MODULATORS OF THE INTEGRATED STRESS RESPONSE PATHWAY
20230391725 · 2023-12-07
Assignee
Inventors
- Holly Victoria ATTON (Oxfordshire, GB)
- Christopher John Brown (Oxfordshire, GB)
- James Lindsay Carr (Oxfordshire, GB)
- Gert-Jan Hofman (Oxfordshire, GB)
- Mohamad Sabbah (Oxfordshire, GB)
- Daryl Simon WALTER (Oxfordshire, GB)
Cpc classification
C07D405/12
CHEMISTRY; METALLURGY
C07D413/12
CHEMISTRY; METALLURGY
C07D401/06
CHEMISTRY; METALLURGY
International classification
C07D401/06
CHEMISTRY; METALLURGY
C07D401/12
CHEMISTRY; METALLURGY
C07D405/12
CHEMISTRY; METALLURGY
Abstract
The present invention relates to compounds of formula (I) or pharmaceutically acceptable salts, solvates, hydrates, tautomers or stereoisomers thereof, wherein R.sup.1, R.sup.2, R.sup.2a, R.sup.3, R.sup.4, R.sup.4a, R.sup.4b, R.sup.4c, R.sup.4d, R.sup.4e, R.sup.4f, R.sup.5, R.sup.6 have the meaning as indicated in the description and claims The invention further relates to pharmaceutical compositions comprising said compounds, their use as medicament and in a method for treating or preventing of one or more diseases or disorders associated with integrated stress response.
##STR00001##
Claims
1. A compound of formula (I) ##STR00095## or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or stereoisomer thereof, wherein: R.sup.1 is H or C.sub.1-4 alkyl, preferably H, wherein C.sub.1-4 alkyl is optionally substituted with one or more halogen, which are the same or different; R.sup.2 is H, F or C.sub.1-4 alkyl, wherein C.sub.1-4 alkyl is optionally substituted with one or more halogen, which are the same or different; R.sup.2a is H or F, preferably H; R.sup.3 is phenyl or 6 membered aromatic heterocyclyl, wherein R.sup.3 is optionally substituted with one or more R.sup.7, which are the same or different; R.sup.7 is halogen, CN, C(O)OR.sup.8, OR.sup.8, C(O)R.sup.8, C(O)N(R.sup.8R.sup.8a), S(O).sub.2N(R.sup.8R.sup.8a), S(O)N(R.sup.8R.sup.8a), S(O).sub.2R.sup.8, S(O)R.sup.8, N(R.sup.8)S(O).sub.2N(R.sup.8aR.sup.8b), SR.sup.8, N(R.sup.8R.sup.8a), NO.sub.2, OC(o)R.sup.8, N(R.sup.8)C(O)R.sup.8a, N(R.sup.8)S(O).sub.2R.sup.8a, N(R.sup.8)S(O)R.sup.8a, N(R.sup.8)C(O)OR.sup.8a, N(R.sup.8)C(o)N(R.sup.8aR.sup.8b), OC(O)N(R.sup.8R.sup.8a), C.sub.1-6 alkyl, C.sub.2-6 alkenyl or C.sub.2-6 alkynyl, wherein C.sub.1-6 alkyl, C.sub.2-6 alkenyl and C.sub.2-6 alkynyl are optionally substituted with one or more R.sup.9, which are the same or different; R.sup.8, R.sup.8a, R.sup.8b are independently selected from the group consisting of H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl and C.sub.2-6 alkynyl, wherein C.sub.1-6 alkyl, C.sub.2-6 alkenyl and C.sub.2-6 alkynyl are optionally substituted with one or more halogen, which are the same or different; R.sup.9 is halogen, CN, C(O)OR.sup.10, OR.sup.10, C(O)R.sup.10, C(O)N(R.sup.10R.sup.10a), S(O).sub.2N(R.sup.10R.sup.10a), S(O)N(R.sup.10R.sup.10a), S(O).sub.2R.sup.10, S(O)R.sup.10, N(R.sup.10S(O).sub.2N(R.sup.10aR.sup.10b), SR.sup.10, N(R.sup.10R.sup.10a), NO.sub.2, OC(O)R.sup.10, N(R.sup.10)C(O )R.sup.10a, N(R.sup.10)SO.sub.2R.sup.10a, N(R).sup.10)S(O)R.sup.10a, N(R.sup.10)C(O)N(R.sup.10aR.sup.10b), N(R.sup.10)C(O)OR.sup.10a or OC(O)N(R.sup.10R.sup.1-a); R.sup.10, R.sup.10a, R.sup.10b are independently selected from the group consisting of H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl and C.sub.2-6 alkynyl, wherein C.sub.1-6 alkyl, C.sub.2-6 alkenyl and C.sub.2-6 alkynyl are optionally substituted with one or more halogen, which are the same or different. R.sup.4 is H, C(O)OC.sub.1-4 alkyl or C.sub.1-4 alkyl, wherein C(O)OC.sub.1-4 alkyl and C.sub.1-4 alkyl are optionally substituted with one or more substituents selected from the group consisting of halogen, OH and O—C.sub.1-3 alkyl, wherein the substituents are the same or different; R.sup.4a, R.sup.4b, R.sup.4c, R.sup.f are independently selected from the group consisting of H, halogen and C.sub.1-4 alkyl; and R.sup.4d, R.sup.43 are independently selected from the group consisting of H, OH, OC.sub.1-4 alkyl, halogen and C.sub.1-4 alkyl; or R.sup.4 and one of R.sup.4d and R.sup.4e form a methylene or ethylene group; or R.sup.4 and R.sup.4c form an ethylene group; or R.sup.4b and R.sup.4d form a covalent single bond; R.sup.5 is H or C.sub.1-6 alkyl, wherein C.sub.1-6 alkyl is optionally substituted with one or more halogen, which are the same or different; and R.sup.6 is R.sup.11; or R.sup.5 and R.sup.6 are joined to form together with the nitrogen atom to which they are attached a ring A.sup.1; R.sup.11 is OR.sup.12, SR.sup.12a, N(R.sup.12R.sup.12a), A.sup.2, C.sub.1-6 alkyl, C.sub.2-6 alkenyl or C.sub.2-6 alkynyl, wherein C.sub.1-6 alkyl, C.sub.2-6 alkenyl and C.sub.2-6 alkynyl are optionally substituted with one or more R.sup.13, which are the same or different; R.sup.12, R.sup.12a are independently selected from the group consisting of H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl and A.sup.2, wherein C.sub.1-6 alkyl, C.sub.2-6 alkenyl and C.sub.2-6 alkynyl are optionally substituted with one or more R.sup.15, which are the same or different; R.sup.13 is halogen, OR.sup.14, CN or A.sup.2; R.sup.14 is H or C.sub.1-4 alkyl, wherein C.sub.1-4 alkyl is optionally substituted with one or more halogen, which are the same or different; R.sup.15 is halogen, CN, OR.sup.14, OA.sup.2 or A.sup.2; A.sup.1 is 3 to 7 membered heterocyclyl or 7 to 12 membered heterobicyclyl, wherein A.sup.1 is optionally substituted with one or more R.sup.16, which are the same or different; A.sup.2 is phenyl, naphthyl, C.sub.3-7 cycloalkyl, C.sub.4-12 bicycloalkyl, 3 to 7 membered heterocyclyl or 7 to 12 membered heterobicyclyl, wherein A.sup.2 is optionally substituted with one or more R.sup.16a, which are the same or different; R.sup.16, R.sup.16a are independently selected from the group consisting of R.sup.17, OH, OR.sup.17, halogen and CN; R.sup.17 is cyclopropyl, C.sub.1-6 alkyl, C.sub.2-6 alkenyl or C.sub.2-6 alkynyl, wherein R.sup.17 is optionally substituted with one or more R.sup.18, which are the same or different; R.sup.18 is halogen, CN or OR.sup.19; R.sup.19 is H or C.sub.1-4 alkyl, wherein C.sub.1-4 alkyl is optionally substituted with one or more halogen, which are the same or different; provided that the following compounds are excluded: ##STR00096##
2. The compound of claim 1 or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or stereoisomer thereof, wherein R.sup.4 is H, CH.sub.3, CH.sub.2CH.sub.3, or CH.sub.2CH.sub.2OCH.sub.3; preferably, H or CH.sub.3; more preferably H.
3. The compound of claim 1 or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or stereoisomer thereof, wherein R.sup.4a, R.sup.4b, R.sup.4c, R.sup.4f are independently selected from the group consisting of H, halogen and C.sub.1-4 alkyl and R.sup.4d, R.sup.4e are independently selected from the group consisting of H, OH, OC.sub.1-4 alkyl, halogen and C.sub.1-4 alkyl; preferably R.sup.4a, R.sup.4b, R.sup.4c, R.sup.4f, R.sup.4d, R.sup.4e are independently selected from the group consisting of H, F and CH.sub.3; more preferably R.sup.4a, R.sup.4b, R.sup.4c, R.sup.4f, R.sup.4d, R.sup.4e are H.
4. The compound of claim 1 any or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or stereoisomer thereof, wherein le is H or CH.sub.3; preferably H.
5. The compound of claim 1 or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or stereoisomer thereof, wherein R.sup.2 is H, F or CH.sub.3, preferably H.
6. The compound of claim 1 or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or stereoisomer thereof, wherein R.sup.1, R.sup.2, R.sup.2a, R.sup.4, R.sup.4a, R.sup.4b, R.sup.4c, R.sup.4f, R.sup.4d, R.sup.4e in formula (I) are H to give formula (Ia): ##STR00097##
7. The compound of claim 1 or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or stereoisomer thereof, wherein R.sup.3 is phenyl or pyridyl, preferably phenyl, wherein R.sup.3 is optionally substituted with one or more R.sup.7, which are the same or different.
8. The compound of claim 1 or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or stereoisomer thereof, wherein R.sup.3 is substituted with one, two or three, preferably one or two, more preferably two, R.sup.7, which are the same or different.
9. The compound of claim 1 or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or stereoisomer thereof, wherein R.sup.7 is F, Cl, Br, CN, CHF.sub.2, CF.sub.3, OCH.sub.3, OCF.sub.3, CH═O, CH.sub.2OH or CH.sub.3; preferably R.sup.7 is CF.sub.3, F or Cl, more preferably F or Cl.
10. The compound of claim 1 or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or stereoisomer thereof, wherein R.sup.1, R.sup.2, R.sup.2a, R.sup.4, R.sup.4a, R.sup.4b, R.sup.4c, R.sup.4f, R.sup.4d, R.sup.4e, R.sup.3 in formula (I) are selected to give formula (Ib): ##STR00098## wherein each R.sup.7 is independently selected from the group consisting of halogen and CF.sub.3.
11. The compound of claim 1 or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or stereoisomer thereof, wherein R.sup.5 is H or CH.sub.3, preferably H.
12. The compound of claim 1 or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or stereoisomer thereof, wherein R.sup.6 is R.sup.11 and R.sup.11 is C.sub.1-6 alkyl or C.sub.1-6 alkenyl, wherein C.sub.1-6 alkyl and C.sub.1-6 alkenyl are substituted with one or more R n , which are the same or different.
13. The compound of claim 1 or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or stereoisomer thereof, wherein R.sup.6 is R.sup.11 and R.sup.11 is C.sub.1-6 alkyl, preferably ethyl or n-propyl, wherein C.sub.1-6 alkyl is substituted with one R.sup.13.
14. The compound of claim 1 or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or stereoisomer thereof, wherein R.sup.6 is R.sup.11 and R.sup.11 is ethyl or n-propyl, each substitiuted with one R.sup.13, wherein R.sup.13 is OR.sup.14, preferably OCF.sub.3.
15. The compound of claim 1 or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or stereoisomer thereof, wherein R.sup.6 is R.sup.11 and R.sup.11 is C.sub.1-6 alkyl, preferably n-propyl or n-pentyl, wherein C.sub.1-6 alkyl is substituted with three F; more preferably R.sup.11 is 3,3,3-trifluoropropyl or 5,5,5-trifluoropentyl.
16. The compound of claim 1 or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or stereoisomer thereof, wherein R.sup.6 is R.sup.11 and R.sup.11 is C.sub.1-6 alkyl, preferably methyl, wherein C.sub.1-6 alkyl is substituted with one R.sup.13, wherein R.sup.13 is A.sup.2, preferably phenyl, pyridyl, pyrazolyl, oxazolyl, cyclobutyl, cyclohexyl, furanyl, bicyclo[3.1.0]hexan-3-yl or 6-oxaspiro[3.4]octan-7-yl.
17. The compound of claim 1 or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or stereoisomer thereof, wherein R.sup.6 is R.sup.11 and R.sup.11 is A.sup.2, preferably phenyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, benzodioxolyl, cyclohexyl, cyclopentyl, cyclobutyl, pyrazolyl, oxazolyl or oxolanyl.
18. The compound of claim 1 or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or stereoisomer thereof, wherein A.sup.2 is unsubstituted or substituted with one or two R.sup.16a.
19. The compound of claim 1 or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or stereoisomer thereof, wherein R.sup.16a is CH.sub.3, CHF.sub.2, CF.sub.3, CH.sub.2CF.sub.3, OCHF.sub.2, OCH.sub.2CF.sub.3, OCF.sub.3, OCH.sub.3, F or Cl.
20. The compound of claim 1 or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or stereoisomer thereof, wherein R.sup.5 and R.sup.6 are joined to form together with the nitrogen atom to which they are attached a ring A.sup.1.
21. The compound of claim 20, wherein A.sup.1 is azetidine, piperidine, oxazepane, indoline, isoindoline, tetrahydroisoquioline azabicyclo[3.1.0]hexane or azaspiro[3.3]heptane and wherein A.sup.1 is optionally substituted with one or more R.sup.16, which are the same or different.
22. The compound of claim 20 or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or stereoisomer thereof, wherein A.sup.1 is unsubstituted or substituted with one R.sup.16.
23. The compound of claim 20 or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or stereoisomer thereof, wherein R.sup.16 is CF.sub.3, OCF.sub.3 or OCH.sub.2CH.sub.2OCF.sub.3.
24. The compound of claim 1 or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or stereoisomer thereof, wherein R.sup.1, R.sup.2, R.sup.2a, R.sup.3, R.sup.4, R.sup.4a, R.sup.4b, R.sup.4c, R.sup.4d, R.sup.4e, R.sup.4f, R.sup.5, R.sup.6 in formula (I) are selected to give: tert-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-{[3-(trifluoromethoxy)propyl]carbamoyl}piperidine-1-carboxylate; (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-[3-(trifluoromethoxy)propyl]piperidine-2-carboxamide hydrochloride; tert-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-({[4-(trifluoromethyl)phenyl]methyl}carbamoyl)piperidine-1-carboxylate; (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-{[4-(trifluoromethyl)phenyl]methyl}piperidine-2-carboxamide; 2-(4-chloro-3-fluorophenoxy)-N-[(3S,6R)-6-{3-[2-(trifluoromethoxy)ethoxy]azetidine-1-carbonyl}piperidin-3-yl]acetamide; (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-[2-(trifluoromethoxy)ethoxy]piperidine-2-carboxamide; tert-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-[(4-chlorophenyl)carbamoyl]piperidine-1-carboxylate; (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-(4-chlorophenyl)piperidine-2-carboxamide; (2S,5R)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-(4-chlorophenyl)piperidine-2-carboxamide; (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-phenylpiperidine-2-carboxamide; (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-(3-chlorophenyl)piperidine-2-carboxamide; (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-[3-(trifluoromethyl)phenyl]piperidine-2-carboxamide; (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-[3-(trifluoromethoxy)phenyl]piperidine-2-carboxamide; (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-[(5-chloropyridin-2-yl)methyl]piperidine-2-carboxamide; (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-[(1s,4s)-4-(trifluoromethoxy)cyclohexyl]piperidine-2-carboxamide; (2R,5S)-N-(4-chloro-2-methoxyphenyl)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]piperidine-2-carboxamide; (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-{[5-(trifluoromethyl)furan-2-yl]methyl}piperidine-2-carboxamide; (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-{[4-(trifluoromethyl)furan-2-yl]methyl}piperidine-2-carboxamide; (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-{[(1s,4s)-4-(trifluoromethyl)cyclohexyl]methyl}piperidine-2-carboxamide; (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-(3-methoxyphenyl)piperidine-2-carboxamide; (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-[4-fluoro-3-(trifluoromethyl)phenyl]piperidine-2-carboxamide; (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-[5-(trifluoromethyl)pyridin-3-yl]piperidine-2-carboxamide; (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-(3-fluorophenyl)piperidine-2-carboxamide; (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-[3-(difluoromethyl)phenyl]piperidine-2-carboxamide; (2R, 5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-(5-chl ropyridin-2-yl)piperidine-2-carboxamide; (2R, 5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-[5-methyl-1-(2,2,2-trifluoroethyl)-1H-pyrazol-4-yl]piperidine-2-carboxamide; (2R, 5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-[(1s,3s)-3-(trifluoromethoxy)cyclobutyl]piperidine-2-carboxamide; (2R, 5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-(3,5-dimethylphenyl)piperidine-2-carboxamide; (2R, 5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-(5,5,5-trifluoropentyl)piperidine-2-carboxamide; (2R, 5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-[3-(difluoromethoxy)phenyl]piperidine-2-carboxamide; (2S,5R)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-[3-(trifluoromethyl)phenyl]piperidine-2-carboxamide; tert-butyl (2R,5S)-2-[[3,5-bis(trifluoromethyl)phenyl]carbamoyl]-5-[[2-(4-chloro-3-fluoro-phenoxy)acetyl]amino]piperidine-1-carboxylate; (2R, 5S)-N-[3,5-bis(trifluoromethyl)phenyl]-5-[2-(4-chloro-3-fluorophenoxy)acetamido]piperidine-2-carboxamide; (2R, 5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-[1-(trifluoromethyl)-1H-pyrazol-3-yl]piperidine-2-carboxamide; (2R, 5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-[2-fluoro-5-(trifluoromethyl)phenyl]piperidine-2-carboxamide; (2R, 5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-[2-fluoro-3-(trifluoromethyl)phenyl]piperidine-2-carboxamide; (2R, 5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-[6-(tri fluoromethoxy)pyridin-2-yl]piperidine-2-carboxamide; (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-[4-(trifluoromethyl)pyridin-2-yl]piperidine-2-carboxamide; tert-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-{3-[2-(trifluoromethoxy)ethoxy]azetidine-1-carbonyl}piperidine-1-carboxylate; tert-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-{[2-(trifluoromethoxy)ethoxy]carbamoyl}piperidine-1-carboxylate; tert-butyl (2S,5R)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-[(4-chlorophenyl)carbamoyl]piperidine-1-carboxylate; trnt-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-(phenylcarbamoyl)piperidine-1-carboxylate; tert-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-[(3-chlorophenyl)carbamoyl]piperidine-1-carboxylate; tert-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-{[3-(trifluoromethyl)phenyl]carbamoyl}piperidine-1-carboxylate; tert-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-{[3-(trifluoromethoxy)phenyl]carbamoyl}piperidine-1-carboxylate; tert-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-{[(5-chloropyridin-2-yl)methyl]carbamoyl}piperidine-1-carboxylate; tert-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-{[(1s,4s)-4-(trifluoromethoxy)cyclohexyl]carbamoyl}piperidine-1-carboxylate; tert-butyl (2R,5S)-2-[(4-chloro-2-methoxyphenyl)carbamoyl]-5-[2-(4-chloro-3-fluorophenoxy)acetamido]piperidine-1-carboxylate; tert-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-({[5-(trifluoromethyl)furan-2-yl]methyl}carbamoyl)piperidine-1-carboxylate; tert-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-({[4-(trifluoromethyl)furan-2-yl]methyl}carbamoyl)piperidine-1-carboxylate; tert-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-({[(1s,4s)-4-(trifluoromethyl)cyclohexyl]methyl}carbamoyl)piperidine-1-carboxylate; tert-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-[(3-methoxyphenyl)carbamoyl]piperidine-1-carboxylate; tert-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-{[4-fluoro-3-(trifluoromethyl)phenyl]carbamoyl}piperidine-1-carboxylate; tert-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-{[5-(trifluoromethyl)pyridin-3-yl]carbamoyl}piperidine-1-carboxylate; tert-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-[(3-fluorophenyl)carbamoyl]piperidine-1-carboxylate; tert-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-{[3-(difluoromethyl)phenyl]carbamoyl}piperidine-1-carboxylate; tert-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-[(5-chloropyridin-2-yl)carbamoyl]piperidine-1-carboxylate; tert-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-{[5-methyl-1-(2,2,2-trifluoroethyl)-1H-pyrazol-4-yl]carbamoyl}piperidine-1-carboxylate; tert-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-{[(1s,3s)-3-(trifluoromethoxy)cyclobutyl]carbamoyl}piperidine-1-carboxylate; tert-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-[(3,5-dimethylphenyl)carbamoyl]piperidine-1-carboxylate; tert-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-[(5,5,5-trifluoropentyl)carbamoyl]piperidine-1-carboxylate; tert-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-{[3-(difluoromethoxy)phenyl]carbamoyl}piperidine-1-carboxylate; tert-butyl (2S,5R)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-{[3-(trifluoromethyl)phenyl]carbamoyl}piperidine-1-carboxylate; tert-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-{[1-(trifluoromethyl)-1H-pyrazol-3-yl]carbamoyl}piperidine-1-carboxylate; tert-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-{[2-fluoro-5-(trifluoromethyl)phenyl]carbamoyl}piperidine-1-carboxylate; tert-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-{[2-fluoro-3-(trifluoromethyl)phenyl]carbamoyl}piperidine-1-carboxylate; tert-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-{[6-(trifluoromethoxy)pyridin-2-yl]carbamoyl}piperidine-1-carboxylate or tert-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-{[4-(trifluoromethyl)pyridin-2-yl]carbamoyl}piperidine-1-carboxylate; (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)piperidine-2-carboxamide; 2-(4-chloro-3-fluorophenoxy)-N-[(3S,6R)-6-[5-(trifluoromethyl)-2,3-dihydro-1H-isoindole-2-carbonyl]piperidin-3-yl]acetamide; 2-(4-chloro-3-fluorophenoxy)-N-[(3S,6R)-6-[5-(trifluoromethoxy)-2,3-dihydro-1H-isoindole-2-carbonyl]piperidin-3-yl]acetamide; (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-{[1-methyl-5-(trifluoromethyl)-1H-pyrazol-3-yl]methyl}piperidine-2-carboxamide; (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-{[5-(trifluoromethyl)-1,2-oxazol-3-yl]methyl}piperidine-2-carboxamide; (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-{[4-(trifluoromethyl)pyridin-2-yl]methyl}piperidine-2-carboxamide; 2-(4-chloro-3-fluorophenoxy)-N-[(3S,6R)-6-[4-(trifluoromethyl)-2,3-dihydro-1H-indole-1-carbonyl]piperidin-3-yl]acetamide; (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-[1-(2,2-difluorocycl opropyl)-1H-pyrazol-3-yl]piperidine-2-carboxamide; (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-[3-(trifluoromethoxy)cyclopentyl]piperidine-2-carboxamide; tert-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-{[2-(trifluoromethyl)pyrimidin-4-yl]carbamoyl}piperidine-1-carboxylate; (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-[2-(trifluoromethyl)pyrimidin-4-yl]piperidine-2-carboxamide; tert-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-{[6-(trifluoromethyl)pyrazin-2-yl]carbamoyl}piperidine-1-carboxylate; (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-[6-(trifluoromethyl)pyrazin-2-yl]piperidine-2-carboxamide; (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-1-methyl-N-[6-(trifluoromethyl)pyrazin-2-yl]piperidine-2-carboxamide; (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-[(1S,3S)-3-(trifluoromethoxy)cyclopentyl]piperidine-2-carboxamide; (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-[(1R,3R)-3-(trifluoromethoxy)cyclopentyl]piperidine-2-carboxamide; tert-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-[(2,2-difluoro-2H-1,3-benzodioxol-5-yl)carbamoyl]piperidine-1-carboxylate; tert-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-[5-(trifluoromethyl)-2,3-dihydro-1H-isoindole-2-carbonyl]piperidine-1-carboxylate; tert-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-[5-(trifluoromethoxy)-2,3-dihydro-1H-isoindole-2-carbonyl]piperidine-1-carboxylate; tert-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-({[1-methyl-5-(trifluoromethyl)-1H-pyrazol-3-yl]methyl}carbamoyl)piperidine-1-carboxylate; tert-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-({[5-(trifluoromethyl)-1,2-oxazol-3-yl]methyl}carbamoyl)piperidine-1-carboxylate; tert-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-({[4-(trifluoromethyl)pyridin-2-yl]methyl}carbamoyl)piperidine-1-carboxylate; tert-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-[4-(trifluoromethyl)-2,3-dihydro-1H-indole-1-carbonyl]piperidine-1-carboxylate; tert-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-{[1-(2,2-difluorocyclopropyl)-1H-pyrazol-3-yl]carbamoyl}piperidine-1-carboxylate; or tert-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-{[3-(trifluoromethoxy)cyclopentyl]carbamoyl}piperidine-1-carboxylate.
25. The compound of claim 1 or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or stereoisomer thereof, wherein formula (I) has a stereochemistry as shown in formula (Ic) ##STR00099##
26. A pharmaceutical composition comprising: (a) at least one compound or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or stereoisomer thereof of claim 1; and (b) at least one pharmaceutically acceptable carrier, optionally in combination with one or more other bioactive compounds or pharmaceutical compositions.
27. (canceled)
28. A method of treating or preventing one or more diseases or disorders associated with integrated stress response comprising administering to a subject in need a compound or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or stereoisomer thereof of claim 1 or a pharmaceutical composition thereof.
29. The method of claim 28, wherein the diseases or disorders are selected from the group consisting of leukodystrophies, intellectual disability syndrome, neurodegenerative diseases and disorders, neoplastic diseases, infectious diseases, inflammatory diseases, musculoskeletal diseases, metabolic diseases, ocular diseases, organ fibrosis, chronic and acute diseases of the liver, chronic and acute diseases of the lung, chronic and acute diseases of the kidney, myocardial infarction, cardiovascular disease, arrhythmias, atherosclerosis, spinal cord injury, ischemic stroke, and neuropathic pain.
Description
EXAMPLES
I Chemical Synthesis
Experimental Procedures:
[0259] The following Abbreviations and Acronyms are used: [0260] aq aqueous [0261] ACN acetonitrile [0262] AgSO.sub.3CF.sub.3 silver trifluoromethanesulfonate [0263] Brine saturated solution of NaCl in water [0264] Bn benzyl [0265] BnONH.sub.2.Math.HCl O-benzylhydroxylamine hydrochloride [0266] Boc tert-butoxycarbonyl [0267] Boc.sub.2O di-tert-butyl dicarbonate [0268] .sup.tBuOK potassium tert-butoxide [0269] CDCl.sub.3 deuterated chloroform [0270] DCM dichloromethane [0271] DMSO dimethylsulfoxide [0272] DMSO-d.sub.6 deuterated dimethylsulfoxide [0273] DIAD diisopropyl azodicarboxylate [0274] DIPEA diisopropylethylamine [0275] DMF dimethyl formamide [0276] DMAP N,N-dimethylpyridin-4-amine [0277] ESI.sup.+ positive ionisation mode [0278] ESI.sup.− negative ionisation mode [0279] EtOAc ethyl acetate [0280] EtOH ethanol [0281] Et.sub.2O diethyl ether [0282] H.sub.2SO.sub.4 sulfuric acid [0283] HATU 1-[bis(dimethylamino)methylidene]-1H-[1,2,3]triazolo[4,5-b]pyridin-1-ium 3-oxide hexafluorophosphate [0284] HCl hydrochloric acid [0285] HPLC high-performance liquid chromatography [0286] h hour(s) [0287] IPA isopropyl alcohol [0288] Josiphos SL-J009-1 {(R)-1-[(Sp)-2-(dicyclohexylphosphino)ferrocenyl]ethyldi-tert-butylphosphine}[2-(2′-amino-1,1′-biphenl)]palladium(II) methanesulfonate [0289] KHCO.sub.3 potassium bicarbonate [0290] KF potassium fluoride [0291] LiOH lithium hydroxide [0292] m multiplet [0293] MeI iodomethane [0294] MeNHNH.sub.2 methylhydrazine [0295] MeOH methanol [0296] MgSO.sub.4 magnesium sulphate [0297] min minutes [0298] MsCl mesyl chloride [0299] MsOH methanesulfonic acid [0300] mL millilitre (s) [0301] N.sub.2 nitrogen atmosphere [0302] Na.sub.2SO.sub.4 sodium sulphate [0303] NaHCO.sub.3 sodium bicarbonate [0304] NH.sub.4Cl ammonium chloride [0305] NMM 4-methylmorpholine [0306] NMR Nuclear Magnetic Resonance [0307] Pd.sub.2(dba).sub.3 tris(dibenzylideneacetone)dipalladium(0) [0308] prep. preparative [0309] POCl.sub.3 phosphoric trichloride [0310] PPh.sub.3 triphenylphosphine [0311] r.t. room temperature [0312] RT retention time [0313] satd saturated [0314] Selectfluor 1-(chloromethyl)-4-fluoro-1,4-diazabicyclo[2.2.2]octane-1,4-diium ditetrafluoroborate [0315] T3P propanephosphonic acid anhydride [0316] THF tetrahydrofuran [0317] TFA 2,2,2-trifluoroacetic acid [0318] TMSOI trimethylsulfoxonium iodide [0319] XPhos dicyclohexyl[2′,4′,6′-tris(propan-2-yl)[1,1′-biphenyl]-2-yl]phosphane [0320] ZnBr.sub.2 zinc dibromide
Analytical LCMS Conditions are as Follows:
System 1 (S1): Acidic IPC Method (MS18 and MS19)
[0321] Analytical (MET/CR/1410) HPLC-MS were performed on a Shimadzu LCMS systems using a Kinetex Core shell C18 column (2.1 mm×50 mm, 5 μm; temperature: 40° C.) and a gradient of 5-100% B (A=0.1% formic acid in H.sub.2O; B=0.1% formic acid in ACN) over 1.2 min then 100% B for 0.1 min. A second gradient of 100-5% B was then applied over 0.01 min with an injection volume of 3 μL at a flow rate of 1.2 mL/min. UV spectra were recorded at 215 nm using a SPD-M20A photo diode array detector spectrum range: 200-400 nm. Mass spectra were obtained using a 2010EV detector. Data were integrated and reported using Shimadzu LCMS-Solutions and PsiPort software.
System 2 (S2): Acidic IPC Method (MSQ1, MSQ2 and MSQ4)
[0322] Analytical (MET/uPLC/1704) uHPLC-MS were performed on a Waters Acquity uPLC system using a Waters UPLC® BEH™ C18 column (2.1 mm×50 mm, 1.7 μm; temperature 40° C.) and a gradient of 5-100% B (A=0.1% formic acid in H.sub.2O: B=0.1% formic acid in ACN) over 1.1 min then 100% B for 0.25 min. A second gradient of 100-5% B was then applied over 0.05 min and held for 0.1 min with an injection volume of 1μL at a flow rate of 0.9 mL/min. UV spectra were recorded at 215 nm on a Waters Acquity PDA with a spectrum range of 200-400 nm. Mass spectra were obtained using a Waters QDa. Data were integrated and reported using Waters MassLynx and OpenLynx software.
System 3 (S3): Basic IPC Method (MS16)
[0323] Analytical (MET/CR/1602) uHPLC-MS were performed on a Waters Acquity uPLC system using Waters UPLC® BEH™ C18 column (2.1 mm×30 mm, 1.7 μm; temperature 40° C.) and a gradient of 5-100% B (A: 2 mM ammonium bicarbonate, buffered to pH 10, B: ACN) over min, then 100% B for 0.1 min. A second gradient of 100-5% B was then applied over 0.05 min and held for 0.1 min with an injection volume of 1 μL at a flow rate of 1 mL/min. UV spectra were recorded at 215 nm on a Waters Acquity PDA with a spectrum range of 200-400 nm. Mass spectra were obtained using a Waters Quattro Premier XE. Data were integrated and reported using Waters MassLynx and OpenLynx software.
System 4 (S4): Acidic Final method (MSQ1 and MSQ2)
[0324] Analytical (MET/uPLC/AB101) uHPLC-MS were performed on a Waters Acquity uPLC system using a Phenomenex Kinetex-XB C18 column (2.1 mm×100 mm, 1.7 μM; temperature: 40° C.) and a gradient of 5-100% B (A=0.1% formic acid in H.sub.2O; B=0.1% formic acid in ACN) over 5.3 min then 100% B for 0.5 min. A second gradient of 100-5% B was then applied over 0.02 min and held for 1.18 min with an injection volume of 1 μL at flow rate of 0.6 mL/min. UV spectra were recorded at 215 nm using a Waters Acquity PDA detector spectrum range: 200-400 nm. Mass spectra were obtained using a Waters SQD (MSQ1) or Waters Acquity QDA (MSQ2). Data were integrated and reported using Waters MassLynx and OpenLynx software.
System 5 (S5): Acidic Final Method (MS18, MS19)
[0325] Analytical (MET/CR/1416) HPLC-MS were performed on Shimadzu LCMS systems using a Waters Atlantis dC18 column (2.1 mm×100 mm, 3 μm; temperature: 40° C.) and a gradient of B (A=0.1% formic acid in H.sub.2O; B=0.1% formic acid in ACN) over 5 min then 100% B for 0.4 min. A second gradient of 100-5% B was then applied over 0.02 min and held for 1.58 min with an injection volume of 3 μL at flow rate of 0.6 mL/min. UV spectra were recorded at 215 nm using a SPD-M20A photo diode array detector spectrum range: 200-400 nm. Mass spectra were obtained using a 2010EV detector. Data were integrated and reported using Shimadzu LCMS-Solutions and PsiPort software.
System 6 (S6): Basic Final Method (MS16)
[0326] Analytical (MET/uHPLC/AB105) uPLC-MS were performed on a Waters Acquity uPLC system using a Waters UPLC® BEH™ C18 column (2.1 mm×100 mm, 1.7 μm column; temperature: 40° C.) and a gradient of 5-100% (A=2 mM ammonium bicarbonate, buffered to pH 10; B=ACN) over 5.3 min then 100% B for 0.5 min. A second gradient of 100-5% B was then applied over 0.02 min and held for 1.18 min with an injection volume of 1 μL and at flow rate of 0.6 mL/min. UV spectra were recorded at 215 nm using a Waters Acquity photo diode array detector Spectrum range: 200-400 nm. Mass spectra were obtained using a Waters Quattro Premier XE mass detector. Data were integrated and reported using Waters MassLynx and OpenLynx software.
Purification Methods are as Follows:
Method 1: Acididc Early Method
[0327] Purifications (P1) LC were performed on a Gilson LC system using a Waters Sunfire C18 column (30 mm×100 mm, 10 μM; temperature: r.t.) and a gradient of 10-95% B (A=0.1% formic acid in H.sub.2O; B=0.1% formic acid in ACN) over 14.44 min then 95% B for 2.11 min. A second gradient of 95-10% B was then applied over 0.2 min with an injection volume of 1500 μL at flow rate of 40 mL/min. UV spectra were recorded at 215 nm using a Gilson detector.
Method 2: Acididc Standard Method
[0328] Purifications (P2) LC were performed on a Gilson LC system using a Waters Sunfire C18 column (30 mm×10 mm, 10 μM; temperature: r.t.) and a gradient of 30-95% B (A=0.1% formic acid in water; B=0.1% formic acid in ACN) over 11.00 min then 95% B for 2.10 min. A second gradient of 95-30% B was then applied over 0.2 min with an injection volume of 1500 μL at flow rate of 40 mL/min. UV spectra were recorded at 215 nm using a Gilson detector.
Method 3: Basic Early Method
[0329] Purifications (P3) LC were performed on a Gilson LC system using a Waters X-Bridge C18 column (30 mm×100 mm, 10 JIM; temperature: r.t.) and a gradient of 10-95% B (A=0.2% NH.sub.4OH in H.sub.2O; B=0.2% NH.sub.4OH in ACN) over 14.44 min then 95% B for 2.11 min. A second gradient of 95-10% B was then applied over 0.2 min with an injection volume of 1500 μL at flow rate of 40 mL/min. UV spectra were recorded at 215 nm using a Gilson detector.
Method 4: Basic Standard Method
[0330] Purifications (P4) LC were performed on a Gilson LC system using a Waters X-Bridge C18 column (30 mm×10 mm, 10 μM; temperature: r.t.) and a gradient of 30-95% B (A=0.2% NH.sub.4OH in water; B=0.2% NH.sub.4OH in ACN) over 11.00 min then 95% B for 2.10 min. A second gradient of 95-30% B was then applied over 0.21 min with an injection volume of 1500 μL at flow rate of 40 mL/min. UV spectra were recorded at 215 nm using a Gilson detector.
Chiral Separation Methods:
NMR Conditions
[0331] Unless otherwise stated, .sup.1H NMR spectra were recorded at 500 MHz, 400 MHz or 250 MHz on either a Bruker Avance III HD 500 MHz spectrometer, Bruker Avance III HD 400 MHz spectrometer or Bruker Avance III HD 250 MHz spectrometer respectively. Chemical shifts, δ, are quoted in parts per million (ppm) and are referenced to the residual solvent peak. The following abbreviations are used to denote the multiplicities and general assignments: s (singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), ddd (doublet of doublet of doublets), dt (doublet of triplets), dq (doublet of quartets), hep (heptet), m (multiplet), pent (pentet), td (triplet of doublets), qd (quartet of doublets), app. (apparent) and br. (broad). Coupling constants, J, are quoted to the nearest 0.1 Hz.
General Synthesis:
[0332] All the compounds have been synthesised with a purity >95% unless otherwise specified.
##STR00009##
Intermediate 1: 2-(4-chloro-3-fluorophenoxy)acetyl Chloride
[0333] ##STR00010##
[0334] To a solution of 2-(4-chloro-3-fluorophenoxy)acetic acid (5.16 g, 22.7 mmol) in DCM (45 mL) at 0° C. was added oxalyl dichloride (10 mL, 0.115 mol) followed by DMF (81 μL, 1.11 mmol) and the mixture was stirred at r.t. for 17 h. The reaction mixture was concentrated in vacuo to afford the title compound (90% purity, 5.30 g, 21.4 mmol, 94% yield) as an orange oil; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.31 (t, J=8.6 Hz, 1H), 6.75 (dt, J=10.2, 2.9 Hz, 1H), 6.66 (ddd, J=8.9, 2.9, 1.2 Hz, 1H), 4.96 (s, 2H).
##STR00011##
Step 2.a: ethyl (2R)-5-[(benzyloxy)imino]-2-{[(tert-butoxy)carbonyl]amino}-6-chlorohexanoate
[0335] ##STR00012##
[0336] DMSO (75 mL) was added to a solution of TMSOI (12.89 g, 58.3 mmol) and .sup.tBuOK (6.27 g, mmol) in anhydrous THF (60 mL) and the mixture was stirred at r.t. for 1 h. The reaction mixture was cooled to −12° C. and a solution of ethyl Boc-D-Pyroglutamate (12.5 g, 48.6 mmol) in anhydrous THF (38 mL) was added and stirred at r.t. for 16 h. The reaction mixture was diluted with satd aq NH.sub.4Cl solution (80 mL), H.sub.2O (15 mL) and EtOAc (200 mL), and the organic layer was isolated, washed with brine, and concentrated in vacuo to approximately 100 mL. A solution of BnONH.sub.2.Math.HCl (8.14 g, 51.0 mmol) in EtOAc (62 mL), was added and the mixture was stirred at reflux for 2 h. The reaction mixture was cooled to r.t., washed with H.sub.2O and brine, and the organic layer was concentrated in vacuo to afford the title compound (85% purity, 19.5 g, 40.1 mmol, 83% yield) as a colourless oil; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.16-7.33 (m, 5H), 5.01-5.06 (m, 2H), 3.95-4.30 (m, 5H), 2.32-2.50 (m, 2H), 1.98-2.13 (m, 1H), 1.75-1.92 (m, 1H),1.30-1.40 (m, 9H), 1.12-1.24 (m, 3H).
Step 2.b: ethyl (2R)-5-[(benzyloxy)imino]piperidine-2-carboxylate
[0337] ##STR00013##
[0338] To a solution of ethyl (2R)-5-[(benzyloxy)imino]-2-{[(tert-butoxy)carbonyl]amino}-6-chlorohexanoate (85% purity, 19.5 g, 40.1 mmol) in EtOAc (157 mL) was added MsOH (7.8 mL, 0.12 mol) and the mixture was stirred at 42° C. for 2 h. The resultant mixture was added to a solution of KHCO.sub.3 (20.1 g, 0.201 mol) in H.sub.2O (100 mL) and stirred at 52° C. for 2 h. The reaction mixture was cooled to r.t. and the organic layer was isolated, washed with brine, dried over Na.sub.2SO.sub.4, and concentrated in vacuo to afford the title compound (85% purity, 13.0 g, 40.0 mmol) in quantitative yield as a dark orange oil; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.20-7.34 (m, 4.99 (d, J=4.8 Hz, 2H), 4.13 (q, J=7.1 Hz, 2H), 3.45-3.56 (m, 1H), 3.25 (dd, J=14.9, 9.8 Hz, 1H), 3.08 (dt, J=14.5, 4.3 Hz, 1H), 2.01-2.32 (m, 3H), 1.55-1.80 (m, 1H), 1.21 (t, J=7.1 Hz, 3H).
Step 2.c: ethyl (2R,5S)-5-[(benzyloxy)amino]piperidine-2-carboxylate Oxalic Acid
[0339] ##STR00014##
[0340] Propanoic acid (23 mL, 0.240 mol) was added to a suspension of NaBH.sub.4 (3.03 g, 80.0 mmol) in EtOAc (95 mL) and the mixture was stirred at r.t. for 1 h. The resultant mixture was added to a solution of ethyl (2R)-5-[(benzyloxy)imino]piperidine-2-carboxylate (85% purity, 13.0 g, 40.0 mmol) in EtOAc (95 mL) and H.sub.2SO.sub.4 (11 mL, 0.20 mol) at −20° C. and stirred at r.t. for 60 h. The reaction mixture was diluted with H.sub.2O (75 mL) and neutralised with aq NH.sub.4OH solution. The organic layer was isolated, washed with brine, dried over Na.sub.2SO.sub.4, and concentrated in vacuo to ˜75 mL volume. The solution was warmed to 45° C., and MeOH (30 mL), followed by a solution of oxalic acid (3.60 g, 40.0 mmol) in MeOH (15 mL) was added. The mixture was cooled to 0° C., and the resultant precipitate was isolated via vacuum filtration, washing with MeOH:EtOH (1:4) and EtOAc to afford the title compound (7.17 g, 19.1 mmol, 48% yield); .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.25-7.42 (m, 5H), 4.59 (s, 2H), 4.17-4.24 (m, 2H), 3.92 (dd, J=12.3, 3.2 Hz, 1H), 3.34-3.40 (m, 1H), 3.10 (ddd, J=15.1, 7.6, 3.9 Hz, 1H), 2.64 (t, J=11.5 Hz, 1H), 2.13 (dt, J=10.2, 3.4 Hz, 1H), 1.87 (dd, J=9.0, 3.8 Hz, 1H), 1.65 (qd, J=13.2, 3.6 Hz, 1H), 1.40 (qd, J=12.8, 3.9 Hz, 1H), 1.23 (t, J=7.1 Hz, 3H); M/Z: 279, [M+H].sup.+, ESI.sup.+, RT=0.81 (S1).
Intermediate 2 (step 2.d): 1-tert-butyl 2-ethyl (2R,5S)-5-[(benzyloxy)amino]piperidine-1,2-dicarboxylate
[0341] ##STR00015##
[0342] To a solution of ethyl (2R,5S)-5-[(benzyloxy)amino]piperidine-2-carboxylate oxalic acid (2.22 g, 6.03 mmol) in anhydrous DCM (30 mL) at 0° C. was added Et.sub.3N (3.6 mL, 25.8 mmol), DMAP (76 mg, 0.622 mmol) and Boc.sub.2O (4.2 mL, 18.3 mmol) and the mixture was stirred at r.t. for 17 h. The reaction mixture was diluted with satd aq NH.sub.4Cl solution and DCM, and the organic layer was isolated, washed with H.sub.2O and brine, dried over Na.sub.2SO.sub.4, and concentrated in vacuo. The residue was purified by chromatography on silica gel (0-20% EtOAc in heptane) to afford the title compound (86% purity, 1.40 g, 3.18 mmol, 53% yield) as a colourless oil; .sup.1H NMR (500 MHz, CDCl.sub.3) δ 7.40-7.26 (m, 5H), 5.51-5.41 (m, 1H), 4.92-4.80 (m, 1H), 4.79-4.62 (m, 2H), 4.19 (q, J=7.0 Hz, 3H), 3.11 (d, J=45.4 Hz, 2H), 1.96 (s, 2H), 1.73-1.60 (m, 1H), 1.55-1.49 (m, 1H), 1.46 (s, 9H), 1.27 (t, J=7.1 Hz, 3H); M/Z: 379, [M+H].sup.+, ESI.sup.+, RT=1.09 (S2).
##STR00016##
Step 3.a: 1-tert-butyl 2-ethyl (2R,5S)-5-aminopiperidine-1,2-dicarboxylate
[0343] ##STR00017##
[0344] To a solution of 1-tert-butyl 2-ethyl (2R,5S)-5-[(benzyloxy)amino]piperidine-1,2-dicarboxylate (93% purity, 8.7 g, 21.3 mmol, Intermediate 2) in anhydrous EtOH (200 mL) under N.sub.2 was added Pd/C (10%, 2.28 g, 2.14 mmol) and the mixture was stirred under H.sub.2 at r.t. for 17 h. The reaction mixture was filtered through a pad of Celite and the filtrate concentrated in vacuo. The residue was purified using an SCX-2 cartridge, first flushing with Me0H and second eluting with 3 M NH.sub.3 in MeOH to afford the title compound (4.88 g, 17.0 mmol, 80% yield) as a pale yellow oil; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 4.98-4.57 (m, 1H), 4.18 (q, J=7.1 Hz, 2H), 3.87-3.64 (m, 1H), 3.35-2.99 (m, 2H), 2.14-1.92 (m, 2H), 1.64-1.52 (m, 2H), 1.45 (s, 11H), 1.26 (t, J=7.1 Hz, 3H).
Step 3.b: 1-tert-butyl 2-ethyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]piperidine-1,2-dicarboxylate
[0345] ##STR00018##
[0346] To a mixture of 1-tert-butyl 2-ethyl (2R,5S)-5-aminopiperidine-1,2-dicarboxylate (4.88 g, 17.0 mmol) and Et.sub.3N (14 mL, 0.103 mol) in DCM (170 mL) at 0° C. was added dropwise a solution of 2-(4-chloro-3-fluoro-phenoxy)acetyl chloride (4.19 g, 18.8 mmol, Intermediate 1) in DCM (10 mL) and stirred at r.t. for 48 h. The reaction mixture was diluted with DCM (250 mL) and washed with satd aq NaHCO.sub.3 solution (2×100 mL) and brine (100 mL), dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The residue was purified by chromatography on silica gel (0-50% EtOAc in heptane) to afford the title compound (7.14 g, 15.6 mmol, 91% yield) as a colourless oil; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.32 (t, J=8.6 Hz, 1H), 6.86-6.72 (m, 2H), 6.69-6.63 (m, 1H), 4.98-4.66 (m, 1H), 4.45 (s, 2H), 4.29-4.13 (m, 3H), 4.09-3.87 (m, 1H), 3.33-3.10 (m, 1H), 2.23-2.02 (m, 1H), 2.00-1.71 (m, 2H), 1.56 (s, 1H), 1.44 (s, 9H), 1.28 (t, J=7.2 Hz, 3H); M/Z: 459, 461 [M+H].sup.+, ESI.sup.+, RT=3.83 (S4).
Intermediate 3 (step 3.c): (2R,5S)-1-[(tert-butoxy)carbonyl]-5-[2-(4-chloro-3-fluorophenoxy) acetamido]piperidine-2-carboxylic Acid
[0347] ##STR00019##
[0348] LiOH (0.78 g, 31.1 mmol) was added to a solution of 1-tert-butyl 2-ethyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]piperidine-1,2-dicarboxylate (7.1 g, 15.6 mmol) in EtOH (80 mL) and H.sub.2O (20 mL) and the mixture was stirred at r.t. for 3 h. The reaction mixture was concentrated in vacuo, dissolved in H.sub.2O (50 mL), and extracted with DCM (2×100 mL). The aqueous layer was then acidified to pH 2 using 2 M aq HCl solution and extracted with EtOAc (3×100 mL). The combined organic extracts were washed with brine (100 mL), dried over anhydrous Na.sub.2SO.sub.4, and concentrated in vacuo to afford the title compound (87% purity, 5.60 g, 11.3 mmol, 73% yield) as a white solid; .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.02 (d, J=7.3 Hz, 1H), 7.47 (t, J=8.9 Hz, 1H), 7.03 (dd, J=11.4, 2.8 Hz, 1H), 6.83-6.75 (m, 1H), 4.59-4.54 (m, 2H), 3.93 (s, 1H), 3.73 (d, J=54.2 Hz, 1H), 3.13-2.94 (m, 1H), 2.06-1.87 (m, 2H), 1.61 (d, J=12.2 Hz, 1H), 1.56-1.43 (m, 1H), 1.37 (s, 10H); M/Z: 429, 431 [M+H].sup.+, ESI.sup.+, RT=0.91 min (S1).
[0349] The intermediate in Table 1 was synthesised according to general route 3 as exemplified by Intermediate 3 using the corresponding starting materials.
TABLE-US-00001 TABLE 1 Inter- Starting LCMS mediate Structure Name material data .sup.1H NMR data 4
##STR00021##
Step 4.a: (1-benzhydrylazetidin-3-yl) methanesulfonate
[0350] ##STR00022##
[0351] To a solution of 1-(diphenylmethyl)azetidin-3-ol (500 mg, 2.09 mmol) in anhydrous DCM (5 mL) was added MsCl (0.19 mL, 2.51 mmol) followed by DIPEA (0.55 mL, 3.13 mmol) and the mixture was stirred at r.t. for 30 min. The reaction mixture was diluted with H.sub.2O (20 mL), the aqueous phase separated and extracted with DCM (2×20 mL). The organic phases were combined, washed with brine, dried using a phase separator cartridge and concentrated in vacuo to afford the title compound (738 mg, 2.05 mmol, 98% yield) as a yellow solid; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.44-7.39 (m, 4H), 7.33-7.27 (m, 4H), 7.24-7.19 (m, 2H), 5.18-5.09 (m, 1H), 4.52-4.44 (m, 1H), 3.80-3.68 (m, 2H), 3.35-3.20 (m, 2H), 2.99 (s, 3H). M/Z: 318 [M+H].sup.+, ESI.sup.+, RT=0.66 (S2).
Step 4.b: 1-benzhydryl-3-[2-(trifluoromethoxy)ethoxy]azetidine
[0352] ##STR00023##
[0353] A solution of (1-benzhydrylazetidin-3-yl) methanesulfonate (590 mg, 1.64 mmol) and 2-(trifluoromethoxy)ethanol (0.80 mL, 8.18 mmol) in anhydrous toluene (0.6 mL) was irradiated at 110° C. in a microwave vial for 30 min. The reaction mixture was diluted with EtOAc (10 mL) and the organic layer was washed with H.sub.2O (10 mL) and satd aq NaHCO.sub.3 solution (10 mL). The organic layer was dried over MgSO.sub.4, concentrated in vacuo, and purified by chromatography on silica gel (5-100% EtOAc in heptane) to afford the title compound (140 mg, 0.319 mmol, 19% yield) as a viscous orange oil; M/Z: 352 [M+H].sup.+, ESI.sup.+, RT=0.70 (S2).
Intermediate 5 (step 4.c): 3-[2-(trifluoromethoxy)ethoxy]azetidine;hydrochloride
[0354] ##STR00024##
[0355] 1-Chloroethyl chloroformate (0.038 mL, 0.351 mmol) was added to a solution of 1-benzhydryl-3-[2-(trifluoromethoxy)ethoxy]azetidine (140 mg, 0.319 mmol) in anhydrous DCM (2.5 mL) at 0° C. and the mixture was stirred at r.t. for 1 h. Anhydrous EtOH (2.5 mL) was added and the resultant mixture was stirred at 45° C. for 1 h. The mixture was then cooled to r.t. and stirred overnight. A second portion of 1-chloroethyl chloroformate (0.017 mL, 0.159 mmol) was added and the reaction mixture stirred at r.t. for 6 h. The solvent was removed in vacuo and the resultant residue was dissolved in anhydrous DCM (2.5 mL) and TFA (0.24 mL, 3.19 mmol) was added. The reaction was stirred at r.t. for 3 h and then concentrated in vacuo to afford the title compound (270 mg, 0.183 mmol, 57% yield) as an orange oil; M/Z: 186 [M+H].sup.+, ESI.sup.+, RT=0.33 (S2).
##STR00025##
Step 5.a: 2-[2-(trifluoromethoxy)ethoxy]-2,3-dihydro-1H-isoindole-1,3-dione
[0356] ##STR00026##
[0357] To a solution of 2-(trifluoromethoxy)ethanol (350 mg, 2.69 mmol), 2-hydroxy-2,3-dihydro-1H-isoindole-1,3-dione (461 mg, 2.83 mmol) and PPh.sub.3 (776 mg, 2.96 mmol) in anhydrous THF (17.5 mL) at 0° C., was added DIAD (556 μL, 2.83 mmol) and the mixture was stirred at r.t. for 3 h. The reaction mixture was concentrated in vacuo and the residue was purified by chromatography on silica gel (10-100% EtOAc in heptane) to afford the title compound (770 mg, 2.66 mmol, 99% yield) as a white solid; .sup.1H NMR (500 MHz, CDCl.sub.3) δ 7.88-7.84 (m, 2H), 7.79-7.75 (m, 2H), 4.48-4.44 (m, 2H), 4.36-4.31 (m, 2H); M/Z: 276 [M+H].sup.+, ESI.sup.+, RT=3.00 (S4).
Intermediate 6 (step 5.b): O-[2-(trifluoromethoxy)ethyl]hydroxylamine
[0358] ##STR00027##
[0359] To solution of 2-[2-(trifluoromethoxy)ethoxy]-2,3-dihydro-1H-isoindole-1,3-dione (770 mg, 2.66 mmol) in DCM (15 mL) was added MeNHNH.sub.2 (122 mg, 2.66 mmol) and the mixture was stirred at r.t. for 1 h. The resultant precipitate was removed via vacuum filtration and the filtrate was concentrated in vacuo (35° C., 700 mbar) to afford the title compound (41% purity, 905 mg, 2.56 mmol, 96% yield) as a light yellow oil; .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 6.12 (s, 2H), 4.22-4.17 (m, 2H), 3.76-3.69 (m, 2H).
##STR00028##
Step 6.a: benzyl N-[3-(trifluoromethoxy)cyclopentyl]carbamate
[0360] ##STR00029##
[0361] 2-fluoropyridine (0.73 mL, 8.50 mmol) and TMS-CF.sub.3 (1.3 mL, 8.50 mmol) were added to a solution of rac-benzyl N-[(1S*,3S*)-3-hydroxycyclopentyl]carbamate (1.00 g, 4.25 mmol), AgSO.sub.3CF.sub.3 (2.19 g, 8.50 mmol), Selectfluor (2.26 g, 6.38 mmol) and KF (0.74 g, 12.8 mmol) in EtOAc (20 mL) at r.t. under nitrogen in a foil-covered flask and the mixture was stirred at r.t. for 6 days. The reaction mixture was filtered through a pad of Celite and washed with EtOAc (100 mL) before concentrating in vacuo to give an orange-brown oil.
[0362] 2-fluoropyridine (0.73 mL, 8.50 mmol) and TMS-CF.sub.3 (1.3 mL, 8.50 mmol) were added to a solution of rac-benzyl N-[(1S*,3R*)-3-hydroxycyclopentyl]carbamate (1.00 g, 4.25 mmol), AgSO.sub.3CF.sub.3 (2.19 g, 8.50 mmol), Selectfluor (2.26 g, 6.38 mmol) and KF (0.74 g, 12.8 mmol) in EtOAc (20 mL) at r.t. under nitrogen in a foil-covered flask and the mixture was stirred at r.t. for 6 days. The reaction mixture was filtered through a pad of Celite and washed with EtOAc (100 mL) before concentrating in vacuo to give an orange-brown oil.
[0363] The crude materials from both reactions were combined and purified by FCC on silica gel (0-100% EtOAc in heptane) to afford the title compound as a mixture of four isomers (90% purity, 1.60 g, 4.75 mmol, 56% yield) as a colourless oil; .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.43 (dd, J=18.2, 7.1 Hz, 1H), 7.39-7.29 (m, 6H), 5.01 (s, 2H), 4.91 (tt, J=6.4, 3.4 Hz, 1H), 4.83-4.76 (m, 1H), 4.05-3.96 (m, 1H), 3.83 (h, J=7.5 Hz, 1H), 2.35 (dt, J=14.4, 7.4 Hz, 1H), 2.16-1.79 (m, 7H), 1.78-1.53 (m, 3H), 1.47 (ddt, J=13.0, 8.9, 6.5 Hz, 1H); M/Z: 304 [M+H].sup.+, ESI.sup.+, RT=0.97 (S2).
Intermediate 7 (step 6.b): 3-(trifluoromethoxy)cyclopentan-1-amine Hydrochloride
[0364] ##STR00030##
[0365] Pd/C (10%, 253 mg, 0.237 mmol) was added to a round-bottomed flask and the flask was evacuated and purged with nitrogen five times. Benzyl N-[3-(trifluoromethoxy)cyclopentyl]carbamate (90% purity, 1.60 g, 4.75 mmol) in EtOH (15 mL) was added, followed by 12 M HCl (0.40 mL, 4.75 mmol). The flask was evacuated and purged with nitrogen five times before purging with H.sub.2 and evacuating five times. The reaction was placed under H.sub.2 and stirred at r.t. for 20 h. The reaction mixture was filtered through Celite and the filtrate was concentrated in vacuo to afford title compound (75% purity, 0.42 g, 1.51 mmol, 32% yield) as a yellow oil; .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 4.97 (tt, J=6.4, 3.6 Hz, 1H), 4.81 (p, J=6.1, 5.5 Hz, 1H), 3.55 (dt, J=13.4, 7.1 Hz, 1H), 3.34 (p, J=7.2 Hz, 1H), 2.38 (dt, J=14.2, 7.3 Hz, 1H), 2.18 (dq, J=14.6, 6.7 Hz, 1H), 2.11-1.82 (m, 2H), 1.82-1.71 (m, 1H), 1.69-1.45 (m, 1H); M/Z: 170 [M+H].sup.+, ESI.sup.+ (S4).
##STR00031##
Example 1 (step 7.a): tert-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-{[3-(trifluoromethoxy)propyl]carbamoyl}piperidine-1-carboxylate
[0366] ##STR00032##
[0367] To a solution of (2R,5S)-1-Rtert-butoxy)carbonyl1-5-[2-(4-chloro-3-fluorophenoxy)acetamido]piperidine-2-carboxylic acid (100 mg, 0.220 mmol, Intermediate 3) in DMSO (1.5 mL) was added DIPEA (120 μL, 0.661 mmol) and HATU (101 mg, 0.265 mmol) and the mixture was stirred at r.t. for 10 min. 3-(Trifluoromethoxy)propan-1-amine hydrochloride (48 mg, 0.265 mmol) was then added and the mixture was stirred at r.t. for 1 h. The reaction mixture was diluted with ACN/H.sub.2O (3:2, 1.5 mL) and purified by prep. HPLC (Method 4) to afford the title compound (97 mg, 0.174 mmol, 79% yield) as a colourless glass; M/Z: 456.2, 458.3 [M-BOC+H].sup.+, ESI.sup.+, RT=1.01 (S2).
Example 2 (step 7.b): (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-[3-(trifluoromethoxy)propyl]piperidine-2-carboxamide Hydrochloride
[0368] ##STR00033##
[0369] To a solution of tert-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-{[3-(trifluoromethoxy)propyl]carbamoyl}piperidine-1-carboxylate (97 mg, 0.174 mmol, Example 1) in anhydrous 1,4-dioxane (3 mL) was added 4 M HCl in 1,4-dioxane (1.0 mL, 4.00 mmol) and the mixture was stirred at r.t. for 16 h. The reaction mixture was concentrated in vacuo to afford the title compound (89 mg, 0.174 mmol, 99% yield) as a white powder; .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 9.42-8.89 (m, 2H), 8.58 (s, 1H), 8.24 (s, 1H), 7.51 (t, J=8.9 Hz, 1H), 7.09 (dd, J=11.3, 2.8 Hz, 1H), 6.92-6.82 (m, 1H), 4.55 (s, 2H), 4.15-4.09 (m, 2H), 4.08-4.01 (m, 1H), 3.77-3.68 (m, 1H), 3.27-3.18 (m, 3H), 2.89-2.76 (m, 1H), 2.22-2.13 (m, 1H), 1.97-1.88 (m, 1H), 1.88-1.80 (m, 2H), 1.70-1.50 (m, 2H); M/Z: 456.2, 458.2 [M+H].sup.+, ESI.sup.+, RT=2.03 (S4).
##STR00034##
Example 3 (step 8.a): tert-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-({[4-(trifluoromethyl)phenyl]methyl}carbamoyl)piperidine-1-carboxylate
[0370] ##STR00035##
[0371] To a solution of (2R,5S)-1-[(tert-butoxy)carbonyl]-5-[2-(4-chloro-3-fluorophenoxy)acetamido]piperidine-2-carboxylic acid (200 mg, 0.464 mmol, Intermediate 3) in anhydrous DMF (2.5 mL) was added DIPEA (163 tiL, 0.933 mmol) and HATU (194 mg, 0.510 mmol) and the mixture was stirred at r.t. for 10 min. 1-[4-(Trifluoromethyl)phenyl]methanamine (73 μL, 0.512 mmol) was added and the mixture was stirred at r.t. for 4 h. The reaction mixture was diluted with EtOAc (20 mL) and H.sub.2O (10 mL). The organic layer was isolated, washed with brine (2×10 mL), dried over MgSO.sub.4, and concentrated in vacuo to afford the title compound (341 mg, 99% yield, 80% purity) as an orange solid; M/Z: 488, 490 [M+H].sup.+, ESI.sup.+, RT=1.13 (S2). The compound was taken forward 15 without further purification.
Example 4 (step 8.b): (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-{[4-(trifluoromethyl)phenyl]methyl}piperidine-2-carboxamide
[0372] ##STR00036##
[0373] To a solution of tert-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-({[4-(trifluoromethyl)phenyl]methyl}carbamoyl)piperidine-1-carboxylate (80% purity, 341 mg, mmol, Example 3) in DCM (3 mL) at 0° C. was added TFA (350 μL, 4.71 mmol) and the mixture was stirred at r.t. for 4 h. The reaction mixture was diluted with DCM (10 mL) and washed with satd aq NaHCO.sub.3 solution (3×10 mL). The organic layer was dried using a phase separation cartridge and concentrated in vacuo. The residue was purified by prep. HPLC (Method 4) to afford the title compound (119 mg, 0.244 mmol, 53% yield) as a white powder; .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.35 (t, J=6.2 Hz, 1H), 7.92 (d, J=8.1 Hz, 1H), 7.67 (d, J=8.1 Hz, 2H), 7.52-7.42 (m, 3H), 7.06 (dd, J=11.4, 2.8 Hz, 1H), 6.85 (ddd, J=9.0, 2.8, 1.1 Hz, 1H), 4.53-4.47 (m, 2H), 4.34 (d, J=6.1 Hz, 2H), 3.70-3.60 (m, 1H), 3.08-3.00 (m, 1H), 3.00-2.91 (m, 1H), 2.47-2.41 (m, 1H), 2.38-2.30 (m, 1H), 1.93-1.79 (m, 2H), 1.48-1.32 (m, 2H); M/Z: 488, 490 [M+H].sup.+, ESI.sup.+, RT=2.18 (S4).
[0374] The example compounds in Table 2 were synthesised according to general route 8 as exemplified by Example 4 using the corresponding intermediates. The corresponding boc protected intermediates of the numbered examples are also examples of the invention.
TABLE-US-00002 TABLE 2 LCMS Ex Structure Name Intermediates data .sup.1H NMR 5
##STR00040##
Example 7 (step 9.a): teat-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-[(4-chlorophenyl)carbamoyl]piperidine-1-carboxylate
[0375] ##STR00041##
[0376] To a solution of (2R,5S)-1-Rtert-butoxy)carbonyll -5-[2-(4-chloro-3-fluorophenoxy)acetamido]piperidine-2-carboxylic acid (90% purity, 200 mg, 0.418 mmol, intermediate 3), T3P (50% in EtOAc, 0.30 mL, 0.501 mmol) and DIPEA (150 μL, 0.836 mmol) in EtOAc (5 mL) was added 4-chloroaniline (53 mg, 0.418 mmol) and stirred at 80° C. for 1 h.
[0377] Further portions of T3P (50% in EtOAc, 99 μL, 0.167 mmol) and DIPEA (58 μL, 0.334 mmol) were added and the mixture was stirred at 80° C. for 1.5 h. The reaction mixture was cooled to r.t., diluted with H.sub.2O (20 mL), and extracted with EtOAc (3×20 mL). The combined organic extracts were washed with brine, dried over MgSO.sub.4, and concentrated in vacuo to afford the title compound (70% purity, 245 mg, 0.317 mmol, 76% yield) as a colourless oil; M/Z: 440, 442, 444 [M-Boc+H].sup.+, ESI.sup.+, RT=1.06 (S2). The product was taken on crude without further purification.
Example 8 (step 9.b): (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-(4-chlorophenyl)piperidine-2-carboxamide
[0378] ##STR00042##
[0379] To a solution of tert-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-[(4-chlorophenyl)carbamoyl]piperidine-1-carboxylate (70% purity, 245 mg, 0.317 mmol, Example 7) in anhydrous 1,4-dioxane (1 mL) was added 4 M HCl in 1,4-dioxane (0.79 mL, 3.17 mmol) and the mixture was stirred at r.t. for 2 h. The reaction mixture was concentrated in vacuo and the residue was purified by prep. HPLC (Method 1), followed by prep. HPLC (Method 3) to afford the title compound (21 mg, 0.0471 mmol, 15% yield) as a white solid; .sup.1H NMR (400 MHz, DMSO-d.sub.6) ϵ 9.70 (s, 1H), 7.82 (d, J=8.0 Hz, 1H), 7.71-7.63 (m, 2H), 7.48 (t, J=8.9 Hz, 1H), 7.36-7.28 (m, 2H), 7.06 (dd, J=11.4, 2.8 Hz, 1H), 6.86 (ddd, J=9.0, 2.8, 1.1 Hz, 1H), 4.51 (s, 2H), 3.73-3.63 (m, 1H), 3.08-3.00 (m, 1H), 2.46-2.36 (m, 2H), 1.99-1.85 (m, 2H), 1.55-1.41 (m, 2H); M/Z: 440, 442, 444 [M+H].sup.+, ESI.sup.+, RT=2.24 (S4).
[0380] The example compound in Table 3 was synthesised according to general route 9 as exemplified by Example 8 using the corresponding intermediates. The corresponding boc protected intermediate of the numbered example is also an example of the invention.
TABLE-US-00003 TABLE 3 LCMS Ex Structure Name Intermediates data .sup.1H NMR 9
##STR00044##
Example 10: (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-phenylpiperidine-2-carboxamide
[0381] ##STR00045##
[0382] To a solution of (2R,5S)-1-[(tert-butoxy)carbonyl]-5-[2-(4-chloro-3-fluorophenoxy)acetamido]piperidine-2-carboxylic acid (100 mg, 0.232 mmol, intermediate 3) in anhydrous 1,4-dioxane (2.5 mL) was added HATU (88 mg, 0.232 mmol) and DIPEA (81 μL, 0.464 mmol) and stirred at r.t. for 45 min. Aniline (163 μL, 0.232 mmol) was added and the mixture was stirred at r.t. for 4 h. The reaction mixture was cooled to 0° C. and 4 M HCl in 1,4-dioxane (1.0 mL, 4.00 mmol) was added dropwise under a flush of N.sub.2. The mixture was stirred at r.t. for 2 h, diluted with EtOAc (10 mL) and washed with H.sub.2O (5 mL). The organic extracts were washed with satd aq NaHCO.sub.3 solution, dried over MgSO.sub.4, and concentrated in vacuo. The residue was purified by prep. HPLC (Method 4) to afford the title compound (12 mg, 0.030 mmol, 13% yield) as a white solid; .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 9.66 (s, 1H), 7.94 (d, J=8.2 Hz, 1H), 7.64 (d, J=7.6 Hz, 2H), 7.51 (t, J=8.9 Hz, 1H), 7.36-7.24 (m, 2H), 7.14-6.97 (m, 2H), 6.90-6.79 (m, 1H), 4.52 (s, 2H), 3.78-3.60 (m, 1H), 3.23-3.14 (m, 1H), 3.12-2.94 (m, 1H), 2.45-2.38 (m, 2H), 2.00-1.86 (m, 2H), 1.47 (t, J=10.0 Hz, 2H); M/Z: 406, 408 [M+H].sup.+, ESI.sup.+, RT=3.10 (S6).
[0383] Example compounds in Table 4 were synthesised according to general route 10 as exemplified by Example 10 using the corresponding intermediates. The corresponding boc protected intermediates of the numbered examples are also examples of the invention.
TABLE-US-00004 TABLE 4 LCMS Ex Structure Name Intermediates data .sup.1H NMR 11
##STR00075##
Example 32 (step 11.a): tert-butyl (2R,5S)-2-[[3,5-bis(trifluoromethyl)phenyl]carbamoyl]-5-[[2-(4-chloro-3-fluoro-phenoxy)acetyl]amino]piperidine-1-carboxylate
[0384] ##STR00076##
[0385] To a solution of (2R,5S)-1-[(tert-butoxy)carbonyl]-5-[2-(4-chloro-3-fluorophenoxy)acetamido]piperidine-2-carboxylic acid (200 mg, 0.441 mmol, Intermediate 3) in anhydrous DCM (4 mL) at 0° C. was added 3,5-bis(trifluoromethyl)aniline (101 mg, 0.441 mmol), pyridine (178 μL, 2.20 mmol) and then POCl.sub.3 (101 mg, 0.661 mmol), and the mixture was stirred at r.t. for 18 h. The reaction mixture was diluted with DCM (6 mL), cooled to 0° C. and then carefully added dropwise to a solution of satd aq NaHCO.sub.3 solution (12 mL) at 0° C. The resultant solution was allowed to warm to r.t. whilst stirring for 1 h. The organic layer was isolated using a phase separator cartridge and then concentrated in vacuo to afford the title compound (70% purity, 312 mg, 0.340 mmol, 77% yield) as an orange solid; M/Z: 488, 490 [M+H].sup.+, RT=1.13 (S2). The product was taken forward without further purification.
Example 33 (step 11.b): (2R,5S)-N-[3,5-bis(trifluoromethyl)phenyl]-5-[2-(4-chloro-3-fluorophenoxy)acetamido]piperidine-2-carboxamide
[0386] ##STR00077##
[0387] To a solution of tert-butyl (2R,5S)-2-[[3,5-bis(trifluoromethyl)phenyl]carbamoyl]-5-[[2-(4-chloro-3-fluoro-phenoxy)acetyl]amino]piperidine-1-carboxylate (70% purity, 312 mg, 0.340 mmol, example 32) in anhydrous 1,4-dioxane (2 mL) was added 4 M HCl in 1,4-dioxane (3.0 mL, 3.00 mmol) and the mixture was stirred at r.t. for 2 h. The reaction mixture was concentrated in vacuo, and the residue was purified by prep. HPLC (Method 3) to afford the title compound (106 mg, 0.196 mmol, 58% yield) as a white powder; .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 10.67-10.11 (m, 1H), 8.42 (s, 2H), 7.96 (d, J=8.1 Hz, 1H), 7.75 (s, 1H), 7.51 (t, J=8.9 Hz, 1H), 7.08 (dd, J=11.4, 2.8 Hz, 1H), 6.87 (ddd, J=9.0, 2.8, 1.1 Hz, 1H), 4.53 (s, 2H), 3.82-3.64 (m, 1H), 3.25-3.19 (m, 1H), 3.09-3.00 (m, 1H), 2.45-2.38 (m, 1H), 2.03-1.85 (m, 2H), 1.57-1.41 (m, 2H); M/Z: 542, 544 [M+H].sup.+, ESI.sup.+, RT=2.61 (S4).
[0388] Example compounds in Table 5 were synthesised according to general route 11 as exemplified by Example 33 using the corresponding intermediates. The corresponding boc protected intermediates of the numbered examples are also examples of the invention.
TABLE-US-00005 TABLE 5 LCMS Ex Structure Name Intermediates data .sup.1H NMR 34
##STR00083##
Intermediate 8 (step 12.a): teat-butyl (2R,5S)-2-carbamoyl-5-[2-(4-chloro-3-fluorophenoxy)acetamido]piperidine-1-carboxylate
[0389] ##STR00084##
[0390] NMM (0.61 mL, 5.57 mmol) and isobutyl chloroformate (0.72 mL, 5.57 mmol) were added to a solution of (2R,5S)-1-[(tert-butoxy)carbonyl]-5-[2-(4-chloro-3-fluorophenoxy)acetamido]piperidine-2-carboxylic acid (Intermediate 3, 2.00 g, 4.64 mmol) in anhydrous THF (32 mL). After 20 mins, NH.sub.4OH (35%, 0.51 mL, 9.28 mmol) was added and the mixture was stirred at r.t. for 2 h. The reaction mixture was concentrated in vacuo and the resultant residue was dissolved in EtOAc (100 mL) and washed with H.sub.2O (50 mL). The organic extracts were dried over MgSO.sub.4 and concentrated in vacuo to afford the title compound (1.82 g, 4.02 mmol, 87% yield) as a white amorphous solid; .sup.1H NMR (500 MHz, CDCl.sub.3) ϵ 7.32 (t, J=8.6 Hz, 1H), 6.89-6.64 (m, 3H), 6.04 (s, 1H), 5.51 (s, 1H), 4.79 (s, 1H), 4.50-4.40 (m, 2H), 4.12 (s, 2H), 3.12 (d, J=13.6 Hz, 1H), 2.21-2.15 (m, 1H), 1.95-1.88 (m, 1H), 1.71-1.63 (m, 2H), 1.44 (s, 9H); M/Z: 452, 454 [M+Na].sup.+, ESI.sup.+, RT=0.82 (S2).
Example 48 (step 12.b): tert-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-{[2-(trifluoromethyl)pyrimidin-4-yl]carbamoyl}piperidine-1-carboxylate
[0391] ##STR00085##
[0392] To a solution of tert-butyl (2R,5S)-2-carbamoyl-5-[2-(4-chloro-3-fluorophenoxy)acetamido]piperidine-1-carboxylate (Intermediate 8, 129 mg, 0.300 mmol) in anhydrous 1,4-dioxane (2 mL) was added 4-bromo-2-(trifluoromethyl)pyrimidine (68 mg, mmol), Pd.sub.2(dba).sub.3 (14 mg, 0.0150 mmol), .sup.tBuOK (47 mg, 0.420 mmol) and XPhos (14 mg, 0.0300 mmol). The reaction vial was degassed under N.sub.2 before being heated at 120° C. under microwave irradiation for 2 h. The reaction mixture was diluted with EtOAc (10 mL) and washed with H.sub.2O (2×10 mL). The organic extracts were concentrated in vacuo and purified by FCC on silica gel (10-100% EtOAc in heptane) to afford the title compound (50% purity, 68 mg, 0.0590 mmol, 20% yield) as a yellow gum; M/Z: 476, 478 [M-Boc+H].sup.+, ESI.sup.+, RT=3.98 (S4).
Example 49 (step 12.c): (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-[2-(trifluoromethyl)pyrimidin-4-yl]piperidine-2-carboxamide
[0393] ##STR00086##
[0394] To a solution of tert-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-{[2-(trifluoromethyl)pyrimidin-4-yl]carbamoyl}piperidine-1-carboxylate (Example 48, 50% purity, 68 mg, 0.0590 mmol) in anhydrous 1,4-dioxane (0.6 mL) was added 4 M HCl in 1,4-dioxane (1.2 mL, 5.00 mmol) and stirred at r.t. overnight. The reaction mixture was quenched with NaHCO.sub.3 (15 mL) and washed with EtOAc (2×15 mL). The organic extracts were combined, dried over MgSO.sub.4, and concentrated in vacuo. The residue was purified by prep. HPLC (Method 3) to afford the title compound (9.0 mg, 0.0189 mmol, 32% yield) as a white powder; .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.88 (d, J=5.8 Hz, 1H), 8.29 (d, J=5.8 Hz, 1H), 7.93 (d, J=8.1 Hz, 1H), 7.50 (t, J=8.9 Hz, 1H), 7.07 (dd, J=11.4, 2.9 Hz, 1H), 6.85 (ddd, J=8.9, 1.8 Hz, 1H), 4.51 (s, 2H), 3.73-3.62 (m, 1H), 3.43-3.37 (m, 2H), 3.03-2.97 (m, 1H), 2.40-2.35 (m, 1H), 1.97-1.85 (m, 2H), 1.49-1.43 (m, 2H), 1.28-1.21 (m, 1H);); M/Z: 476, 478 [M+H].sup.+, ESI.sup.+, RT=1.98 (S4).
##STR00087##
Example 50 (step 13.a): tert-butyl (2R,5S)-5-[2-(4-chloro-3-11uorophenoxy)acetamido]-2-{[6-(trifluoromethyl)pyrazin-2-yl]carbamoyl}piperidine-1-carboxylate
[0395] ##STR00088##
[0396] Vial A was charged with Josiphos SL-J009-1 (12 mg, 0.0221 mmol) and Pd.sub.2(dba).sub.3 (10 mg, 0.0110 mmol). Vial B was charged with tert-butyl (2R,5S)-2-carbamoyl-5-[2-(4-chloro-3-fluorophenoxy)acetamido]piperidine-1-carboxylate (Intermediate 8, 95 mg, 0.220 mmol), .sup.tBuONa (42 mg, 0.441 mmol) and 2-bromo-6-(trifluoromethyl)pyrazine (50 mg, 0.220 mmol). Both vials were sealed and purged with N.sub.2. Degassed DME (4 mL) was added to vial A and the solution was stirred for 5 mins to form the active catalyst solution. This solution was then added to vial B and the mixture was stirred at 100° C. for 18 h. The reaction mixture was allowed to cool to r.t., diluted with H.sub.2O (10 mL) and extracted with EtOAc (2×10 mL). The combined organic extracts were washed with brine (10 mL), dried over Na.sub.2SO.sub.4 and concentrated in vacuo to afford the title compound (34% purity, 160 mg, 0.0945 mmol, 43% yield) as a brown oil; M/Z: 476, 478 [M-Boc+H].sup.+, ESI.sup.+, RT=1.05 (S2).
Example 51 (step 13.b): (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-[6-(trifluoromethyl)pyrazin-2-yl]piperidine-2-carboxamide
[0397] ##STR00089##
[0398] To a solution of tert-butyl (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-2-{[6-(trifluoromethyl)pyrazin-2-yl]carbamoyl}piperidine-1-carboxylate (Example 50, 34% purity, 160 mg, 0.0945 mmol) in anhydrous 1,4-dioxane (1 mL) was added 4 M HCl in 1,4-dioxane (2.0 mL, 8.00 mmol) and the mixture was stirred at r.t. for 24 h. The reaction mixture was quenched with NaHCO.sub.3 (15 mL) and washed with EtOAc (2×15 mL). The combined organic extracts were dried over MgSO.sub.4, concentrated in vacuo, and purified by prep. HPLC (Method 3) to afford the title compound (26 mg, 0.0544 mmol, 58% yield) as a yellow powder; .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 9.62 (s, 1H), 8.88 (s, 1H), 7.94 (d, J=8.2 Hz, 1H), 7.50 (t, J=8.9 Hz, 1H), 7.07 (dd, J=11.4, 2.8 Hz, 1H), 6.86 (ddd, J=9.0, 2.8, 1.1 Hz, 1H), 4.51 (s, 2H), 3.73-3.65 (m, 1H), 3.40 (dd, J=10.2, 2.9 Hz, 1H), 3.01 (dd, J=12.3, 3.0 Hz, 1H), 2.44-2.38 (m, 1H), 2.00-1.86 (m, 2H), 1.53-1.44 (m, 2H); M/Z: 476, 478 [M+H].sup.+, ESI.sup.+, RT=1.96 (S4).
##STR00090##
Example 52: (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-1-methyl-N-[6-(trifluoromethyl)pyrazin-2-yl]piperidine-2-carboxamide
[0399] ##STR00091##
[0400] To a solution of (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-[6-(trifluoromethyl)pyrazin-2-yl]piperidine-2-carboxamide (Example 51, 90% purity, 33 mg, 0.0624 mmol) and K.sub.2CO.sub.3 (17 mg, 0.125 mmol) in DMF (1 mL) was added Mel (3.5 !IL, 0.0562 mmol) and the mixture was stirred at r.t. for 2 h. The reaction mixture was diluted with EtOAc (20 mL) and washed with H.sub.2O (10 mL). The organic extracts were dried over MgSO.sub.4, concentrated in vacuo, and purified by prep. HPLC (Method 3) to afford the title compound (18 mg, 0.0357 mmol, 57% yield) as a white amorphous solid; .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 11.01 (s, 1H), 9.63 (s, 1H), 8.88 (s, 1H), 8.03 (d, J=8.2 Hz, 1H), 7.50 (t, J=8.9 Hz, 1H), 7.08 (dd, J=11.4, 2.8 Hz, 1H), 6.88-6.83 (m, 1H), 4.52 (s, 2H), 4.00-3.82 (m, 1H), 2.94 (dd, J=20 10.7, 3.6 Hz, 1H), 2.80 (dd, J=10.8, 2.8 Hz, 1H), 2.17 (s, 3H), 1.94-1.79 (m, 3H), 1.76-1.64 (m, 1H), 1.39-1.27 (m, 1H); M/Z: 490, 492 [M+H].sup.+, ESI.sup.+, RT=2.10 (S4).
##STR00092##
Example 53 and 54: Chiral separation of (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-[3-(trifluoromethoxy)cyclopentyl]piperidine-2-carboxamide (Example 47)
[0401] (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy) acetamido]-N-[3-(trifluoromethoxy)cyclopentyl]piperidine-2-carboxamide (148 mg, Example 47) was subjected to chiral purification using the following method:
[0402] Purification method=90:10 heptane: EtOH+0.2% DEA; Amylose-2, 21.2×250 mm, 5 μm, at 18 ml/min. Sample diluent: MeOH, IPA.
[0403] Followed by:
[0404] Purification method=90:10 heptane: IPA; Chiralpak AD-H, 20×250 mm, 5 μm, at 18 ml/min. Sample diluent: MeOH, IPA.
[0405] This afforded (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-[(1S,3S)-3-(trifluoromethoxy)cyclopentyl]piperidine-2-carboxamide (Example 53, 4 mg) and (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-[(1R,3R)-3-(trifluoromethoxy)cyclopentyl]piperidine-2-carboxamide (Example 54, 18 mg), as well as two isomers (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N-[(1R,3S)-3-(trifluoromethoxy)cyclopentyl]piperidine-2-carboxamide and (2R,5S)-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-N[(1S,3R)-3-(trifluoromethoxy)cyclopentyl]piperidine-2-carboxamide as white powders. The stereochemistry in the pentyl ring of each compound was arbitrarily assigned.
[0406] Example compounds in Table 6 were chirally purified according to the general route 15 as exemplified by Example 53 and 54, using the corresponding intermediates and methods.
TABLE-US-00006 TABLE 6 Intermediate LCMS Ex Structure Name and Method data 1H NMR 53
II Assays
HEK-ATF4 High Content Imaging Assay
[0407] Example compounds were tested in the HEK-ATF4 High Content Imaging assay to assess their pharmacological potency to prevent Tunicamycin induced ISR. Wild-type HEK293 cells were plated in 384-well imaging assay plates at a density of 12,000 cells per well in growth medium (containing DMEM/F12, 10% FBS, 2 mM L-Glutamine, 100 U/mL Penicillin—100 μg/mL Streptomycin) and incubated at 37° C., 5% CO.sub.2. 24 h later, the medium was changed to 50 μL assay medium per well (DMEM/F12, 0.3% FBS, 2mM L-Glutamine, 100 U/mL Penicillin—100 μg/mL Streptomycin). Example compounds were serially diluted in DMSO, spotted into intermediate plates and prediluted with assay medium containing 3.3 1,1M Tunicamycin to give an 11-fold excess of final assay concentration. In addition to the example compound testing area, the plates also contained multiples of control wells for assay normalization purposes, wells containing Tunicamycin but no example compounds (High control), as well as wells containing neither example compound nor Tunicamycin (Low control). The assay was started by transferring 5 μL from the intermediate plate into the assay plates, followed by incubation for 6 h at 37° C., 5% CO.sub.2. Subsequently, cells were fixed (4% PFA in PBS, 20 min at r.t.) and submitted to indirect ATF4 immunofluorescence staining (primary antibody rabbit anti ATF4, clone D4B8, Cell Signaling Technologies; secondary antibody Alexa Fluor 488 goat anti-rabbit IgG (H+L), Thermofisher Scientific). Nuclei were stained using Hoechst dye (Thermofisher Scientific), and plates were imaged on an Opera Phenix High Content imaging platform equipped with 405 nm and 488 nm excitation. Finally, images were analyzed using script based algorithms. The main readout HEK-ATF4 monitored the ATF4 signal ratio between nucleus and cytoplasm. Tunicamycin induced an increase in the overall ATF4 ratio signal, which was prevented by ISR modulating example compounds. In addition, HEK-CellCount readout was derived from counting the number of stained nuclei corresponding to healthy cells. This readout served as an internal toxicity control. The example compounds herein did not produce significant reduction in CellCount.
[0408] HEK ATF4 Activity of the tested example compounds is provided in Table 6 as follows: [0409] +++=IC.sub.50 1-500 nM; ++=IC.sub.50>500-2000 nM; +=IC50>2000-15000 nM.
TABLE-US-00007 TABLE 6 Example number HEK-ATF4 Activity 2 + 4 +++ 5 + 6 + 8 +++ 9 + 10 + 11 +++ 12 +++ 13 +++ 14 + 15 + 16 + 17 + 18 +++ 19 + 20 + 21 +++ 22 +++ 23 ++ 24 +++ 25 ++ 26 + 27 ++ 28 + 29 + 30 ++ 31 +++ 33 ++ 34 ++ 35 ++ 36 +++ 37 +++ 38 ++ 39 +++ 40 +++ 41 +++ 42 +++ 43 ++ 44 + 45 + 46 + 47 ++ 49 +++ 51 +++ 52 +++ 53 +++ 54 ++
REFERENCES
[0410] (1) Pakos-Zebrucka K, Koryga I, Mnich K, Ljujic M, Samali A, Gorman A M. The integrated stress response. EMBO Rep. 2016 October; 17(10):1374-1395. Epub 2016 Sep. 14. [0411] (2) Wek R C, Jiang H Y, Anthony T G. Coping with stress: eIF2 kinases and translational control. Biochem Soc Trans. 2006 February; 34(Pt 1):7-11. [0412] (3) Donnelly N, Gorman A M, Gupta S, Samali A. The eIF2alpha kinases: their structures and functions. Cell Mol Life Sci. 2013 October; 70(19):3493-511 [0413] (4) Jackson R J, Hellen C U, Pestova T V. The mechanism of eukaryotic translation initiation and principles of its regulation. Nat Rev Mol Cell Biol. 2010 February; 11(2):113-27 [0414] (5) Lomakin I B, Steitz T A. The initiation of mammalian protein synthesis and mRNA scanning mechanism. Nature. 2013 Aug. 15; 500(7462):307-11 [0415] (6) Pain V M. Initiation of protein synthesis in eukaryotic cells. Eur J Biochem. 1996 Mar. 15; 236(3):747-71 [0416] (7) Pavitt G D. Regulation of translation initiation factor eIF2B at the hub of the integrated stress response. Wiley Interdiscip Rev RNA. 2018 November; 9(6):e1491. [0417] (8) Krishnamoorthy T, Pavitt G D, Zhang F, Dever T E, Hinnebusch A G. Tight binding of the phosphorylated alpha subunit of initiation factor 2 (eIF2alpha) to the regulatory subunits of guanine nucleotide exchange factor eIF2B is required for inhibition of translation initiation. Mol Cell Biol. 2001 August; 21(15):5018-30. [0418] (9) Hinnebusch, A. G., Ivanov, I. P., & Sonenberg, N. (2016). Translational control by 5′-untranslated regions of eukaryotic mRNAs. Science, 352(6292), 1413-1416. [0419] (10) Young, S. K., & Wek, R. C. (2016). Upstream open reading frames differentially regulate gene-specific translation in the integrated stress response. The Journal of Biological Chemistry, 291(33), 16927-16935. [0420] (11) Lin J H, Li H, Zhang Y, Ron D, Walter P (2009) Divergent effects of PERK and IREl signaling on cell viability. PLoS ONE 4: e4170 [0421] (12) Tabas I, Ron D. Nat Cell Biol. 2011 March; 13(3):184-90. Integrating the mechanisms of apoptosis induced by endoplasmic reticulum stress. [0422] (13) Shore G C, Papa F R, Oakes S A. Curr Opin Cell Biol. 2011 April; 23(2):143-9. Signaling cell death from the endoplasmic reticulum stress response. [0423] (14) Bi M, Naczki C, Koritzinsky M, Fels D, Blais J, Hu N, Harding H, Novoa I, Varia M, Raleigh J, Scheuner D, Kaufman R J, Bell J, Ron D, Wouters B G, Koumenis C. EMBO J. 2005 Oct. 5; 24(19):3470-81 ER stress-regulated translation increases tolerance to extreme hypoxia and promotes tumor growth. [0424] (15) Bobrovnikova-Marjon E, Grigoriadou C, Pytel D, Zhang F, Ye J, Koumenis C, Cavener D, Diehl J A. Oncogene. 2010 Jul. 8; 29(27):3881-95 PERK promotes cancer cell proliferation and tumor growth by limiting oxidative DNA damage. [0425] (16) Avivar-Valderas A, Salas E, Bobrovnikova-Marjon E, Diehl J A, Nagi C, Debnath J, Aguirre-Ghiso J A. Mol Cell Biol. 2011 September; 31(17):3616-29. PERK integrates autophagy and oxidative stress responses to promote survival during extracellular matrix detachment. [0426] (17) Blais, J. D.; Addison, C. L.; Edge, R.; Falls, T.; Zhao, H.; Kishore, W.; Koumenis, C.; Harding, H. P.; Ron, D.; Holcik, M.; Bell, J. C. Mol. Cell. Biol. 2006, 26, 9517-9532.PERK-dependent translational regulation promotes tumor cell adaptation and angiogenesis in response to hypoxic stress. [0427] (18) Taalab Y M, Ibrahim N, Maher A, Hassan M, Mohamed W, Moustafa A A, Salama M, Johar D, Bernstein L. Rev Neurosci. 2018 Jun. 27; 29(4):387-415. Mechanisms of disordered neurodegenerative function: concepts and facts about the different roles of the protein kinase RNA-like endoplasmic reticulum kinase (PERK). [0428] (19) Remondelli P, Renna M. Front Mol Neurosci. 2017 Jun. 16; 10:187. The Endoplasmic Reticulum Unfolded Protein Response in Neurodegenerative Disorders and Its Potential Therapeutic Significance. [0429] (20) Halliday M, Mallucci G R. Neuropathol Appl Neurobiol. 2015 June; 41(4):414-27.Review: Modulating the unfolded protein response to prevent neurodegeneration and enhance memory. [0430] (21) Halliday M, Radford H, Sekine Y, Moreno J, Verity N, le Quesne J, Ortori C A, Barrett D A, Fromont C, Fischer P M, Harding H P, Ron D, Mallucci G R. Cell Death Dis. 2015 Mar. 5; 6:e1672.Partial restoration of protein synthesis rates by the small molecule ISRIB prevents neurodegeneration without pancreatic toxicity. [0431] (22) Moreno J A, Radford H, Peretti D, Steinert J R, Verity N, Martin M G, Halliday M, Morgan J, Dinsdale D, Ortori C A, Barrett D A, Tsaytler P, Bertolotti A, Willis A E, Bushell M, Mallucci G R. Nature 2012; 485: 507-11. Sustained translational repression by eIF2alpha-P mediates prion neurodegeneration. [0432] (23) Skopkova M, Hennig F, Shin B S, Turner C E, Stanikova D, Brennerova K, Stanik J, Fischer U, Henden L, Muller U, Steinberger D, Leshinsky-Silver E, Bottani A, Kurdiova T, Ukropec J, Nyitrayova O, Kolnikova M, Klimes I, Borck G, Bahlo M, Haas S A, Kim J R, Lotspeich-Cole L E, Gasperikova D, Dever T E, Kalscheuer V M. Hum Mutat. 2017 April; 38(4):409-425. EIF2S3 Mutations Associated with Severe X-Linked Intellectual Disability Syndrome MEHMO. [0433] (24) Hamilton E M C, van der Lei H D W, Vermeulen G, Gerver J A M, Lourenco C M, Naidu S, Mierzewska H, Gemke R J B J, de Vet H C W, Uitdehaag B M J, Lissenberg-Witte B I; VWM Research Group, van der Knaap MS. Ann Neurol. 2018 August; 84(2):274-288. Natural History of Vanishing White Matter. [0434] (25) Bugiani M, Vuong C, Breur M, van der Knaap MS. Brain Pathol. 2018 May; 28(3):408-421. Vanishing white matter: a leukodystrophy due to astrocytic dysfunction. [0435] (26) Wong Y L, LeBon L, Edalji R, Lim H B, Sun C, Sidrauski C. Elife. 2018 Feb. 28; 7. The small molecule ISRIB rescues the stability and activity of Vanishing White Matter Disease eIF2B mutant complexes. [0436] (27) Wong Y L, LeBon L, Basso A M, Kohlhaas K L, Nikkel A L, Robb H M, Donnelly-Roberts D L, Prakash J, Swensen A M, Rubinstein N D, Krishnan S, McAllister F E, Haste N V, O'Brien J J, Roy M, Ireland A, Frost J M, Shi L, Riedmaier S, Martin K, Dart M J, Sidrauski C. Elife. 2019 Jan. 9; 8. eIF2B activator prevents neurological defects caused by a chronic integrated stress response. [0437] (28) Nguyen H G, Conn CS, Kye Y, Xue L, Forester C M, Cowan J E, Hsieh A C, Cunningham J T, Truillet C, Tameire F, Evans M J, Evans C P, Yang J C, Hann B, Koumenis C, Walter P, Carroll P R, Ruggero D. Sci Transl Med. 2018 May 2; 10(439). Development of a stress response therapy targeting aggressive prostate cancer. [0438] (29) Waring M, Expert Opinion on Drug Discovery Volume 5, 2010—Issue 3, 235-248. Lipophilicity in Drug Discovery. [0439] (30) Alelyunas Y W, et. al. Bioorg. Med. Chem. Lett., 20(24) 2010, 7312-7316. Experimental solubility profiling of marketed CNS drugs, exploring solubility limit of CNS discovery candidate. [0440] (31) Redfern W S, et.al., Cardiovascular Research 58(2003), 32-45. Relationships between preclinical cardiac electrophysiology, clinical QT interval prolongation and torsade de pointes for a broad range of drugs.