HETEROCYCLIC TRPML1 AGONISTS
20230010299 · 2023-01-12
Inventors
- Paolo PEVARELLO (Bresso (MI), IT)
- Valentina CUSANO (Bresso (MI), IT)
- Mariangela SODANO (Bresso (MI), IT)
- Domenica TORINO (Bresso (MI), IT)
- Rocco VITALONE (Bresso (MI), IT)
- Chiara LIBERATI (Bresso (MI), IT)
- Francesco PISCITELLI (Bresso (MI), IT)
Cpc classification
C07D409/12
CHEMISTRY; METALLURGY
C07D295/135
CHEMISTRY; METALLURGY
C07C311/21
CHEMISTRY; METALLURGY
C07D409/04
CHEMISTRY; METALLURGY
C07D211/46
CHEMISTRY; METALLURGY
C07D333/24
CHEMISTRY; METALLURGY
C07D295/04
CHEMISTRY; METALLURGY
C07D277/32
CHEMISTRY; METALLURGY
C07D211/14
CHEMISTRY; METALLURGY
C07D209/08
CHEMISTRY; METALLURGY
C07D217/04
CHEMISTRY; METALLURGY
C07D207/06
CHEMISTRY; METALLURGY
C07D401/12
CHEMISTRY; METALLURGY
C07D307/79
CHEMISTRY; METALLURGY
A61P25/28
HUMAN NECESSITIES
International classification
C07D409/12
CHEMISTRY; METALLURGY
C07C311/21
CHEMISTRY; METALLURGY
C07D209/08
CHEMISTRY; METALLURGY
C07D211/14
CHEMISTRY; METALLURGY
C07D217/04
CHEMISTRY; METALLURGY
C07D295/135
CHEMISTRY; METALLURGY
C07D307/79
CHEMISTRY; METALLURGY
C07D401/12
CHEMISTRY; METALLURGY
Abstract
Disclosed is a compound of formula (I)
##STR00001## or a stereoisomer thereof, or a salt of any of the foregoing and to processes for its preparation. The compounds of formula (I) are useful in the treatment TRPML1-mediated disorders or diseases.
Claims
1. A compound of formula (I) ##STR00298## or a stereoisomer thereof, or a salt of any of the foregoing, wherein: A is a six membered aromatic, heteroaromatic or aliphatic ring, optionally substituted by one or more substituents selected from the group consisting of C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkyloxy, C.sub.3-C.sub.6 cycloalkyl, halogen, and cyano. Y is chosen from CH, or N; each of R.sub.1 and R.sub.2 independently is a C.sub.1-C.sub.4 alkyl group or R.sub.1 and R.sub.2 taken together with Y form a cyclic compound being a four, five, six or seven membered heteroaliphatic compound, containing at least one nitrogen atom, optionally fused with a six membered aromatic ring, optionally substituted by one or more substituents selected from the group consisting of: halogen; C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkyloxy, C.sub.1-C.sub.4 alkylcarbonyl, C.sub.1-C.sub.4 alkoxycarbonyl, wherein the C.sub.1-C.sub.4 alkyl group is optionally substituted by one or more halogen or C.sub.1-C.sub.4-alkyloxy; phenyl, benzyl, phenoxy, benzyloxy, pyrazin-2-yl-oxy and pyridin-2-yl-oxy, thiophene-2-carbonyl, 1,3 thiazole-5-carbonyl, thiophene-2-yl-acetyl, wherein the aromatic ring is optionally substituted by one or more halogen, C.sub.1-C.sub.4 alkyl, or C.sub.1-C.sub.4-alkyloxy; a C.sub.1-C.sub.2 alkylene bridge; R.sub.11R.sub.12N—CO— wherein each of R.sub.11 and R.sub.12 is C.sub.1-C.sub.4 alkyl; X is —CO— or —SO.sub.2—; each of R.sub.3, R.sub.4, R.sub.6 and R.sub.7 independently is hydrogen or C.sub.1-C.sub.4 alkyl; R.sub.5 is independently hydrogen or C.sub.1-C.sub.4 alkyl; each of m and n independently is 0 or 1; R.sub.8 is a five or six membered monocyclic or a nine or ten membered bicyclic aromatic compound, optionally substituted by one or more substituents selected from the group consisting of halogen, bromo, or pentafluorosulfanyl (SF.sub.5), or trifluoromethylthio (SCF.sub.3), C.sub.1-C.sub.4 alkyl or C.sub.1-C.sub.6cycloalkyl optionally substituted by a cyano group, —NO.sub.2, C.sub.1-C.sub.4 alkyloxy, C.sub.1-C.sub.4 alkylthio, —SO.sub.2NR.sub.9R.sub.10 or —NR.sub.9R.sub.10, wherein each of R.sub.9 and R.sub.10 independently is hydrogen or C.sub.1-C.sub.4 alkyl or R.sub.9 and R.sub.10 are taken together to form a five or six membered heteroaliphatic ring, optionally containing one or two oxygen atoms; provided that: when X is —CO— then R.sub.5 is C1-C4 alkyl when X is —SO.sub.2— then R.sub.5 is hydrogen. when A is phenyl, X is —SO.sub.2, m and n are both 0 and R.sub.1 and R.sub.2 taken together form a piperazine substituted by phenyl optionally substituted as defined above, then R.sub.8 is not phenyl optionally substituted as defined above; when A is phenyl, X is —SO.sub.2, m and n are both 0 and R.sub.1 and R.sub.2 taken together form an unsubstituted piperidine, pyrrolidine, or azepane, then R.sub.8 is not thiophene optionally substituted as defined above; when A is cyclohexyl, X is —SO.sub.2, m and n are both 0 and R.sub.1 and R.sub.2 taken together form an unsubstituted morpholine, then R.sub.8 is not phenyl optionally substituted as defined above.
2. A compound of formula (I) according to claim 1 wherein both m and n are 0.
3. A compound of formula (I) or a stereoisomer or a salt thereof according to claim 1 wherein: A is phenyl or 1,2 disubstituted cyclohexyl; Y is N and R.sub.1 and R.sub.2 taken together form a cyclic compound selected from piperidine, pyrrolidine, azete, azepane, 2-azabicyclo[2.2.1]heptane, 4-phenylpiperidine, tert-butyl piperazine-1-carboxylate, 4-phenoxypiperidine, 4-(benzyloxy)piperidine, 4-methoxypiperidine, morpholine, dimethylamine, pyrrolidine, 4,4-difluoropiperidine, 3-phenylpiperidine, 3-methoxypiperidine, 1-(4-chloro-2-fluorophenyl)piperazine, 3-phenylpyrrolidine, 2,3-dihydro-1H-indole, 1,2,3,4-tetrahydroquinoline, methyl piperazine-1-carboxylate, 2,2,2-trifluoroethyl piperazine-1-carboxylate, ethyl piperazine-1-carboxylate, 1-(piperazin-1-yl)propan-1-one, 3-methoxy-1-(piperazin-1-yl)propan-1-one, 1-benzoylpiperazine, 2-(piperidin-4-yloxy)pyridine, 2-(piperidin-4-yloxy)pyrazine, tert-butyl piperidine-4-carboxylate, tert-butyl piperidine-1-carboxylate, 4-(pyridin-2-yl)piperidine, 4-(pyrazin-2-yl)piperidine, 4-(thiophene-2-carbonyl)piperazine, 4-(4-methyl-1,3-thiazole-5-carbonyl)piperazine, 4-(piperazin-1-yl)-2-(thiophen-2-yl)ethan-1-one, N-methyl-N-propylpiperazine-1-carboxamide, 4-(thiophene-2-carbonyl)piperazine, 4-(4-methyl-1,3-thiazole-5-carbonyl)piperazine, 4-(piperazin-1-yl)-2-(thiophen-2-yl)ethan-1-one, N-methyl-N-propylpiperazine-1-carboxamide, 3-(piperidin-4-yloxy)pyridine, azepan-1-yl, 4-methoxyazepan-4-yl, 1,4-oxazepan-4-yl, N-propylpiperidine-4-carboxamide, N-(2-methylpropyl)piperidine-4-carboxamide, 2-(piperidin-4-yloxy)pyrimidine, piperidin-4-yl 2,2-dimethylpropanoate. both m and n are 0; R.sub.3, R.sub.4, R.sub.6 and R.sub.7 are hydrogen; X is —CO— or —SO.sub.2—; R.sub.5 is hydrogen or methyl; R.sub.8 is selected from phenyl, thiophene or benzothiophene, optionally substituted by dimethylsulfamoyl, methyl, ethyl, propyl, tert-butyl, 1,3-dioxolan-2-yl, cyanomethyl, monoalkylamino, dialkylamino, piperidine-1-sulfonyl, pyrrolidine-1-sulfonyl, morpholine-4-sulfonyl, fluoro, chloro, bromo, SF.sub.5, SCF.sub.3, nitro, and C.sub.1-C.sub.4 alkoxy. provided that: provided that: when X is —CO— then R.sub.5 is C1-C4 alkyl when X is —SO.sub.2— then R.sub.5 is hydrogen. when A is phenyl, X is —SO.sub.2, m and n are both 0 and R.sub.1 and R.sub.2 taken together form a piperazine substituted by phenyl optionally substituted as defined above, then R.sub.8 is not phenyl optionally substituted as defined above; when A is phenyl, X is —SO.sub.2, m and n are both 0 and R.sub.1 and R.sub.2 taken together form an unsubstituted piperidine, pyrrolidine, or azepane, then R.sub.8 is not thiophene optionally substituted as defined above; when A is cyclohexyl, X is —SO.sub.2, m and n are both 0 and R.sub.1 and R.sub.2 taken together form an unsubstituted morpholine, then R.sub.8 is not phenyl optionally substituted as defined above.
4. A compound of formula (I) or a stereoisomer or a salt thereof according to claim 1 wherein: A is phenyl; Y is N; R.sub.1 and R.sub.2 taken together with Y form a cyclic compound selected from piperidine, homopiperidine, pyrrolidine, morpholine, homomorpholine, 2,3-dihydro-1H-indole, 1,2,3,4-tetrahydroisoquinoline optionally substituted by one or more substituents selected from the group consisting of: halogen; C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkyloxy, C.sub.1-C.sub.4 alkylcarbonyl, C.sub.1-C.sub.4 alkoxycarbonyl, wherein the C.sub.1-C.sub.4 alkyl group is optionally substituted by one or more halogen or C.sub.1-C.sub.4-alkyloxy; phenyl, benzyl, phenoxy, benzyloxy, pyrazin-2-yl-oxy and pyridin-2-yl-oxy, thiophene-2-carbonyl, 1,3 thiazole-5-carbonyl, thiophene-2-yl-acetyl, wherein the aromatic ring is optionally substituted by one or more halogen, C.sub.1-C.sub.4 alkyl, or C.sub.1-C.sub.4-alkyloxy; C.sub.1-C.sub.2 alkylene bridge; R.sub.11R.sub.12N—CO— wherein each of R.sub.11 and R.sub.12 is C.sub.1-C.sub.4 alkyl; or R.sub.1 and R.sub.2 taken together with Y form a piperazine optionally substituted with —R.sub.11R.sub.12N—CO— wherein each of R.sub.11 and R.sub.12 is C.sub.1-C.sub.4 alkyl; n and m are both 0; R.sub.3, R.sub.4, R.sub.6 and R.sub.7 are hydrogen; X is —SO.sub.2— and R.sub.5 is hydrogen; or X is —CO— and R.sub.5 is methyl; R.sub.8 is selected from phenyl, thiophene or benzothiophene, thiazole, furan, pyridine, isoxazole, indoline, dihydrobenzofuran, quinolone, optionally substituted by one or more groups selected from dimethylsulfamoyl, methyl, ethyl, propyl, tert-butyl, 1,3-dioxolan-2-yl, mono(C.sub.1-C.sub.6)alkylamino, di (C.sub.1-C.sub.6)alkylamino, methylsulfonyl, and C.sub.1-C.sub.4 alkoxy; provided that: when X is —SO.sub.2, and R.sub.1 and R.sub.2 taken together form an unsubstituted piperidine, pyrrolidine, or azepane, then R.sub.8 is not thiophene optionally substituted as defined above.
5. A compound of formula (I) or a stereoisomer or a salt thereof according to claim 1 wherein: A is cyclohexyl; Y is N; R.sub.1 and R.sub.2 taken together with Y form a cyclic compound selected from piperidine, homopiperidine, piperazine, pyrrolidine, morpholine, homomorpholine, 2,3-dihydro-1H-indole, 1,2,3,4-tetrahydroisoquinoline optionally substituted by one or more substituents selected from the group consisting of: halogen; C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkyloxy, C.sub.1-C.sub.4 alkylcarbonyl, C.sub.1-C.sub.4 alkoxycarbonyl, wherein the C.sub.1-C.sub.4 alkyl group is optionally substituted by one or more halogen or C.sub.1-C.sub.4-alkyloxy; phenyl, benzyl, phenoxy, benzyloxy, pyrazin-2-yl-oxy and pyridin-2-yl-oxy, thiophene-2-carbonyl, 1,3 thiazole-5-carbonyl, thiophene-2-yl-acetyl, wherein the aromatic ring is optionally substituted by one or more halogen, C.sub.1-C.sub.4 alkyl, or C.sub.1-C.sub.4-alkyloxy; C.sub.1-C.sub.2 alkylene bridge; R.sub.11R.sub.12N—CO— wherein each of R.sub.11 and R.sub.12 is C.sub.1-C.sub.4 alkyl; or n and m and n are both 0; R.sub.3, R.sub.4, R.sub.6 and R.sub.7 are hydrogen; X is —SO.sub.2— and R.sub.5 is hydrogen; or X is —CO— and R.sub.5 is methyl; R.sub.8 is selected from phenyl, thiophene or benzothiophene, thiazole, furan, pyridine, isoxazole, indoline, dihydrobenzofuran, quinolone, substituted by one or more groups selected from dimethylsulfamoyl, methyl, ethyl, propyl, tert-butyl, 1,3-dioxolan-2-yl, mono(C1-C6)alkylamino, di (C1-C6)alkylamino, methylsulfonyl, and C.sub.1-C.sub.4 alkoxy; when X is —SO.sub.2, and R.sub.1 and R.sub.2 taken together form an unsubstituted morpholine, then R.sub.8 is not phenyl optionally substituted as defined above.
6. A compound of formula (I) according to claim 1 or a stereoisomer thereof, or a salt of any of the foregoing, selected from the group consisting of: TABLE-US-00019 N-[2-(piperidin-1-yl)phenyl]-1-benzothiophene-2-sulfonamide 4-(dimethylamino)-N-[2-(piperidin-1-yl)phenyl] benzene-1-sulfonamide N4-[2-(dimethylamino)phenyl]-N1,N1-dimethylbenzene-1,4-disulfonamide N4-(2-{2-azabicyclo[2.2.1]heptan-2-yl}phenyl)-N1,N1-dimethylbenzene-1,4-disulfonamide tert-butyl 4-(2-(4-(N,N-dimethylsulfamoyl)-N-methylbenzamido)phenyl)piperazine-1-carboxylate N1,N1-dimethyl-N4-[2-(piperidin-1-yl)phenyl]benzene-1,4-disulfonamide 2-[4-(dimethylsulfamoyl)phenyl]-N-[2-(pyrrolidin-1-yl)phenyl] acetamide N4-[2-(2,3-dihydro-1 H-indol-1-yl)phenyl]-N1,N1-dimethylbenzene-1,4-disulfonamide N1,N1-dimethyl-N4-[2-( 1,2,3,4-tetrahydroisoquinolin-2-yl)phenyl]benzene-1,4-disulfonamide 4-(N,N-dimethylsulfamoyl)-N-methyl-N-(2-(4-(pyridin-2-yloxy)piperidin-1-yl)phenyl)benzamide 3-(l,3-dioxolan-2-yl)-N-[2-(piperidin-1-yl)phenyl]thiophene-2-sulfonamide N1,N1-dimethyl-N4-[2-(4-phenylpiperidin-1-yl)phenyl] benzene-1,4-disulfonamide tert-butyl 4-{2-[4-(dimethylsulfamoyl)benzenesulfonamido]phenyl}piperazine-1-carboxylate 1-(2-((4-(N,N-dimethylsulfamoyl)phenyl)sulfonamido)phenyl)-N-methyl-N-propylpiperidine-4-carboxamide 4-(dimethylsulfamoyl)-N-methyl-N-[2-(piperidin-1-yl)phenyl]benzamide N-(2-{2-azabicyclo[2.2.1]heptan-2-yl}phenyl)-5-tert-butylthiophene-2-sulfonamide N-(2-{2-azabicyclo[2.2.1]heptan-2-yl}phenyl)-5-ethylthiophene-2-sulfonamide tert-butyl 4-(2-((4-ethylphenyl)sulfonamido)phenyl)piperazine-1-carboxylate tert-butyl 1-(2-((4-ethylphenyl)sulfonamido)phenyl)piperidine-4-carboxylate tert-butyl 4-(2-(4-(N,N-dimethylsulfamoyl)-N-ethylbenzamido)phenyl)piperazine-1-carboxylate N-(2-{2-azabicyclo[2.2.1]heptan-2-yl}phenyl)-5-ethyl-N-methylthiophene-2-sulfonamide N1,N1-dimethyl-N4-[2-(piperidin-1-yl)cyclohexyl]benzene-1,4-disulfonamide 3-methyl-N-[2-(piperidin-1-yl)phenyl]benzene-1-sulfonamide N1,N1-dimethyl-N4-[2-(4-phenoxypiperidin-1-yl)phenyl] benzene-1,4-disulfonamide N4-{2-[4-(benzyloxy)piperidin-1-yl]phenyl}-N1,N1-dimethylbenzene-1,4-disulfonamide N4-[2-(4-methoxypiperidin-1-yl)phenyl]-N1,N1-dimethylbenzene-1,4-disulfonamide N1,N1-dimethyl-N4-[2-(morpholin-4-yl)phenyl]benzene-1,4-disulfonamide tert-butyl 4-(2-((4-isopropylphenyl)sulfonamido)phenyl)piperazine-1-carboxylate tert-butyl 1-(2-((4-isopropylphenyl)sulfonamido)phenyl)piperidine-4-carboxylate N1,N1-dimethyl-N4-(2-(piperidin-1-ylmethyl)phenyl)benzene-1,4-disulfonamide tert-butyl 1-(2-((4-isopropoxyphenyl)sulfonamido)phenyl)piperidine-4-carboxylate N1,N1-dimethyl-N4-[2-(pyrrolidin-1-yl)phenyl]benzene-1,4-disulfonamide N4-[2-(4,4-difluoropiperidin-1-yl)phenyl]-N1,N1-dimethylbenzene-1,4-disulfonamide N1,N1-dimethyl-N4-[2-(3-phenylpiperidin-1-yl)phenyl]benzene-1,4-disulfonamide N4-[2-(3-methoxypiperidin-1-yl)phenyl]-N1,N1-dimethylbenzene-1,4-disulfonamide N1,N1-dimethyl-N4-[2-(3-phenylpyrrolidin-1-yl)phenyl]benzene-1,4-disulfonamide N1-(2-(4-hydroxypiperidin-1-yl)phenyl)-N4,N4-dimethylbenzene-1,4-disulfonamide 4-isopropoxy-N-(2-(piperidin-1-yl)phenyl)benzenesulfonamide N1-(2-(4-methoxyazepan-1-yl)phenyl)-N4,N4-dimethylbenzene-1,4-disulfonamide N1-(2-(4-methoxyazepan-1-yl)phenyl)-N4,N4-dimethylbenzene-1,4-disulfonamide N1-(2-(4-methoxyazepan-1-yl)phenyl)-N4,N4-dimethylbenzene-1,4-disulfonamide N1-(2-(l,4-oxazepan-4-yl)phenyl)-N4,N4-dimethylbenzene-1,4-disulfonamide N1-(2-(4-fluoropiperidin-1-yl)phenyl)-N4,N4-dimethylbenzene-1,4-disulfonamide N1,N1-dimethyl-N4-(2-(4-propoxypiperidin-1-yl)phenyl)benzene-1,4-disulfonamide tert-butyl 4-(2-(4-(N,N-dimethylsulfamoyl)-N-isopropylbenzamido)phenyl)piperazine-1-carboxylate tert-butyl 4-(2-((2-methylphenyl)sulfonamido)phenyl)piperazine-1-carboxylate N-(2-{2-azabicyclo[2.2.1]heptan-2-yl}phenyl)-4-(dimethylsulfamoyl)-N-methylbenzamide tert-butyl 4-(2-((2,4-dimethylphenyl)sulfonamido)phenyl)piperazine-1-carboxylate tert-butyl 4-(2-((3,4-dimethylphenyl)sulfonamido)phenyl)piperazine-1-carboxylate methyl4-{2-[4-(dimethylsulfamoyl)benzenesulfonamido]phenyl}piperazine-1-carboxylate 2,2,2-trifluoroethyl 4-{2-[4-(dimethylsulfamoyl)benzenesulfonamido]phenyl}piperazine-1-carboxylate tert-butyl 4-(2-((3,5-dimethylphenyl)sulfonamido)phenyl)piperazine-1-carboxylate N1,N1-dimethyl-N4-[2-(4-phenylpiperidin-1-yl)cyclohexyl]benzene-1,4-disulfonamide tert-butyl 4-(2-((2,4-dimethylthiazole)-5-sulfonamido)phenyl)piperazine-1-carboxylate N1,N1-dimethyl-N4-[2-(4-phenoxypiperidin-1-yl)cyclohexyl]benzene-1,4-disulfonamide tert-butyl 4-(2-((2,5-dimethylfuran)-3-sulfonamido)phenyl)piperazine-1-carboxylate tert-butyl4-(2-((2,5-dimethylthiophene)-3-sulfonamido)phenyl)piperazine-1-carboxylate tert-butyl 4-(2-((5-methylpyridine)-3-sulfonamido)phenyl)piperazine-1-carboxylate N1,N1-dimethyl-N4-[2-(morpholin-4-yl)cyclohexyl]benzene-1,4-disulfonamide N1,N1-dimethyl-N4-[2-(4-phenoxypiperidin-1-yl)cyclohexyl]benzene-1,4-disulfonamide N1,N1-dimethyl-N4-[2-(4-phenoxypiperidin-1-yl)cyclohexyl]benzene-1,4-disulfonamide tert-butyl 4-(2-((3,5-dimethylisoxazole)-4-sulfonamido)phenyl)piperazine-1-carboxylate 2,4-dimethyl-N-(2-(4-(pyridin-2-yloxy)piperidin-1-yl)phenyl)benzenesulfonamide 2,4-dimethyl-N-(2-(4-propoxypiperidin-1-yl)phenyl)benzenesulfonamide 2,5-dimethyl-N-(2-(4-propoxypiperidin-1-yl)phenyl)furan-3-sulfonamide 2,5-dimethyl-N-(2-(4-propoxypiperidin-1-yl)phenyl)thiophene-3-sulfonamide ethyl 4-{2-[4-(dimethylsulfamoyl)benzenesulfonamido]phenyl}piperazine-1-carboxylate N1,N1-dimethyl-N4-[2-(4-propanoylpiperazin-1-yl)phenyl]benzene-1,4-disulfonamide tert-butyl 4-(2-((l-methylindoline)-5-sulfonamido)phenyl)piperazine-1-carboxylate N4-[2-(4-benzoylpiperazin-1-yl)phenyl]-N1,N1-dimethylbenzene-1,4-disulfonamide N1,N1-dimethyl-N4-{[2-(piperidin-1-yl)phenyl]methyl}benzene-1,4-disulfonamide N1,N1-dimethyl-N4-{2-[4-(pyridin-2-yloxy)piperidin-1-yl]phenyl}benzene-1,4-disulfonamide t-butyl 1-{2-[4-(dimethylsulfamoyl)benzenesulfonamido]phenyl}piperidine-4-carboxylate N1,N1-dimethyl-N4-{2-[4-(pyrazin-2-yloxy)piperidin-1-yl]phenyl}benzene-1,4-disulfonamide t-butyl 4-{2-[4-(dimethylsulfamoyl)benzenesulfonamido]phenyl}piperidine-1-carboxylate N1,N1-dimethyl-N4-{2-[4-(thiophene-2-carbonyl)piperazin-1-yl] phenyl}benzene-1,4-disulfonamide N1,N1-dimethyl-N4-{2-[4-(4-methyl-l,3-thiazole-5-carbonyl)piperazin-1-yl]phenyl}benzene-1,4-disulfonamide N1,N1-dimethyl-N4-(2-{4-[2-(thiophen-2-yl)acetyl]piperazin-1-yl}phenyl)benzene-1,4-disulfonamide 4-{2-[4-(dimethylsulfamoyl)benzenesulfonamido]phenyl}-N-methyl-N-propylpiperazine-1-carboxamide t-butyl 4-[2-(4-methylbenzenesulfonamido)phenyl]piperazine-1-carboxylate t-butyl 4-[2-(4-methoxybenzenesulfonamido)phenyl]piperazine-1-carboxylate N4-[2-(azepan-1-yl)phenyl]-N1,N1-dimethylbenzene-1,4-disulfonamide N1,N1-dimethyl-N4-{2-[4-(pyridin-3-yloxy)piperidin-1-yl]phenyl}benzene-1,4-disulfonamide 1-{2-[4-(dimethylsulfamoyl)benzenesulfonamido]phenyl}-N-propylpiperidine-4-carboxamide 1-{2-[4-(dimethylsulfamoyl)benzenesulfonamido]phenyl}-N-(2-methylpropyl)piperidine-4-carboxamide N1,N1-dimethyl-N4-{2-[4-(pyrimidin-2-yloxy)piperidin-1-yl]phenyl}benzene-1,4-disulfonamide 1-[2-(4-methylbenzenesulfonamido)phenyl]piperidin-4-yl 2,2-dimethylpropanoate 1-[2-(4-methoxybenzenesulfonamido)phenyl]piperidin-4-yl 2,2-dimethylpropanoate tert-butyl 4-(2-((2-methyl-2,3-dihydrobenzofuran)-5-sulfonamido)phenyl)piperazine-1-carboxylate tert-butyl 4-(2-((2-methyl-2,3-dihydrobenzofuran)-5-sulfonamido)phenyl)piperazine-1-carboxylate tert-butyl 4-(2-((2-methyl-2,3-dihydrobenzofuran)-5-sulfonamido)phenyl)piperazine-1-carboxylate tert-butyl 4-(2-((8-methylquinoline)-5-sulfonamido)phenyl)piperazine-1-carboxylate tert-butyl 4-(2-(N-isopropyl-4-(methylsulfonyl)benzamido)phenyl)piperazine-1-carboxylate 2,5-dimethyl-N-(2-(4-(pyridin-2-yloxy)piperidin-1-yl)phenyl)thiophene-3-sulfonamide 6-methyl-N-(2-(4-(pyridin-2-yloxy)piperidin-1-yl)phenyl)pyridine-3-sulfonamide 2,5-dimethyl-N-(2-(4-(pyridin-2-yloxy)piperidin-1-yl)phenyl)thiazole-4-sulfonamide N1,N1-dimethyl-N4-(2-(2-phenylmorpholino)phenyl)benzene-1,4-disulfonamide N1-(2-(2-benzylmorpholino)phenyl)-N4,N4-dimethylbenzene-1,4-disulfonamide N-isopropyl-4-(methylsulfonyl)-N-(2-(4-(pyrazin-2-yloxy)piperidin-1-yl)phenyl)benzamide methyl 1-(2-((4-(N,N-dimethylsulfamoyl)phenyl)sulfonamido)phenyl)piperidine-4-carboxylate N1,N1-dimethyl-N4-(2-(4-(pyridin-4-yloxy)piperidin-1-yl)phenyl)benzene-1,4-disulfonamide
7. A process for preparing a compounds of formula (I) according to claim 1 or a stereoisomer thereof, or a salt of any of the foregoing comprising the step of reacting a compound of formula (II) or formula (III): ##STR00299## wherein Y, R.sub.1, R.sub.2, R.sub.3, R.sub.4 and n are as defined in claim 1, with a compound of formula (IV) or formula (V) ##STR00300## wherein R.sub.6, R.sub.7, m and R.sub.8 are as defined in claim 1.
8. A process for preparing a compounds of formula (I) according to claim 1 or a stereoisomer thereof, or a salt of any of the foregoing comprising the step of reacting a compound of formula (VI): ##STR00301## wherein R.sub.6, R.sub.7, m and R.sub.8 are as defined above; with a suitably substituted amine.
9. A process for preparing a compounds of formula (I) according to claim 1 or a stereoisomer thereof, or a salt of any of the foregoing comprising the step of reacting a compound of formula (VII) or compound of formula (VIII) with CH.sub.3I or CH.sub.3CH.sub.2I or (CH.sub.3).sub.2CHI ##STR00302## wherein A, Y, R.sub.1, R.sub.2, R.sub.6, R.sub.7, m and R.sub.8 are as defined in claim 1.
10. A process for preparing a compounds of formula (I) according to claim 1 or a stereoisomer thereof, or a salt of any of the foregoing comprising the step of reacting a compound of formula (IX) ##STR00303## wherein A, Y, R.sub.6, R.sub.7, m and R.sub.8 are a fined above, with a compound of formula (X) ##STR00304## wherein R.sub.11 and R.sub.12 is as defined above.
11. A pharmaceutical composition comprising a compound of formula (I) according to claim 1, or a stereoisomer thereof, or a salt of any of the foregoing and a pharmaceutically acceptable carrier.
12. A medicament comprising the compound of formula (I) according to claim 1, or a stereoisomer thereof, or a salt of any of the foregoing.
13. A method for treatment of a TRPML1-mediated disorder or disease, comprising administering an effective amount of the compound of formula (I) according to claim 1, or a stereoisomer thereof, or a salt of any of the foregoing, to a patient in need thereof.
14. The method of claim 13, wherein the TRPML1-mediated disorder or disease is selected from diseases of aging, bone diseases, cardiovascular diseases, congenital developmental disorders, eye diseases, hematological and solid malignancies, infectious diseases, inflammatory diseases, liver diseases, metabolic diseases, neurological or neurodegenerative diseases, pancreatitis, renal diseases, skeletal muscle disorders, obesity, lysosomal storage diseases and pulmonary Diseases
15. The method of claim 13, wherein the TRPML1-mediated disorder or disease is selected from Aicardi-Goutiéres syndrome, Alzheimer's Disease, amyotrophic lateral sclerosis, ataxia-telangiectasia, autism spectrum disorders, Batten Disease, bipolar disorder, cerebral ataxia, Charcot-Marie-Tooth variant diseases, Chronic Wasting Disease, corticobasal degeneration, corticobasal syndrome, bovine spongiform encephalopathy, Creutzfeldt-Jacob Disease, Danon Disease, Duchenne Muscular Dystrophy, Exotic ungulate encephalopathy, Fabre Disease, Fatal Famial insomnia, Fridreich Ataxia, Feline spongiform Encephalopathy, Fragile X, Frontal temporal dementia, Gaucher Disease, Gerstmann-Straussler-Scheinker Disease, Giant axonal neuropathy, GM1 and GM2 gangliosidosis, Huntington's Disease, Infantile Refsum Disease, JUNQ and IPOD, Kuru, Leukoencephalopathy, Lewy Body Dementia, locomotor ataxia, Lyme disease, Machado Joseph Disease, major depressive disorder, MPS-III, Mucolipidosis, multiple sulfatase deficiency, multiple systems atrophy, myofibrillar myopathies, myotonic dystrophy, Niemann-Pick Disease, Parkinson's Disease, Parkinsonism, Pick's disease, polyglutamine diseases, Pompe Disease, pontocerebellar hypoplasia, prion diseases, progressive nuclear palsy, pyruvate dehydrogenase deficiency, Sandhoff Disease, Schizophrenia, Scrapie, Shy-Drager syndrome, spinal muscular atrophy, spinocerebellar ataxias, sporadic familial insomnia, subacute degeneration of the spinal cord, subacute sclerosing panencephalitis, Tay-Sachs disease, transneuronal degeneration, and vascular dementia.
Description
EXAMPLES
[0263] The invention is further illustrated by the following examples.
Example 1
[0264] The title compound was prepared by the general method A, starting from commercially available amine CAS: 39643-31-7 and sulphonyl chloride CAS: 90001-64-2, using TEA as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 10%. (y=63%).
Example 2
[0265] The title compound was prepared by the general method A, starting from commercially available amine CAS: 39643-31-7 and sulphonyl chloride CAS: 19715-49-2, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 10%. (y=89%).
Example 3
[0266] The title compound was prepared by the general method A, starting from commercially available amine CAS: 2836-03-5 and sulphonyl chloride CAS: 677782-39-7, using TEA as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 50%. (y=23%).
Example 4
[0267] The title compound was prepared by the general method A, starting from commercially available amine CAS: 1250695-37-4 and sulphonyl chloride (CAS: 677782-39-7), using TEA as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 20% (y=11%).
Example 5
[0268] The title compound was prepared by the general method C, from intermediate VIIIa and iodomethane (CAS: 74-88-4) at rt for 2 h and purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 50% (y=60%).
Example 6
[0269] The title compound was prepared by the general method A, starting from commercially available amine CAS 39643-31-7 and sulphonyl chloride CAS 677782-39-7, using TEA as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 30% (y=69%).
Example 7
[0270] The title compound was prepared by the general method E, starting from commercially available carboxylic acid CAS: 91013-44-4 and amine CAS: 21627-58-7. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 45%. (y=87%).
Example 8
[0271] The title compound was prepared by the general method A, starting from commercially available amine CAS: 180629-70-3 and sulphonyl chloride CAS: 677782-39-7, using TEA as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 50%. (y=41%).
Example 9
[0272] The title compound was prepared by the general method A, starting from commercially available amine CAS: 246247-91-6 and sulphonyl chloride CAS: 677782-39-7, using TEA as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 50%. (y=47%).
Example 10
[0273] The title compound was prepared by the general method C, from intermediate VIIIb and iodomethane (CAS: 74-88-4) at rt for 2 h and purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 50% (y=75%).
Example 11
[0274] The title compound was prepared by the general method A, starting from commercially available amine CAS: 39643-31-7 and sulphonyl chloride CAS: 103011-38-7, using TEA as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 20% (y=46%).
Example 12
[0275] The title compound was prepared by the general method A, starting from commercially available amine CAS: 252758-95-5 and sulphonyl chloride CAS: 677782-39-7, using TEA as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 70% (y=60%).
Example 13
[0276] The title compound was prepared by the general method A, starting from commercially available amine CAS: 170017-74-0 and sulphonyl chloride CAS: 677782-39-7, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 50% (y=96%).
Example 14
[0277] The title compound was prepared by the general method E, starting from intermediate XIIa and commercially available amine CAS: 627-35-0. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 40% (y=88%).
Example 15
[0278] The title compound was prepared by the general method C, from intermediate VIIIe and iodomethane (CAS: 74-88-4) at rt for 2 h and purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 50% (y=54%).
Example 16
[0279] The title compound was prepared by the general method A, starting from commercially available amine CAS: 1250695-37-4 and sulphonyl chloride CAS: 179400-17-0, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 10% (y=72%).
Example 17
[0280] The title compound was prepared by the general method A, starting from commercially available amine CAS: 1250695-37-4 and sulphonyl chloride CAS: 56921-00-7, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 10% (y=62%).
Example 18
[0281] The title compound was prepared by the general method A, starting from commercially available amine CAS: 170017-74-0 and sulphonyl chloride CAS: 16712-69-9, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 40% (y=95%).
Example 19
[0282] The title compound was prepared by the general method A, starting from intermediate IId and sulphonyl chloride CAS: 16712-69-9, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 20% (y=87%).
Example 20
[0283] The title compound was prepared by the general method C, from intermediate VIIIa and iodoethane (CAS: 75-03-6) at rt for 2 h and purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 50% (y=67%).
Example 21
[0284] The title compound was prepared by the general method F, starting from Example 17. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 50% (y=56%).
Example 22
[0285] The title compound was prepared by the general method B, starting from intermediate VIa and commercially available amine CAS: 110-89-4. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 70%. (y=87%).
Example 23
[0286] The title compound was prepared by the general method A, starting from commercially available amine CAS: 39643-31-7 and sulphonyl chloride CAS: 1899-93-0, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 10% (y=75%).
Example 24
[0287] The title compound was prepared by the general method A, starting from intermediate IIa and sulphonyl chloride CAS: 677782-39-7, using TEA as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 70% (y=9%).
Example 25
[0288] The title compound was prepared by the general method A, starting from commercially available amine CAS: 1018636-14-0 and sulphonyl chloride CAS: 677782-39-7, using TEA as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 70% (y=13%).
Example 26
[0289] The title compound was prepared by the general method A, starting from commercially available amine CAS: 1018624-48-0 and sulphonyl chloride CAS: 677782-39-7, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 80% (y=90%).
Example 27
[0290] The title compound was prepared by the general method A, starting from commercially available amine CAS: 5585-33-1 and sulphonyl chloride CAS: 677782-39-7, using TEA as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 50% (y=39%).
Example 28
[0291] The title compound was prepared by the general method A, starting from commercially available amine CAS: 170017-74-0 and sulphonyl chloride CAS: 54997-90-9, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 40% (y=87%).
Example 29
[0292] The title compound was prepared by the general method A, starting from intermediate IId and sulphonyl chloride CAS: 54997-90-9, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 20% (y=96%).
Example 30
[0293] The title compound was prepared by the general method A, starting from commercially available amine CAS: 19577-83-4 and sulphonyl chloride CAS: 677782-39-7, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 20% to 100% (y=96%).
Example 31
[0294] The title compound was prepared by the general method A, starting from intermediate IId and sulphonyl chloride CAS: 98995-40-5, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 25% (y=66%).
Example 32
[0295] The title compound was prepared by the general method A, starting from commercially available amine CAS: 21627-58-7 and sulphonyl chloride CAS: 677782-39-7, using TEA as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 40% (y=15%).
Example 33
[0296] The title compound was prepared by the general method A, starting from commercially available amine CAS: 854044-39-6 and sulphonyl chloride CAS: 677782-39-7, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 40% (y=65%).
Example 35
[0297] The title compound was prepared by the general method A, starting from intermediate IIb and sulphonyl chloride CAS: 677782-39-7, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 60% (y=90%).
Example 36
[0298] The title compound was prepared by the general method A, starting from commercially available amine CAS: 1602318-01-3 and sulphonyl chloride CAS: 677782-39-7, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 40% (y=91%).
Example 37
[0299] The title compound was prepared by the general method A, starting from commercially available amine CAS: 1272785-78-0 and sulphonyl chloride CAS: 677782-39-7, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 40% (y=74%).
Example 38
[0300] The title compound was prepared by the general method A, starting from commercially available amine CAS: 252758-96-6 and sulphonyl chloride CAS: 677782-39-7, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 30% to 100% (y=98%).
Example 39
[0301] The title compound was prepared by the general method A, starting from commercially available amine CAS: 39643-31-7 and sulphonyl chloride CAS: 98995-40-5, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 25% (y=60%).
Example 40
[0302] The title compound was prepared by the general method A, starting from commercially available amine CAS: 1996133-97-1 and sulphonyl chloride CAS: 677782-39-7, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 50% (y=55%).
Example 41 and Example 42
[0303] The title compounds, were obtained by enantiomeric separation of the racemate of Example 40; for separation procedures, see analytical methods.
Example 43
[0304] The title compound was prepared by the general method A, starting from commercially available amine CAS: 955428-44-1 and sulphonyl chloride CAS: 677782-39-7, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of methanol in dichloromethane from 0% to 5% (y=68%).
Example 44
[0305] The title compound was prepared by the general method A, starting from commercially available amine CAS: 1554044-58-4 and sulphonyl chloride CAS: 677782-39-7, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 70% (y=57%).
Example 45
[0306] The title compound was prepared by the general method A, starting from commercially available amine CAS: 1018624-51-5 and sulphonyl chloride CAS: 677782-39-7, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 70% (y=50%).
Example 46
[0307] The title compound was prepared by the general method C, from intermediate VIIIa and 2-iodopropane (CAS: 75-30-9) at rt overnight and purified by flash silica gel chromatography using a linear gradient of ethyl acetate in hexane from 0% to 70% (y=22%).
Example 47
[0308] The title compound was prepared by the general method A, starting from commercially available amine CAS: 170017-74-0 and sulphonyl chloride CAS: 133-59-5, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 70% (y=85%).
Example 49
[0309] The title compound was prepared by the general method C, from intermediate VIIIf and iodomethane (CAS: 74-88-4) at rt for 2 h and purified by flash silica gel chromatography using a linear gradient of ethyl acetate in hexane from 0% to 50% (y=80%).
Example 50
[0310] The title compound was prepared by the general method A, starting from commercially available amine CAS: 170017-74-0 and sulphonyl chloride CAS: 609-60-9, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 70% (y=94%).
Example 51
[0311] The title compound was prepared by the general method A, starting from commercially available amine CAS: 170017-74-0 and sulphonyl chloride CAS: 2905-30-8, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 70% (y=86%).
Example 52
[0312] The title compound was prepared by the general method D, starting from intermediate IXa and commercially available acyl chloride CAS: 79-22-1. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 70% (y=33%).
Example 53
[0313] The title compound was prepared by the general method D, starting from intermediate IXa and commercially available acyl chloride CAS: 27746-99-2. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 40% (y=40%).
Example 54
[0314] The title compound was prepared by the general method A, starting from commercially available amine CAS: 170017-74-0 and sulphonyl chloride CAS: 2905-27-3, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 70% (y=82%).
Example 55
[0315] The title compound was prepared by the general method B, starting from intermediate VIa and commercially available amine CAS: 771-99-3. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 40% (y=33%).
Example 56
[0316] The title compound was prepared by the general method A, starting from commercially available amine CAS: 170017-74-0 and sulphonyl chloride CAS: 80466-80-4, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 60% (y=59%).
Example 57
[0317] The title compound was prepared by the general method B, starting from intermediate VIa and commercially available amine CAS: 3202-33-3. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 50% (y=49%).
Example 58
[0318] The title compound was prepared by the general method A, starting from commercially available amine CAS: 170017-74-0 and sulphonyl chloride CAS: 166964-26-7, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 50% (y=95%).
Example 59
[0319] The title compound was prepared by the general method A, starting from commercially available amine CAS: 170017-74-0 and sulphonyl chloride CAS: 97272-04-3, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 50% (y=96%).
Example 60
[0320] The title compound was prepared by the general method A, starting from commercially available amine CAS: 170017-74-0 and sulphonyl chloride CAS: 166337-57-1, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 40% (y=50%).
Example 61
[0321] The title compound was prepared by the general method B, starting from intermediate VIa and commercially available amine CAS: 110-91-8. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 50% (y=65%).
Example 62 and Example 63
[0322] The title compounds, were obtained by enantiomeric separation of the racemate of Example 57; for separation procedures, see analytical methods.
Example 64
[0323] The title compound was prepared by the general method A, starting from commercially available amine CAS: 170017-74-0 and sulphonyl chloride CAS: 80466-79-1, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 35% (y=93%).
Example 65
[0324] The title compound was prepared by the general method A, starting from intermediate IIc and sulphonyl chloride CAS: 609-60-9, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 40% (y=95%).
Example 66
[0325] The title compound was prepared by the general method A, starting from commercially available amine CAS: 1018624-51-5 and sulphonyl chloride CAS: 609-60-9, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 35% (y=99%).
Example 67
[0326] The title compound was prepared by the general method A, starting from commercially available amine CAS: 1018624-51-5 and sulphonyl chloride CAS: 166964-26-7, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 35% (y=98%).
Example 68
[0327] The title compound was prepared by the general method A, starting from commercially available amine CAS: 1018624-51-5 and sulphonyl chloride CAS: 97272-04-3, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 35% (y=99%).
Example 70
[0328] The title compound was prepared by the general method D, starting from intermediate IXa and commercially available acyl chloride CAS: 541-41-3. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 60% (y=68%).
Example 71
[0329] The title compound was prepared by the general method D, starting from intermediate IXa and commercially available acyl chloride CAS: 79-03-8. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 100% (y=75%).
Example 72
[0330] The title compound was prepared by the general method A, starting from commercially available amine CAS: 170017-74-0 and sulphonyl chloride CAS: 173669-61-9, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 50% (y=99%).
Example 73
[0331] The title compound was prepared by the general method D, starting from intermediate IXa and commercially available acyl chloride CAS: 98-88-4. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 70% (y=92%).
Example 74
[0332] The title compound was prepared by the general method A, starting from commercially available amine CAS: 72752-54-6 and sulphonyl chloride CAS: 677782-39-7, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 50% (y=83%).
Example 75
[0333] The title compound was prepared by the general method A, starting from intermediate IIc and sulphonyl chloride CAS: 677782-39-7, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 50% (y=98%).
Example 76
[0334] The title compound was prepared by the general method A, starting from intermediate IId and sulphonyl chloride CAS: 677782-39-7, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 40% (y=80%).
Example 77
[0335] The title compound was prepared by the general method A, starting from intermediate IIe and sulphonyl chloride CAS: 677782-39-7, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 50% (y=96%).
Example 78
[0336] The title compound was prepared by the general method A, starting from commercially available amine CAS: 199105-03-8 and sulphonyl chloride CAS: 677782-39-7, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 80% (y=80%).
Example 79
[0337] The title compound was prepared by the general method D, starting from intermediate IXa and commercially available acyl chloride CAS: 5271-67-0. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 80% (y=66%).
Example 80
[0338] The title compound was prepared by the general method D, starting from intermediate IXa and commercially available acyl chloride CAS: 54237-09-1. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 20% to 100% (y=59%).
Example 81
[0339] The title compound was prepared by the general method D, starting from intermediate IXa and commercially available acyl chloride CAS: 39098-97-0. (y=55%). It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 20% to 100% (y=55%)
Example 82
[0340] The title compound was prepared by the general method D, starting from intermediate IXa and commercially available acyl chloride CAS: 51493-02-8. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 20% to 100% (y=45%).
Example 83
[0341] The title compound was prepared by the general method A, starting from commercially available amine CAS: 170017-74-0 and sulphonyl chloride CAS: 98-59-9, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 50% (y=55%).
Example 84
[0342] The title compound was prepared by the general method A, starting from commercially available amine CAS: 170017-74-0 and sulphonyl chloride CAS: 98-68-0, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 50% (y=61%).
Example 85
[0343] The title compound was prepared by the general method A, starting from commercially available amine CAS: 51627-46-4 and sulphonyl chloride CAS: 677782-39-7, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 20% (y=40%).
Example 86
[0344] The title compound was prepared by the general method A, starting from intermediate IIf and sulphonyl chloride CAS: 677782-39-7, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 20% to 100% (y=44%).
Example 87
[0345] The title compound was prepared by the general method E, starting from intermediate XIIa and commercially available amine CAS: 107-10-8. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 20% to 100% (y=98%).
Example 88
[0346] The title compound was prepared by the general method E, starting from intermediate XIIa and commercially available amine CAS: 78-81-9. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 20% to 100% (y=98%).
Example 89
[0347] The title compound was prepared by the general method A, starting from intermediate IIg and sulphonyl chloride CAS: 677782-39-7, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 20% to 100% (y=97%).
Example 90
[0348] The title compound was prepared by the general method A, starting from intermediate IId and sulphonyl chloride CAS: 98-59-9, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 70% (y=98%).
Example 91
[0349] The title compound was prepared by the general method A, starting from intermediate IId and sulphonyl chloride CAS: 98-68-0, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 70% (y=98%).
Example 92
[0350] The title compound was prepared by the general method A, starting from commercially available amine CAS: 170017-74-0 and sulphonyl chloride CAS: 369638-66-4, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 50% (y=94%).
Example 93 and Example 94
[0351] The title compounds, were obtained by enantiomeric separation of the racemate of Example 92; for separation procedures, see analytical methods.
Example 95
[0352] The title compound was prepared by the general method A, starting from commercially available amine CAS: 170017-74-0 and sulphonyl chloride CAS: 120164-05-8, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 50% (y=90%).
Example 96
[0353] The title compound was prepared by the general method C, from intermediate VIIIc and 2-iodopropane (CAS: 75-30-9) at 100° C. overnight and purified by flash silica gel chromatography using a linear gradient of ethyl acetate in hexane from 0% to 70% (y=13%).
Example 97
[0354] The title compound was prepared by the general method A, starting from intermediate IIc and sulphonyl chloride CAS: 97272-04-3, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 40% (y=95%).
Example 98
[0355] The title compound was prepared by the general method A, starting from intermediate IIc and sulphonyl chloride CAS: 478264-00-5, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 50% (y=70%).
Example 99
[0356] The title compound was prepared by the general method A, starting from intermediate IIc and sulphonyl chloride CAS: 955085-16-2, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 50% (y=78%).
Example 100
[0357] The title compound was prepared by the general method A, starting from intermediate IIh and sulphonyl chloride CAS: 677782-39-7, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 10% to 50% (y=32%).
Example 101
[0358] The title compound was prepared by the general method A, starting from intermediate IIi and sulphonyl chloride CAS: 677782-39-7, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 10% to 50% (y=34%).
Example 103
[0359] The title compound was prepared by the general method C, from intermediate VIIId and 2-iodopropane (CAS: 75-30-9) at 100° C. overnight and purified by flash silica gel chromatography using a linear gradient of ethyl acetate in hexane from 0% to 50% (y=53%).
Example 105
[0360] The title compound was prepared by the general method A, starting from intermediate IIj and sulphonyl chloride CAS: 677782-39-7, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 70% (y=92%).
Example 106
[0361] The title compound was prepared by the general method A, starting from intermediate IIk and sulphonyl chloride CAS: 677782-39-7, using pyridine as the base. It was purified by flash silica gel column chromatography using a linear gradient of ethyl acetate in hexane from 0% to 70% (y=76%).
[0362] Table 2 lists final compounds that were prepared according to the experimental procedures described before.
TABLE-US-00011 TABLE 2 Example Structure 1
[0363] Analytical Procedures and Data
[0364] Purification System
[0365] Flash Chromatography (FC)
[0366] FC separations were performed on Biotage Isolera F our equipped with UV detector. Type of silica columns: Claricep Screw-on, Irregular, 40-60 μm, 12-80 g.
[0367] System Purification
[0368] Preparative HPLC
[0369] HPLC system WATERS Quaternary Gradient Mobile 2535 equipped with WATERS UVNisible Detector 2489 set to dual-wavelength UV detection. Two mobile phases were used, mobile phase A: water (MilliQ) 0,05% TFA; mobile phase B: acetonitrile (Chromasolv Sigma-Aldrich) 0,05% TFA, and the run gradient conditions were set specifically for each compound. The purifications were achieved on a LUNA 5 μm C18 150×21.2 Colum. An injection volume between 100 and 500 μl was applied, and the flow was 15 ml/min.
[0370] Racemate Separation
[0371] The two enantiomers Example 62 and 63 were obtained by resolution of the racemic mixture (Example 57) using a WATERS Quaternary Gradient Mobile 2535 equipped with WATERS UVNisible Detector 2489 set to a dual-wavelength UV detection at 245 and 275 nm. The chiral resolution was achieved on the Lux Amylose-1 column (250 mm×21.6 mm) using Hexane-Isopropanol 68:32 (v/v) as isocratic mobile phase; The sample was eluted from the column at a flow rate of 1.0 ml/min at room temperature (Pressure: ≈800 psi). The racemic mixture was dissolved in Ethanol and heated, at concentration of 1.0% (w/v) and the injection volume was 100 μL.
[0372] LCMS
[0373] LCMS—Procedure 1
[0374] The HPLC measurement was performed using a Dionex 3000 module comprising a quaternary pump with degasser, an autosampler, a column oven (set at 29° C.), a diode-array detector DAD and a column as specified in the respective methods below. Flow from the column was split to a MS spectrometer. The MS detector (LCQ Fleet Thermo Scientific) was configured with an electrospray ionization source. Mass spectra were acquired by scanning from 50 to 800 in 0.48 second. The capillary needle voltage was 5 kV in positive and negative ionization mode and the source temperature was maintained at 275° C. Nitrogen was used as the nebulizer gas, the flow was 8 l/min. Data acquisition was performed with Thermo Xcalibur Qual Browser. Reversed phase HPLC was carried out on a Kinetex XB-C18 column Phenomenex (1.7 μm, 50×2.1 mm) with a flow rate of 0.300 ml/min. Two mobile phases were used, mobile phase A: ammonium formate buffer solution at pH 3.5; mobile phase B: acetonitrile (Chromasolv Sigma-Aldrich), and they were employed to run a gradient conditions from 15% B for 0.5 minutes, from 15% to 98% in 4.0 minutes, 98% B for 1.35 minutes and 15% B in 0.10 minutes and hold these conditions for 2.75 minutes in order to re-equilibrate the column (Total Run Time 8.7 minutes). An injection volume of 1 μl was used.
[0375] LCMS—Procedure 2
[0376] The HPLC measurement was performed using a Vanquish module comprising a quaternary pump with degasser, an autosampler, a column oven (set at 29° C.), a diode-array detector DAD and a column as specified in the respective methods below. Flow from the column was split to a MS spectrometer. The MS detector (ISQEC) was configured with an electrospray ionization source. Mass spectra were acquired by scanning from 50 to 600 in 0.2 second. The capillary needle voltage was 3 kV in positive and 2 kV in negative ionization mode and the source temperature was maintained at 250° C. Nitrogen was used as the nebulizer gas. Data acquisition was performed with Chromeleon 7. Reversed phase HPLC was carried out on a Luna Omega Polar C18 column (50×2.1 mm 1.6 μm) with a flow rate of 0.5 mL/min. Two mobile phases were used, mobile phase A: ammonium formate buffer solution at pH 3.5; mobile phase B: acetonitrile (Chromasolv Sigma-Aldrich), and they were employed to run a gradient conditions from 15% B for 0.2 minutes, from 15% to 95% in 1.6 minutes, 98% B for 0.65 minutes and 15% B in 0.15 minutes and hold these conditions for 1.9 minutes in order to re-equilibrate the column (Total Run Time 4.5 minutes). An injection volume of 0.8 μl was used.
[0377] Table 3 shows retention time (R.sub.t) in minutes, [M+H].sup.+ and/or [M−H].sup.− peak and LCMS procedure.
TABLE-US-00012 TABLE 3 Example R.sub.t (min) [M + H].sup.+ [M − H].sup.− LC-MS 1 6.47 373.3 371.4 1 2 6.11 360.2 358.5 1 3 5.13 384.2 382.2 1 4 5.48 436.0 434.4 1 5 2.30 503.4 — 2 6 5.80 424.1 422.3 1 7 5.06 388.4 — 1 8 5.74 458.1 456.6 1 9 5.86 472.3 470.6 1 10 2.31 495.2 — 2 11 5.87 395.3 393.4 1 12 6.31 500.2 498.7 1 13 5.77 525.0 523.6 1 14 2.28 523.2 521.2 2 15 5.48 402.4 — 1 16 6.53 391.3 389.6 1 17 6.08 363.2 361.5 1 18 2.66 446.2 — 2 19 2.66 446.2 — 2 20 2.40 517.3 — 2 21 6.46 377.3 — 1 22 3.94 430.3 428.3 1 23 6.25 331.2 329.4 1 24 6.12 516.1 514.6 1 25 6.19 530.2 528.8 1 26 5.41 454.3 542.6 1 27 5.09 426.3 424.5 1 28 2.74 460.2 — 2 29 2.92 459.3 — 2 30 2.35 438.2 436.1 2 31 2.84 475.2 — 2 32 5.28 410.3 408.5 1 33 5.58 460.2 458.6 1 35 6.28 500.3 498.7 1 36 5.58 454.3 542.6 1 37 5.88 486.4 484.7 1 38 2.01 440.1 438.1 2 39 2.64 375.2 — 2 40 2.32 468.2 466.2 2 41 2.32 468.2 466.2 2 42 2.32 468.2 466.2 2 43 2.18 440.2 438.2 2 44 2.31 442.2 440.1 2 45 2.50 482.2 480.0 2 46 2.48 531.3 — 2 47 2.57 432.2 430.2 2 49 5.44 414.4 — 1 50 2.65 446.2 444.2 2 51 2.63 446.3 — 2 52 5.18 483.3 481.6 1 53 5.60 — 549.7 1 54 2.66 446.2 — 2 55 4.62 506.3 504.6 1 56 2.42 453.2 451.1 2 57 4.67 522.2 520.7 1 58 2.56 436.2 — 2 59 2.64 452.2 — 2 60 2.35 433.2 431.2 2 61 2.71 432.6 430.4 1 62 4.67 522.4 520.8 1 63 4.67 522.4 520.7 1 64 2.45 437.2 435.1 1 65 2.70 438.2 — 2 66 2.76 403.3 — 2 67 2.65 393.2 — 2 68 2.75 409.2 — 2 70 2.90 497.2 495.2 2 71 2.74 481.2 479.2 2 72 2.59 473.2 — 2 73 2.86 529.3 527.2 2 74 3.03 438.2 436.0 2 75 3.10 517.2 515.2 2 76 3.21 524.3 522.3 2 77 3.00 518.2 516.3 2 78 2.98 — 522.2 2 79 2.85 533.2 535.2 2 80 2.70 550.2 548.3 2 81 2.86 549.2 547.2 2 82 2.90 524.3 522.3 2 83 2.56 432.2 430.3 2 84 2.49 448.2 446.2 2 85 2.47 438.2 436.2 2 86 1.98 517.2 515.2 2 87 2.26 509.2 507.2 2 88 2.22 521.3 523.3 2 89 2.24 518.3 516.3 2 90 2.74 431.2 — 2 91 2.62 447.2 445.5 2 92 2.55 474.2 — 2 93 2.53 474.2 — 2 94 2.53 474.2 — 2 95 2.58 483.2 481.2 2 96 2.33 502.3 — 2 97 2.69 444.1 — 2 98 2.35 425.2 423.2 2 99 2.48 445.2 — 2 100 2.43 502.1 500.1 2 101 2.46 517.2 — 2 103 2.25 495.3 — 2 105 2.96 482.2 480.1 2 106 1.82 517.2 — 2
[0378] NMR Characterization
[0379] .sup.1H NMR spectra were recorded on a Varian Mercury NMR 400 MHz spectrometer using CDCl.sub.3. DMSO-d6 or CD.sub.3OD as solvents Chemical shifts (δ) are reported in parts per million (ppm) relative to residual signal of non-fully deuterated solvents pick for .sup.1H NMR assigned as 7.26 ppm for CHCl.sub.3, 3.31 ppm for CHD.sub.2OD and 2.50 ppm for DMSO-d.sub.5.
TABLE-US-00013 TABLE 4 NMR data of compounds Example Structure NMR 1
[0380] Pharmacology
[0381] Cellular TRPML1 Assay
[0382] Buffers and reagents [0383] PBS (D-PBS without calcium and magnesium; EuroClone) [0384] Trypsin (Trypsin 0.05%, EDTA 0.02% in PBS; EuroClone) [0385] DMSO (Sigma) [0386] Ca.sup.2+ free Tyrode's buffer: in-house solution (130 mM NaCl, 5 mM KCl, 1 mM MgCl2, 5 mM NaHCO.sub.3, 20 mM HEPES in water at pH 7.4; sterile filtered). [0387] Opti-MEM (Gibco) [0388] Agonist: ML-SA1 (Sigma); stock: 60 mM in DMSO, stored at −20° C. [0389] Blocker: ML-SI3 (in house synthesis); stock: 20 mM in DMSO, stored at −20° C.
[0390] Cell Line
[0391] The final clone for the TRPML1 assay is HEK T-REx/GCaMP6f/TRPML1.
[0392] GCaMP6f is a genetically encoded calcium indicator that is stably expressed in this cell line and used as a fluorescent read-out.
[0393] Assay Protocol
[0394] Experiments are performed in 384 MTP format. Cells are seeded at 15000 cells/well either in 25 μl/well of growth medium or in 20 μl/well of Optimem+0.5% FBS without selection antibiotics. Twenty-four hours later, cells are assayed for the response to various compounds using the Ca.sup.2+ sensitive GCaMP6f protein stably expressed in the cells as readout.
[0395] The experiment is performed in a 384-well format according to the following procedures for either Ca.sup.2+ free or Optimem conditions: [0396] Ca.sup.2+ free condition (in absence of extracellular Ca.sup.2+): [0397] 24 h after seeding, pre-incubate the cells at room temperature for about 10′. [0398] Then remove the culture medium and replace it with 20 μL of Ca.sup.2+ free Tyrode's buffer. [0399] Start the experiment at the FLIPR.sup.TETRA by injecting 10 μL/w of 3× concentrated test compounds and controls in Ca.sup.2+ free Tyrode's buffer. Monitor the kinetic response over a period of 300 seconds. [0400] Final DMSO concentration: 0.5% [0401] Optimem condition (in presence of extracellular Ca.sup.2+): [0402] 24 h after seeding, pre-incubate the cells at room temperature for about 10′. [0403] Start the experiment at the FLIPR.sup.TETRA by injecting 10 μL/w of 3× concentrated test compounds and controls in Ca.sup.2+ free Tyrode's buffer. Monitor the kinetic response over a period of 300 seconds. [0404] Final DMSO concentration: 0.5%
[0405] Data from FLIPR.sup.TETRA measurements are analyzed with the Genedata Screener® software.
[0406]
[0407] Stimulator controls (Col 2): injection of ML-SA1 EC.sub.100 (50 μM) in alternated wells positions
[0408] Neutral controls (Col 2): injection of 0 mM Ca.sup.2+ free Tyrode's buffer in alternated wells positions.
[0409] Test wells (Col 3-22): test compounds dose response curve from either 100 μM with 1:2 dilution steps or from 50 μM with 1:3.16 dilution steps.
[0410] Data Analysis
TABLE-US-00014 Compound Calculate the Kinetic Response Value (KRV) as: % Activity MAX.sub.CA/Baseline = [max(sec5 . . . 290)/Mean (see 1 . . . sec2)] Normalize the KRV to the median (< >) of Neutral and Stimulator control wells:
TABLE-US-00015 TABLE 5 Activity of TRPML1 agonists Example Ec.sub.50 (μM) 1 91.1 2 58.9 3 50.2 4 8.4 5 6.0 6 1.7 7 85.4 8 64.0 9 47.4 10 8.4 11 33.5 12 2.8 13 2.1 14 19.7 15 59.1 16 14.6 17 18.2 18 3.5 19 2.1 20 3.4 21 55.6 22 98.0 23 15.5 24 2.1 25 1.8 26 4.5 27 13.3 28 2.9 29 2.3 30 50.0 31 6.0 32 19.2 33 10.8 35 7.7 36 7.3 37 6.3 38 34.5 39 54.5 40 3.0 41 4.4 42 5.6 43 7.0 44 2.6 45 1.9 46 9.4 47 5.2 49 29.3 50 2.2 51 3.0 52 7.2 53 3.6 54 20.0 55 6.1 56 3.6 57 3.0 58 2.3 59 1.6 60 7.3 61 39.5 62 15.9 63 1.7 64 36.4 65 3.5 66 22.1 67 37.8 68 4.6 70 3.2 71 17.3 72 3.0 73 6.4 74 3.2 75 1.6 76 1.3 77 1.7 78 6.9 79 8.8 80 18.9 81 11.1 82 5.9 83 1.8 84 4.2 85 1.6 86 5.7 87 18.5 88 6.5 89 3.2 90 2.5 91 3.3 92 2.4 93 4.9 94 3.8 95 10.7 96 7.3 97 2.8 98 3.3 99 5.0 100 6.8 101 4.0 103 14.8 105 18.0 106 8.6
[0411] TRPML1—Automated Patch-Clamp
[0412] Cell Culture Conditions
[0413] HEK T-REx/GcaMP6f/TRPML1 cells were cultured in DMEM High Glucose (Lonza BioWhittaker cat. BE12-604F/U1; 500 mL) supplemented with Fetal Bovine Serum TET-FREE (Euroclone cat. EC S0182L; 50 mL, Penicillin-Streptomycin (BioWhittaker, cat. DE17-602E; 5 mL of 100× Solution), G418 (Sigma, cat. G8168; 0.25 mg/ml) and Zeocyn (InvivoGen, cat. ant-zn-1; 50 μg/ml).
[0414] Experimental Protocol
[0415] HEK T-REx/GcaMP6f/TRPML1 cells were seeded 48 or 72 hours before experiment, at a concentration of 8*10.sup.6/4*10.sup.6 cells onto a T225 flask. Just before the experiments, cells were washed twice with D-PBS w/o Ca.sup.2+/Mg.sup.2+ (Euroclone cat. ECB4004L) and detached from the flask with trypsin-EDTA (Sigma cat. T4174; diluted 1/10). Cells were then re-suspended in the suspension solution: 25 mL EX-CELL ACF CHO medium(Sigma cat. C5467); 0.625 mL HEPES (Sigma cat. H0887); 0.25 mL of 100× Penicillin/Streptomycin (Euroclone cat. ECB3001D), 0.1 mL of Soybean Trypsin Inhibitor 10 mg/mL (Sigma cat. T6522) and placed on the QPatch 16X.
[0416] Ligands and Buffer
[0417] Compounds:
[0418] 2 (two) agonists solubilized 100% DMSO (20 mM) were tested in three concentrations (0.5, 5, 50 μM). Dilution from stock were prepared just before the experiments in the extracellular solution (0.25% final DMSO concentration). DMSO solution was obtained from AppliChem (cat. A3672)
[0419] Patch Clamp Solutions: [0420] Intracellular solution (mM): 120 CsMeSO3, 4 NaCl, 2 MgCl2, 10 EGTA, 20 HEPES, 2 Mg-ATP (pH 7.2 with CsOH). [0421] Extracellular solution (mM): 140 NaGluconate, 5 KCl, 1 MgCl2, 2 CaCl.sub.2), 10 Glucose, 10 MES (pH 4.6 with HCl).
[0422] Patch Clamp Analysis—QPatch 16X
[0423] For the voltage clamp experiments on TRPML1, data were sampled at 5 KHz. After establishment of the seal and the passage in the whole cell configuration, the cells were held at −20 mV and the TRPML1 current was evoked using the voltage protocol illustrated in
[0424] Data Analysis
[0425] Agonists: For data collection, the Sophion proprietary software was used and the analysis was performed off-line using Excel and GraphPad Prism (V 8.00). Results are reported in Table 6, expressed as TRPML1 current fold increase evoked by the compound under investigation (i.e. current in the presence of the compound/current in vehicle).
TABLE-US-00016 TABLE 6 QPatch activity of TRPML1 agonists Qpatch Assay TRPML1 agonism Current fold increase Example Mean ± SEM (n) 6 0.3 μM: 1.0 ± 0.77 (3) 3.0 μM: 32.7 ± 8.9 (3) 13 0.3 μM: 13.5 ± 4.8 (4) 3.0 μM: 208 ± 15.7 (4) 30 μM: 244 ± 27.6 (4) 24 0.3 μM: 4.6 ± 1.8 (4) 3 μM: 168.8 ± 96.1 (4) 30 μM: 687.7 (1) 25 0.3 μM: 3.5 ± 1.8 (3) 3 μM: 177.1 ± 38.2 (3) 30 μM: 1013.8 (1) 26 0.3 μM: 0.46 ± 0.16 (3) 3.0 μM: ) 41.4 ± 21.1 (3 30 μM: 390 ± 158 (3) 52 0.3 μM: 1.3 ± 1.0 (3) 3 μM: 10.4 ± 3.8 (3) 30 μM: 472.5 ± 267.9 (3) 53 0.3 μM: 1.9 ± 1.6 (3) 3 μM: 88.3 ± 59.9 (3) 30 μM: 257.4 ± 190.7 (3) 57 0.3 μM: 0.39 ± 0.14 (2) 3.0 μM: 0.86 ± 0.68 (2) 30 μM: 4.3 ± 2.2 (2) 63 0.3 μM: 0.042 ± 0.153 (2) 3.0 μM: 0.86 ± 0.60 (2) 30 μM: 4.3 ± 0.9 (2) 70 0.3 μM: 0.5 ± 0.3 (3) 3 μM: 22.5 ± 10.6 (3) 30 μM: 656.7 ± 150.5 (3) 73 0.3 μM: −0.070 3.0 μM: −0.31 30 μM: 11.3 74 0.3 uM: 4.3 ± 2.0 (6) 3.0 uM: 356 ± 69.7 (6) 30 uM: 639 + 241 (3) 75 0.3 uM: 310 ± 147 (4) 3.0 uM: 435 ± 172 (4) 30 uM: 603 ± 155 (3) 76 0.3 uM: 35.6 ± 26.7 (3) 3.0 uM: 38.5 ± 24.8 (2) 30 uM: 52.7 ± 36.3 (2) 77 0.3 uM: 74.4 ± 13.8 (3) 3.0 uM: 167 ± 10.7 (3) 30 uM: 168 ± 9.8 (3) 78 0.3 uM: 0.68 ± 0.34 (3) 3.0 uM: 16.5 ± 9.1 (3) 30 uM: 166 ± 75.7 (3) 82 0.3 uM: 2.5 ± 1.3 (3) 3.0 uM: 12.5 ± 8.6 (3) 30 uM: 138 ± 55.7 (3) 83 0.3 uM: 70.2 ± 13.8 (4) 3 uM: 125.0 ± 31.0 (4) 30 uM: 140.3 ± 36.8 (4) 84 0.3 uM: 8.6 ± 1.3 (2) 3.0 uM: 32.9 ± 3.5 (2) 30 uM: 42.7 ± 1.4 (2) 85 0.3 uM: 17.7 ± 5.5 (4) 3.0 uM: 206 ± 167 (2) 30 uM: 260 ± 202 (2) 88 0.3 uM: −0.066 ± 0.080 (3) 3.0 uM: 3.9 ± 2.5 (3) 30 uM: 125 ± 75.3 (3) 90 0.3 uM: 140 ± 44.1 (4) 3.0 uM: 83.1 ± 34.0 (2) 30 uM: 112 ± 38.1 (2) 91 0.3 uM: 3.1 ± 1.2 (3) 3.0 uM: 21.7 ± 7.9 (3) 30 uM: 58.8 ± 16.6 (3)
[0426] TRPML1 —Manual Patch Clamp
[0427] Endolysosomal electrophysiology was performed in isolated enlarged endolysosomes using a modified patch-clamp method (Dong et al., 2010). HEK T-REx/GcaMP6f/TRPML1 cells were treated with 0.8 mM PIKfive inhibitor overnight. Whole-endolysosome recordings were performed on manually isolated enlarged endolysosomes (Dong et al., 2010). In brief, a patch pipette was pressed against a cell and quickly pulled away to slice the cell membrane. Enlarged endolysosomes were released into a dish and a gigaseal between the patch pipette and the enlarged endolysosome was obtained. Negative pressure or voltage steps of several hundred millivolts with millisecond duration were then applied to break into the vacuolar membrane.
[0428] Bath (internal/cytoplasmic) solution contained 140 mM K-gluconate, 4 mM NaCl, 1 mM EGTA, 2 mM MgCl2, 0.39 mM CaCl.sub.2), 20 mM HEPES (pH was adjusted with KOH to 7.2). The pipette (luminal) solution was standard extracellular solution (modified Tyrode's: 145 mM NaCl, 5 mM KCl, 2 mM CaCl.sub.2), 1 mM MgCl2, 10 mM HEPES, 10 mM MES, 10 mM glucose; the pH was adjusted with NaOH to pH 4.6).
[0429] Agonists solubilized 100% DMSO (20 mM) were tested in single concentration (2 μM). Dilution from stock were prepared just before the experiments in the extracellular solution (0.1% final DMSO concentration). DMSO solution was obtained from AppliChem (cat. A3672).
[0430] All bath solutions were applied via a perfusion system that allowed us to achieve complete solution exchange within a few seconds.
[0431] Data were collected using an HEKA EPC 10 patch-clamp amplifier, and PatchMaster v2x73.5 software (HEKA Elektronik). Whole-endolysosome currents were digitized at 20 kHz. All experiments were conducted at room temperature, and all recordings were analyzed with pClamp 10.7 (Molecular Devices) and GraphPad Prism (V8.00).
[0432] TRPML1 current was evoked using the voltage protocol illustrated in
TABLE-US-00017 TABLE 7 Secondary Assays Agonism-Manual Patch Lysosomal Patch- Clamp Current Concentration fold increase Example (uM) Mean ± SEM (n) 6 2.0 3.3 ± 0.67 (3) 13 30.0 27.5 ± 10.8 (2) 57 2.0 18.0 ± 8.5 (3)
[0433] Autophagy Assay
[0434] Buffers and Reagents [0435] Tyrode's buffer with 2 mM Ca.sup.2+ in-house solution (130 mM NaCl, 5 mM KCl, 2 mM CaCl2), 1 mM MgCl2, 5 mM NaHCO.sub.3, 20 mM HEPES in water at pH 7.4; sterile filtered) [0436] DMSO (Sigma) [0437] Bis-benzimide H 33342 trihydrochloride (Hoechst 33342; Sigma-Aldrich) [0438] Reference molecule (agonist): Torin-1, (#4247, Tocris) stock 1.2 mM in DMSO, stored at −20° C.
[0439] Reporter of Autophagy and Cell Line
[0440] Reporter Rosella: a fluorescent-based chimera constitutes by two fluorescent proteins, Green and Red variants, acting as a bimodal indicator of pH. The green fluorescent variant of the constructs is pH sensitive (pKa=6.9) while the red variant is not pH sensitive and it is used as reference. The reporter is fused to sequence for autophagosome localization (LC3 tag).
[0441] The reporter Rosella have been stably expressed in HeK293 cell line and used as read-out.
[0442] Assay Protocol
[0443] Experiments have been performed in 384 MTP poly-lysine coated well format. Cells have been seeded in 384-w at a density of 6000 cells/well in 25 μl/well complete growth medium without antibiotics. Twenty-four hours later, cells have been treated with compounds and incubated for further 18 hours. Then the cells have been imaged and assayed for the response to various compounds using the reporter for autophagy expressed in the cells as readout.
[0444] The experiments were performed in a 384-well format according to the following procedure: [0445] 24 h after seeding, cells were incubated with compounds at the desired concentration, and with the reference molecule (agonist) Torin-1 at a top concentration of 1 μM (max signal), for 18 hours at 37° C. and 5% CO2. The final percentage of DMSO was 0.3% in all the conditions. [0446] Then the culture medium was carefully removed to avoid cells detachment and to discard red phenol into the medium and that can interfere with the measurements. [0447] Staining of the nuclei was obtained by incubating the cells with 8 μM/well of Hoechst 3342 in standard Tyrode's buffer for 20 min at RT [0448] Then the cells were carefully washed two times with standard Tyrode's buffer [0449] Finally, the samples were acquired by recording three fluorescence emission channels (green, red and blue) at 20× magnification and with at least 3-4 fields of view per well in an Operetta CLS microscope (PerkinElmer). [0450] Image analysis was performed by using Harmony software (PerkinElmer). The image analysis involved the following steps: flat-field illumination correction, nuclei segmentation, cell segmentations and identification of vesicles/granules within the cytosol compartments and representing the autophagy vesicles. Measurements of signal intensity ratio (Green to Red) and number of autophagy vesicles per cells were used to obtain information on the effects of compounds on the autophagy flux. [0451] Data from image analysis measurements were finally loaded and analyzed for normalization and fitting procedures in Genedata Screener© software.
TABLE-US-00018 TABLE 8 Autophagy assay results for TRPML1 agonists Secondary Assays Autophagy Example qAC50 − (μM) 6 4.4 13 0.52 19 0.60 20 0.46 33 1.3 40 1.3 46 1.3 57 1.8 58 22.4 65 31.4 68 15.3 75 0.17 76 0.059 77 0.25 96 11.2 99 6.0