MODULATORS OF THE INTEGRATED STRESS RESPONSE PATHWAY
20230129907 · 2023-04-27
Inventors
- Holly Victoria Atton (Abingdon, GB)
- Christopher John Brown (Abingdon, GB)
- James Lindsay CARR (Abingdon, GB)
- Scott Alexander SADLER (Abingdon, GB)
- Jonathan Paul SHINE (Abingdon, GB)
- Daryl Simon WALTER (Abingdon, GB)
Cpc classification
A61K45/06
HUMAN NECESSITIES
A61P25/18
HUMAN NECESSITIES
C07D413/04
CHEMISTRY; METALLURGY
A61K31/357
HUMAN NECESSITIES
A61K31/4439
HUMAN NECESSITIES
C07D405/04
CHEMISTRY; METALLURGY
International classification
C07D413/04
CHEMISTRY; METALLURGY
C07D405/04
CHEMISTRY; METALLURGY
Abstract
##STR00001##
The present invention relates to compounds of formula (I) or pharmaceutically acceptable salts, solvates, hydrates, tautomers or stereoisomers thereof, wherein R.sup.1, R.sub.2, R.sup.2a, R.sup.3, R.sup.a1, R.sup.a2, R.sup.a3, R.sup.a4, R.sup.a5, R.sup.a6, A.sup.1 and A.sup.2 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 and preventing of one or more diseases or disorders associated with integrated stress response.
Claims
1-26. (canceled)
27. A compound of formula (I) ##STR00042## wherein: R.sup.a1, R.sup.a2, R.sup.a3, R.sup.a4, R.sup.a5, and R.sup.a6 are independently selected from the group consisting of H, a halogen, C.sub.1-4 alkyl, and A.sup.2a, wherein C.sub.1-4 alkyl is unsubstituted or substituted with substituents selected from the group consisting of a halogen, OH, and O—C.sub.1-3 alkyl, and wherein only one of R.sup.a1, R.sup.a2, R.sup.a3, R.sup.a4, R.sup.a5, and R.sup.a6 is A.sup.2a; ring A.sup.1 is C.sub.5 cycloalkylene, C.sub.5 cycloalkenylene, or a nitrogen-containing 5-membered heterocyclene where the atom of ring A.sup.1 marked with an asterisk is a carbon atom, and wherein ring A.sup.1 is unsubstituted or substituted with one or more R.sup.4; each R.sup.4 is independently oxo (═O), thiooxo (═S), a halogen, CN, OR.sup.5, or C.sub.1-6alkyl, wherein each C.sub.1-6 alkyl is independently unsubstituted or substituted with one or more halogen, which are the same or different; R.sup.5 is H or C.sub.1-6 alkyl that is unsubstituted or substituted with one or more halogen, which are the same or different; A.sup.2 is R.sup.6a or A.sup.2a; R.sup.6a is OR.sup.6a1, SR.sup.6a1, N(R.sup.6a1R.sup.6a2), 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 unsubstituted or substituted with one or more substituents selected from the group consisting of a halogen, OR.sup.6a3, CN, and A.sup.2a, wherein the substituents are the same or different; R.sup.6a1 and R.sup.6a2 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.2a, wherein C.sub.1-6 alkyl, C.sub.2-6 alkenyl, and C.sub.2-6 alkynyl are unsubstituted or substituted with one or more substituents selected from the group consisting of a halogen, CN, OR.sup.6a3, A.sup.2a, and OA.sup.2a, wherein the substituents are the same or different; R.sup.6a3 is H or C.sub.1-4 alkyl, wherein C.sub.1-4 alkyl is unsubstituted or substituted with one or more halogen, which are the same or different; each A.sup.2a is independently phenyl, C.sub.3-7 cycloalkyl, C.sub.4-12 bicycloalkyl, or 3- to 7-membered heterocyclyl, wherein each A.sup.2a is independently unsubstituted or substituted with one or more R.sup.6, which are the same or different; each R.sup.6 is independently R.sup.6b, OH, OR.sup.6b, a halogen, or CN, wherein each R.sup.6b is independently cyclopropyl, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, or C.sub.2-6 alkynyl, wherein each R.sup.6b is independently unsubstituted or substituted with one or more halogen, which are the same or different, or two R.sup.6 are joined together with the atoms to which they are attached to form a ring A.sup.2b; A.sup.2b is independently phenyl, C.sub.3-7 cycloalkyl, or a 3- to 7-membered heterocyclyl, wherein A.sup.2b is independently unsubstituted or substituted with one or more R.sup.7, which are the same or different; each R.sup.7 is independently 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 independently unsubstituted or substituted with one or more halogen, which are the same or different; R.sup.1 is H or C.sub.1-4 alkyl, wherein C.sub.1-4 alkyl is unsubstituted or 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 unsubstituted or substituted with one or more halogen, which are the same or different, and R.sup.3 is A.sup.3, 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 unsubstituted or substituted with one or more R.sup.8, which are the same or different, or R.sup.2 and R.sup.3 are joined together with the oxygen atom and carbon atom to which they are attached to form a ring A.sup.3a, wherein ring A.sup.3a is a 7- to 12-membered heterobicyclyl, wherein the 7- to 12-membered heterobicyclyl is unsubstituted or substituted with one or more R.sup.10, which are the same or different; R.sup.2a is H or F; each R.sup.8 is independently a halogen, CN, C(O)OR.sup.9, OR.sup.9, C(O)R.sup.9, C(O)N(R.sup.9R.sup.9a), S(O).sub.2N(R.sup.9R.sup.9a), S(O)N(R.sup.9R.sup.9a), S(O).sub.2R.sup.9, S(O)R.sup.9, N(R.sup.9)S(O).sub.2N(R.sup.9aR.sup.9b), SR.sup.9, N(R.sup.9R.sup.9a), NO.sub.2, OC(O)R.sup.9, N(R.sup.9)C(O)R.sup.9a, N(R.sup.9)SO.sub.2R.sup.9a, N(R.sup.9)S(O)R.sup.9a, N(R.sup.9)C(O)N(R.sup.9aR.sup.9b), N(R.sup.9)C(O)OR.sup.9a, OC(O)N(R.sup.9R.sup.9a), or A.sup.3; each R.sup.9, R.sup.9a, and R.sup.9b 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 unsubstituted or substituted with one or more halogen, which are the same or different, or one OH, or one OC.sub.1-4 alkyl, or one A.sup.3; each A.sup.3 is independently phenyl, naphthyl, C.sub.3-7 cycloalkyl, a 3- to-7 membered heterocyclyl, or a 7-to 12-membered heterobicyclyl, wherein each A.sup.3 is independently unsubstituted or substituted with one or more R.sup.10, which are the same or different; each R.sup.10 is independently a halogen, CN, C(O)OR.sup.11, OR.sup.11, C(O)R.sup.11, C(O)N(R.sup.11R.sup.11a), S(O).sup.2N(R.sup.11R.sup.11a), S(O)N(R.sup.11R.sup.11a), S(O).sub.2R.sup.11, S(O)R.sup.11, N(R.sup.11)S(O).sub.2N(R.sup.11aR.sup.11b), SR.sup.11, N(R.sup.11R.sup.11a), NO.sub.2, OC(O)R.sup.11, N(R.sup.11)C(O)R.sup.11a, N(R.sup.11)S(O).sub.2R.sup.11a, N(R.sup.11)S(O)R.sup.11a, N(R.sup.11)C(O)OR.sup.11a, N(R.sup.11)C(O)N(R.sup.11aR.sup.11b), OC(O)N(R.sup.11R.sup.11a), oxo (═O), C.sub.1-6alkyl, 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 unsubstituted or substituted with one or more R.sup.12, which are the same or different; each R.sup.11, R.sup.11a, and R.sup.11b 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 unsubstituted or substituted with one or more halogen, which are the same or different; each R.sup.12 is independently a halogen, CN, C(O)OR.sup.13, OR.sup.13, C(O)R.sup.13, C(O)N(R.sup.13R.sup.13a), S(O).sub.2N(R.sup.13R.sup.13a), S(O)N(R.sup.13R.sup.13a), S(O).sub.2R.sup.13, S(O)R.sup.13, N(R.sup.13)S(O).sub.2N(R.sup.13aR.sup.13b), SR.sup.3, N(R.sup.13R.sup.13a), NO.sub.2, OC(O)R.sup.13, N(R.sup.13)C(O)R.sup.13a, N(R.sup.13)SO.sub.2R.sup.13a, N(R.sup.13)S(O)R.sup.13a, N(R.sup.13)C(O)N(R.sup.13aR.sup.13b), NR.sup.13)C(O)OR.sup.13a, or OC(O)N(R.sup.13R.sup.13a); and each R.sup.13, R.sup.13a, and R.sup.13b 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 unsubstituted or substituted with one or more halogen, which are the same or different.
28. The compound of claim 27, wherein R.sup.a1, R.sup.a2, R.sup.a3, R.sup.a4, R.sup.a5, and R.sup.a6 are H.
29. The compound of claim 27, wherein the ring A.sup.1 is a nitrogen-containing 5-membered heterocyclene and wherein the ring A.sup.1 is unsubstituted or substituted with one or more R.sup.4, which are the same or different.
30. The compound of claim 27, wherein the ring A.sup.1 is a nitrogen-containing 5-membered heterocyclene selected from the group of bivalent heterocycles consisting of oxadiazole, imidazole, imidazolidine, pyrazole, and triazole, and wherein A.sup.1 is unsubstituted or substituted with one or more R.sup.4, which are the same or different.
31. The compound of claim 27, wherein the ring A.sup.1 is unsubstituted or substituted with one or two R.sup.4, which are the same or different.
32. The compound of claim 27, wherein R.sup.4 is oxo (═O).
33. The compound of claim 27, wherein the ring A.sup.1 is ##STR00043##
34. The compound of claim 27, wherein A.sup.2 is R.sup.6a.
35. The compound of claim 34, wherein: R.sup.6a is OR.sup.6a1 and R.sup.6a1 is A.sup.2a or C.sub.1-6 alkyl, wherein C.sub.1-6 alkyl is unsubstituted or substituted with one or more halogen, which are the same or different, and/or one A.sup.2a, and/or one OR.sup.6a3; or R.sup.6a is C.sub.1-6 alkyl, wherein the C.sub.1-6 alkyl unsubstituted or substituted with one or more halogen, which are the same or different, and/or one A.sup.2a, and/or one OR.sup.6a3.
36. The compound of claim 34, wherein: R.sup.6a is OR.sup.6a1 and R.sup.6a1 is C.sub.1-6 alkyl, wherein the C.sub.1-6 alkyl unsubstituted or substituted with one or more F and/or one OR.sup.6a3; or R.sup.6a is C.sub.1-6 alkyl, wherein the C.sub.1-6 alkyl unsubstituted or substituted with one or more halogen, which are the same or different, and/or one OR.sup.6a3.
37. The compound of claim 27, wherein A.sup.2 is A.sup.2a.
38. The compound of claim 37, wherein A.sup.2a is phenyl, cyclobutyl, azetidinyl, or a 5- to 6-membered aromatic heterocyclyl, and wherein A.sup.2a is unsubstituted or substituted with one or more R.sup.6, which are the same or different.
39. The compound of claim 27, wherein A.sup.2a is substituted with one or two R.sup.6, which are the same or different.
40. The compound of claim 27, wherein each R.sup.6 is independently F, Cl, CF.sub.3, OCH.sub.3, OCF.sub.3, CH.sub.3, CH.sub.2CH.sub.3, or cyclopropyl.
41. The compound of claim 27, wherein R.sup.2 is H.
42. The compound of claim 27, wherein R.sup.3 is A.sup.3.
43. The compound of claim 27, wherein A.sup.3 is phenyl, pyridyl, pyrazinyl, pyrimidazyl, cyclopropyl, cyclobutyl, or cyclohexyl and wherein A.sup.3 is unsubstituted or substituted with one or more R.sup.10, which are the same or different.
44. The compound of claim 27, wherein A.sup.3 is substituted with one or two R.sup.10, which are the same or different.
45. The compound of claim 27, wherein R.sup.2 and R.sup.3 are joined together with the oxygen and carbon atom to which they are attached to form a dihydrobenzopyran ring, wherein the ring is unsubstituted or substituted with one or more R.sup.10, which are the same or different.
46. The compound of claim 27, wherein each R.sup.10 is independently F, Cl, Br, CHF.sub.2, CF.sub.3, OCF.sub.3, CH═O, CH.sub.2OH, or CH.sub.3.
47. The compound of claim 27, wherein the compound is selected from the group consisting of: 2-(4-chloro-3-fluorophenoxy)-N-[trans-2-[5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl]-1,3-dioxan-5-yl]acetamide; 2-(4-chloro-3-fluorophenoxy)-N-[trans-2-{5-[cis-3-(trifluoromethoxy)cyclobutyl]-1,3,4-oxadiazol-2-yl}-1,3-dioxan-5-yl]acetamide; 2-(4-chloro-3-fluorophenoxy)-N-[cis-2-[1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl]-1,3-dioxan-5-yl]acetamide; 2-(4-chloro-3-fluorophenoxy)-N-[trans-2-[1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl]-1,3-dioxan-5-yl]acetamide; 2-(4-chloro-3-fluorophenoxy)-N-[trans-2-[5-(4,4,4-trifluorobutoxy)-1,3,4-oxadiazol-2-yl]-1,3-dioxan-5-yl]acetamide; 2-(4-chloro-3-fluorophenoxy)-N-[trans-2-{5-[2-(trifluoromethoxy)ethoxy]-1,3,4-oxadiazol-2-yl}-1,3-dioxan-5-yl]acetamide; 2-(4-chloro-3-fluorophenoxy)-N-[trans-2-{5-[3-(trifluoromethoxy)azetidin-1-yl]-1,3,4-oxadiazol-2-yl}-1,3-dioxan-5-yl]acetamide; 2-[(6-chloro-5-fluoro-3-pyridyl)oxy]-N-[trans-2-[5-[cis-3-(trifluoromethoxy)cyclobutyl]-1,3,4-oxadiazol-2-yl]-1,3-dioxan-5-yl]acetamide; 2-[3-chloro-4-(difluoromethyl)phenoxy]-N-[trans-2-{5-[3-(trifluoromethoxy)azetidin-1-yl]-1,3,4-oxadiazol-2-yl}-1,3-dioxan-5-yl]acetamide; and 2-(3,4-dichlorophenoxy)-N-[trans-2-{5-[2-(trifluoromethoxy)ethoxy]-1,3,4-oxadiazol-2-yl}-1,3-dioxan-5-yl]acetamide.
48. The compound of claim 27, wherein the compound is of formula (Ia) ##STR00044##
49. A pharmaceutical composition comprising one or more compounds of claim 27 and a pharmaceutically acceptable carrier.
50. A method for treating one or more diseases or disorders associated with integrated stress response in a patient in need thereof, wherein the method comprises administering to the patient a therapeutically effective amount of a pharmaceutical composition comprising one or more compounds of claim 27.
51. A method for treating one or more diseases or disorders in a patient in need thereof, wherein the method comprises administering to the patient a therapeutically effective amount of a pharmaceutical composition comprising one or more compounds of claim 27, wherein the one or more 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
[0141] The following Abbreviations and Acronyms are used:
[0142] aq aqueous
[0143] ACN acetonitrile
[0144] BF.sub.3.OEt.sub.2 boron trifluoride diethyl etherate
[0145] Brine saturated solution of NaCl in water
[0146] Burgess reagent 1-methoxy-N-triethylammoniosulfonyl-methanimidate
[0147] CDI di-1H-imidazol-1-ylmethanone
[0148] CSA Camphorsulfonic acid
[0149] CV column volume
[0150] δ chemical shifts in parts per million
[0151] DAST N,N-diethylaminosuflur trifluoride
[0152] DCM dichloromethane
[0153] DMSO dimethylsulfoxide
[0154] DMSO-d.sub.6 deuterated dimethylsulfoxide
[0155] DIPEA diisopropylethylamine
[0156] DMF dimethyl formamide
[0157] EDCI 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride salt
[0158] ELS evaporative light scattering
[0159] ESI+ positive ionisation mode
[0160] ESI− negative ionisation mode
[0161] EtOAc ethyl acetate
[0162] EtOH ethanol
[0163] FCC flash column chromatography
[0164] g gram(s)
[0165] HATU 1-[bis(dimethylamino)methylidene]-1H-[1,2,3]triazolo[4,5-b]pyridin-1-ium 3-oxide hexafluorophosphate
[0166] HCl hydrochloric acid
[0167] HOAt 1-hydroxy-7-azabenzotriazole
[0168] HPLC high-performance liquid chromatography
[0169] h hour(s)
[0170] H.sub.2 hydrogen atmosphere
[0171] J NMR coupling constant
[0172] LC liquid chromatography
[0173] LCMS liquid chromatography mass spectrometry
[0174] MgSO.sub.4 magnesium sulphate
[0175] M molar
[0176] mg milligram(s)
[0177] MHz megahertz(s)
[0178] mL millilitre (s)
[0179] min minute(s)
[0180] mm millimetre(s)
[0181] mM millimolar
[0182] mol mole(s)
[0183] nm nanometre(s)
[0184] MW microwave
[0185] MTBE methyl tent-butyl ether
[0186] M/Z mass to charge ratio
[0187] N.sub.2 nitrogen atmosphere
[0188] Na.sub.2SO.sub.4 sodium sulfate
[0189] NaBH.sub.4 sodium borahydride
[0190] NaClO.sub.2 sodium chlorite
[0191] NaHCO.sub.3 sodium bicarbonate
[0192] NH.sub.4Cl ammonium chloride
[0193] NaH.sub.2PO.sub.4 sodium dihydrogen phosphate
[0194] NMM 4-methylmorpholine
[0195] NMR nuclear magnetic resonance
[0196] NaOCl sodium hypochlorite
[0197] PDA photometric diode array
[0198] Pd/C Palladium on carbon
[0199] prep. preparative
[0200] PTSA para-toluenesulfonic acid monohydrate
[0201] r.t. room temperature
[0202] RT retention time
[0203] satd saturated
[0204] sec second(s)
[0205] TEMPO (2,2,6,6-Tetramethylpiperidin-1-yl)oxidanyl
[0206] THF Tetrahydrofuran
[0207] Tol Toluene
[0208] TsCl 4-toluenesulfonyl chloride
[0209] uPLC ultra performance liquid chromatography
[0210] UV ultraviolet
[0211] μL microlitre(s)
Analytical LCMS conditions are as follows:
System 1 (S1): Acidic IPC Method (MS18 and MS19)
[0212] 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 (MSQ2 and MSQ4)
[0213] 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 1 μL 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)
[0214] 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 0.75 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)
[0215] 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)
[0216] 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 5-100% 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)
[0217] 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.
System 7 (S7): Basic Final Method (MS10)
[0218] Analytical (MET/CR/1603) (M6) HPLC-MS were performed on a Agilent G1312A system using a Phenomenex Gemini NX C18 column (2.0 mm×100 mm, 3 μm; temperature: 40° C.) and a gradient of 5-100% B (A=2 mM ammonium bicarbonate, buffered to pH 10; B=ACN) over 5.5 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 1.08 min with an injection volume of 3 μL and at flow rate of 0.6 mL/min. UV spectra were recorded at 215 nm using a Waters 2996 photo diode array detector spectrum range: 200-400 nm. ELS data was collected using a Water 2420 detector when reported. Mass spectra were obtained using a Waters ZQ mass detector. Data were integrated and reported using Waters MassLynx and OpenLynx software.
Purification methods are as follows:
Method 1: Acidic Early Method
[0219] 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: Acidic Standard Method
[0220] 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
[0221] Purifications (P3) LC were performed on a Gilson LC system using a Waters X-Bridge C18 column (30 mm×100 mm, 10 μM; temperature: r.t.) and a gradient of 10-95% B (A=0.2% ammonium hydroxide in H.sub.2O; B=0.2% ammonium hydroxide 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
[0222] 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% ammonium hydroxide in water; B=0.2% ammonium hydroxide 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.
Method 5: Reverse Phase Chromatography using Acidic pH, Standard Elution Method
[0223] Purifications by FCC on reverse phase silica (acidic pH, standard elution method) were performed on Biotage Isolera systems using the appropriate SNAP C18 cartridge and a gradient of 10% B (A=0.1% formic acid in H.sub.2O; B=0.1% formic acid in ACN) over 1.7 CV then 10-100% B over 19.5 CV and 100% B for 2 CV.
Method 6: Reverse Phase Chromatography using Neutral pH, Standard Elution Method
[0224] Purifications by FCC on reverse phase silica (neutral pH, standard elution method) were performed on Biotage Isolera systems using the appropriate SNAP C18 cartridge and a gradient of 10% B (A=H.sub.2O; B=ACN) over 1.7 CV then 10-100% B over 19.5 CV and 100% B for 2 CV.
NMR Conditions
[0225] 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, 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
[0226] All the compounds have been synthesised with a purity >95% unless otherwise specified.
##STR00008##
Intermediate 1 (Step 1.a): 2-[2-hydroxy-1-(hydroxymethyl)ethyl]isoindoline-1,3-dione
[0227] A solution of isobenzofuran-1,3-dione (854 mg, 5.8 mmol) and 2-aminopropane-1,3-diol (521 mg, 5.7 mmol) in toluene (20 mL) was heated at 110° C. for 24 h. The reaction mixture was cooled to 62° C. and treated with MTBE (20 mL), resulting in precipitation of a white powder. The suspension was stirred for 1 h, before being filtered hot to collect the precipitate. The precipitate was washed with warm MTBE (20 mL) and dried in vacuo to afford the title compound (786 mg, 3.45 mmol, 60% yield) as a white powder; .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ7.87-7.81 (m, 4H), 4.89-4.84 (m, 2H), 4.27-4.20 (m, 1H), 3.83-3.76 (m, 2H), 3.69-3.62 (m, 2H); M/Z: 222 [M+H].sup.+, ESI.sup.+, RT=0.73 (S1).
##STR00009##
Intermediate 2 (Step 2.a): 2-(4-chloro-3-fluorophenoxy)acetyl chloride
[0228] 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, 0.12 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, chloroform-d) 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).
[0229] The intermediate in Table 1 was synthesised according to the general route 2 as exemplified by Intermediate 2 using the corresponding starting material.
TABLE-US-00001 TABLE 1 Intermediate Structure Name Starting material LCMS data NMR data 3
##STR00011##
Step 3.a: 4-chloro-N′-(2,2-diethoxyacetyl)benzohydrazide
[0230] A suspension of 4-chlorobenzohydrazide (250 mg, 1.47 mmol) in ethyl 2,2-diethoxyacetate (2.7 mL, 14.7 mmol) was stirred at 110° C. for 16 h. The reaction mixture was cooled to r.t., diluted with MeOH, and the precipitate was removed by filtration. The filtrate was concentrated in vacuo and purified by chromatography on silica gel (0-100% EtOAc in heptane) to afford the title compound (80% purity, 135 mg, 0.36 mmol, 25% yield) as a pale yellow oil; .sup.1H NMR (400 MHz, chloroform-d) δ 9.24 (d, J=5.3 Hz, 1H), 8.93 (d, J=4.8 Hz, 1H), 7.81-7.76 (m, 2H), 7.48-7.41 (m, 2H) 3.82-3.64 (m, 4H), 1.32-1.24 (m, 6H); M/Z: 299, 301, [M−H].sup.−, ESI.sup.−, RT=0.98 (S1).
Intermediate 4 (Step 3.b): 2-(4-chlorophenyl)-5-(diethoxymethyl)-1,3,4-oxadiazole
[0231] A suspension of 4-chloro-N′-(2,2-diethoxyacetyl)benzohydrazide (135 mg, 0.45 mmol) and methoxycarbonyl-(triethylammonio)sulfonylazanide (0.43 g, 1.80 mmol) in anhydrous THF (4.4 mL) was stirred at 120° C. for 10 min under microwave irradiation. The reaction mixture was partitioned between EtOAc and satd aq NaHCO.sub.3 solution, and the organic layer was isolated, washed with brine, dried over MgSO.sub.4, and concentrated in vacuo. The residue was purified by chromatography on silica gel (0-100% EtOAc in heptane) to afford the title compound (92% purity, 68 mg, 0.22 mmol, 49% yield) as an off-white solid; .sup.1H NMR (400 MHz, chloroform-d) δ 8.10-8.03 (m, 2H), 7.54-7.49 (m, 2H), 5.79 (s, 1H), 3.90-3.70 (m, 4H), 1.32 (t, J=7.1, 6H); M/Z: 283, 285 [M+H].sup.+, ESI.sup.+, RT=1.21 (S1).
##STR00012##
Step 4.a: 2-[trans-2-[(benzyloxy)methyl]-1,3-dioxan-5-yl]-2,3-dihydro-1H-isoindole-1,3-dione
[0232] A solution of benzyloxyacetaldehyde (0.25 mL, 1.77 mmol), PTSA (28 mg, 0.15 mmol) and 2-[2-hydroxy-1-(hydroxymethyl)ethyl]isoindoline-1,3-dione (336 mg, 1.47 mmol, Intermediate 1) in toluene (30 mL) was heated at reflux under Dean—Stark conditions for 18 h. The reaction mixture was cooled to r.t. and washed sequentially with satd aq NaHCO.sub.3 solution (2×10 mL) and brine (10 mL). The organic layer was dried over Na.sub.2SO.sub.4, concentrated in vacuo, and purified by chromatography on silica gel (0-35% EtOAc in heptane) to afford the title compound (303 mg, 0.82 mmol, 55% yield) as a colourless oil; .sup.1H NMR (400 MHz, chloroform-d) δ 7.90-7.82 (m, 2H), 7.79-7.71 (m, 2H), 7.41-7.34 (m, 4H), 7.34-7.29 (m, 1H), 4.91 (t, J=4.5 Hz, 1H), 4.74-4.66 (m, 1H), 4.64 (s, 2H), 4.52-4.43 (m, 2H), 4.09 (dd, J=10.8, 4.9 Hz, 2H), 3.60 (d, J=4.5 Hz, 2H).
Intermediate 5 (Step 4.b): 2-[trans-2-(hydroxymethyl)-1,3-dioxan-5-yl]-2,3-dihydro-1H-isoindole-1,3-dione
[0233] A suspension of 2-[trans-2-[(benzyloxy)methyl]-1,3-dioxan-5-yl]-2,3-dihydro-1H-isoindole-1,3-dione (393 mg, 1.06 mmol) and Pd/C (10%, 112 mg, 0.11 mmol) in EtOH (10 mL) and EtOAc (6 mL) was stirred under H.sub.2 for 5 h. The reaction mixture was purged with N.sub.2, warmed to near reflux, and then filtered through a pad of Celite. The filtrate was concentrated in vacuo to afford the title compound (94% purity, 260 mg, 0.93 mmol, 88% yield) as a white solid; .sup.1H NMR (400 MHz, chloroform-d) δ 7.90-7.80 (m, 2H), 7.79-7.66 (m, 2H), 4.78 (t, J=4.3 Hz, 1H), 4.69-4.58 (m, 1H), 4.47 (dd, J=10.8 Hz, 2H), 4.07 (dd, J=10.7, 4.8 Hz, 2H), 3.68 (d, J=4.2 Hz, 2H), 1.92 (s, 1H).
##STR00013##
Intermediate 6 (Step 5.a): trans-5-(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)-1,3-dioxane-2-carboxylic acid
[0234] TEMPO (0.13 g, 0.80 mmol) was added to a solution of 2-[trans-2-(hydroxymethyl)-1,3-dioxan-5-yl]-2,3-dihydro-1H-isoindole-1,3-dione (89% purity, 2.37 g, 8.01 mmol, Intermediate 5) in ACN (66 mL) and 0.67 M aq NaH.sub.2PO.sub.4 solution (66 mL). The reaction mixture was warmed to 35° C. A solution of NaClO.sub.2 (80%, 1.83 g, 16.0 mmol) in H.sub.2O (15 mL) was added, followed by NaOCl (5.0%, 0.5 mL, 0.41 mmol). The reaction mixture was stirred at 35° C. for 20 h. Additional TEMPO (0.13 g, 0.80 mmol), NaClO.sub.2 (80%, 1.83 g, 16.0 mmol) and NaOCl (5.0%, 0.5 mL, 0.41 mmol) was added and the reaction mixture was stirred at 35° C. for 24 h. Additional TEMPO (0.13 g, 0.80 mmol), NaClO.sub.2 (80%, 1.83 g, 16.0 mmol) and NaOCl (5.0%, 0.5 mL, 0.41 mmol) was added and the mixture was stirred at 35° C. for 24 h. The reaction mixture was concentrated in vacuo. The aqueous residue was basified to pH 9 using satd aq NaHCO.sub.3 solution, and washed with EtOAc (2×20 mL). The aqueous layer was cooled to 0° C. and acidified to pH 2 by the slow addition of 1 M aq HCl solution. The aqueous layer was re-extracted with EtOAc (3×20 mL). The combined organic extracts were washed with H.sub.2O (50 mL), dried over Na.sub.2SO.sub.4 and concentrated in vacuo to afford the title compound (2.10 g, 7.50 mmol, 94% yield) as an off-white powder; .sup.1H NMR (400 MHz, methanol-d) δ 7.90-7.86 (m, 2H), 7.85-7.81 (m, 2H), 5.11 (s, 1H), 4.63-4.56 (m, 1H), 4.56-4.49 (m, 2H), 4.20-4.14 (m, 2H).
##STR00014##
Intermediate 7 (Step 6.a): [trans-5-amino-1,3-dioxan-2-yl]methanol
[0235] A suspension of 2-[trans-2-(hydroxymethyl)-1,3-dioxan-5-yl]-2,3-dihydro-1H-isoindole-1,3-dione (93% purity, 904 mg, 3.19 mmol, Intermediate 5) and hydrazine hydrate (0.6 mL, 12.8 mmol) in EtOH (49 mL) was stirred at 50° C. for 2 h. The reaction mixture was cooled to r.t. and concentrated in vacuo. The residue was suspended in MeOH (20 mL), resulting in precipitation of a white solid. The solid was filtered under vacuum and the filtrate was concentrated in vacuo. The residue was dissolved in MeOH and loaded onto an SCX cartridge (2 g). The product was washed with MeOH, then eluted with 7 M NH.sub.3 in MeOH. The elution was collected and concentrated in vacuo to afford the title compound (299 mg, 2.25 mmol, 70% yield) as a viscous, yellow oil; .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 4.76 (s, 1H), 4.34 (t, J=4.7 Hz, 1H), 3.98-3.86 (m, 2H), 3.41-3.19 (m, 2H), 3.19-3.07 (m, 2H), 2.75 (tt, J=10.2, 5.0 Hz, 1H).
Step 6.b: 2-(4-chloro-3-fluorophenoxy)-N-[trans-2-(hydroxymethyl)-1,3-dioxan-5-yl]acetamide
[0236] A solution of 2-(4-chloro-3-fluorophenoxy)acetic acid (459 mg, 2.3 mmol) and DIPEA (1.2 mL, 6.7 mmol) in anhydrous DMF (10 mL) was treated with HATU (807 mg, 2.1 mmol). The reaction mixture was stirred at r.t. for 10 min. [trans-5-Amino-1,3-dioxan-2-yl]methanol (299 mg, 2.25 mmol, Intermediate 7) was added and the reaction mixture was stirred at r.t. for 19 h. The reaction mixture was diluted with H.sub.2O (30 mL) and then extracted with EtOAc (30 mL). The combined organic extracts were washed with brine (30 mL), dried over anhydrous Na.sub.2SO.sub.4 and concentrated in vacuo. Purification by chromatography on silica gel (0-100% EtOAc in heptane) afforded the title compound (393 mg, 1.20 mmol, 54% yield) as an off-white solid; .sup.1H NMR (400 MHz, chloroform-d) δ 7.35 (t, J=8.6 Hz, 1H), 6.76 (dd, J=10.2, 2.9 Hz, 1H), 6.71-6.63 (m, 1H), 6.12 (d, J=8.0 Hz, 1H), 4.61 (t, J=4.4 Hz, 1H), 4.45 (s, 2H), 4.42-4.31 (m, 1H), 4.28 (dd, J=11.2, 4.8 Hz, 2H), 3.67 (dd, J=6.6, 4.4 Hz, 2H), 3.51 (t, J=10.7 Hz, 2H), 2.81 (s, 4H), 1.80 (t, J=6.6 Hz, 1H); M/Z: 320, 322 [M+H].sup.+, ESI.sup.+, RT=0.74 (S2).
Intermediate 8 (Step 6.c): trans-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-1,3-dioxane-2-carboxylic acid
[0237] NaOCl (5%, 0.23 mL, 0.18 mmol), NaClO.sub.2 (80%, 843 mg, 7.38 mmol) and TEMPO (58 mg, 0.369 mmol) were added to a solution of 2-(4-chloro-3-fluorophenoxy)-N-[trans-2-(hydroxymethyl)-1,3-dioxan-5-yl]acetamide (393 mg, 1.23 mmol) in ACN (10 mL) and NaH.sub.2PO.sub.4 (0.67 M, 10 mL, 6.8 mmol) at r.t. The reaction mixture was heated at 35° C. for 22 h. The reaction mixture was cooled to r.t. and diluted with EtOAc (30 mL) and H.sub.2O (30 mL). The aqueous layer was acidified to pH 2 using 2 M aq HCl solution and extracted with EtOAc (3×20 mL). The combined organic layers were washed with brine (30 mL), dried over Na.sub.2SO.sub.4 and concentrated in vacuo to afford the title compound (88% purity, 274 mg, 0.723 mmol, 59% yield) as an off-white solid; .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.12 (d, J=7.8 Hz, 1H), 7.49 (t, J=8.9 Hz, 1H), 7.07 (dd, J=11.4, 2.8 Hz, 1H), 6.85 (ddd, J=9.0, 2.8, 1.2 Hz, 1H), 4.91 (s, 1H), 4.54 (s, 2H), 4.12-3.91 (m, 3H), 3.66-3.53 (m, 2H); M/Z: 334, 336 [M+H].sup.+, ESI.sup.+, RT=0.76 (S2).
[0238] The intermediate in Table 2 was synthesised according to the general route 6 as exemplified by Intermediate 8 using the corresponding starting material.
TABLE-US-00002 TABLE 2 Intermediate Structure Name Starting material LCMS data NMR data 9
##STR00016##
Step 7.a: benzyl cis-3-hydroxycyclobutane-1-carboxylate
[0239] NaBH.sub.4 (0.51 g, 13.5 mmol) was added to a solution of benzyl 3-oxocyclobutanecarboxylate (2.76 g, 13.5 mmol) in MeOH (25 mL) at −40° C. under N.sub.2 and stirred for 1 h. The reaction mixture was warmed to 0° C. and quenched with satd aq NH.sub.4Cl solution (10 mL). The reaction mixture was warmed to r.t. and concentrated in vacuo. The residue was dissolved in H.sub.2O (50 mL) and extracted with EtOAc (2×50 mL). The combined organic extracts were washed with brine (100 mL), dried over Na.sub.2SO.sub.4 and concentrated in vacuo to afford the title compound (2.73 g, 13.2 mmol, 98% yield) as a colourless oil; .sup.1H NMR (400 MHz, chloroform-d) δ7.41-7.29 (m, 5H), 5.14 (s, 2H), 4.27-4.15 (m, 1H), 2.74-2.53 (m, 3H), 2.28-2.12 (m, 2H), 1.86 (d, J=7.6 Hz, 1H).
Step 7.b: benzyl cis-3-(trifluoromethoxy)cyclobutane-1-carboxylate
[0240] 2-Fluoropyridine (3.5 mL, 40.4 mmol) and trifluoromethyltrimethylsilane (6.0 mL, 40.4 mmol) were successively added dropwise to a solution of benzyl cis-3-hydroxycyclobutane-1-carboxylate (2.78 g, 13.5 mmol), silver trifluoromethanesulfonate (10.43 g, 40.4 mmol), Selectfluor (7.16 g, 20.2 mmol) and potassium fluoride (3.13 g, 53.9 mmol) in EtOAc (120 mL) at r.t. under N.sub.2 in a foil-covered flask. The reaction mixture was stirred at r.t. for 20 h before being filtered through Celite, washing with EtOAc (50 mL). The filtrate was concentrated in vacuo and purified by chromatography on silica gel (5-30% EtOAc in heptane) to afford the title compound (1.69 g, 6.16 mmol, 46% yield) as a colourless oil; .sup.1H NMR (400 MHz, chloroform-d) δ 7.44-7.29 (m, 5H), 5.15 (s, 2H), 4.58 (p, J=7.6 Hz, 1H), 2.83-2.73 (m, 1H), 2.65 (dtd, J=10.0, 7.3, 2.6 Hz, 2H), 2.60-2.47 (m, 2H).
Intermediate 10 (Step 7.c): cis-3-(trifluoromethoxy)cyclobutanecarboxylic acid
[0241] A suspension of benzyl cis-3-(trifluoromethoxy)cyclobutane-1-carboxylate (1.70 g, 6.20 mmol) and Pd/C (10%, 0.66 g, 0.31 mmol) in EtOH (50 mL) was stirred at r.t. under H.sub.2 for 18 h. The reaction mixture was purged with N.sub.2, then filtered through Celite, washing with EtOH (50 mL). The filtrate was concentrated in vacuo to afford the title compound (1.06 g, 5.64 mmol, 91% yield) as a pale yellow oil; .sup.1H NMR (400 MHz, chloroform-d) δ 9.11 (s, 1H), 4.60 (p, J=7.4 Hz, 1H), 2.84-2.62 (m, 3H), 2.55 (q, J=10.2, 9.5 Hz, 2H). Contains 10% of the trans-isomer, as observed by .sup.1H NMR.
##STR00017##
Step 8.a: 4,4,4-trifluorobutyl imidazole-1-carboxylate
[0242] A solution of 4,4,4-trifluorobutan-1-ol (3.0 g, 23.4 mmol) in DCM (50 mL) was added to a solution of CDI (5.70 g, 35.1 mmol) in THF (100 mL) at 0° C. under N.sub.2 and stirred for 1 h. The reaction mixture was warmed to r.t. and stirred for 19 h. The reaction mixture was concentrated in vacuo and purified by chromatography on silica gel (5-50% EtOAc in heptane) to afford the title compound (4.92 g, 22.1 mmol, 95% yield) as a colourless oil; .sup.1H NMR (400 MHz, chloroform-d) δ 8.14 (s, 1H), 7.42 (t, J=1.4 Hz, 1H), 7.14-7.04 (m, 1H), 4.50 (t, J=6.4 Hz, 2H), 2.37-2.19 (m, 2H), 2.11 (dq, J=10.3, 6.6 Hz, 2H); .sup.19F{.sup.1H} NMR (376 MHz, chloroform-d) δ −66.31 (3F, s); M/Z: 223 [M+H].sup.+, ESI.sup.+, RT=0.73 (S2).
Intermediate 11 (Step 8.b): 4,4,4-trifluorobutyl N-aminocarbamate
[0243] A solution of 4,4,4-trifluorobutyl imidazole-1-carboxylate (4.92 g, 22.1 mmol) and hydrazine hydrate (4.4 mL, 88.6 mmol) in DCM (70 mL) was stirred at r.t. for 1.5 h. Isopropanol (15 mL) was added and the organic layer was washed with H.sub.2O (100 mL), satd aq NaHCO.sub.3 solution (100 mL), brine (100 mL), dried over Na.sub.2SO.sub.4, and concentrated in vacuo to afford the title compound (2.54 g, 13.7 mmol, 62% yield) as a colourless oil; .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.15 (s, 1H), 4.15-3.89 (m, 4H), 2.40-2.15 (m, 2H), 1.84-1.67 (m, 2H); .sup.19F{.sup.1H} NMR (376 MHz, chloroform-d) δ −64.89 (3F, s).
[0244] The following intermediates were prepared using route 8.
Intermediate 12: 2-(trifluoromethoxy)ethyl N-aminocarbamate
[0245] ##STR00018##
[0246] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.30 (s, 1H), 4.23 (s, 4H), 4.07 (s, 2H); .sup.19F{.sup.1H} NMR (376 MHz, DMSO-d.sub.6) δ −58.99 (3F, s).
Intermediate 13: 3-(trifluoromethoxy)azetidine-1-carbohydrazide
[0247] ##STR00019##
[0248] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.92 (s, 2H), 9.45 (s, 1H), 5.22 (tt, J=6.9, 4.0 Hz, 1H), 4.33 (dd, J=10.1, 6.7 Hz, 2H), 4.01 (dd, J=10.1, 3.8 Hz, 2H); .sup.19F{.sup.1H} NMR (376 MHz, chloroform-d) δ −58.43 (3F, s).
##STR00020##
Step 9.a: tert-butyl 2-(3-chloro-4-formylphenoxy)acetate
[0249] ##STR00021##
[0250] To a solution of 2-chloro-4-hydroxybenzaldehyde (1.50 g, 9.58 mmol) in anhydrous DMF (15 mL) was added tert-butyl bromoacetate (1.6 mL, 10.5 mmol) followed by K.sub.2CO.sub.3 (2.65 g, 19.2 mmol) and the mixture was stirred at 65° C. for 2 h. The reaction mixture was cooled to r.t. and then poured onto water (100 mL). The resultant solution was extracted with EtOAc (2×70 mL), and the combined organic extracts were washed with brine (100 mL), dried over MgSO.sub.4, and concentrated in vacuo to afford the title compound in quantitative yield (2.76 g, 9.89 mmol) as an off-white solid; .sup.1H NMR (400 MHz, chloroform-d) δ 10.34 (s, 1H), 7.90 (d, J=8.7 Hz, 1H), 6.93 (d, J=2.4 Hz, 1H), 6.88 (dd, J=8.7, 2.2 Hz, 1H), 4.58 (s, 2H), 1.49 (s, 9H); M/Z: 271, 273 [M+H].sup.+, ESI.sup.+, RT=0.98 (S2).
Step 9.b: tert-butyl 2-[3-chloro-4-(difluoromethyl)phenoxy]acetate
[0251] ##STR00022##
[0252] To a solution of tert-butyl 2-(3-chloro-4-formylphenoxy)acetate (2.76 g, 9.89 mmol) in anhydrous DCM (20 mL) at 0° C. was added DAST (2.6 mL, 19.8 mmol) dropwise and the mixture was stirred at r.t. for 3 h. Satd aq NaHCO.sub.3 solution (50 mL) was added slowly and stirred at r.t. for 1 h. The solution was extracted with DCM (2×50 mL) and the combined 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 chromatography on silica gel (5-40% EtOAc in heptane) to afford the title compound (2.02 g, 6.62 mmol, 67% yield) as a yellow oil; .sup.1H NMR (400 MHz, chloroform-d) δ 7.58 (d, J=8.6 Hz, 1H), 7.05-6.73 (m, 3H), 4.53 (s, 2H), 1.49 (s, 9H); M/Z: 291, 293 [M−H].sup.−, RT=1.06 (S2).
Intermediate 14 (Step 9.c): 2-[3-chloro-4-(difluoromethyl)phenoxy]acetic acid
[0253] ##STR00023##
[0254] To a solution of tent-butyl 2-[3-chloro-4-(difluoromethyl)phenoxy]acetate (2.02 g, 6.62 mmol) in 1,4-dioxane (5 mL) was added 4 M HCl in 1,4-dioxane (17 mL, 66.2 mmol) and the mixture was stirred at 60° C. for 4 h. The reaction mixture was cooled to r.t. and concentrated in vacuo. H.sub.2O was added and the resultant precipitate was filtered under vacuum, washing with H.sub.2O. The solid was then dissolved in MeCN, filtered under vacuum and the filtrate concentrated in vacuo to afford the title compound (1.51 g, 6.26 mmol, 95% yield) as a cream solid; .sup.1H NMR (400 MHz, chloroform-d) δ 7.61 (d, J=8.7 Hz, 1H), 7.05-6.74 (m, 3H), 4.73 (s, 2H); .sup.19F NMR (376 MHz, chloroform-d) δ −113.69; M/Z: 235, 237 [M−H].sup.−, ESI.sup.−, RT=0.74 (S2).
##STR00024##
Step 10.a: 2-(3,4-dichlorophenoxy)-N-[trans-2-(hydroxymethyl)-1,3-dioxan-5-yl]acetamide and [trans-5-[2-(3,4-dichlorophenoxy)acetamido]-1,3-dioxan-2-yl]methyl 2-(3,4-dichlorophenoxy)acetate
[0255] A solution of [trans-5-amino-1,3-dioxan-2-yl]methanol (495 mg, 3.72 mmol, Intermediate 7) and DIPEA (3.9 mL, 22.3 mmol) in DCM (10 mL) was cooled to 0° C. and treated dropwise with a solution of 2-(3,4-dichlorophenoxy)acetyl chloride (1.87 g, 7.81 mmol, Intermediate 3) in DCM (15 mL). The mixture was allowed to warm to r.t. and stirred for 3 h. The reaction mixture was cooled to 0° C. and quenched slowly with H.sub.2O (10 mL). The organic layer was separated and washed with satd aq NaHCO.sub.3 solution (20 mL) and brine (20 mL), dried over Na.sub.2SO.sub.4, and concentrated in vacuo. The residue was purified by chromatography on silica gel (20-100% EtOAc in heptane) to afford the title compounds 2-(3,4-dichlorophenoxy)-N-[trans-2-(hydroxymethyl)-1,3-dioxan-5-yl]acetamide (405 mg, 1.14 mmol, 31% yield) and [trans-5-[2-(3,4-dichlorophenoxy)acetamido]-1,3-dioxan-2-yl]methyl 2-(3,4-dichlorophenoxy)acetate (92% purity, 800 mg, 1.37 mmol, 37% yield) as off-white solids.
2-(3,4-dichlorophenoxy)-N-[trans-2-(hydroxymethyl)-1,3-dioxan-5-yl]acetamide
[0256] ##STR00025##
[0257] (405 mg, 1.14 mmol, 31% yield); .sup.1H NMR (400 MHz, chloroform-d) δ 7.39 (d, J=8.9 Hz, 1H), 7.04 (d, J=2.9 Hz, 1H), 6.78 (dd, J=8.9, 2.9 Hz, 1H), 6.11 (d, J=8.1 Hz, 1H), 4.60 (t, J=4.4 Hz, 1H), 4.44 (s, 2H), 4.40-4.30 (m, 1H), 4.30-4.23 (m, 2H), 3.66 (dd, J=6.6, 4.4 Hz, 2H), 3.54-3.46 (m, 2H), 1.81 (t, J=6.6 Hz, 1H).
[trans-5-[2-(3,4-dichlorophenoxy)acetamido]-1,3-dioxan-2-yl]methyl 2-(3,4-dichlorophenoxy)acetate
[0258] ##STR00026##
[0259] (92% purity, 800 mg, 1.37 mmol, 37% yield); .sup.1H NMR (500 MHz, chloroform-d) δ 7.36 (m, 2H), 7.02 (m, 2H), 6.81-6.75 (m, 2H), 6.14 (d,J=8.1 Hz, 1H), 4.71 (t,J=4.4 Hz, 1H), 4.66 (s, 2H), 4.44 (s, 2H), 4.38-4.30 (m, 1H), 4.30-4.21 (m, 4H), 3.52-3.44 (m, 2H).
Step 10.b: 2-(3,4-dichlorophenoxy)-N-[trans-2-(hydroxymethyl)-1,3-dioxan-5-yl]acetamide
[0260] ##STR00027##
[0261] A solution of [trans-5-[2-(3,4-dichlorophenoxy)acetamido]-1,3-dioxan-2-yl]methyl 2-(3,4-dichlorophenoxy)acetate (92% purity, 800 mg, 1.37 mmol) in H.sub.2O (2.8 mL), THF (2.8 mL) and MeOH (2.8 mL) was treated with LiOH (43 mg, 1.78 mmol) and stirred at r.t. for 1 h. The organic solvent was removed in vacuo to afford an aqueous residue which was diluted with H.sub.2O (10 mL) and extracted with EtOAc (2×10 mL). The combined organic extracts were washed with brine (20 mL), dried over Na.sub.2SO.sub.4, and concentrated in vacuo to afford the title compound (385 mg, 1.09 mmol, 73% yield) as an off-white solid; .sup.1H NMR (400 MHz, chloroform-d) δ 7.39 (d, J=8.9 Hz, 1H), 7.05 (d, J=2.9 Hz, 1H), 6.78 (dd, J=8.9, 2.9 Hz, 1H), 6.11 (d, J=8.2 Hz, 1H), 4.60 (t, J=4.4 Hz, 1H), 4.44 (s, 2H), 4.40-4.31 (m, 1H), 4.30-4.20 (m, 2H), 3.66 (d, J=4.4 Hz, 2H), 3.55-3.43 (m, 2H), 1.80 (s, 1H).
Intermediate 15 (Step 10.c): trans-5-[2-(3,4-dichlorophenoxy)acetamido]-1,3-dioxane-2-carboxylic acid
[0262] ##STR00028##
[0263] NaOCl (5.0%, 0.44 mL, 0.353 mmol), NaClO.sub.2 (80%, 1.61 g, 14.1 mmol) and TEMPO (111 mg, 0.705 mmol) were added to a solution of 2-(3,4-dichlorophenoxy)-N-[trans-2-(hydroxymethyl)-1,3-dioxan-5-yl]acetamide (790 mg, 2.35 mmol) in ACN (38 mL) and 0.67 M NaH.sub.2PO.sub.4 (19 mL, 12.9 mmol) at r.t. and the mixture was stirred at 35° C. for 48 h. The reaction mixture was concentrated in vacuo at r.t. to remove the organic solvent, and the resultant aqueous solution was basified to pH 8/9 using satd aq NaHCO.sub.3 solution. The aqueous solution was washed with EtOAc (2×30 mL) and the organic extracts were discarded. The aqueous solution was then cooled to 0° C. and acidified to pH 2/3 by slow addition of 1 M aq HCl solution. The resultant solution was then extracted with EtOAc (2×50 mL) and the combined organic extracts were washed with H.sub.2O (50 mL), dried over Na.sub.2SO.sub.4, and concentrated in vacuo to afford the title compound (90% purity, 760 mg, 1.95 mmol, 83% yield) as a foamy off-white solid; .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.11 (d, J=7.8 Hz, 1H), 7.54 (d, J=8.9 Hz, 1H), 7.25 (d, J=2.9 Hz, 1H), 6.98 (dd, J=8.9, 2.9 Hz, 1H), 4.91 (s, 1H), 4.55 (s, 2H), 4.08-3.95 (m, 3H), 3.59 (m, 2H); M/Z: 348, 350, 352 [M−H].sup.−, ESI.sup.−, RT=2.61 (S4).
##STR00029##
Step 11.a: 2-[trans-2-[5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl]-1,3-dioxan-5-yl]isoindoline-1,3-dione
[0264] A solution of BF.sub.3.OEt.sub.2 (0.12 mL, 0.97 mmol) in anhydrous ACN (3 mL) was stirred at 80° C. A separate solution of 2-[2-hydroxy-1-(hydroxymethyl)ethyl]isoindoline-1,3-dione (100 mg, 0.44 mmol, Intermediate 1) and 2-(4-chlorophenyl)-5-(diethoxymethyl)-1,3,4-oxadiazole (94 μL, 0.44 mmol, Intermediate 4) in ACN (2 mL) was added dropwise. The reaction mixture was stirred for 1 h, then additional BF.sub.3.OEt.sub.2 (0.12 mL, 0.97 mmol) was added and the reaction mixture was heated at 80° C. for a further 1 h. The reaction was cooled to r.t., quenched with satd aq NaHCO.sub.3 solution (0.1 mL) and concentrated in vacuo. The residue was dissolved in EtOAc (10 mL) and washed sequentially with satd aq NaHCO.sub.3 solution (10 mL) and brine (10 mL). The organic layer was dried over anhydrous Na.sub.2SO.sub.4, concentrated in vacuo and purified by chromatography on silica gel (10-100% EtOAc in heptane) to afford the title compound (70% purity, 33 mg, 0.06 mmol, 13% yield) as an off-white solid, as a mixture of diastereomers; .sup.1H NMR (400 MHz, chloroform-d) δ 8.12-8.08 (m, 1H), 8.08-8.04 (m, 2H), 7.89-7.82 (m, 4H), 7.79-7.71 (m, 4H), 7.54-7.49 (m, 3H), 6.04 (s, 1H), 4.90-4.80 (m, 2H), 4.73-4.65 (m, 3H), 4.57-4.49 (m, 1H), 4.35-4.24 (m, 3H), 4.19 (dd, J=11.0, 5.0 Hz, 1H), 4.12 (q, J=7.1 Hz, 3H), 2.04 (s, 4H), 1.73-1.59 (m, 4H); M/Z: 412, 414 [M+H].sup.+, ESI.sup.+, RT=3.52, 3.67 (S4).
Step 11.b: trans-2-[5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl]-1,3-dioxan-5-amine
[0265] A suspension of hydrazine hydrate (0.02 mL, 0.14 mmol) and 2-[trans-2-[5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl]-1,3-dioxan-5-yl]isoindoline-1,3-dione (33 mg, 0.06 mmol) in EtOH (0.7 mL) was stirred at r.t. for 2 h. The reaction mixture was heated at 40° C. for 25 h, then heated at 50° C. for a further 6 h. The reaction mixture was cooled to r.t., filtered through a pad of Celite and concentrated in vacuo to afford the title compound (50% purity, 17 mg, 0.03 mmol, 54% yield) as a white solid; 41 NMR (400 MHz, chloroform-d) δ 8.04 (m, 2H), 7.49 (m, 2H), 5.80 (s, 1H), 4.35 (dd, J=11.4, 4.6 Hz, 2H), 4.09 (d, J=12.0 Hz, 1H), 3.51 (t, J=10.8 Hz, 2H), 3.36-3.25 (m, 2H); M/Z: 282 [M+H].sup.+, ESI.sup.+, RT=0.82 (S1).
Example 1 (Step 11.c): 2-(4-chloro-3-fluorophenoxy)-N-[trans-2-[5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl]-1,3-dioxan-5-yl]acetamide
[0266] A solution of trans-2-[5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl]-1,3-dioxan-5-amine (50%, 17 mg, 0.03 mmol) in DCM (0.5 mL) was treated with DIPEA (0.02 mL, 0.09 mmol) and cooled to 0° C. A solution of 2-(4-chloro-3-fluorophenoxy)acetyl chloride (7.4 mg, 0.03 mmol, Intermediate 2) in DCM (0.5 mL) was added slowly. The reaction mixture was allowed to warm to r.t. and stirred for 1 h. 2-(4-chloro-3-fluorophenoxy)acetyl chloride (3.0 mg, 0.01 mmol, Intermediate 2) was added and the reaction was stirred at r.t. for 1 h. The reaction mixture was cooled to 0° C., quenched with H.sub.2O and concentrated in vacuo. The residue was purified by prep. HPLC (Method 4) to afford the title compound (5.0 mg, 0.01 mmol, 35% yield) as an off-white powder; .sup.1H NMR (400 MHz, chloroform-d) δ 8.07-8.01 (m, 2H), 7.54-7.48 (m, 2H), 7.36 (t, J=8.6 Hz, 1H), 6.84-6.76 (m, 2H), 6.72 (ddd, J=8.9, 2.9, 1.3 Hz, 1H), 6.03 (s, 1H), 4.53-4.44 (m, 4H), 4.39-4.32 (m, 1H), 3.86 (dd, J=11.6, 6.6 Hz, 2H); M/Z: 468, 470, 472 [M+H].sup.+, ESI.sup.+, RT=3.65 (S4).
##STR00030## ##STR00031##
Step 12.a: 2-[trans-2-[({[(benzyloxy)carbonyl]amino}amino)carbonyl]-1,3-dioxan-5-yl]-2,3-dihydro-1H-isoindole-1,3-dione
[0267] A solution of trans-5-(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)-1,3-dioxane-2-carboxylic acid (2.07 g, 7.39 mmol, Intermediate 6) in anhydrous THF (49 mL) was treated with sequential addition of isobutyl carbonochloridate (0.9 mL, 7.02 mmol) and NMM (0.8 mL, 7.39 mmol) at 0° C. under N.sub.2. Benzyl N-aminocarbamate (1.17 g, 7.02 mmol) was added and the reaction was stirred at 0° C. for 30 min, then at r.t. for 1 h. The reaction was cooled to 0° C. and quenched with H.sub.2O (1 mL). The resultant mixture was concentrated in vacuo and the residue was partitioned between EtOAc (50 mL) and H.sub.2O (50 mL). The layers were separated and the aqueous layer was re-extracted with EtOAc (20 mL). The combined organic extracts were washed with satd aq NaHCO.sub.3 solution (80 mL), brine (80 mL), dried over anhydrous Na.sub.2SO.sub.4, and concentrated in vacuo to afford the title compound (86%, 2.86 g, 5.78 mmol, 78% yield) as an off-white powder; .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 10.16-9.91 (m, 1H), 9.27 (s, 1H), 7.93-7.82 (m, 4H), 7.44-7.27 (m, 5H), 5.09 (s, 2H), 5.03 (s, 1H), 4.44-4.28 (m, 3H), 4.22-4.12 (m, 2H).
Step 12.b: trans-5-(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)-1,3-dioxane-2-carbohydrazide
[0268] A suspension of 2-[trans-2-[({[(benzyloxy)carbonyl]amino}amino)carbonyl]-1,3-dioxan-5-yl]-2,3-dihydro-1H-isoindole-1,3-dione (86% purity, 2.86 g, 5.78 mmol) and Pd/C (10%, 0.62 g, 0.578 mmol) in EtOH (160 mL) and EtOAc (50 mL) was stirred under H.sub.2 for 3 h. Additional Pd/C (10%, 0.62 g, 0.578 mmol) was added and the reaction mixture was stirred under H.sub.2 for a further 4 h. The reaction vessel was flushed with N.sub.2. The reaction mixture was heated to near reflux and filtered through a pad of Celite, washing with hot EtOH. The reaction mixture was concentrated in vacuo and purified by chromatography on silica gel (0-7% MeOH in DCM) to afford the title compound (90% purity, 290 mg, 0.90 mmol, 15% yield) as a pale yellow powder; .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 9.32 (s, 1H), 7.93-7.79 (m, 4H), 4.93 (s, 1H), 4.41-4.25 (m, 5H), 4.17-4.08 (m, 2H).
Step 12.c: trans-5-(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)-N′-[cis-3-(trifluoromethoxy)cyclobutanecarbonyl]-1,3-dioxane-2-carbohydrazide
[0269] A solution of cis-3-(trifluoromethoxy)cyclobutanecarboxylic acid (170 mg, 0.90 mmol, Intermediate 10) in anhydrous THF (7.5 mL) was treated with sequential addition of isobutyl carbonochloridate (0.11 mL, 0.85 mmol) and NMM (0.1 mL, 0.90 mmol) at 0° C. The mixture was stirred for 15 min, then a suspension of trans-5-(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)-1,3-dioxane-2-carbohydrazide (90% purity, 290 mg, 0.90 mmol) in anhydrous THF (5 mL) was added. The reaction was warmed to r.t. and stirred for 3 h. The reaction mixture was cooled to 0° C. and quenched by the slow addition of H.sub.2O (5 mL). The reaction mixture was concentrated in vacuo and the resultant residue was partitioned between H.sub.2O and EtOAc. The aqueous layer was re-extracted with EtOAc. The combined organic extracts were washed with satd aq NaHCO.sub.3 solution and brine, and then concentrated in vacuo to afford the title compound (90% purity, 382 mg, 0.75 mmol, 84% yield) as an off-white powder; .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 10.04 (s, 1H), 9.96 (s, 1H), 7.88 (s, 4H), 5.04 (s, 1H), 4.89-4.71 (m, 1H), 4.46-4.26 (m, 3H), 4.26-4.10 (m, 2H), 2.77-2.63 (m, 2H), 2.36-2.18 (m, 4H).
Step 12.d: 2-[trans-2-{5-[cis-3-(trifluoromethoxy)cyclobutyl]-1,3,4-oxadiazol-2-yl}-1,3-dioxan-5-yl]-2,3-dihydro-1H-isoindole-1,3-dione
[0270] A suspension of trans-5-(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)-N′-[cis-3-(trifluoromethoxy)cyclobutanecarbonyl]-1,3-dioxane-2-carbohydrazide (85%, 365 mg, 0.68 mmol) and Burgess reagent (647 mg, 2.71 mmol) in anhydrous THF (11 mL) was irradiated at 120° C. in a microwave vial for 3 min. The reaction mixture was concentrated in vacuo and purified by prep. HPLC (Method 6) to afford the title compound (190 mg, 0.41 mmol, 61% yield) as an off-white solid; .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.98-7.73 (m, 4H), 6.09 (s, 1H), 4.97-4.87 (m, 1H), 4.55-4.39 (m, 3H), 4.29-4.20 (m, 2H), 3.56-3.46 (m, 1H), 2.92-2.83 (m, 2H), 2.57-2.51 (m, 2H).
Intermediate 16 (Step 12.e): trans-2-{5-[cis-3-(trifluoromethoxy)cyclobutyl]-1,3,4-oxadiazol-2-yl}-1,3-dioxan-5-amine
[0271] A solution of 2-[trans-2-{5-[cis-3-(trifluoromethoxy)cyclobutyl]-1,3,4-oxadiazol-2-yl}-1,3-dioxan-5-yl]-2,3-dihydro-1H-isoindole-1,3-dione (90% purity, 330 mg, 0.68 mmol) and hydrazine hydrate (0.13 mL, 2.70 mmol) in EtOH (7.8 mL) was heated at 40° C. for 6 h, then cooled to r.t. and stirred for 18 h. The resultant precipitate was filtered under vacuum, washing with EtOH. The combined filtrates were collected and concentrated in vacuo to afford the title compound (77% purity, 250 mg, 0.62 mmol, 92% yield) as an off-white solid; .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 5.78 (s, 1H), 4.97-4.83 (m, 1H), 4.14-4.02 (m, 2H), 3.52-3.39 (m, 5H), 2.95 (tt, J=10.2, 5.0 Hz, 1H), 2.90-2.80 (m, 2H), 2.55-2.51 (m, 2H).
Example 2 (Step 12.f): 2-(4-chloro-3-fluorophenoxy)-N-[trans-2-{5-[cis-3-(trifluoromethoxy)cyclobutyl]-1,3,4-oxadiazol-2-yl}-1,3-dioxan-5-yl]acetamide
[0272] A solution of trans-2-{5-[cis-3-(trifluoromethoxy)cyclobutyl]-1,3,4-oxadiazol-2-yl}-1,3-dioxan-5-amine (77% purity, 255 mg, 0.64 mmol, Intermediate 16) and DIPEA (0.33 mL, 1.90 mmol) in DCM (8 mL) was cooled to 0° C. and treated with 2-(4-chloro-3-fluorophenoxy)acetyl chloride (90% purity, 173 mg, 0.70 mmol, Intermediate 2). The reaction was warmed to r.t. and stirred for 1 h. The reaction was cooled to 0° C. and quenched with H.sub.2O (0.1 mL), then the reaction mixture was concentrated in vacuo. The residue was purified by prep. HPLC (Method 6) and the resulting solid was dissolved in ACN (10 mL) and H.sub.2O (10 mL). The cloudy solution was heated to solubilise the material, then left to cool for 4 days. The resultant solid was collected and purified by prep. HPLC (Method 3) to afford the title compound (62 mg, 0.13 mmol, 20% yield) as a white solid; .sup.1H NMR (500 MHz, chloroform-d) δ 7.35 (t, J=8.6 Hz, 1H), 6.79 (dd, J=10.2, 2.9 Hz, 1H), 6.75 (d, J=8.4 Hz, 1H), 6.71 (ddd, J=8.9, 2.9, 1.2 Hz, 1H), 5.93 (s, 1H), 4.72 (p, J=7.6 Hz, 1H), 4.49 (s, 2H), 4.43 (dd, J=11.6, 3.8 Hz, 2H), 4.35 (dtt, J=10.8, 7.4, 3.8 Hz, 1H), 3.82 (dd, J=11.6, 7.0 Hz, 2H), 3.38 (tt, J=10.1, 7.8 Hz, 1H), 2.94-2.86 (m, 2H), 2.77-2.68 (m, 2H); M/Z: 496, 498 [M+H].sup.+, ESI.sup.+, RT=3.50 (S4).
##STR00032##
Step 13.a: 2-(4-chloro-3-fluorophenoxy)-N-[2-hydroxy-1-(hydroxymethyl)ethyl]-acetamide
[0273] 2-(4-Chloro-3-fluorophenoxy)acetic acid (200 mg, 0.98 mmol), 2-aminopropane-1,3-diol (107 mg, 1.17 mmol) and HOAt (160 mg, 1.18 mmol) were dissolved in anhydrous DMF (5 mL). EDCI (225 mg, 1.17 mmol) was then added in small portions at 0° C. The reaction mixture was warmed to r.t. and stirred for 17 h. The reaction mixture was quenched with H.sub.2O (20 mL) and extracted with EtOAc (2×30 mL). The combined organic extracts were washed with brine (20 mL), dried over MgSO.sub.4 and concentrated in vacuo. Purification by chromatography on silica gel (0-100% EtOAc in heptane) afforded the title compound (278 mg, 1.0 mmol) in quantitative yield as a white powder; .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.67 (d, J=8.3 Hz, 1H), 7.49 (t, J=8.9 Hz, 1H), 7.08 (dd, J=11.4, 2.9 Hz, 1H), 6.85 (ddd, J=9.0, 2.9, 1.1 Hz, 1H), 4.74-4.63 (m, 2H), 4.54 (s, 2H), 3.83-3.74 (m, 1H), 3.46-3.40 (m, 4H); M/Z: 278, 280 [M+H].sup.+, ESI.sup.+, RT=0.88 (S1).
Example 3 (Step 13.b): 2-(4-chloro-3-fluorophenoxy)-N-[cis-2-[1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl]-1,3-dioxan-5-yl]acetamide
[0274] A suspension of 1-(4-chlorophenyl)-1H-1,2,3-triazole-4-carbaldehyde (50 mg, 0.241 mmol), CSA (6 mg, 0.03 mmol) and 2-(4-chloro-3-fluorophenoxy)-N-[2-hydroxy-1-(hydroxymethyl)ethyl]acetamide (67 mg, 0.24 mmol) in anhydrous toluene (2 mL) was heated at 110° C. for 18 h. Additional 2-(4-chloro-3-fluorophenoxy)-N-[2-hydroxy-1-(hydroxymethyl)ethyl]acetamide (67 mg, 0.24 mmol) and CSA (6 mg, 0.03 mmol) were added and the reaction mixture was heated at 110° C. for 5 h. The reaction mixture was cooled to r.t. and brine was added. The aqueous layer was extracted with EtOAc (2×50 mL), dried over MgSO.sub.4, and concentrated in vacuo. Purification by chromatography on silica gel (0-100% EtOAc in heptane) followed by prep. HPLC (Method 3) afforded the title compound (10 mg, 0.02 mmol, 8.6% yield) as a white powder, as a mixture of cis and trans isomers (93:7); .sup.1H NMR (500 MHz, chloroform-d) δ 7.93 (s, 1H), 7.70-7.65 (m, 2H), 7.55-7.49 (m, 2H), 7.40-7.34 (m, 1H), 7.30 (t, J=8.6 Hz, 1H), 6.78 (dd, J=10.3, 2.9 Hz, 1H), 6.69 (dd, J=8.9, 1.6 Hz, 1H), 5.91 (s, 1H), 4.53 (s, 2H), 4.28-4.15 (m, 4H), 4.11 (d, J ═8.3 Hz, 1H); M/Z: 467, 469 [M+H].sup.+, ESI.sup.+, RT=3.65 (S6).
##STR00033##
Step 14.a: 2-[trans-2-[1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl]-1,3-dioxan-5-yl]-2,3-dihydro-1H-isoindole-1,3-dione
[0275] A suspension of 1-(4-chlorophenyl)-1H-1,2,3-triazole-4-carbaldehyde (270 mg, 1.30 mmol), CSA (32 mg, 0.14 mmol) and 2-[2-hydroxy-1-(hydroxymethyl)ethyl]isoindoline-1,3-dione, Intermediate 1 (288 mg, 1.30 mmol) in anhydrous toluene (11 mL) was heated at 110° C. for 1 h. The reaction mixture was allowed to cool to r.t. and diluted with cold DCM. The resultant precipitate was filtered under vacuum to afford the title compound (305 mg, 0.74 mmol, 57% yield) as a colourless solid; .sup.1H NMR (400 MHz, chloroform-d) δ 8.11 (s, 1H), 7.89 (dd, J=5.5, 3.1 Hz, 2H), 7.79 (dd, J=5.5, 3.1 Hz, 2H), 7.75-7.68 (m, 2H), 7.57-7.50 (m, 2H), 6.02 (s, 1H), 4.86-4.67 (m, 3H), 428-4.19 (m, 2H); M/Z: 411, 413 [M+H].sup.+, ESI.sup.+, RT=1.29 (S1).
Step 14.b: trans-2-[1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl]-1,3-dioxan-5-amine
[0276] A suspension of hydrazine hydrate (0.46 mL, 3.71 mmol) and 2-[trans-2[1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl]-1,3-dioxan-5-yl]-2,3-dihydro-1H-isoindole-1,3-dione (305 mg, 0.74 mmol) in EtOH (7.4 mL) was heated at 80° C. for 1 h. The reaction mixture was cooled to r.t. and the resulting precipitate was filtered under vacuum. The filtrate was then concentrated in vacuo to afford the title compound (200 mg, 0.69 mmol, 93% yield) as a colourless solid; .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.88 (s, 1H), 8.06-7.89 (m, 2H), 7.72 — 7.58 (m, 2H), 5.66 (s, 1H), 4.09 (dd, J=11.3, 5.0 Hz, 2H), 3.52-3.39 (m, 2H), 2.94 (tt, J=10.3, 5.0 Hz, 1H); M/Z: 281, 283, 285 [M+H].sup.+, ESI.sup.+, RT=0.66 (S1).
Example 4 (Step 14.c): 2-(4-chloro-3-fluorophenoxy)-N-[trans-2-[1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl]-1,3-dioxan-5-yl]acetamide
[0277] A solution of 2-(4-chloro-3-fluorophenoxy)acetyl chloride (0.05 mL, 0.77 mmol, Intermediate 2) in DCM (1 mL) was added to a solution of trans-2-[1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl]-1,3-dioxan-5-amine (72 mg, 0.26 mmol) and Et.sub.3N (0.21 mL, 1.54 mmol) in DCM (1.2 mL) at 0° C. The reaction mixture was warmed to r.t. and stirred for 15 min. The reaction mixture was diluted with satd aq NaHCO.sub.3, with the insoluble material isolated by filtration. The organic layer was collected, dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The residual material (along with the solid isolated via suction filtration) was triturated with ACN to afford the title compound (87 mg, 0.18 mmol, 70% yield) as an off-white solid; .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.94 (s, 1H), 8.17 (d, J=7.6 Hz, 1H), 7.99 (d, J=8.9 Hz, 2H), 7.67 (d, J=8.9 Hz, 2H), 7.52 (t, J=8.9 Hz, 1H), 7.10 (dd, J=11.3, 2.8 Hz, 1H), 6.88 (dd, J=8.9, 1.8, 1H), 5.80 (s, 1H), 4.57 (s, 2H), 4.23-4.08 (m, 3H), 3.85-3.68 (m, 2H); M/Z: 511, 513, 515 [M+H].sup.+, ESI.sup.+, RT=3.58 (S4).
##STR00034##
Step 15.a: 2-(4-chloro-3-fluorophenoxy)-N-[trans-2-{N′-[(4,4,4-trifluorobutoxy)carbonyl]hydrazinecarbonyl}-1,3-dioxan-5-yl]acetamide
[0278] NMM (80 μL, 0.7 mmol) and isobutyl carbonochloridate (90 μL, 0.7 mmol) were added to a solution of trans-5-[2-(4-chloro-3-fluorophenoxy)acetamido]-1,3-dioxane-2-carboxylic acid (88%, 274 mg, 0.7 mmol, Intermediate 8) in anhydrous THF (6.5 mL) at 0° C. under N.sub.2 and stirred for 15 min. 4,4,4-Trifluorobutyl N-aminocarbamate (135 mg, 0.7 mmol, Intermediate 11) was added and the reaction mixture was warmed to r.t. and stirred for 17 h. The reaction mixture was cooled to 0° C. and additional NMM (40 μL, 0.35 mmol) and isobutyl carbonochloridate (45 μL, 0.35 mmol) were added. The reaction mixture was warmed to r.t. and stirred for 1 h. The reaction mixture was diluted with EtOAc (30 mL) and washed with H.sub.2O (30 mL), satd aq NaHCO.sub.3 solution (30 mL), brine (30 mL), dried over anhydrous Na.sub.2SO.sub.4 and concentrated in vacuo. The resulting solid was triturated with ACN to afford the title compound (222 mg, 0.442 mmol, 61% yield) as a white solid; .sup.1E1 NMR (400 MHz, DMSO-d.sub.6) δ 9.92 (s, 1H), 9.25-8.65 (m, 1H), 8.10 (d, J=7.6 Hz, 1H), 7.50 (t, J=8.9 Hz, 1H), 7.08 (dd, J=11.3, 2.8 Hz, 1H), 6.85 (ddd, J=9.0, 2.8, 1.1 Hz, 1H), 4.89 (s, 1H), 4.54 (s, 2H), 4.14-3.96 (m, 5H), 3.67-3.51 (m, 2H), 2.39-2.19 (m, 2H), 1.87-1.61 (m, 2H); .sup.19F{.sup.1H} NMR (376 MHz, chloroform-d) δ −64.91-65.02 (m, 3F), −113.96 (s, 1F); M/Z: 502, 504 [M+H].sup.+, ESI.sup.+, RT=0.98 (S2).
Example 5 (Step 15.b): 2-(4-chloro-3-fluorophenoxy)-N-[trans-2-[5-(4,4,4-trifluorobutoxy)-1,3,4-oxadiazol-2-yl]-1,3-dioxan-5-yl]acetamide
[0279] ##STR00035##
[0280] To a suspension of 2-(4-chloro-3-fluorophenoxy)-N-[trans-2-{N′-[(4,4,4-trifluorobutoxy)carbonyl]hydrazinecarbonyl}-1,3-dioxan-5-yl]acetamide (162 mg, 0.32 mmol) in ACN (5 mL) was added K.sub.2CO.sub.3 (223 mg, 1.6 mmol) and TsCl (154 mg, 0.81 mmol) at r.t. The resulting mixture was heated to 80° C. and stirred for 1.5 h. The reaction mixture was cooled to r.t. and diluted with DCM (30 mL), and washed with H.sub.2O (30 mL), satd aq NaHCO.sub.3 solution (30 mL), brine (30 mL), dried over anhydrous Na.sub.2SO.sub.4 and concentrated in vacuo. Purification by prep. HPLC (Method 3) afforded the title compound (65 mg, 0.13 mmol, 41% yield) as a viscous, colourless gum; .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.19 (d, J=7.6 Hz, 1H), 7.50 (t, J=8.9 Hz, 1H), 7.08 (dd, J=11.3, 2.8 Hz, 1H), 6.85 (dd, J=8.9, 1.8 Hz, 1H), 5.82 (s, 1H), 4.62-4.45 (m, 4H), 4.20-3.99 (m, 3H), 3.82-3.67 (m, 2H), 2.10-1.92 (m, 2H); .sup.19F{.sup.1H} NMR (376 MHz, chloroform-d) δ −64.89 (3F, s), −113.95 (1F, s); M/Z: 484, 486 [M+H].sup.+, ESI.sup.+, RT=3.42 (S4).
[0281] The following examples were prepared using route 15.
Example 6: 2-(4-chloro-3-fluorophenoxy)-N-[trans-2-{5-[2-(trifluoromethoxy)ethoxy]-1,3,4-oxadiazol-2-yl}-1,3-dioxan-5-yl]acetamide
[0282] ##STR00036##
[0283] Using Intermediate 8 and Intermediate 12; .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.22 (d, J=7.7 Hz, 1H), 7.51 (t, J=8.9 Hz, 1H), 7.08 (dd, J=11.4, 2.8 Hz, 1H), 6.85 (ddd, J=8.9, 2.8, 1.1 Hz, 1H), 5.84 (s, 1H), 4.79-4.66 (m, 2H), 4.56 (s, 2H), 4.50-4.42 (m, 2H), 4.22-4.01 (m, 3H), 3.75 (t, J=10.1 Hz, 2H); M/Z: 486, 488 [M+H].sup.+, ESI.sup.+, RT=3.35 (S4).
Example 7: 2-(4-chloro-3-fluorophenoxy)-N-[trans-2-{5-[3-(trifluoromethoxy)azetidin-1-yl]-1,3,4-oxadiazol-2-yl}-1,3-dioxan-5-yl]acetamide
[0284] ##STR00037##
[0285] Using Intermediate 8 and Intermediate 13; .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.18 (d, J=7.6 Hz, 1H), 7.50 (t, J=8.9 Hz, 1H), 7.08 (dd, J=11.3, 2.8 Hz, 1H), 6.85 (ddd, J=9.0, 2.8, 1.2 Hz, 1H), 5.78 (s, 1H), 5.32 (tt,J=7.0, 4.1 Hz, 1H), 4.59-4.43 (m, 4H), 4.26 (dd,J=9.7, 4.0 Hz, 2H), 4.18-4.01 (m, 3H), 3.74 (t, J=9.6 Hz, 2H); M/Z: 497, 499 [M+H].sup.+, ESI.sup.+, RT=3.26 (S4).
Example 8: 2-[3-chloro-4-(difluoromethyl)phenoxy]-N-[trans-2-{5-[3-(trifluoromethoxy)azetidin-1-yl]-1,3,4-oxadiazol-2-yl}-1,3-dioxan-5-yl]acetamide
[0286] ##STR00038##
[0287] Using Intermediate 9 and Intermediate 13; .sup.1H NMR (400 MHz, DMSO-d6) δ 8.21 (d, J=7.5 Hz, 1H), 7.63 (d, J=8.8 Hz, 1H), 7.28-6.97 (m, 3H), 5.79 (s, 1H), 5.32 (tt, J=7.0, 4.1 Hz, 1H), 4.62 (s, 2H), 4.51 (dd, J=9.6, 6.8 Hz, 2H), 4.26 (dd, J=9.6, 4.0 Hz, 2H), 4.18-4.01 (m, 3H), 3.81-3.67 (m, 2H); .sup.19F NMR (376 MHz, DMSO-d.sub.6) δ −58.47, −112.49 (d, J=54.6 Hz); M/Z: 529, 531 [M+H].sup.+, ESI.sup.+, RT=3.38 (S4).
Example 9: 2-(3,4-dichlorophenoxy)-N-[trans-2-{5-[2-(trifluoromethoxy)ethoxy]-1,3,4-oxadiazol-2-yl}-1,3-dioxan-5-yl]acetamide
[0288] ##STR00039##
[0289] Using Intermediate 15 and Intermediate 12: .sup.1H NMR (500 MHz, chloroform-d) δ 7.40 (d, J=8.9 Hz, 1H), 7.08 (d, J=2.9 Hz, 1H), 6.85-6.79 (m, 2H), 5.83 (s, 1H), 4.77-4.71 (m, 2H), 4.49 (s, 2H), 4.41 (dd, J=11.7, 3.6 Hz, 2H), 4.37-4.33 (m, 2H), 4.33-4.26 (m, 1H), 3.82 (dd, J=11.7, 6.4 Hz, 2H); M/Z: 502, 504, 506 [M+H].sup.+, ESI.sup.+, RT=3.56 (S4).
##STR00040##
Step 16.a: tert-butyl 2-[(6-chloro-5-fluoropyridin-3-yl)oxy]acetate
[0290] To a solution of 6-chloro-5-fluoropyridin-3-ol (4.90 g, 33.2 mmol) in DMF (50 mL) was added tert-butyl 2-bromoacetate (4.5 mL, 34.9 mmol) and K.sub.2CO.sub.3 (13.8 g, 0.0996 mol) and the resultant mixture was stirred at 65° C. for 2 h. The reaction mixture was cooled to r.t., suspended in EtOAc (100 mL), and washed with H.sub.2O (2×50 mL) and brine (50 mL). The combined organic extracts were dried over Na.sub.2SO.sub.4 and concentrated in vacuo to afford the title compound (9.00 g, 32.7 mmol, 98% yield) as a brown oil; .sup.1H NMR (500 MHz, chloroform-d) δ 7.91 (d, J=2.6 Hz, 1H), 7.07 (dd, J=9.1, 2.6 Hz, 1H), 4.55 (s, 2H), 1.53-1.39 (m, 9H); M/Z: 262, 264 [M+H].sup.+, ESI.sup.+, RT=1.00 min (S2).
Step 16.b: 2-[(6-chloro-5-fluoropyridin-3-yl)oxy]acetic acid
[0291] 4 M HCl in 1,4-dioxane (25 mL, 98.0 mmol) was added to tert-butyl 2-[(6-chloro-5-fluoropyridin-3-yl)oxy]acetate (9.00 g, 32.7 mmol) and the resultant mixture was stirred at r.t. for 2 h. A further portion of 4 M HC.sub.1 in 1,4-dioxane (25 mL, 98.0 mmol) was added and the reaction mixture was stirred at 50° C. for 5 h. The reaction mixture was concentrated in vacuo and then triturated using Et.sub.2O and heptane. The resultant precipitate was filtered under vacuum to afford the title compound (6.48 g, 31.2 mmol, 96% yield) as an off white solid; .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 13.22 (s, 1H), 8.07 (d, J=2.6 Hz, 1H), 7.76 (dd, J=10.4, 2.6 Hz, 1H), 4.85 (s, 2H); M/Z: 206, 208 [M+H].sup.+, ESI.sup.+, RT=0.60 min (S2).
Example 10 (Step 16.c): 2-[(6-chloro-5-fluoro-3-pyridyl)oxy]-N-[trans-2-[5-[cis-3-(trifluoromethoxy)cyclobutyl]-1,3,4-oxadiazol-2-yl]-1,3-dioxan-5-yl]acetamide
[0292] ##STR00041##
[0293] A solution of 2-[(6-chloro-5-fluoropyridin-3-yl)oxy]acetic acid (34 mg, 0.163 mmol) in THF (2 mL) was cooled to 0° C. and treated with isobutyl carbonochloridate (20 μL, 0.155 mmol) and NMM (18 μL, 0.163 mmol). The reaction was stirred for 15 min then a solution of trans-2-{5-[cis-3-(trifluoromethoxy)cyclobutyl]-1,3,4-oxadiazol-2-yl}-1,3-dioxan-5-amine (50 mg, 0.163 mmol, Intermediate 16, described in route 12) in THF (1 mL) was added dropwise. The reaction was stirred at r.t. for 1 h, quenched with H.sub.2O (2 drops) and concentrated in vacuo. Purification by prep. HPLC (Method 3) afforded the title compound (37 mg, 0.0745 mmol, 46% yield) as a white powder; .sup.1H NMR (500 MHz, chloroform-d) δ 8.03 (d, J=2.6 Hz, 1H), 7.17 (dd, J=8.8, 2.6 Hz, 1H), 6.78 (d, J=8.4 Hz, 1H), 5.96 (s, 1H), 4.77-4.67 (m, 1H), 4.57 (s, 2H), 4.49-4.41 (m, 2H), 4.39-4.28 (m, 1H), 3.85 (dd, J=11.7, 6.4 Hz, 2H), 3.44-3.32 (m, 1H), 2.95-2.84 (m, 2H), 2.78-2.66 (m, 2H); M/Z: 497, 499 [M+H].sup.+, ESI.sup.+, RT=3.15 (S4).
II Biological Assay
HEK-ATF4 High Content Imaging Assay
[0294] 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, 2mM L-Glutamine, 100 U/mL Penicillin—100 μg/mL Streptomycin) and incubated at 37° C., 5% CO.sub.2. 24-hrs later, the medium was changed to 50 μl assay medium per well (DMEM/F12, 0.3% FBS, 2 mM L-Glutamine, 100 U/mL Penicillin—100 μg/mL Streptomycin). Example compounds were serially diluted in dimethyl sulfoxide (DMSO), spotted into intermediate plates and prediluted with assay medium containing 3.3 μM 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 hrs at 37° C., 5% CO.sub.2. Subsequently, cells were fixed (4% PFA in PBS, 20 min at room temperature) 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.
[0295] Activity of the tested example compounds is provided in Table 3 as follows: +++=IC.sub.50 1-500 nM; ++=IC.sub.50>500-2000 nM; +=IC.sub.50>2000-15000 nM.
TABLE-US-00003 TABLE 3 Example number Activity 1 +++ 2 +++ 3 + 4 ++ 5 +++ 6 +++ 7 +++ 8 +++ 9 +++ 10 +
Protocol—Measure of the Effect on hERG Channel by Tail Current Recording using in Vitro Rapid ICE
[0296] The potency of the example compounds in inhibiting human ERG potassium channel (hERG) tail current was assessed in a recombinant HEK293 cell line stably transfected with hERG cDNA under an inducible promoter, using Rapid ICE (rapid ion channel electrophysiology) assay. Rapid ICE is an automated patch-clamp assay utilizing the QPatch HTX system (Sophion Bioscience A/S). Briefly, inducible HEK hERG cells were cultivated in minimum essential medium supplemented with 10% FBS, 1% non-essential amino acids, 1% sodium pyruvate, 2 mM 1-glutamine, 15 μg/mL blasticidin, and 100 μg/mL hygromycin. hERG channel expression induction was obtained by adding 10 μg/mL tetracycline for 24, 48, or 72 h before recordings.
[0297] On the day of the experiment, cells were detached with TrypLE and prepared to be loaded on the instrument. Cells were resuspended in 7 mL of Serum-Free Media containing 25 mM Hepes and soybean trypsin inhibitor and immediately placed in the cell storage tank of the machine. The composition of the extracellular buffer was (mM): NaCl 137, KCl 4, CaCl.sub.2 1.8, MgCl.sub.2 1.0, d-glucose 10, N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) 10, pH 7.4 with 1 M NaOH. The composition of the intracellular solution was (mM): KCl.sub.130, MgCl.sub.2 1.0, ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) 5, MgATP 5, HEPES 10, pH 7.2 with 1 M KOH. The voltage protocol included the following steps: step from −80 to −50 mV for 200 ms, +20 mV for 4.8 s, step to −50 mV for 5 s, then step to the holding potential of ÷80 mV. Example compounds were dissolved in DMSO and diluted in extracellular buffer to achieve final test concentrations (0.3, 3, and 30 μM) in 0.3% DMSO. The voltage protocol was run and recorded continuously during the experiment. The vehicle, corresponding to 0.3% DMSO in extracellular buffer was then applied for 3 min, followed by the example compound in triplicate. The standard combined exposure time was 5 min. The average of tail current amplitude values recorded from four sequential voltage pulses was used to calculate for each cell the effect of the example compound by calculating the residual current (% control) compared with vehicle pretreatment. Data were reported as % inhibition for each concentration tested, and IC.sub.50 values were estimated using QPatch software. At least two cells were tested, and even more if results diverged.
TABLE-US-00004 TABLE 5 Example number hERG Activity* 2 + 5 ++ 6 + 7 + 8 + 9 + 10 + *hERG Activity category: +++ = IC.sub.50 1-1000 nM; ++ = IC.sub.50 >1000-5000 nM; + = IC.sub.50 >5000 nM
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