NGUYEN VAN (US)
CAVITT MARCHELLO (US)
ZHU BIN (US)
HAWKINS MICHAEL J (US)
LIU ZHIJIE (US)
ZHANG WEI (US)
WO2022136509A1 | 2022-06-30 | |||
WO2013030802A1 | 2013-03-07 | |||
WO2013030802A1 | 2013-03-07 |
US5023252A | 1991-06-11 | |||
US4992445A | 1991-02-12 | |||
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US5451233A | 1995-09-19 | |||
US5040548A | 1991-08-20 | |||
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US5496346A | 1996-03-05 | |||
US5292331A | 1994-03-08 | |||
US5674278A | 1997-10-07 | |||
US3657744A | 1972-04-25 | |||
US4739762A | 1988-04-26 | |||
US5195984A | 1993-03-23 | |||
US5879382A | 1999-03-09 | |||
US6344053B1 | 2002-02-05 |
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MARTINDALE: "The Extra Pharmacopoeia", 1999, THE PHARMACEUTICAL PRESS
What is claimed: 1. A compound of formula (I): or pharmaceutically acceptable salts thereof, wherein R2 is optionally substituted pyridinyl, optionally substituted pyrazolyl; optionally substituted 2,3-dihydro-[1,4]dioxino[2,3-b]pyridinyl; optionally substituted 6,7- dihydro-5H-pyrazolo[5,1-b][1,3]oxazinyl; optionally substituted 6,7-dihydro-4H- pyrazolo[5,1-c][1,4]oxazinyl; optionally substituted 4,5,6,7-tetrahydro-pyrazolo[1,5- a]pyrimidinyl; optionally substituted 5,6-dihydro-4H-pyrrolo[1,2-b]pyrazolyl; or optionally substituted pyridin-2(1H)-one-yl; R3 and R4 are each independently optionally substituted alkyl, optionally substituted cycloalkyl, or one of R3 or R4 may be H; or R3 and R4, together with the nitrogen atom to which they are both attached, form an optionally substituted 3-12-membered heterocycloalkyl ring, an optionally substituted 5-12-membered bridged heterocycloalkyl ring, an optionally substituted 4- 12-membered fused heterocycloalkyl ring system, or an optionally substituted 5-12- membered spiroheterocycloalkyl ring system, wherein said 3-12-membered heterocycloalkyl ring, 5-12-membered bridged heterocycloalkyl ring, 4-12-membered fused heterocycloalkyl ring system, or 5-12-membered spiroheterocycloalkyl ring system may include, in addition to the nitrogen atom to which both R3 and R4 are attached, 1-3 other heteroatoms that are each independently O, S, or N; each R5 and each R6 is independently H, C1-C4alkyl, or C3-C5cycloalkyl; or an R5 or R6, together with an R3 or R4 may form an optionally substituted 3-12- membered heterocycloalkyl ring, an optionally substituted 5-12-membered bridged heterocycloalkyl ring, an optionally substituted 4-12-membered fused heterocycloalkyl ring system, or an optionally substituted 5-12-membered spiroheterocycloalkyl ring system; or when L is NHC(O)NR7-, an R5 or an R6 attached to a carbon atom, together with R7, form a heterocycloalkyl ring; n is 1 or 2; and L is -NHC(O)- when n is 1; or -NHC(O)-, -NHC(O)O-, -C(O)NH-, or -NHC(O)NR7- when n is 2. 2. The compound of claim 1, wherein R2 is an optionally substituted pyrazolyl. 3. The compound of claim 2, wherein the optionally substituted pyrazolyl is an optionally substituted pyrazol-4-yl. 4. The compound of claim 2 or claim 3, wherein the optionally substituted pyrazolyl is substituted with one or more of optionally substituted -C1-C6alkyl, optionally substituted -O-C1-C6alkyl; and optionally substituted -C1-C6alkyl-O-C1-C6alkyl. 5. The compound of claim 4, wherein the optionally substituted -C1-C6alkyl is -CH3, - CH2OH, -CH2CH2F, or -CH2CH2OH. 6. The compound of claim 4 or claim 5, wherein the optionally substituted -O-C1- C6alkyl is -OCH3, or -OCH2CH2F. 7. The compound of any one of claims 4, 5, or 6, wherein the optionally substituted C1- C6alkyl-O-C1-C6alkyl is -CH2CH2OCH3. 8. The compound of claim 1, wherein R2 is an optionally substituted optionally substituted pyridinyl. 9. The compound of claim 8, wherein the optionally substituted pyridinyl is substituted with one or more of halo or optionally substituted -O-C1-C6alkyl. 10. The compound of claim 9, wherein the halo is -F. 11. The compound of claim 9, wherein the optionally substituted -O-C1-C6alkyl is -OCH3, or -OCH2CH2F. 12. The compound of claim 1, wherein R2 is an optionally substituted 5,6-dihydro-4H- pyrrolo[1,2-b]pyrazolyl. 13. The compound of claim 12, wherein R2 is unsubstituted 5,6-dihydro-4H-pyrrolo[1,2- b]pyrazol-3-yl. 14. The compound of claim 1, wherein R2 is an optionally substituted 6,7-dihydro-5H- pyrazolo[5,1-b][1,3]oxazinyl. 15. The compound of claim 14, wherein R2 is an optionally substituted 6,7-dihydro-5H- pyrazolo[5,1-b][1,3]oxazin-3-yl. 16. The compound of claim 15, wherein the optionally substituted 6,7-dihydro-5H- pyrazolo[5,1-b][1,3]oxazin-3-yl is substituted with one or more of -OH or optionally substituted -C1-C6alkyl. 17. The compound of claim 16, wherein the 6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazin- 3-yl is substituted with -OH. 18. The compound of claim 16 or claim 17, wherein the optionally substituted -C1- C6alkyl is -CH3 or -CH2OH. 19. The compound of claim 15, wherein the 6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazin- 3-yl is unsubstituted. 20. The compound of claim 1, wherein R2 is an optionally substituted 6,7-dihydro-4H- pyrazolo[5,1-c][1,4]oxazinyl. 21. The compound of claim 20, wherein R2 is an optionally substituted 6,7-dihydro-4H- pyrazolo[5,1-c][1,4]oxazin-3-yl. 22. The compound of claim 1, wherein R2 is an optionally substituted 2,3-dihydro- [1,4]dioxino[2,3-b]pyridinyl. 23. The compound of claim 22, wherein the 2,3-dihydro-[1,4]dioxino[2,3-b]pyridinyl is unsubstituted 2,3-dihydro-[1,4]dioxino[2,3-b]pyridine-8-yl. 24. The compound of claim 1, wherein R2 is an optionally substituted 4,5,6,7-tetrahydro- pyrazolo[1,5-a]pyrimidinyl. 25. The compound of claim 24, wherein R2 is unsubstituted 4,5,6,7-tetrahydro- pyrazolo[1,5-a]pyrimidin-3-yl. 26. The compound of claim 1, wherein R2 is an optionally substituted pyridin-2(1H)-one- yl. 27. The compound of claim 26, wherein R2 is an optionally substituted pyridin-2(1H)- one-3-yl. 28. The compound of claim 27, wherein the pyridin-2(1H)-one-3-yl is substituted with optionally substituted -C1-C6alkyl. 29. The compound of claim 28, wherein the optionally substituted -C1-C6alkyl is - CH2CH2F. 30. The compound according to any one of the preceding claims, wherein L is -C(O)NH-. 31. The compound according to any one of claims 1-29, wherein L is -NHC(O)-. 32. The compound according to any one of claims 1-29, wherein L is or -NHC(O)O-. 33. The compound according to claim 31, wherein n is 1. 34. The compound according to any one of claims 1-32, wherein n is 2. 35. The compound according to any one of the preceding claims, wherein each R5 and each R6 is H. 36. The compound according to any one of claims 1-29, wherein L is or -NHC(O)NR7-. 37. The compound according to claim 36, wherein an R5 together with R7, form a heterocycloalkyl ring. 38. The compound according to claim 37, wherein an R5 together with R7, form an azetidinyl ring. 39. The compound according to any one of the preceding claims, wherein R3 is H and R4 is C1-C6alkyl or C5-C6cycloalkyl. 40. The compound according to any one of the preceding claims, wherein R3 and R4, together with the nitrogen atom to which they are both attached, form an optionally substituted 3-12-membered heterocycloalkyl ring, an optionally substituted 5-12- membered bridged heterocycloalkyl ring, an optionally substituted 4-12-membered fused heterocycloalkyl ring system, or an optionally substituted 5-12-membered spiroheterocycloalkyl ring system, wherein said 3-12-membered heterocycloalkyl ring, 5-12-membered bridged heterocycloalkyl ring, 4-12-membered fused heterocycloalkyl ring system, or 5-12-membered spiroheterocycloalkyl ring system may include, in addition to the nitrogen atom to which both R3 and R4 are attached, 1- 3 other heteroatoms that are each independently O, S, or N; 41. The compound according to claim 40, wherein R3 and R4, together with the nitrogen atom to which they are both attached, form an optionally substituted 4-6-membered heterocycloalkyl ring. 42. The compound according to claim 40, wherein R3 and R4, together with the nitrogen atom to which they are both attached, form an optionally substituted 8-membered bridged heterocycloalkyl ring. 43. The compound according to claim 40, wherein R3 and R4, together with the nitrogen atom to which they are both attached, form an optionally substituted 7-9-membered spiroheterocycloalkyl ring. 44. The compound according to claim 40, wherein R3 and R4, together with the nitrogen atom to which they are both attached, form an optionally substituted 6-7-membered fused heterocycloalkyl ring system. 45. The compound of claim 1, wherein an R5 or R6, together with an R3 or R4 form an optionally substituted 3-12-membered heterocycloalkyl ring, an optionally substituted 5-12-membered bridged heterocycloalkyl ring, an optionally substituted 4-12- membered fused heterocycloalkyl ring system, or an optionally substituted 5-12- membered spiroheterocycloalkyl ring system. 46. The compound of claim 45, wherein an R5 or R6, together with an R3 or R4 form an optionally substituted 4- or 5-membered heterocycloalkyl ring. 47. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is: N-(5-((2-(2,2-dimethylpyrrolidin-1-yl)ethyl)carbamoyl)-2-methylpyridin-3-yl)-2-(3- methoxy-1-methyl-1H-pyrazol-4-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; (S)-2-(5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-N-(2-methyl-5-((2-(2- methylpyrrolidin-1-yl)ethyl)carbamoyl)pyridin-3-yl)pyrazolo[5,1-b]thiazole-7- carboxamide; N-(5-((2-(3-azabicyclo[3.1.0]hexan-3-yl)ethyl)carbamoyl)-2-methylpyridin-3-yl)-2- (5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; N-(5-((2-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)ethyl)carbamoyl)-2-methylpyridin-3- yl)-2-(5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)pyrazolo[5,1-b]thiazole-7- carboxamide; N-(5-((2-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)ethyl)carbamoyl)-2-methylpyridin-3- yl)-2-(1,3-dimethyl-1H-pyrazol-4-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; N-(5-((2-(3-azabicyclo[3.2.0]heptan-3-yl)ethyl)carbamoyl)-2-methylpyridin-3-yl)-2- (6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazin-3-yl)pyrazolo[5,1-b]thiazole-7- carboxamide; N-(5-((2-(5-azaspiro[2.4]heptan-5-yl)ethyl)carbamoyl)-2-methylpyridin-3-yl)-2-(5,6- dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; N-(5-((2-(4-azaspiro[2.4]heptan-4-yl)ethyl)carbamoyl)-2-methylpyridin-3-yl)-2-(1,5- dimethyl-1H-pyrazol-4-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; N-(5-((2-(4-azaspiro[2.4]heptan-4-yl)ethyl)carbamoyl)-2-methylpyridin-3-yl)-2-(1,3- dimethyl-1H-pyrazol-4-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; N-(5-((2-(6-azaspiro[3.4]octan-6-yl)ethyl)carbamoyl)-2-methylpyridin-3-yl)-2-(1,5- dimethyl-1H-pyrazol-4-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; N-(5-((2-(6-azaspiro[3.4]octan-6-yl)ethyl)carbamoyl)-2-methylpyridin-3-yl)-2-(1,3- dimethyl-1H-pyrazol-4-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; 2-(1,3-dimethyl-1H-pyrazol-4-yl)-N-(2-methyl-5-((2-(piperidin-1- yl)ethyl)carbamoyl)pyridin-3-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; 2-(5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-N-(2-methyl-5-((2-(piperidin-1- yl)ethyl)carbamoyl)pyridin-3-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; N-(5-((2-(2-oxa-5-azaspiro[3.4]octan-5-yl)ethyl)carbamoyl)-2-methylpyridin-3-yl)-2- (6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazin-3-yl)pyrazolo[5,1-b]thiazole-7- carboxamide; N-(5-((2-(2-oxa-5-azaspiro[3.4]octan-5-yl)ethyl)carbamoyl)-2-methylpyridin-3-yl)-2- (5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; N-(5-((2-(4-azaspiro[2.4]heptan-4-yl)ethyl)carbamoyl)-2-methylpyridin-3-yl)-2-(5,6- dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; N-(5-((2-(5-azaspiro[3.4]octan-5-yl)ethyl)carbamoyl)-2-methylpyridin-3-yl)-2-(1-(2- fluoroethyl)-3-methyl-1H-pyrazol-4-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; N-(5-((2-(5-azaspiro[3.4]octan-5-yl)ethyl)carbamoyl)-2-methylpyridin-3-yl)-2-(1-(2- fluoroethyl)-5-methyl-1H-pyrazol-4-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; N-(5-((2-(3-azabicyclo[3.2.0]heptan-3-yl)ethyl)carbamoyl)-2-methylpyridin-3-yl)-2- (1,3-dimethyl-1H-pyrazol-4-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; N-(5-((2-(3-azabicyclo[3.2.0]heptan-3-yl)ethyl)carbamoyl)-2-methylpyridin-3-yl)-2- (5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; N-(5-((2-(4-azaspiro[2.4]heptan-4-yl)ethyl)carbamoyl)-2-methylpyridin-3-yl)-2-(3- methoxy-1-methyl-1H-pyrazol-4-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; (S)-2-(3-methoxy-1-methyl-1H-pyrazol-4-yl)-N-(2-methyl-5-((2-(2-methylpyrrolidin- 1-yl)ethyl)carbamoyl)pyridin-3-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; N-(5-((2-(5-azaspiro[3.4]octan-5-yl)ethyl)carbamoyl)-2-methylpyridin-3-yl)-2-(3- methoxy-1-methyl-1H-pyrazol-4-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; N-(5-((2-(4-azaspiro[2.4]heptan-4-yl)ethyl)carbamoyl)-2-methylpyridin-3-yl)-2-(6,7- dihydro-5H-pyrazolo[5,1-b][1,3]oxazin-3-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; 2-(6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazin-3-yl)-N-(2-methyl-5-((2-(piperidin-1- yl)ethyl)carbamoyl)pyridin-3-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; N-(5-((2-(1-oxa-7-azaspiro[3.5]nonan-7-yl)ethyl)carbamoyl)-2-methylpyridin-3-yl)- 2-(6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazin-3-yl)pyrazolo[5,1-b]thiazole-7- carboxamide; N-(5-((2-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)ethyl)carbamoyl)-2-methylpyridin-3- yl)-2-(6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazin-3-yl)pyrazolo[5,1-b]thiazole-7- carboxamide; (S)-2-(5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-N-(2-methyl-5-((2-(3- methylpyrrolidin-1-yl)ethyl)carbamoyl)pyridin-3-yl)pyrazolo[5,1-b]thiazole-7- carboxamide; N-(5-((2-(1-oxa-7-azaspiro[3.5]nonan-7-yl)ethyl)carbamoyl)-2-methylpyridin-3-yl)- 2-(5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)pyrazolo[5,1-b]thiazole-7- carboxamide; N-(5-((2-(8-oxa-5-azaspiro[3.5]nonan-5-yl)ethyl)carbamoyl)-2-methylpyridin-3-yl)- 2-(5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)pyrazolo[5,1-b]thiazole-7- carboxamide; N-(5-((2-(1-oxa-7-azaspiro[3.5]nonan-7-yl)ethyl)carbamoyl)-2-methylpyridin-3-yl)- 2-(1,3-dimethyl-1H-pyrazol-4-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; N-(5-((2-(2,2-dimethylpyrrolidin-1-yl)ethyl)carbamoyl)-2-methylpyridin-3-yl)-2-(5- methoxy-1-methyl-1H-pyrazol-4-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; N-(5-((2-(5-azaspiro[3.4]octan-5-yl)ethyl)carbamoyl)-2-methylpyridin-3-yl)-2-(1-(2- hydroxyethyl)-1H-pyrazol-4-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; N-(5-((2-(5-azaspiro[3.4]octan-5-yl)ethyl)carbamoyl)-2-methylpyridin-3-yl)-2-(5- methoxy-1-methyl-1H-pyrazol-4-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; (S)-2-(5-methoxy-1-methyl-1H-pyrazol-4-yl)-N-(2-methyl-5-((2-(2-methylpyrrolidin- 1-yl)ethyl)carbamoyl)pyridin-3-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; N-(5-((2-(4-azaspiro[2.4]heptan-4-yl)ethyl)carbamoyl)-2-methylpyridin-3-yl)-2-(5- methoxy-1-methyl-1H-pyrazol-4-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; 2-(5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-N-(5-((2- (isopropylamino)ethyl)carbamoyl)-2-methylpyridin-3-yl)pyrazolo[5,1-b]thiazole-7- carboxamide; N-(5-((2-(cyclohexylamino)ethyl)carbamoyl)-2-methylpyridin-3-yl)-2-(5,6-dihydro- 4H-pyrrolo[1,2-b]pyrazol-3-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; N-(5-((2-(cyclopentylamino)ethyl)carbamoyl)-2-methylpyridin-3-yl)-2-(5,6-dihydro- 4H-pyrrolo[1,2-b]pyrazol-3-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; N-(5-((2-(3-azabicyclo[3.1.0]hexan-3-yl)ethyl)carbamoyl)-2-methylpyridin-3-yl)-2- (6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazin-3-yl)pyrazolo[5,1-b]thiazole-7- carboxamide; N-(5-((2-(4-azaspiro[2.4]heptan-4-yl)ethyl)carbamoyl)-2-methylpyridin-3-yl)-2-(6,7- dihydro-4H-pyrazolo[5,1-c][1,4]oxazin-3-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; N-(5-((2-(3-azabicyclo[3.1.0]hexan-3-yl)ethyl)carbamoyl)-2-methylpyridin-3-yl)-2- (1,3-dimethyl-1H-pyrazol-4-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; N-(5-((2-(2,2-dimethylpyrrolidin-1-yl)ethyl)carbamoyl)-2-methylpyridin-3-yl)-2-(2- (2-fluoroethoxy)pyridin-3-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; N-(5-((2-(5-azaspiro[3.4]octan-5-yl)ethyl)carbamoyl)-2-methylpyridin-3-yl)-2-(2-(2- fluoroethoxy)pyridin-3-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; 2-(2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-8-yl)-N-(5-((2-(2,2-dimethylpyrrolidin-1- yl)ethyl)carbamoyl)-2-methylpyridin-3-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; N-(5-((2-(2-azabicyclo[2.2.2]octan-2-yl)ethyl)carbamoyl)-2-methylpyridin-3-yl)-2- (2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-8-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; N-(5-(2-(2,2-dimethylpyrrolidin-1-yl)acetamido)-2-methylpyridin-3-yl)-2-(6- fluoropyridin-3-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; N-(5-(2-(2,2-dimethylpyrrolidin-1-yl)acetamido)-2-methylpyridin-3-yl)-2-(2- methoxypyridin-4-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; N-(5-(2-(2,2-dimethylpyrrolidin-1-yl)acetamido)-2-methylpyridin-3-yl)-2-(2- fluoropyridin-4-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; N-(5-(2-(2,2-dimethylpyrrolidin-1-yl)acetamido)-2-methylpyridin-3-yl)-2-(2- fluoropyridin-3-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; N-(5-(2-(2,2-dimethylpyrrolidin-1-yl)acetamido)-2-methylpyridin-3-yl)-2-(6- fluoropyridin-2-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; N-(5-(2-(5-azaspiro[3.4]octan-5-yl)acetamido)-2-methylpyridin-3-yl)-2-(1-(2- fluoroethyl)-2-oxo-1,2-dihydropyridin-3-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; (S)-(1-methylpyrrolidin-2-yl)methyl (5-(2-(1-(2-methoxyethyl)-1H-pyrazol-4- yl)pyrazolo[5,1-b]thiazole-7-carboxamido)-6-methylpyridin-3-yl)carbamate; N-(5-(2-(3,3-dimethylazetidin-1-yl)acetamido)-2-methylpyridin-3-yl)-2-(5- (hydroxymethyl)-3-methoxy-1-methyl-1H-pyrazol-4-yl)pyrazolo[5,1-b]thiazole-7- carboxamide; N-(5-(2-(3,3-dimethylazetidin-1-yl)acetamido)-2-methylpyridin-3-yl)-2-(2- (hydroxymethyl)-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazin-3-yl)pyrazolo[5,1- b]thiazole-7-carboxamide; N-(5-(2-(3,3-dimethylazetidin-1-yl)acetamido)-2-methylpyridin-3-yl)-2-(5-methoxy- 1,3-dimethyl-1H-pyrazol-4-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; N-(5-(2-(2,2-dimethylazetidin-1-yl)acetamido)-2-methylpyridin-3-yl)-2-(5-methoxy- 1,3-dimethyl-1H-pyrazol-4-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; N-(5-(2-(2,2-dimethylazetidin-1-yl)acetamido)-2-methylpyridin-3-yl)-2-(5-methoxy- 1-methyl-1H-pyrazol-4-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; N-(5-(2-(3,3-dimethylazetidin-1-yl)acetamido)-2-methylpyridin-3-yl)-2-(5-methoxy- 1-methyl-1H-pyrazol-4-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; 2-(6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazin-3-yl)-N-(5-(2-(2,2-dimethylazetidin-1- yl)acetamido)-2-methylpyridin-3-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; N-(5-(2-(2,2-dimethylazetidin-1-yl)acetamido)-2-methylpyridin-3-yl)-2-(3-methoxy- 1-methyl-1H-pyrazol-4-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; N-(5-(2-(3,3-dimethylazetidin-1-yl)acetamido)-2-methylpyridin-3-yl)-2-(6-hydroxy- 2-methyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazin-3-yl)pyrazolo[5,1-b]thiazole-7- carboxamide; N-(5-(2-(2,2-dimethylazetidin-1-yl)acetamido)-2-methylpyridin-3-yl)-2-(6-hydroxy- 2-methyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazin-3-yl)pyrazolo[5,1-b]thiazole-7- carboxamide; N-(5-(2-(3,3-Dimethylazetidin-1-yl)acetamido)-2-methylpyridin-3-yl)-2-(4,5,6,7- tetrahydropyrazolo[1,5-a]pyrimidin-3-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; N-(5-(1-(2,2-dimethylcyclopentyl)azetidine-3-carboxamido)-2-methylpyridin-3-yl)-2- (1-methyl-1H-pyrazol-4-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; N-(5-(1-cyclohexylazetidine-3-carboxamido)-2-methylpyridin-3-yl)-2-(1-methyl-1H- pyrazol-4-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; N-(5-(1-(cyano(cyclopentyl)methyl)azetidine-3-carboxamido)-2-methylpyridin-3-yl)- 2-(1-methyl-1H-pyrazol-4-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; N-(5-(1-(cyclopentylmethyl)azetidine-3-carboxamido)-2-methylpyridin-3-yl)-2-(1- methyl-1H-pyrazol-4-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; 2-(2-methoxypyridin-3-yl)-N-(2-methyl-5-(3-(piperidin-1-yl)azetidine-1- carboxamido)pyridin-3-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; N-(5-((2-(2,2-dimethylpyrrolidin-1-yl)ethyl)carbamoyl)-2-methylpyridin-3-yl)-2-(6- (2-fluoroethoxy)pyridin-2-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; N-(5-((2-(5-azaspiro[3.4]octan-5-yl)ethyl)carbamoyl)-2-methylpyridin-3-yl)-2-(1-(2- fluoroethyl)-1H-pyrazol-4-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; N-(5-((2-(5-azaspiro[3.4]octan-5-yl)ethyl)carbamoyl)-2-methylpyridin-3-yl)-2-(1H- pyrazol-4-yl)pyrazolo[5,1-b]thiazole-7-carboxamide; or 18F-N-(5-((2-(5-azaspiro[3.4]octan-5-yl)ethyl)carbamoyl)-2-methylpyridin-3-yl)-2-(1- (2-fluoroethyl)-1H-pyrazol-4-yl)pyrazolo[5,1-b]thiazole-7-carboxamide. 48. A compound according to any one of claims 1-47, wherein the compound has an IC50< 20 nM in a PDGFR cellular assay. 49. The compound according to claim 48, wherein the compound has an IC50< 5 nM in a PDGFR cellular assay. 50. A compound of any one of claims 1 to 49, or a pharmaceutically acceptable salt thereof, wherein a fluorine atom in the compound has been enriched in 18F, or a carbon atom in the compound has been enriched in 11C, for use in positron emission tomography (PET) imaging. 51. A pharmaceutical composition comprising a compound according to any one of claims 1-49, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. 52. A pharmaceutical composition comprising a compound according to 50, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, for use in positron emission tomography (PET) imaging. 53. A method of treating a disease or disorder in which PDGFR signaling is implicated in a subject in need thereof, comprising administering to said subject an amount of a compound according to any one of claims 1-49, or a pharmaceutically acceptable salt thereof, that is effective to treat said disease or disorder. 54. The method according to claim 53, wherein said disease or disorder is pulmonary hypertension (PH). 55. The method according to claim 54, wherein said pulmonary hypertension is pulmonary arterial hypertension (PAH); PH secondary to heart failure; PH secondary to lung diseases and/or hypoxia; PH due to pulmonary artery obstruction; or PH due to unknown or rare diseases. 56. The method according to claim 55, wherein said pulmonary hypertension is pulmonary arterial hypertension (PAH). 57. A method of positron emission tomography (PET) imaging of a tissue in a subject, the method comprising administering to the subject a 18F- or 11C-containing compound of any one of claims 1-49, allowing the compound to penetrate into the tissue of the subject; and then collecting a PET image of the tissue of the subject. 58. The method of claim 57, wherein the tissue is heart or lung tissue. 59. The method of claim 57 or 58, wherein the compound is 18F-N-(5-((2-(5- azaspiro[3.4]octan-5-yl)ethyl)carbamoyl)-2-methylpyridin-3-yl)-2-(1-(2-fluoroethyl)- 1H-pyrazol-4-yl)pyrazolo[5,1-b]thiazole-7-carboxamide. 103693.003965 (ACT6046WOPCT1) ABSTRACT The disclosure is directed to compounds of formula (I) R2 N S O R5 R6 R3 N L n N N H N R4 (I), and pharmaceutically acceptable salts thereof. Pharmaceutical compositions comprising compounds of formula (I), as well as methods of their use and preparation, are also described. |
, or a pharmaceutically acceptable salt thereof. [00271] Compounds of formula (I) in the present invention can be synthesized in accordance with general synthetic methods familiar to those who are skilled in the art. The following reaction schemes are only meant to represent examples of the invention and are in no way meant to be a limit of the invention.
[00272] The schemes below illustrate synthesis of compounds of formula (I). [00273] Scheme 1 illustrated the synthesis of key intermediate A.2-methylthiazole (A-1) treated with NBS in DMF at room temperature to give 5-bromo-2-methylthiazole (A- 2), 5-bromo-2methylthiazole was then reacted with LiHMDS and diethyl carbonate in THF yielded ethyl 2-(5-bromothiazol-2-yl)acetate (A-2), subsequently treated with ethyl (Z)-N- ((mesitylsulfonyl)oxy)acetimidate (A-4) and TFA in dichloromethane to give A-5, reacted A-5 with triethyl orthoformate resulted ethyl 2-bromopyrazolo[5,1-b]thiazole-7-carboxylate (A). [00274] Scheme 2 show the synthesis of Formula I while L = CONH, n = 2. Ethyl 2-bromopyrazolo[5,1-b]thiazole-7-carboxylate (A) hydrolyzed to 2-bromopyrazolo[5,1- b]thiazole-7-carboxylic acid (I-1) under a base such as NaOH in a solvent such as ethanol- water, 2-bromopyrazolo[5,1-b]thiazole-7-carboxylic acid (I-1) then converted into acid chloride with SOCl 2 or oxalyl dichloride in a solvent such as toluene or methylene chloride, the acid chloride reacted with amine (I-2) and a base such as DIEA or pyridine in a solvent such as methylene chloride or pyridine to give ester compound (I-3), ester compound (I-3) hydrolyzed to acid compound (I-4) under a base such as NaOH in a solvent such as ethanol- water, then treated with amine (I-5), a coupling reagent such as HATU, a base such as DIEA in a solvent such as DMF to produce compound (I-6), cross coupling compound (I-6) with a coupling reagent such a boronic acid or a boronate or a stannyl reagent (I-7) with a catalyst such as Pd(dppf)Cl 2 DCM, a base such as Cs 2 CO 3 or K 3 PO 4 in a solvent such as DMF-water or dioxane-water to give Formula I compound while L = CONH, n = 2. [00275] Scheme 3 show the alternative synthesis of Formula I while L = CONH, n = 2. Ethyl 2-bromopyrazolo[5,1-b]thiazole-7-carboxylate (A) was coupled with a coupling reagent such a boronic acid or a boronate or a stannyl reagent (I-7) with a catalyst such as Pd(dppf)Cl2 DCM, a base such as Cs 2 CO 3 or K 3 PO 4 in a solvent such as DMF-water or dioxane-water to give ester (I-8), ester (I-8) then hydrolyzed to acid (I-9) under a base such as NaOH in a solvent such as ethanol-water, the acid (I-9) then converted into acid chloride with SOCl 2 or oxalyl dichloride in a solvent such as toluene or methylene chloride, the acid chloride reacted with amine (I-2) and a base such as DIEA or pyridine in a solvent such as methylene chloride or pyridine to give ester compound (I-10), ester compound (I-10) hydrolyzed to acid compound (I-11) under a base such as NaOH in a solvent such as ethanol- water, then treated with amine (I-5), a coupling reagent such as HATU, a base such as DIEA in a solvent such as DMF to produce Formula I compound while L = CONH, n = 2. [00276] Scheme 4 show the synthesis of Formula I while L = NHCO, n = 1 or 2. Acid (I-9) was first converted into acid chloride with SOCl 2 or Oxalyl dichloride in a solvent such as toluene or methylene chloride, the acid chloride reacted with amine (I-12) and a base such as DIEA or pyridine in a solvent such as methylene chloride or pyridine to give a Boc- protected compound (I-13). Alternatively, acid (I-1) was converted into acid chloride with SOCl 2 or oxalyl dichloride in a solvent such as toluene or methylene chloride, the acid chloride reacted with amine (I-12) and a base such as DIEA or pyridine in a solvent such as methylene chloride or pyridine to give compound (I-14), compound (I-14) was then coupled with a coupling reagent such a boronic acid or a boronate or a stannyl reagent (I-7) with a catalyst such as Pd(dppf)Cl 2 DCM, a base such as Cs 2 CO 3 or K 3 PO 4 in a solvent such as DMF-water or dioxane-water to give a Boc-protected compound (I-13). Deprotecting Boc by treating compound (I-13) with acid such as TFA in a solvent such as methylene chloride followed by treatment with chloroacetyl chloride (I-16) and a base such as NaHCO 3 in a solvent such as DMF to give compound (I-17). Compound (I-17) was then reacted with amine (I-18) and a base such as K 2 CO 3 in a solvent such as DMF to produce Formula I while L = NHCO, n = 0 or 1. Alternatively, compound (I-15) treated with an acid (I-19), a coupling agent such as HATU, a base such as DIEA in a solvent such as DMF to yield Formula I compound while L = NHCO, n = 1 or 2. Further alternatively, compound (I-15) treated with 3-chloropropanoyl chloride (I-20) and a base such as triethylamine in a solvent such as dichloromethane to give compound (I-21) which then reacted with amine (I-18) and a base such as K 2 CO 3 in a solvent such as DMF to produce Formula I while L = NHCO, n = 1 or 2. [00278] Scheme 5 shows the synthesis of Formula I while L = NHC(O)O, n = 2. The Boc-protected compound (I-14) was treated with an acid such as TFA in a solvent such as methylene chloride to give de-protected compound (I-28). The compound (I-28) was first treated with 1,1′-carbonyldiimidazole (CDI), in the presence of a base such as Et 3 N or DIEA, in a solvent such as DMF, and was then reacted with alcohol (II-5) to give carbamate compound (II-6). Compound (II-6) was then coupled with a coupling reagent such a boronic acid or a boronate or a stannyl reagent (I-7) with a catalyst such as Pd(dppf)Cl 2 DCM, a base such as Cs 2 CO 3 or K 3 PO 4 in a solvent such as DMF-water or dioxane-water to produce Formula I while L = NHC(O)O, n = 2. Alternatively, compound (I-14) was coupled with a coupling reagent such a boronic acid or a boronate or a stannyl reagent (I-7) with a catalyst such as Pd(dppf)Cl 2 DCM, a base such as Cs 2 CO 3 or K 3 PO 4 in a solvent such as DMF-water or dioxane-water to give a Boc-protected compound (I-13). Deprotecting Boc by treating compound (I-13) with acid such as TFA in a solvent such as methylene chloride gave compound (I-15). Compound (I-15) was first treated with 1,1′-carbonyldiimidazole (CDI), in the presence of a base such as Et 3 N or DIEA, in a solvent such as DMF, and was then reacted with alcohol (II-4) to produce Formula I while L = NHC(O)O, n = 2. [00279] Examples Intermediate A: ethyl 2-bromopyrazolo[3,2-b][1,3]thiazole-7-carboxylate Step a: 5-bromo-2-methyl-1,3-thiazole [00280] Into a 2-L 4-necked round-bottom flask, was placed 2-methyl-thiazole (150.00 g, 1361.56 mmol), DMF (1.17 L), NBS (290.8 g, 1633.89 mmol). The resulting solution was stirred for 8 h at room temperature. The reaction was then quenched by the addition of 1500 mL of water. The resulting solution was extracted with 3x500 mL of Et 2 O and the organic layer was concentrated. The product was precipitated by the addition of n- heptane (300 mL). The solids were collected by filtration. This resulted in 99 g (40.8%) of 5- bromo-2-methyl-1,3-thiazole as a brown solid. LC-MS: (ES, m/z): [M+H] + =178 Step b: ethyl 2-(5-bromo-1,3-thiazol-2-yl)acetate [00281] Into a 2-L 4-necked round-bottom flask, was placed 5-bromo-2-methyl-1,3- thiazole (99.00 g, 556.02 mmol), THF (1100 mL). This was followed by the addition of LiHMDS (667.23 mL, 667.23 mmol) dropwise with stirring at -60°C in 30 min. Diethyl carbonate (75.64 g, 667.23 mmol) was added dropwise to the mixture with stirring at -60°C in 30 min. The resulting solution was stirred for 1 h at room temperature. The reaction was then quenched by the addition of 1100 mL of water. The resulting solution was extracted with 3x500 mL of ethyl acetate and the organic layer was concentrated. The residue was purified by a silica gel column with ethyl acetate/petroleum ether (1:50). The fractions were combined and concentrated to give 40.1 g (28.8%) of ethyl 2-(5-bromo-1,3-thiazol-2-yl)acetate as a yellow solid. LC-MS: (ES, m/z): [M+H] + =250 Step c: 3-amino-5-bromo-2-(2-ethoxy-2-oxoethyl)-1,3-thiazol-3-ium 2,4,6- trimethylbenzenesulfonate [00282] (Z)-(ethyl N-[(2,4,6-trimethylbenzenesulfonyl)oxy]ethanimidate) (51.34 g, 179.92 mmol) was added at 0°C was added to a mixture of TFA (235.15 g, 2398.9 mmol) and ice water (50 mL) at 0°C and stirred for 1.5 h. Ice water (300 mL) was then added. The solid was collected by filtration. The solid was dissolved into DCM and dried with anhydrous Na 2 SO 4 . Then the organic phase was collected through filtration, a solution of ethyl 2-(5- bromo-1,3-thiazol-2-yl)acetate (40.1 g, 160.32 mmol) in DCM (300 mL) was added dropwise. Then the resulting solution was stirred at r.t for 1.5 h. The white precipitate was collected by filtration, washed with MTBE (1x50 mL), dried to give 39.5 g (91.3%) of 3- amino-5-bromo-2-(2-ethoxy-2-oxoethyl)-1,3-thiazol-3-ium 2,4,6-trimethylbenzenesulfonate as a white solid. LC-MS: (ES, m/z): [M+H] + =265 Step d: ethyl 2-bromopyrazolo[3,2-b][1,3]thiazole-7-carboxylate (Intermediate A) [00283] 3-amino-5-bromo-2-(2-ethoxy-2-oxoethyl)-1,3-thiazol-3-ium 2,4,6- trimethylbenzenesulfonate (39.50 g, 84.87 mmol), triethyl orthoformate (150 mL) was placed into a 500-mL round-bottom flask. The resulting solution was stirred for 2 h at 120°C and concentrated. The residue was purified by a silica gel column with ethyl acetate/petroleum ether (1:50). The fractions were combined, concentrated, dried to give 9.0 g (38.6%) of ethyl 2-bromopyrazolo[3,2-b][1,3]thiazole-7-carboxylate as a light pink solid. LC-MS: (ES, m/z): 275 [M+H] + ; 1 H-NMR: (300 MHz, CDCl 3 , ppm): δ 8.21 (s, 1H), 7.86 (s, 1H), 4.36 (q, J = 7.1 Hz, 2H), 1.40 (t, J = 7.1 Hz, 3H). Example 1, N-(5-((2-(2,2-dimethylpyrrolidin-1-yl)ethyl)carbamoyl)-2-met hylpyridin-3- yl)-2-(3-methoxy-1-methyl-1H-pyrazol-4-yl)pyrazolo[5,1-b]thi azole-7-carboxamide Step a: 2-(2, 2-dimethylpyrrolidin-1-yl)acetonitrile [00284] To a solution of 2,2-dimethylpyrrolidine (20 g, 201 mmol) and potassium carbonate (55.74 g, 403 mmol) in DMF (130 mL) was added 2-bromoacetonitrile (15.4 mL, 221 mmol) at room-temperature. The resulting mixture was stirred at 30 °C for 12 h. The reaction was concentrated under reduced pressure to give crude product, which was purified by column chromatography over silica gel (eluent: ethyl acetate: methanol = 7:3) to give the title compound 2-(2, 2-dimethylpyrrolidin-1-yl)acetonitrile (22 g, 79%) as a pale yellow oil. 1 H NMR (400 MHz, DMSO-d6) δ 3.50 (s, 2H), 2.81 - 2.86 (m, 2H), 1.69 - 1.77 (m, 2H), 1.56 - 1.62 (m, 2H), 0.98 (s, 6 H). Step b: 2-(2,2-dimethylpyrrolidin-1-yl)ethanamine [00285] To a solution of 2-(2, 2-dimethylpyrrolidin-1-yl)acetonitrile (22 g, 159.18 mmol) in THF (400 mL) was added lithium aluminium hydride (7.25 g, 191.01 mmol) by portions at 0 °C (ice/water).The resultant mixture was stirred at 20 °C for 4 hours before quenched with water (7.25 g) at 0 °C. The reaction mixture was filtered. And the filtration was concentrated to dryness under reduced pressure to afford the crude product 2-(2, 2- dimethylpyrrolidin-1-yl)ethanamine a yellow oil. 1 H NMR (400 MHz, DMSO-d6) δ 2.71 - 2.81 (m, 2H), 2.63 - 2.71 (m, 2H), 2.44 - 2.50 (m, 2H), 1.71 - 1.82 (m, 2H), 1.59 - 1.69 (m, 2H), 0.96 - 1.02 (m, 6H). Step c: 2-Bromopyrazolo[5,1-b]thiazole-7-carboxylic acid [00286] To a solution of ethyl 2-bromopyrazolo[5,1-b]thiazole-7-carboxylate (3.1 g, 11.3 mmol) in ethanol (6 mL) was added sodium hydroxide (11.3 ml, 2M in water, 22.6 mmol) at room temperature. The reaction mixture was stirred at 40 °C for 16 h before cooling to room-temperature. The mixture was adjusted to pH=3~4 with HCl (aq, 2 M). The mixture was filtered and washed with water (10 mL x 3). The solid was evaporated under vacuum to give the desired product 2- bromopyrazolo[5,1-b]thiazole-7-carboxylic acid (2.8 g, 97%) as white solid. LCMS (ESI): mass calcd. for C 6 H 3 BrN 2 O 2 S, 245.9; m/z found, 247 [M+H] + . Step d: Ethyl 5-(2-bromopyrazolo[5,1-b]thiazole-7-carboxamido)-6-methylnic otinate [00287] A mixture of 2-bromopyrazolo[5,1-b]thiazole-7-carboxylic acid (1g, 4.04mmol) in thionyl chloride (28ml, 393mmol) was stirred at 70 °C. After stirred for 1 h at 70 °C, the reaction mixture was concentrated under vacuum to give the crude product 2- bromopyrazolo[5,1-b]thiazole-7-carbonyl chloride as white solid. To a solution of ethyl 5- amino-6-methylnicotinate (680 mg, 3.8 mmol), TEA (2.1 ml, 15.0 mmol) in THF (10 mL) was added 2-bromopyrazolo[5,1-b]thiazole-7-carbonyl chloride (1 g, 3.8 mmol) at room- temperature. The resulting mixture was stirred at room-temperature for 1 h before quenched with cooled H 2 O. The mixture was extracted with ethyl acetate (30ml*3). The organic extracts were dried over anhydrous Na 2 SO 4 and filtered. The filtration was concentrated under reduced pressure to give crude product, which was purified by column chromatography over silica gel (eluent: petroleum ether: ethyl acetate = 3:1) to give the title compound ethyl 5-(2-bromopyrazolo[5,1-b]thiazole-7-carboxamido)-6-methylnic otinate (800 mg, 52%) as a yellow solid. LCMS (ESI): mass calcd. for C 15 H 13 BrN 4 O 3 S, 409; m/z found, 411 [M+H] + . 1 H NMR (400 MHz, CDCl 3 ) δ 8.93 (d, J=1.76 Hz, 1H), 8.79(d, J=1.76 Hz, 1H), 8.08 (s, 1H), 7.88 (s, 1H), 7.33 (s, 1H), 4.34 - 4.43 (m, 2H), 2.64 (s, 3H), 1.39 (t, J=7.17 Hz, 3H). Step e: 3-methoxy-1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan -2-yl)-1H-pyrazole [00288] To 40 mL of thread vial with magnetic stirrer was added 4-bromo-3- methoxy-1-methyl-1H-pyrazole (0.8 g, 4.19 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'- bi(1,3,2-dioxaborolane) (1.276 g, 5.03 mmol), KOAc (1.356 g, 13.82 mmol), Xphos-Pd-G4 (0.288 g, 0.335 mmol) and Dioxane (15 mL) at room temperature. The reaction mixture was purged with N2 for 2 minutes. Then the reaction mixture was stirred at 95°C overnight. The mixture was concentrated under vacuum to give the product as black solid. The black solid was purified by silica gel column chromatography (eluent: petroleum ether: ethyl acetate=100/0 to petroleum ether: ethyl acetate=40/60). The fractions were collected and the solvent was removed to give the title compound as brown solid (300 mg, 30.1 % yield). LCMS (ESI): mass calcd. for C 11 H 19 BN 2 O 3 , 238.1; m/z found, 239.2 [M+H] + . Step f: 5-(2-(3-methoxy-1-methyl-1H-pyrazol-4-yl)pyrazolo[5,1-b]thia zole-7- carboxamido)-6-methylnicotinic acid [00289] Methyl 5-(2-bromopyrazolo[5,1-b]thiazole-7-carboxamido)-6- methylnicotinate (400 mg, 1.01 mmol), 3-methoxy-1-methyl-4-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)-1H-pyrazole (289.16 mg, 1.21 mmol), cesium carbonate (989.25 mg, 3.04 mmol), Pd(dppf)Cl 2 ·CH 2 Cl 2 (247.95 mg, 0.30 mmol), dioxane (16 mL) and H 2 O (4 mL) were added to 50 mL three-necked round bottom flask equipped with magnetic stirrer under an atmosphere of nitrogen. The resulting mixture was heated at 95 °C overnight. The mixture was concentrated under vacuum to give the product as black solid. The mixture was adjusted to pH=3~4 with HCl (aq, 2 M). The mixture was filtered. The residue was washed with H 2 O (10 mL x 3). [00290] The solid was evaporated under vacuum to give the title compound as black solid. LCMS (ESI): mass calcd. for C 17 H 16 N 6 O 4 S, 412.4; m/z found, 413.1 [M+H] + . Step g: N-(5-((2-(2,2-dimethylpyrrolidin-1-yl)ethyl)carbamoyl)-2-met hylpyridin- 3-yl)-2-(3-methoxy-1-methyl-1H-pyrazol-4-yl)pyrazolo[5,1-b]t hiazole-7-carboxamide [00291] 5-(2-(3-methoxy-1-methyl-1H-pyrazol-4-yl)pyrazolo[5,1-b]thia zole-7- carboxamido)-6-methylnicotinic acid (100 mg, 0.24 mmol),2-(2,2-dimethylpyrrolidin-1- yl)ethanamine (39.28 mg, 0.28 mmol), N-ethyl-N-isopropylpropan-2-amine (94.53 mg, 0.73 mmol), N,N-dimethylformamide (4 mL) and 2-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)- 1,1,3,3-tetramethylisouronium hexafluorophosphate(V) (140.24 mg, 0.37 mmol) were added a 8 mL thread vial equipped with magnetic stirrer at r.t.. The reaction was stirred at r.t. for 0.5h. The reaction was concentrated under vacuum to give the crude product as a yellow solid. Then the yellow solid was purified by Prep-HPLC: Column:Phenomenex C1875*30 mm*3um Condition: A: water(FA) B: MeCN; at the beginning: A (95%)and B (5%),at the end: A (65%) and B (35%), Gradient Time(min) 7; 100%B Hold Time(min) 1.8; Flow Rate(ml/min) 25. The pure fractions were collected and the solvent was evaporated under vacuum, lyophilized to dryness to give the title compound (28.1 mg, 20.1% yield) as a white solid. LCMS (ESI): mass calcd. for C 26 H 32 N 8 O 3 S, 536.6; m/z found, 537.3 [M+H] + . 1 H NMR (400MHz, METHANOL-d4) δ = 8.80 (d, J=1.8 Hz, 1H), 8.41 - 8.34 (m, 2H), 8.10 (s, 1H), 7.84 (s, 1H), 4.02 (s, 3H), 3.79 (s, 3H), 3.34 - 3.32 (m, 2H), 2.63 (s, 3H), 2.18 - 2.02 (m, 4H), 1.57 - 1.19 (m, 10H). [00292] The following tabulated Examples were prepared by a similar method of Example 1 from the appropriate starting materials Example 47. N-(5-(2-(2,2-dimethylpyrrolidin-1-yl)acetamido)-2-methylpyri din-3-yl)-2- (6-fluoropyridin-3-yl)pyrazolo[5,1-b]thiazole-7-carboxamide Step a: tert-butyl (6-methyl-5-nitropyridin-3-yl)carbamate [00293] To a solution of 5-bromo-2-methyl-3-nitropyridine (22.0 g, 101 mmol), tert-butyl carbamate (14.2 g, 122 mmol) and cesium carbonate (46.2 g, 142 mmol) in dioxane (500 mL) was added dicyclohexyl (2',4',6'-triisopropyl-[1,1'-biphenyl]-2-yl)phosphine (21.7 g, 45.6 mmol) and tris(dibenzylideneacetone) dipalladium(0) (13.9 g, 15.2 mmol) under nitrogen at room-temperature. The resulting mixture was stirred at 100 °C for 16 h. the mixture was cooled to room temperature and evaporated in vacuum to afford crude product as black solid. The residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate=100:0 to 70:30). The desired fractions were collected, and the solvent was concentrated to dryness under vacuum to afford the crude product. Petroleum ether (500 mL) was added to the crude product. The mixture was stirred at room temperature for 30 min. The mixture was filtered, the filtered cake was washed with petroleum ether (200 mL*2). The filter cake was dried in vacuum to afford desired product tert-butyl (6-methyl-5- nitropyridin-3-yl)carbamate (19.9 g, 100%)as white solid. LCMS (ESI): mass calcd. for C 11 H 15 N 3 O 4 , 253.2; m/z found, 254.0 [M+H]+. Step b: tert-butyl (5-amino-6-methylpyridin-3-yl)carbamate [00294] To a solution of tert-butyl (6-methyl-5-nitropyridin-3-yl)carbamate (5.0 g, 19.7 mmol) in methanol (50 mL) was added palladium 10% on activated carbon (1.66 g, 1.56 mmol) under nitrogen at room-temperature. The resulting mixture was hydrogenated at 25°C (atmospheric pressure) for 16 h. The reaction mixture was filtered and the filtrate was evaporated under vacuum to afford desired product tert-butyl (5-amino-6-methylpyridin-3-yl)carbamate (4.8 g, 92%) as white solid. LCMS (ESI): mass calcd. for C 11 H 17 N 3 O 2 , 223.2; m/z found, 224.1 [M+H]+. Step c: tert-butyl (5-(2-bromopyrazolo[5,1-b]thiazole-7-carboxamido)-6-methylpy ridin- 3-yl)carbamate [00295] To a solution of 2-bromopyrazolo[5,1-b]thiazole-7-carboxylic acid (4.5 g, 18.08 mmol) in thionyl chloride(40 mL), The resulting mixture was stirred at 70 °C for 1 h before cooling to room-temperature. The reaction mixture was concentrated under vacuum to give the crude product as white solid 2-bromopyrazolo[5,1-b]thiazole-7-carbonyl chloride. To a solution of tert-butyl (5-amino-6-methylpyridin-3-yl)carbamate (3.6 g, 16.1 mmol) and TEA (6.73 ml, 48.3 mmol) in THF (720 mL) was added 2-bromopyrazolo[5,1-b]thiazole-7- carbonyl chloride at room-temperature, The resulting mixture was stirred at 95 °C for 16 h before cooling to room-temperature. The resulting mixture was evaporated in vacuum to afford crude product. The residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate/methanol=100:0:0 to 0:90:10). The desired fractions were collected and the solvent was concentrated to dryness under vacuum to afford desired product tert-butyl (5-(2-bromopyrazolo[5,1-b]thiazole-7-carboxamido)-6-methylpy ridin-3- yl)carbamate (6.4 g, 85 %) as a yellow solid. LCMS (ESI): mass calcd. for C 17 H 18 BrN 5 O 3 S, 452.3; m/z found, 453. [M+H]+. Step d: tert-butyl (5-(2-(6-fluoropyridin-3-yl)pyrazolo[5,1-b]thiazole-7-carbox amido)-6- methylpyridin-3-yl)carbamate [00296] A 100 ml three-necked round bottom flask equipped with magnetic stirrer. To a solution of tert-butyl (5-(2-bromopyrazolo[5,1-b]thiazole-7-carboxamido)-6- methylpyridin-3-yl)carbamate (500 mg, 1.11 mmol), 2-fluoro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyri dine (300 mg, 1.35 mmol), Pd(dppf)Cl 2 ·CH 2 Cl 2 (275 mg, 0.34 mmol) and K 2 CO 3 (462.5 mg, 3.35 mmol) at room temperature. Then dioxane (20 mL) and H 2 O (5 mL) was added. The reaction mixture was under N2 atmosphere and stirred at 80°C for 14 hrs. The product was purified by silica gel chromatography (DCM/MeOH from 100/0 to 95/5). The product fractions were collected and the solvent was evaporated to give the desired product tert-butyl (5-(2-(6-fluoropyridin-3- yl)pyrazolo[5,1-b]thiazole-7-carboxamido)-6-methylpyridin-3- yl)carbamate as a brown solid. LCMS (ESI): mass calcd. for C 22 H 21 FN 6 O 3 S, 468.5; m/z found, 469.0 [M+H] + . Step e: N-(5-amino-2-methylpyridin-3-yl)-2-(6-fluoropyridin-3-yl)pyr azolo[5,1- b]thiazole-7-carboxamide [00297] To the mixture of tert-butyl (5-(2-(6-fluoropyridin-3-yl)pyrazolo[5,1- b]thiazole-7-carboxamido)-6-methylpyridin-3-yl)carbamate (260 mg, 0.56 mmol) in DCM (10 mL) and HCl/dioxane (1.67 mL, 6.66 mmol) was added, and the vial was sealed with a rubber septum. The resulting mixture was stirred at r.t. for 7 h. The reaction mixture was concentrated to give yellow solid. The yellow solid was used next step without purification. LCMS (ESI): mass calcd. for C 17 H 13 FN 6 OS, 368.4; m/z found, 369.1 [M+H] + . Step f: N-(5-(2-chloroacetamido)-2-methylpyridin-3-yl)-2-(6-fluoropy ridin-3- yl)pyrazolo[5,1-b]thiazole-7-carboxamide [00298] To the mixture of N-(5-amino-2-methylpyridin-3-yl)-2-(6-fluoropyridin-3- yl)pyrazolo[5,1-b]thiazole-7-carboxamide (250 mg, 0.57 mmol) in DMF (10 mL) was added NaHCO 3 (105 mg, 1.25 mmol) ,then 2-chloroacetyl chloride (74 µL, 0.94 mmol) was added at 0 °C. The mixture was warmed at 25 °C 2h. The reaction mixture was extracted with ethyl acetate (40 mL). The resulting solution was concentrated to give brown solid. LCMS (ESI): mass calcd. for C 19 H 14 ClFN 6 O 2 S, 444.9; m/z found, 445.1 [M+H] + . Step g: N-(5-(2-(2,2-dimethylpyrrolidin-1-yl)acetamido)-2-methylpyri din-3-yl)-2- (6-fluoropyridin-3-yl)pyrazolo[5,1-b]thiazole-7-carboxamide [00299] To a solution of N-(5-(2-chloroacetamido)-2-methylpyridin-3-yl)-2-(6- fluoropyridin-3-yl)pyrazolo[5,1-b]thiazole-7-carboxamide (400 mg, 0.36 mmol), 2,2- dimethylpyrrolidine (118 mg, 1.19 mmol), K 2 CO 3 (317 mg, 2.29 mmol) and NaI (73 mg, 0.487 mmol) in DMF (10 mL) was stirred at 50°C for 12 hours. The reaction mixture was concentrated to give black oil. The black oil was purified by silica gel column chromatography (eluent: DCM/MeOH from 100:0 to 95:5). The fractions were collected and the solvent was removed to give brown oil. The brown oil was purified with high-performance liquid chromatography: (Column: Xtimate C18 150*40mm*5um, Condition: water (HCl)-CAN, Begin B: 1%, End B: 28%, Gradient Time(min): 10, 100%B Hold Time(min): 2, FlowRate(ml/min): 60). The eluent was concentrated to remove organic solvents. The residual aqueous solution was lyophilized to give N-(5-(2-(2,2-dimethylpyrrolidin-1-yl)acetamido)-2-methylpyri din-3-yl)-2-(6- fluoropyridin-3-yl)pyrazolo[5,1-b]thiazole-7-carboxamide as a brown solid (25 mg, 13.4% yield). LCMS (ESI): mass calcd. for C 25 H 26 CFN 7 O 2 S, 507.6; m/z found, 508.2 [M+H] + . 1 H NMR (400 MHz, METHANOL-d4) δ ppm 9.11 (d, J=2.03 Hz, 1 H), 8.87 (d, J=2.03 Hz, 1 H), 8.68 (s, 1 H), 8.57 - 8.61 (m, 2 H), 8.31 (ddd, J=8.55, 7.30, 2.62 Hz, 1 H), 7.25 (dd, J=8.64, 2.56 Hz, 1 H), 4.44 (d, J=15.97 Hz, 1 H), 4.05 - 4.18 (m, 2 H), 3.42 (br dd, J=10.37, 5.01 Hz, 1 H), 2.80 (s, 3 H), 2.24 (br d, J=4.77 Hz, 1 H), 2.10 - 2.20 (m, 3 H), 1.59 (s, 3 H), 1.42 (s, 3 H) [00300] The following tabulated Examples were prepared by a similar method of Example 47 from the appropriate starting materials Example 53, (S)-(1-methylpyrrolidin-2-yl)methyl (5-(2-(1-(2-methoxyethyl)-1H-pyrazol- 4-yl)pyrazolo[5,1-b]thiazole-7-carboxamido)-6-methylpyridin- 3-yl)carbamate Step a: tert-butyl (5-(2-(1-(2-methoxyethyl)-1H-pyrazol-4-yl)pyrazolo[5,1- b]thiazole-7-carboxamido)-6-methylpyridin-3-yl)carbamate [00301] To a mixture of tert-butyl (5-(2-bromopyrazolo[5,1-b]thiazole-7- carboxamido)-6-methylpyridin-3-yl)carbamate(250 mg, 0.55 mmol), 1-(2-methoxyethyl)-4- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (209 mg, 0.83 mmol), and K 2 CO 3 (229.16 mg, 1.66 mmol) in 1,4-dioxane (2.4 mL) and water (0.6 mL) was added 1,1'- bis(diphenylphosphino)ferrocene-palladium(II)dichloride dichloromethane complex (90.27 mg, 0.11 mmol). The reaction was thoroughly flushed with argon before being capped and heated at 100 °C for 25 h. The reaction was diluted with MeOH (25 mL) and silica gel (3 g) was added. All solvents were removed in vacuo. The silica gel mesh was loaded on a Redi Sep Rf silica gel cartridge (40 g) eluting with EtOAc over 10 min then MeOH/EtOAc (0 - 30%) over 15 min to afford the product, tert-butyl (5-(2-(1-(2-methoxyethyl)-1H-pyrazol-4- yl)pyrazolo[5,1-b]thiazole-7-carboxamido)-6-methylpyridin-3- yl)carbamate, as a tan solid (170 mg). LCMS (ESI): mass calcd. for C 23 H 27 N 7 O 4 S, 497.2; m/z found, 498.2 [M+H] + . Step b: N-(5-amino-2-methylpyridin-3-yl)-2-(1-(2-methoxyethyl)-1H-py razol-4- yl)pyrazolo[5,1-b]thiazole-7-carboxamide [00302] To a solution of tert-butyl (5-(2-(1-(2-methoxyethyl)-1H-pyrazol-4- yl)pyrazolo[5,1-b]thiazole-7-carboxamido)-6-methylpyridin-3- yl)carbamate (170 mg, 0.34 mmol) in DCM (2 mL) was added HCl (4M in dioxane) (0.43 mL, 4 M, 1.71 mmol). The reaction mixture was stirred at 25 °C for 96 h. All solvents were removed in vacuo to afford the HCl salt of the product, N-(5-amino-2-methylpyridin-3-yl)-2-(1-(2-methoxyethyl)-1H- pyrazol-4-yl)pyrazolo[5,1-b]thiazole-7-carboxamide, as a tan solid (quantitative yield). The product was used without any further purification. LCMS (ESI): mass calcd. for C 18 H 19 N 7 O 2 S, 397.1; m/z found, 398.2 [M+H] + ; 1 H NMR (400 MHz, DMSO-d6) δ ppm 2.52 (s, 3 H), 3.26 (s, 3 H), 3.73 (t, J=1.00 Hz, 2 H), 4.30 (t, J=1.00 Hz, 2 H), 6.47 (br s, 3 H), 7.77 (d, J=1.00 Hz, 1 H), 7.84 (d, J=1.00 Hz, 1 H), 7.91 (s, 1 H), 8.23 (s, 1 H), 8.62 (s, 1 H), 8.65 (s, 1 H), 10.32 (s, 1 H). Step c. (S)-(1-methylpyrrolidin-2-yl)methyl (5-(2-(1-(2-methoxyethyl)-1H- pyrazol-4-yl)pyrazolo[5,1-b]thiazole-7-carboxamido)-6-methyl pyridin-3-yl)carbamate [00303] A 2-5 mL microwave vial, equipped with a stir bar, was charged with N-(5- amino-2-methylpyridin-3-yl)-2-(1-(2-methoxyethyl)-1H-pyrazol -4-yl)pyrazolo[5,1- b]thiazole-7-carboxamide (77.6 mg, 0.18 mmol), THF (3 mL, 0.886 g/mL, 36.862 mmol), N,N-diisopropylethylamine (0.2 mL, 0.742 g/mL, 1.137 mmol), and 1,1'-carbonyldiimidazole (59.1 mg, 0.364 mmol). A nitrogen atmosphere was established [N 2 gas blown over the headspace]. The vial was sealed with a cap. An argon inlet was used, and the reaction proceeded at 21 °C. After 23 h, Solvent was removed in vacuo. The residue was dissolved in DMF (3 mL), and the mixture was transferred to a 2-5 mL microwave vial, equipped with a stir bar. A nitrogen atmosphere was established [N 2 gas blown over the headspace]. The vial was sealed with a cap. N-Methyl-l-prolinol (0.2 mL, 1.03 g/mL, 1.753 mmol) was added. The mixture was irradiated in a Biotage Initiator+ microwave reactor at 70 °C for 1 h and allowed to cool to room temperature. Solvent was removed under reduced pressure. The crude was purified by HPLC to give the title compound (40.7 mg, 41.7% yield) as white solid. LCMS (ESI): mass calcd. for C 25 H 30 N 8 O 4 S, 538.6; m/z found, 539.2 [M+H] + ; 1 H NMR (DMSO-d6, 400 MHz) δ (ppm) 9.8-9.9 (m, 2H), 8.59 (s, 1H), 8.50 (s, 1H), 8.42 (d, 1H, J=2.4 Hz), 8.22 (d, 1H, J=0.7 Hz), 7.96 (d, 1H, J=1.7 Hz), 7.90 (d, 1H, J=0.7 Hz), 4.30 (t, 2H, J=5.3 Hz), 4.0- 4.1 (m, 2H), 3.72 (t, 2H, J=5.3 Hz), 3.25 (s, 3H), 2.95 (br s, 1H), 2.4-2.5 (m, 1H), 2.38 (s, 3H), 2.33 (s, 3H), 2.1-2.2 (m, 1H), 1.8-2.0 (m, 1H), 1.5-1.7 (m, 3H) Example 54. N-(5-(2-(3,3-dimethylazetidin-1-yl)acetamido)-2-methylpyridi n-3-yl)-2-(5- (hydroxymethyl)-3-methoxy-1-methyl-1H-pyrazol-4-yl)pyrazolo[ 5,1-b]thiazole-7- carboxamide Step a: Methyl 3-methoxy-1-methyl-1H-pyrazole-5-carboxylate [00304] To a mixture of methyl 5-hydroxy-1H-pyrazole-3-carboxylate (1.01 g, 7.11 mmol) and cesium carbonate (5.0 g, 15.41 mmol) under nitrogen was added DMF (10 mL) and the yellow-orange mixture was stirred at rt for 10 min, after which methyl iodide (1 mL, 16.06 mmol) was added rapidly dropwise to yield a milky mixture. After 50 min the reaction was poured into 10% LiCl and extracted 3x EtOAc. The combined organics were washed 3x brine, filtered through cotton, and concentrated to a yellow solid which was purified by flash column (0 - 100% EtOAc/heptane) to yield methyl 3-methoxy-1-methyl-1H-pyrazole-5- carboxylate as a white solid (0.92 g, 76%). LCMS (ESI): mass calcd. for C 7 H 10 N 2 O 3 , 170.2; m/z found, 171.1 [M+H] + . Step b: (3-Methoxy-1-methyl-1H-pyrazol-5-yl)methanol [00305] A clear yellow solution of methyl 3-methoxy-1-methyl-1H-pyrazole-5- carboxylate (0.92 g, 5.41 mmol) in THF (20 mL) under nitrogen was chilled on an ice bath and LAH (1 M/THF, 8 mL, 8 mmol) was added dropwise. The ice bath was removed, and the reaction was stirred at rt overnight. The reaction was chilled in an ice bath, diluted with an equal volume of ether, and cold water (0.49 mL) was added carefully.15% NaOH (0.49 ml) was added followed by cold water (1.5 mL) with vigorous stirring. The ice bath was removed, and the stirred mixture was allowed to warm to rt. MgSO 4 was added and stirring continued for 30 min. The mixture was filtered, the salts were washed 2x Et2O, and the clear colorless filtrate was concentrated to a crude oil which was purified by flash column (0 - 5% MeOH/DCM) to yield (3-methoxy-1-methyl-1H-pyrazol-5-yl)methanol (215 mg, 28%). LCMS (ESI): mass calcd. for C 6 H 10 N 2 O 2 , 142.2; m/z found, 171.1 [M+H] + . Step c: 5-(((tert-Butyldimethylsilyl)oxy)methyl)-3-methoxy-1-methyl- 1H- pyrazole [00306] To a clear colorless solution of (3-methoxy-1-methyl-1H-pyrazol-5- yl)methanol (200 mg, 1.41 mmol) in DCM (50 mL) was added imidazole (287 mg, 4.22 mmol), tert-butyldimethylsilyl chloride (656 mg, 4.22 mmol), and DMAP (10 mg, 0.082 mmol) to yield a thick suspension which was stirred at rt for 1.25 h. The reaction was poured into water. The layers were separated, and organics washed once with water, once with brine, filtered through cotton, concentrated, and purified by flash column (0 - 50% EtOAc/heptane) to yield 5-(((tert-butyldimethylsilyl)oxy)methyl)-3-methoxy-1-methyl- 1H- pyrazole (257 mg, 71%) as a clear colorless oil. LCMS (ESI): mass calcd. for C 12 H 24 N 2 O 2 Si, 256.4; m/z found, 257.2 [M+H] + . Step d: 5-(((tert-Butyldimethylsilyl)oxy)methyl)-3-methoxy-1-methyl- 4-(4,4,5,5- tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole [00307] A mixture of bis(pinacolato)diboron (87 mg, 0.34 mmol), bis(1,5- cyclooctadiene)dimethoxydiiridium (9 mg, 0.014 mmol), and 3,4,7,8-tetramethyl-1,10- phenanthroline (8 mg, 0.034 mmol) in heptane (1.3 mL) under nitrogen in a capped 5 mL microwave vial was sparged with nitrogen for 10 min, then heated at 80°C for 1 h. A nitrogen-sparged solution of 5-(((tert-butyldimethylsilyl)oxy)methyl)-3-methoxy-1-methyl- 1H-pyrazole (57 mg, 0.22 mmol) in heptane (0.7 mL) was added and the reaction was heated for 20 h. The reaction was blown down under nitrogen and the crude 5-(((tert- butyldimethylsilyl)oxy)methyl)-3-methoxy-1-methyl-4-(4,4,5,5 -tetramethyl-1,3,2- dioxaborolan-2-yl)-1H-pyrazole was used without purification in the next reaction. LCMS (ESI): mass calcd. for C 18 H 35 BN 2 O 4 Si, 382.4; m/z found, 383.3 [M+H] + . Step e: N-(5-amino-2-methylpyridin-3-yl)-2-bromopyrazolo[5,1-b]thiaz ole-7- carboxamide [00308] A solution of tert-butyl (5-(2-bromopyrazolo[5,1-b]thiazole-7- carboxamido)-6-methylpyridin-3-yl)carbamate (777 mg, 1.72 mmol) in 25% TFA/CH 2 Cl 2 (25 mL) was stirred at 25 °C for 3 h. All solvents were removed in vacuo. The residue was taken up in toluene (25 mL) and all solvents were removed in vacuo (2x). The residue was dried under high vacuum to afford the TFA salt of the product, N-(5-amino-2-methylpyridin-3-yl)- 2-bromopyrazolo[5,1-b]thiazole-7-carboxamide, as a tan solid (quantitative yield). The product was used without further purification. LCMS (ESI): mass calcd. for C 12 H 10 BrN 5 OS, 351.0/353.0; m/z found, 352.0/354.0 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ ppm 2.45 (s, 3 H), 6.26 (br s, 2 H), 7.80 (d, J=1.00 Hz, 1 H), 7.83 (d, J=1.00 Hz, 1 H), 8.64 (s, 1 H), 8.82 (s, 1 H), 10.18 - 10.33 (m, 1 H). Step f: 2-bromo-N-(5-(2-chloroacetamido)-2-methylpyridin-3-yl)pyrazo lo[5,1- b]thiazole-7-carboxamide [00309] To a suspension of N-(5-amino-2-methylpyridin-3-yl)-2- bromopyrazolo[5,1-b]thiazole-7-carboxamide (605.10 mg, 1.72 mmol) in DCM (10 mL) was added Et 3 N (0.72 mL, 0.73 g/mL, 5.15 mmol) followed by the addition of chloroacetyl chloride (0.15 mL, 1.42 g/mL, 1.89 mmol). The reaction was stirred at 25 °C under argon for 17 h. Another 0.5 equivalent of chloroacetyl chloride (0.076 mL, 1.42 g/mL, 0.95 mmol) was added. The reaction was maintained stirring at 25 °C under argon for 2 h. All solvents were removed in vacuo. The residue was taken up in toluene (25 mL) and all solvents were removed in vacuo. The residue was taken up in 20% MeOH/CH 2 Cl 2 (25 mL) and silica gel (6 g). All solvents were removed in vacuo. The silica gel mesh was loaded on a SiliCycle silica gel cartridge (40 g) eluting with MeOH/CH 2 Cl 2 (0 - 30%) over 30 min to afford a mixture of products. The mixture was taken up in 20% MeOH/CH 2 Cl 2 (25 mL) and silica gel (6 g) and concentrated in vacuo. The silica gel mesh was loaded on a SiliCycle silica gel cartridge (40 g) eluting with 100% EtOAc over 30 min to afford the product, 2-bromo-N-(5- (2-chloroacetamido)-2-methylpyridin-3-yl)pyrazolo[5,1-b]thia zole-7-carboxamide, as a tan solid (402.9 mg). LCMS (ESI): mass calcd. for C 14 H 11 BrClN 5 O 2 S, 427.0/429.0; m/z found, 428.0/429.9 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ ppm 2.40 (s, 3 H), 4.29 (s, 2 H), 8.14 (d, J=1.00 Hz, 1 H), 8.52 (d, J=1.00 Hz, 1 H), 8.62 (s, 1 H), 8.79 (s, 1 H), 9.93 (s, 1 H), 10.53 (s, 1 H). Step g: 2-bromo-N-(5-(2-(3,3-dimethylazetidin-1-yl)acetamido)-2-meth ylpyridin- 3-yl)pyrazolo[5,1-b]thiazole-7-carboxamide [00310] A mixture of 2-bromo-N-(5-(2-chloroacetamido)-2-methylpyridin-3- yl)pyrazolo[5,1-b]thiazole-7-carboxamide (2 g, 4.67 mmol), 3,3-dimethylazetidine hydrochloride (0.68 g, 5.60 mmol), K 2 CO 3 (2.58 g, 18.66 mmol), and NaI (250 mg) in DMF (25 mL) was heated at 50 °C for 23 h. The reaction was poured into water (450 mL) with stirring. The reaction was filtered and the collected precipitate was air dried then dried under high vacuum to afford the product, 2-bromo-N-(5-(2-(3,3-dimethylazetidin-1-yl)acetamido)- 2-methylpyridin-3-yl)pyrazolo[5,1-b]thiazole-7-carboxamide, as a tan solid (2.181 g). The product was used without further purification. LCMS (ESI): mass calcd. for C 19 H 21 BrN 6 O 2 S, 476.1/478.1; m/z found, 477.1/479.1 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.20 (s, 6 H), 2.38 (s, 3 H), 3.04 (s, 4 H), 3.20 (s, 2 H), 8.13 (d, J=1.00 Hz, 1 H), 8.55 (d, J=1.00 Hz, 1 H), 8.60 (s, 1 H), 8.78 (s, 1 H), 9.81 (s, 1 H), 9.95 (s, 1 H). Step h: N-(5-(2-(3,3-dimethylazetidin-1-yl)acetamido)-2-methylpyridi n-3-yl)-2- (5-(hydroxymethyl)-3-methoxy-1-methyl-1H-pyrazol-4-yl)pyrazo lo[5,1-b]thiazole-7- carboxamide [00311] To crude 5-(((tert-butyldimethylsilyl)oxy)methyl)-3-methoxy-1-methyl- 4- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (85 mg, 0.22 mmol) in a 5 mL microwave vial was added 2-bromo-N-(5-(2-(3,3-dimethylazetidin-1-yl)acetamido)-2- methylpyridin-3-yl)pyrazolo[5,1-b]thiazole-7-carboxamide (54 mg, 0.11 mmol), cesium carbonate (111 mg, 0.34 mmol), 1,1'-bis(diphenylphosphino)ferrocene- palladium(II)dichloride dichloromethane complex (17.5 mg, 0.021 mmol), 1,4-dioxane (1.4 mL), DMF (0.5 mL), and water (0.4 mL). The mixture was placed under vacuum, the vial was back-filled with nitrogen, and the mixture was sparged with nitrogen for 10 min then heated at 130°C in the microwave for 1 h. Tetrabutylammonium fluoride (0.57 mL, 1 M, 0.57 mmol) was added and the reaction was heated at 60°C for 90 min then cooled to rt, stirred with Si-trisamine for 20 min, and filtered. The crude product was purified by prep-HPLC (5% - 25% MeCN/water/0.1% TFA) followed by subsequent purification by prep-HPLC (15% - 40% MeCN/10 mM NH 4 OH) to yield N-(5-(2-(3,3-dimethylazetidin-1-yl)acetamido)- 2-methylpyridin-3-yl)-2-(5-(hydroxymethyl)-3-methoxy-1-methy l-1H-pyrazol-4- yl)pyrazolo[5,1-b]thiazole-7-carboxamide (1.6 mg, 2.6%). LCMS (ESI): mass calcd. for C 25 H 30 N 8 O 4 S, 538.6; m/z found, 539.2 [M+H] + . 1 H NMR (METHANOL-d4) δ: 8.57 (d, J=2.4 Hz, 1H), 8.38 (s, 1H), 8.20 (d, J=2.4 Hz, 1H), 8.12 (s, 1H), 4.71 (s, 2H), 3.98 (s, 3H), 3.83 (s, 3H), 3.35 (s, 2H), 3.19 (s, 4H), 2.49 (s, 3H), 1.26 (s, 6H). Example 55. N-(5-(2-(3,3-dimethylazetidin-1-yl)acetamido)-2-methylpyridi n-3-yl)-2-(2- (hydroxymethyl)-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazin-3- yl)pyrazolo[5,1- b]thiazole-7-carboxamide Step a: Methyl 6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-2-carboxylate [00312] To a mixture of methyl 5-hydroxy-1H-pyrazole-3-carboxylate (520 mg, 3.66 mmol) and cesium carbonate (4.77 g, 14.64 mmol) in MeCN (25 mL) under nitrogen was added 1,3-dibromopropane (0.41 mL, 4.02 mmol) and the reaction was heated to 80°C overnight. The reaction was cooled to rt, filtered, and concentrated to an orange oil. The crude product was partitioned between EtOAc/water and the layers were separated. The organics were washed once with water, once with brine, filtered through cotton, and concentrated to methyl 6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-2-carboxylate (421 mg, 63%) as a pale yellow solid which was used without purification in the next step. LCMS (ESI): mass calcd. for C 8 H 10 N 2 O 3 , 182.2; m/z found, 183.1 [M+H] + . Step b: N-(5-(2-(3,3-dimethylazetidin-1-yl)acetamido)-2-methylpyridi n-3-yl)-2- (2-(hydroxymethyl)-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazin -3-yl)pyrazolo[5,1- b]thiazole-7-carboxamide [00313] From methyl 6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-2-carboxylate was prepared N-(5-(2-(3,3-dimethylazetidin-1-yl)acetamido)-2-methylpyridi n-3-yl)-2-(2- (hydroxymethyl)-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazin-3- yl)pyrazolo[5,1-b]thiazole-7- carboxamide according to Example 54, steps b – e. LCMS (ESI): mass calcd. for C 26 H 30 N 8 O 4 S, 550.6; m/z found, 551.3 [M+H] + . 1 H NMR (METHANOL-d4) δ: 8.41 (s, 2H), 8.13-8.31 (m, 1H), 4.47 (br s, 2H), 4.33 (s, 2H), 3.94-4.22 (m, 6H), 2.50-2.70 (m, 3H), 2.29 (br s, 2H), 1.27-1.56 (m, 7H). Example 56. N-(5-(2-(3,3-dimethylazetidin-1-yl)acetamido)-2-methylpyridi n-3-yl)-2-(5- methoxy-1,3-dimethyl-1H-pyrazol-4-yl)pyrazolo[5,1-b]thiazole -7-carboxamide Step a: 5-Methoxy-1,3-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxabor olan-2-yl)- 1H-pyrazole [00314] A mixture of bis(pinacolato)diboron (617 mg, 2.43 mmol), 3,4,7,8- tetramethyl-1,10-phenanthroline (47 mg, 0.2 mmol), and bis(1,5- cyclooctadiene)dimethoxydiiridium (59 mg, 0.089 mmol) under nitrogen in a capped 5 mL microwave vial was added 5 mL heptane and the mixture was sparged with nitrogen for 10 min. The reaction was heated at 80°C for one hour. A solution of 5-methoxy-1,3-dimethyl- 1H-pyrazole (200 mg, 1.59 mmol) in 2 mL heptane was sparged with nitrogen for 10 min and added to the reaction, which was heated at 80°C overnight, cooled to rt, filtered, concentrated, and purified by flash column (0 - 50% EtOAc/heptane) to yield 5-methoxy-1,3- dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H- pyrazole (368 mg, 92%) as a yellow viscous oil. LCMS (ESI): mass calcd. for C 12 H 21 BN 2 O 3 , 252.1; m/z found, 253.1 [M+H] + . Step b: N-(5-(2-(3,3-dimethylazetidin-1-yl)acetamido)-2-methylpyridi n-3-yl)-2- (5-methoxy-1,3-dimethyl-1H-pyrazol-4-yl)pyrazolo[5,1-b]thiaz ole-7-carboxamide [00315] A mixture of 2-bromo-N-(5-(2-(3,3-dimethylazetidin-1-yl)acetamido)-2- methylpyridin-3-yl)pyrazolo[5,1-b]thiazole-7-carboxamide (52 mg, 0.11 mmol), 5-methoxy- 1,3-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) -1H-pyrazole (55 mg, 0.22 mmol), cesium carbonate (117 mg, 0.36 mmol), and 1,1'-bis(diphenylphosphino)ferrocene- palladium(II)dichloride dichloromethane complex (19 mg, 0.023 mmol) in 1,4-dioxane (2 mL) and water (0.5 mL) under nitrogen in a capped 5 mL microwave vial was sparged with nitrogen for 10 min then heated at 130°C in the microwave for 1 h. An additional charge of 5-methoxy-1,3-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxabor olan-2-yl)-1H-pyrazole and 1,1'-bis(diphenylphosphino)ferrocene-palladium(II)dichloride dichloromethane complex was added, the reaction vial was evacuated, back-filled with nitrogen, and returned to microwave at 130°C for 1 h. The reaction was stirred with Si-trisamine for 25 min, then purified by prep- HPLC (5% - 25% MeCN/water/0.1% TFA). The product fractions were concentrated, dissolved in DMSO, basified with drops 10% NH 4 OH and purified by prep-HPLC (20% - 40% MeCN/10 mM NH 4 OH) to yield N-(5-(2-(3,3-dimethylazetidin-1-yl)acetamido)-2- methylpyridin-3-yl)-2-(5-methoxy-1,3-dimethyl-1H-pyrazol-4-y l)pyrazolo[5,1-b]thiazole-7- carboxamide (9.5 mg, 17%). LCMS (ESI): mass calcd. for C 25 H 30 N 8 O 3 S, 522.6; m/z found, 523.2 [M+H] + . 1 H NMR (METHANOL-d4) δ: 8.57 (d, J=1.5 Hz, 1H), 8.41 (s, 1H), 8.21 (d, J=2.0 Hz, 1H), 8.09 (s, 1H), 3.96 (s, 3H), 3.67 (s, 3H), 3.35 (s, 2H), 3.20 (s, 4H), 2.49 (s, 3H), 2.29 (s, 3H), 1.26 (s, 6H). [00316] The following tabulated Examples were prepared by a similar method of Example 56 from the appropriate starting materials
Example 62. N-(5-(2-(3,3-dimethylazetidin-1-yl)acetamido)-2-methylpyridi n-3-yl)-2-(6- hydroxy-2-methyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazin-3 -yl)pyrazolo[5,1- b]thiazole-7-carboxamide Step a: 2-((1,3-Dibromopropan-2-yl)oxy)tetrahydro-2H-pyran [00317] To a mixture of 1,3-dibromo-2-propanol (1 g, 4.59 mmol) and pyridinium p-toluenesulfonate (116.5 mg, 0.46 mmol) in THF (10 mL) under nitrogen in a 20 mL vial was added 3,4-dihydro-2H-pyran (0.78 mL, 9.18 mmol) and the reaction was stirred at rt overnight. The reaction was diluted with 50 mL EtOAc, washed 3x water then 1x brine, filtered through cotton, and concentrated to a clear colorless oil. The crude oil was purified by flash column (0 - 10% EtOAc/heptane) to yield 2-((1,3-dibromopropan-2- yl)oxy)tetrahydro-2H-pyran (1.23 g, 88%) as a clear colorless oil. 1 H NMR (CHLOROFORM-d) δ: 4.79 (dd, J=4.2, 3.2 Hz, 1H), 3.99-4.06 (m, 1H), 3.90-3.98 (m, 1H), 3.62-3.72 (m, 2H), 3.51-3.60 (m, 3H), 1.71-1.91 (m, 2H), 1.55-1.70 (m, 3H), 1.50-1.54 (m, 1H). Step b: 2-Methyl-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,7-dihydro-5H- pyrazolo[5,1-b][1,3]oxazine [00318] A mixture of 3-methyl-1h-pyrazol-5-ol (275 mg, 2.8 mmol), 2-((1,3- dibromopropan-2-yl)oxy)tetrahydro-2H-pyran (1 g, 3.31 mmol), and K 2 CO 3 (1.33 g, 9.65 mmol) in (15 mL) under nitrogen in a 50 mL rbf was heated at 100° for 15 h. The reaction was cooled to rt, poured into 10% LiCl, and extracted 4x EtOAc. The combined organics were washed 3x 10% LiCl, 3x brine, filtered through cotton, and concentrated to an orange oil which was purified by flash column (0 - 100% EtOAc/heptane) to yield 2-methyl-6- ((tetrahydro-2H-pyran-2-yl)oxy)-6,7-dihydro-5H-pyrazolo[5,1- b][1,3]oxazine as a clear oil (163 mg, 24%) which crystallizes overnight. 1 H NMR (CHLOROFORM-d) δ: 5.30-5.33 (m, 1H), 4.79-4.89 (m, 1H), 4.09-4.35 (m, 5H), 3.76-3.93 (m, 1H), 3.51-3.60 (m, 1H), 2.19 (s, 3H), 1.68-1.89 (m, 2H), 1.47-1.67 (m, 6H). Step c: 2-Methyl-6-((tetrahydro-2H-pyran-2-yl)oxy)-3-(4,4,5,5-tetram ethyl-1,3,2- dioxaborolan-2-yl)-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazin e [00319] A mixture of bis(pinacolato)diboron (263 mg, 1.03 mmol), bis(1,5- cyclooctadiene)dimethoxydiiridium (26.5 mg, 0.04 mmol), and 3,4,7,8-tetramethyl-1,10- phenanthroline (18.5 mg, 0.078 mmol) in Me-THF (3 mL) under nitrogen in a capped 5 mL microwave vial was sparged with nitrogen for 10 min and heated for 1 h at 80°C in a heating block. A nitrogen-sparged solution of 2-methyl-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,7- dihydro-5H-pyrazolo[5,1-b][1,3]oxazine (163 mg, 0.68 mmol) in 2 mL Me-THF was added and heating was continued overnight. The reaction was cooled to rt, concentrated, and purified by flash column, (0 - 100% EtOAc/heptane) to yield 2-methyl-6-((tetrahydro-2H- pyran-2-yl)oxy)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2- yl)-6,7-dihydro-5H- pyrazolo[5,1-b][1,3]oxazine (228 mg, 92%) as an amber gum. LCMS (ESI): mass calcd. for C 18 H 29 BN 2 O 5, 364.3; m/z found, 365.2 [M+H]+. Step d: N-(5-(2-(3,3-dimethylazetidin-1-yl)acetamido)-2-methylpyridi n-3-yl)-2- (6-hydroxy-2-methyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazi n-3-yl)pyrazolo[5,1- b]thiazole-7-carboxamide [00320] A mixture of 2-bromo-N-(5-(2-(3,3-dimethylazetidin-1-yl)acetamido)-2- methylpyridin-3-yl)pyrazolo[5,1-b]thiazole-7-carboxamide (97.21 mg, 0.27 mmol), 2- methyl-6-((tetrahydro-2H-pyran-2-yl)oxy)-3-(4,4,5,5-tetramet hyl-1,3,2-dioxaborolan-2-yl)- 6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine (97.21 mg, 0.27 mmol), cesium carbonate (169 mg, 0.52 mmol), and 1,1'-bis(diphenylphosphino)ferrocenedichloro palladium(II) (25.2 mg, 0.034 mmol) in 1,4-dioxane (1.6 mL) and water (0.5 mL) under nitrogen in a capped 5 mL microwave vial was sparged with nitrogen for 10 min then heated at 130°C for 1.25 h. An additional portion of 1,1'-bis(diphenylphosphino)ferrocenedichloro palladium(II) was added, the reaction was placed under vacuum, back-filled with nitrogen, and heated at 130°C in the microwave for 1 h. The reaction was stirred with Si-trisamine over the weekend, filtered, and purified by prep-HPLC, (5% - 30% MeCN/water/0.1% TFA). The product was dissolved in DMSO, basified with 10% NH 4 OH, and purified by prep-HPLC (12% - 37% MeCN/10mM NH 4 OH) to yield N-(5-(2-(3,3-dimethylazetidin-1-yl)acetamido)-2-methylpyridi n-3-yl)-2-(6- hydroxy-2-methyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazin-3 -yl)pyrazolo[5,1-b]thiazole- 7-carboxamide (7 mg, 10%) as an off-white solid. LCMS (ESI): mass calcd. for C 26 H 30 N 8 O 4 S, 550.6; m/z found, 551.2 [M+H]+. 1 H NMR (METHANOL-d4) δ: 8.58 (br s, 1H), 8.37 (s, 1H), 8.20 (s, 1H), 8.01 (s, 1H), 4.34-4.50 (m, 3H), 4.27 (dd, J=12.7, 2.9 Hz, 1H), 4.04 (br d, J=12.2 Hz, 1H), 3.36 (br s, 2H), 3.20 (br s, 4H), 2.49 (s, 3H), 2.38 (s, 3H), 1.26 (s, 6H). [00321] Examples 63 was prepared by a similar method of Example 62 from the appropriate starting material Example 64. N-(5-(2-(3,3-Dimethylazetidin-1-yl)acetamido)-2-methylpyridi n-3-yl)-2- (4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-3-yl)pyrazolo[5, 1-b]thiazole-7- carboxamide Step a: 4,5,6,7-Tetrahydropyrazolo[1,5-a]pyrimidine [00322] A solution of 1H-pyrazol-5-amine (611 mg, 7.35 mmol), 1,3- dibromopropane (0.75 mL, 7.35 mmol) and TEA (3.06 mL, 22.06 mmol) in 1,4-dioxane (12 mL) under nitrogen was heated at 100°C for 5 h. The reaction was filtered of a gummy precipitate, washing with dioxane, and concentrated to an oil which crystallizes to an off- white solid. The crude product was purified by flash column (0 - 5% MeOH/DCM) to yield 4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine (373 mg, 41%) as a white solid. 1 H NMR (CHLOROFORM-d) δ: 7.25 (d, J=2.0 Hz, 1H), 5.34 (d, J=2.0 Hz, 1H), 4.13 (t, J=6.4 Hz, 2H), 3.98 (br s, 1H), 3.29-3.34 (m, 2H), 2.15 (quin, J=5.9 Hz, 2H). Step b: tert-Butyl 6,7-dihydropyrazolo[1,5-a]pyrimidine-4(5H)-carboxylate [00323] To a solution of 4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine (373 mg, 3.03 mmol) in THF (5 mL) under nitrogen was added NaH (60% dispersion in mineral oil, 181.7 mg, 4.54 mmol) and the mixture was stirred at rt for 1 h. BOC-anhydride (0.99 g, 4.54 mmol) was added and the reaction was stirred at rt overnight. The reaction was poured into water and extracted 3x EtOAc. The combined organics were washed with brine, filtered through cotton and concentrated to yield a yellow oil which was purified by flash column (0 - 100% EtOAc/heptane) to yield tert-butyl 6,7-dihydropyrazolo[1,5-a]pyrimidine-4(5H)- carboxylate (162 mg, 24 %) as a colorless oil that crystalizes to an off-white solid. 1 H NMR (CHLOROFORM-d) δ: 7.36 (d, J=2.0 Hz, 1H), 6.01-6.50 (m, 1H), 4.18 (t, J=6.1 Hz, 2H), 3.75-3.88 (m, 2H), 2.10-2.23 (m, 2H), 1.56 (s, 9H). Step c: (4-(tert-Butoxycarbonyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]py rimidin-3- yl)boronic acid [00324] A mixture of bis(pinacolato)diboron (286 mg, 1.13 mmol), bis(1,5- cyclooctadiene)dimethoxydiiridium (26 mg, 0.039 mmol), and 3,4,7,8-tetramethyl-1,10- phenanthroline (22 mg, 0.093 mmol) in 2-MeTHF (3.5 mL) under nitrogen in a capped 5 mL nitrogen vial was sparged with nitrogen for 10 min and heated at 80°C for 1 h in a heating block. A nitrogen-sparged solution of tert-butyl 6,7-dihydropyrazolo[1,5-a]pyrimidine- 4(5H)-carboxylate (162 mg, 0.73 mmol) in 1.5 mL 2-MeTHF was added and the reaction was heated overnight. The reaction was cooled to rt, concentrated, taken up in DMSO, and stirred with Si-trisamine for 1 h, filtered, and purified by prep-HPLC (5% - 25% MeCN/10 mM NH4OH) to yield (4-(tert-butoxycarbonyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]py rimidin-3- yl)boronic acid (33 mg, 17%) as a white solid. LCMS (ESI): mass calcd. for C 11 H 18 BN 3 O 4 , 276.1; m/z found, 212.1 [M-tBu]. Step d: N-(5-(2-(3,3-Dimethylazetidin-1-yl)acetamido)-2-methylpyridi n-3-yl)-2- (4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-3-yl)pyrazolo[5, 1-b]thiazole-7- carboxamide [00325] A mixture of 2-bromo-N-(5-(2-(3,3-dimethylazetidin-1-yl)acetamido)-2- methylpyridin-3-yl)pyrazolo[5,1-b]thiazole-7-carboxamide (35 mg, 0.073 mmol), (4-(tert- butoxycarbonyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-3 -yl)boronic acid (32 mg, 0.12 mmol), cesium carbonate (72 mg, 0.22 mmol), and 1,1'- bis(diphenylphosphino)ferrocenedichloro palladium(II) (13 mg, 0.018 mmol) in 1,4-dioxane (1.6 mL) and water (0.5 mL) under nitrogen in a capped 5 mL microwave vial was sparged with nitrogen for 10 min and heated at 130°C in the microwave for 1 h. The reaction was stirred with Si-trisamine for 40 min, filtered, and purified by prep-HPLC (10% - 30% MeCN/water/0.1% TFA) to yield a pale yellow solid which was purified by prep-HPLC (5% - 25% MeCN/water/0.1% TFA) to yield N-(5-(2-(3,3-dimethylazetidin-1-yl)acetamido)-2- methylpyridin-3-yl)-2-(4,5,6,7-tetrahydropyrazolo[1,5-a]pyri midin-3-yl)pyrazolo[5,1- b]thiazole-7-carboxamide (8 mg, 17%) as a white solid. LCMS (ESI): mass calcd. for C 25 H 29 N 9 O 2 S, 519.6; m/z found, 520.2 [M+H]+. 1 H NMR (DMSO-d6) δ: 10.72 (s, 1H), 10.39-10.52 (m, 1H), 9.81 (s, 1H), 8.69 (s, 1H), 8.55 (d, J=2.4 Hz, 1H), 8.53 (s, 1H), 8.16 (d, J=2.0 Hz, 1H), 5.73 (s, 1H), 4.31 (br d, J=5.9 Hz, 2H), 4.00 (br t, J=6.1 Hz, 2H), 3.93 (br d, J=5.9 Hz, 4H), 3.15-3.21 (m, 2H), 2.46 (s, 3H), 1.96-2.06 (m, 2H), 1.35 (s, 3H), 1.25 (s, 3H). Example 65, N-(5-(1-(2,2-dimethylcyclopentyl)azetidine-3-carboxamido)-2- methylpyridin-3-yl)-2-(1-methyl-1H-pyrazol-4-yl)pyrazolo[5,1 -b]thiazole-7- carboxamide Step a. tert-butyl (6-methyl-5-(2-(1-methyl-1H-pyrazol-4-yl)pyrazolo[5,1- b]thiazole-7-carboxamido)pyridin-3-yl)carbamate [00326] To a mixture of 2-(1-methyl-1H-pyrazol-4-yl)pyrazolo[5,1-b]thiazole-7- carboxylic acid (430 mg, 1.73 mmol) and tert-butyl (5-amino-6-methylpyridin-3- yl)carbamate (500 mg, 2.24 mmol) in pyridine (10 mL) was added EDCI (500 mg, 2.61 mmol). The reaction was stirred room temperature for 4 days. The reaction mixture was concentrated and the residue was diluted with EtOAc and washed with aq. NaHCO 3 . The organic layer was dried over Na 2 SO 4 and concentrated. The residue was purified with silica gel column (7% MeOH/CH 2 Cl 2 ) to give tert-butyl (6-methyl-5-(2-(1-methyl-1H-pyrazol-4- yl)pyrazolo[5,1-b]thiazole-7-carboxamido)pyridin-3-yl)carbam ate (717 mg, yield 91.3%). LCMS (ESI): mass calcd. for C 21 H 23 N 7 O 3 S, 453.2; m/z found, 454.2 [M+H] + . Step b. N-(5-amino-2-methylpyridin-3-yl)-2-(1-methyl-1H-pyrazol-4- yl)pyrazolo[5,1-b]thiazole-7-carboxamide hydrochloride [00327] To a suspension of tert-butyl (6-methyl-5-(2-(1-methyl-1H-pyrazol-4- yl)pyrazolo[5,1-b]thiazole-7-carboxamido)pyridin-3-yl)carbam ate (717 mg, 1.58 mmol) in CH 2 Cl 2 (16 mL) was added a solution of 4 N HCl in dioxane (8 mL, 32 mmol). The reaction was stirred at room temperature for 6 h. The reaction mixture was diluted with diethyl ether and the resulting mixture was filtered. The solid was washed with diethyl ether and dried to give N-(5-amino-2-methylpyridin-3-yl)-2-(1-methyl-1H-pyrazol-4-yl )pyrazolo[5,1- b]thiazole-7-carboxamide hydrochloride, which will be used in next step without purification. LCMS (ESI): mass calcd. for C 16 H 15 N 7 OS, 353.1; m/z found, 354.1 [M+H] + . Step c. tert-butyl 3-((6-methyl-5-(2-(1-methyl-1H-pyrazol-4-yl)pyrazolo[5,1- b]thiazole-7-carboxamido)pyridin-3-yl)carbamoyl)azetidine-1- carboxylate [00328] A mixture of N-(5-amino-2-methylpyridin-3-yl)-2-(1-methyl-1H-pyrazol-4- yl)pyrazolo[5,1-b]thiazole-7-carboxamide hydrochloride (100 mg, 0.256 mmol), 1-(tert- butoxycarbonyl)azetidine-3-carboxylic acid (75 mg, 0.37 mmol), and EDCI (80 mg, 0.42 mmol) in pyridine (2 mL) was stirred at room temperature for 2 h. The reaction mixture was concentrated and the residue was purified with silica gel column (9%MeOH/CH 2 Cl 2 with 0.45%NH 4 OH) to give tert-butyl 3-((6-methyl-5-(2-(1-methyl-1H-pyrazol-4-yl)pyrazolo[5,1- b]thiazole-7-carboxamido)pyridin-3-yl)carbamoyl)azetidine-1- carboxylate (128 mg, yield 93.0%). LCMS (ESI): mass calcd. for C 25 H 28 N 8 O 4 S, 536.2; m/z found, 537.2 [M+H] + . Step d. N-(5-(azetidine-3-carboxamido)-2-methylpyridin-3-yl)-2-(1-me thyl-1H- pyrazol-4-yl)pyrazolo[5,1-b]thiazole-7-carboxamide [00329] A solution of tert-butyl 3-((6-methyl-5-(2-(1-methyl-1H-pyrazol-4- yl)pyrazolo[5,1-b]thiazole-7-carboxamido)pyridin-3-yl)carbam oyl)azetidine-1-carboxylate (128 mg, 0.24 mmol) in TFA (0.5 mL) and CH 2 Cl 2 (2.5 mL) was stirred at room temperature for 2 h. The reaction was concentrated to give crude N-(5-(azetidine-3-carboxamido)-2- methylpyridin-3-yl)-2-(1-methyl-1H-pyrazol-4-yl)pyrazolo[5,1 -b]thiazole-7-carboxamide trifluoroacetate, which will be used in next step without purification. LCMS (ESI): mass calcd. for C 20 H 20 N 8 O 2 S, 436.1; m/z found, 437.1 [M+H] + . Step e. N-(5-(1-(2,2-dimethylcyclopentyl)azetidine-3-carboxamido)-2- methylpyridin-3-yl)-2-(1-methyl-1H-pyrazol-4-yl)pyrazolo[5,1 -b]thiazole-7- carboxamide [00330] To a solution of N-(5-(azetidine-3-carboxamido)-2-methylpyridin-3-yl)-2- (1-methyl-1H-pyrazol-4-yl)pyrazolo[5,1-b]thiazole-7-carboxam ide trifluoroacetate (44 mg, 0.08 mmol) and 2,2-dimethylcyclopentan-1-one (40 mg, 0.36 mmol) in MeOH (2 mL) was added NaCNBH 3 (25 mg, 0.40 mmol) at room temperature. The reaction was then heated at 55°C for 7 h. Additional 2,2-dimethylcyclopentan-1-one (20 mg, 0.178 mmol) and NaCNBH 3 (15 mg, 0.24 mmol) was added. The reaction was heated at 55°C for another 2 h. The reaction was quenched with aq. NaHCO3 and extracted with CH2Cl2. The organic layer was dried over Na 2 SO 4 and concentrated. The residue was purified with silica gel column (10%MeOH/CH 2 Cl 2 with 0.5%NH4OH) to give N-(5-(1-(2,2-dimethylcyclopentyl)azetidine- 3-carboxamido)-2-methylpyridin-3-yl)-2-(1-methyl-1H-pyrazol- 4-yl)pyrazolo[5,1-b]thiazole- 7-carboxamide (21 mg, yield 49.3%). LCMS (ESI): mass calcd. for C 27 H 32 N 8 O 2 S, 532.2; m/z found, 533.3 [M+H] + . 1 H NMR (METHANOL-d4) δ: 8.52 (d, J=2.4 Hz, 1H), 8.39 (s, 1H), 8.28 (d, J=2.4 Hz, 1H), 8.20 (s, 1H), 8.01 (s, 1H), 7.81 (s, 1H), 3.94 (s, 3H), 3.63-3.71 (m, 1H), 3.55-3.61 (m, 1H), 3.35-3.53 (m, 3H), 2.49 (s, 3H), 2.42 (t, J=7.8 Hz, 1H), 1.87 (dtd, J=12.7, 8.3, 4.4 Hz, 1H), 1.36-1.70 (m, 5H), 1.04 (s, 3H), 0.94 (s, 3H). [00331] Example 66 was prepared by a similar method of Example 65 from the appropriate starting material Example 67, N-(5-(1-(cyano(cyclopentyl)methyl)azetidine-3-carboxamido)-2 - methylpyridin-3-yl)-2-(1-methyl-1H-pyrazol-4-yl)pyrazolo[5,1 -b]thiazole-7- carboxamide and Example 68, N-(5-(1-(cyclopentylmethyl)azetidine-3-carboxamido)-2- methylpyridin-3-yl)-2-(1-methyl-1H-pyrazol-4-yl)pyrazolo[5,1 -b]thiazole-7- carboxamide [00332] To a solution of N-(5-(azetidine-3-carboxamido)-2-methylpyridin-3-yl)-2- (1-methyl-1H-pyrazol-4-yl)pyrazolo[5,1-b]thiazole-7-carboxam ide trifluoroacetate (44 mg, 0.08 mmol) and cyclopentanecarbaldehyde (80 mg, 0.82 mmol) in MeOH (2 mL) was added NaCNBH 3 (30 mg, 0.48 mmol). The reaction was stirred at room temperature for 3 days (over weekend). The reaction was quenched with 1N NaOH and the resulting mixture was extracted with CH 2 Cl 2 (2x). The organic layer was dried over Na 2 SO 4 and concentrated. The residue was purified with silica gel column (8-14% MeOH/CH 2 Cl 2 with 0.4-0.7%NH 4 OH) to give N-(5-(1-(cyano(cyclopentyl)methyl)azetidine-3-carboxamido)-2 -methylpyridin-3-yl)-2- (1-methyl-1H-pyrazol-4-yl)pyrazolo[5,1-b]thiazole-7-carboxam ide (9.8 mg, yield 22.5%). LCMS (ESI): mass calcd. for C 27 H 29 N 9 O 2 S, 543.2; m/z found, 544.2 [M+H] + . 1 H NMR (METHANOL-d4) δ: 8.45-8.62 (m, 1H), 8.39 (s, 1H), 8.28 (d, J=2.4 Hz, 1H), 8.20 (s, 1H), 8.01 (s, 1H), 7.82 (s, 1H), 3.95 (s, 3H), 3.54-3.66 (m, 5H), 3.42-3.54 (m, 1H), 2.50 (s, 3H), 1.92-2.20 (m, 1H), 1.79-1.92 (m, 2H), 1.53-1.75 (m, 4H), 1.33-1.51 (m, 2H), and then N-(5- (1-(cyclopentylmethyl)azetidine-3-carboxamido)-2-methylpyrid in-3-yl)-2-(1-methyl-1H- pyrazol-4-yl)pyrazolo[5,1-b]thiazole-7-carboxamide (7.4 mg, yield 17.8 %), LCMS (ESI): mass calcd. for C 26 H 30 N 8 O 2 S, 518.2; m/z found, 519.3 [M+H] + . 1 H NMR (METHANOL-d4) δ: 8.56 (d, J=2.4 Hz, 1H), 8.40 (s, 1H), 8.24-8.28 (m, 1H), 8.22 (s, 1H), 8.03 (s, 1H), 7.83 (s, 1H), 3.96 (s, 3H), 3.59-3.71 (m, 2H), 3.51 (t, J=7.6 Hz, 1H), 3.38-3.45 (m, 2H), 2.46-2.55 (m, 5H), 1.85-1.95 (m, 1H), 1.79 (br dd, J=11.2, 3.9 Hz, 2H), 1.50-1.70 (m, 4H), 1.18 (br dd, J=12.0, 7.6 Hz, 2H). Example 69, 2-(2-methoxypyridin-3-yl)-N-(2-methyl-5-(3-(piperidin-1-yl)a zetidine-1- carboxamido)pyridin-3-yl)pyrazolo[5,1-b]thiazole-7-carboxami de Step a. tert-butyl (5-(2-(2-methoxypyridin-3-yl)pyrazolo[5,1-b]thiazole-7- carboxamido)-6-methylpyridin-3-yl)carbamate [00333] A mixture of tert-butyl (5-(2-bromopyrazolo[5,1-b]thiazole-7- carboxamido)-6-methylpyridin-3-yl)carbamate (500 mg, 1.11 mmol), (2-methoxypyridin-3- yl)boronic acid (240 mg, 1.57 mmol), Cs 2 CO 3 (750 mg, 2.30 mmol), and PdCl 2 (dppf) (100 mg, 0.14 mmol) in 1,4-dioxane (8 mL) and H 2 O (2 mL) was de-gassed and then heated at 130°C by microwave for 1 h. The reaction mixture was diluted with 10% MeOH/CH 2 Cl 2 and filtered. The solution was concentrated and the residue was purified by silica gel column with MeOH/CH 2 Cl 2 (6%) to give tert-butyl (5-(2-(2-methoxypyridin-3-yl)pyrazolo[5,1- b]thiazole-7-carboxamido)-6-methylpyridin-3-yl)carbamate (360 mg, yield 67.8%). LCMS (ESI): mass calcd. for C 23 H 24 N 6 O 4 S, 480.2; m/z found, 480.8 [M+H] + . Step b. N-(5-amino-2-methylpyridin-3-yl)-2-(2-methoxypyridin-3- yl)pyrazolo[5,1-b]thiazole-7-carboxamide [00334] To a suspension of tert-butyl (5-(2-(2-methoxypyridin-3-yl)pyrazolo[5,1- b]thiazole-7-carboxamido)-6-methylpyridin-3-yl)carbamate (286 mg, 0.60 mmol) in CH 2 Cl 2 (5 mL) was added TFA (1 mL). The reaction was stirred at room temperature for 2 h. The reaction mixture was concentrated, and the residue was dissolved in MeOH/CH 2 Cl 2 = 1/1. To the resulting solution was added aq. NaHCO 3 . The resulting mixture was stirred for 10 min and was then concentrated to remove most of the organic solvents. The remaining mixture was filtered and the solid was collected and dried to give N-(5-amino-2- methylpyridin-3-yl)-2-(2-methoxypyridin-3-yl)pyrazolo[5,1-b] thiazole-7-carboxamide which was used in the next step without purification. LCMS (ESI): mass calcd. for C 18 H 16 N 6 O 2 S, 380.1; m/z found, 380.9 [M+H] + . Step c.2-(2-methoxypyridin-3-yl)-N-(2-methyl-5-(3-oxoazetidine-1- carboxamido)pyridin-3-yl)pyrazolo[5,1-b]thiazole-7-carboxami de [00335] A mixture of N-(5-amino-2-methylpyridin-3-yl)-2-(2-methoxypyridin-3- yl)pyrazolo[5,1-b]thiazole-7-carboxamide (90 mg, 0.24 mmol), CDI (70 mg, 0.432 mmol), and Et 3 N (0.07 mL, 0.73 g/mL, 0.50 mmol) in DMF (2 mL) was stirred at room temperature for 1.5 h. Additional CDI (35 mg, 0.22 mmol) was added and the reaction was stirred for another 1 h. Azetidin-3-one hydrochloride (100 mg, 0.93 mmol) was added followed by Et 3 N (0.13 mL, 0.94 mmol). The reaction was stirred at room temperature for 1 h. The reaction was quenched with aq. NaHCO 3 and extracted with 10% MeOH/CH 2 Cl 2 (4x). The organic layer was dried over Na 2 SO 4 and concentrated. The residue was purified by silica gel column with MeOH/CH 2 Cl 2 (10%) to give 2-(2-methoxypyridin-3-yl)-N-(2-methyl-5-(3- oxoazetidine-1-carboxamido)pyridin-3-yl)pyrazolo[5,1-b]thiaz ole-7-carboxamide (50 mg, yield 44.3%). LCMS (ESI): mass calcd. for C 22 H 19 N 7 O 4 S, 477.1; m/z found, 478.1 [M+H] + . Step d.2-(2-methoxypyridin-3-yl)-N-(2-methyl-5-(3-(piperidin-1-yl )azetidine-1- carboxamido)pyridin-3-yl)pyrazolo[5,1-b]thiazole-7-carboxami de [00336] A mixture of 2-(2-methoxypyridin-3-yl)-N-(2-methyl-5-(3-oxoazetidine-1- carboxamido)pyridin-3-yl)pyrazolo[5,1-b]thiazole-7-carboxami de (50 mg, 0.11 mmol), piperidine (30 mg, 0.35 mmol), and NaBH(OAc) 3 (50 mg, 0.24 mmol) in HOAc (0.2 mL) and DCE (2 mL) was stirred at room temperature overnight. Additional piperidine (30 mg, 0.35 mmol), NaBH(OAc) 3 (50 mg, 0.24 mmol), and HOAc (0.2 mL) was added. The reaction was heated at 65°C for 2.5 h. Additional NaBH(OAc) 3 (50 mg, 0.24 mmol) was added and the reaction was heated at 65°C for another 1.5 h. The reaction was cooled to room temperature and was quenched with aq. NaHCO 3 . The resulting mixture was extracted with 10% MeOH/CH 2 Cl 2 (3x). The organic layer was dried over Na 2 SO 4 and concentrated. The residue was purified by silica gel column (8%MeOH/CH 2 Cl 2 with 0.4%NH 4 OH) to give 2-(2-methoxypyridin-3-yl)-N-(2-methyl-5-(3-(piperidin-1-yl)a zetidine-1- carboxamido)pyridin-3-yl)pyrazolo[5,1-b]thiazole-7-carboxami de (17.5 mg, yield 30.6%). LCMS (ESI): mass calcd. for C 27 H 30 N 8 O 3 S, 546.2; m/z found, 547.2 [M+H] + . 1 H NMR (METHANOL-d4) δ: 8.43-8.56 (m, 1H), 8.39-8.42 (m, 1H), 8.37 (d, J=2.4 Hz, 1H), 8.18 (dd, J=4.9, 1.5 Hz, 1H), 7.98-8.09 (m, 1H), 7.90 (dd, J=7.3, 1.5 Hz, 1H), 7.00-7.14 (m, 1H), 4.14 (s, 3H), 4.08-4.13 (m, 2H), 3.96 (dd, J=8.3, 5.4 Hz, 2H), 3.18 (quin, J=6.4 Hz, 1H), 2.49 (s, 3H), 2.30-2.43 (m, 4H), 1.58-1.71 (m, 4H), 1.46-1.58 (m, 2H). Example 70, N-(5-((2-(2,2-dimethylpyrrolidin-1-yl)ethyl)carbamoyl)-2-met hylpyridin-3- yl)-2-(6-(2-fluoroethoxy)pyridin-2-yl)pyrazolo[5,1-b]thiazol e-7-carboxamide
Step a. ethyl 2-(6-methoxypyridin-2-yl)pyrazolo[5,1-b]thiazole-7-carboxyla te [00337] A mixture of ethyl 2-bromopyrazolo[5,1-b]thiazole-7-carboxylate (300 mg, 1.09 mmol), 2-methoxy-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyr idine (320 mg, 1.36 mmol). K 2 CO 3 (340 mg, 2.46 mmol), and PdCl 2 (dppf) (75 mg, 0.102 mmol) in 1,4- dioxane (4 mL) and water (1 mL) was de-gassed and heated at 130°C by microwave for 3 h. The reaction mixture was diluted with CH 2 Cl 2 and filtered. The solution was concentrated and the residue was purified by silica gel column (30% EtOAc/heptane) to give ethyl 2-(6- methoxypyridin-2-yl)pyrazolo[5,1-b]thiazole-7-carboxylate (140 mg, yield 42.3%). LCMS (ESI): mass calcd. for C 14 H 13 N 3 O 3 S, 303.1; m/z found, 304.0 [M+H] + . Step b. ethyl 2-(6-hydroxypyridin-2-yl)pyrazolo[5,1-b]thiazole-7-carboxyla te [00338] A mixture of ethyl 2-(6-methoxypyridin-2-yl)pyrazolo[5,1-b]thiazole-7- carboxylate (120 mg, 0.40 mmol) in 4 M HCl in dioxane (8 mL, 32 mmol) was heated at 120°C by microwave for 10 h. The reaction mixture was concentrated to remove most of 1,4- dioxane and was then diluted with diethyl ether. The resulting mixture was filtered and the solid was collected and dried to give ethyl 2-(6-hydroxypyridin-2-yl)pyrazolo[5,1-b]thiazole- 7-carboxylate which was used in next step without purification. LCMS (ESI): mass calcd. for C 13 H 11 N 3 O 3 S, 289.1; m/z found, 290.1 [M+H] + . Step c. ethyl 2-(6-(2-fluoroethoxy)pyridin-2-yl)pyrazolo[5,1-b]thiazole-7- carboxylate [00339] A mixture of ethyl 2-(6-hydroxypyridin-2-yl)pyrazolo[5,1-b]thiazole-7- carboxylate (15 mg, 0.052 mmol), 1-fluoro-2-iodoethane (20 mg, 0.115 mmol), and Cs 2 CO 3 (50 mg, 0.15 mmol) in DMF (0.7 mL) was stirred at room temperature overnight. The reaction mixture was diluted with CH 2 Cl 2 and filtered. The solution was concentrated and the residue was purified with silica gel column (30% EtOAc/heptane) to give ethyl 2-(6-(2- fluoroethoxy)pyridin-2-yl)pyrazolo[5,1-b]thiazole-7-carboxyl ate (14 mg, yield 80.5%). LCMS (ESI): mass calcd. for C 15 H 14 FN 3 O 3 S, 335.1; m/z found, 336.0 [M+H] + . Step d.2-(6-(2-fluoroethoxy)pyridin-2-yl)pyrazolo[5,1-b]thiazole- 7-carboxylic acid [00340] A mixture of ethyl 2-(6-(2-fluoroethoxy)pyridin-2-yl)pyrazolo[5,1- b]thiazole-7-carboxylate (31 mg, 0.094 mmol) and LiOH (15 mg, 0.63 mmol) in H 2 O (1 mL) and THF (2 mL) for 3 days. The reaction mixture was diluted with H 2 O (2 mL) and extracted with CH 2 Cl 2 (4 mL x 2). The aqueous layer was acidified with 2N HCl solution to adjust pH to ~3. The resulting mixture was filtered and the solid was dried to give 2-(6-(2- fluoroethoxy)pyridin-2-yl)pyrazolo[5,1-b]thiazole-7-carboxyl ic acid (25 mg, yield 87.0%) which was used in next step without purification. LCMS (ESI): mass calcd. for C 13 H 10 FN 3 O 3 S, 307.0; m/z found, 308.0 [M+H] + . Step e. N-(5-((2-(2,2-dimethylpyrrolidin-1-yl)ethyl)carbamoyl)-2-met hylpyridin- 3-yl)-2-(6-(2-fluoroethoxy)pyridin-2-yl)pyrazolo[5,1-b]thiaz ole-7-carboxamide [00341] A solution of 2-(6-(2-fluoroethoxy)pyridin-2-yl)pyrazolo[5,1-b]thiazole-7- carboxylic acid (25 mg, 0.081 mmol), 5-amino-N-(2-(2,2-dimethylpyrrolidin-1-yl)ethyl)-6- methylnicotinamide (23 mg, 0.083 mmol), and EDCI (25 mg, 0.13 mmol) in pyridine (2 mL) was heated at 70°C for 7 h. The reaction mixture was concentrated and the residue was first purified with silica gel column (12% MeOH/CH 2 Cl 2 with 0.6% NH 4 OH). The purified product was purified again with prep-HPLC [Mobile phase A: H 2 O with 10 mM NH 4 OH, Mobile phase B: ACN; Gradient: 40%-90% in 7 min] to give N-(5-((2-(2,2- dimethylpyrrolidin-1-yl)ethyl)carbamoyl)-2-methylpyridin-3-y l)-2-(6-(2- fluoroethoxy)pyridin-2-yl)pyrazolo[5,1-b]thiazole-7-carboxam ide. LCMS (ESI): mass calcd. for C 28 H 32 FN 7 O 3 S, 565.2; m/z found, 566.3 [M+H] + . 1 H NMR (METHANOL-d4) δ: 8.67- 8.69 (m, 1H), 8.65-8.66 (m, 1H), 8.29-8.42 (m, 1H), 8.22 (d, J=2.0 Hz, 1H), 7.60-7.74 (m, 1H), 7.44 (d, J=7.3 Hz, 1H), 6.72 (d, J=8.3 Hz, 1H), 4.72-4.75 (m, 1H), 4.60-4.64 (m, 1H), 4.54-4.58 (m, 1H), 4.46-4.53 (m, 1H), 3.42 (t, J=6.8 Hz, 2H), 2.74-2.86 (m, 2H), 2.58 (t, J=6.8 Hz, 2H), 2.52 (s, 3H), 1.66-1.81 (m, 2H), 1.55-1.65 (m, 2H), 0.95 (s, 6H). Example 71, N-(5-((2-(5-azaspiro[3.4]octan-5-yl)ethyl)carbamoyl)-2-methy lpyridin-3- yl)-2-(1-(2-fluoroethyl)-1H-pyrazol-4-yl)pyrazolo[5,1-b]thia zole-7-carboxamide Step a. methyl 5-(2-(1-(2-fluoroethyl)-1H-pyrazol-4-yl)pyrazolo[5,1-b]thiaz ole-7- carboxamido)-6-methylnicotinate [00342] To a mixture of methyl 5-(2-bromopyrazolo[5,1-b]thiazole-7- carboxamido)-6-methylnicotinate (960 mg, 2.43 mmol) in 1,4-dioxane (20 mL) and H 2 O (5 mL) was added 1-(2-fluoroethyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan- 2-yl)-1H- pyrazole (1.2 g, 5.00 mmol), Cs 2 CO 3 (2.4 g, 7.37 mmol), and Pd(dppf)Cl 2 ·CH 2 Cl 2 (480 mg, 0.85 mmol). The system was degassed, and the reaction mixture was stirred at 90 °C under N 2 for 12 hours. The mixture was concentrated under vacuum to give crude methyl 5-(2-(1- (2-fluoroethyl)-1H-pyrazol-4-yl)pyrazolo[5,1-b]thiazole-7-ca rboxamido)-6-methylnicotinate which was used in next step without purification. Step b.5-(2-(1-(2-fluoroethyl)-1H-pyrazol-4-yl)pyrazolo[5,1-b]thi azole-7- carboxamido)-6-methylnicotinic acid [00343] To a solution of methyl 5-(2-(1-(2-fluoroethyl)-1H-pyrazol-4- yl)pyrazolo[5,1-b]thiazole-7-carboxamido)-6-methylnicotinate (2.80 mmol) in THF (9 mL) and MeOH (9 mL) was added a solution of LiOH (0.3 g, 7.15 mmol) in H 2 O (6 mL). The reaction was stirred at 20°C for 1 hour. The mixture was concentrated under vacuum to afford a brown oil. The brown oil was poured into water (30 mL) carefully. The aqueous layer was washed with DCM (20 mL × 3) and acidified with 1N HCl to pH=3. The resulting precipitate was collected by filtration. The solid was rinsed with H 2 O (10 mL) and dried in vacuo to give 5-(2-(1-(2-fluoroethyl)-1H-pyrazol-4-yl)pyrazolo[5,1-b]thiaz ole-7- carboxamido)-6-methylnicotinic acid (800 mg, 1.31 mmol, 46.9%) as a brown solid. LCMS (ESI): mass calcd. for C 18 H 15 FN 6 O 3 S, 414.1; m/z found, 415.2 [M+H] + . Step c. N-(5-((2-(5-azaspiro[3.4]octan-5-yl)ethyl)carbamoyl)-2-methy lpyridin-3- yl)-2-(1-(2-fluoroethyl)-1H-pyrazol-4-yl)pyrazolo[5,1-b]thia zole-7-carboxamide [00344] To a mixture of 5-(2-(1-(2-fluoroethyl)-1H-pyrazol-4-yl)pyrazolo[5,1- b]thiazole-7-carboxamido)-6-methylnicotinic acid (400 mg, 0.66 mmol) in DMF (15 mL) was added 2-(5-azaspiro[3.4]octan-5-yl)ethanamine (180 mg, 1.17 mmol), DIEA (0.4 mL, 2.42 mmol), and HATU (374 mg, 0.98 mmol). The reaction mixture was stirred at room temperature for 2 hours. The mixture was concentrated under vacuum and the residue was first purified by silica gel column chromatography with MeOH/CH 2 Cl 2 (0-20%). The resulting product was purified again by prep-HPLC [Column: Boston Green ODS 150*30mm*5µm; Mobile phase A: water (FA), Mobile phase B: ACN; Gradient: 18% B to 48% B in 7 min; Flow Rate: 25 mL/min] to give N-(5-((2-(5-azaspiro[3.4]octan-5- yl)ethyl)carbamoyl)-2-methylpyridin-3-yl)-2-(1-(2-fluoroethy l)-1H-pyrazol-4- yl)pyrazolo[5,1-b]thiazole-7-carboxamide (32.1 mg, yield 8.04%) as white solid. LCMS (ESI): mass calcd. for C 27 H 31 FN 8 O 2 S, 550.2; m/z found, 551.3 [M+H] + . 1 H NMR (400MHz, METHANOL-d4) δ = 8.82 (d, J=2.0 Hz, 1H), 8.52 (br s, 1H), 8.44 (s, 1H), 8.37 (d, J=2.0 Hz, 1H), 8.30 (s, 1H), 8.14 (s, 1H), 7.92 (s, 1H), 4.87 - 4.85 (m, 1H), 4.74 (t, J=4.6 Hz, 1H), 4.55 (t, J=4.7 Hz, 1H), 4.49 (t, J=4.6 Hz, 1H), 3.70 (t, J=6.6 Hz, 2H), 3.20 (br t, J=6.9 Hz, 2H), 3.12 (br t, J=6.3 Hz, 2H), 2.64 (s, 3H), 2.49 - 2.41 (m, 2H), 2.18 - 2.13 (m, 2H), 2.01 - 1.83 (m, 6H). Example 72, N-(5-((2-(5-azaspiro[3.4]octan-5-yl)ethyl)carbamoyl)-2-methy lpyridin-3- yl)-2-(1H-pyrazol-4-yl)pyrazolo[5,1-b]thiazole-7-carboxamide Step a. N-(5-((2-(5-azaspiro[3.4]octan-5-yl)ethyl)carbamoyl)-2-methy lpyridin-3- yl)-2-bromopyrazolo[5,1-b]thiazole-7-carboxamide [00345] To a mixture of 5-(2-bromopyrazolo[5,1-b]thiazole-7-carboxamido)-6- methylnicotinic acid (0.9 g, 2.36 mmol) in DMF (15 mL) was added 2-(5-azaspiro[3.4]octan- 5-yl)ethanamine ( 648 mg, 4.20 mmol), DIEA (1.4 mL, 8.47 mmol), and HATU (1.3 g, 3.42 mmol). The resulting mixture was stirred at room temperature for 12 hours. The reaction mixture was concentrated under vacuum. The residue was purified by silica gel column chromatography with MeOH/CH 2 Cl 2 (0-20%) to give N-(5-((2-(5-azaspiro[3.4]octan-5- yl)ethyl)carbamoyl)-2-methylpyridin-3-yl)-2-bromopyrazolo[5, 1-b]thiazole-7-carboxamide as a yellow solid. LCMS (ESI): mass calcd. for C 22 H 25 BrN 6 O 2 S, 516.1; m/z found, 517.2 [M+H] + . Step b. N-(5-((2-(5-azaspiro[3.4]octan-5-yl)ethyl)carbamoyl)-2-methy lpyridin-3- yl)-2-(1H-pyrazol-4-yl)pyrazolo[5,1-b]thiazole-7-carboxamide [00346] A mixture of N-(5-((2-(5-azaspiro[3.4]octan-5-yl)ethyl)carbamoyl)-2- methylpyridin-3-yl)-2-bromopyrazolo[5,1-b]thiazole-7-carboxa mide (300 mg, 0.57 mmol), 4- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (272 mg, 1.40 mmol), Pd(dppf)Cl 2 ·CH 2 Cl 2 (418 mg, 0.74 mmol), and K 2 CO 3 (470 mg, 3.40 mmol) in 1,4-dioxane (12 mL) and H 2 O (3 mL) was degassed by applying alternating N 2 atmosphere and was then heated at 85 °C for 12 hours. The mixture was concentrated under vacuum. The resulting residue was first purified by silica gel column chromatography with MeOH/CH 2 Cl 2 (0-10%). The purified product was then purified by prep-HPLC [Column: Welch Xtimate C18 150*30mm*5µm; Mobile phase A: water (NH 3 H 2 O+NH 4 HCO 3 ), Mobile phase B: ACN; Gradient: 24% B to 54% B in 7 min, Flow rate: 25 mL/min] to give N-(5-((2-(5- azaspiro[3.4]octan-5-yl)ethyl)carbamoyl)-2-methylpyridin-3-y l)-2-(1H-pyrazol-4- yl)pyrazolo[5,1-b]thiazole-7-carboxamide. The purified product was purified again by Supercritical Fluid Chromatography [Column: Chiralpak AS-3, 100mm, 4.6mm I.D., 3µm; Mobile phase A: CO 2 , Mobile phase B: ethanol (0.05% DEA); Gradient: A:B = 60:40; Flow rate: 2.8 mL/min] to give N-(5-((2-(5-azaspiro[3.4]octan-5-yl)ethyl)carbamoyl)-2- methylpyridin-3-yl)-2-(1H-pyrazol-4-yl)pyrazolo[5,1-b]thiazo le-7-carboxamide as a white solid. LCMS (ESI): mass calcd. for C 25 H 28 N 8 O 2 S, 504.2; m/z found, 505.2 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ = 13.25 (br s, 1H), 9.99 (s, 1H), 8.79 (d, J=2.0 Hz, 1H), 8.65 (br s, 1H), 8.61 (s, 1H), 8.53 (s, 1H), 8.28 (br s, 1H), 8.22 (s, 1H), 7.95 (s, 1H), 3.40 - 3.38 (m, 2H), 2.68 (br s, 4H), 2.49 (s, 3H), 2.09 (br d, J=12.5 Hz, 2H), 1.84 (br s, 2H), 1.73 - 1.51 (m, 6H). Example 73, PET tracer synthesis: 18 F- N-(5-((2-(5-azaspiro[3.4]octan-5- yl)ethyl)carbamoyl)-2-methylpyridin-3-yl)-2-(1-(2-fluoroethy l)-1H-pyrazol-4- yl)pyrazolo[5,1-b]thiazole-7-carboxamide HPLC conditions Semiprep HPLC 1 for 2- 18 F-fluoroethyl tosylate ( 18 F-FETs) Column: Agilent Eclipse XDB C18250x9.4mm, mobile phase: 50% CH 3 CN in water UV detector: 254 nm flow rate: 4 ml/min Retention time: 9-10 min Semiprep HPLC 2 for 18 F- N-(5-((2-(5-azaspiro[3.4]octan-5-yl)ethyl)carbamoyl)-2- methylpyridin-3-yl)-2-(1-(2-fluoroethyl)-1H-pyrazol-4-yl)pyr azolo[5,1-b]thiazole-7- carboxamide Column: Agilent Eclipse XDB C18250x9.4mm, mobile phase: 20% CH 3 CN in 50 mM acetate buffer, pH4.0 UV detector: 254 nm flow rate: 4 ml/min Retention time: about 19 min Analytical HPLC for quality control for 18 F- N-(5-((2-(5-azaspiro[3.4]octan-5- yl)ethyl)carbamoyl)-2-methylpyridin-3-yl)-2-(1-(2-fluoroethy l)-1H-pyrazol-4- yl)pyrazolo[5,1-b]thiazole-7-carboxamide Column: Kinetex EVO 5 µm C18150x4.6mm, Flow rate: 1 ml/min UV detector: 254 nm Mobile phase A: CH 3 CN, Mobile phase B: 50 mM acetate buffer, pH4.0, Gradient: 5% A for 0-1 min, 5-75% A for 1-12 min Retention time: 8 min Radiochemistry: Step a.2- 18 F-fluoroethyl tosylate ( 18 F-FETs) [00347] 2- 18 F-Fluoroethyl tosylate ( 18 F-FETs) was synthesized in GE Tracerlab FX2N, GE Healthcare. Specifically, the 18 F-F- was trapped on a QMA cartridge and eluted with a solution of K 2 CO 3 /K 222 (3 mg/15mg in 900 µl CH 3 CN and 100 µl water). The solvent was evaporated under a stream of nitrogen at 80-120 °C and azeotropic drying was repeated again with 1 ml portion of dry CH 3 CN. To the dried 18 F-K222/K + complex ([K222] + / 18 F-) was added 7.5-8.5 mg of ethylene di(p-toluenesulfonate) in 1 ml anhydrous CH 3 CN. The reaction was remained at 80 °C for 5 min to produce 18 F-FETs crude. After cooling down to room temperature, 1 ml of CH 3 CN and 3 ml of 10 mM NH 4 OAc in water were added to the reaction crude before loading onto Semi-prep HPLC 1. The HPLC fractions containing 18 F- FETs were collected in a round bottom flask with 30 ml water. 18 F-FETs was trapped onto a C18 Sep-Pak light cartridge and eluted with 0.5 ml DMF to a 2 nd reaction vial. Step b. 18 F- N-(5-((2-(5-azaspiro[3.4]octan-5-yl)ethyl)carbamoyl)-2- methylpyridin-3-yl)-2-(1-(2-fluoroethyl)-1H-pyrazol-4-yl)pyr azolo[5,1-b]thiazole-7- carboxamide [00348] About 2.5 mg of precursor N-(5-((2-(5-azaspiro[3.4]octan-5- yl)ethyl)carbamoyl)-2-methylpyridin-3-yl)-2-(1H-pyrazol-4-yl )pyrazolo[5,1-b]thiazole-7- carboxamide was dissolved in 300 µl DMF. To the precursor solution was added 3.5-4.5 mg of Cs 2 CO 3 . After the precursor and Cs 2 CO 3 was stirred at room temperature for 20-30 min, the precursor and the 18 F-FETs solution were mixed together. The reaction remained at 110 °C with mild stirring for 10 min. After cooling down to room temperature, 1.5 ml of acetate buffer (pH4) was added to the reaction mixture before loading onto semi-prep HPLC 2. The factions containing 18 F- N-(5-((2-(5-azaspiro[3.4]octan-5-yl)ethyl)carbamoyl)-2- methylpyridin-3-yl)-2-(1-(2-fluoroethyl)-1H-pyrazol-4-yl)pyr azolo[5,1-b]thiazole-7- carboxamide were collected in a flask with 30 ml water. Then, 18 F- N-(5-((2-(5- azaspiro[3.4]octan-5-yl)ethyl)carbamoyl)-2-methylpyridin-3-y l)-2-(1-(2-fluoroethyl)-1H- pyrazol-4-yl)pyrazolo[5,1-b]thiazole-7-carboxamide was trapped on to a C18 Sep-Pak light cartridge and eluted with 0.5 ml ethanol and 4.5 ml 0.9% saline consequently into an intermediate vial. At last, 18 F-N-(5-((2-(5-azaspiro[3.4]octan-5-yl)ethyl)carbamoyl) -2- methylpyridin-3-yl)-2-(1-(2-fluoroethyl)-1H-pyrazol-4-yl)pyr azolo[5,1-b]thiazole-7- carboxamide solution passed through a 0.22 µm Millex filter into the final product vial for imaging purpose. [00349] The 18 F-compound was authenticated using the analytical HPLC system by co-injecting with the 19 F-cold standard. The entire process was completed in approximately 2.5 h including QC. The radiochemical yield was 5-15% (decay corrected to EOB) with the radiochemical purity of >95% and the specific activity was >1000 Ci/mmol (decay corrected to end of synthesis). Biological Assays [00350] PDGFRβ HTRF assay [00351] I. Materials [00352] Reagents [00353] Instrumentation: a. Compound liquid handling: LabCyte Echo; b. Reagent liquid handling: Thermo Scientific Multidrop Combi; c. BMG PHERAStar multilabel plate reader. [00354] Protein Reagent: His6-TEV-PDGFRβ Protein Prep prepared at Accelagen. [00355] II. Methods and Procedures [00356] Stock solutions: [00357] Assay buffer stock solution, contains 50 mM Hepes, 10 mM MgCl 2 , 1 mM EGTA, and 0.01% Brij-35, 0.01% ovalbumin, 2 mM DTT at pH 7.5, in molecular biology grade water. Store at room temperature. [00358] DTT, 2 M in molecular biology grade water, store at -20°C in aliquots. [00359] Ovalbumin, 10% or 100 mg/mL, prepare fresh on experimental day. [00360] PDGFRβ, 116 µM (PDGFRb_08 Prep 02), produced at Accelagen. Store at -80°C in aliquots. [00361] TK-biotin peptide, 0.5 µM in molecular biology grade water, store at - 20°C in aliquots. [00362] ATP, 100 mM in molecular biology grade water, store at -20°C in aliquots. [00363] HTRF KinEASE-TK kit: Allow the contents of the Cisbio kit to warm up to room temperature before use. This kit contains HTRF detection buffer, TK-Antibody labeled with Eu 3+- cryptate, TK-substrate biotin and Streptavidin-XL665. [00364] TK Substrate-Biotin, reconstitute 500 µg lyopholized with 574 µL molecular biology grade water to prepare a 500 µM stock; After use, aliquot the rest and store at -20°C. [00365] TK Antibody-Cryptate, reconstitute lyophilized with 1 mL of molecular biology grade water (100x solution) then add 99 mL detection buffer to prepare a ready to use TK-antibody-cryptate solution; the concentration of the TK-antibody-cryptate reagent is not known. After use, aliquot the rest and store at -20°C. [00366] Streptavidin-XL665, reconstitute 3 mg lyophilized with 3 mL molecular biology grade water to prepare a 1 mg/mL or 16.67 µM stock; MW = 60 kDa; After use, aliquot the rest and store at -20°C. [00367] Freshly prepared solutions: [00368] Assay buffer. Dilute 5x Kinase buffer 5-fold with molecular biology grade water and add DTT to 2 mM and ovalbumin to 0.1 mg/mL (or 0.01%). [00369] 2X protein solution. Make a working solution of 100 pM PDGFRβ in assay buffer. Keep on ice until use to maintain enzyme stability. [00370] 2X substrate solution. Make a working solution of 1.6 mM ATP and 1 µM TK-substrate biotin peptide in assay buffer. [00371] 3X quench/detection solution. Make a working solution of 0.1875 µM SA-XL665 and the TK-antibody cryptate diluted by ½ of total quench/detection volume in assay buffer. [00372] Keep final streptavidin/biotin ratio at 1 to 8. [00373] Example of 3x quench/detection solution preparation: 8 mL total volume. [00374] 1x assay buffer – 3910 µL [00375] TK antibody-cryptate in detection buffer – 4000 µL [00376] 0.1875 µM SA-XL665 – 90 uL [00377] The kinase reaction is stopped by the addition of the detection reagents which contain EDTA (detection step). [00378] Assay Procedure: [00379] Assay in white ProxiPlate 384-well [00380] Step 1. Dispensing inhibitors/DMSO and low control: Using the ECHO 555 acoustic dispenser, spot desired compound serial dilutions in DMSO, NEAT DMSO to represent the uninhibited enzyme control, and 10 µM final [imatinib] to the represent the 100% inhibited enzyme control [00381] Step 2. PDGFRβ E + I pre-incubation: Add 2 µL 2x protein solution to columns 1-24 using the Multidrop Combi. Centrifuge at 1000 rpm for 1 min. Incubate 30 min at RT [00382] Step 2. Enzymatic reaction: Add 2 µL substrate solution to columns 1-24 to initiate the reaction using the Multidrop Combi; cover/seal the assay plate to reduce evaporation. Centrifuge at 1000 rpm for 1 min. Incubate at room temperature for 3 hours. [00383] Final concentrations of components in PDGFRβ cascade assay: [00384] 50 mM Hepes, pH 7.5 [00385] 10 mM MgCl2 [00386] 0.01% Brij-35 [00387] 1 mM EGTA [00388] 2 mM DTT [00389] 0.01% Ovalbumin [00390] 50 pM inactive PDGFRβ [00391] 0.5 µM TK-substrate biotin peptide [00392] 62.5 nM SA-XL-665 [00393] TK antibody-Eu3+-cryptate (diluted by 1/3 final from stock) [00394] 800 µM ATP [00395] ≤ 1% DMSO [00396] Step 3. Quench/Detection: Add 2 µl 3x quench/detection solution to columns 1-24 using the Multidrop Combi; cover/seal the plate. Centrifuge 1 min 1000 rpm. Incubate at RT for 60 min. Read the plate in PHERAstar (or similar instrument) on HTRF setting at excitation 337nm - dual emission - 665/620 nm ratio. [00397] III. Calculations and Formulas [00398] HTRF ratio values calculated by the instrument (Ratio is acceptor counts/donor counts * 10,000) is exported from the plate reader and used in data analysis. The exported data will be used to calculate 1) compound activity and 2) assay statistics. Compound activity is represented by % Inhibition when testing a single dose of a compound or IC50 when testing a dose response of a compound. Assay statistics can include Robust Z’ and Signal to Background. [00399] % Inhibition Calculation: Percent inhibition will be calculated for sample wells based on the equation: [00400] Where, x: sample activity; cr: central reference is calculated based on wells containing all assay components and no compound (DMSO only); sr: scale reference is calculated based on wells inhibited with 10 µM Imatinib (these wells will contain the enzyme and substrate solutions) [00401] IC 50 Calculation: For IC 50 determination, full 11- point dose response data will be processed using the following equation: [00402] Where S0=Activity level at zero concentration of test compound; SInf=Activity level at infinite concentration; IC50: Concentration at which activity reaches 50% of maximum level; c= Concentration in logarithmic units corresponding to the values on the x-axis of the dose-response curve plot; Hill coefficient n= Measure of the slope at IC50. See Table 1, below. [00403] Registered Parameters (when applicable): % Activity, IC 50 , nHill Slope, S inf , S 0 , and Comments [00404] Robust Z’ Calculation: Robust Z prime (RZ’) value will be calculated as defined by the following equation: [00405] Where, RSD: Robust standard deviation; cr: central reference is calculated based on wells containing all assay components and no compound (DMSO only); sr: scale reference is calculated based on wells inhibited with 10 µM Imatinib compound (these wells will contain the enzyme and substrate solutions) [00406] Signal to Background, S/B, Calculation [00407] Where, CR, Central Reference (no compound wells); SR, Scale Reference (inhibitor control wells). [00408] PDGFRβ LanthaScreen assay [00409] I. Materials [00410] II. Methods and Procedures [00411] Stock solutions: [00412] Assay buffer stock contains 50 mM HEPES pH7.5, 10 mM MgCl 2 , 0.01% Brij- 35, 1 mM EGTA. [00413] Tb-labeled inactive PDGFRβ.3.6 µM in 50 mM HEPES, pH 7.4, 150 mM NaCl, 0.005% Tween-20 and 10% glycerol. Store at -80 °C in aliquots. [00414] Tracer 222, 50 µM in DMSO, store at -20 °C. [00415] Freshly prepared solutions: [00416] Assay buffer. Add DTT to 2 mM and ovalbumin to 0.1 mg/mL to Assay buffer stock. [00417] Kinase-Tracer solution. Make a working solution of 0.2 nM Tb-labeled inactive PDGFRβ and 40 nM Tracer 222 in Assay buffer. Keep on ice until use. [00418] Assay Procedure: [00419] Step 1. Dispensing inhibitors: Using Echo, dispense 40nL/well (or less) compound serial dilutions in DMSO onto the assay plate. [00420] Step 2. Dispensing Kinase-Tracer solution: Add 4 µL/well Kinase-Tracer solution. Seal the plate with optically transparent plate seal. Centrifuge at 1000 rpm for 1 min. [00421] Final concentrations of components in the assay: [00422] [Tb-PDGFRβ] = 0.2 nM; [00423] [Tracer 222] = 40 nM; [00424] [DMSO] ≤ 1%. [00425] Step 3. Detection: Read TR-FRET signals after 18 hours incubation at room temperature. [00426] III. Calculations and Formulas [00427] % Inhibition: % Inhibition = (NC – sample) / (NC – PC) * 100 where NC is the mean of negative control (reactions without inhibitor), and PC is the mean of positive control (1µM sunitinib). [00428] IC50 determination: Compounds are serially diluted 3-fold and tested in an 11- point dose response. IC 50 values are determined from a 4-parameter fit, using the following equation: Y = Bottom + (Top – Bottom) / (1+10((Log IC 50 -X)*Hill slope)), where X = log 10 of the compound concentration; top can be defined by PC; bottom defined by NC. See Table 1, below. [00429] PDGFRβ cellular assay [00430] I. Materials [00431] II. Methods and Procedures [00432] Cell Culture and Preparation: Cells are cultured according to ATCC procedure (5) with the addition of the antibiotic penicillin-streptomycin. If working from frozen, cells should be thawed according to ATCC procedure. Depending on the cell density of frozen vial, cells will need time to recover from thaw. An 80% confluent T75 flask should be enough for one 384 well plate. [00433] Stock Solutions: Rat PDGFBB. A 100ug/mL stock is prepared by reconstituting 50ug in 500uL of 4mM HCl and 0.1% BSA. It can be stored for a month at 4°C, or aliquoted out and frozen in the -20/-80°C to avoid multiple free-thaw cycles. [00434] Freshly Prepared Solutions: [00435] 1x Cisbio cell lysis buffer. The cell lysis buffer is diluted 4-fold with molecular grade water. The blocking agent is then diluted 25-fold in the diluted lysis buffer. [00436] Antibody solutions. Equal amounts d2 and cryptate antibody are diluted 20-fold in detection buffer. [00437] Rat PDGFBB. A working stock of 100ng/mL is created from the stock solution in 10%FBS culture media. [00438] Assay Steps [00439] Step 1: Plating Cells: Media from the A10 cell flask is aspirated. The cells are rinsed with PBS and then trypsinized to disperse the cell layer. The cells are then pelleted are resuspended to 1.25e5 cells/mL. 40uL of cells are then plated in 384 Greiner TC treated plates at a density of 5000 cells/well using the Combi. Plates are covered and placed in the incubator (37°C 5% CO2) overnight to allow cells to adhere. [00440] Step 2: Compound Dispense: Approximately 18 hours after plating, dispense 40nL compound onto cells using Echo. Column 12 is the neutral control DMSO, column 24 is the inhibitor control 10 mM Imatinib (10uM final assay concentration). The plate is returned to the incubator for 3 hours. [00441] Step 3: Activation by PDGFbb: 6uL of the working stock of 100ng/mL PDGFbb is dispensed using the Tempest to give a final assay concentration of 15ng/mL (EC80). After 10 minutes the media is removed by flicking the plate. [00442] Step 4: Cell lysis and antibody addition: 20uL lysis buffer per well is added to the plate via Tempest. 5uL antibody solution is added per well via the Tempest. The plate is placed on the shaker at 230 rpm for 1 hour at room temperature. [00443] Step 5: Detection: The plate is read using the HTRF module on the BMG Pherastar. Data is analyzed using Genedata Screener. [00444] III. Calculations and Formulas: [00445] % Inhibition: % Inhibition = (NC – sample) / (NC – PC) * 100 where NC is the mean of negative control (reactions without inhibitor), and PC is the mean of positive control (10µM imatinib). [00446] IC50 determination: Compounds are serially diluted 3-fold and tested in an 11- point dose response. IC 50 values are determined from a 4-parameter fit, using the following equation: Y = Bottom + (Top – Bottom) / (1+10((Log IC 50 -X)*Hill slope)), where X = log 10 of the compound concentration; top can be defined by PC; bottom defined by NC. See Table 1, below. [00447] VEGFR ADP GLO assay [00448] I. Materials [00449] II. Methods and Procedures [00450] Stock solutions: [00451] Assay buffer stock contains 50mM HEPES pH7.5, 10mM MgCl 2 , 0.01% Brij-35, and 1mM EGTA. [00452] Unphosphorylated VEGFR2.52.6 µM in 50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 5% Glycerol, 0.5 mM TCEP. Store at -80 °C in aliquots. [00453] 10mg/mL srctide solution, prepared in Assay buffer (Assay buffer stock with 2mM DTT, 0.1% Pluronic F-127, and 0.1mg/mL ovalbumin). Sonicate 10 mg/ml Srctide solution for 10 minutes and then vacuum filter. [00454] Freshly prepared solutions [00455] Assay buffer. Add DTT to 2mM, Pluronic F-127 to 0.1% and ovalbumin to 0.1mg/mL to Assay buffer stock. [00456] 2X kinase solution. Make a working solution of 10 nM unphosphorylated VEGFR2 in Assay buffer. Vacuum filter 2x kinase solution prior to running assay. Keep on ice until use. [00457] 2X substrate/ATP solution. Make a working solution of 2mg/mL srctide and 2.4mM ATP in Assay buffer. Keep on ice until use. [00458] Assay Procedure: [00459] Step 1. Dispensing inhibitors/controls: Using Echo, dispense 10nL/well compound serial dilutions in DMSO to columns 1-22 (in 384-well plates) or columns 1-44 (in 1536-well plates). Dilution series = 11 pt, 3-fold dilutions. The top compound concentration in the source plate is 4 mM. The top compound concentration in the assay plate is 10 uM. Using Echo, dispense 10 nl/well DMSO to column 23 (in 384-well plates) or columns 45-47 (in 1536-well plates). These wells will serve as negative control wells Using Echo, dispense 10 nl/well 400 uM TAK-593 in DMSO to column 24 (in 384-well plates) or column 48 (in 1536-well plates). The final concentration of TAK-593 in the assay should be 1 uM. These wells will serve as positive control wells. [00460] Step 2. Pre-incubation of inhibitors with kinase: Add 2 µL/well 2X kinase solution. Centrifuge at 1000 rpm for 1 min. Incubate at room temperature for 30 min. [00461] Step 3. Kinase cascade reaction: Add 2 µL/well 2X substrate/ATP solution to initiate kinase reactions. Centrifuge at 1000 rpm for 1 min. Incubate at room temperature for 180 min. [00462] Final concentrations of components in the assay: [00463] [VEGFR2] = 5 nM; [00464] [ATP] = 1.2 mM; [00465] [Srctide] = 1 mg/mL; [00466] [DMSO] ≤ 1%. [00467] Step 4. Quench: Add 2 uL/well ADP Glo Reagent + 0.05% CHAPS. Centrifuge at 1000 rpm for 1 min; Incubate at room temperature for one hour. [00468] Step 5. Detection: Add 2uL/well Kinase Detection Reagent + 0.05% CHAPS. Centrifuge at 1000 rpm for 1 min; Incubate at room temperature for 1 hour; Read Luminescence on a plate reader. [00469] III. Calculations and Formulas [00470] % Inhibition: % Inhibition = (NC – sample) / (NC – PC) * 100 where NC is the mean of negative control (reactions without inhibitor), and PC is the mean of positive control (1µM TAK-593). [00471] IC 50 determination: Compounds are serially diluted 3-fold and tested in an 11- point dose response. IC 50 values are determined from a 4-parameter fit, using the following equation: Y = Bottom + (Top – Bottom) / (1+10 (Log IC50-X)*Hill slope ), where X = log 10 of the compound concentration; top can be defined by PC; bottom defined by NC. See Table 1, below. Table 1. Biology chemical and cellular activity
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