, NR.sup.4, NR.sup.4C(O), C(O)NR.sup.4, NR.sup.4C(O)NR.sup.5, C(O), C(O)O, OC(O), OC(O)O, SO.sub.2NR.sup.4, NR.sup.4SO.sub.2, and NR.sup.4SO.sub.2NR.sup.5; R.sup.4 and R.sup.5 are each independently 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; group ‘A’ is selected from a 3 to 10-membered carbocyclic ring, and a 3 to 10-membered heterocyclic ring comprising 1 to 4 heteroatoms independently selected from N, O, and S; with the proviso that for the compound of formula (Ia) when L.sup.1 is a covalent bond, ‘A’ is linked to the sulfonamide via a ring C-atom; group ‘B’ is selected from a 3 to 10-membered carbocyclic ring, and a 3 to 10-membered heterocyclic ring comprising 1 to 4 heteroatoms independently selected from N, O, and S; and each carbocyclic and heterocyclic ring may be optionally substituted with 1 to 4 substituents independently selected from halo, cyano, hydroxy, oxo, (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.

2. The compound according claim 1, wherein m is 1 or 2.

3. The compound according claim 1, wherein each other R.sup.1 is independently selected from fluoro, cyano, methyl, methoxy, and methoxymethyl.

4. The compound according claim 1, wherein L.sup.1 is selected from a covalent bond, methylene, and ethylene.

5. The compound according claim 1, wherein L.sup.2 is selected from a covalent bond, an oxygen atom, methylene, OCH.sub.2, and NHC(O).

6. The compound according claim 1, wherein group ‘A’ is selected from indanyl, phenyl, tetrahydrofuranyl, tetrahydropyranyl, tetralinyl, benzothiazolyl, imidazolyl, isoxazolyl, piperidinyl, pyrazolyl, pyridyl, pyrimidinyl, thiazolyl, 1,2,4-triazolyl, and quinolinyl.

7. The compound according claim 1, wherein group ‘B’ is selected from phenyl, oxazolyl, piperazinyl, piperidinyl, pyrazolyl, pyridyl, pyrimidinyl, pyrrolidinyl, 1,2-thiazolidinyl, and thiazolyl.

8. The compound according claim 1, wherein each carbocyclic and heterocyclic ring may be optionally substituted with 1 to 2 substituents independently selected from halo, cyano, hydroxy, oxo, (C.sub.1-C.sub.6)alkyl, (C.sub.1-C.sub.6)alkoxy, CF.sub.3, OCF.sub.3, and NHC(O)(C.sub.1-C.sub.6)alkyl.

9. The compound according to claim 1, wherein each carbocyclic and heterocyclic ring may be optionally substituted with 1 to 2 substituents independently selected from chloro, fluoro, cyano, hydroxy, oxo, methyl, isopropyl, methoxy, CF.sub.3, OCF.sub.3, NHC(O)isobutyl.

10. The compound according to claim 1, a tautomer thereof, or a pharmaceutically acceptable salt of said compound or tautomer, wherein: m is 1; L.sup.1 is selected from a covalent bond, methylene, and ethylene; L.sup.2 is a covalent bond; group ‘A’ is phenyl; group ‘B’ is pyridyl; and each phenyl and pyridyl ring may be optionally substituted with 1 to 2 substituents independently selected from chloro, fluoro, cyano, hydroxy, methyl, isopropyl, methoxy, CF.sub.3, OCF.sub.3, NHC(O)isobutyl.

11. The compound according to claim 1, a tautomer thereof, or a pharmaceutically acceptable salt of said compound or tautomer, wherein: n is 1; and group ‘B’ is a substituted 3 to 10-membered heterocyclic ring comprising 1 to 4 heteroatoms independently selected from N, O, and S.

12. A compound, wherein said compound is: (3aR,6aR)-1-((4-(pyridin-3-yl)phenyl)sulfonyl)hexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile, or a tautomer thereof, or a pharmaceutically acceptable salt of said compound or tautomer.

13. A pharmaceutical composition, comprising the compound according to claim 1, or a pharmaceutically acceptable salt of said compound or tautomer, together with a pharmaceutically acceptable diluent or carrier.

14. A pharmaceutical composition, comprising the compound according to claim 12, or a pharmaceutically acceptable salt of said compound or tautomer, together with a pharmaceutically acceptable diluent or carrier.

Description

EXAMPLE 20

(R)—N-(1-cyanopyrrolidin-3-yl)-N-methyl-4-(pyridin-3-yl)benzenesulfonamide

(1) ##STR00037##

Step 1. tert-Butyl (R)-3-((4-bromo-N-methylphenyl)sulfonamido) pyrrolidine-1-carboxylate)

(2) ##STR00038##

(3) To a stirred solution of tert-butyl (R)-3-(methylamino)pyrrolidine-1-carboxylate (CAS no. 199336-83-9, Available from Combi Blocks) (0.500 g, 2.50 mmol) in THF was added TEA (0.756 g, 7.49 mmol) at 0° C. under nitrogen and stirred for 10 min at the same temperature. A solution of 4-bromobenzenesulfonyl chloride (CAS No. 98-58-8, available from Combi-blocks) (0.636 g, 2.50 mmol) in THF (1 mL) was slowly added to the reaction mixture at 0° C. The reaction mixture was stirred for 1 h at room temperature. The resulting reaction mixture was diluted with water (30 mL) and was extracted with EtOAc (3×20 mL). The combined organic phases were dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure to yield tert-butyl (R)-3-((4-bromo-N-methylphenyl)sulfonamido)-pyrrolidine-1-carboxylate [1.00 g, 95.5% (crude)]. LCMS: Method C, 2.427 min, MS: ES+363.1, 365.1 (M−56).

Step 2. tert-Butyl (R)-3-((N-methyl-4-(pyridin-3-yl)phenyl)sulfonamido)pyrrolidine-1-carboxylate

(4) ##STR00039##

(5) To a mixture of tert-butyl (R)-3-((4-bromo-N-methylphenyl)sulfonamido)pyrrolidine-1-carboxylate (1.0 g, 2.39 mmol) and pyridine-3-boronic acid (CAS No. 1692-25-7, available from Combi-blocks) (0.293 g, 2.39 mmol) in DMF-water (3:2; 5 mL) was added Na.sub.2CO.sub.3 (0.505 g, 4.77 mmol). Resulting mixture was degassed (by purging nitrogen through the reaction solution) for 15 to 20 min. Tetrakis(triphenylphosphine)palladium (O) (0.275 g, 0.24 mmol) was added into the reaction solution and the resulting mixture was stirred at 110° C. for 16 h. The reaction mixture was cooled to room temperature and diluted with water (110 mL) and was extracted with EtOAc (3×50 mL). The combined organic extracts were dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure to yield tert-butyl (R)-3-((N-methyl-4-(pyridin-3-yl)phenyl)sulfonamido)pyrrolidine-1-carboxylate (0.5 g, 1.199 mmol) as crude mass. LCMS: Method C, 2.035 min, MS: ES+418.3.

Step 3. (R)—N-methyl-4-(pyridin-3-yl)-N-(pyrrolidin-3-yl)benzenesulfonamide TFA salt

(6) ##STR00040##

(7) To a solution of (R)-3-((N-methyl-4-(pyridin-3-yl)phenyl)sulfonamido)pyrrolidine-1-carboxylate (0.4 g, 0.96 mmol) in DCM (5 mL) was added TFA (1 mL) at 0° C. and the resulting solution was stirred at room temperature for 4 h. Reaction mixture was concentrated under reduced pressure. The crude was azeotropically distilled with diethyl ether (2×10 mL) and dried under reduced pressure to afford (R)—N-methyl-4-(pyridin-3-yl)-N-(pyrrolidin-3-yl)benzenesulfonamide TFA salt (0.4 g, quantitative). LCMS: Method C, 1.377 min, MS: ES+318.3.

Step 4. (R)—N-(1-cyanopyrrolidin-3-yl)-N-methyl-4-(pyridin-3-yl)-benzenesulfonamide

(8) To a stirred solution (R)—N-methyl-4-(pyridin-3-yl)-N-(pyrrolidin-3-yl)-benzenesulfonamide TFA salt (0.400 g, 0.93 mmol) in THF (5 mL) was added K.sub.2CO.sub.3 (0.384 g, 2.78 mmol) at 0° C. CNBr (0.118 g, 1.11 mmol) was added into the reaction mixture at 0° C. The reaction mixture was stirred at room temperature for 1 h. The resulting reaction mixture was poured in to water (50 mL) and was extracted with EtOAc (3×50 mL). Combined organic extracts were dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The resulting residue was purified by Combi-flash Column chromatography (silica; eluted with 2.5% MeOH in DCM) yielding title compound (0.080 g, 0.23 mmol). LCMS: Method B, 3.096 min, MS: ES+343.1 [M+1]; .sup.1H NMR (400 MHz, CDCl3) δ ppm: 9.00 (d, J=2 Hz, 1H), 8.66 (dd, J=4.8, 1.2 Hz, 1H), 8.19 (dt, J=8.0, 1.6 Hz, 1H), 8.01 (d, J=8.8 Hz, 2H), 7.93 (d, J=8.4 Hz, 2H), 7.57-7.54 (m, 1H), 4.64-4.60 (m, 1H), 3.45-3.40 (m, 2H), 3.33-3.29 (m, 1H), 3.20-3.15 (m, 1H), 2.72 (s, 3H), 1.88-1.83 (m, 1H), 1.79-1.73 (m, 1H).

EXAMPLE 21

(3aR,6aR)-1-((4-(pyridin-3-yl)phenyl)sulfonyl)hexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile

(9) ##STR00041##

Step 1. tert-butyl rac-(3aR,6aR)-1-((4-bromophenyl)sulfonyl)hexahydropyrrolo-[3,4-b]pyrrole-5(1H)-carboxylate

(10) ##STR00042##

(11) To a stirred solution of 4-bromobenzene sulfonyl chloride (CAS No. 98-58-8, available from Alfa Aesar) (0.2 g, 0.78 mmol) and tert-butyl rac-(3aS,6aS)-hexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate (CAS No. 180975-51-3, available from Enamine) (0.166 g, 0.78 mmol) in THF (10 mL) was added K.sub.2CO.sub.3 (0.324 g, 2.35 mmol) at ambient temperature. The reaction mixture was stirred at room temperature for 8 h. The resulting reaction mixture was diluted with water (200 mL) and was extracted with diethyl ether (3×100 mL).

(12) Combined organic extracts were dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure to yield tert-butyl rac-(3aR,6aR)-1-((4-bromophenyl)sulfonyl)hexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate (0.350 g, Quantitative) as crude mass. LCMS: Method C, 2.667 min, MS: ES+448.3, 450.3 (M+18).

Step 2. tert-butyl (3aR,6aR)-1-((4-(pyridin-3-yl)phenyl)sulfonyl)hexahydropyrrolo-[3,4-b]pyrrole-5(1H)-carboxylate

(13) ##STR00043##

(14) To a mixture of tert-butyl rac-(3aR,6aR)-1-((4-bromophenyl)sulfonyl)-hexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate (0.350 g, 0.81 mmol), pyridine-3-boronic acid (CAS No. 1692-25-7, available from Combiblocks) (0.120 g, 0.98 mmol) and in DMF-water (4:1; 17.5 mL) was added K.sub.2CO.sub.3 (0.224 g, 1.62 mmol). The reaction mixture was degassed (by purging nitrogen through the solution) for 30 min. PdCl.sub.2(dppf) (0.060 g, 0.08 mmol) was added into the reaction mixture and the resulting mixture was stirred at 80° C. for 2 h. Reaction mixture was cooled to room temperature, diluted with saturated aqueous NaHCO.sub.3 (350 mL) and was extracted with EtOAc (3×200 mL). The combined organic phase was dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The resulting residue was purified by Flash column chromatography (eluting with 3% MeOH in DCM) to yield tert-butyl rac-(3aR,6aR)-1-((4-(pyridin-3-yl)phenyl)sulfonyl)hexa-hydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate (0.350 g, 0.82 mmol). LCMS: Method C, 2.160 min, MS: ES+430.5

(15) Step 3. rac-(3aR,6aR)-1-((4-(pyridin-3-yl)phenyl)sulfonyl)octahydropyrrolo[3,4-b]pyrrole TFA salt

(16) ##STR00044##

(17) To a stirred solution of tert-butyl rac-(3aR,6aR)-1-((4-(pyridin-3-yl)phenyl)sulfonyl)hexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate (0.350 g, 0.82 mmol) in DCM (20 mL) was added TFA (2 mL) at 0° C. The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was concentrated under reduced pressure. The crude residue was further azetropically distilled with diethyl ether (3×10 mL) and dried under reduced pressure to afford rac-(3aR,6aR)-1-((4-(pyridin-3-yl)phenyl)sulfonyl) octahydropyrrolo[3,4-b]pyrrole TFA salt (0.210 g, quantitative) as crude mass. LCMS: Method C, 1.472 min, MS: ES+330.29

Step 4. (3aR,6aR)-1-((4-(pyridin-3-yl)phenyl)sulfonyl)hexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile

(18) ##STR00045##

(19) To a stirred solution rac-(3aR,6aR)-1-((4-(pyridin-3-yl)phenyl)sulfonyl) octahydropyrrolo[3,4-b]pyrrole TFA salt (0.200 g, 0.45 mmol) in THF (20 mL) was added K.sub.2CO.sub.3 (0.333 g, 2.41 mmol) at 0° C. followed by the addition of CNBr (0.051 g, 0.48 mmol) at the same temperature. The reaction mixture was stirred at room temperature for 1 h. The resulting reaction mixture was concentrated under reduced pressure and resulting residue was purified by flash column chromatography (eluting with 3.0% MeOH in DCM) yielding the title compound (0.150 g, 0.423 mmol). LCMS: Method A, 3.698 min, MS: ES+354.9 [M+1]; .sup.1H NMR (400 MHz, DMSO-D6): 8.96 (d, J=2 Hz, 1H), 8.65-8.64 (m, 1H), 8.19-8.17 (m, 1H), 8.02 (d, J, 8.4 Hz, 2H), 7.95 (d, J=8.8 Hz, 2H), 7.54-7.57 (m, 1H), 4.05-4.01 (m, 1H), 3.58 (d, J=7.2 Hz, 2H), 3.56-3.48 (m, 1H), 3.41-3.28 (m, 1H), 3.35 (dd, J=10.0 & 6.0 Hz, 1H), 3.18-3.14 (m, 1H), 1.74-1.62 (m, 1H), 1.65-1.71 (m, 2H).

EXAMPLE 22

rac-(4aR,7aR)-4-tosylhexahydropyrrolo[3,4-b][1,4]oxazine-6(2H)-carbonitrile

(20) ##STR00046##

(21) The title compound was synthesised via general method A using pyrrolo[3,4-b]-1,4-oxazine-6(2H)-carboxylic acid, hexahydro-1,1-dimethylethyl ester, (4aR,7aS) in step a. LCMS: Method D, 2.591 min, MS: ES+345.1 [M+1].

EXAMPLE 23

rac-(4aR,7aS)-4-(4-methylbenzyl)sulfonyl)hexahydropyrrolo[3,4-b][1,4]oxazine-6(2H)-carbonitrile

(22) ##STR00047##

(23) The title compound was synthesised via general method A using pyrrolo[3,4-b]-1,4-oxazine-6(2H)-carboxylic acid, hexahydro-1,1-dimethylethyl ester, (4aR,7aS) in step a. LCMS: Method D, 2.591 min, MS: ES+322.4 [M+1].

EXAMPLE 24

N-(5-(N-(1-cyanopyrrolidin-3-yl)sulfamoyl)pyridin-2-yl)-1-methyl-1H-pyrazole-5-carboxamide

(24) ##STR00048##

(25) Step 1. pyridin-2-amine was heated to 150° C. in chlorosulfonic acid to afford 6-aminopyridine-3-sulfonyl chloride which was used crude in the next reaction.

(26) Step 2. 6-aminopyridine-3-sulfonyl chloride was added to tert-butyl 3-aminopyrrolidine-1-carboxylate using general method A, step a to afford t-butyl 3-((6-aminopyridine)-3-sulfonamido)pyrrolidine-1-carboxylate.

(27) Step 3. t-butyl 3-((6-aminopyridine)-3-sulfonamido)pyrrolidine-1-carboxylate and 1-methyl-1H-pyrazole-5-carboxylic acid were heated to 70° C. with 50% T3P in ethyl acetate, triethylamine and THF for 30 h to afford tert-butyl 3-((6-(1-methyl-1H-pyrazole-5-carboxamido)pyridine)-3-sulfonamido)pyrrolidine-1-carboxylate.

(28) Step 4. The title compound was synthesised following the procedure of general method A, steps b-c. LCMS: Method B, 2.998 min, MS: ES+376.13 [M+1]; .sup.1H NMR (400 MHz, DMSO-d6) δ 11.32 (s, 1H), 8.77 (d, J=2.4 Hz, 1H), 8.37 (d, J=8.4 Hz, 1H), 8.24-8.26 (m, 2H), 7.55 (d, J=2 Hz, 1H), 7.34 (d, J=2 Hz, 1H), 4.1 (s, 3H), 3.78-3.82 (m, 1H), 3.31-3.46 (m, 3H), 3.09-3.12 (m, 1H), 1.88-1.99 (m, 1H), 1.66-1.74 (m, 1H).

EXAMPLE 25

N-(1-cyanopyrrolidin-3-yl)-N-methyl-4-((5-(trifluoromethyl)pyridin-2-yl)oxy)benzenesulfonamide

(29) ##STR00049##

(30) The title compound was synthesised via general method C using tert-butyl 3-(methylamino)pyrrolidine-1-carboxylate in step a. LCMS: Method D, 3.046 min, MS: ES+427.2 [M+1].

(31) Compounds in Table 4 were synthesised according to General Method D.

(32) ##STR00050##

(33) TABLE-US-00010 TABLE 4 LCMS LCMS RT MS Ex R′R″N— Name Method (min) (ES+) 26 N-([1,1′-biphenyl]-4-yl)-1-cyanopyrrolidine- 3-sulfonamide B 4.193 326.2 (ES−) 27 3-((4-(4-fluorophenyl)piperazin-1- yl)sulfonyl)pyrrolidine-1-carbonitrile D 2.603 339.1 28 (3-((4-(pyridin-2-yl)piperazin-1- yl)sulfonyl)pyrrolidine-1-carbonitrile E 2.162 322.1 29 3-((4-(pyrimidin-2-yl)piperazin-1- yl)sulfonyl)pyrrolidine-1-carbonitrile E 1.889 323.1 30 3-((4-(4-chlorophenyl)piperidin-1- yl)sulfonyl)pyrrolidine-1-carbonitrile D 2.824 354.1 31 (S)-3-((4-(4-chlorophenyl)piperidin-1- yl)sulfonyl)pyrrolidine-1-carbonitrile B 4.586 370.5 32 (R)-3-((4-(4-chlorophenyl)piperidin-1- yl)sulfonyl)pyrrolidine-1-carbonitrile B 4.561 370.5 33 3-((4-benzylpiperidin-1- yl)sulfonyl)pyrrolidine-1-carbonitrile D 3.107 334.2 34 3-((3-(4-chlorophenyl)azetidin-1- yl)sulfonyl)pyrrolidine-1-carbonitrile B 4.129 326.3 35 0 (S)-3-((3-(4-chlorophenyl)azetidin-1- yl)sulfonyl)pyrrolidine-1-carbonitrile B 4.211 326.3 36 (R)-3-((3-(4-chlorophenyl)azetidin-1- yl)sulfonyl)pyrrolidine-1-carbonitrile B 4.220 326.3 37 3-((3-phenoxyazetidin-1- yl)sulfonyl)pyrrolidine-1-carbonitrile A 4.239 308.0
Biological Activity of Compounds of the Invention

Abbreviations

(34) TAMRA carboxytetramethylrhodamine PCR polymerase chain reaction PBS phosphate buffered saline EDTA ethylenediaminetetraacetic acid Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol NP-40 Nonidet P-40, octylphenoxypolyethoxyethanol BSA bovine serum albumin PNS peripheral nervous system BH3 Bcl-2 homology domain 3 PTEN phosphatase and tensin homologue
In Vitro UCHL1 Inhibition Assay
Expression and Purification of UCHL1

(35) The UCHL1 construct was PCR amplified and cloned into a pFLAG-CMV-6a vector (Sigma-Aldrich) with an N-terminal FLAG tag. HEK293T cells were transfected with FLAG-UCHL1 using TransIT-LT1 transfection reagent (Mirus) according to the manufacturer's instructions. Cells were harvested 40 hours after transfection. Cells were washed once with PBS and scraped in lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 3 mM EDTA, 0.5% NP40, 10% glycerol, 5 mM beta-mercaptoethanol, protease inhibitors (complete mini, Roche) and phosphatase inhibitors (PhosSTOP mini, Roche). Lysates were incubated for 30 min on ice and centrifuged at 1200 rpm for 10 min at 4° C. Soluble supernatant was added to FLAG affinity resin (EZview Rad ANTI-FLAG M2 affinity gel, Sigma-Aldrich) equilibrated in low salt buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 5 mM beta-mercaptoethanol) and incubated at 4° C. for 3 hours rotating. The resin was spun at 2000 rpm for 2 min and the supernatant was removed. The resin was washed two times with low salt buffer and one time with high salt buffer (20 mM Tris, pH 7.5, 500 mM NaCl, 0.5 mM EDTA, 5 mM beta-mercaptoethanol, protease inhibitors (complete mini, Roche) and phosphatase inhibitors (PhosSTOP mini, Roche). To elute the bound UCHL1, elution buffer (10 mM Tris, pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 10% glycerol, 0.5% NP40, 5 mM beta-mercaptoethanol, 0.15 mg/ml 3× FLAG peptide (Sigma-Aldrich)) was added to the resin and incubated at 4° C. for 2.5 hours rotating. The resin was centrifuged at 4000 rpm for 30 seconds, and the supernatant containing purified FLAG-UCHL1 was removed and stored at −80° C.

(36) UCHL1 Biochemical Kinetic Assay

(37) Reactions were performed in duplicate in black 384 well plates (small volume, Greiner 784076) in a final reaction volume of 21 μl. UCHL1 was diluted in reaction buffer (20 mM Tris, pH 7.5, 100 mM NaCl, 0.05% Tween 20, 0.5 mg/ml BSA, 5 mM beta-mercaptoethanol) to the equivalent of 0, 0.01, 0.05, 0.1, 0.5, and 1 μl/well. Buffer was optimised for optimal temperature, pH, reducing agent, salts, time of incubation, and detergent. 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 incubated at room temperature and read every 2 min for 120 min. Readings were performed on a Pherastar Plus (BMG Labtech). λ Excitation 540 nm; λ Emission 590 nm.

(38) UCHL1 Biochemical IC50 Assay

(39) Dilution plates were prepared at 21 times the final concentration (2100 μM for a final concentration of 100 μM) in 50% DMSO in a 96-well polypropylene V-bottom plate (Greiner #651201). A typical 8-point dilution series to be 30, 10, 3, 1, 0.3, 0.1, 0.03, 0.01 μM final. Reactions were performed in duplicate in black 384 well plates (small volume, Greiner 784076) in a final reaction volume of 21 μl. Either 1 μl of 50% DMSO or diluted compound was added to the plate. UCHL1 was diluted in reaction buffer (20 mM Tris, pH 7.5, 100 mM NaCl, 0.05% Tween 20, 0.5 mg/ml BSA, 5 mM beta-mercaptoethanol) to the equivalent of 0.05 μl/well and 10 μl of diluted UCHL1 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). λ Excitation 540 nm; λ Emission 590 nm.

(40) Activity of Exemplary Compounds in UCHL1 Biochemical 1050 Assay

(41) Ranges:

(42) A<0.1 μM; 0.1<B<1 μM; 1<C<10 μM; 10 μM<D<100 μM

(43) TABLE-US-00011 Example IC50 range 1 D 2 C 3 D 4 C 5 D 6 D 8 C 9 C 11 D 12 C 13 C 14 D 15 C 16 D 18 D 19 B 20 C 24 D 25 C 26 D
In Vitro USP30 Inhibition Assay

(44) USP30 biochemical kinetic assay. Reactions were performed in duplicate in black 384 well plates (small volume, Greiner 784076) in a final reaction volume of 21 μl. USP30 CD (57-517, #64-0057-050 Ubiquigent) 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, 0.005, 0.01, 0.05, 0.1 and 0.5 μl/well. Buffer was optimised for optimal temperature, pH, reducing agent, salts, time of incubation, and detergent. 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 incubated at room temperature and read every 2 min for 120 min. Readings were performed on a Pherastar Plus (BMG Labtech). λ Excitation 540 nm; λ Emission 590 nm.

(45) USP30 Biochemical 1050 Assay

(46) Dilution plates were prepared at 21 times the final concentration (2100 μM for a final concentration of 100 μM) 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 μM final. Reactions were performed in duplicate in black 384 well plates (small volume, Greiner 784076) in a final reaction volume of 21 μl. Either 1 μl 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 μl/well and 10 μl 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). λ Excitation 540 nm; λ Emission 590 nm.

(47) Activity of Exemplary Compounds in USP30 Biochemical IC50 Assay

(48) Ranges:

(49) A<0.1 μM; 0.1<B<1 μM; 1<C<10 μM; 10 μM<D<100 μM

(50) TABLE-US-00012 Example IC50 range 1 B 2 B 3 B 4 B 6 C 7 C 8 B 9 B 10 C 11 B 13 B 14 B 15 B 16 B 17 C 18 C 19 B 20 B 21 B 22 D 23 B 24 B 25 B 26 B 27 D 28 B 29 C 30 A 31 A 32 A 33 B 34 B 35 B 36 B 37 D