[go: up one dir, main page]

WO2024255765A1 - Nouveaux composés, compositions les comprenant et leurs utilisations - Google Patents

Nouveaux composés, compositions les comprenant et leurs utilisations Download PDF

Info

Publication number
WO2024255765A1
WO2024255765A1 PCT/CN2024/098708 CN2024098708W WO2024255765A1 WO 2024255765 A1 WO2024255765 A1 WO 2024255765A1 CN 2024098708 W CN2024098708 W CN 2024098708W WO 2024255765 A1 WO2024255765 A1 WO 2024255765A1
Authority
WO
WIPO (PCT)
Prior art keywords
ring
substituted
alkyl
cycloalkyl
halo
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2024/098708
Other languages
English (en)
Inventor
Kun Chin Liu
Weiguo QUAN
Xiangqing JIANG
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nuphase Therapeutics Hangzhou Ltd Co
Original Assignee
Nuphase Therapeutics Hangzhou Ltd Co
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nuphase Therapeutics Hangzhou Ltd Co filed Critical Nuphase Therapeutics Hangzhou Ltd Co
Publication of WO2024255765A1 publication Critical patent/WO2024255765A1/fr
Anticipated expiration legal-status Critical
Pending legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00

Definitions

  • the invention relates to the field of medicinal chemistry, and in particular, it provides bicyclic compounds and analogues and derivatives thereof, the use thereof for inhibiting Werner Syndrome RecQ DNA helicase (WRN) and methods of treating disease using said compounds, in particular the use in treating cancer, and in particular the treatment of cancer characterized as microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) , including colorectal, gastric and endometrial cancer.
  • WRN Werner Syndrome RecQ DNA helicase
  • MSI-H microsatellite instability-high
  • dMMR mismatch repair deficient
  • the invention also provides the use of said compounds as research chemicals, intermediate compounds, combinations, processes and formulations.
  • WRN Werner Syndrome helicase
  • RecQ helicases and WRN are involved in multiple DNA processing steps, including DNA replication, double-strand break repair, transcription, and telomere maintenance, and are therefore considered as “genome guardians” (Chu and Hickson, 2009) .
  • Defects in DNA mismatch repair (MMR) promote microsatellite instability (MSI) , characterized by frequent insertion and/or deletion mutations in repetitive nucleotide sequences.
  • MSI-induced genomic instability causes the occurrence and development of various cancers, including colorectal cancer, gastric cancer, endometrial cancer, and ovarian cancer (Kim et al., 2013; Kunitomi, H et al., 2017) .
  • CRISPR-CAS9-mediated large-scale silencing revealed that RecQ DNA helicase WRN is selectively essential in MSI models but not necessary in microsatellite-stable models. Knocking out WRN in MSI models induces DNA double-strand breaks and selectively promotes cell apoptosis and cell cycle arrest (Chan et al., 2019) .
  • the invention provides compounds, pharmaceutically acceptable salts thereof, pharmaceutical compositions thereof and combinations thereof, said compounds being inhibitors of Werner Syndrome RecQ DNA Helicase (WRN) .
  • WRN Werner Syndrome RecQ DNA Helicase
  • the invention further provides methods of treating, preventing, or ameliorating a disease or condition, comprising administering to a subject in need thereof an effective amount of a WRN inhibitor.
  • the invention also provides compounds, pharmaceutically acceptable salts thereof, pharmaceutical compositions thereof and combinations thereof, said compounds being useful for the treatment of cancer, in particular cancers characterized as microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) . Also provided are compounds that bind to, and/or inhibit WRN, and are therefore useful as research chemicals, e.g. as a chemical probe, and as tool compounds. Various embodiments of the invention are described herein.
  • A is a linker selected from -C (O) -, -S (O) -, -S (O) 2 -, and
  • Y is N, C, or CH
  • y is 0, 1, 2, 3 or 4;
  • Y is linked via a single bond to the adjacent carbon atom when Y is CH, or Y is linked via a double bond to the adjacent atom when Y is C, and when is a single bond, Y is carbon unsubstituted or substituted by OH or F;
  • J is N or CH
  • R 5 substituents on the same ring carbon atom may join, together with the carbon atom to which they are attached, to form a (C 3 -C 4 ) cycloalkyl spiro ring or a 3 or 4-membered heterocyclyl spiro ring, wherein said heterocyclyl spiro ring contains ring carbon ring atoms and one ring heteroatom selected from O, N and S,
  • ring C is a fused (C 3 -C 6 ) cycloalkyl ring, a fused (C 3 -C 6 ) heterocyclyl ring or a fused phenyl ring, wherein said fused (C 3 -C 6 ) heterocyclyl ring contains ring carbon atoms and one ring heteroatom selected from O, N and S,
  • A is a linker selected from -S (O) -, -S (O) 2 -, and aR 5 substituent form ring C:
  • two R 5 substituents may join to form a (C 1 -C 3 ) alkylene bridge or a heteroalkylene bridge, wherein said heteroalkylene bridge is one heteroatom selected from N and O, or is -CH 2 -O-CH 2 -;
  • R 1 is:
  • ⁇ (C 3 -C 4 ) cycloalkyl is unsubstituted or substituted by 1 or 2 R 33 , wherein R 33 is halo, and wherein said (C 3 -C 4 ) cycloalkyl or halo-substituted (C 3 -C 4 ) cycloalkyl is substituted by 0, 1 or 2 R 15 substituents,
  • cycloalkyl has 2 substituents at the same ring carbon atom which join to form a (C 3 -C 6 ) cycloalkyl spiro ring, or 3, 4, 5, or 6 membered heterocyclyl spiro ring contains ring carbon atoms and 1 or 2 ring heteroatom selected from O, N and S, and wherein said cycloalkyl or heterocyclyl is substituted by 0, 1 or 2 substituents independently selected from R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 22 and R 23 ,
  • cycloalkyl is fused to (C 3 -C 6 ) cycloalkyl ring, or 3, 4, 5, or 6 membered heterocyclyl ring contains ring carbon atoms and 1-4 ring heteroatom selected from O, N and S, and wherein said cycloalkyl or heterocyclyl is substituted by 0, 1 or 2 substituents independently selected from R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 22 and R 23 ,
  • C 4 cycloalkyl it can be bridged by 1 or 2 carbon atoms, and wherein said bridged C 4 cycloalkyl is substituted by 0, 1 or 2 substituents independently selected from R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 22 and R 23 ;
  • bridged is bridged, and said bridge contains 1 or 2 carbon atoms, and substituted by 0, 1 or 2 substituents independently selected from R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 22 and R 23 ;
  • ⁇ heterocyclyl is a 4 or 7 membered fully saturated or partially unsaturated group comprising ring carbon atoms and 1 or 2 ring heteroatoms independently selected from N, O and S, wherein said heterocyclyl is unsubstituted or substituted by 1, 2, 3 or 4, preferably 1 or 2, R 33 , wherein R 33 is halo, wherein said heterocyclyl or halo-substituted heterocyclyl
  • cycloalkyl ring is fused to (C 3 -C 6 ) cycloalkyl ring, or 3, 4, 5, or 6 membered heterocyclyl ring contains ring carbon atoms and 1-4 ring heteroatoms selected from O, N and S, and wherein said cycloalkyl or heterocyclyl is substituted by 0, 1 or 2 substituents independently selected from R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 22 and R 23 ;
  • phenyl is fused to phenyl, wherein said phenyl is substituted by 0, 1 or 2 substituents independently selected from R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 22 and R 23 ;
  • heterocyclyl is a 5 or 6 membered fully saturated or partially unsaturated group comprising ring carbon atoms and 1 or 2 ring heteroatoms independently selected from N, O and S, wherein said heterocyclyl is unsubstituted or substituted by 1, 2, 3 or 4, preferably 1 or 2 R 33 , wherein R 33 is halo, wherein said heterocyclyl or halo-substituted heterocyclyl
  • cycloalkyl ring is fused to (C 4 -C 6 ) cycloalkyl ring, or 4, 5, or 6 membered heterocyclyl ring containing ring carbon atoms and 1-3 ring heteroatoms selected from N and S, or 5-6 membered heteroaryl ring containing ring carbon atoms and 1-3 ring heteroatoms selected from N and S, or 1 ring O atom and 1 or 2 ring heteroatoms selected from N and S; and wherein said cycloalkyl, heterocyclyl or heteroaryl is substituted by 0, 1 or 2 substituents independently selected from R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 22 and R 23 ;
  • phenyl is fused to phenyl, wherein said phenyl is substituted by 0, 1 or 2 substituents independently selected from R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 22 and R 23 ;
  • heteroaryl is a 5 or 6 membered fully unsaturated monocyclic group comprising ring carbon atoms and 1, 2, 3 or 4 ring heteroatoms independently selected from N, O and S, preferably 1 or 2 ring heteratoms, wherein the total number of ring S atoms does not exceed 1, and the total number of ring O atoms does not exceed 1, the heteroaryl is substituted by one or more substituents selected from CN, -S (O) 2 C 1-4 alkyl, -S (O) C 1-4 alkyl, R 25 (R 24 ) N-and C 1-4 alkoxy and contains 0, 1 or 2 R 15 substituents which is not CN, or -S (O) 2 C 1-4 alkyl, -S (O) C 1-4 alkyl, R 25 (R 24 ) N-or C 1-4 alkoxy;
  • heteroaryl is a 5 or 6 membered fully unsaturated monocyclic group comprising ring carbon atoms and 1, 2, 3 or 4 ring heteroatoms independently selected from N, O and S, preferably 1 or 2 ring heteratoms, wherein the total number of ring S atoms does not exceed 1, and the total number of ring O atoms does not exceed 1; said heteroaryl contains 0, 1 or 2 R 15 substituents and is fused to 4, 5 or 6 membered heterocyclyl or 5-6 membered heteroaryl, wherein the heterocyclyl ring contains ring carbon atoms and 1-4 ring heteroatom selected from N, O and S and the heteroaryl ring contains ring carbon atoms and 1, 2, 3 or 4 ring heteroatoms independently selected from N, O and S, and wherein the heterocyclyl and heteroaryl are substituted by 0, 1 or 2 substituents independently selected from R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 22 and R
  • phenyl wherein said phenyl is unsubstituted or substituted by 1, 2, 3 or 4, preferably 1 or 2, R 33 , wherein R 33 is halo, and wherein said phenyl or halo-substituted phenyl contains 1 or 2 substituents selected from CN, -S (O) 2 C 1-4 alkyl, -S (O) C 1-4 alkyl, halogenated -S (O) C 1-4 alkyl, halogenated -S (O) 2 C 1-4 alkyl, -S (O) C 3-6 cycloalkyl and -PO (C 1-4 alkyl) 2 ,
  • phenyl is unsubstituted or substituted by 1, 2, 3 or 4 R 15 , and is fused to 4, 5 or 6 membered monocyclic ring or 6, 7, 8, 9 or 10 membered di-cyclic ring, wherein said 4, 5 or 6 membered monocyclic ring or 6, 7, 8, 9 or 10 membered di-cyclic ring contains ring carbon atoms and 0-3 ring heteroatoms selected from N, O and S and is substituted by 0, 1 or 2 substituents independently selected from R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 22 and R 23 ;
  • the 4, 5 or 6 membered ring or 6, 7, 8, 9 or 10 membered di-cyclic ring is saturated, partially saturated or unsaturated heterocycle or carbocycle
  • each R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 22 and R 23 is independently selected from:
  • azetidinyl or pyrrolidinyl wherein said azetidinyl and pyrrolidinyl are linked to the rest of the molecule via the N atom, and are each unsubstituted or substituted by 1 or 2 F,
  • R 25 (R 24 ) N- wherein R 24 is H or (C 1 -C 4 ) alkyl unsubstituted or substituted by 1, 2 or 3 halo, R 25 is H or (C 1 -C 4 ) alkyl unsubstituted or substituted by 1, 2 or 3 halo,
  • R 27 is CH 3 , H, halogen or deuterium
  • R 2 is the moiety selected from:
  • R 6 is selected from:
  • R 8 is selected from H, halo, and (C 1 -C 4 ) alkyl unsubstituted or substituted by 1, 2 or 3 halo;
  • R 9 is selected from H, O-CH 3 , OH, CN, CH 3 and halo;
  • R 28 is selected from:
  • X is selected from C-R 7 and N, wherein R 7 is H or halo, or R 7 can join, together with R 28 or R 6 , and the atoms to which they are attached, to form a fused (C 4 -C 6 ) cycloalkyl ring, wherein said fused (C 4 -C 6 ) cycloalkyl ring is unsubstituted or substituted by 1, 2 or 3 halo, or
  • R 2 is selected from:
  • R 31 is selected from H, halo and CH 3 ,
  • R 32 is selected from H, halo and CH 3 ,
  • R 3 is:
  • R 4 is selected from:
  • R 10 , R 11 , R 12 , R 13 and R 14 are each independently selected from:
  • R 34 and R 35 are each independently selected from:
  • (C 1 -C 4 ) alkyl wherein said (C 1 -C 4 ) alkyl is unsubstituted or substituted by OH or -O (C 1 -C 2 ) alkyl,
  • R 34 and R 35 can join, together with the atom to which they are attached, to form an azetidinyl, pyrrolidinyl or piperidinyl ring, wherein said azetidinyl, pyrrolidinyl and piperidinyl are unsubstituted or substituted with CH 3 ;
  • R 1 is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • ring E is (C 3 -C 4 ) cycloalkyl which contains 0, 1, or 2 R 15 substituents;
  • E is a phenyl substituted by 1 or 2 substituents selected from CN, -S (O) 2 C 1-4 alkyl, -S (O) C 1-4 alkyl, -S (O) C 3-6 cycloalkyl, halogenated -S (O) C 1-4 alkyl, halogenated -S (O) 2 C 1-4 alkyl and -PO (C 1-4 alkyl) 2 and 0, 1, or 2 R 15 substituents which are not CN, -S (O) 2 C 1-4 alkyl, -S (O) C 1-4 alkyl, -S (O) C 3-6 cycloalkyl, halogenated -S (O) C 1-4 alkyl, halogenated -S (O) 2 C 1-4 alkyl or –PO (C 1-4 alkyl) 2 ;
  • E is a 5-6-membered heteroaryl substituted by 1 or 2 substituents selected from CN, R 25 (R 24 ) N-and -OC 1-4 alkyl and 0, 1, or 2 R 15 substituents which are not CN, R 25 (R 24 ) N-or -OC 1-4 alkyl;
  • ring E' is independently selected from partially unsaturated monocyclic ring C5-C7 cycloalkenyl
  • ring E 1 ' is independently selected from partially unsaturated monocyclic ring C7 cycloalkenyl
  • ring E is independently selected from 4 or 7 membered fully saturated or partially unsaturated heterocyclyl comprising ring carbon atoms and 1 or 2 ring heteroatoms independently selected from N, O and S;
  • ring E”' is independently selected from 5 or 6 membered fully saturated or partially unsaturated heterocyclyl
  • ring E is independently selected from 5-6 membered heteroaryl comprising ring carbon atoms and 1, 2, 3 or 4 ring heteroatoms independently selected from N, O and S, preferably 1 or 2 ring heteraoms, wherein the total number of ring S atoms does not exceed 1, and the total number of ring O atoms does not exceed 1;
  • ring E””' is independently selected from phenyl
  • ring F is independently selected from (C 3 -C 6 ) cycloalkyl, or 3, 4, 5, or 6 membered heterocyclyl comprising ring carbon atoms and 1 or 2 ring heteroatom selected from O, N and S;
  • ring F' is independently selected from (C 4 -C 5 ) cycloalkyl, or 4, 5, 6 or 7 membered heterocyclyl comprising ring carbon atoms and 1 or 2 ring heteroatoms selected from O, N and S;
  • ring G is independently selected from (C 3 -C 6 ) cycloalkyl, or 3, 4, 5, or 6 membered heterocyclyl comprising ring carbon atoms and 1-4 ring heteroatom selected from O, N and S;
  • ring G' is independently selected from (C 4 -C 6 ) cycloalkyl, or 4, 5, or 6 membered heterocyclyl comprising ring carbon atoms and 1-3 ring heteroatoms selected from N and S, or 5-6 membered heteroaryl comprising ring carbon atoms and 1-3 ring heteroatom selected from N and S, or 1 ring O atom and 1 or 2 ring heteroatoms selected from N and S;
  • ring G is independently selected from 4, 5 or 6 membered heterocyclyl or 5-6 membered heteroaryl, wherein the heterocyclyl ring contains ring carbon atoms and 1-4 ring heteroatoms selected from N, O and S and the heteroaryl ring contains ring carbon atoms and 1, 2, 3 or 4 ring heteroatoms independently selected from N, O and S;
  • ring G”' is independently selected from 4, 5 or 6 membered ring contains ring carbon atoms and 0-3 ring heteroatoms selected from N, O and S; preferably G”' is independently selected from (C 4 -C 6 ) cycloalkyl, 4, 5, or 6 membered heterocyclyl comprising ring carbon atoms and 1-3 ring heteroatom selected from N and S, or 1 ring O atom and 1 or 2 ring heteroatoms selected from N and S, or 5-6 membered heteroaryl comprising ring carbon atoms and 1-3 ring heteroatoms selected from N and S, or 1 ring O atom and 1 or 2 ring heteroatoms selected from N and S;
  • R 33 is halo
  • each R 15 and R 16 is independently selected from:
  • azetidinyl or pyrrolidinyl wherein said azetidinyl and pyrrolidinyl are linked to the rest of the molecule via the N atom, and are each unsubstituted or substituted by 1 or 2 F,
  • R 25 (R 24 ) N- wherein R 24 is H or (C 1 -C 4 ) alkyl unsubstituted or substituted by 1, 2 or 3 halo, R 25 is H or (C 1 -C 4 ) alkyl unsubstituted or substituted by 1, 2 or 3 halo,
  • n 0, 1 or 2
  • f1 0, 1 or 2
  • f2 is 0, 1 or 2
  • f3 is 0, 1 or 2.
  • R 1 is:
  • bridged is bridged, and said bridge contains 1 or 2 carbon atoms, and substituted by 0, 1 or 2 substituents independently selected from R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 22 and R 23 ;
  • cycloalkyl ring is fused to (C 3 -C 6 ) cycloalkyl ring, or 3, 4, 5, or 6 membered heterocyclyl ring contains ring carbon atoms and 1-4 ring heteroatoms selected from O, N and S, and wherein said cycloalkyl or heterocyclyl is substituted by 0, 1 or 2 substituents independently selected from R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 22 and R 23 ;
  • ⁇ heterocyclyl is a 4 or 7 membered fully saturated or partially unsaturated group comprising ring carbon atoms and 1 or 2 ring heteroatoms independently selected from N, O and S, wherein said heterocyclyl is unsubstituted or substituted by 1, 2, 3 or 4, preferably 1 or 2, R 33 , wherein R 33 is halo, wherein said heterocyclyl or halo-substituted heterocyclyl
  • cycloalkyl ring is fused to (C 3 -C 6 ) cycloalkyl ring, or 3, 4, 5, or 6 membered heterocyclyl ring contains ring carbon atoms and 1-4 ring heteroatoms selected from O, N and S, and wherein said cycloalkyl or heterocyclyl is substituted by 0, 1 or 2 substituents independently selected from R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 22 and R 23 ;
  • phenyl is fused to phenyl, wherein said phenyl is substituted by 0, 1 or 2 substituents independently selected from R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 22 and R 23 ;
  • heterocyclyl is a 5 or 6 membered fully saturated or partially unsaturated group comprising ring carbon atoms and 1 or 2 ring heteroatoms independently selected from N, O and S, wherein said heterocyclyl is unsubstituted or substituted by 1, 2, 3 or 4, preferably 1 or 2 R 33 , wherein R 33 is halo, wherein said heterocyclyl or halo-substituted heterocyclyl
  • cycloalkyl ring is fused to (C 4 -C 6 ) cycloalkyl ring, 4, 5, or 6 membered heterocyclyl ring contains ring carbon atoms and 1-3 ring heteroatoms selected from N and S, or 5-6 membered heteroaryl ring containing ring carbon atoms and 1-3 ring heteroatoms selected from N and S, or 1 ring O atom and 1 or 2 ring heteroatoms selected from N and S; and wherein said cycloalkyl, heterocyclyl or heteroaryl is substituted by 0, 1 or 2 substituents independently selected from R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 22 and R 23 ;
  • phenyl is fused to phenyl, wherein said phenyl is substituted by 0, 1 or 2 substituents independently selected from R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 22 and R 23 ;
  • heteroaryl is a 5 or 6 membered fully unsaturated monocyclic group comprising ring carbon atoms and 1, 2, 3 or 4 ring heteroatoms independently selected from N, O and S, preferably 1 or 2 ring heteratoms, wherein the total number of ring S atoms does not exceed 1, and the total number of ring O atoms does not exceed 1, the heteroaryl is substituted by one or more substituents selected from CN, -S (O) 2 C 1-4 alkyl, -S (O) C 1-4 alkyl, R 25 (R 24 ) N-and C 1-4 alkoxy and contains 0, 1 or 2 R 15 substituents which is not CN, or -S (O) 2 C 1-4 alkyl, -S (O) 2 C 1-4 alkyl or R 25 (R 24 ) N-or C 1-4 alkoxy;
  • heteroaryl is a 5 or 6 membered fully unsaturated monocyclic group comprising ring carbon atoms and 1, 2, 3 or 4 ring heteroatoms independently selected from N, O and S, preferably 1 or 2 ring heteratoms, wherein the total number of ring S atoms does not exceed 1, and the total number of ring O atoms does not exceed 1; said heteroaryl contains 0, 1 or 2 R 15 substituents and is fused to 4, 5 or 6 membered heterocyclyl or 5-6 membered heteroaryl, wherein the heterocyclyl ring contains ring carbon atoms and 1-4 ring heteroatom selected from N, O and S and the heteroaryl ring contains ring carbon atoms and 1, 2, 3 or 4 ring heteroatoms independently selected from N, O and S, and wherein the heterocyclyl and heteroaryl are substituted by 0, 1 or 2 substituents independently selected from R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 22 and R
  • phenyl wherein said phenyl is unsubstituted or substituted by 1, 2, 3 or 4, preferably 1 or 2, R 33 , wherein R 33 is halo, and wherein said phenyl or halo-substituted phenyl contains 1 or 2 substituents selected from CN, -S (O) 2 C 1-4 alkyl, -S (O) C 1-4 alkyl, -S (O) C 3-6 cycloalkyl and -PO (C 1-4 alkyl) 2 ,
  • phenyl is unsubstituted or substituted by 1, 2, 3 or 4 R 15 , and is fused to 4, 5 or 6 membered monocyclic ring or 6, 7, 8, 9 or 10 membered di-cyclic ring, wherein said 4, 5 or 6 membered monocyclic ring or 6, 7, 8, 9 or 10 membered di-cyclic ring contains ring carbon atoms and 0-3 ring heteroatoms selected from N, O and S, and is substituted by 0, 1 or 2 substituents independently selected from R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 22 and R 23 ;
  • the 4, 5 or 6 membered ring or 6, 7, 8, 9 or 10 membered di-cyclic ring is saturated, partially saturated or unsaturated heterocycle or carbocycle
  • each R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 22 and R 23 is independently selected from:
  • azetidinyl or pyrrolidinyl wherein said azetidinyl and pyrrolidinyl are linked to the rest of the molecule via the N atom, and are each unsubstituted or substituted by 1 or 2 F,
  • R 25 (R 24 ) N- wherein R 24 is H or (C 1 -C 4 ) alkyl unsubstituted or substituted by 1, 2 or 3 halo, R 25 is H or (C 1 -C 4 ) alkyl unsubstituted or substituted by 1, 2 or 3 halo,
  • n 0, 1 or 2.
  • R 2 is selected from:
  • X is selected form: N, or CH;
  • R 6 is selected from:
  • R 8 is selected from H, halo, and (C 1 -C 4 ) alkyl unsubstituted or substituted by 1, 2 or 3 halo;
  • R 9 is selected from H, O-CH 3 , OH, CN, CH 3 and halo;
  • R 28 is selected from:
  • A is -C (O) -.
  • R 33 is F.
  • the compound has a structure of formula (II)
  • R 1 , R 2 , R 3 , R 4 , R 5 and y is defined as above.
  • R 4 is preferably R 4 is
  • R 2 is
  • R 2 is
  • the compound has a structure of formula (III)
  • M is N or CH
  • Rf is selected from H, halogen, C 1-6 alkyl, and halogenated C 1-6 alkyl; preferably Rf is halogen;
  • Rg is selected from H, halogen, C 1-6 alkyl, and halogenated C 1-6 alkyl;
  • R 1 , R 3 , R 5 and y are defined as above.
  • R 3 is methyl, ethyl, or propyl.
  • the compound when M is N, the compound has a structure of formula (III-A) :
  • Rf, Rg, R 1 , R 3 , R 5 and y are defined as above.
  • the compound when M is CH, the compound has a structure of formula (III-B ) :
  • Rf, Rg, R 1 , R 3 , R 5 and y are defined as above.
  • Rf is selected from Cl and F.
  • Rg is selected from H, methyl, Cl, F, CHF 2 and CF 3 .
  • R 1 is independently selected from wherein n 1 and n 2 are each independently selected from 1, 2, or 3;
  • n 3 independently selected from 0, 1, or 2;
  • R 15 is defined as above.
  • R 1 is independently selected from oxacycloheptenyl, wherein said oxacycloheptenyl is unsubstituted or substituted by 1, 2, 3 or 4, preferably 1 or 2, R 33 , wherein R 33 is halo, wherein said oxacycloheptenyl or halo-substituted oxacycloheptenyl
  • cycloalkyl ring is fused to (C 3 -C 6 ) cycloalkyl ring, or 3, 4, 5 , 6 membered heterocyclyl ring containing ring carbon atoms and one ring heteroatom selected from O, N and S, and wherein said cycloalkyl or heterocyclyl is substituted by 0, 1 or 2 substituents independently selected from R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 22 and R 23 .
  • R 1 is independently selected from:
  • R 1 is independently selected from:
  • the compound is selected from:
  • said heterocyclyl and carbocyclyl can be saturated or partially unsaturated, or unsaturated, substituted or unsubstituted, aromatic or non aromatic.
  • said heterocyclyl and carbocyclyl may be in fused, bridged or spiro-connected fashion.
  • each group is the corresponding group in the specific compound in the example.
  • the compound is the compound prepared in examples 1-328.
  • the compound is the compound of examples 1, 7, 45, 46, 51, 52, 77, 78, 127, 131, 200, 201, 202, 224, 232, 238, 245, 246, 250, 254, 256, 257, 259, 260, 264, 265, 270, 272, 273, 274, 276, 277, 278, 279a, 279b, 281, 282a, 282b, 283, 284, 285, 286a, 286b, 287a, 287b, 302, 304, 308, 311, 314, 316, 317, 325, 326, 327, 328.
  • the present application relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the compound of the first aspect of the present invention, or a pharmaceutically acceptable salt, a hydrate, a solvate, a prodrug, a stereoisomer, a deuterated product or a tautomer thereof and one or more pharmaceutically acceptable carriers or excipients.
  • compositions can be used to prepare individual, single-unit dosage forms.
  • single unit dosage forms provided herein are suitable for oral, mucosal, parenteral, topical, transdermal, or transcutaneous administration to a patient.
  • dosage forms include, but are not limited to: tablets; caplets; capsules, such as soft elastic gelatin capsules; cachets; troches; lozenges; dispersions; suppositories; powders; aerosols; gels; liquid dosage forms suitable for oral or mucosal administration to a patient, including suspensions, solutions, and elixirs; liquid dosage forms suitable for parenteral administration to a patient; eye drops or other ophthalmic preparations suitable for topical administration; and sterile solids that can be reconstituted to provide liquid dosage forms suitable for parenteral administration to a patient.
  • dosage forms comprise the compound provided herein, or the enantiomer, stereoisomer, solvate or pharmaceutically acceptable salt thereof in an amount of 1 to about 1000 mg, from about 5 to about 500 mg, from about 10 to about 350 mg, or from about 50 to about 200 mg.
  • the specific amount of the compound provided herein, or the enantiomer, stereoisomer, solvate or pharmaceutically acceptable salt thereof will depend on the specific agent used, the diseases or conditions being treated or prevented.
  • the compounds of formula (I) of the present invention in free form or in pharmaceutically acceptable salt form, exhibit valuable pharmacological properties, e.g. WRN inhibiting properties, e.g. as indicated in vitro and in vivo tests as provided in the next sections, and are therefore indicated for therapy, or for use as research chemicals, e.g. as a chemical probe, and as tool compounds.
  • WRN inhibiting properties e.g. as indicated in vitro and in vivo tests as provided in the next sections, and are therefore indicated for therapy, or for use as research chemicals, e.g. as a chemical probe, and as tool compounds.
  • compositions comprise a compound provided herein, or an enantiomer, a stereoisomer, a solvate, a deuterated product or a pharmaceutically acceptable salt thereof, and optionally a second active agent used for treating cancer, and in particular the treatment of cancer characterized as microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) , including colorectal, gastric and endometrial cancer.
  • MSI-H microsatellite instability-high
  • dMMR mismatch repair deficient
  • the present application relates to a method of:
  • ⁇ modulating WRN activity in a subject comprises administering to the subject a therapeutically effective amount of the compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof,
  • ⁇ inhibiting WRN in a subject comprises administering to the subject a therapeutically effective amount of the compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof,
  • ⁇ treating a disorder or disease which can be treated by WRN inhibition in a subject comprising administering to the subject a therapeutically effective amount of the compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof,
  • treating cancer in a subject comprising administering to the subject a therapeutically effective amount of the compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof,
  • ⁇ treating cancer in a subject comprising administering a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, wherein the cancer is characterized as microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) .
  • the cancer characterized as microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) is selected from colorectal, gastric, prostate, endometrial, adrenocortical, uterine, cervical, esophageal, breast, kidney and ovarian cancer.
  • the cancer characterized as microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) is selected from colorectal, gastric, prostate and endometrial cancer.
  • MSI-H microsatellite instability-high
  • dMMR mismatch repair deficient
  • examples include uterine corpus endometrial carcinoma, colon adenocarcinoma, stomach adenocarcinoma, rectal adenocarcinoma, adrenocortical carcinoma, uterine carcinosarcoma, cervical squamous cell carcinoma, endocervical adenocarcinoma, esophageal carcinoma, breast carcinoma, kidney renal clear cell carcinoma, prostate cancer and ovarian serous cystadenocarcinoma.
  • MSI-H microsatellite instability-high
  • dMMR mismatch repair deficient
  • MSI-H microsatellite instability-high
  • dMMR mismatch repair deficient
  • ⁇ for the treatment of cancer that is characterized as microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) is selected from colorectal, gastric, prostate and endometrial cancer, or
  • the cancer characterized as microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) is selected from uterine corpus endometrial carcinoma, colon adenocarcinoma, stomach adenocarcinoma, rectal adenocarcinoma, adrenocortical carcinoma, uterine carcinosarcoma, cervical squamous cell carcinoma, endocervical adenocarcinoma, esophageal carcinoma, breast carcinoma, kidney renal clear cell carcinoma, prostate cancer and ovarian serous cystadenocarcinoma.
  • MSI-H microsatellite instability-high
  • dMMR mismatch repair deficient
  • said cancer is characterized as microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) ,
  • the cancer is characterized by microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) , for example colorectal, gastric, prostate, endometrial, adrenocortical, uterine, cervical, esophageal, breast, kidney and ovarian cancer, in particular, colorectal, gastric, prostate or endometrial cancer, or uterine corpus endometrial carcinoma, colon adenocarcinoma, stomach adenocarcinoma, rectal adenocarcinoma, adrenocortical carcinoma, uterine carcinosarcoma, cervical squamous cell carcinoma, endocervical adenocarcinoma, esophageal carcinoma, breast carcinoma, kidney renal clear cell carcinoma and ovarian serous cystadenocarcinoma.
  • MSI-H microsatellite instability-high
  • dMMR mismatch repair deficient
  • the subject has or is identified as having a microsatellite instable (MSI-H) cancer, e.g., in reference to a control, e.g., a normal, subject.
  • MSI-H microsatellite instable
  • the subject has MSI-H advanced solid tumors, a colorectal cancer (CRC) , endometrial, uterine, stomach or other MSI-H cancer.
  • CRC colorectal
  • endometrial or stomach cancer which cancer has or is identified as having a microsatellite instability (MSI-H) , e.g., in reference to a control, e.g., a normal, subject.
  • MSI-H microsatellite instable
  • the invention provides a combination, in particular a pharmaceutical combination, comprising a compound of the first aspect of the present invention, or a pharmaceutically acceptable salt, a hydrate, a solvate, a deuterated product, a prodrug, a stereoisomer, or a tautomer thereof and one or more therapeutically active agents.
  • the invention provides a compound of the first aspect of the present invention or a pharmaceutically acceptable salt, a hydrate, a solvate, a deuterated product, a prodrug, a stereoisomer, or a tautomer thereof for use as a research chemical, for example as a chemical probe or as a tool compound.
  • the invention provides a solid form, process or intermediate as described herein.
  • alkyl refers to a branched or straight chain saturated aliphatic hydrocarbon group. In one embodiment, the alkyl contains from 1 to about 12 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) carbon atoms, more generally from 1 to about 6 carbon atoms ( (C 1 -C 6 ) alkyl) or from 1 to about 4 carbon atoms ( (C 1 -C 4 ) alkyl) .
  • the specified ranges as used herein indicate an alkyl group having each member of the range described as an independent species.
  • (C 1 -C 4 ) alkyl indicates a straight or branched alkyl group having from 1, 2, 3 or 4 carbon atoms and is intended to mean that each of these is described as an independent species.
  • alkyl include, but are not limited to, methyl, ethyl, npropyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl.
  • the alkyl group is optionally substituted as described herein.
  • alkenyl refers to a branched or straight chain aliphatic hydrocarbon group having one or more carbon-carbon double bonds that may occur at a stable point along the chain.
  • Nonlimiting example is (C 2 -C 4 ) alkenyl (such as C 2 , C 3 , C 4 ) .
  • the specified ranges as used herein indicate an alkenyl group having each member of the range described as an independent species, as described herein for the alkyl moiety.
  • alkenyl include, but are not limited to, ethenyl, propenyl, and butadienyl (including 1, 2-butadienyl and 1, 3-butadienyl) .
  • the alkenyl group is optionally substituted as described herein.
  • alkynyl refers to a branched or straight chain aliphatic hydrocarbon group having one or more carbon-carbon triple bonds that may occur at any stable point along the chain, for example, (C 2 -C 4 ) alkynyl (such as C 2 , C 3 , C 4 ) .
  • the specified ranges as used herein indicate an alkynyl group having each member of the range described as anindependent species, as described herein for the alkyl moiety.
  • alkynyl include, but are not limited to, ethynyl, propynyl, 1-butynyl, 2-butynyl, and 3-butynyl.
  • the alkynyl group is optionally substituted as described herein.
  • cycloalkyl refers to a saturated cyclic alkyl group having a single ring or multiple rings including fused, bridged, and spiro ring systems.
  • C3-8 cycloalkyl has from 3 to 8 ring carbon atoms (such as, 3, 4, 5, 6, 7 or 8 ring carbon atoms) .
  • Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl.
  • cycloalkenyl refers to a partially unsaturated monocyclic ring alkyl group having a single ring or multiple rings including fused, bridged, and spiro ring systems, for example, 5 or 6 ring carbon atoms.
  • Examples of cycloalkenyl include, but are not limited to, cyclopentenyl and cyclohexenyl.
  • haloalkyl refers to both branched and straight-chain alkyl groups substituted with 1 or more halogen atoms, up to the maximum allowable number of halogen atoms.
  • haloalkyl include, but are not limited to, trifluoromethyl, monofluoromethyl, difluoromethyl, 2-fluoroethyl, and penta-fluoroethyl.
  • heterocyclyl refers to a saturated or a partially unsaturated (i.e., having one or more double and/or triple bonds within the ring without aromaticity) monocyclic or bicyclic radical of 4, 5, 6, 7, 8, 9, or 10 ring atoms in which at least one ring atom is a heteroatom selected from nitrogen, oxygen, and sulfur, the remaining ring atoms being C, where one or more ring atoms is optionally substituted independently with one or more substituents described above.
  • the only heteroatom is oxygen.
  • a heterocyclyl may be a monocycle having 4 to 7 ring members (2 to 6 carbon atoms and 1 to 4 heteroatoms selected from N, O, and S) or a bicycle having 6 to 10 ring members (4 to 9 carbon atoms and 1 to 6 heteroatoms selected from N, O, and S) , for example: a bicyclo [4, 5] , [5, 5] , [5, 6] , or [6, 6] system.
  • the only heteroatom is sulfur.
  • Spiro moieties are also included within the scope of this definition.
  • Examples of a heterocyclic group wherein 1 or 2 ring carbon atoms are substituted with oxo ( ⁇ O) moieties are pyrimidinonyl and 1, 1-dioxothiomorpholinyl.
  • the heterocycle groups herein are optionally substituted independently with one or more substituents described herein.
  • the compound of the present invention refers to the compound represented by the formula I, and further comprises the pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.
  • the invention therefore provides a compound of formula (I) :
  • each R 1 ring atom adjacent to the R 1 ring atom to which said R 1 ring is joined to the remainder of the molecule is independently unsubstituted or substituted by halo only, in particular, independently unsubstituted or substituted with one F substituent, and
  • the salt of the compound in the present invention may be formed which are also within the scope of the present invention. Unless otherwise stated, the compound in the present invention is understood to include its salt.
  • the term "salt” as used herein refers to a salt formed in the form of acid or base from inorganic or organic acid and base. Further, when the compound in the present invention contains a base fragment which includes, but is not limited to pyridine or imidazole, when contains an acid segment which includes, but is not limited to carboxylic acid.
  • the zwitter-ion that may form “inner salt” is included within the range of the term “salt” .
  • compositions of the present invention may form a salt, for example, compound I is reacted with a certain amount (such as an equivalent amount) of an acid or base, and precipitated in a medium, or freeze-dried in aqueous solution.
  • the compounds in the present invention containing base fragment which includes but is not limited to amines or pyridine or imidazole rings, may form salt with organic or inorganic acid.
  • Typical acids that form salts include acetate (such as acetate or trihalogenated acetic acid, such as trifluoroacetic acid) , adipate, alginate, ascorbate, aspartate, benzoate, benzene sulfonate, disulfate, borate, butyrate, citrate, camphorate, camphor sulfonate, cyclopentane propionate, diethylene glycolate, lauryl sulfate, ethanesulphonate, fumarate, gluceptate, glycerophosphate, hemisulphate, enanthate, caproate, hydrochloride, hydrobromide, hydriodate, isethionate (e.g., 2-hydroxy-ethesulfonate) , lactate, male
  • Some compounds of the invention may contain acidic fragments including, but not limited to carboxylic acid may form salts with various o rg anic or inorganic bases.
  • Salt formed by typical base includes ammonium salt, alkali metal salt (such as sodium, lithium and potassium salts) , alkaline earth metal salt (such as calcium and magnesium salts) , and salt formed by organic bases (such as organic amines) , such as benzathine, dicyclohexylamine, hydrabamine (salt formed with N, N-bis (dehydroabietyl) ethylenediamine) , N-methyl-D-glucanamine, N-methyl-D-glucoamide, tert-butyllamine, and the salt formed with amino acids such as arginine, lysine, etc..
  • alkali metal salt such as sodium, lithium and potassium salts
  • alkaline earth metal salt such as calcium and magnesium salts
  • organic bases such as organic amines
  • organic bases such as organic amine
  • Basic nitrogen-containing groups can form quaternary ammonium salts with halides, such as small molecular alkyl halides (such as chlorides, bromides and iodides of methyl, ethyl, propyl and butyl) , dialkyl sulfate (such as dimethyl, diethyl, dibutyl, and dipentyl sulfates) , long chain halides (such as asuch as chlorides, bromides and iodides of decyl, dodecyl, tetradecyl, and tetradecyl) , aralkyl halides (such as bromides of benzyl and phenyl) , etc..
  • halides such as small molecular alkyl halides (such as chlorides, bromides and iodides of methyl, ethyl, propyl and butyl) , dialkyl sulfate (such as dimethyl
  • prodrug and solvate of the compound in the present invention are also included within the scope of the present invention.
  • prodrug herein refers to a compound resulting from the chemical transformation of a metabolic or chemical process to produce a compound, salt, or solvate in the present invention for the treatment of an associated disease.
  • the compounds of the invention include solvates such as hydrates.
  • Compound, salt or solvate in the present invention may be present in tautomeric forms such as amide and imino ether. All of these tautomers are part of the present invention.
  • Stereisomers of all compounds e.g., those asymmetric carbon atoms that may be present due to various substitutions
  • the independent stereoisomer in the present invention may not coexist with other isomers (e.g., as a pure or substantially pure optical isomer with special activity) , or may be a mixture (e.g., racemate) , or a mixture formed with all other stereoisomers or a part thereof.
  • the chiral center of the present invention has two configurations of S or R, which is defined by International Union of Pure and Applied Chemistry (IUPAC) founded in 1974.
  • racemization form can be solved by physical methods, such as fractional crystallization, or separation crystallization by derivation into diastereomers, or separation by chiral column chromatography.
  • Individual optical isomer can be obtained from racemate by appropriate methods, including but not limited to conventional methods, such as recrystallization after salting with optically active acids.
  • Weight content of compound in the present invention obtained by preparation, separation and purification in turn is equal to or greater than 90%, such as equal to or greater than 95%, equal to or greater than 99% ( “very pure” compound) , and listed in the description of the text.
  • “very pure” compound of the present invention is also part of the present invention.
  • All configuration isomers of the compound of the present invention are within the scope, whether in mixture, pure or very pure form.
  • the definition of the compound of the present invention comprises cis (Z) and trans (E) olefin isomers, and cis and trans isomers of carbocyclic and heterocyclic.
  • Some compounds of the present invention may exist in specific geometric or stereoisomer forms.
  • the present invention covers all compounds, including their cis and trans isomers, R and S enantiomers, diastereomers, (D) type isomers, (L) type isomers, racemic mixtures and other mixtures.
  • asymmetric carbon atom can represent substituent, such as alkyl. All isomers and mixtures thereof are included in the present invention.
  • mixtures of isomers may contain a variety ratios of isomers.
  • mixtures with only two isomers may have the following combinations: 50: 50, 60: 40, 70: 30, 80: 20, 90: 10, 95: 5, 96: 4, 97: 3, 98: 2, 99: 1, or 100: 0, all ratios of the isomers are within the scope of the present invention.
  • Similar ratio and the ratio of mixtures of more complex isomers, which are readily understood by general skill of the art are also within the scope of the invention.
  • the present invention also includes the isotope labeled compound, which is equivalent to the original compound herein. However, in fact, the substitution of one or more atoms by an atom with a different atomic weight or mass number usually occurs.
  • Examples of compound isotopes that may be listed in the present invention include hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine isotopes such as 2 H, 3 H, 13 C, 11 C, 14 C, 15 N, 18 O, 17 O, 31 P, 32 P, 35 S, 18 F and 36 Cl, respectively.
  • the synthesis of the compound of the invention can be prepared by asymmetric synthesis, or derivatized with chiral auxiliary reagent, separating the resulting diastereomeric mixture and removing the chiral adjunct to obtain a pure enantiomer.
  • a molecule contains a basic functional group, such as an amino acid, or an acidic functional group, such as a carboxyl group
  • a diastereomer can be formed with a salt of suitable optically active acids or bases, which can be separated by conventional means, such as crystallization or chromatography, to obtain a pure enantiomer.
  • the preparation method of the compound of the formula (I) of the present invention is more specifically described below, but these specific methods do not constitute any limitation of the invention.
  • the compound of the invention may also optionally be conveniently prepared by combining the various synthetic methods described in this specification or known in the art, such a combination may be easily performed by a skilled person in the art to which the invention belongs.
  • the preparation process for the compounds of the present invention is as follows, in which the raw materials and reagents used may be commercially purchased unless otherwise specified.
  • Step 1 the compound I-6 was obtained by cyclization of compound I-4 and tert-butyl 4- (1-ethoxy-1, 3-dioxopentan-2-yl) piperazine-1-carboxylate (compound I-5) .
  • Step 2 the compound I-6 was alkylated with the various intermediate in the base condition to afford compound I-7.
  • Step 3 the compound I-7 was then deprotected and the resulting amine was coupled with the corresponding acid to get the final compound I-8.
  • compositions of the present invention are used to prevent and /or treat cancer.
  • the compounds of the formula (I) may be used in combination with other drugs known to treat or improve similar conditions.
  • the original administration for the drug can remain unchanged, while compound of formula I may be administered simultaneously or subsequently.
  • Pharmaceutical composition containing one or more known drugs and the compound of formula I may be preferred when administered in combination with one or more other drugs.
  • the drug combination also includes administering the compound of formula I and other one or more known drugs at overlapping time. When the compound of formula I is combined with other one or more drugs, the dose of the compound or known drug may be lower than that of their individual use.
  • a compound provided herein, or an enantiomer, a stereoisomer, a solvate or a pharmaceutically acceptable salt thereof is administered in combination with another drug ( "second active agent” ) or treatment.
  • Second active agents include small molecules and large molecules (e.g., proteins and antibodies) .
  • Other therapies that can be used in combination with the administration of compounds provided herein include, but are not limited to, surgery, immunotherapy, biological therapy, radiation therapy, and other non-drug-based therapies useful for treating or preventing various diseases described herein.
  • the dosage forms of the pharmaceutical composition of the present invention include (but are not limited to) : injection, tablet, capsule, aerosol, suppository, pellicle, pill, liniment for external use, controlled release or sustained-release or nano formulation.
  • the pharmaceutical composition of the present invention comprises a compound of the present invention or a pharmaceutically acceptable salt and a pharmaceutically acceptable excipient or carrier with safe and effective amount.
  • safe and effective amount refers to the amount of compound is sufficient to significantly improve the condition, not to produce severe side effects.
  • the pharmaceutical composition contains 1-2000 mg of the compound /dosage of the present invention, and preferably contains 10-1000 mg of the compound /dosage of the present invention.
  • "one dosage" is a capsule or a pill.
  • “Pharmaceutically acceptable carrier” refers to one or more compatible solid or liquid filler or gel substances, which are suitable for human use, and must be sufficiently pure and of sufficiently low toxicity. "Compatible” herein refers to ability of each component of a composition can be mixed with the compound of the present invention and can be mixed with each other without appreciably reducing the efficacy of the compound.
  • pharmaceutically acceptable carrier include cellulose and derivatives thereof (such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc. ) , gelatin, talc, solid lubricant (such as stearic acid, magnesium stearate) , calcium sulfate, vegetable oil (such as soybean oil, sesame oil, peanut oil, olive oil, etc.
  • polyol such as propylene glycol, glycerol, mannitol, sorbitol, etc.
  • emulsifier such as )
  • wetting agent such as lauryl sodium sulfate
  • colorant such as flavoring, stabilizer, antioxidant, preservative, pyrogen-free water, etc.
  • administration mode for the compound or pharmaceutical compositions of the present invention
  • representative administration mode includes (but is not limited to) : oral, intratumorally, rectal, parenteral (intravenous, intramuscular or subcutaneous) , and topical administration.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.
  • the active compounds are mixed with at least one conventional inert excipient (or carrier) , such as sodium citrate or dicalcium phosphate, or mixed with any of the following components: (a) fillers or compatibilizer, such as starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and arabic gum; (c) humectant, such as glycerol; (d) disintegrating agent, such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain composite silicates, and sodium carbonate; (e) dissolution-retarding agents, such as paraffin; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetylene glyco
  • the solid dosage forms such as tablets, sugar pills, capsules, pills and granules can be prepared by using coating and shell materials, such as enteric coatings and any other materials known in the art. They can contain an opaque agent.
  • the release of the active compounds or compounds in the compositions can be released in a delayed mode in a given portion of the digestive tract.
  • the embedding components include polymers and waxes. If necessary, the active compounds and one or more above excipients can form microcapsules.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures.
  • the liquid dosage forms may contain any conventional inert diluents known in the art such as water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butanediol, dimethyl formamide, as well as oil, in particular, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, or the combination thereof.
  • composition may also contain additives such as wetting agents, emulsifiers, and suspending agent, sweetener, flavoring agents and perfume.
  • additives such as wetting agents, emulsifiers, and suspending agent, sweetener, flavoring agents and perfume.
  • the dosage forms for topical administration of compounds of the invention include ointments, powders, patches, aerosol, and inhalants.
  • the active ingredients are mixed with physiologically acceptable carriers and any preservatives, buffers, or propellant if necessary, under sterile conditions.
  • Compounds of the present invention can be administrated alone, or in combination with any other pharmaceutically acceptable compounds.
  • the present invention also provides a preparation method of pharmaceutical composition comprising the step of mixing a pharmaceutically acceptable carrier with the compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof of the present invention.
  • the main advantages of the present invention include:
  • the compound of the present invention is a new class of Werner Syndrome helicase inhibitor.
  • the compound of the present disclosure shows a good inhibitory effect on Werner Syndrome helicase, which can be used as Werner Syndrome helicase (WRN) inhibitor to treat cancers.
  • WRN Werner Syndrome helicase
  • the compound of the present disclosure possesses good druggability (including but not limited to: solubility, safety, etc. ) .
  • the disclosed compounds can generally be synthesized by the above general procedure or by an appropriate combination of generally well-known synthetic methods. Techniques useful in synthesizing these compounds are both readily apparent and accessible to those of skill in the relevant art, based on the instant disclosure. Many of the optionally substituted starting compounds and other reactants are commercially available or can be readily prepared by those skilled in the art using commonly employed synthetic method.
  • Step 1 To the solution of tert-butyl 4-oxopiperidine-1-carboxylate (1.25 g, 6.2 mmol) and 2- (difluoromethane) sulfonylpyridine (1 g, 5.2 mmol) in DMF (30 mL) was added the solution of t-BuOK (1.05 g, 9.3 mmol) in DMF (10 mL) dropwise at -40°C. Then the mixture was stirred under nitrogen at -40°C for 2 hrs. The reaction mixture was warmed to room temperature, poured into water (50 mL) , adjusted PH to 3 with 2N HCl, then extracted with EtOAc (50 mL *3) .
  • Step 2 To a solution of tert-butyl 4- (difluoromethylene) piperidine-1-carboxylate (1.1 g, 4.7 mmol) in DCM (10 mL) stirred under nitrogen was added 4M HCl in dioxane (10 mL, 40 mmol) dropwise. The reaction mixture was stirred at 25°C for 2 hrs. The mixture was concentrated under reduced pressure to afford 4- (difluoromethylene) piperidine hydrochloride (0.6 g, 3.5 mmol, yield: 74.47 %) as a white solid. The product was used directly without any further purification.
  • 1 H NMR 400 MHz, DMSO-d 6 ) ⁇ ppm 9.31–9.17 (m, 2H) , 3.06–3.05 (m, 4H) , 2.38–2.37 (m, 4H) .
  • Step 3 The mixture of 4- (difluoromethylidene) piperidine hydrochloride (133 mg, 1 mmol) , [bis (methylsulfanyl) methylidene] (cyano) amine (146 mg, 1 mmol) and TEA (106 mg, 1.04 mmol) in MeCN (5 mL) was stirred at 90°C for 16 hrs. Then it was cooled to 0°C. Hydrazine hydrate (625.12 mg, 10 mmol) was added dropwise and the resulting mixture was stirred at 90°C for 20 hrs.
  • Step 5 The mixture of ethyl 2-bromo-3-oxopentanoate (19 g, 85.2 mmol) , tert-butyl piperazine-1-carboxylate (31.74 g, 170.4 mmol) and K 2 CO 3 (35.33 g, 0.25 mmol) in CH 3 CN (100 mL) was stirred at 50°C for 2 hrs. The mixture was filtered, the cake was washed with CH 3 CN.
  • Step 7 To a solution of 2- [4- (difluoromethylidene) piperidin-1-yl] -5-ethyl-6- (piperazin-1-yl) -4H- [1, 2, 4] triazolo [1, 5-a] pyrimidin-7-one (140 mg, 0.37 mmol) in DCM (10 mL) were added Boc 2 O (96.64 mg, 0.44 mmol) and TEA (112.02 mg, 1.1 mmol) . The mixture was stirred at room temperature for 16 hrs.
  • Step 8 To a solution of 2-chloro-4- (trifluoromethyl) aniline (10 g, 50.9 mmol) in DCM (50 mL) were added DIPEA (19.73 g, 152.7 mmol) and 2-bromoacetyl bromide (30.82 g, 152.7 mmol) at 0°C. The mixture was stirred at room temperature for 16 hrs. The mixture was diluted with water (50 mL) , extracted with DCM (50 mL *3) .
  • Step 10 To the solution of tert-butyl 4- [4- ( ⁇ [2-chloro-4- (trifluoromethyl) phenyl] carbamoyl ⁇ methyl) -2- [4- (difluoromethylidene) piperidin-1-yl] -5-ethyl-7-oxo- [1, 2, 4] triazolo [1, 5-a] pyrimidin-6 -yl] piperazine-1-carboxylate (133 mg, 0.186 mmol) in DCM (5 mL) was added HCl in dioxane (4 mL) . The mixture was stirred at room temperature for 2 hrs.
  • Step 11 The mixture of 4, 6-dichloro-5-methoxypyrimidine (20 g, 111.7 mmol) , methylboranediol (10.03 g, 167.55 mmol) , Pd (dppf) Cl 2 (3.27 g, 4.46 mmol) and K 3 PO 4 (47.42 g. 223.4 mmol) in tuluene (60 mL) , dioxane (8 mL) and H 2 O (20 mL) was stirred at 105°C for 16 hrs under N 2 atmosphere. The mixture was diluted with water (100 mL) , extracted with EtOAc (100 mL*3) .
  • Step 13 A mixture of methyl 5-methoxy-6-methylpyrimidine-4-carboxylate (7.9 g, 43.4 mmol) in HBr (25 mL, 48 wt. %in H 2 O) was stirred at 40°C for 16 hrs. Then HI (25 mL, 55-57 wt. %aqueous) was added to the solution and stirred for 16 hrs. The PH was adjusted to 3-4 with NaOH (50%in water) at 0 -20°C. The mixture was filtered to give the product 5-hydroxy-6-methylpyrimidine-4-carboxylic acid (3.1 g, 20.1 mmol, 46%yield) as yellow solid.
  • 1 H NMR 400 MHz, DMSO-d 6 ) ⁇ 8.50 (s, 1H) , 6.31 (s, 3H) , 2.42 (s, 3H) .
  • Step 14 To a solution of 5-hydroxy-6-methylpyrimidine-4-carboxylic acid (27.06 mg, 0.17 mmol) in DCM (5 mL) was added (1-chloro-2-methylprop-1-en-1-yl) dimethylamine (29.32 mg, 0.22 mmol) . The mixture was stirred at room temperature for 30 min.
  • Step 1 To a solution of ethyl 3-oxopentanoate (30 g, 208.1 mmol) in DCM (200 mL) were added N-Bromosuccinimide (38.89 g, 218.5 mmol) and TsOH. H 2 O (7.91 g, 41.6 mmol) . The mixture was stirred at room temperature for 2.5 hrs. The mixture was diluted with water (200 mL) , extracted with DCM (200 mL *3) .
  • Step 2 The mixture of ethyl 2-bromo-3-oxopentanoate (19 g, 85.2 mmol) , tert-butyl piperazine-1-carboxylate (31.74 g, 170.4 mmol) and K 2 CO 3 (35.33 g, 0.25 mmol) in CH 3 CN (100 mL) was stirred at 50°C for 2 hrs. The mixture was filtered, the cake was washed with CH 3 CN.
  • Step 4 To a solution of tert-butyl 4- ⁇ 2-bromo-5-ethyl-7-oxo-4H- [1, 2, 4] triazolo [1, 5-a] pyrimidin-6-yl ⁇ piperazine-1-carboxylate (310 mg, 0.72 mmol) in DMF (6 mL) were added 2-bromo-N- [2-chloro-4- (trifluoromethyl) phenyl] acetamide (252.58 mg, 0.79 mmol) and DIPEA (281.29 mg, 2.17 mmol) . The mixture was stirred at room temperature for 16 hrs. The mixture was diluted with water (20 mL) , extracted with EtOAc (20 mL *3) .
  • Step 5 The mixture of tert-butyl 4- [2-bromo-4- ( ⁇ [2-chloro-4- (trifluoromethyl) phenyl] carba moyl ⁇ methyl) -5-ethyl-7-oxo- [1, 2, 4] triazolo [1, 5-a] pyrimidin-6-yl] piperazine-1-carboxylate (100 mg, 0.15 mmol) , 1, 4-oxazepane hydrochloride (208 mg, 1.5 mmol) and DIEA (97 mg, 0.75 mmol) in DMSO (5 mL) was stirred at 100°C for 48 hrs.
  • Step 6 To a solution of tert-butyl 4- (4- (2- ( (2-chloro-4- (trifluoromethyl) phenyl) amino) -2-oxoethyl) -5-ethyl-2- (1, 4-oxazepan-4-yl) -7-oxo-4, 7-dihydro- [1, 2, 4] triazolo [1, 5-a] pyrimidin-6-yl) piperazine-1-carboxylate (20 mg, 0.03 mmol) in DCM (3 mL) was added HCl/dioxane (1 mL) .
  • Step 7 To a solution of 5-hydroxy-6-methylpyrimidine-4-carboxylic acid (7 mg, 0.04 mmol) in DCM (2 mL) was added 1-chloro-N, N, 2-trimethyl-1-propen-1-amine (7 mg, 0.05 mmol) , the mixture was stirred at room temperature for 30 mins, then N- [2-chloro-4- (trifluoromethyl) phenyl] -2- [5-ethyl-2- (1, 4-oxazepan-4-yl) -7-oxo-6- (piperazin-1-yl) - [1, 2, 4] triazolo [1, 5-a] pyrimidin-4-yl] acetamide (20 mg, 0.03 mmol) was added and the mixture was stirred at room temperature for 30 mins.
  • Step 1 To a solution of 1, 4-dioxaspiro [4.5] decan-8-one (5000 mg, 32.0 mmol) and DFMPS (5565 mg, 28.8 mmol) in DMF (20 mL) stirred under nitrogen at -60 °C was added t-BuOK (6466 mg, 57.6 mmol) . The reaction mixture was stirred at -60 to -25 °C for 2 hrs. The reaction was quenched with water (20 mL) .
  • Step 2 To a solution of 8- (difluoromethylene) -1, 4-dioxaspiro [4.5] decane (2000 mg, 10.5 mmol) in THF (10 mL) was added 3N HCl (10 mL) . The reaction mixture was stirred at room temperature for 16 hrs. The crude mixture was extracted with DCM (50 mL *3) , and the combined organic layers were washed with brine (20 mL) , dried over Na 2 SO 4 , filtered and concentrated in vacuo to get the crude product.
  • Step 3 To a solution of 4- (difluoromethylene) cyclohexan-1-one (600 mg, 4.1 mmol) in THF (20 mL) stirred at -78 °C was added LDA (2 M, 4.1 mL, 8.2 mmol) . The solution was stirred at -78 °C 30 mins under nitrogen. Then N-phenyl-O- ( (trifluoromethyl) sulfonyl) -N- ( ( (trifluoromethyl) sulfonyl) oxy) hydroxylamine (1591 mg, 4.1 mmol) was added to the solution. The mixture was stirred at room temperature for 16 hrs. The reaction was quenched with NH 4 Cl.
  • Step 4 To a solution of 4- (difluoromethylene) cyclohex-1-en-1-yl trifluoro methanesulfonate (350 mg, 1.25 mmol) , 4, 4, 4', 4', 5, 5, 5', 5'-octamethyl-2, 2'-bi (1, 3, 2-dioxaborolane) (383 mg, 1.5 mmol) and KOAc (247 mg, 2.5 mmol) in dioxane (3 mL) was added Pd (dppf) Cl 2 (92 mg, 0.12 mmol) . The reaction mixture was stirred at 80°C for 5 hrs under nitrogen.
  • Step 5 To a solution of 2- (4- (difluoromethylene) cyclohex-1-en-1-yl) -4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolane (90 mg, 0.35 mmol) , tert-butyl 4- (2-bromo-5-ethyl-7-oxo-4, 7-dihydro- [1, 2, 4] triazolo [1, 5-a] pyrimidin-6-yl) piperazine-1-carboxylate (165 mg, 0.38 mmol) and Na 2 CO 3 in Dioxane (3 mL) /H 2 O (0.5 mL) was added Pd (dppf) Cl 2 (25.17 mg, 0.1 mmol) .
  • Step 6 To a solution of tert-butyl 4- (2- (4- (difluoromethylene) cyclohex-1-en-1-yl) -5-ethyl-7-oxo-4, 7-dihydro- [1, 2, 4] triazolo [1, 5-a] pyrimidin-6-yl) piperazine-1-carboxylate (95 mg, 0.20 mmol) , 2-bromo-N- (2-chloro-4- (trifluoromethyl) phenyl) acetamide (80 mg, 0.25 mmol) in DMF (3 mL) was added DIEA (81 mg, 0.63 mmol) . The mixture was stirred at room temperature for 16 hrs.
  • Step 7 A solution of tert-butyl 4- (4- (2- ( (2-chloro-4- (trifluoromethyl) phenyl) amino) -2-oxoethyl) -2- (4- (difluoromethylene) cyclohex-1-en-1-yl) -5-ethyl-7-oxo-4, 7-dihydro- [1, 2, 4] triazolo [1, 5-a] pyrimidin-6-yl) piperazine-1-carboxylate (100 mg, 0.14 mmol) in DCM (1 mL) was added HCl/Dioxane (4 M, 1 mL) . The mixture was stirred at room temperature for 16 hrs.
  • Step 8 To a solution of 5-hydroxy-6-methylpyrimidine-4-carboxylic acid (20 mg, 0.13 mmol) in DCM (3 mL) was added (1-chloro-2-methylprop-1-en-1-yl) dimethylamine (22 mg, 0.17 mmol) . The mixture was stirred at room temperature for 30 mins.
  • Step 1 To a solution of NaH (1.68 g, 60%, 0.04 mol) in Et 2 O (30 mL) was added pent-4-en-1-ol (3 g, 0.035 mol) dropwise, the reaction mixture was stirred at 30°C for 1 hr. 3-bromoprop-1-yne was added dropwise at room temperature, the reaction mixture was refluxed overnight. The mixture was quenched with ice-water (20 mL) , extracted with EtOAc (20 mL*3) .
  • Step 2 To a solution of 5- (prop-2-yn-1-yloxy) pent-1-ene (2 g, 0.016 mol) in toluene (10 mL) , 4, 4, 5, 5-tetramethyl-2- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1, 3, 2-dioxaborolane (4.91 g, 0.019 mol) , CuCl (0.16 g, 1.61 mmol) , NaOtBu (0.46 g, 4.83 mmol) and (tBu) 3 PHBF 4 (0.56 g, 1.93 mmol) were added at room temperature under argon, then methanol (2 mL) was added, the reaction mixture was stirred at room temperature for 3 hrs.
  • Step 3 A solution of 4, 4, 5, 5-tetramethyl-2- [3- (pent-4-en-1-yloxy) prop-1-en-2-yl] -1, 3, 2-dioxaborolane (1.1 g, 4.4 mmol) in toluene (10 mL) was refluxed for 1 hr under argon, then cool to room temperature. Grubbs 2 nd generation catalyst (190 mg, 0.2 mmol) was added, the reaction mixture was refluxed overnight.
  • Step 5 The mixture of tert-butyl 4- [5-ethyl-7-oxo-2- (2, 5, 6, 7-tetrahydrooxepin-3-yl) -4H- [1, 2, 4] triazolo [1, 5-a] pyrimidin-6-yl] piperazine-1-carboxylate (80 mg, 0.18 mmol) , 2-bromo-N- [2-chloro-4- (trifluoromethyl) phenyl] acetamide (68 mg, 0.21 mmol) and DIEA (70 mg, 0.54 mmol) in DMF (5 mL) was stirred at room temperature for 16 hrs.
  • Step 1 To a solution of Triphenyl phosphite (1631 mg, 5.257 mmol) in DCM (15 mL) stirred under nitrogen at -60 °C was added Br 2 (728 mg, 4.556 mmol) . The reaction mixture was stirred at -60°C for 30 minutes. Then added TEA (709 mg, 7.009 mmol) and stirred at -60°C for 30 minutes. Then added oxepan-4-one (400 mg, 3.504 mmol) . The reaction mixture was stirred at the room temperature for 16 hrs. The reaction mixture was quenched with water (20 mL) .
  • Step 2 To a solution of mixture 5-bromo-2, 3, 4, 7-tetrahydrooxepine and 4-bromo-2, 3, 6, 7-tetrahydrooxepine (150 mg, 0.847 mmol) , 4, 4, 5, 5-tetramethyl-2- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolane-2-yl) -1, 3, 2-dioxaborolane (322.74 mg, 1.271 mmol) and [1, 1'-Bis (diphenylphos phino) ferrocene] dichloropalladium (II) (61.49 mg, 0.085 mmol) in dioxane (10 mL) was added KOAc (166.31 mg, 1.695 mmol) .
  • Step 3 To a solution of mixture 4, 4, 5, 5-tetramethyl-2- (2, 3, 6, 7-tetrahydrooxepin-4-yl) -1, 3, 2-dioxaborolane and 4, 4, 5, 5-tetramethyl-2- (2, 5, 6, 7-tetrahydrooxepin-4-yl) -1, 3, 2-dioxaborolane (160 mg, 0.714 mmol) , tert-butyl 4- ⁇ 2-bromo-5-ethyl-7-oxo-4H- [1, 2, 4] triazolo [1, 5-a] pyrimidin-6-yl ⁇ piperazine-1-carboxylate (152.61 mg, 0.357 mmol) and Na 2 CO 3 (151.41 mg, 1.429 mmol) in dioxane (6 mL) /H 2 O (1 mL) was added Pd (dppf) Cl 2 (0.5 mg, 0.0714 mmol) .
  • Step 4 To a solution of mixture tert-butyl 4- [5-ethyl-7-oxo-2- (2, 3, 6, 7-tetrahydrooxepin-4-yl) -4H- [1, 2, 4] triazolo [1, 5-a] pyrimidin-6-yl] piperazine-1-carboxylate and tert-butyl 4- [5-ethyl-7-oxo-2- (2, 5, 6, 7-tetrahydrooxepin-4-yl) -4H- [1, 2, 4] triazolo [1, 5-a] pyrimidin-6-yl] piperazine-1-carboxylate (120 mg, 0.270 mmol) and 2-bromo-N- [2-chloro-4- (trifluoromethyl) phenyl] acetamide (85.42 mg, 0.270 mmol) in DMF (10 mL) was added DIPEA (104.65 mg, 0.270 mmol) .
  • Step 5 To a solution of mixture tert-butyl 4- [4- ( ⁇ [2-chloro-4- (trifluoromethyl) phenyl] carbamoyl ⁇ methyl) -5-ethyl-7-oxo-2- (2, 3, 6, 7-tetrahydrooxepin-4-yl) - [1, 2, 4] triazolo [1, 5-a] pyrimidin-6-yl] piperazine-1-carboxylate and tert-butyl 4- [4- ( ⁇ [2-chloro-4- (trifluoromethyl) phenyl] carbamoyl ⁇ methyl) -5-ethyl-7-oxo-2- (2, 5, 6, 7-tetrahydrooxepin-4-yl) - [1, 2, 4] triazolo [1, 5-a] pyrimidin-6-yl] piperazine-1-carboxylate (30 mg, 0.044 mmol) in DCM (3 mL) was added HCl in 1,
  • Step 6 To a solution of 5-hydroxy-6-methylpyrimidine-4-carboxylic acid (11.15 mg, 0.072 mmol) in DCM (4 mL) was added (1-chloro-2-methylprop-1-en-1-yl) dimethylamine (12.09 mg, 0.0904 mmol) . The mixture was stirred at the room temperature for 30 minutes.
  • DIPEA (23.38 mg, 0.181 mmol) in DCM (1 mL) was added dropwise and the mixture was stirred at the room temperature for 1 hr. The mixture was diluted with water (10 mL) , extracted with DCM (10 mL *3) .
  • Step 1 To a solution of tert-butyl 4- (2-bromo-4- (2- ( (2-chloro-4- (trifluoromethyl) phenyl) amino) -2-oxoethyl) -5-ethyl-7-oxo-4, 7-dihydro- [1, 2, 4] triazolo [1, 5-a] pyrimidin-6-yl) piperazine-1-carboxylate (480 mg, 0.72 mmol) in DCM (5 mL) was added HCl/dioxane (2 mL) and stirred at room temperature for 1 hr.
  • Step 2 The mixture of 5-hydroxy-6-methylpyrimidine-4-carboxylic acid (248 mg, 1.6 mmol) , HOAt (163 mg, 1.2 mmol) and EDCI (229 mg, 1.2 mmol) in ACN (10 mL) was stirred at room temperature for 1 hr under argon, then 2- (2-bromo-5-ethyl-7-oxo-6- (piperazin-1-yl) - [1, 2, 4] triazolo [1, 5-a] pyrimidin-4 (7H) -yl) -N- (2-chloro-4- (trifluoromethyl) phenyl) acetamide (450 mg, 0.8 mmol) and DIEA (310 mg, 2.4 mmol) in ACN (5 mL) was added, the mixture was stirred at room temperature for 1 hr.
  • Step 1 To a solution of 2-oxaspiro [3.5] nonan-7-one (200 mg, 1.42 mmol) and phenyl [ (trifluoromethane) sulfonyloxy] amino trifluoromethanesulfonate (555 mg, 1.42 mmol) in THF (5 mL) was added LDA (1.5 mL, 3 mmol) at -78°C, the mixture was stirred at room temperature for 16 hrs under argon, then concentrated under vacuum to afford the crude product, which was purified by flash chromatography (EtOAc/hexanes: 0 ⁇ 5%) to afford the compound 2-oxaspiro [3.5] non-6-en-7-yl trifluoromethanesulfonate (200 mg, 0.73 mmol, yield: 51.7%) as a yellow oil.
  • Step 2 The mixture of 2-oxaspiro [3.5] non-6-en-7-yl trifluoromethanesulfonate (200 mg, 0.73 mmol) , 4, 4, 5, 5-tetramethyl-2- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1, 3, 2-dioxaborolane (205 mg, 0.8 mmol) , Pd (dppf) Cl 2 (52 mg, 0.07 mmol) and KOAc (144 mg, 1.46 mmol) in dioxane (5 mL) was stirred at 80°C for 16 hrs under argon, then concentrated under vacuum to afford the crude product, which was purified by flash chromatography (EtOAc/hexanes: 0 ⁇ 5%) to afford the compound 4, 4, 5, 5-tetramethyl-2- (2-oxaspiro [3.5] non-6-en-7-yl) -1, 3, 2-dioxaborolane (100 mg, 0.4 m
  • Step 3 The mixture of 4, 4, 5, 5-tetramethyl-2- (2-oxaspiro [3.5] non-6-en-7-yl) -1, 3, 2-dioxaborolane (60 mg, 0.24 mmol) , tert-butyl 4- ⁇ 2-bromo-5-ethyl-7-oxo-4H- [1, 2, 4] triazolo [1, 5-a] pyrimidin-6-yl ⁇ piperazine-1-carboxylate (68 mg, 0.16 mmol) , X-Phos Pd G2 (12 mg, 0.016 mmol) and K 3 PO 4 (101 mg, 0.48 mmol) in DMF (5 mL) and H 2 O (0.5 mL) was stirred at 110°C for 16 hrs under argon.
  • Step 4 The mixture of tert-butyl 4- (5-ethyl-7-oxo-2- (2-oxaspiro [3.5] non-6-en-7-yl) -4, 7-dihydro- [1, 2, 4] triazolo [1, 5-a] pyrimidin-6-yl) piperazine-1-carboxylate (30 mg, 0.06 mmol) , 2-bromo-N- (2-chloro-4- (trifluoromethyl) phenyl) acetamide (23 mg, 0.07 mmol) and DIPEA (23 mg, 0.18 mmol) in DMF (3 mL) was stirred at room temperature for 6 hrs.
  • Step 5 To a solution of tert-butyl 4- (4- (2- ( (2-chloro-4- (trifluoromethyl) phenyl) amino) -2-oxoethyl) -5-ethyl-7-oxo-2- (2-oxaspiro [3.5] non-6-en-7-yl) -4, 7-dihydro- [1, 2, 4] triazolo [1, 5-a] pyrimidin-6-yl) piperazine-1-carboxylate (20 mg, 0.03 mmol) in DCM (2 mL) was added TFA (1 mL) and stirred at room temperature for 1 hr. The mixture was quenched by NaHCO 3 and extracted with EtOAc (10 mL *3) .
  • Step 6 The mixture of 5-hydroxy-6-methylpyrimidine-4-carboxylic acid (8 mg, 0.048 mmol) , HOAt (5 mg, 0.036 mmol) and EDCI (7 mg, 0.036 mmol) in ACN (5 mL) was stirred at room temperature for 1 hr under argon, then N- (2-chloro-4- (trifluoromethyl) phenyl) -2- (5-ethyl-7-oxo-6- (piperazin-1-yl) -2- (2-oxaspiro [3.5] non-6-en-7-yl) - [1, 2, 4] triazolo [1, 5-a] pyrimidin-4 (7H) -yl) acetamide (15 mg, 0.024 mmol) and DIEA (10 mg, 0.072 mmol) in ACN (3 mL) was added, the mixture was stirred at room temperature for 1 hr.
  • DIEA 10 mg, 0.072 mmol
  • Step 4 To a solution of tert-butyl 4- ⁇ 2-cyclopropyl-5-ethyl-7-oxo-4H- [1, 2, 4] triazolo [1, 5-a] pyrimidin-6-yl ⁇ piperazine-1-carboxylate (50 mg, 0.13 mmol) in DMF (4 mL) were added 2-bromo-N- (2-chloro-4- (trifluoromethyl) phenyl) acetamide (49 mg, 0.15 mmol) and DIPEA (50 mg, 0.39 mmol) . The mixture was diluted with water (20 mL) , extracted with EtOAc (20 mLx3) .
  • Step 5 A mixture of tert-butyl 4- [4- ( ⁇ [2-chloro-4- (trifluoromethyl) phenyl] carbamoyl ⁇ methyl) -2-cyclopropyl-5-ethyl-7-oxo- [1, 2, 4] triazolo [1, 5-a] pyrimidin-6-yl] piperazine-1-carboxylate (50 mg, 0.08 mmol) in HCl/Dioxane (1 mL) and DCM (1 mL) was stirred at 25°C for 1 hr.
  • Step 6 The mixture of 5-hydroxy-6-methylpyrimidine-4-carboxylic acid (18 mg, 0.11 mmol) , HOAt (10 mg, 0.076 mmol) and EDCI (22 mg, 0.11 mmol) was stirred in ACN at 25°C for 1 hr under N 2 , then N- [2-chloro-4- (trifluoromethyl) phenyl] -2- [2-cyclopropyl-5-ethyl-7-oxo-6- (piperazin-1-yl) - [1, 2, 4] triazolo [1, 5-a] pyrimidin-4-yl] acetamide (40 mg, 0.076 mmol) and DIEA (49 mg, 0.38 mmol) was added and further stirred for 15 hrs.
  • the following compounds were synthesized by using the similar methods as above procedure or reference.
  • the start material is commercially available or prepared in house.
  • Step 2 The mixture of 2- (2-bromo-5-ethyl-6- (4- (5-hydroxy-6-methylpyrimidine-4-carbonyl) piperazin-1-yl) -7-oxo- [1, 2, 4] triazolo [1, 5-a] pyrimidin-4 (7H) -yl) -N- (2-chloro-4- (trifluoromethyl) phenyl) acetamide (70 mg, 0.1 mmol) , 5- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolane-2-yl) pyrazolo [1, 5-a] pyridine (37 mg, 0.15 mmol) , Pd (PPh 3 ) 4 (12 mg, 0.01 mmol) and Na 2 CO 3 (32 mg, 0.3 mmol) in dioxane (5 mL) and H 2 O (0.5 mL) was stirred at 90°C for 16 hrs under N 2 atmosphere.
  • Step 1 The mixture of ethyl 2-bromo-3-oxopentanoate (800 mg, 3.60 mmol) , trans tert-butyl 2, 5-diazabicyclo [4.2.0] octane-2-carboxylate (761 mg, 3.58 mmol) and K 2 CO 3 (1.48 g, 10.75 mmol) in CH 3 CN (20 mL) was stirred at 50°C for 2 hrs. The mixture was filtered, the cake was washed with CH 3 CN.
  • Step 4 To the solution of trans tert-butyl 5- (2-bromo-5-ethyl-7-oxo-4, 7-dihydro- [1, 2, 4] triazolo [1, 5-a] pyrimidin-6-yl) -2, 5-diazabicyclo [4.2.0] octane-2-carboxylate (157 mg, 0.34 mmol) in MeCN (10 mL) were added 2-bromo-N- [2-chloro-4- (trifluoromethyl) phenyl] acetamide (164 mg, 0.51 mmol) and DIPEA (137 mg, 1.03 mmol) . The mixture was stirred at 25°C for 16 hrs.
  • Step 6 The mixture of 5-hydroxy-6-methylpyrimidine-4-carboxylic acid (54 mg, 0.35 mmol) , HOAt (60 mg, 0.44 mmol) and EDCI (84 mg, 0.44 mmol) in MeCN (80 mL) was stirred at room temperature for 1 hr under argon, then trans 2- (6- (2, 5-diazabicyclo [4.2.0] octan-2-yl) -2-bromo -5-ethyl-7-oxo- [1, 2, 4] triazolo [1, 5-a] pyrimidin-4 (7H) -yl) -N- (2-chloro-4- (trifluoromethyl) phenyl) acetamide (110 mg, 0.187 mmol) and DIEA (136 mg, 1.05 mmol) in MeCN (20 mL) was added, the mixture was stirred at room temperature for 1 hr.
  • DIEA 136 mg, 1.05 mmol
  • Step 7 The mixture of trans 2- (2-bromo-5-ethyl-6- (5- (5-hydroxy-6-methylpyrimidine-4-carbonyl) -2, 5-diazabicyclo [4.2.0] octan-2-yl) -7-oxo- [1, 2, 4] triazolo [1, 5-a] pyrimidin-4 (7H) -yl) -N- (2-chloro-4- (trifluoromethyl) phenyl) acetamide (90 mg, 0.124 mmol) , 5- (4, 4, 5, 5-tetramethyl-1, 3, 2 -dioxaborolan -2-yl) pyrazolo [1, 5-a] pyridine (45.48 mg, 0.1863 mmol) , Pd (PPh 3 ) 4 (14.35 mg, 0.0124 mmol) and Na 2 CO 3 (39.49 mg, 0.37 mmol) in dioxane (10 mL) and H 2 O (1 mL) was stirred at
  • Step 1 The mixture of trans 2- (2-bromo-5-ethyl-6- (5- (5-hydroxy-6-methylpyrimidine-4-carbonyl) -2, 5-diazabicyclo [4.2.0] octan-2-yl) -7-oxo- [1, 2, 4] triazolo [1, 5-a] pyrimidin-4 (7H) -yl) -N- (2-chloro-4- (trifluoromethyl) phenyl) acetamide (97 mg, 0.1338 mmol) , 2- [4- (difluoromethylidene) cyclohex-1-en-1-yl] -4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolane (51.4 mg, 0.2 mmol) , Pd (dppf) Cl 2 (9.79 mg, 0.0133 mmol) and Na 2 CO 3 (42.54 mg, 0.4 mmol) in dioxane (10 mL) and H 2 O (1 mL)
  • Step 2 The trans N- (2-chloro-4- (trifluoromethyl) phenyl) -2- (2- (4- (difluoromethylene) cyclohex-1-en-1-yl) -5-ethyl-6- (5- (5-hydroxy-6-methylpyrimidine-4-carbonyl) -2, 5-diazabicyclo [4.2.0] octan-2-yl) -7-oxo- [1, 2, 4] triazolo [1, 5-a] pyrimidin-4 (7H) -yl) acetamide (25 mg, 0.032 mmol) was purified by SFC (Apparatus: SFC Thar prep 80, Column: CHIRALPAK AD-H 250mm ⁇ 20 mm, 5 ⁇ m, Modifier: 40%IPA (NH 4 OH 0.2%) : 60%CO 2 , Total Flow: 40g/min) to give N- (2-chloro-4- (trifluoromethyl) phenyl) -2- (2- (4- (d
  • Step 1 To a solution of trans tert-butyl 5- (1-ethoxy-1, 3-dioxopentan-2-yl) -2, 5-diazabicyclo [4.2.0] octane-2-carboxylate (66 mg, 0.186 mmol) in EtOH (5 mL) were added 5- [4- (difluoro methylidene) piperidin-1-yl] -4H-1, 2, 4-triazol-3-amine (40 mg, 0.186 mmol) and H 3 PO 4 (182.48 mg, 1.86 mmol) . The reaction mixture was stirred at 85°C for 48 hrs under N 2 . Then the mixture was added Sat.
  • Step 2 To a solution of trans tert-butyl 5- (2- (4- (difluoromethylene) piperidin-1-yl) -5-ethyl-7-oxo-4, 7-dihydro- [1, 2, 4] triazolo [1, 5-a] pyrimidin-6-yl) -2, 5-diazabicyclo [4.2.0] octane-2-car boxylate (20 mg, 0.04 mmol) in MeCN (10 mL) were added 2-bromo-N- [2-chloro-4- (trifluoromethyl) phenyl] acetamide (18.86 mg, 0.06 mmol) and DIPEA (15.35 mg, 0.11 mmol) .
  • Step 3 To the solution of trans tert-butyl 5- (4- (2- ( (2-chloro-4- (trifluoromethyl) phenyl) amino) -2-oxoethyl) -2- (4- (difluoromethylene) piperidin-1-yl) -5-ethyl-7-oxo-4, 7-dihydro- [1, 2, 4] triazolo [1, 5-a] pyrimidin-6-yl) -2, 5-diazabicyclo [4.2.0] octane-2-carboxylate (21 mg, 0.0283 mmol) in DCM (5 mL) was added HCl in dioxane (2 mL, 4M) .
  • Step 4 The mixture of 5-hydroxy-6-methylpyrimidine-4-carboxylic acid (97.13 mg, 0.63 mmol) , HOAt (107.22 mg, 0.78 mmol) and EDCI (151.01 mg, 0.78 mmol) in MeCN (80 mL) was stirred at room temperature for 1 hr under argon, then the reagent trans 2- (6- (2, 5-diazabicyclo [4.2.0] octan-2-yl) -2- (4- (difluoromethylene) piperidin-1-yl) -5-ethyl-7-oxo- [1, 2, 4] triazolo [1, 5-a] pyr imidin-4 (7H) -yl) -N- (2-chloro-4- (trifluoromethyl) phenyl) acetamide (202 mg, 0.31 mmol) and DIEA (407.24 mg, 3.15 mmol) in MeCN (10 mL) was added, the mixture was stirred at room temperature for
  • Step 2 To a mixture of 5-bromo-1- (methyl-d3) -1H-indazole (1.0 g, 4.67 mmol) in 1, 4-Dioxane (30.0 mL) was added potassium acetate (1.37 g, 14.01 mmol) , 4, 4, 4', 4', 5, 5, 5', 5'-octamethyl-2, 2'-bi (1, 3, 2-dioxaborolane) (1.30 g, 5.14 mmol) and Pd (dppf) Cl 2 (190 mg, 0.23 mmol) in portions at room temperature under N 2 . The resulting mixture was stirred for 16 h at 100 °C for 16 hours under N 2 .
  • Step 3 The mixture of 2- (2-bromo-5-ethyl-6- (4- (5-hydroxy-6-methylpyrimidine-4-carbonyl) piperazin-1-yl) -7-oxo-4H, 7H- [1, 2, 4] triazolo [1, 5-a] pyrimidin-4-yl) -N- (2-chloro-4- (trifluoromethyl) phenyl) acetamide (100 mg, 0.14 mmol) , 1- (methyl-d3) -5- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1H-indazole (73 mg, 0.28 mmol) , Pd (PPh 3 ) 4 (16 mg, 0.014 mmol) and sodium carbonate (45 mg, 0.42 mmol) in dioxane (12 mL) and H 2 O (1 mL) was stirred at 90 °C for 16 hours under N 2 .
  • Step 1 To a mixture of 5-bromo-2- (methyl-d3) -2H-indazole (1.0 g, 4.67 mmol) in 1, 4-Dioxane (30.0 mL) was added potassium acetate (1.37 g, 14.01 mmol) , 4, 4, 4', 4', 5, 5, 5', 5'-octamethyl-2, 2'-bi (1, 3, 2-dioxaborolane) (1.30 g, 5.14 mmol) and Pd (dppf) Cl 2 (190 mg, 0.23 mmol) at room temperature under N 2 . The resulting mixture was stirred at 100 °C for 16 hours under N 2 .
  • Step 2 The mixture of 2- (2-bromo-5-ethyl-6- (4- (5-hydroxy-6-methylpyrimidine-4-carbonyl) piperazin-1-yl) -7-oxo- [1, 2, 4] triazolo [1, 5-a] pyrimidin-4 (7H) -yl) -N- (2-chloro-4- (trifluoromethyl) phenyl) acetamide (100 mg, 0.14 mmol) , 2- (methyl-d3) -5- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolane -2-yl) -2H-indazole (73 mg, 0.28 mmol) , Pd (PPh 3 ) 4 (16 mg, 0.014 mmol) and sodium carbonate (45 mg, 0.42 mmol) in dioxane (12 mL) and H 2 O (1 mL) was stirred at 90 °C for 16 hours under N 2 .
  • Step 1 To a solution of methyl 2-methylthiophene-3-carboxylate (5 g, 32.01 mmol) in DMF (24 mL) was added NBS (5.70 g, 32.01 mmol) at room temperature and stirred for 16 hours. LCMS indicated completion of reaction. The reaction mixture was diluted with water (100 mL) and extracted with PE (50 mL *3) . The organic layer was washed with saturated aqueous NaHCO 3 and brine and dried over Na 2 SO 4 .
  • Step 3 To a solution of 5-bromo-2-methylthiophene-3-carboxylic acid (6.3 g, 28.5 mmol) , HATU (16.2 g, 42.8 mmol) and DIEA (7.37 g, 57 mmol) in DMF (60 mL) was added NH 4 Cl (2.29 g, 42.8 mmol) . The mixture was stirred at room temperature for 2 hours. LCMS indicated completion of reaction. The mixture was diluted with water (10 mL) and extracted with EtOAc (50 mL *3) . The combined organic layer was washed with brine (50 mL *2) , dried over Na 2 SO 4 and concentrated in vacuum.
  • Step 4 To a mixture of 5-bromo-2-methylthiophene-3-carboxamide (2 g, 9.09 mmol) in THF (30 mL) was added pyridine (2.75 g, 34.81 mmol) and trifluoroacetic anhydride (3.40 g, 16.18 mmol) at 0°C. The mixture was stirred at room temperature for 24 hours. The mixture was concentrated and diluted with water (100 mL) and extracted with DCM (300 mL *3) . The combined organic layer was washed with brine (50 mL *3) , dried over Na 2 SO 4 and concentrated in vacuum.
  • Step 5 A mixture of 5-bromo-2-methylthiophene-3-carbonitrile (500 mg, 2.47 mmol) , 4, 4, 4', 4', 5, 5, 5', 5'-octamethyl-2, 2'-bi (1, 3, 2-dioxaborolane) (820 mg, 3.21 mmol) , potassium acetate (800 mg, 8.2 mmol) and Pd (dppf) Cl 2 (130 mg, 0.17 mmol) in anhydrous dioxane (10 mL) was stirred at 100 °C for 16 hours under N 2 .
  • Step 6 The mixture of 2- (2-bromo-5-ethyl-6- (4- (5-hydroxy-6-methylpyrimidine-4-carbonyl) piperazin-1-yl) -7-oxo- [1, 2, 4] triazolo [1, 5-a] pyrimidin-4 (7H) -yl) -N- (2-chloro-4- (trifluoromethyl) phenyl) acetamide (100 mg, 0.14 mmol) , 2-methyl-5- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) thiophene-3-carbonitrile (25 mg, 0.21 mmol) , Pd (dppf) Cl 2 (14 mg, 0.014 mmol) and sodium carbonate (44.25 mg, 0.42 mmol) in dioxane (10 mL) and H 2 O (1 mL) was stirred at 90°C for 16 hours under N 2 .
  • Step 2 The mixture of 4-chloro-3-hydroxypicolinic acid (88 mg, 0.51 mmol) , HOAt (69 mg, 0.51 mmol) and EDCI (98 mg, 0.51 mmol) in ACN (40 mL) was stirred at room temperature for 1 hour under N 2 , then N- (2-chloro-4- (trifluoromethyl) phenyl) -2- (5-ethyl-7-oxo-6- (piperazin-1-yl) -2- (pyrazolo [1, 5-a] pyridin-5-yl) - [1, 2, 4] triazolo [1, 5-a] pyrimidin-4 (7H) -yl) acetamide (100 mg, 0.17 mmol) and DIEA (0.17 mL, 1.02 mmol) in ACN (10 mL) was added, the mixture was stirred at room temperature for 12 hours.
  • Step 1 The 4, 4, 5, 5-tetramethyl-2- (4- (methylsulfinyl) phenyl) -1, 3, 2-dioxaborolane (300 mg, 1.13 mmol) was separated by chiral SFC (colum: 15 10%IPA DEA C1 WHELK) to obtain (S) -4, 4, 5, 5-tetramethyl-2- (4- (methylsulfinyl) phenyl) -1, 3, 2-dioxaborolane and (R) -4, 4, 5, 5-tetramethyl -2- (4- (methylsulfinyl) phenyl) -1, 3, 2-dioxaborolane as a white solid.
  • Step 2 A mixture of 2- (2-bromo-5-ethyl-6- (4- (5-hydroxy-6-methylpyrimidine-4-carbonyl) piperazin-1-yl) -7-oxo- [1, 2, 4] triazolo [1, 5-a] pyrimidin-4 (7H) -yl) -N- (2-chloro-4- (trifluoromethyl) phenyl) acetamide (60 mg, 0.086 mmol) , (S) -4, 4, 5, 5-tetramethyl-2- (4- (methylsulfinyl) phenyl) -1, 3, 2-dioxaborolane or (R) -4, 4, 5, 5-tetramethyl-2- (4- (methylsulfinyl) phenyl) -1, 3, 2-dioxaborolane (23 mg, 0.086 mmol) , Pd (dppf) Cl 2 (6 mg, 0.009 mmol) and K 2 CO 3 (36 mg, 0.260 mmol) in di
  • Example 279a (12.77 mg, 0.017 mmol, 19.6%yield) was synthesized from Compound of Peak 1 using the above procedure.
  • Example 279b (32.39 mg, 0.043 mmol, 49.8%yield) was synthesized from Compound of Peak 2 using the above procedure.
  • Step 1 The mixture of 3-hydroxypicolinic acid (71 mg, 0.51 mmol) , HOAt (69 mg, 0.51 mmol) and EDCI (98 mg, 0.51 mmol) in ACN (40 mL) was stirred at room temperature for 1 hour under N 2 , then N- (2-chloro-4- (trifluoromethyl) phenyl) -2- (5-ethyl-7-oxo-6- (piperazin-1-yl) -2- (pyrazolo [1, 5-a] pyridin-5-yl) - [1, 2, 4] triazolo [1, 5-a] pyrimidin-4 (7H) -yl) acetamide (100 mg, 0.17 mmol) and DIEA (0.17 mL, 1.02 mmol) in ACN (10 mL) was added, the mixture was stirred at room temperature for 12 hours.
  • Step 1 To a mixture of 4-chloro-3-hydroxypicolinic acid (41.6 mg, 0.24 mmol) in DMF (3 mL) was added DIEA (62.04 mg, 0.48 mmol) and HATU (91.3 mg, 0.24 mmol) at room temperature.
  • Step 1 To a solution of tert-butyl 4- (2-bromo-4- (2- ( (2-chloro-4- (trifluoromethyl) phenyl) amino) -2-oxoethyl) -5-ethyl-7-oxo-4, 7-dihydro- [1, 2, 4] triazolo [1, 5-a] pyrimidin-6-yl) piperazine-1-c arboxylate (300 mg, 0.45 mmol) in DCM (3 mL) was added HCl/dioxane (3 mL, 4 M) and the mixture was stirred at room temperature for 2 hours.
  • Step 2 To a solution of 2- (2-bromo-5-ethyl-7-oxo-6- (piperazin-1-yl) - [1, 2, 4] triazolo [1, 5-a] pyrimidin-4 (7H) -yl) -N- (2-chloro-4- (trifluoromethyl) phenyl) acetamide (240 mg, 0.43 mmol) , 4-chloro-3-hydroxypicolinic acid (90 mg, 0.52 mmol) , HATU (245 mg, 0.65 mmol) in DMF (5 mL) was added DIEA (111 mg, 0.86 mmol) and the mixture was stirred at room temperature for 16 hours.
  • Step 3 A mixture of 2- (2-bromo-6- (4- (4-chloro-3-hydroxypicolinoyl) piperazin-1-yl) -5-ethyl-7-oxo- [1, 2, 4] triazolo [1, 5-a] pyrimidin-4 (7H) -yl) -N- (2-chloro-4- (trifluoromethyl) phenyl) acet amide (90 mg, 0.130 mmol) , (R) -4, 4, 5, 5-tetramethyl-2- (4- (methylsulfinyl) phenyl) -1, 3, 2-dioxaborolane or (S) -4, 4, 5, 5-tetramethyl-2- (4- (methylsulfinyl) phenyl) -1, 3, 2-dioxaborolane (35 mg, 0.130 mmol) , Pd (dppf) Cl 2 (10 mg, 0.013 mmol) and K 2 CO 3 (54 mg, 0.390 mmol) in dio
  • Example 282a (36.10 mg, 0.047 mmol, 37.1%yield) was synthesized from Compound of Peak 1 (see the reference of example 279a) using the above procedure.
  • Example 282b (22.08 mg, 0.028 mmol, 22.7%yield) was synthesized from Compound of Peak 2 (see the reference of example 279b) using the above procedure.
  • Step 1 The mixture of 2- (2-bromo-5-ethyl-6- (4- (5-hydroxy-6-methylpyrimidine-4-carbonyl) piperazin-1-yl) -7-oxo- [1, 2, 4] triazolo [1, 5-a] pyrimidin-4 (7H) -yl) -N- (2-fluoro-4- (trifluoromethyl) phenyl) acetamide (100 mg, 0.15 mmol) , 2-methyl-5- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -2H-indazole (58.08 mg, 0.22 mmol) , Pd (dppf) Cl 2 (10.98 mg, 0.015mmol) and sodium carbonate (47.7 mg, 0.45 mmol) in dioxane (10 mL) and H 2 O (1 mL) was stirred at 90 °C for 16 hours under N 2 .
  • Step 1 To a mixture of 4-chloro-3-hydroxypicolinic acid (133.29 mg, 0.77 mmol) in N, N-dimethylformamide (5 mL) was added DIEA (248.14 mg, 1.92 mmol) and HATU (365.02 mg, 0.96 mmol) .
  • Step 2 To a mixture of 2- (2-bromo-6- (4- (4-chloro-3-hydroxypicolinoyl) piperazin-1-yl) -5-ethyl-7-oxo- [1, 2, 4] triazolo [1, 5-a] pyrimidin-4 (7H) -yl) -N- (2-fluoro-4- (trifluoromethyl) phenyl) acet amide (90 mg, 0.14 mmol) in 1, 4-dioxane (10 mL) and water (1 mL) was added 2- (methyl-d 3 ) -5- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -2H-indazole (73.12 mg, 0.28 mmol) , sodium carbonate (44.52 mg, 0.42 mmol) and Pd (dppf) Cl 2 (5.72 mg, 0.007 mmol) .
  • the mixture was degassed with N 2 and stirred at 85 °C for 12 hours.
  • the mixture was diluted with water (20 mL) and extracted with EtOAc (15 mL *3) .
  • the combined organic layer was washed with brine (15 mL *3) , dried over Na 2 SO 4 and concentrated in vacuum.
  • Step 1 To a mixture of 2- (2-bromo-6- (4- (4-chloro-3-hydroxypicolinoyl) piperazin-1-yl) -5-ethyl-7-oxo- [1, 2, 4] triazolo [1, 5-a] pyrimidin-4 (7H) -yl) -N- (2-fluoro-4- (trifluoromethyl) phenyl) acet amide (90 mg, 0.13 mmol) in 1, 4-dioxane (10 mL) and water (1 mL) was added 2-methyl-5- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -2H-indazole (67.11 mg, 0.26 mmol) , sodium carbonate (41.34 mg, 0.39 mmol) and Pd (dppf) Cl 2 (5.31 mg, 0.0065 mmol) .
  • the mixture was degassed with N 2 and stirred at 85 °C for 12 hours.
  • the mixture was diluted with water (20 mL) and extracted with EtOAc (15 mL *3) .
  • the combined organic layer was washed with brine (15 mL *3) , dried over Na 2 SO 4 and concentrated in vacuum.
  • Step 1 To a mixture of cis tert-butyl 2, 5-diazabicyclo [4.2.0] octane-2-carboxylate (1.8 g, 8.48 mmol) in acetonitrile (45 mL) was added ethyl 2-bromo-3-oxopentanoate (1.9 g, 8.48 mmol) and potassium carbonate (3.5 g, 25.44 mmol) . The mixture was stirred at 50 °C for 2 hours. The mixture was filtered, the filter cake was washed with CH 3 CN.
  • Step 4 To a solution of cis tert-butyl 5- (2-bromo-5-ethyl-7-oxo-4, 7-dihydro- [1, 2, 4] triazolo [1, 5-a] pyrimidin-6-yl) -2, 5-diazabicyclo [4.2.0] octane-2-carboxylate (870 mg, 1.92 mmol) in acetonitrile (55 mL) were added 2-bromo-N- (2-chloro-4- (trifluoromethyl) phenyl) acetamide (668.45 mg, 2.11 mmol) and DIEA (545.91 mg, 4.22 mmol) . The mixture was stirred at 25°C for 16 hours.
  • Step 5 To the mixture of cis tert-butyl 5- (2-bromo-4- (2- ( (2-chloro-4- (trifluoromethyl) phenyl) amino) -2-oxoethyl) -5-ethyl-7-oxo-4, 7-dihydro- [1, 2, 4] triazolo [1, 5-a] pyrimidin-6-yl) -2, 5-diazabicyclo [4.2.0] octane-2-carboxylate (650 mg, 0.94 mmol) in dichloromethane (20 mL) was added 4 M HCl/1, 4-dioxane (10 mL) . The mixture was stirred at room temperature for 2 hours.
  • Step 6 To a mixture of 5-hydroxy-6-methylpyrimidine-4-carboxylic acid (148 mg, 0.96 mmol) in acetonitrile (220 mL) was added HOAt (163.33 mg, 1.2 mmol) and EDCI (230.04 mg, 1.2 mmol) .
  • the mixture was concentrated in vacuum and diluted with water (50 mL) .
  • the mixture was extracted with DCM (50 mL *3) .
  • the combined organic layer was washed with brine (30 mL *3) , dried over Na 2 SO 4 and concentrated in vacuum.
  • Step 7 To the mixture of cis 2- (2-bromo-5-ethyl-6- (5- (5-hydroxy-6-methylpyrimidine-4-carbonyl) -2, 5-diazabicyclo [4.2.0] octan-2-yl) -7-oxo- [1, 2, 4] triazolo [1, 5-a] pyrimidin-4 (7H) -yl) -N- (2-chloro-4- (trifluoromethyl) phenyl) acetamide (130 mg, 0.18 mmol) in 1, 4-dioxane (10 mL) and water (1 mL) was added 5- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyrazolo [1, 5-a] pyridine (70.30 mg, 0.29 mmol) , Pd (PPh 3 ) 4 (20.80 mg, 0.018 mmol) and sodium carbonate (57.23 mg, 0.54 mmol) .
  • Step 8 the example 286 was further purified by SFC to afford the example 286a and example 286b as N- (2-chloro-4- (trifluoromethyl) phenyl) -2- (5-ethyl-6- ( (1S, 6R) -5- (5-hydroxy-6-methylpyrimidine-4-carbonyl) -2, 5-diazabicyclo [4.2.0] octan-2-yl) -7-oxo-2- (pyrazolo [1, 5-a] pyridin -5-yl) - [1, 2, 4] triazolo [1, 5-a] pyrimidin-4 (7H) -yl) acetamide or N- (2-chloro-4- (trifluoromethyl) phenyl) -2- (5-ethyl-6- ( (1R, 6S) -5- (5-hydroxy-6-methylpyrimidine-4-carbonyl) -2, 5-diazabicyclo [4.2.0] octan-2-yl) -7-o
  • Step 1 To a solution of benzo [b] thiophen-3 (2H) -one (2.0 g, 13.3 mmol) in EtOH (10 mL) was added Hydrazinium hydroxide (10.3 g, 205.8 mmol) . The solution was stirred at 80 °C for 1 hour. The solution was concentrated and the residue was added to Diethylene glycol (11.2 g, 105.49 mmol) , then potassium hydroxide (2.24 g, 39.96mmol) was added. The new solution was stirred at 120 °C for 16 hours. GCMS indicated completion of reaction. EtOAc (30 mL) was added washed with brine (15 mL *2) .
  • Step 2 To a mixture of 2, 3-dihydrobenzo [b] thiophene (830 mg, 6.09 mmol) in dichloromethane (20 mL) was added bromine (973.24 mg, 6.09 mmol) at 0 °C. The solution was stirred at room temperatures for 16 hours. Then NaHSO 3 (20 mL) was added, extracted with DCM (20 mL *2) , washed with brine. Concentrated and the residue was purified by gel chromatography (PE) to give 5-bromo-2, 3-dihydrobenzo [b] thiophene (900 mg, 4.18 mmol, 68.7%yield) as a yellow solid.
  • PE gel chromatography
  • Step 3 To a solution of 5-bromo-2, 3-dihydrobenzo [b] thiophene (866 mg, 4.03 mmol) in dichloromethane (20 mL) was added 3-chloroperbenzoic acid (981.82 mg, 4.84 mmol, Purity 85%) at 0 °C. The impon was stirred at room temperatures for 16 hours. LCMS indicated completion of reaction. NaOH (10 mL, 2N) was added. Extracted with DCM (20 mL *3) . The organic phase was washed with brine (10 mL) , dried over Na 2 SO 4 , filtered and the filtrate was concentrated under reduced pressure.
  • Step 4 To a mixture of 5-bromo-2, 3-dihydrobenzo [b] thiophene 1-oxide (120 mg, 0.52 mmol) in 1, 4-Dioxane (20.0 mL) was added potassium acetate (153.1 g, 1.56 mmol) , B 2 pin 2 (150 mg, 0.57mmol) and Pd (dppf) Cl 2 (43 mg, 0.052 mmol) at room temperature under N 2 . The resulting mixture was stirred at 90 °C for 16 hours under N 2 .
  • Step 5 A mixture of 2- (2-bromo-6- (4- (4-chloro-3-hydroxypicolinoyl) piperazin-1-yl) -5-ethyl-7-oxo- [1, 2, 4] triazolo [1, 5-a] pyrimidin-4 (7H) -yl) -N- (2-chloro-4- (trifluoromethyl) phenyl) acetamide (100 mg, 0.14 mmol) , 5- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -2, 3-dihydrobenzo [b] thiophene 1-oxide (47 mg, 0.17 mmol) , Pd (dppf) Cl 2 (11 mg, 0.014 mmol) and Na 2 CO 3 (45 mg, 0.42 mmol) in dioxane (10 mL) and H 2 O (1 mL) was stirred at 85°C for 16 hours under N 2 .
  • Step 6 the compound example 287 was further purified by SFC to afford the example 287a and example 287b as (S) -2- (6- (4- (4-chloro-3-hydroxypicolinoyl) piperazin-1-yl) -5-ethyl-2- (1-oxido-2, 3-dihydrobenzo [b] thiophen-5-yl) -7-oxo- [1, 2, 4] triazolo [1, 5-a] pyrimidin-4 (7H) -yl) -N- (2-c hloro-4- (trifluoromethyl) phenyl) acetamide or (R) -2- (6- (4- (4-chloro-3-hydroxypicolinoyl) piperazi n-1-yl) -5-ethyl-2- (1-oxido-2, 3-dihydrobenzo [b] thiophen-5-yl) -7-oxo- [1, 2, 4] triazolo [1, 5-a] pyrimid in-4 (7H
  • the following compounds were synthesized by using the similar methods as above procedure or reference.
  • the start material is commercial or prepared in house.
  • Step 1 To a solution of 6-bromoimidazo [1, 2-a] pyridine (300 mg, 1.523 mmol) , 4, 4, 5, 5-tetramethyl-2- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1, 3, 2-dioxaborolane (502 mg, 1.979 mmol) and KOAc (448 mg, 4.568 mmol) in dioxane (20 mL) stirred under nitrogen was added Pd (dppf) Cl 2 (111 mg, 0.152 mmol) . The reaction mixture was stirred at 90 °C for 16 hrs. The mixture was concentrated under vacuum.
  • Step 1 To a solution of PPh 3 (6.71 g, 25.6 mmol) in MeCN (10 mL) stirred under nitrogen was added dropwise the solution of 1, 4-dioxaspiro [4.5] decan-8-one hydrate (1 g, 6.4 mmol) and CCl 4 (1.97 g, 12.8 mmol) in MeCN (10 mL) . Following the addition, the reaction mixture was stirred at 25 °C for 16 hrs.
  • Step 3 To a solution of 4- (dichloromethylene) cyclohexan-1-one (700 mg, 3.910 mmol) in THF (20 mL) was added LDA (3.9 mL, 7.819 mmol) at -78°C under N 2 , the mixture was stirred at this temperature for 0.5 hr, then 1, 1, 1-trifluoro-N-phenyl-N- ( (trifluoromethyl) sulfonyl) methanesulfonamide (1396 mg, 3.910 mmol) was added. The mixture was stirred at 25°C for 16 hrs. The reaction was quenched by Sat.
  • Step 4 Amixture of 4- (dichloromethylene) cyclohex-1-en-1-yl trifluoromethanesulfonate (500 mg, 1.607 mmol) , 4, 4, 5, 5-tetramethyl-2- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1, 3, 2-dioxaborolane (449 mg, 1.768 mmol) , Pd (dppf) Cl 2 (117 mg, 0.161 mmol) and KOAc (473 mg, 4.822 mmol) in dioxane (20 mL) was stirred at 80 °C for 10 hrs.
  • Step 1 To a solution of 6-bromopyrazolo [1, 5-a] pyridine (150 mg, 0.76 mmol) , 4, 4, 5, 5-tetramethyl-2- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1, 3, 2-dioxaborolane (251 mg, 0.989 mmol) and KOAc (149 mg, 1.52 mmol) in dioxane (3 mL) was added Pd (dppf) Cl 2 (55 mg, 0.076 mmol) . The reaction mixture was stirred under nitrogen at 80°C for 16 hrs. The mixture was concentrated under vacuum.
  • Step 1 A mixture of methanesulfonamide (7.2 g, 75.6 mmol) and SOCl 2 (13.09 g, 110 mmol) in toluene (10 mL) was stirred at 120 °C for 18 hrs under nitrogen. After cooling to the room temperature, toluene was removed under reduced pressure and the residue was used directly in the next step. To a solution of 4-bromo-2-methylaniline (2.0 g, 10.7 mmol) in toluene (40 mL) was added SOCl 2 (1.41 g, 11.8 mmol) dropwise at 0°C. After the addition was complete, the reaction mixture was heated at 120°C for 18 hrs.
  • Step 2 To a solution of 5-bromobenzo [c] isothiazole (800 mg, 3.737 mmol) , 4, 4, 5, 5-tetramethyl-2- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1, 3, 2-dioxaborolane (1043 mg, 4.111 mmol) and KOAc (733 mg, 7.474 mmol) in dioxane (15 mL) was added Pd (dppf) Cl 2 (273 mg, 0.374 mmol) . The reaction mixture was stirred at 80 °C for 10 hrs under nitrogen. The mixture was concentrated under vacuum.
  • Step 2 A mixture of 1-oxaspiro [3.5] non-6-en-7-yl trifluoromethanesulfonate (170 mg, 0.70 mmol) , 4, 4, 5, 5-tetramethyl-2- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1, 3, 2-dioxaborolane (213 mg, 0.84 mmol) , Pd (dppf) Cl 2 (51 mg, 0.07 mmol) and Potassium acetate (137 mg, 1.40 mmol) in dioxane (8 mL) was stirred at 80 °C overnight.
  • Step 1 To a solution of 2-oxaspiro [3.4] octan-6-one (200 mg, 1.6 mmol ) in THF (5 mL) was added LDA (1.19 mL, 2.4 mmol) at -78°C under N 2 , the mixture was stirred at this temperature for 0.5 hr, then 1, 1, 1-trifluoro-N-phenyl-N- ( (trifluoromethyl) sulfonyl) methanesulfonamide (740 mg, 1.9 mmol) was added. The mixture was stirred at 25°C for 16 hrs. The reaction was quenched by Sat.
  • Step 1 A solution of Deoxo-Fluor (16 g, 72.4 mmol) in DCM (100 mL) was added to a cold (0 °C) solution of hexahydropentalene-2, 5-dione (5 g, 36.2 mmol) in DCM (250 mL) followed by addition of a catalytic amount of EtOH (0.50 g, 10.8 mmol) . The reaction mixture was stirred at the room temperature for 16 hrs. The reaction was quenched by Sat.
  • Step 2 To a solution of 5, 5-difluoro-hexahydropentalen-2-one (400 mg, 2.498 mmol) in THF (20 mL) was added LDA (2.5 mL, 4.995 mmol) at -78°C under N 2 , the mixture was stirred at this temperature for 0.5 hr, then 1, 1, 1-trifluoro-N-phenyl-N- ( (trifluoromethyl) sulfonyl) methane sulfonamide (892.23 mg, 2.4975 mmol) was added. The mixture was stirred at 25°C for 16 hrs. The reaction was quenched by Sat.
  • Step 3 A mixture of 5, 5-difluoro-1, 3a, 4, 5, 6, 6a-hexahydropentalen-2-yl trifluoromethane sulfonate (300 mg, 1.027 mmol) , 4, 4, 5, 5-tetramethyl-2- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2 -yl) -1, 3, 2-dioxaborolane (286.76 mg, 1.129 mmol) , Pd (dppf) Cl 2 (75.12 mg, 0.103 mmol) and KOAc (302.26 mg, 3.080 mmol) in dioxane (20 mL) was stirred at 80 °C for 10 hrs.
  • Step 1 To a solution of 1-oxaspiro [4.5] decan-8-one (200 mg, 1.3 mmol ) in THF (5 mL) was added LDA (1.5 mL, 2.6 mmol) at -78°C under N 2 , the mixture was stirred at this temperature for 0.5 hr, then 1, 1, 1-trifluoro-N-phenyl-N- ( (trifluoromethyl) sulfonyl) methanesulfonamide (505 mg, 1.3 mmol) was added. The mixture was stirred at 25°C for 16 hrs. The reaction was quenched by Sat.
  • Step 2 A mixture of 1-oxaspiro [4.5] dec-7-en-8-yl trifluoromethanesulfonate (150 mg, 0.52 mmol) , 4, 4, 5, 5-tetramethyl-2- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1, 3, 2-dioxaborolane (146 mg, 0.57 mmol) , Pd (dppf) Cl 2 (38 mg, 0.05 mmol) and KOAc (103 mg, 1.04 mmol) in dioxane (5 mL) was stirred at 80 °C for 3 hrs.
  • Step 1 To a solution of 5-oxaspiro [3.4] octan-7-one (300 mg, 2.38 mmol ) in THF (5 mL) was added LDA (2.5 mL, 4.75 mmol) at -78°C under N 2 , the mixture was stirred at this temperature for 0.5 hr, then 1, 1, 1-trifluoro-N-phenyl-N- ( (trifluoromethyl) sulfonyl) methane sulfonamide (926 mg, 2.38 mmol) was added. The mixture was stirred at 25°C for 16 hrs. The reaction was quenched by Sat.
  • Step 2 A mixture of 5-oxaspiro [3.4] oct-7-en-7-yl trifluoromethanesulfonate (100 mg, 0.38 mmol) , 4, 4, 5, 5-tetramethyl-2- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1, 3, 2-dioxaborolane (108 mg, 0.42 mmol) , Pd (dppf) Cl 2 (28 mg, 0.038 mmol) and KOAc (76 mg, 0.77 mmol) in dioxane (5 mL) was stirred at 80 °C for 3 hrs.
  • Step 1 To a solution of Triphenyl phosphite (737.88 mg, 2.378 mmol) in DCM (15 mL) stirred under nitrogen at -60 °C was added Br 2 (329.37 mg, 2.061 mmol) . The reaction mixture was stirred at -60 °C for 30 minutes. Then added TEA (320.85 mg, 3.171 mmol) and stirred at -60 °C for 30 minutes. Then added tetrahydro-1H-cyclopenta [c] furan-5 (3H) -one (200 mg, 1.585 mmol) . The reaction mixture was stirred at the room temperature for 16 hrs. The reaction mixture was quenched with water (20 mL) .
  • Step 2 To a solution of 5-bromo-3, 3a, 4, 6a-tetrahydro-1H-cyclopenta [c] furan (143 mg, 0.756 mmol) , 4, 4, 5, 5-tetramethyl-2- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1, 3, 2-dioxaborolane (211.29 mg, 0.832 mmol) and KOAc (148.47 mg, 1.513 mmol) in dioxane (15 mL) was added Pd (dppf) Cl 2 (54.89 mg, 0.0756 mmol) . The reaction mixture was stirred under nitrogen at 80 °Cfor 3 hrs. The mixture was concentrated under vacuum.
  • Step 2 ethyl 5, 5-difluoro-2-oxocycloheptane-1-carboxylate (5.6 g, 25.4 mmol) in 3 M aqueous HCl (13.5 mL) was stirred at 100 °C for 16 hrs.
  • Step 3 To a solution of Triphenyl phosphite (1256.63 mg, 4.05 mmol) in DCM (15 mL) stirred under nitrogen at -60°C was added Br 2 (560.93 mg, 3.51 mmol) . The reaction mixture was stirred at -60°C for 30 minutes. Then added TEA (546.43 mg, 5.4 mmol) and stirred at -60°C for 30 minutes. Then added 4, 4-difluorocycloheptan-1-one (400 mg, 2.7 mmol) . The reaction mixture was stirred at the room temperature for 16 hrs. The reaction mixture was quenched with water (20 mL) .
  • Step 4 To a solution of the mixture of 1-bromo-4, 4-difluorocyclohept-1-ene and 1-bromo-5, 5-difluorocyclohept-1-ene (320 mg, 1.516 mmol) , 4, 4, 5, 5-tetramethyl-2- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1, 3, 2-dioxaborolane (423.52 mg, 1.668 mmol) and KOAc (297.61 mg, 3.032 mmol) in dioxane (10 mL) was added Pd (dppf) Cl 2 (110.02 mg, 0.152 mmol) .
  • Step 1 To a solution of pyrrolidine-2-carboxylic acid (250 mg, 2.17 mmol) , NaNO 2 (210 mg, 3.04 mmol) in H 2 O (1 mL) was added con. HCl (0.5 mL) at 0°C. The mixture was stirred at 25°Cfor 6 hrs. The mixture was diluted with H 2 O (10 mL) , extracted with DCM (15 mL *8) . The combined organic was dried over Na 2 SO 4 , filtered, then concentrated to get the product nitrosoproline (140 mg, 0.87 mmol, 40.3%yield) as a white solid.
  • Step 2 To a solution of 1-nitrosopyrrolidine-2-carboxylic acid (1.6 g, 11.1 mmol) in MeCN (20 mL) was added TFAA (4.66g, 22.2 mmol) at 0°C. The mixture was stirred at 25°C for 2 hrs. Then K 2 CO 3 (3.07 g, 22.2 mmol) was added, the mixture was further stirred at 25°C for 1 hr.
  • Step 3 A mixture of 5, 6-dihydro-4H-pyrrolo [1, 2-c] [1, 2, 3] oxadiazol-7-ium-3-olate (820 mg, 6.5 mmol) , 2-ethynyl-4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolane (1.98 g, 13.0 mmol) in Xylene (15 mL) was stirred at 160 °C for 20 hrs.
  • Step 1 Add prop-2-enal (3.2 g, 20.6 mmol) and camphorsulfonic acid (0.96 g, 4 mmol) to a cooled ice water bath solution of tert-butyl N- (prop-2-yn-1-yl) carbamate (11.5 g, 206 mmol) in DCM (60 mL) , stirring the resulting mixture at 0 °C for 15 minutes, then stirring the resulting mixture for 4 hours at room temperature. The mixture was diluted with water (50 mL) , extracted with DCM (50 mL *3) .
  • Step 2 Add hydroxylamine hydrochloride (615 mg, 8.85 mmol) to cooled ice water bath suspension of tert-butyl N- (3-oxopropyl) -N- (prop-2-yn-1-yl) carbamate (1.7 g, 8 mmol) in H 2 O/EtOH (30 mL/10 mL) , then a solution of NaOH (708 mg, 17.7 mmol) dissolved in EtOH /H 2 O (5 mL/10 mL) was added to the mixture dropwise over 15 minutes. The resulting mixture was stirred for 3 hours at room temperature.
  • Step 3 To a solution of tert-butyl N- [ (3Z) -3- (hydroxyimino) propyl] -N- (prop-2-yn-1-yl) carbamate (1500 mg, 6.63 mmol) in MeCN (15 mL) was added a suspension of chloramine-T trihydrate (1640 mg, 6.63 mmol) . The reaction mixture was stirred at 85°C for 6 hrs. The mixture was diluted with water (30 mL) , extracted with MTBE (30 mL *3) .
  • Step 2 To a solution of tert-butyl 4- ⁇ 2- [4- (difluoromethylidene) piperidin-1-yl] -5-ethyl-7-oxo-4H- [1, 2, 4] triazolo [1, 5-a] pyrimidin-6-yl ⁇ piperazine-1-carboxylate (80 mg, 0.1668 mmol) in DMF (10 mL) were added 2-bromo-N- [2-methyl-4- (trifluoromethyl) phenyl] acetamide (74.08 mg, 0.25 mmol) and DIPEA (64.67 mg, 0.5 mmol) . The mixture was stirred at room temperature for 16 hrs.
  • Step 1 To a solution of 2-methyl-6- (trifluoromethyl) pyridin-3-amine (150 mg, 0.85 mmol) in DCM (10 mL) were added TEA (257 mg, 2.55 mmol) and 2-bromoacetyl bromide (515 mg, 2.55 mmol) . The reaction mixture was stirred at room temperature for 5 hrs. The mixture was diluted with water (10 mL) , extracted with DCM (10 mL *3) .
  • Step 2 To a solution of tert-butyl 4- ⁇ 2- [4- (difluoromethylidene) piperidin-1-yl] -5-ethyl-7-oxo-4H- [1, 2, 4] triazolo [1, 5-a] pyrimidin-6-yl ⁇ piperazine-1-carboxylate (50 mg, 0.1 mmol) in DMF (5 mL) were added 2-bromo-N- [2-methyl-6- (trifluoromethyl) pyridin-3-yl] acetamide (46.48 mg, 0.15 mmol) and DIPEA (40.44 mg, 3 mmol) . The mixture was stirred at room temperature for 16 hrs.
  • Step 1 To a solution of 4- (trifluoromethyl) aniline (322 mg, 2 mmol) in DCM (20 mL) were added TEA (606 mg, 6 mmol) and 2-bromoacetyl bromide (1.21 g, 6 mmol) . The reaction mixture was stirred at room temperature for 16 hrs. The mixture was diluted with water (20 mL) , extracted with DCM (20 mL *3) .
  • Step 2 To a solution of tert-butyl 4- ⁇ 2- [4- (difluoromethylidene) piperidin-1-yl] -5-ethyl-7-oxo-4H- [1, 2, 4] triazolo [1, 5-a] pyrimidin-6-yl ⁇ piperazine-1-carboxylate (80 mg, 0.17 mmol) in DMF (5 mL) were added 2-bromo-N- [4- (trifluoromethyl) phenyl] acetamide (70 mg, 0.25 mmol) and DIPEA (65 mg, 0.5 mmol) . The mixture was stirred at room temperature for 16 hrs.
  • Step 1 To a solution of tert-butyl 4- ⁇ 2- [4- (difluoromethylidene) piperidin-1-yl] -5-ethyl-7-oxo-4H- [1, 2, 4] triazolo [1, 5-a] pyrimidin-6-yl ⁇ piperazine-1-carboxylate (100 mg, 0.21 mmol) in DMF (10 mL) were added 2-bromo-N- [2-chloro-4- (pentafluoro- ⁇ 6 -sulfanyl) phenyl] acetamide (117 mg, 0.31 mmol) and DIPEA (81 mg, 0.63 mmol) . The mixture was stirred at room temperature for 16 hrs.
  • Step 2 A mixture of tert-butyl 4- ⁇ 2-bromo-5-ethyl-7-oxo-4H- [1, 2, 4] triazolo [1, 5-a] pyrimidin-6-yl ⁇ piperazine-1-carboxylate (100 mg, 0.23 mmol) , 2-bromo-N- [2-chloro-6- (trifluoro methyl) pyridin-3-yl] acetamide (89 mg, 0.28 mmol) and DIEA (90 mg, 0.7 mmol) in DMF (3 mL) was stirred at room temperature for 6 hrs.
  • Step 3 To a solution of tert-butyl 4- (2-bromo-4- (2- ( (2-chloro-6- (trifluoromethyl) pyridin-3-yl) amino) -2-oxoethyl) -5-ethyl-7-oxo-4, 7-dihydro- [1, 2, 4] triazolo [1, 5-a] pyrimidin-6-yl) piperazine-1-carboxylate (100 mg, 0.15 mmol) in DCM (2 mL) was added HCl/dioxane (1 mL, 4M) , the mixture was stirred at room temperature for 1 hr.
  • Step 4 A mixture of 5-hydroxy-6-methylpyrimidine-4-carboxylic acid (49 mg, 0.32 mmol) , HOAt (54 mg, 0.39 mmol) and EDCI (76 mg, 0.39 mmol) in MeCN (6 mL) was stirred at room temperature for 1 hr under nitrogen atmosphere, then 2- (2-bromo-5-ethyl-7-oxo-6- (piperazin-1-yl) - [1, 2, 4] triazolo [1, 5-a] pyrimidin-4 (7H) -yl) -N- (2-chloro-6- (trifluoromethyl) pyridin-3-yl) acetamide (90 mg, 0.16 mmol) and DIPEA (103 mg, 0.79 mmol) in MeCN (3 mL) was added, the mixture was stirred at room temperature for 1 hr.
  • Step 1 To a solution of 4- (trifluoromethyl) aniline (1 g, 6.2 mmol) in DCM (20 mL) were added TEA (1.87 g, 18.6 mmol) and 2-bromoacetyl bromide (3.75 g, 18.6 mmol) at 0°C. The reaction mixture was stirred at room temperature for 16 hrs. The mixture was diluted with water (20 mL) , extracted with DCM (20 mL *3) .
  • Step 2 To a solution of tert-butyl 4- ⁇ 2-bromo-5-ethyl-7-oxo-4H- [1, 2, 4] triazolo [1, 5-a] pyrimidin-6-yl ⁇ piperazine-1-carboxylate (1.5 g, 3.5 mmol) in MeCN (20 mL) were added 2-bromo-N- [4- (trifluoromethyl) phenyl] acetamide (1.48 g, 5.2 mmol) and DIPEA (1.36 g, 10.5 mmol) . The mixture was stirred at 50°C for 16 hrs.
  • Step 3 To a solution of tert-butyl 4- [2-bromo-5-ethyl-7-oxo-4- ( ⁇ [4- (trifluoromethyl) phenyl] carbamoyl ⁇ methyl) - [1, 2, 4] triazolo [1, 5-a] pyrimidin-6-yl] piperazine-1-carboxylate (335 mg, 0.53 mmol) in DCM (5 mL) was added HCl in dioxane (5 mL, 4M) . The mixture was stirred at room temperature for 2 hrs.
  • Step 4 The mixture of 5-hydroxy-6-methylpyrimidine-4-carboxylic acid (164 mg, 1.06 mmol) , HOAt (181 mg, 1.32 mmol) and EDCI (254 mg, 1.3 mmol) in MeCN (25 mL) was stirred at room temperature for 1 hr under argon, then 2- [2-bromo-5-ethyl-7-oxo-6- (piperazin-1-yl) - [1, 2, 4] triazolo [1, 5-a] pyrimidin-4-yl] -N- [4- (trifluoromethyl) phenyl] acetamide (300 mg, 0.53 mmol) and DIEA (412 mg, 3.18 mmol) in MeCN (5 mL) was added, the mixture was stirred at room temperature for 1 hr.
  • Step 1 To a solution of 4- (pentafluoro- ⁇ 6 -sulfanyl) aniline (1700 mg, 7.75 mmol) in MeCN (20mL) were added NCS (1035 mg, 7.75 mmol) , the mixture was stirred at 45°C for 16 hrs. The mixture was diluted with water (20 mL) , extracted with DCM (20 mL *3) .
  • Step 2 To a solution of 2-chloro-4- (pentafluoro- ⁇ 6 -sulfanyl) aniline (1500 mg, 5.91 mmol) in DCM (15 mL) were added 2-bromoacetyl bromide (3581 mg, 17.74 mmol) and TEA (1795 mg, 17.74 mmol) . The mixture was stirred at room temperature for 1 hr. The mixture was diluted with water (20 mL) , extracted with DCM (20 mL *3) .
  • Step 3 To a solution of tert-butyl 4- ⁇ 2- [4- (difluoromethylidene) piperidin-1-yl] -5-ethyl-7-oxo-4H- [1, 2, 4] triazolo [1, 5-a] pyrimidin-6-yl ⁇ piperazine-1-carboxylate (450 mg, 1.053 mmol) in DMF (10 mL) were added 2-bromo-N- [2-chloro-4- (pentafluoro- ⁇ 6 -sulfanyl) phenyl] acetamide (512 mg, 1.37 mmol) and DIPEA (408 mg, 3.16 mmol) . The mixture was stirred at room temperature for 16 hrs.
  • Step 4 To a solution of tert-butyl 4- (2-bromo-4- (2- ( (2-chloro-4- (pentafluoro- ⁇ 6 -sulfaneyl) phenyl) amino) -2-oxoethyl) -5-ethyl-7-oxo-4, 7-dihydro- [1, 2, 4] triazolo [1, 5-a] pyrimidin-6-yl) piperazine-1-carboxylate (100 mg, 0.138 mmol) in DCM (5 mL) was added HCl in dioxane (3 mL, 4M) . The mixture was stirred at room temperature for 2 hrs.
  • Step 5 The mixture of 5-hydroxy-6-methylpyrimidine-4-carboxylic acid (60 mg, 0.387 mmol) , HOAt (43 mg, 0.32 mmol) and EDCI (62 mg, 0.32 mmol) in MeCN (15 mL) was stirred at room temperature for 1 hr under argon, then 2- (2-bromo-5-ethyl-7-oxo-6- (piperazin-1-yl) - [1, 2, 4] triazolo [1, 5-a] pyrimidin-4 (7H) -yl) -N- (2-chloro-4- (pentafluoro- ⁇ 6 -sulfaneyl) phenyl) acetam ide (80 mg, 0.13 mmol) and DIEA (83 mg, 0.64 mmol) in MeCN (5 mL) was added, the mixture was stirred at room temperature for 1 hr.
  • Step 1 To a solution of 3-oxaspiro [5.5] undecan-9-one (250 mg, 1.49 mmol ) in THF (8 mL) was added LDA (1.2 mL, 2.40 mmol, 2 M in THF) at -78°C under N 2 . The mixture was stirred at this temperature for 0.5 hr. Then 1, 1, 1-trifluoro-N-phenyl-N- ( (trifluoromethyl) sulfonyl) methanesulfonamide (637 mg, 1.78 mmol) was added. The mixture was stirred at 25°C for 16 hrs. The reaction was quenched by Sat. NH 4 Cl and diluted with ethyl acetate (40 mL) .
  • Step 2 A mixture of 3-oxaspiro [5.5] undec-8-en-9-yl trifluoromethanesulfonate (310 mg, 1.03 mmol) , 4, 4, 5, 5-tetramethyl-2- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1, 3, 2-dioxaboro lane (315 mg, 1.24 mmol) , Pd (dppf) Cl 2 (75 mg, 0.10 mmol) and Potassium acetate (203 mg, 2.06 mmol) in dioxane (8 mL) was stirred at 80 °C overnight under N 2 .
  • Step 2 To a solution of 5-bromo-3-fluoropyrazolo [1, 5-a] pyridine (100 mg, 0.465 mmol) , 4, 4, 5, 5-tetramethyl-2- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1, 3, 2-dioxaborolane (130 mg, 0.5116 mmol) and KOAc (91 mg, 0.930 mmol) in dioxane (3 mL) was added Pd (dppf) Cl 2 (34 mg, 0.046 mmol) . The reaction mixture was stirred under nitrogen at 80°C for 16 hrs.
  • Step 2 To a solution of 5-bromo-1- ( (2- (trimethylsilyl) ethoxy) methyl) -1H-indazole (500 mg, 1.523 mmol) , 4, 4, 5, 5-tetramethyl-2- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1, 3, 2-dioxaborolane (425 mg, 1.675 mmol) and KOAc (299 mg, 3.046 mmol) in dioxane (3 mL) was added Pd (dppf) Cl 2 (110 mg, 0.152 mmol) . The reaction mixture was stirred under nitrogen at 80 °C for 16 hrs under N 2 .
  • Step 1 To a solution of 4-bromo-2-methylpyridine (2 g, 11.6 mmol) in THF (20 mL) was added LDA (11.5 mL, 23.2 mmol) at -78°C under N 2 , the mixture was stirred at this temperature for 0.5 hr, then N-methoxy-N-methylacetamide (1.2 g, 11.6 mmol) was added. The mixture was stirred at 25°C for 16 hrs. The reaction was quenched by Sat.
  • Step 2 To a solution of Ethyl O-mesitylsulfonylacetohydroxamate (2 g, 7 mmol) in dioxane (3 mL) was added HClO 4 (0.8 mL) at 0°C, the mixture was stirred at 25°C for 0.5 hr, ice water was added to the mixture then filtered. The filter cake collected was dissolved in DCM (3 mL) , 1- (4-bromopyridin-2-yl) propan-2-one (800 mg, 3.74 mmol) was added, the mixture was stirred at 25°C for 1 hr.
  • Step 3 A mixture of 5-bromo-2-methylpyrazolo [1, 5-a] pyridine (110 mg, 0.52 mmol) , 4, 4, 5, 5-tetramethyl-2- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1, 3, 2-dioxaborolane (147 mg, 0.58 mmol) , Pd (dppf) Cl 2 (37 mg, 0.052 mmol) and KOAc (154 mg, 1.57 mmol) in dioxane (5 mL) was stirred at 90 °C for 16 hrs under Ar atmosphere.
  • Step 3 A mixture of 1-amino-4-bromo-2-methylpyridin-1-ium (1 g, 5.3 mmol) , methyl prop-2-ynoate (0.45 g, 5.3 mmol) , K 2 CO 3 (1.47 g, 10.6 mmol) in DMF (15 mL) was stirred at 25°C for 16 hrs.
  • Step 4 A mixture of methyl 5-bromo-7-methylpyrazolo [1, 5-a] pyridine-3-carboxylate (290 mg, 1.08 mmol) in 50%H 2 SO 4 (8 mL) was stirred at 110°C for 20 hrs. The mixture was neutralized by Sat. NaOH, then extracted with EtOAc (50 mL *2) . The combined organic layer was washed by water (30 mL *2) , brine (20 mL) , dried over Na 2 SO 4 .
  • Step 5 A mixture of 5-bromo-7-methylpyrazolo [1, 5-a] pyridine (190 mg, 0.9 mmol) , 4, 4, 5, 5-tetramethyl-2- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1, 3, 2-dioxaborolane (251 mg, 1.0 mmol) , Pd (dppf) Cl 2 (65 mg, 0.09 mmol) and KOAc (177 mg, 1.8 mmol) in dioxane (6 mL) was stirred at 90 °C for 6 hrs.
  • Step 1 A mixture of 7-bromoimidazo [1, 5-a] pyridine (100 mg, 0.51 mmol) , 4, 4, 5, 5-tetramethyl-2- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1, 3, 2-dioxaborolane (142 mg, 0.56 mmol) , Pd (dppf) Cl 2 (37 mg, 0.051 mmol) and KOAc (100 mg, 1.01 mmol) in dioxane (6 mL) was stirred at 80°C for 16 hrs under N 2 .
  • Step 1 To a solution of 5-bromo-2-nitrobenzaldehyde (690 mg, 3 mmol) in MeOH-EtOAc (1: 1, 20 mL) was added SnCl 2 ⁇ 2H 2 O (2.03 g, 9 mmol) . The mixture was stirred at room temperature for 24 hrs. The reaction was quenched by saturated NaHCO 3 (20 mL) and filtered. The aqueous phase was extracted with EA (20 mL *3) .
  • Step 2 The mixture of 5-bromobenzo [c] isoxazole (417 mg, 2.1 mmol) , (BPin) 2 (641.73 mg, 2.52 mmol) , KOAc (620.02 mg, 6.3 mmol) and Pd (dppf) Cl 2 (154.09 mg. 0.21 mmol) in dioxane (20 mL) was stirred at 90°C for 1 hr under N 2 atmosphere.
  • Step 2 A mixture of 2-bromo-N- [2-fluoro-4- (trifluoromethyl) phenyl] acetamide (300 mg, 1 mmol) , tert-butyl 4- (2-bromo-5-ethyl-7-oxo-4, 7-dihydro- [1, 2, 4] triazolo [1, 5-a] pyrimidin-6-yl) piperazine-1-carboxylate (428 mg, 1 mmol) , DIPEA (388 mg, 3 mmol) in ACN (5 mL) was stirred at 25°C for 16 hrs.
  • Step 3 To a solution of tert-butyl 4- (2-bromo-5-ethyl-4- (2- ( (2-fluoro-4- (trifluoromethyl) phenyl) amino) -2-oxoethyl) -7-oxo-4, 7-dihydro- [1, 2, 4] triazolo [1, 5-a] pyrimidin-6-yl) piperazine-1-carboxylate (320 mg, 0.49 mmol) in DCM (3 mL) was added HCl/Dioxane (1 mL, 4M) , then stir the mixture at 25°C for 1 hr.
  • Step 4 The mixture of 5-hydroxy-6-methylpyrimidine-4-carboxylic acid (141 mg, 0.91 mmol) , HOAt (156 mg, 1.14 mmol) and EDCI (219 mg, 1.14 mmol) was stirred in ACN (20 mL) at 25°C for 1h under N 2 , then 2- [2-bromo-5-ethyl-7-oxo-6- (piperazin-1-yl) - [1, 2, 4] triazolo [1, 5-a] pyrimidin-4-yl] -N- [2-fluoro-4- (trifluoromethyl) phenyl] acetamide (250 mg, 0.46 mmol) and DIPEA (296 mg, 2.3 mmol) in MeCN (5 mL) was added and further stirred for 1 h.
  • Step 1 The mixture of ethyl 2-bromo-3-oxopentanoate (574 mg, 2.57 mmol) , tert-butyl piperazine-1-carboxylate-2, 2, 3, 3, 5, 5, 6, 6-d 8 (500 mg, 2.57 mmol) and K 2 CO 3 (1.06 g, 7.71 mmol) in ACN (10 mL) was stirred at 50°C for 2 hrs. The mixture was filtered, the cake was washed with ACN.
  • Step 4 To a solution of tert-butyl 4- (2-bromo-5-ethyl-7-oxo-4, 7-dihydro- [1, 2, 4] triazolo [1, 5-a] pyrimidin-6-yl) piperazine-1-carboxylate-2, 2, 3, 3, 5, 5, 6, 6-d 8 (93 mg, 0.15 mmol) in ACN (10 mL) were added 2-bromo-N- [2-chloro-4- (trifluoromethyl) phenyl] acetamide (33.23 mg, 0.1 mmol) and DIPEA (57.96 mg, 0.44 mmol) . The mixture was stirred at 25°C for 16 hrs.
  • Step 5 To a solution of tert-butyl 4- (2-bromo-4- (2- ( (2-chloro-4- (trifluoromethyl) phenyl) amino) -2-oxoethyl) -5-ethyl-7-oxo-4, 7-dihydro- [1, 2, 4] triazolo [1, 5-a] pyrimidin-6-yl) piperazine-1-carboxylate-2, 2, 3, 3, 5, 5, 6, 6-d 8 (47 mg, 0.07 mmol) in DCM (5 mL) was added HCl in dioxane (2 mL) . The mixture was stirred at room temperature for 1 hr.
  • Step 6 The mixture of 5-hydroxy-6-methylpyrimidine-4-carboxylic acid (21.58 mg, 0.14 mmol) , HOAt (23.82 mg, 0.17 mmol) and EDCI (33.55 mg, 0.17 mmol) in ACN (25 mL) was stirred at 25°C for 1 hr under argon, then 2- (2-bromo-5-ethyl-7-oxo-6- (piperazin-1-yl-2, 2, 3, 3, 5, 5, 6, 6-d 8 ) - [1, 2, 4] triazolo [1, 5-a] pyrimidin-4 (7H) -yl) -N- (2-chloro-4- (trifluoromethyl) phenyl) acetamide (40 mg, 0.07 mmol) and DIEA (45.23 mg, 0.35 mmol) in ACN (5 mL) was added, the mixture was stirred at room temperature for 1 hr.
  • Step 1 To a suspension of 5-bromo-2, 3-dihydrobenzo [d] isothiazole 1, 1-dioxide (400 mg, 1.61 mmol) and Potassium carbonate (222.52 mg, 1.61 mmol) in DMF (4 mL) was added MeI (799.83 mg, 5.64 mmol) . The reaction mixture was stirred at room temperature for 16 hours. The reaction was cooled and concentrated in vacuum.
  • Step 1 To a solution of 4- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1, 2, 3, 6-tetrahydropyridine hydrochloride (0.5 g, 2.04 mmol) in DMF was added cesium carbonate (1.10 g, 3.39 mmol) at 0°C. The mixture was stirred for 15 min at 0°C and cyanogen bromide solution (0.22 g, 2.04 mmol) in THF (1 mL) was added dropwise. The mixture was stirred for 16 hours. LCMS indicated completion of reaction. The reaction mixture was quenched with ice water and diluted with DCM. The aqueous layer was separated and extracted with DCM (10 mL *2) .
  • Step 1 To a mixture of 3-chloro-4-nitrobenzaldehyde (1900 mg, 10.24 mmol) in DCM (20 mL) was added diethylaminosulfur trifluoride (1980 mg, 12.29 mmol) at -78°C. The mixture was stirred at the same temperature for 10 min. The cooling bath was removed, and the mixture was stirred overnight. Concentrated aqueous NaHCO 3 solution was added slowly to the mixture and the mixture was extracted with DCM (50 mL *3) . The combined organic layers were dried over anhydrous Na 2 SO 4 , filtered and concentrated to dryness.
  • Step 3 To the mixture of 2-chloro-4- (difluoromethyl) aniline (3.89 g, 21.91 mmol) and ethyldiisopropylamine (8.5 g, 65.73 mmol) in dichloromethane (50 mL) was added 2-bromoacetyl bromide (13.2 g, 65.73 mmol) dropwise at 0°C under N 2 . The resulting mixture was stirred at room temperature for 16 hours under N 2 . The mixture was diluted with water (50 mL) , extracted with DCM (50 mL *3) .
  • Step 4 To the mixture of tert-butyl 4- (2-bromo-5-ethyl-7-oxo-4, 7-dihydro- [1, 2, 4] triazolo [1, 5-a] pyrimidin-6-yl) piperazine-1-carboxylate (500 mg, 1.17 mmol) in DMF (8 mL) was added 2-bromo-N- (2-chloro-4- (difluoromethyl) phenyl) acetamide (349 mg, 1.17 mmol) and ethyldiisopropylamine (453 mg, 3.51 mmol) . The mixture was stirred at room temperature for 12 hours under N 2 . LCMS indicated completion of reaction.
  • Step 5 To the mixture of tert-butyl 4- (2-bromo-4- (2- ( (2-chloro-4- (difluoromethyl) phenyl) amino) -2-oxoethyl) -5-ethyl-7-oxo-4, 7-dihydro- [1, 2, 4] triazolo [1, 5-a] pyrimidin-6-yl) piperazine-1-c arboxylate (384 mg, 0.59 mmol) in dichloromethane (5 mL) was added 4 M HCl/dioxane (5 mL) . The mixture was stirred at room temperature for 2 hours.
  • Step 6 The mixture of 5-hydroxy-6-methylpyrimidine-4-carboxylic acid (254 mg, 1.65 mmol) , 1-Hydroxy-7-azabenzotriazole (224 mg, 1.65 mmol) and 3- (ethyliminomethylideneamino) -N, N-dimethylpropan-1-amine (316 mg, 1.65mmol) in acetonitrile (40 mL) was stirred at room temperature for 1 hour under N 2 , then 2- (2-bromo-5-ethyl-7-oxo-6- (piperazin-1-yl) - [1, 2, 4] triazolo [1, 5-a] pyrimidin-4 (7H) -yl) -N- (2-chloro-4- (difluoromethyl) phenyl) acetamide (300 mg, 0.55 mmol) and ethyldiisopropylamine (0.55 mL, 3.30 mmol) in acetonitrile (10
  • Step 1 To the mixture of tert-butyl 4- (2-bromo-4- (2- ( (2-chloro-4- (trifluoromethyl) phenyl) amino) -2-oxoethyl) -5-ethyl-7-oxo-4, 7-dihydro- [1, 2, 4] triazolo [1, 5-a] pyrimidin-6-yl) piperazine-1-carboxylate (1000 mg, 1.51 mmol) in 1, 4-Dioxane (20 mL) and water (2 mL) was added 5- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyrazolo [1, 5-a] pyridine (552 mg, 2.27 mmol) , sodium carbonate (480 mg, 4.53 mmol) and Pd (dppf) Cl 2 (110 mg, 0.15 mmol) .
  • Step 2 To a solution of tert-butyl 4- (4- (2- ( (2-chloro-4- (trifluoromethyl) phenyl) amino) -2-oxoethyl) -5-ethyl-7-oxo-2- (pyrazolo [1, 5-a] pyridin-5-yl) -4, 7-dihydro- [1, 2, 4] triazolo [1, 5-a] pyrimidin-6-yl) piperazine-1-carboxylate (689 mg, 0.98 mmol) in DCM (10 mL) was added hydrogen chloride (7 mL, 4M in dioxane) . The mixture was stirred at room temperature for 2 hours.
  • Step 2 To a solution of 2-chloro-4-fluoro-3- [ (4-methoxyphenyl) methoxy] pyridine (1200 mg, 4.48 mmol) in TEA (1360 mg, 13.44 mmol) and methanol (10 mL) was added [1, 1'-Bis (diphenyl phosphino) ferrocene] dichloropalladium (II) , complex with dichloromethane (365 mg, 0.45 mmol) . The mixture was stirred at 80°C for 16 hours under 10 bars of CO. The reaction mixture was filtered through celite and concentrated under reduced pressure.
  • the 2.0 Assay provides a homogeneous method for quantifying the number of viable cells in culture by measuring the metabolite ATP.
  • SW-48 a human colon cancer cell line used in this study, has the microsatellite instability-high (MSI-H) phenotype, and was obtained from Roche.
  • SW-48 cells were cultured in 1640 basal media (BasalMedia, L210KJ) supplemented with 10%fetal bovine serum (Gibco, 10091-148) and 1%penicillin-streptomycin (Solarbio, P1400) at 37°C in a 5%CO 2 incubator.
  • the Z’ factor was used to measure the success of the experiment, with a value greater than 0.7 indicating reliable experimental results.
  • Day0 represents the initial cell viability level when the compounds were added
  • High and Sample represent the cell viability levels of the solvent control group (0.1%DMSO) and the compound treatment group, respectively, at the end of the compound incubation period
  • Low represents the cell-free PBS group.
  • Percentages of 100%, 0%, and -100% represent no growth inhibition, growth arrest, and complete cell killing, respectively.
  • Table 1-1 Biology data A: ⁇ 0.5 uM; B: 0.5 uM-1 uM; C: 1 ⁇ M ⁇ C ⁇ 5 ⁇ M; D: > 5 ⁇ M.
  • WRN is an ATP-dependent helicase that can bind and hydrolyze ATP during the unwinding process, producing ADP, which provides energy to unwind double-stranded DNA. Therefore, we can use the ADP-Glo method to determine the inhibitory effect of compounds on the WRN helicase by detecting the amount of ADP produced.
  • the WRN (500-946) protein was purchased from ICE (S2201T-H03H) .
  • the double-stranded DNA substrate was obtained by annealing equal amounts of primers SEQ ID No: 1 (TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
  • the final reaction system consisted of 10 nM WRN protein, 0.2 nM DNA substrate, and 300 ⁇ M ATP.
  • the buffer of the reaction system was prepared with deionized water to a final concentration of 30 mM Tris (pH 7.5) , 2 mM MgCl 2 , 0.02%BSA, 50 mM NaCl, and 0.10%Pluronic F-68.
  • a gradient dilution of the compounds was first performed using DMSO (10-point 3-fold dilution) .
  • the diluted compounds (with a starting concentration of 10 mM) were transferred to a 384-well plate (PerkinElmer, 6007290) in 5 nL using an Echo (Beckman, Echo 650) .
  • Each compound was tested in duplicate, and 2.5 ⁇ L of buffer containing 2X ATP (600 ⁇ M) and 2X WRN (20 nM) was added, followed by preincubation at room temperature for 3 hours.
  • the positive control was a DMSO group without compound treatment but containing WRN (no inhibitory effect)
  • the negative control was a DMSO group without WRN (maximum inhibitory effect)
  • 2.5 ⁇ L of buffer containing 2X DNA 0.4 nM
  • 5 ⁇ L of ADP-Glo reagent was added and incubated at room temperature for 40 minutes to terminate the enzyme reaction and consume excess ATP.
  • 10 ⁇ L of ATP detection reagent was added to convert ADP to ATP, and the newly formed ATP was detected by a luciferase reaction using a plate reader (TECAN, Spark) with a detection time of 300 ms.
  • the Z’ factor was used to measure the success of the experiment, with a value greater than 0.6 indicating reliable experimental results.
  • Curve fitting was performed based on the concentration of the samples and the percentage of inhibition (%Inhibition) , calculated using the following formula:
  • HC represents the positive control (high control) , which is a DMSO group without compound treatment but containing WRN (no inhibitory effect)
  • LC represents the negative control (low control) , which is a DMSO group without WRN (maximum inhibitory effect) .
  • Table 2-1 Biology data A: ⁇ 0.5 uM; B: 0.5 uM-1 uM; C: 1 ⁇ M ⁇ C ⁇ 5 ⁇ M; D: > 5 ⁇ M.
  • Fluorescence quantification is used to measure the helicase activity of WRN, where DNA single strands are labeled with fluorescence and quenching groups, annealed and then unwound by WRN at specific sites, exposing the labeled fluorescence group and producing fluorescence. The fluorescence intensity of different compounds treated is measured to determine their inhibitory effect on WRN helicase.
  • the WRN (500-946) protein is purchased from ICE (S2201T-H03H) .
  • the fluorescence-labeled double-stranded DNA substrate is annealed by equal amounts of BHQ2 and TAMRA in 2 ⁇ Phanta Max Buffer, and the primers are synthesized by Xianghongbio.
  • Pre-experiments were conducted to determine optimal enzyme assay conditions, including buffer, reaction time, and protein, ATP, and DNA substrate concentrations.
  • the final reaction system includes 20 nM WRN protein, 200 nM DNA substrate, and 1 mM ATP in a buffer composed of 25 mM Tris (pH 7.5) , 2 mM MgCl 2 , 1 mM DTT, 50 mM NaCl, and 0.01%Tween-20.
  • DMSO 3-fold dilution, 10 points
  • the diluted compounds (initial concentration point 10 mM) were transferred to a 384-well plate (Greiner, 781090) using the Echo liquid handler (Beckman, Echo 650) at 50 nL per well. Each compound was treated in duplicate, and the high control was treated with WRN-conjugated DMSO without any inhibitor, and the low control was treated with DMSO without WRN (maximum inhibition effect) . After adding 22.5 ⁇ L buffer containing DNA double-stranded substrate and WRN, the plate was incubated at room temperature for 15 minutes. Then, after adding 2.5 ⁇ L buffer containing 10X ATP (10 mM) , the plate was incubated for 1 hour at room temperature. Finally, the fluorescence intensity was measured using a plate reader (TECAN, Spark) with an excitation filter of 535 ⁇ 25 nm and an emission filter of 595 ⁇ 35 nm.
  • the Z’ factor was used to measure whether the experiment was successful. If Z’ > 0.7, the experiment result is considered reliable.
  • the Z’ factor is calculated as follows:
  • HC high control, which is the WRN-conjugated DMSO without any inhibitor
  • LC low control, which is the DMSO without WRN (maximum inhibition effect) .
  • %Inhibition is calculated using the following formula:
  • Readout_HC is the fluorescence intensity of high control
  • Readout_Sample is the fluorescence intensity of the sample well
  • Readout_LC is the fluorescence intensity of low control.
  • the four-parameter curve fitting method (XLfit, equation 205) was used to calculate the absolute IC50 (AbIC50) , which is the concentration where the inhibition rate reaches 50%.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne de nouveaux composés, des compositions les comprenant et leurs utilisations. En particulier, l'invention concerne des composés bicycliques et des analogues et des dérivés de ceux-ci, leur utilisation pour inhiber l'hélicase d'ADN RecQ du syndrome de Werner (WRN) et des méthodes de traitement d'une maladie à l'aide desdits composés, en particulier l'utilisation dans le traitement du cancer.
PCT/CN2024/098708 2023-06-12 2024-06-12 Nouveaux composés, compositions les comprenant et leurs utilisations Pending WO2024255765A1 (fr)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
CNPCT/CN2023/099754 2023-06-12
CN2023099754 2023-06-12
CNPCT/CN2023/124042 2023-10-11
CN2023124042 2023-10-11
CN2024087799 2024-04-15
CNPCT/CN2024/087799 2024-04-15

Publications (1)

Publication Number Publication Date
WO2024255765A1 true WO2024255765A1 (fr) 2024-12-19

Family

ID=93851398

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2024/098708 Pending WO2024255765A1 (fr) 2023-06-12 2024-06-12 Nouveaux composés, compositions les comprenant et leurs utilisations

Country Status (1)

Country Link
WO (1) WO2024255765A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12344609B2 (en) 2023-06-08 2025-07-01 Nimbus Wadjet, Inc. WRN inhibitors
WO2025152932A1 (fr) * 2024-01-15 2025-07-24 上海齐鲁制药研究中心有限公司 Dérivé de triazolopyrimidine, son procédé de préparation et son utilisation
WO2025162253A1 (fr) * 2024-02-02 2025-08-07 中国科学院上海药物研究所 Composé spiro, composition pharmaceutique le contenant et son utilisation
WO2025215527A2 (fr) 2024-04-10 2025-10-16 Novartis Ag Combinaisons pharmaceutiques et leurs utilisations

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102439011A (zh) * 2009-02-11 2012-05-02 加利福尼亚大学校务委员会 Toll样受体调节剂和疾病的治疗
WO2018229683A1 (fr) * 2017-06-15 2018-12-20 Novartis Ag Composés 5,6-fusionnés-bicycliques et compositions pour le traitement de maladies parasitaires
WO2022249060A1 (fr) * 2021-05-26 2022-12-01 Novartis Ag Analogues de triazolo-pyrimidine pour le traitement de maladies liées à l'inhibition de l'hélicase recq du syndrome de werner (wrn)
WO2024105610A1 (fr) * 2022-11-18 2024-05-23 Novartis Ag Combinaisons pharmaceutiques et leurs utilisations
WO2024105553A1 (fr) * 2022-11-16 2024-05-23 Novartis Ag Hétérocycles bicycliques et leur utilisation en tant qu'inhibiteurs de wrn

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102439011A (zh) * 2009-02-11 2012-05-02 加利福尼亚大学校务委员会 Toll样受体调节剂和疾病的治疗
WO2018229683A1 (fr) * 2017-06-15 2018-12-20 Novartis Ag Composés 5,6-fusionnés-bicycliques et compositions pour le traitement de maladies parasitaires
WO2022249060A1 (fr) * 2021-05-26 2022-12-01 Novartis Ag Analogues de triazolo-pyrimidine pour le traitement de maladies liées à l'inhibition de l'hélicase recq du syndrome de werner (wrn)
WO2024105553A1 (fr) * 2022-11-16 2024-05-23 Novartis Ag Hétérocycles bicycliques et leur utilisation en tant qu'inhibiteurs de wrn
WO2024105610A1 (fr) * 2022-11-18 2024-05-23 Novartis Ag Combinaisons pharmaceutiques et leurs utilisations

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12344609B2 (en) 2023-06-08 2025-07-01 Nimbus Wadjet, Inc. WRN inhibitors
US12421233B2 (en) 2023-06-08 2025-09-23 Nimbus Wadjet, Inc. WRN inhibitors
WO2025152932A1 (fr) * 2024-01-15 2025-07-24 上海齐鲁制药研究中心有限公司 Dérivé de triazolopyrimidine, son procédé de préparation et son utilisation
WO2025162253A1 (fr) * 2024-02-02 2025-08-07 中国科学院上海药物研究所 Composé spiro, composition pharmaceutique le contenant et son utilisation
WO2025215527A2 (fr) 2024-04-10 2025-10-16 Novartis Ag Combinaisons pharmaceutiques et leurs utilisations

Similar Documents

Publication Publication Date Title
WO2024255765A1 (fr) Nouveaux composés, compositions les comprenant et leurs utilisations
JP5972964B2 (ja) 抗腫瘍剤としての三環式および四環式ピラゾロ［３，４−ｂ］ピリジン化合物
ES2935746T3 (es) Inhibidores de desmetilasa-1 específica de lisina
EP2464647B1 (fr) Azaindoles en tant qe modulateurs du recepteur btk et leur utilisation
AU2018224488A1 (en) Modulators of the cystic fibrosis transmembrane conductance regulator protein and methods of use
AU2012310168B2 (en) 6 - substituted 3 - (quinolin- 6 - ylthio) - [1,2,4] triazolo [4, 3 -a] pyradines as tyrosine kinase
BRPI0708615A2 (pt) compostos de pirazol heterobicìclicos e métodos de uso
AU2003221500B2 (en) Heteroaryl substituted 2-pyridinyl and 2-pyrimidinyl -6,7,8,9-tetrahydropyrimido[1,2-a] pyrimidin-4-one derivatives
CA2698753A1 (fr) Pyrazolo-pyridines en tant qu'inhibiteurs de tyrosine kinase
JP2015508784A (ja) アミド−ベンジルスルホン及びスルホキシド誘導体
CA3085460A1 (fr) Derives d'amine aryl-bipyridine utilises en tant qu'inhibiteurs de la phosphatidylinositol phosphate kinase
JP2017518344A (ja) カゼインキナーゼ１デルタ／イプシロン阻害剤としてのイミダゾ−ピリダジン誘導体
JP6776272B2 (ja) Ｔｎｆ活性のモジュレーターとしての縮合三環式イミダゾピラジン類
WO2024249888A2 (fr) Composés et compositions pouvant être utilisés en tant qu'agents de dégradation de cbp/p300 et leurs utilisations
WO2024222866A1 (fr) Dérivés cycliques, compositions et utilisations de ceux-ci
WO2009154870A1 (fr) Inhibiteurs de l'intégrase vih
IL320626A (en) Fused tricyclic parp1 inhibitor, method of preparation thereof, and use thereof
TWI848608B (zh) Wee1激酶抑制劑的製備及其應用
WO2025077896A1 (fr) Inhibiteur d'adn polymérase thêta et son utilisation
WO2025232693A1 (fr) Nouveaux composés, compositions les comprenant et leurs utilisations
CN116375728A (zh) 噻吩吡咯并三嗪酮类化合物及其用途
WO2021239727A1 (fr) Dérivés de 4-(7h-pyrrolo[2,3-d]pyrimidin-4-yl)-3,6-dihydropyridine-1-(2h)-carboxamide servant d'inhibiteurs de kinases limk et/ou rock destinés à être utilisés dans le traitement du cancer
TW202542120A (zh) 化合物及其用途
EP4441034A1 (fr) Utilisation d'analogues de pyrazole urée fusionnés en tant qu'inhibiteurs de la glucosylcéramide synthase
CN119899186A (zh) 氮杂双环类衍生物及其制备方法和用途

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24822714

Country of ref document: EP

Kind code of ref document: A1