WRN inhibitor, and pharmaceutical composition thereof and use thereof

Abstract

A WRN inhibitor, and a composition and the use thereof. Particularly, a compound as represented by formula (I), a pharmaceutical composition containing same and the use thereof as a WRN inhibitor in the treatment of cancer-related diseases.

Description

WRN inhibitors and pharmaceutical compositions and applications thereof Technical Field

[0001] The present invention relates to the field of medicine and provides pharmaceutically active compounds, compositions thereof, and methods of use. Specifically, the compounds of the present invention can modulate WRN-related factors and are used to treat various diseases, including cancer. Background Art

[0002] Werner helicase (WRN) is a synthetic lethal target in dMMR / MSI-H cancers, with a high proportion of sensitivity in colorectal cancer cell lines. WRN is a member of the RecQ family of DNA helicases and the only protein in this family with both helicase and exonuclease activities. It plays an important role in maintaining genomic stability, DNA repair, replication, transcription, and telomere maintenance.

[0003] Mismatch Repair (MMR), an important DNA repair mechanism, can repair incorrectly paired bases in DNA. The human genome contains many short tandem repeat regions. These repetitive DNA regions are called "microsatellites" and are prone to slippage and errors during replication. Therefore, they are highly dependent on the MMR system for repair. When the MMR system is abnormal, dMMR cannot recognize and repair microsatellite replication errors, resulting in microsatellite instability (MSI), which may lead to frameshift mutations, abnormalities in tumor-related genes, and further induce the occurrence and development of cancer. Mismatch repair genes are conserved genes in the process of biological evolution and are housekeeping genes. They can accurately recognize and repair base mismatches, small base deletions or insertions generated during DNA replication or recombination, and play an important role in maintaining genome stability and the accuracy of hereditary offspring.

[0004] Microsatellite instability (MSI) occurs when DNA mismatch repair (MMR) is dysfunctional, leading to the accumulation of uncorrected replication errors in microsatellites and altered microsatellite sequence length or base composition. MMR genes are DNA mismatch repair genes. Common MMR proteins include MLH1, MSH2, MSH6, and PMS2. Loss of their expression can lead to the accumulation of mismatches during DNA replication, resulting in microsatellite instability. MSI is generally diagnosed by measuring the expression of these four proteins. dMMR (deficient Mismatch Repair) is characterized by loss of MMR expression, while pMMR (proficient Mismatch Repair) is characterized by normal MMR expression without loss. dMMR manifests as high-frequency microsatellite instability (MSI-H), while pMMR manifests as low-frequency microsatellite instability (MSI-L) or microsatellite stability (MSS). The presence of MSI in tumor tissue is a key marker of deficient DNA mismatch repair (dMMR).

[0005] DNA mismatch repair (dMMR) can identify and repair spontaneous mismatched bases during DNA replication. Microsatellite instability (MSI) is caused by damaged dMMR and is a common phenomenon in cancer. Thousands of MSI cancers are diagnosed worldwide each year, and approximately 10-15% of sporadic colorectal cancers (CRC) show dMMR / MSI.

[0006] Studies have shown that dMMR / MSI-H cells depend on WRN for growth and proliferation, indicating that dMMR tumor cells have a synthetic lethal relationship with WRN. In the MSI-H model, knocking out WRN induces double-strand DNA breaks and selectively promotes apoptosis and cell cycle arrest. An analysis of 60 dMMR colorectal cancer clinical samples revealed that 55 were WRN-dependent. These findings indicate that WRN is a synthetic lethal partner and a promising drug target for MSI-H cancers. However, the development of drugs that can effectively target WRN remains an obstacle, which has greatly affected the treatment process of dMMR / MSI-H cancers.

[0007] In recent years, WRN has been considered an important target for dMMR / MSI-H cancers. Currently, there are no tumor drugs targeting WRN on the market, and research on this target is urgently needed. Therefore, it is of great research significance to develop small molecule inhibitor drugs that can target WRN and provide patients with safer and more effective WRN inhibitors.

[0008] Summary of the Invention

[0009] In order to solve the above problems, the present invention provides a compound that can serve as a WRN inhibitor for treating cancer.

[0010] In one aspect, the present invention provides a compound as shown in Formula I, or a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt or deuterated form thereof:

[0011] Indicates whether the bond on the ring is a single bond or a double bond; represents a single bond or a double bond;

[0012] Z and M are independently selected from CR x , CR x R x NR x , N, O or S;

[0013] Q, W, J, T, K, G are independently selected from C, CR x or N;

[0014] X is a bond or C 1-4 A divalent saturated or unsaturated straight or branched hydrocarbon group, wherein one, two or three methylene groups in the hydrocarbon group may be optionally and independently replaced by: -CH(R e )-、-C(R e )2-、C 3-5 Cycloalkylene, -N(R e )-、-N(R e )C(=O)-、-C(=O)N(R e )-、-N(R e )C(=S)-、-C(=S)N(R e )-、-N(R e )S(=O)2-、-S(=O)2N(R e )-, -O-, -C(=O)-, -C(=S)-, -OC(=O)-, -C(=O)O-, -S-, -S(=O)- or -S(=O)2-;

[0015] Ring A is C 6-14 Aryl, C 3-14 carbocyclyl, 5-14 membered heteroaryl or 3-14 membered heterocyclyl;

[0016] R1 is selected from halogen, -CN, -OR e 、-NR e R f 、-SR e 、-C(O)R e 、-N(R e )C(O)Rf 、-C(O)NR e R f 、-N(R e )C(O)OR f 、-OC(O)NR e R f 、-N(R e )C(O)NR f R j 、-S(O)R e 、-S(O)2R e 、-S(O)(=NRe)R f 、-S(O)NR e R f 、-S(O)2NR e R f 、-N(R e )S(O)R f 、-N(R e )S(O)2R f 、-C(O)OR e 、-OC(O)R f 、-OC(O)OR j 、C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, -C 0-6 Alkylene-C 3-14 Carbocyclic group, -C 0-6 Alkylene-C 6-14 Aryl, -C 0-6 Alkylene-3-14 membered heterocyclic group or -C 0-6 Alkylene-5-14 membered heteroaryl or Among them, the C 1- 6 alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, -C 0-6 Alkylene-C 3-14 Carbocyclic group, -C 0-6 Alkylene-C 6-14 Aryl, -C 0-6 Alkylene-3-14 membered heterocyclic group, -C 0-6 Alkylene-5-14 membered heteroaryl, R e 、R f 、R j 、R 1’ and R 1” Optionally replaced by one or more hydrogen, deuterium, halogen, CN, oxo, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C2-6 Alkenyl, C 2-6 Alkynyl, -OR h 、-NR h R i , substituted or unsubstituted -C 0-6 Alkylene-C 3- 14 Carbocyclyl, substituted or unsubstituted -C 0-6 Alkylene-3-14 membered heterocyclic group, -C(O)OR h 、-C(O)NR h R i or R h replace;

[0017] R 1’ and R 1” are independently selected from C 1-6 Alkyl and C 1-6 haloalkyl; or

[0018] R 1’ and R 1” Together with the atoms to which it is attached, it forms C 3-6 Carbocyclic group, 3-7 membered heterocyclic group; wherein, the C 3-6 Carbocyclic group, 3-7 membered heterocyclic group are optionally substituted by one or more R h replace;

[0019] Each R x Same or different, R x , R2 is selected from hydrogen, deuterium, halogen, CN, OH, NH2, -COOH, -NH(C 1-6 Alkyl), -N(C 1-6 alkyl)2, oxo, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, -NHCO-C 1-6 Alkyl, -NHCO-C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, -OC 3-14 Cycloalkyl, -O-3-14 membered heterocyclic group; wherein said OH, NH2, -COOH, -NH(C 1-6 Alkyl), -N(C 1-6 Alkyl)2, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, -NHCO-C 1-6 Alkyl, -NHCO-C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6Alkynyl, -OC 3- 14 Cycloalkyl, -O-3-14 membered heterocyclic group is optionally replaced by one or more hydrogen, deuterium, halogen, CN, OH, NH2, -COOH, oxo, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy, -NH(C 1-6 Alkyl), -N(C 1-6 Alkyl)2, -COO(C 1- 6 alkyl), -CONH(C 1-6 Alkyl) or -S(O)2-C 1-6 Alkyl substitution;

[0020] Ring B is C 6-14 Aryl, C 3-14 carbocyclyl, 5-14 membered heteroaryl or 3-14 membered heterocyclyl;

[0021] Each R4 is the same or different and is independently selected from hydrogen, deuterium, halogen, CN, OH, NH2, -COOH, -NH(C 1-6 Alkyl), -N(C 1-6 alkyl)2, oxo, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, -NHCO-C 1-6 Alkyl, -NHCO-C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, -OC 3-14 Cycloalkyl or -O-3-14 membered heterocyclic group; wherein said OH, NH2, -COOH, -NH(C 1-6 Alkyl), -N(C 1-6 Alkyl)2, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, -NHCO-C 1-6 Alkyl, -NHCO-C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, -OC 3- 14 Cycloalkyl and -O-3-14 membered heterocyclyl are optionally replaced by one or more hydrogen, deuterium, halogen, CN, OH, NH2, -CF3, -COOH, oxo, C 1- 6 alkyl, C 1-6 Halogenated alkyl, C 2-6Alkenyl, C 2-6 Alkynyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy, -NH(C 1-6 Alkyl), -N(C 1-6 Alkyl)2, -COO(C 1-6 alkyl), -CONH(C 1-6 Alkyl) or -S(O)2-C 1-6 Alkyl substitution;

[0022] L is a key, C 1-3 Alkylene, C 2-4 Alkenylene, C 2-4 Alkynylidene, C 1-4 Heteroalkylene, -OC 1-6 Alkylene-, -N(R e )C(O)-、-C(O)N(R e )-, -C(O)-, -S(O)-, -S(O)2-, -N(R e )S(=O)2-、-S(=O)2N(R e )-or Among them, the C 1-3 Alkylene, C 2-4 Alkenylene, C 2-4 Alkynylidene, C 1-4 Heteroalkylene and -OC 1-6 Alkylene - optionally substituted with one or more R h replace;

[0023] Ring C is C 6-14 Aryl, C 3-14 carbocyclyl, 5-14 membered heteroaryl or 3-14 membered heterocyclyl;

[0024] Each R3, R5 is the same or different and is independently selected from H, deuterium, halogen, -CN, oxo, thioxo, -OR e 、-NR e R f ,-BR e R f 、-SR e 、-C(O)R e 、-N(R e )C(O)R f 、-C(O)NR e R f 、-N(R e )C(O)OR f 、-OC(O)NR e R f 、-N(R e )C(O)NR fR j 、-S(O)R e 、-S(O)2R e 、-S(O)(=NRe)R f 、-S(O)NR e R f 、-S(O)2NR e R f 、-N(R e )S(O)R f 、-N(R e )S(O)2R f 、-C(O)OR e 、-OC(O)R f 、-OC(O)OR j 、C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, -C 0-6 Alkylene-C 3-14 Carbocyclic group, -C 0-6 Alkylene-C 6-14 Aryl, -C 0-6 Alkylene-3-14 membered heterocyclic group or -C 0-6 Alkylene-5-14 membered heteroaryl; wherein, the C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, -C 0- 6-alkylene-C 3-14 Carbocyclic group, -C 0-6 Alkylene-C 6-14 Aryl, -C 0-6 Alkylene-3-14 membered heterocyclic group, -C 0-6 Alkylene-5-14 membered heteroaryl, R e 、 R f and R j Optionally replaced by one or more hydrogen, deuterium, halogen, CN, oxo, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, -OR h 、-NR h R i , substituted or unsubstituted -C 0-6 Alkylene-C 3-14 Carbocyclyl, substituted or unsubstituted -C 0-6 Alkylene-3-14 membered heterocyclic group, -C(O)OR h 、-OC(O)R h、-NC(O)R h R i 、-C(O)NR h R i or R h replace;

[0025] R e 、R f 、R h 、R i 、R j are independently selected from hydrogen, deuterium, halogen, CN, OH, NH2, -COOH, -NH(C 1-6 Alkyl), -N(C 1-6 alkyl)2, oxo, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, -OC 3-14 Cycloalkyl, -O-3-14 membered heterocyclic group, -S-3-14 membered heterocyclic group, -SC 3-14 Cycloalkyl, -SC 1-6 Alkyl, -CONH2, -CONH(C 1-6 alkyl), -CON(C 1-6 alkyl)2, -CONH(C 2-6 alkenyl), -CON(C 2-6 alkenyl)2, -CONH(C 2-6 Alkynyl), -CON(C 2-6 Alkynyl)2, -S(O)-C 1-6 Alkyl, -S(O)2-C 1-6 Alkyl, -S(O)-C 2-6 Alkenyl, -S(O)2-C 2-6 Alkenyl, -S(O)-C 2-6 Alkynyl, -S(O)2-C 2-6 Alkynyl, -S(O)-C 3-14 Cycloalkyl, -S(O)2-3-14 membered heterocyclic, -S(O)NH2, -S(O)NHC 1-6 Alkyl, -S(O)N(C 1-6 Alkyl)2, -CHO, -C(O)-C 1-6 Alkyl, -C(O)-C 2-6 Alkenyl, -C 0-6 Alkylene-C 6-14 Aryl, -C 0-6 Alkylene-5-14 membered heteroaryl, -C 0-6 Alkylene-C 3-14 Carbocyclic group, -C 0-6Alkylene-3-14 membered heterocyclic group, -CO-C 6-14 Aryl, -CO-5-14 membered heteroaryl, -CO-C 3-14 Carbocyclic group, -CO-3-14 membered heterocyclic group, -NHCO-C 6-14 Aryl, -NHCO-5-14 membered heteroaryl, -NHCO-C 3-14 Carbocyclic group or -NHCO-3-14 membered heterocyclic group; wherein, the -NH(C 1-6 Alkyl), -N(C 1-6 Alkyl)2, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, -OC 3-14 Cycloalkyl, -O-3-14 membered heterocyclic group, -S-3-14 membered heterocyclic group, -SC 3-14 Cycloalkyl, -SC 1-6 Alkyl, -CONH(C 1-6 alkyl), -CON(C 1-6 alkyl)2, -CONH(C 2-6 alkenyl), -CON(C 2-6 alkenyl)2, -CONH(C 2-6 Alkynyl), -CON(C 2-6 Alkynyl)2, -S(O)-C 1-6 Alkyl, -S(O)2-C 1-6 Alkyl, -S(O)-C 2-6 Alkenyl, -S(O)2-C 2-6 Alkenyl, -S(O)-C 2-6 Alkynyl, -S(O)2-C 2-6 Alkynyl, -S(O)-C 3-14 Cycloalkyl, -S(O)2-3-14 membered heterocyclic group, -S(O)NHC 1-6 Alkyl, -S(O)N(C 1- 6 alkyl) 2, -C(O)-C 1-6 Alkyl, -C(O)-C 2-6 Alkenyl, -C 0-6 Alkylene-C 6-14 Aryl, -C 0-6 Alkylene-5-14 membered heteroaryl, -C 0-6 Alkylene-C 3-14 Cycloalkyl, -C 0-6 Alkylene-3-14 membered heterocyclic group, -CO-C 6-14 Aryl, -CO-5-14 membered heteroaryl, -CO-C 3-14Carbocyclic group, -CO-3-14 membered heterocyclic group, -NHCO-C 6-14 Aryl, -NHCO-5-14 membered heteroaryl, -NHCO-C 3-14 The carbocyclyl and -NHCO-3-14 membered heterocyclyl are optionally substituted with one or more hydrogen, deuterium, halogen, CN, OH, NH2, -COOH, oxo, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl, C 2- 6 alkynyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy, -NH(C 1-6 Alkyl), -N(C 1-6 Alkyl)2, -COO(C 1-6 alkyl), -CONH(C 1-6 alkyl), -CON(C 1-6 Alkyl)2, -S(O)-C 1-6 Alkyl, -S(O)2-C 1-6 Alkyl, -C 0-6 Alkylene-NHC(=O)C 1-6 Alkyl, -C 0-6 Alkylene-NHC(=O)C 1- 6-halogenated alkyl, -C 0-6 Alkylene-NHC(=O)C 2-6 Alkenyl, -CON(C 1-6 Alkyl)2, substituted or unsubstituted -C 0-6 Alkylene-C 3-14 Carbocyclyl, substituted or substituted-C 0-6 Alkylene-3-14 membered heterocyclic substitution;

[0026] m is 0, 1, 2, 3 or 4;

[0027] n is 0, 1, 2, 3 or 4;

[0028] q is 0, 1, 2, 3 or 4.

[0029] In some embodiments, in Formula I, Indicates whether the bond on the ring is a single bond or a double bond.

[0030] In some embodiments, in Formula I, Z is selected from N.

[0031] In some embodiments, in Formula I, K is selected from N.

[0032] In some embodiments, in Formula I, M is selected from N.

[0033] In some embodiments, in Formula I, G is selected from C.

[0034] In some embodiments, in Formula I, T is selected from C or N.

[0035] In some embodiments, in Formula I, Q is selected from C.

[0036] In some embodiments, in Formula I, W is selected from C.

[0037] In some embodiments, in Formula I, J is selected from N.

[0038] In some embodiments, in Formula I, Ring A is phenyl, naphthyl, 5-6 membered monocyclic heteroaryl, 5-6 membered monocyclic heterocyclyl, 9-10 membered bicyclic heterocyclyl or 9-10 membered bicyclic heteroaryl.

[0039] In some embodiments, in Formula I, Ring A is phenyl, naphthyl, a 5-membered heteroaryl, a 6-membered heteroaryl, a 10-membered bicyclic heterocyclyl, or a 10-membered bicyclic heteroaryl.

[0040] In some embodiments, in Formula I, for wherein r is selected from 0, 1, 2, 3 or 4; p is selected from 0, 1, 2, 3 or 4; r+p=m.

[0041] In some embodiments, in Formula I, R3 is H, halogen, CN, oxo, thio, NH2, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, -COOH, -C(O)NH2, -NHCOOC 1-6 Alkyl, -NHCOC 1-6 Alkyl, -S(O)2-C 1-6 Alkyl, C 3-6 Cycloalkyl, -OC 3- 6-membered cycloalkyl, 3-7-membered heterocyclic group, phenyl or 5-6-membered heteroaryl; wherein, the NH2, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, -COOH, -C(O)NH2, -NHCOOC 1-6 Alkyl, -NHCOC 1-6 Alkyl, -S(O)2-C 1-6 Alkyl, C 3-6 Cycloalkyl, -OC 3-6 Cycloalkyl, 3-7 membered heterocyclyl, phenyl and 5-6 membered heteroaryl are optionally substituted with one or more halogen, C 1-6 Alkyl, C 1-6Halogenated alkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkoxy, C 3-6 The cycloalkyl group or the 3-6 membered heterocyclic group is substituted.

[0042] In some embodiments, in Formula I, R3 is H, F, Cl, Br, CN, NH2, oxo, thio, CH3, -CF3, -CHF2, -CH2F, -C2F5,

[0043] In some embodiments, in Formula I, X is a bond,

[0044] In some embodiments, in Formula I, for

[0045] In some embodiments, in Formula I, R1 is halogen, -NH2, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, -C(O)-C 1-6 Alkyl, -C(O)OC 1-6 Alkyl, -C(O)NH2, -C(O)NH-C 1-6 Alkyl, -C(O)N(C 1-6 alkyl)2, 5-6 membered heteroaryl, 5-6 membered heterocyclic group, 8-9 membered heteroaryl, 7-9 membered heterocyclic group, -NH-3-6 membered heterocyclic group, -C(O)-3-6 membered heterocyclic group, -C(O)NH-3-6 membered heterocyclic group, -5-6 membered heterocyclic group-C 1-3 Alkyl or Wherein, the -NH2, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, -C(O)-C 1-6 Alkyl, -C(O)OC 1-6 Alkyl, -C(O)NH2, -C(O)NH-C 1-6 Alkyl, -C(O)N(C 1-6 alkyl)2, 5-6 membered heteroaryl, 5-6 membered heterocyclyl, 8-9 membered heteroaryl, 7-9 membered heterocyclyl, -NH-3-6 membered heterocyclyl, -C(O)-3-6 membered heterocyclyl, -C(O)NH-3-6 membered heterocyclyl and -5-6 membered heterocyclyl-C 1-3 The alkyl group is optionally substituted with one or more halogen, substituted or unsubstituted C 1-6 Alkyl, substituted or unsubstituted C 1-6 Alkoxy, substituted or unsubstituted C 3-6Cycloalkyl, substituted or unsubstituted 3-6 membered heterocyclic group or -S(O)2-C 1-6 Alkyl substitution.

[0046] In some embodiments, in Formula I, R1 is Br, NH2,

[0047] In some embodiments, in Formula I, R2 is H, CN, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl or C 1-6 Halogenated alkoxy; wherein the C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkyl, C 1-6 The haloalkoxy group is optionally substituted with one or more halogen, CN, C 1-6 Alkoxy, C 1-6 Halogenated alkoxy, C 1-6 Haloalkyl or -S(=O)2C 1-6 Alkyl substitution.

[0048] In some embodiments, in Formula I, R2 is H, CN, CH2CH3,

[0049] In some embodiments, in Formula I, Ring B is phenyl, a 4-6 membered monocyclic heterocyclyl, a 5-6 membered heteroaryl, or a 7-8 membered bicyclic heterocyclyl.

[0050] In some embodiments, in Formula I, for wherein j is selected from 0, 1, 2, 3 or 4; k is selected from 0, 1, 2, 3 or 4; j+k=q.

[0051] In some embodiments, in Formula I, R4 is H, halogen or C 1-6 alkyl.

[0052] In some embodiments, in Formula I, R4 is H, Cl or Br.

[0053] In some embodiments, in Formula I, L is a bond, C 1-3 Alkylene, C 2-4 Alkenylene, C 2-4 Alkynylidene, -C(O)NH-, -NHC(O)-, -C(O)-, -OC 1-6 Alkylene- or

[0054] In some embodiments, in Formula I, L is a bond, CH2,

[0055] In some embodiments, in Formula I, Ring C is phenyl, 5-membered heteroaryl, 6-membered heteroaryl, 5-membered heterocyclyl, or 6-membered heterocyclyl.

[0056] In some embodiments, in Formula I, R5 is H, deuterium, halogen, CN, oxo, -OH, -NH2, -N(C 1-6 Alkyl)2, -COOH, boric acid, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, -C(O)OC 1-6 Alkyl, phenyl, 3-10 membered heterocyclic group, C 3-6 Carbocyclic group, 5-6 membered heteroaryl, -SC 1-6 Alkyl, -S(O)-C 1-6 Alkyl or -S(O)2-C 1-6 Alkyl; wherein the -OH, -NH2, -N(C 1-6 Alkyl)2, -COOH, boric acid, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, -C(O)OC 1-6 Alkyl, phenyl, 3-10 membered heterocyclic group, C 3-6 Carbocyclic group, 5-6 membered heteroaryl, -SC 1-6 Alkyl, -S(O)-C 1-6 Alkyl and -S(O)2-C 1-6 The alkyl group is optionally substituted with one or more OH, halogen, C 1-6 Alkyl, C 1- 6 alkoxy, C 1-6 Halogenated alkyl, C 1-6 Halogenated alkoxy, 3-6 membered heterocyclic group, -S(O)-C 1-6 Alkyl or -S(O)2-C 1-6 Alkyl substitution.

[0057] In some embodiments, in Formula I, R5 is H, F, Cl, OH, CH3, -C≡CH, CN, NH2, COOH,

[0058] In some embodiments, in Formula I, for

[0059] In some embodiments, in Formula I, for

[0060] In some embodiments, the compound of formula I is selected from the compound of formula II shown below:

[0061] The definitions of R1, R2, R3, R4, R5, Z, K, G, M, X, L, ring A, ring B, ring C, m, n and q in the compound of formula II are the same as those of formula I above.

[0062] In some embodiments, the compound of formula I is selected from the compound of formula III shown below:

[0063] The definitions of R1, R2, R3, R4, R5, Z, K, G, M, X, L, ring A, ring B, ring C, m, n and q in the compound of formula III are the same as those of formula I above.

[0064] In some embodiments, the compound of formula I is selected from the compounds of formula IV-1, formula IV-2, formula IV-3 or formula IV-4 as shown below:

[0065] The definitions of R1, R2, R3, R4, R5, X, L, ring A, ring B, ring C, m, n and q in the compounds of formula IV-1, formula IV-2, formula IV-3 and formula IV-4 are the same as those of the aforementioned formula I.

[0066] In some embodiments, the compound of formula I is selected from the compound of formula VI shown below:

[0067] in,

[0068] R1, R2, R3, R4, R5, X, L, Ring A, Ring B, m, n and q in the compound of Formula VI are as defined above in Formula I.

[0069] In some embodiments, the compound of formula I is selected from the compound of formula VII shown below:

[0070] Ring A1 is selected from phenyl or 5-6 membered heteroaryl;

[0071] Ring A2 is selected from C 5-6 Carbocyclyl, phenyl, 5-6 membered heterocyclyl or 5-6 membered heteroaryl;

[0072] represents a single bond or a double bond;

[0073] r is selected from 0, 1, 2, 3, 4, p is selected from 0, 1, 2, 3, 4, r + p = m;

[0074] The definitions of R1, R2, R3, R4, R5, X, Ring B, Ring C, m, n and q in the compound of formula VII are the same as those of formula I above.

[0075] In some embodiments, the compound of formula I is selected from the compound of formula VIII shown below:

[0076] Ring A1 is selected from phenyl or 5-6 membered heteroaryl;

[0077] Ring A2 is selected from C 5-6 Carbocyclyl, phenyl, 5-6 membered heterocyclyl or 5-6 membered heteroaryl;

[0078] represents a single bond or a double bond;

[0079] r is selected from 0, 1, 2, 3, 4, p is selected from 0, 1, 2, 3, 4, r+p=m.

[0080] R1, R2, R3, R5, X, ring C, m and n in the compound of formula VIII are as defined above for formula I.

[0081] In some embodiments, the compound of formula I is selected from the compounds of formula IX-1 or formula IX-2 as shown below:

[0082] in,

[0083] represents a single bond or a double bond;

[0084] X1, X2, X3 are independently selected from C or N;

[0085] Y1, Y2 and Y3 are independently selected from C, N, O, or S;

[0086] r is selected from 0, 1, 2, 3, 4, p is selected from 0, 1, 2, 3, 4, r + p = m;

[0087] The definitions of R1, R2, R3, R4, R5, L, ring B, ring C, m, n and q in the compounds of formula IX-1 and formula IX-2 are the same as those of formula I above.

[0088] In some embodiments, the compound of formula I is selected from the compounds of formula X-1 or formula X-2 shown below:

[0089] in,

[0090] Expressed as a single or double bond;

[0091] Y1, Y2 and Y3 are independently selected from C, N, O, or S;

[0092] r is 0, 1, 2, 3 or 4; p is 0, 1, 2, 3 or 4; r + p = m;

[0093] R1, R2, R3, R4, R5, L, Ring B, Ring C, m, n and q in the compound of Formula X-1 or Formula X-2 are as defined above in Formula I.

[0094] In some embodiments, the compound of formula I is selected from the compounds of formula XI-1, formula XI-2 or formula XI-3 as shown below:

[0095] r is 0, 1, 2, 3 or 4; p is 0, 1, 2, 3 or 4; r + p = m;

[0096] The definitions of R1, R2, R3, R4, R5, L, ring B, ring C, m, n and q in the compound of formula XI-1, formula XI-2 or formula XI-3 are the same as those of formula I above.

[0097] In some embodiments, the compound of formula I is selected from the compounds of formula XI-1', formula XI-2' or formula XI-3' as shown below:

[0098] r is 0, 1, 2, 3 or 4; p is 0, 1, 2, 3 or 4; r + p = m;

[0099] The definitions of R1, R2, R3, R4, R5, L, ring C, m, n and q in the compound of formula XI-1′, formula XI-2′ or formula XI-3′ are as defined above for formula I.

[0100] In some embodiments, the compound of formula I is selected from the compound of formula XII shown below:

[0101] in,

[0102] Indicates whether the bond on the ring is a single bond or a double bond;

[0103] Y1, Y2, Y3, Y4 and Y5 are independently selected from the group consisting of: C, N, O, or S;

[0104] R1, R2, R3, R4, R5, L, Ring B, Ring C, m, n and q in the compound of Formula XII are as defined above in Formula I.

[0105] In some embodiments, the compound of formula I is selected from the compounds in Table 1:

[0106] Table 1

[0107] In another aspect, the present invention further provides a method for preparing the compound of Formula Z, Formula Z12, Formula Z10, Formula Z8, Formula Z6, Formula Z4 or Formula Z3, characterized in that it comprises the following steps:

[0108] in,

[0109] Y is selected from halogen; preferably, Y is selected from chlorine or bromine;

[0110] R 100 is a hydroxyl protecting group; preferably, R 100 Selected from tert-butyldimethylsilyl, methyl, benzyl, p-methoxybenzyl, methoxymethyl;

[0111] The groups at other positions are as defined above in formula I.

[0112] The present invention also provides a method for preparing the compound of formula Z, formula Z19, formula Z18, Z17, Z16 or Z15, characterized in that it comprises the following steps:

[0113] Y is selected from halogen; preferably, Y is selected from chlorine or bromine; the groups at other positions are as defined in formula I above.

[0114] The present invention also provides a method for preparing the compound of formula Z, formula Z23, formula Z22 or formula Z21, characterized in that it comprises the following steps:

[0115] in,

[0116] Y is selected from halogen; preferably, Y is selected from chlorine or bromine;

[0117] R 100 is a hydroxyl protecting group; preferably, R 100 Selected from tert-butyldimethylsilyl, methyl, benzyl, p-methoxybenzyl, methoxymethyl;

[0118] The groups at other positions are as defined above in formula I.

[0119] In another aspect, the present invention further provides an intermediate compound, wherein the intermediate compound is selected from the following compounds:

[0120] in,

[0121] Y is selected from halogen; preferably, Y is selected from chlorine or bromine;

[0122] R 100 is a hydroxyl protecting group; preferably, R 100 Selected from tert-butyldimethylsilyl, methyl, benzyl, p-methoxybenzyl, methoxymethyl;

[0123] R1, R2, R3, R4, Ring A, m and q are as defined above in Formula I.

[0124] The present invention also provides an intermediate compound, which is selected from the following compounds:

[0125] In another aspect, the present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of at least one compound of formula I, or a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt or deuterated form thereof and at least one pharmaceutically acceptable excipient.

[0126] In another aspect, the present invention also provides the use of the above-mentioned compound, or its stereoisomers, tautomers, solvates, pharmaceutically acceptable salts or deuterated substances, and / or pharmaceutical compositions in the preparation of anti-MSI-H / dMMR tumor drugs.

[0127] In certain embodiments, the MSI-H / dMMR tumor is a microsatellite instability-high (MSI-H) or mismatch repair-deficient (dMMR) cancer selected from prostate cancer, endometrial cancer, colon adenocarcinoma, gastric adenocarcinoma, rectal adenocarcinoma, adrenocortical carcinoma, uterine carcinosarcoma, cervical squamous cell carcinoma, endocervical adenocarcinoma, esophageal cancer, breast cancer, renal clear cell carcinoma, and ovarian serous cystadenocarcinoma.

[0128] In another aspect, the present invention provides a method for modulating WRN activity in a subject, comprising administering to the subject a therapeutically effective amount of a compound of Formula I, or a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt or deuterated form thereof, or a pharmaceutical composition as described above.

[0129] In another aspect, the present invention provides a method for inhibiting WRN in a subject, comprising administering to the subject a therapeutically effective amount of the compound of formula I, or its stereoisomers, tautomers, solvates, pharmaceutically acceptable salts or deuterated substances, or the pharmaceutical composition described above.

[0130] In another aspect, the present invention provides a method for treating a disease by inhibiting WRN in a subject, comprising administering to the subject a therapeutically effective amount of the compound of formula I, or its stereoisomers, tautomers, solvates, pharmaceutically acceptable salts or deuterated substances, or the pharmaceutical composition described above.

[0131] In certain embodiments, the compound is a WRN inhibitor.

[0132] Definition and Description

[0133] General chemical terms used in the above structural formulae have their usual meanings.

[0134] For example, unless otherwise indicated, the terms "halo" and "halogen" as used herein refer to fluorine, chlorine, bromine or iodine. Preferred halogen groups include fluorine, chlorine and bromine.

[0135] In the present invention, unless otherwise specified, the term "alkyl" includes a linear or branched monovalent saturated hydrocarbon group, preferably a C 1-6 Alkyl, more preferably C 1-3 Alkyl. The alkyl groups used herein may be optionally substituted with one or more substituents. Non-limiting examples of alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 3-(2-methyl)butyl, 2-pentyl, 2-methylbutyl, neopentyl, n-hexyl, 2-hexyl, 2-methylpentyl, and the like. Similarly, “C 1-6 "C" in "alkyl" 1-6 ” refers to a group containing 1, 2, 3, 4, 5 or 6 carbon atoms in a straight or branched chain.

[0136] The terms "alkenyl" and "alkynyl" include straight or branched chain alkenyl and alkynyl groups. 2-6 Alkenyl" and "C 2-6 "Alkynyl" refers to an alkenyl or alkynyl group containing 2, 3, 4, 5 or 6 carbon atoms arranged in a straight chain or branched form.

[0137] The term "alkoxy" refers to the oxygen ether form of the aforementioned straight-chain or branched alkyl groups.

[0138] The term "haloalkyl" refers to the aforementioned "alkyl" substituted by one or more halogens.

[0139] The term "heteroalkyl" refers to an alkyl group in which one or more -CH2- atoms are replaced by heteroatoms selected from N, O and S, preferably C 1-6 Heteroalkyl, more preferably C 1-3 Heteroalkyl. A heteroalkyl group may be substituted or unsubstituted.

[0140] The term "heteroalkylene" refers to an alkylene group in which one or more -CH2- atoms are replaced by heteroatoms selected from N, O and S, preferably C 1-6 Heteroalkylene, more preferably C 1-3 Heteroalkylene. Heteroalkylene can be substituted or unsubstituted.

[0141] The term "hydroxy protecting group" refers to a group that is introduced on a hydroxy group and is easily removed, and is used to block or protect the hydroxy group while reacting on other functional groups of the compound. Non-limiting examples include trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), methyl, tert-butyl, allyl, benzyl, methoxymethyl (MOM), ethoxyethyl, 2-tetrahydropyranyl (THP), formyl, acetyl, benzoyl, p-nitrobenzoyl, etc.

[0142] In the present invention, "a", "an", "the", "at least one" and "one or more" are used interchangeably. Thus, for example, a composition comprising "a" pharmaceutically acceptable excipient can be interpreted to mean that the composition includes "one or more" pharmaceutically acceptable excipients.

[0143] The term "aryl" in the present invention, unless otherwise specified, refers to an unsubstituted or substituted monocyclic, fused or condensed aromatic group containing carbon atoms, preferably C 6-14 Aryl, more preferably C 6-10 Examples of these aromatic rings include, but are not limited to, phenyl and naphthyl.

[0144] The term "heteroaryl" refers to a monocyclic or polycyclic (e.g., fused bicyclic) aromatic heterocycle having at least one heteroatom ring member (e.g., 1 to 4 heteroatoms, or preferably 1 to 3 heteroatoms), wherein the heteroatoms are selected from N, O, and / or S. The nitrogen or sulfur heteroatom may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized. The heteroaryl group may be attached to any heteroatom or carbon atom to produce a stable structure. The heteroaryl group is preferably a 5-14 membered heteroaryl group, more preferably an 8-9 membered heteroaryl group, and more preferably a 5-6 membered heteroaryl group. Examples of heteroaryl groups include, but are not limited to, thienyl, furanyl, imidazolyl, isoxazolyl, oxazolyl, pyrazolyl, pyrrolyl, thiazolyl, thiadiazolyl, triazolyl, pyridinyl, pyridazinyl, indolyl, azaindolyl, indazolyl, benzimidazolyl, benzofuranyl, benzothienyl, benzisoxazolyl, benzoxazolyl, benzopyrazolyl, benzothiazolyl, benzothiadiazolyl, benzotriazolyladeninyl, quinolinyl, or isoquinolinyl.

[0145] The term "carbocyclyl" refers to a saturated or unsaturated cyclic group that is not aromatic. Depending on its degree of saturation, it may include "cycloalkyl", "cycloalkenyl" or "cycloalkynyl". Monocyclic carbocyclyl groups include, but are not limited to, cyclopropane, cyclobutane, cyclopentane, cyclohexane or cyclohexene and the like; polycyclic carbocyclyl groups include spirocyclic, fused ring, bridged ring carbocyclyl groups. The carbocyclyl is preferably C 3-14 Carbocyclic group, more preferably C 3-8 Carbocyclic group, more preferably C 3-6 Carbocyclic group.

[0146] The term "cycloalkyl" refers to saturated monocyclic and polycyclic ring systems containing only carbon atoms in the ring and may be optionally substituted with one or more substituents. "Cycloalkyl" may have ring systems including bridged rings, fused rings, and spiro rings. Non-limiting examples of cycloalkyl include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, spiro[3.4]octyl, bicyclo[2.2.1]heptane, and the like. The cycloalkyl is preferably C 3-14 Cycloalkyl, more preferably C 3-8 Cycloalkyl, more preferably C 3-6 Cycloalkyl.

[0147] The term "heterocyclic group" as used herein, unless otherwise indicated, refers to unsubstituted or substituted monocyclic and polycyclic ring systems consisting of carbon atoms and 1-3 heteroatoms selected from N, O or S, and includes saturated or unsaturated ring systems and polycyclic ring systems with partially unsaturated and / or aromatic moieties. Nitrogen or sulfur heteroatoms may be selectively oxidized, and nitrogen heteroatoms may be selectively quaternized. The heterocyclic group may be attached to any heteroatom or carbon atom to form a stable structure. It should be understood that polycyclic heterocycloalkyl groups may have ring systems including fused rings, bridged rings and spirocycles. The heterocyclic group is preferably a 3-14 membered heterocyclic group, more preferably a 3-8 membered heterocyclic group, and more preferably a 3-6 membered heterocyclic group. The heterocycloalkyl group used herein may optionally be substituted by one or more substituents. Examples of such heterocyclic groups include, but are not limited to, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, oxopiperazinyl, oxopiperidinyl, tetrahydrofuranyl, dioxolanyl, tetrahydroimidazolyl, tetrahydrothiazolyl, tetrahydrooxazolyl, tetrahydropyranyl, morpholinyl, thiomorpholinyl, thiomorpholinylsulfoxide, thiomorpholinylsulfone, and tetrahydrooxadiazolyl.

[0148] However, in no case will heterocyclyls and carbocyclyls intersect or include each other. Thus, according to the above definition, if at least one all-carbocycle is fused with a heterocyclyl to form a bi-, poly- or spiro-ring, it will still be defined as a heterocyclyl.

[0149] Additionally, if a heteroaryl group is fused with a heterocyclyl group to form a bi-, poly-, or spiro-ring, it will be defined as a heterocyclyl group rather than a heteroaryl group.

[0150] The term "substituted" means that one or more hydrogen atoms in a group are replaced by the same or different substituents. Typical substituents include, but are not limited to, deuterium, halogen (F, Cl, Br or I), oxo, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 6-14 Aryl, 5-14 membered heteroaryl, C 3-14 Cycloalkyl, 3-14 membered heterocyclic group, -OR 1 、-SR 1 , =O, =S, -C(O)R 1 、-C(S)R 1 、=NR 1 、-C(O)OR 1 、-C(S)OR 1 、-NR 1 R 2 、-C(O)NR 1 R 2 , cyano, nitro, -S(O)2R 1 、-OS(O2)OR 1、-OS(O)2R 1 、-OP(O)(OR 1 )(OR 2 ); where R 1 and R 2 Independently selected from H, C 1-6 Alkyl, C 1-6 haloalkyl; wherein said C 6-14 Aryl, 5-14 membered heteroaryl, C 3-14 Cycloalkyl, 3-14 membered heterocyclic group may be further optionally substituted with H, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkyl, C 1-6 Haloalkoxy, hydroxy, cyano, nitro, NH2, NHC 1-6 Alkyl, NHC 1-6 Haloalkyl, N(C 1-6 Alkyl)2, N(C 1-6 In some embodiments, the substituents are independently selected from the group consisting of -F, -Cl, -Br, -I, -OH, trifluoromethoxy, ethoxy, propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, -SCH 3 , -SC 2 H 5 , formaldehyde, -C(OCH 3 ), cyano, nitro, -CF 3 , -OCF 3 , amino, dimethylamino, methylthio, sulfonyl, and acetyl.

[0151] The term "pharmaceutically acceptable salt" refers to a salt prepared from a pharmaceutically acceptable base or acid. When the compound provided by the present invention is an acid, its corresponding salt can be conveniently prepared from a pharmaceutically acceptable base, including inorganic bases and organic bases.

[0152] Since the compound represented by Formula I is to be used as a medicine, it is preferably used with a certain purity, for example, at least 60% purity, more preferably at least 75% purity, and particularly preferably at least 98% purity (% is weight ratio).

[0153] As used herein, "deuterated" refers to a compound or chemical group formed when some or all of the hydrogen atoms in its structure are replaced by its isotope, deuterium. Any position specifically designated as "D" or "deuterium" is understood to have a deuterium enrichment of 50%, 80%, 90%, 95%, 98%, or 99%. "Deuterium enrichment" is a molar percentage determined by dividing the number of deuterium-containing compounds at the designated position by the total number of all compounds. When a position is designated as "H" or "hydrogen," hydrogen is present at its natural abundance at that position. When a position is silent regarding the presence of hydrogen or deuterium, hydrogen is present at its natural abundance at that position. A specific alternative embodiment relates to compounds of the present disclosure having a deuterium enrichment of at least 5%, 10%, 25%, 50%, 80%, 90%, 95%, 98%, or 99% at one or more positions not specifically designated as "D" or "deuterium." In some embodiments, one or more hydrogen atoms of any compound described herein can be replaced with deuterium to provide the corresponding labeled or enriched compound.

[0154] Prodrugs of the compounds of the present invention are included within the scope of the present invention. Generally, the prodrug refers to a functional derivative that is easily converted into the desired compound in vivo. For example, any pharmaceutically acceptable salt, ester, salt of an ester, or other derivative of the compounds of the present invention, which, upon administration to a recipient, can directly or indirectly provide the compounds of the present invention or their pharmaceutically active metabolites or residues. Particularly preferred derivatives or prodrugs are those that can increase the bioavailability of the compounds of the present invention when administered to a patient (e.g., by making orally administered compounds more readily absorbed into the blood), or those that promote the delivery of the parent compound to biological organs or sites of action (e.g., the brain or lymphatic system). Therefore, the term "administration" in the treatment methods provided herein refers to the administration of the compounds of the present invention that can treat various diseases, or, although not explicitly disclosed, can be converted into the compounds of the present invention in vivo upon administration to a subject. Conventional methods for selecting and preparing suitable prodrug derivatives are described in books such as "Design of Prodrugs" (ed. H. Bundgaard, Elsevier, 1985).

[0155] Obviously, the definition of any substituent or variable at a particular position in a molecule is independent of the definition of any other position in the molecule. It is readily understood that one skilled in the art can select substituents or substitution patterns for the compounds of the present invention using existing techniques and the methods described herein to obtain chemically stable and readily synthesized compounds.

[0156] The compounds of the present invention may contain one or more asymmetric centers and may thus produce diastereomers and optical isomers. The present invention includes all possible diastereomers and racemic mixtures thereof, their substantially pure resolved enantiomers, all possible geometric isomers and pharmaceutically acceptable salts thereof.

[0157] The above-described Formula I does not precisely define the stereostructure of a particular position of the compound. The present invention encompasses all stereoisomers of the compound represented by Formula I and pharmaceutically acceptable salts thereof. Furthermore, mixtures of stereoisomers and isolated specific stereoisomers are also encompassed by the present invention. During the synthetic process for preparing such compounds, or using racemization or epimerization processes known to those skilled in the art, the resulting product may be a mixture of stereoisomers.

[0158] When the compound represented by Formula I exists in tautomers, unless otherwise stated, the present invention includes any possible tautomers and pharmaceutically acceptable salts thereof, and mixtures thereof.

[0159] When the compound represented by Formula I and its pharmaceutically acceptable salts exist as solvates or polymorphs, the present invention includes any possible solvates and polymorphs. The type of solvent that forms the solvate is not particularly limited, as long as the solvent is pharmaceutically acceptable.

[0160] The term "composition," as used herein, is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product produced, directly or indirectly, from combination of the specified ingredients in the specified amounts. Thus, pharmaceutical compositions containing a compound of the invention as an active ingredient, as well as methods for preparing the compounds of the invention, are also part of this invention.

[0161] In addition, some crystalline forms of the compound can exist as polymorphs, and such polymorphs are included in the present invention. In addition, some compounds can form solvates with water (i.e. hydrates) or common organic solvents, and such solvates also fall within the scope of the present invention.

[0162] The pharmaceutical composition provided by the present invention includes the compound shown in formula I as active ingredient (or its pharmaceutically acceptable salt), a pharmaceutically acceptable excipient and other optional therapeutic components or adjuvants. Although in any given case, the most suitable active ingredient administration mode depends on the specific main body, main body property and the severity of the disease being administered, the pharmaceutical composition of the present invention includes a pharmaceutical composition suitable for oral, rectal, local and parenteral (including subcutaneous administration, intramuscular injection, intravenous administration) administration. The pharmaceutical composition of the present invention can be easily prepared in any preparation method known in the art and in unit dosage form.

[0163] The pharmaceutical compositions of the present invention can be prepared by any pharmaceutical method. Generally, such methods include the step of combining the active ingredient with a carrier comprising one or more essential ingredients. Typically, the pharmaceutical compositions are prepared by uniformly and intimately mixing the active ingredient with a liquid carrier, a finely divided solid carrier, or a mixture thereof. Furthermore, the product can be readily prepared to a desired appearance.

[0164] Therefore, the pharmaceutical composition of the present invention comprises a pharmaceutically acceptable carrier and a compound of Formula I or its stereoisomers, tautomers, polymorphs, solvates, pharmaceutically acceptable salts, or prodrugs. Combinations of the compound of Formula I or its pharmaceutically acceptable salts with one or more other therapeutically active compounds are also included in the pharmaceutical composition of the present invention.

[0165] The drug carrier used in the present invention can be, for example, a solid carrier, a liquid carrier or a gaseous carrier.

[0166] The compounds of the present invention have excellent biological activities, such as in vitro enzymatic activity against WRN ATPase, cell proliferation inhibitory activity against MSI-H, and pharmacokinetic activity, demonstrating their potential as WRN inhibitors. They have broad application prospects and can provide patients with more and better treatment options. BRIEF DESCRIPTION OF THE DRAWINGS

[0167] Figure 1: Changes in expression of the biomarker γH2AX corresponding to compound A192 and comparative compound A;

[0168] Figure 2: Changes in expression of biomarkers WRN protein, γH2AX, and p21 corresponding to compound A231 and comparative compound A;

[0169] Figure 3: Changes in tumor volume after administration of Compound A192 and control Compound A in mice bearing SW48 xenograft models;

[0170] Figure 4: Body weight changes after administration of Compound A192 and Comparative Compound A in SW48 xenograft model mice. DETAILED DESCRIPTION

[0171] To make the above content clearer and more specific, the present invention will further illustrate the technical solutions of the present invention with the following examples. The following examples are only used to illustrate the specific embodiments of the present invention so that those skilled in the art can understand the present invention, but are not intended to limit the scope of protection of the present invention. In the specific embodiments of the present invention, technical means or methods not specifically described are conventional technical means or methods in the art.

[0172] Unless otherwise indicated, all parts and percentages herein are by weight and all temperatures are in degrees Celsius.

[0173] The following abbreviations are used in the examples:

[0174] AcOH: acetic acid;

[0175] ACN: acetonitrile;

[0176] BINAP: 1,1'-binaphthyl-2,2'-bis(diphenylphosphine);

[0177] CbzCl: benzyl chloroformate;

[0178] EDCI: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride;

[0179] ESI-MS: electrospray ionization mass spectrometry;

[0180] DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene;

[0181] DCM: dichloromethane;

[0182] DIEA: N,N-diisopropylethylamine;

[0183] DMAP: 4-dimethylaminopyridine;

[0184] DMF: N,N-dimethylformamide;

[0185] EtOAc: ethyl acetate;

[0186] HATU: O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate;

[0187] HOBt: 1-hydroxybenzotriazole;

[0188] LCMS: liquid chromatography-mass spectrometry;

[0189] mCPBA: meta-chloroperbenzoic acid;

[0190] MeOH: methanol;

[0191] NBS: N-bromosuccinimide;

[0192] NMP: N-methylpyrrolidone;

[0193] TFA: trifluoroacetic acid;

[0194] TMSOTf: trimethylsilyl trifluoromethanesulfonate;

[0195] TsOH·H2O: p-toluenesulfonic acid monohydrate;

[0196] THF: tetrahydrofuran;

[0197] PE: petroleum ether;

[0198] PLC: thin layer chromatography;

[0199] PMBCl: 4-methoxybenzyl chloride;

[0200] Pd(PPh3)2Cl2: bis(triphenylphosphine)palladium dichloride;

[0201] PyBOP: 1H-benzotriazol-1-yloxytripyrrolidino hexafluorophosphate;

[0202] Py.: pyridine;

[0203] Pd2dba3: tris(dibenzylideneacetone)dipalladium;

[0204] PTSA: p-toluenesulfonic acid;

[0205] pre-HPLC: preparative high performance liquid chromatography;

[0206] Xphos Pd G3: methanesulfonate (2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)(2'-amino-1,1'-biphenyl-2-yl)palladium(II).

[0207] The raw materials and reagents used in the examples can be obtained from commercial sources or prepared by conventional methods in the art.

[0208] Synthesis of intermediate B1

[0209] 3-Bromo-1H-1,2,4-triazol-5-amine

[0210] In a 3000 mL single-necked flask, 3,5-diamino-1H-1,2,4-triazole (130.0 g, 1.51 mol) was added to a 48% aqueous hydrobromic acid solution (700 mL). The mixture was cooled to 0°C, and a solution of NaNO2 (156.3 g, 2.26 mol) and H2O (300 mL) was slowly added dropwise over 1 hour. The mixture was then allowed to react at room temperature for 1 hour. The reaction was continued at 100°C for 15 hours. The mixture was cooled to room temperature and filtered. The filtrate was adjusted to a neutral pH of 7 (1N NaOH), extracted with EtOAc (3 x 20 L), dried over anhydrous sodium sulfate, and concentrated to afford Intermediate B1 (113.9 g, 50% yield) as a white solid. ESI-MS m / z = 162.98 [M+H] + .

[0211] Synthesis of intermediate B2

[0212] tert-Butyl 4-(1-methoxy-1,3-dioxolane-2-yl)piperazine-1-carboxylate

[0213] Step 1: Synthesis of intermediate B2-1

[0214] In a 3000 mL single-necked flask, the substrate, methyl 3-oxopentanoate (130 g, 1 mol), was added to the reaction flask. DCM (700 mL) was then added. NBS (178 g, 1 mol) and TsOH·H2O (38 g, 0.2 mol) were then added at 0°C. The reaction was stirred at room temperature for 3 hours. LCMS was used to monitor the reaction. Upon completion, stirring was discontinued. The mixture was filtered, and the filter cake was rinsed with 300 mL of dichloromethane. The filtrate was extracted with water (300 mL x 2). The aqueous phase was then back-extracted with DCM (300 mL x 2). The organic phases were combined, dried, and the solvent removed by rotary evaporation to obtain crude product B2-1 (180 g, 90% yield). ESI-MS m / z = 209.17, 211.17 [M+H] + .

[0215] Step 2: Synthesis of intermediate B2

[0216] In a 3000 mL single-necked flask, substrate B2-1 (180 g, 0.86 mol) was added to the reaction flask. Acetonitrile (1.8 L) was then added, along with potassium carbonate (298 g, 2.16 mol) and N-Boc-piperazine (192 g, 1.03 mol). The reaction was stirred at room temperature for 3 hours. LCMS was used to monitor the reaction. Upon completion, stirring was discontinued. The mixture was filtered, and the filter cake was rinsed with ethyl acetate (0.5 L) and the solvent removed by rotary evaporation. The sample was mixed with silica gel and passed through a column. The eluent was changed from pure petroleum ether to a ratio of petroleum ether:ethyl acetate (2:1). The product B2 (150 g, 95% yield) was obtained by concentration. ESI-MS m / z = 315.18 [M+H] + .

[0217] Synthesis of intermediate B3

[0218] 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

[0219] In a 1000 mL single-necked bottle, B1 (20.00 g, 122.72 mmol), B2 (36.65 g, 116.58 mmol), DBU (37.37 g, 245.44 mmol), and ACN (300 mL) were mixed and allowed to react at room temperature for 3 days. The starting material disappeared after monitoring by LCMS. The product was concentrated under reduced pressure and purified by column chromatography using DCM / MeOH = 12 / 1 to afford product B3 (29.10 g, 55% yield). ESI-MS m / z = 371.10 [M+H-56] + .

[0220] Synthesis of intermediate B4

[0221] tert-Butyl 4-(2-(3,6-dihydro-2H-pyran-4-yl)-5-ethyl-7-oxo-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)piperazine-1-carboxylate

[0222] In a 100 mL single-necked vial, B3 (1.02 g, 2.39 mmol), 3,6-dihydro-2H-pyran-4-boronic acid pinacol ester (0.75 g, 3.58 mmol), Xphos Pd G3 (0.20 g, 0.24 mmol), potassium phosphate (1.52 g, 7.16 mmol), 1,4-dioxane (20 mL), and H2O (10 mL) were mixed thoroughly. After replacing the atmosphere with N2, the mixture was reacted at 80°C for 16 h. LCMS indicated that the starting material was nearly complete. The mixture was concentrated under reduced pressure and purified by column chromatography using DCM / MeOH = 15 / 1 to afford product B4 (0.81 g, 79% yield). ESI-MS m / z = 431.23 [M+H] + .

[0223] Synthesis of intermediate B5:

[0224] 2-(6-(4-(tert-Butoxycarbonyl)piperazin-1-yl)-2-(3,6-dihydro-2H-pyran-4-yl)-5-ethyl-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)acetic acid

[0225] Step 1: Synthesis of intermediate B5-1

[0226] In a 250 mL single-necked flask, Intermediate B3 (7.0 g, 16.38 mmol) was added to DMF (100.0 mL), followed by K2CO3 (11.3 g, 81.91 mmol), and then tert-butyl bromoacetate (9.6 g, 49.15 mmol) was added dropwise. The reaction was allowed to proceed at 80°C for 12 h. The mixture was cooled to room temperature, added to ice water, and extracted with EtOAc (3 x 500 mL). The mixture was washed with saturated brine, and the organic phases were combined, concentrated under reduced pressure, and purified by column chromatography using DCM / MeOH = 15 / 1 to afford Intermediate B5-1 (7.0 g, 79% yield). ESI-MS m / z = 541.45 [M+H] + .

[0227] Step 2: Synthesis of intermediate B5-2

[0228] In a 250 mL single-necked bottle, intermediate B5-1 (7.0 g, 12.94 mmol), 3,6-dihydro-2H-pyran-4-boronic acid pinacol ester (5.4 g, 25.87 mmol), XPhos Pd G3 (2.2 g, 2.59 mmol), and K3PO4 (8.2 g, 38.81 mmol) were added to 1,4-dioxane (60.0 mL) and H2O (15.0 mL) in sequence and reacted at 80°C for 2 h. The reaction solution was added with water, saturated sodium bicarbonate, and extracted with EtOAc (3 x 100 mL). The mixture was washed with saturated brine and dried over anhydrous sodium sulfate. The organic phases were combined, concentrated under reduced pressure, and purified by column chromatography using DCM / MeOH = 15 / 1 to obtain the intermediate B5-2 (5.5 g, yield: 78%). ESI-MS m / z = 544.65 [M+H] + .

[0229] Step 3: Synthesis of intermediate B5

[0230] In a 250 mL single-necked flask, B5-3 (5.0 g, 9.17 mmol) and LiOH·H2O (5.8 g, 137.55 mmol) were added to THF (60.0 mL) and H2O (20.0 mL) and allowed to react at room temperature for 12 h. The THF was removed by distillation under reduced pressure, and the mixture was dissolved in 20 mL of water and washed with EtOAc (2 x 50 mL). The aqueous phase was adjusted to a neutral pH of 3 (1N HCl) and extracted with EtOAc (3 x 100 mL). The mixture was dried over anhydrous sodium sulfate and distilled under reduced pressure to afford intermediate B5 (3.0 g, 67% yield) as a yellow solid. ESI-MS m / z = 488.55 [M+H] + .

[0231] Synthesis of intermediate B6:

[0232] 2-Iodo-N-(2-methyl-4-(trifluoromethyl)phenyl)acetamide

[0233] Step 1: Synthesis of intermediate B6-1

[0234] In a 250 mL single-necked flask, 2-methyl-4-(trifluoromethyl)aniline (10.00 g, 57.09 mmol) was dissolved in DCM (100 mL). Triethylamine (14.44 g, 142.73 mmol) was added, and the temperature was maintained below 10°C with ice water. 2-Chloroacetyl chloride (7.74 g, 68.51 mmol) was added dropwise. After addition, the mixture was allowed to warm to room temperature and react for 2.0 h. The reaction system became clear, as monitored by LCMS. The reaction was then diluted with DCM, the separated liquid was washed with water, dried, and concentrated to afford Intermediate B6-1 (13.20 g, 92% yield). ESI-MS m / z = 250.03 [MH] - .

[0235] Step 2: Synthesis of intermediate B6

[0236] In a 250 mL single-necked bottle, intermediate B6-1 (13.00 g, 51.66 mmol) was dissolved in acetone (130 mL), and potassium iodide (12.86 g, 77.49 mmol) was added. The mixture was heated to 75°C and reacted for 4-5 h. The reaction was monitored by LCMS. The mixture was cooled, filtered, concentrated, and column chromatography was performed using PE:EtOAc = 100:0 to 70:30 to obtain intermediate B6 (17.12 g, yield: 96%). ESI-MS m / z = 341.97 [MH] - .

[0237] Synthesis of intermediate B7

[0238] tert-Butyl 4-(2-bromo-5-ethyl-4-(2-(2-methyl-4-(trifluoromethyl)phenyl)amino)-2-oxoethyl)-7-oxo-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)piperazine-1-carboxylate

[0239] In a 100 mL single-necked vial, Intermediate B3 (1.38 g, 3.23 mmol), Intermediate B6 (1.22 g, 3.55 mmol), DIEA (1.25 g, 9.69 mmol), and 1,4-dioxane (17 mL) were mixed and reacted at 80°C for 3 h. The reaction was complete as monitored by LCMS. The product was then concentrated under reduced pressure. The crude product was diluted with EtOAc and washed once with water. The EA phase was separated and concentrated under reduced pressure. Purification by column chromatography using a PE / EtOAc ratio of 1 / 1 afforded Intermediate B7 (1.66 g, 80% yield). ESI-MS m / z = 586.16 [M+H-56] + .

[0240] Synthesis of intermediate B8

[0241] 2-(6-Bromo-2-(3,6-dihydro-2H-pyran-4-yl)-5-ethyl-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)-N-(2-methyl-4-(trifluoromethyl)phenyl)acetamide

[0242] Step 1: Synthesis of intermediate B8-1

[0243] In a 100 mL single-necked bottle, intermediate B1 (6.20 g, 38.04 mmol) was dissolved in glacial acetic acid (40.0 mL). Methyl propionylacetate (6.44 g, 49.46 mmol) was added and the mixture was heated to 80°C and stirred for 16 h. The mixture was cooled to room temperature and dried by spin drying. The mixture was then purified by column chromatography using DCM / MeOH (100 / 1 to 95 / 5) to obtain intermediate B8-1 (5.26 g, 57% yield). ESI-MS m / z = 242.98 [M+H] + .

[0244] Step 2: Synthesis of intermediate B8-2

[0245] The synthesis of intermediate B8-2 was prepared by referring to the synthesis method of intermediate B7.

[0246] Step 3: Synthesis of intermediate B8-3

[0247] The synthesis of intermediate B8-2 was prepared by referring to the synthesis method of intermediate B5-2.

[0248] Step 4: Synthesis of intermediate B8

[0249] In a 100 mL single-necked vial, intermediate B8-3 (1.07 g, 2.32 mmol) was dissolved in acetonitrile (11.00 mL). N-bromosuccinimide (0.45 g, 2.55 mmol) was added and stirred at room temperature overnight. LCMS monitored the reaction until the starting material was almost completely reacted. 50 mL of DCM / MeOH (10:1) and 20 mL of water were added and separated. The aqueous phase was washed twice with 50 mL of DCM / MeOH (10:1). The combined organic phases were washed with 20 mL of saturated brine, dried over anhydrous sodium sulfate, and dried by spin drying. The product, intermediate B8 (0.88 g, 70% yield), was obtained by column chromatography with DCM / MeOH (100:1 to 9:1). ESI-MS m / z = 560.02 [M+H] + .

[0250] Synthesis of intermediate B9

[0251] 2-(2-(3,6-dihydro-2H-pyran-4-yl)-5-ethyl-7-oxo-6-(piperazin-1-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)-N-(2-methyl-4-(trifluoromethyl)phenyl)acetamide

[0252] Step 1: Synthesis of intermediate B9-1

[0253] In a 100 mL single-necked bottle, Intermediate B7 (1.63 g, 2.54 mmol), 3,6-dihydro-2H-pyran-4-boronic acid pinacol ester (0.80 g, 3.81 mmol), Xphos Pd G3 (0.11 g, 0.13 mmol), potassium phosphate (1.62 g, 7.61 mmol), 1,4-dioxane (20 mL), and H2O (10 mL) were mixed. After replacing the atmosphere with N2, the mixture was reacted at 80°C for 1 h. LCMS indicated that the starting materials had almost completely reacted. DCM (40 mL) and water (20 mL) were added for separation. The aqueous phase was extracted with DCM (40 mL) two to three times. The combined organic phases were concentrated under reduced pressure and purified by column chromatography using a DCM / MeOH ratio of 15:1 to afford Intermediate B9-1 (1.62 g, 99% yield). ESI-MS m / z=590.29[M+H-56] + .

[0254] Step 2: Synthesis of intermediate B9

[0255] To a 100 mL reaction flask, Intermediate 9-1 (1.60 g, 2.48 mmol), DCM (15 mL), and a 4 M HCl solution in 1,4-dioxane (15 mL, 60.00 mmol) were added and allowed to react at room temperature for 1 h. LCMS monitored the reaction completion of the starting material. The mixture was then concentrated under reduced pressure to afford Intermediate B9 (1.33 g, 98% yield). ESI-MS m / z = 546.10 [M+H] + .

[0256] Synthesis of intermediate B10

[0257] 2-(6-(4-(tert-Butoxycarbonyl)piperazin-1-yl)-5-ethyl-2-(1-methyl-1H-pyrazol-4-yl)-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)acetic acid

[0258] Step 1: Synthesis of intermediate B10-1

[0259] In a 100 mL reaction flask, Intermediate B5-1 (2.00 g, 3.69 mmol), 1-methylpyrazole-4-boronic acid pinacol ester (0.92 g, 4.43 mmol), XPhos Pd G3 (0.47 g, 0.55 mmol), and potassium phosphate (2.35 g, 11.08 mmol) were dissolved in 1,4-dioxane (25 mL) and water (5 mL). The atmosphere in the reaction system was replaced with nitrogen three times. Under nitrogen protection, the reaction was incubated at 99°C for 4 hours. LCMS monitored the reaction to be nearly complete. The mixture was cooled to room temperature and concentrated. The residue was dissolved in dichloromethane (50 mL) and washed sequentially with tap water (50 mL) and saturated brine (50 mL). The organic layer was concentrated to dryness, and the residue was directly used in the next step.

[0260] Step 2: Synthesis of intermediate B10

[0261] In a 100 mL reaction flask, compound intermediate B10-1 (2.00 g, 3.69 mmol) and lithium hydroxide monohydrate (1.55 g, 36.86 mmol) were dissolved in tetrahydrofuran (25 mL) and water (8 mL) and stirred at 25°C for 16 h. LCMS monitored the reaction as nearly complete. The THF was evaporated after concentration, and the remaining aqueous phase was diluted to 40 mL and extracted with EtOAc (25 mL x 3). The aqueous phase was adjusted to pH 4-5 with 1.4 M dilute hydrochloric acid, resulting in a viscous material. This was extracted with EtOAc (30 mL x 3). The EtOAc phases were combined and washed sequentially with tap water (30 mL) and saturated brine (30 mL). The EtOAc phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated to dryness, and the residue was sonicated with anhydrous ether (30 mL). The resulting filter cake was filtered and dried to obtain the desired product. ESI-MS m / z=485.29[MH] - .

[0262] Example 2 Synthesis of Compound A2

[0263] 2-(2-(3,6-dihydro-2H-pyran-4-yl)-5-ethyl-6-(4-(2-hydroxybenzoyl)piperazin-1-yl)-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)-N-(2-methyl-4-(trifluoromethyl)phenyl)acetamide

[0264] Synthesis of compound A2

[0265] In a 25 mL single-necked vial, 1-chloro-N,N,2-trimethylpropenamine (27 mg, 0.20 mmol) was added to a solution of 2-hydroxybenzoic acid (25 mg, 0.18 mmol) in DCM (5 mL). The mixture was allowed to react at room temperature for 1 h. A solution of Intermediate B9 (50 mg, 0.09 mmol) and DIEA (118 mg, 0.92 mmol) in DCM (5 mL) was then added and the mixture was allowed to react at room temperature for 2 h. The reaction was complete as monitored by LCMS. Water (5 mL) was added to quench the reaction. The aqueous phase was extracted with DCM (5 mL) two to three times. The organic phases were combined, concentrated under reduced pressure, and purified by column chromatography using a DCM / MeOH ratio of 15:1 to afford product A2 (5 mg, 8% yield). ESI-MS m / z = 666.26 [M+1] + .

[0266] Example 22 Synthesis of Compound A22

[0267] 2-(5-ethyl-6-(4-(3-hydroxypyridinoyl)piperazin-1-yl)-2-(1-methyl-1H-pyrazol-4-yl)-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)-N-(2-methyl-4-(trifluoromethyl)phenyl)acetamide

[0268] Step 1: Synthesis of compound A22-1

[0269] In a 100 mL single-necked bottle, (1-methyl-1H-pyrazol-4-yl)boronic acid (78.4 mg, 0.62 mmol), tert-butyl 4-[8-bromo-4-ethyl-5-[[2-methyl-4-(trifluoromethyl)phenyl]carbamoylmethyl]-2-oxo-1,5,7,9-tetraazabicyclo[4.3.0]nona-3,6,8-trien-3-yl]piperazine-1-carboxylate (200 mg, 0.31 mmol), XPhos Pd G3 (79 mg, 0.09 mmol) and K3PO4 (198 mg, 0.93 mmol) were added 1,4-dioxane (10.00 mL) and water (2.00 mL). After nitrogen replacement, the temperature was raised to 100 ° C and the reaction was reacted for 3 hours. The reaction was completed by LCMS. Water (20 mL) was added to the reaction solution and extracted with ethyl acetate (50 mL). x2), the organic phase was dried by spin drying, and column chromatography was performed using PE / EA = 2 / 1 to obtain product A22-1 (150.00 mg, yield: 75%). ESI-MS m / z = 644.21 [M+H] + .

[0270] Step 2: Synthesis of compound A22-2

[0271] In a 50 mL single-necked vial, A22-1 (150 mg, 0.23 mmol) was dissolved in ethyl acetate (10 mL). A 4 M HCl solution in 1,4-dioxane (4 mL) was added and the mixture was allowed to react at room temperature for 4 hours. The reaction was monitored by LCMS until complete. The product A22-2 (107 mg, 85% yield) was obtained by spin drying. ESI-MS m / z = 544.15 [M+H] + .

[0272] Step 3: Synthesis of compound A22

[0273] In a 25 mL single-necked vial, 3-hydroxypyridinic acid (15 mg, 0.11 mmol) was suspended in DCM (2 mL). 1-Chloro-N,N,2-trimethylpropenamine (20 mg, 0.15 mmol) was added and allowed to react at room temperature for 1.5 hours. A mixture of A22-2 (54 mg, 0.1 mmol) and DIEA (52 mg, 0.3 mmol) in DCM (2 mL) was then added. The reaction was continued at room temperature for 1 hour. LCMS monitored the reaction for completion. 10 mL of water was added to the reaction solution, and the mixture was extracted with dichloromethane (10 mL x 2). The organic phase was spin-dried and reversed to afford product A22 (20.2 mg, 33% yield). ESI-MS m / z = 665.35 [M+H] + .

[0274] 1 H NMR(500MHz, DMSO-d6)δ:10.45(s,1H),10.04(s,1H),8.30(d,J=2.5Hz,1H),8.07-8.05(m,1H),7.91(d,J=2.5H z,1H),7.75-7.70(m,1H),7.63(s,1H),7.53(d,J=8.5Hz,1H),7.33-7.25(m,2H),5.27(s,2H),4.56(d,J=12.7H z,1H),3.91(d,J=2.5Hz,3H),3.50(s,1H),3.47(d,J=11.4Hz,1H),3.42(d,J=12.9Hz,1H),3.23(t,J=12.2Hz,1 H),3.04-2.92(m,3H),2.81(d,J=11.3Hz,1H),2.63(d,J=10.8Hz,1H),2.37(d,J=2.4Hz,3H),1.21-1.19(m,3H).

[0275] Example 28 Synthesis of Compound A28

[0276] 2-(2-(3,6-dihydro-2H-pyran-4-yl)-5-ethyl-6-(4-((3-hydroxypyridin-2-yl)methoxy)phenyl)-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)-N-(2-methyl-4-(trifluoromethyl)phenyl)acetamide

[0277] Step 1: Synthesis of compound A28-1

[0278] In a 100 mL single-necked flask, 3-hydroxypyridine-2-methanol (400 mg, 3.2 mmol) was dissolved in DMF (10.0 mL). PMBCl (0.52 mL, 3.84 mmol) and Cs2CO3 (2.08 g, 6.39 mmol) were added. The temperature was raised to 60°C and the reaction was allowed to react overnight. LCMS confirmed the reaction was complete. The reaction solution was poured into water and extracted with ethyl acetate (50 mL x 2). The organic phase was dried by spin drying and purified by column chromatography using PE / EtOAc = 1 / 1 to obtain product A28-1 (480.0 mg, yield: 61%). ESI-MS m / z = 246.11 [M+H] + .

[0279] Step 2: Synthesis of compound A28-2

[0280] In a 100 mL single-necked flask, triphenylphosphine (209 mg, 0.8 mmol) was dissolved in toluene (10 mL). DIEA (138 mg, 0.8 mmol), A28-1 (130 mg, 0.53 mmol), and 4-hydroxyphenylboronic acid pinacol ester (116 mg, 0.53 mmol) were added. The mixture was heated to 100°C and reacted for 4 hours. LCMS monitored the reaction for completion. The reaction solution was poured into water and extracted with EtOAc (50 mL x 2). The organic phase was dried by spin drying and purified by column chromatography using a PE / EA ratio of 1 / 3 to afford A28-2 (185 mg, 78% yield). ESI-MS m / z = 448.20 [M+H] + .

[0281] Step 3: Synthesis of compound A28-3

[0282] In a 100 mL single-necked bottle, (2-[3-bromo-8-(3,6-dihydro-2H-pyran-4-yl)-4-ethyl-2-oxo-1,5,7,9-tetraazabicyclo[4.3.0]nona-3,6,8-trien-5-yl]-N-[2-methyl-4-(trifluoromethyl)phenyl]acetamide (150 mg, 0.28 mmol), A28-2 (186 mg, 0.42 mmol), XPhos Pd G3 (26 mg, 0.03 mmol) and K3PO4 (176 mg, 0.83 mmol) were added 1,4-dioxane (10.00 mL) and water (2.00 mL). After nitrogen replacement, the temperature was raised to 100 ° C and the reaction was reacted for 3 hours. The reaction was completed by liquid chromatography-mass spectrometry. The mixture was extracted with EtOAc (50 mL) x2), the organic phase was dried by spin drying, and column chromatography was performed using PE / EtOAc = 1 / 10 to obtain product A28-3 (160 mg, yield: 74%). ESI-MS m / z = 781.15 [M+H] + .

[0283] Step 4: Synthesis of Compound A28

[0284] In a 50 mL single-necked vial, A28-3 (100 mg, 0.13 mmol) was dissolved in DCM (4 mL). TFA (2.0 mL) was added and the mixture was allowed to react at room temperature for 2 hours. The reaction was monitored by LCMS for completion. The mixture was then spin-dried and the product A28 (20.5 mg, yield: 24%) was obtained by reverse phase chromatography. ESI-MS m / z = 661.39 [M+H] + .

[0285] 1 H NMR(500MHz,DMSO-d6)δ:10.23(s,1H),10.03(s,1H),8.10-8.05(m,1H),7.77-7.71(m,2H),7.70-7.67( m,1H),7.63(s,1H),7.54(d,J=8.6Hz,1H),7.32-7.23(m,2H),7.22-7.17(m,2H),7.14-7.09(m,2H),6.86 (s,1H),5.28(s,2H),5.15(s,2H),4.28(q,J=2.8Hz,2H),3.82(s,3H),2.58-2.54(m,4H),1.09(t,J=7.5Hz,3H).

[0286] Example 35 Synthesis of Compound A35

[0287] N-(4-(2-(3,6-dihydro-2H-pyran-4-yl)-5-ethyl-4-(2-((2-methyl-4-(trifluoromethyl)phenyl)amino)-2-oxoethyl)-7-oxo-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3-fluorophenyl)-3-hydroxypyridine-2-amide

[0288] Step 1: Synthesis of compound A35-1

[0289] In a 50 mL single-necked vial, compound intermediate B8 (120 mg, 0.22 mmol), 4-amino-2-fluorophenylboronic acid pinacol ester (74 mg, 0.31 mmol), XPhos Pd G3 (38 mg, 0.04 mmol), and potassium phosphate (141 mg, 0.67 mmol) were dissolved in 1,4-dioxane (6 mL) and water (1.2 mL). The mixture was reacted at 93°C under nitrogen for 1.5 hours. LCMS indicated that the reaction was nearly complete. The mixture was cooled to room temperature and concentrated. The residue was dissolved in dichloromethane (30 mL) and washed sequentially with tap water (30 mL) and saturated brine (30 mL). The organic layer was concentrated and purified by PLC using a 20 / 1 ratio of DCM / MeOH to afford A35-1 (126 mg, 99% yield).

[0290] Step 2: Synthesis of compound A35

[0291] In a 50 mL single-necked vial, compound A35-1 (70 mg, 0.12 mmol), 3-hydroxypyridine-2-carboxylic acid (34 mg, 0.25 mmol), HOBt (25 mg, 0.18 mmol), EDCI (35 mg, 0.18 mmol), and DMAP (15 mg, 0.12 mmol) were dissolved in anhydrous DCM (1.5 mL) and anhydrous DMF (0.15 mL). DIEA (127 mg, 0.98 mmol) was then added. After addition, the mixture was stirred at room temperature for 4 h and at 35°C for 2 days. LCMS monitoring was used to monitor the reaction mixture. The reaction mixture was dissolved in dichloromethane (30 mL), washed sequentially with tap water (30 mL) and saturated brine (30 mL). The organic layer was concentrated, and the residue was purified by prep-HPLC to afford product A35 (26 mg, 29% yield). ESI-MS m / z = 692.42 [M+H] + .

[0292] 1H NMR(500MHz,DMSO-d6)δ11.94(s,1H),11.26(s,1H),10.08(s,1H),8.29(s,1H),7.96-7.89( m,1H),7.83(d,J=8.2Hz,1H),7.75(d,J=8.4Hz,1H),7.63(dd,J=8.3,4.4Hz,2H),7.53(dd,J= 14.3,8.4Hz,2H),7.38(t,J=8.4Hz,1H),6.88(s,1H),5.32(s,2H),4.28(d,J=2.3Hz,2H),3.8 2(t,J=5.4Hz,2H),3.39-3.23(m,2H),2.57-2.51(m,2H),2.37(s,3H),1.09(t,J=7.5Hz,3H).

[0293] Example 39 Synthesis of Compound A39

[0294] 2-(2-(Dimethylamino)-5-ethyl-6-(4-(3-hydroxypyridinoyl)piperazin-1-yl)-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)-N-(2-methyl-4-(trifluoromethyl)phenyl)acetamide

[0295] Step 1: Synthesis of compound A39-1:

[0296] Intermediate B7 (200 mg, 0.31 mmol), dimethylamine hydrochloride (50 mg, 0.62 mmol), DIEA (0.26 mL, 1.56 mmol), and DMF (5 mL) were mixed and reacted at 120°C for 6 h. LCMS monitored the reaction completion. The reaction solution was added dropwise to 20 mL of water, filtered, and dried under vacuum to obtain compound A39-1 (150 mg, 80% yield). ESI-MS m / z = 607.29 [M+H] + .

[0297] Step 2: Synthesis of compound A39-2:

[0298] Compound A39-1 (150 mg, 0.25 mmol) was added to a 4 M HCl solution in 1,4-dioxane (5 mL) and allowed to react at room temperature for 2 h. LCMS monitored the reaction completion and the product was directly dried and used in the next step. ESI-MS m / z = 507.24 [M+H] + .

[0299] Step 3: Synthesis of compound A39:

[0300] The synthesis method of compound A39 was prepared by referring to the synthesis method of compound A35. ESI-MS m / z=628.25[M+H] + .

[0301] Example 41 Synthesis of Compound A41

[0302] 2-(2-(1-ethoxyvinyl)-5-ethyl-6-(4-(3-hydroxypyridinoyl)piperazin-1-yl)-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)-N-(2-methyl-4-(trifluoromethyl)phenyl)acetamide

[0303] Step 1: Synthesis of compound A41-1

[0304] To a 50 mL single-necked flask was added intermediate B7 (499.97 mg, 0.78 mmol), tributyl(1-ethoxyethylene)tin (562.08 mg, 1.56 mmol), bistriphenylphosphine palladium dichloride (54.61 mg, 0.08 mmol) and DMF (5 mL). The mixture was reacted at 100°C for 16 h. The reaction was complete as monitored by LCMS. The mixture was diluted with EtOAc (5 mL), washed with water, and then with saturated aqueous sodium chloride solution (5 mL). The organic phase was dried, spin-dried, and purified by reverse phase to give compound A41-1 (420 mg, yield: 85%). ESI-MS m / z = 634.29 [M+H] + .

[0305] Step 2: Synthesis of compound A41-2

[0306] A41-1 (100.00 mg, 0.16 mmol), N,N-diisopropylethylamine (0.08 mL, 0.47 mmol), dichloromethane (1.00 mL), and trimethylsilyl trifluoromethanesulfonate (0.06 mL, 0.32 mmol) were added to a 10 mL single-necked bottle and reacted at room temperature for 16 h. The reaction was complete as monitored by LCMS. The system was poured into a NaHCO3 solution (5 mL), extracted with DCM (5 mL), washed with water (5 mL), and then with sodium chloride solution (5 mL). The organic phase was dried and spin-dried to give compound A41-2 (80 mg, yield: 95%). ESI-MS m / z = 534.24 [M+H] + .

[0307] Step 3: Synthesis of compound A41

[0308] A41-2 (30 mg, 0.06 mmol), 3-hydroxy-2-pyridinecarboxylic acid (8 mg, 0.06 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (17 mg, 0.09 mmol), 1-hydroxybenzotriazole (12 mg, 0.09 mmol), N,N-dimethylformamide (1.0 mL), and N,N-diisopropylethylamine (0.10 mL, 0.59 mmol) were added to a 10 mL single-necked vial and reacted at 60°C for 1 h. The reaction was complete as monitored by LCMS. The mixture was diluted with EtOAc (5 mL), washed with water (5 mL), and then washed with sodium chloride solution (5 mL). The organic phase was dried, spin-dried, and reversed to obtain compound A41 (36.00 mg, yield: 98%). ESI-MS m / z = 655.25 [M+H] + .

[0309] Example 45 Synthesis of Compound A45

[0310] 2-(2-(3,6-dihydro-2H-pyran-4-yl)-6-(4-(3-hydroxy-2-pyridinyl)piperazin-1-yl)-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)-N-(2-methyl-4-(trifluoromethyl)phenyl)acetamide

[0311] Step 1: Synthesis of compound A45-1

[0312] The synthesis of intermediate A45-1 was prepared by referring to the synthesis method of intermediate B8-1.

[0313] Step 2: Synthesis of compound A45-2

[0314] The synthesis of intermediate A45-2 was prepared by referring to the synthesis method of intermediate B7.

[0315] Step 3: Synthesis of compound A45-3

[0316] The synthesis of intermediate A45-3 was prepared by referring to the synthesis method of intermediate B5-2.

[0317] Step 4: Synthesis of compound A45-4

[0318] In a 50 mL single-necked flask, compound A45-3 (200 mg, 0.46 mmol) was dissolved in anhydrous DMF (8 mL). After cooling to 2°C in an ice-water bath, Br2 (221 mg, 1.38 mmol) was slowly added dropwise in portions. After addition, the mixture was stirred at this temperature for 10 minutes and then at room temperature for 2 hours. The reaction mixture was diluted with dichloromethane (50 mL) and washed sequentially with 1 M aqueous sodium hydroxide solution (10 mL), tap water (50 mL), and saturated brine (50 mL). The organic layer was concentrated, and the residue was purified by prep-HPLC to afford product A45-4 (58 mg, 25% yield). ESI-MS m / z = 512.18 [M+H] + .

[0319] Step 5: Synthesis of compound A45-5

[0320] In a 50 mL single-necked vial, compound A45-4 (30 mg, 0.06 mmol) and piperazine (152 mg, 1.78 mmol) were dissolved in anhydrous DMSO (5 mL). The reaction was carried out in a microwave oven at 140°C for 1.5 h under nitrogen protection. LCMS monitoring was used. The reaction solution was diluted with dichloromethane (30 mL) and washed sequentially with saturated aqueous sodium bicarbonate (10 mL x 3) and saturated brine (30 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness to obtain product A45-5 (22 mg, 72% yield).

[0321] Step 6: Synthesis of Compound A45

[0322] In a 25 mL single-necked vial, compound A45-5 (11 mg, 0.02 mmol), 3-hydroxypyridine-2-carboxylic acid (9 mg, 0.06 mmol), HOBt (6 mg, 0.04 mmol), EDCI (8 mg, 0.04 mmol), and DMAP (3 mg, 0.02 mmol) were dissolved in anhydrous DCM (1.5 mL) and anhydrous DMF (0.1 mL). DIEA (22 mg, 0.17 mmol) was then added. After addition, the mixture was stirred at 37°C for 0.5 h and then at 45°C for 0.5 h. LCMS monitoring was used to monitor the reaction mixture. The reaction mixture was dissolved in dichloromethane (20 mL), washed sequentially with tap water (10 mL) and saturated brine (10 mL). The organic layer was concentrated, and the residue was purified by prep-HPLC to afford product A45 (5.5 mg, 40% yield). ESI-MS m / z = 639.37 [M+H] +

[0323] Example 54 Synthesis of Compound A54

[0324] N-(4-(Difluoromethoxy)phenyl)-2-(2-(3,6-dihydro-2H-pyran-4-yl)-5-ethyl-6-(4-(3-hydroxypyridinoyl)piperazin-1-yl)-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)acetamide

[0325] Step 1: Synthesis of compound A54-1

[0326] In a 25 mL single-necked vial, the substrate 4-difluoromethoxyaniline (239 mg, 1.00 mmol) and intermediate B5 (488 mg, 1.00 mmol) were added to the reaction flask. DCM (6 mL) was added, and pyridine (237 mg, 3.00 mmol) was added dropwise. Phosphorus oxychloride (185 mg, 1.50 mmol) was added dropwise at 0°C. The reaction was stirred at room temperature for 1 hour. LCMS was used to monitor the reaction. Upon completion, stirring was stopped. The reaction mixture was poured into purified water (30 mL) for quenching, extracted with dichloromethane (50 mL), separated, and dried. The organic phase was mixed with silica gel and passed through a column with PE:EtOAc = 1:0-1:1. Concentration afforded the product A54-1 (503 mg, 80% yield), ESI-MS m / z = 630.28 [M+H]. + .

[0327] Step 2: Synthesis of compound A54-2

[0328] In a 25 mL single-necked vial, compound A54-1 (503 mg, 0.80 mmol) was added to the reaction flask. DCM (5 mL) was added, and trifluoroacetic acid (912 mg, 8.00 mmol) was added dropwise at room temperature. The reaction was stirred for 2 h. LCMS was used to monitor the reaction. Upon completion, stirring was stopped. The reaction solution was evaporated to remove the solvent, yielding product A54-2 (370 mg, 87% yield). ESI-MS m / z = 530.28 [M+H] + .

[0329] Step 3: Synthesis of compound A54

[0330] In a 25 mL single-necked flask, compound A54-2 (370 mg, 0.7 mmol) and 3-hydroxy-2-pyridinecarboxylic acid (97.3 mg, 0.7 mmol) were added to the reaction flask. DMF (5 mL) was added, followed by the addition of HOBT (135 mg, 1 mmol) and EDCI (192 mg, 1 mmol). Finally, DIPEA (271 mg, 2.1 mmol) was added dropwise and the mixture was reacted at 85°C for 2 hours. LCMS was used to monitor the reaction. Upon completion, the reaction was stopped and the mixture was cooled. The reaction mixture was poured into water (15 mL) and extracted with ethyl acetate (50 mL). The organic phase was dried, the solvent removed by rotary evaporation, and the product A54 (325 mg, 71% yield) was obtained by dissolving in DMF (3 mL). ESI-MS m / z = 651.25 [M+H] + .

[0331] 1 H NMR(500MHz,DMSO-d6)δ10.57(s,1H),10.39(s,1H),8.06(dd,J=3.6,2.3Hz,1H),7.63-7.56(m,2H ),7.32-7.28(m,2H),7.18-7.13(m,3H),6.80(tt,J=3.1,1.7Hz,1H),5.24-5.12(m,2H),4.55(d,J= 12.5Hz,1H),4.24(q,J=2.8Hz,3H),3.79(t,J=5.4Hz,3H),3.55-3.37(m,3H),3.22(t,J=11.8Hz,1 H),2.96(dt,J=13.0,5.1Hz,3H),2.80(d,J=11.2Hz,1H),2.68-2.58(m,1H),1.17(t,J=7.5Hz,3H).

[0332] Example 59 Synthesis of Compound A59

[0333] 2-(2-(3,6-dihydro-2H-pyran-4-yl)-5-ethyl-6-(4-(3-hydroxy-2-pyridinyl)piperazin-1-yl)-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)-N-(7-methylbenzofuran-4-yl)acetamide

[0334] Step 1: Synthesis of compound A59-1

[0335] In a 25 mL single-necked vial, compound B5 (80 mg, 0.16 mmol), 7-methyl-4-aminobenzofuran (29 mg, 0.20 mmol), HATU (124 mg, 0.33 mmol), and DMAP (20 mg, 0.16 mmol) were dissolved in anhydrous DCM (8 mL) and anhydrous DMF (0.8 mL). DIEA (170 mg, 1.31 mmol) was then added. After addition, the mixture was stirred at room temperature for 5 min and then at 35°C for 16 h. LCMS monitoring was used. The reaction solution was diluted with dichloromethane (30 mL) and washed sequentially with tap water (30 mL x 3) and saturated brine (30 mL). The organic layer was concentrated, and the residue was separated and purified by PLC using a developing solvent of DCM:MeOH = 20:1 to obtain A59-1 (70 mg, 69% yield).

[0336] Step 2: Synthesis of compound A59-2

[0337] In a 25 mL single-necked vial, compound A59-1 (70 mg, 0.11 mmol) was dissolved in DCM (5 mL) and MeOH (1.5 mL). A 4 M solution of hydrogen chloride in 1,4-dioxane (2 mL) was added and the mixture was stirred at room temperature for 40 min. LCMS confirmed the reaction was complete. The reaction solution was concentrated to dryness, and the residue was directly carried to the next step to obtain A59-2 (58 mg, 99% yield).

[0338] Step 3: Synthesis of compound A59

[0339] In a 25 mL single-necked vial, compound A59-2 (58 mg, 0.11 mmol), 3-hydroxypyridine-2-carboxylic acid (20 mg, 0.15 mmol), HATU (85 mg, 0.22 mmol), and DMAP (14 mg, 0.11 mmol) were dissolved in anhydrous DCM (2 mL) and anhydrous DMF (0.2 mL). DIEA (145 mg, 1.12 mmol) was then added. After addition, the mixture was stirred at room temperature for 10 minutes and then at 35°C for 16 hours. LCMS monitoring was used. The reaction solution was diluted with dichloromethane (20 mL), washed sequentially with tap water (30 mL) and saturated brine (30 mL). The organic layer was concentrated, and the residue was purified by prep-HPLC to yield product A59 (15 mg, 21%). ESI-MS m / z = 639.42 [M+H] + .

[0340] Example 62 Synthesis of Compound A62

[0341] 2-(2-(3,6-dihydro-2H-pyran-4-yl)-5-ethyl-6-(4-(5-fluoro-2-hydroxybenzoyl)piperazin-1-yl)-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)-N-(2-methyl-4-(trifluoromethyl)phenyl)acetamide

[0342] Synthesis of compound A62

[0343] In a 50 mL single-necked bottle, 3-fluorosalicylic acid (16 mg, 0.10 mmol) was dissolved in DMF (4 mL), and 2-[8-(3,6-dihydro-2H-pyran-4-yl)-4-ethyl-2-oxo-3-piperazin-1-yl-1,5,7,9-tetraazabicyclo[4.3.0]nona-3,6,8-trien-5-yl]-N-[2-methyl-4-(trifluoromethyl)phenyl]acetamide (55 mg, 0.10 mmol), 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (79 mg, 0.15 mmol), HOBt (20.43 mg, 0.15 mmol) and DIEA (52 mg, 0.40 mmol) were added. The mixture was reacted at room temperature for 1 h. The reaction was completed under LCMS monitoring. Water (10 mL) and ethyl acetate (10 mL) were added to the reaction solution. x2) was extracted, the organic phase was dried by rotary evaporation, and the product A62 (23.3 mg, yield: 34%) was obtained by reverse phase preparation. ESI-MS m / z = 684.27 [M+H] + .

[0344] Example 64 Synthesis of Compound A64

[0345] N-(4-chloronaphth-1-yl)-2-(2-(3,6-dihydro-2H-pyran-4-yl)-5-ethyl-6-(4-(3-hydroxypyridinoyl)piperazin-1-yl)-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)acetamide

[0346] Step 1: Synthesis of compound A64-1

[0347] In a 10 mL single-necked vial, substrate B6 (75.0 mg, 0.15 mmol) and 1-amino-4-chloronaphthalene (32.7 mg, 0.18 mmol) were added to DMF (2 mL). HOBt (41.5 mg, 0.31 mmol) and EDCI (88.3 mg, 0.46 mmol) were then added dropwise. Finally, DIPEA (59.5 mg, 0.46 mmol) was added dropwise. The mixture was reacted at 80°C for 2 h. After cooling, the reaction mixture was poured into ice water (10 mL). A large amount of solid precipitated, and the filter cake was collected by filtration to obtain A64-1 (60.0 mg, 60% yield) as a yellow solid. ESI-MS m / z = 648.16 [M+H] + .

[0348] Step 2: Synthesis of compound A64-2

[0349] In a 25 mL single-necked vial, substrate A64-1 (55 mg, 0.08 mmol) was added to DCM (5 mL). Trifluoroacetic acid (484.0 mg, 4.25 mmol) was added dropwise at room temperature. The reaction was stirred at room temperature for 1 h. Evaporation under reduced pressure afforded A64-2 (45 mg, 97% yield) as a yellow solid. ESI-MS m / z = 548.04 [M+H] + .

[0350] Step 3: Synthesis of compound A64

[0351] In a 10 mL single-necked vial, substrate A64-2 (40 mg, 0.07 mmol) and 3-hydroxy-2-pyridinecarboxylic acid (10.1 mg, 0.07 mmol) were added to DMF (2 mL). HOBt (11.8 mg, 0.09 mmol) and EDCI (16.8 mg, 0.09 mmol) were then added dropwise. Finally, DIPEA (28.3 mg, 0.22 mmol) was added dropwise. The mixture was reacted at 80°C for 1 h. After cooling, the reaction mixture was poured into water (10 mL) and extracted with ethyl acetate (50 mL). The organic phase was dried, evaporated under reduced pressure, dissolved in DMF (3 mL), and reversed phase prepared. After appropriate vacuum distillation and lyophilization, A64 (16.6 mg, 33% yield) was obtained as a white solid. ESI-MS m / z = 669.14 [M+H] + .

[0352] Example 67 Synthesis of Compound A67

[0353] 2-(2-(3,6-dihydro-2H-pyran-4-yl)-5-ethyl-6-(4-(5-hydroxy-2-morpholinopyrimidine-4-carbonyl)piperazin-1-yl)-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)-N-(2-methyl-4-(trifluoromethyl)phenyl)acetamide

[0354] Step 1: Synthesis of compound A67-1

[0355] 2-Chloro-5-methoxypyrimidine (1.44 g, 30 mmol), K2CO3 (4.13 g, 30 mmol), and water (30 mL) were added to a 100 mL single-necked flask and heated to 100°C for 8 hours. The reaction was monitored for completion by LCMS. 1 M dilute hydrochloric acid was added to adjust the pH to 7, and the mixture was extracted with ethyl acetate (100 mL x 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and the organic phase was spin-dried to obtain product A67-1 (1.3 g, yield: 67%). ESI-MS m / z = 196.10 [M+H] + .

[0356] Step 2: Synthesis of compound A67-2

[0357] Ethyl pyruvate (1.87 mg, 16.10 mmol) was added to a 100 mL single-necked bottle, and the mixture was cooled to -10°C, and glacial acetic acid (7.00 mL) and 30% hydrogen peroxide (1.22 g, 10.76 mmol) were added. In another reactor, A67-1 (700 mg, 3.59 mmol) was added to a mixed solvent of toluene (42.00 mL) and water (7.00 mL). The reaction solution was cooled to -10°C, and concentrated sulfuric acid (0.58 mL, 10.80 mmol) and ferrous sulfate heptahydrate (3.04 g, 11.00 mmol) were added. After vigorous stirring, the reaction solution was slowly added dropwise to a glacial acetic acid solution of ethyl pyruvate containing hydrogen peroxide, maintained below 0°C, and reacted for another 1 hour. LCMS detected that the reaction was complete, and 1N sodium hydroxide aqueous solution was added to adjust the pH to 7. The mixture was filtered through celite and extracted with dichloromethane (100 mL x 3). The organic phase was washed with 5% NaHSO3 aqueous solution and saturated brine, respectively, and then dried over anhydrous sodium sulfate, filtered, and the organic phase was spin-dried. Column chromatography PE:EtOAc = 80:20 gave the product A67-2 (260 mg, yield: 27%). ESI-MS m / z=254.20[M+H] + .

[0358] Step 3: Synthesis of compound A67-3

[0359] In a 50 mL single-necked flask, A67-2 (253 mg, 1.00 mmol) was dissolved in a mixture of ethanol (10.00 mL) and water (5.00 mL). Sodium hydroxide (171 mg, 4.30 mmol) was added and allowed to react at room temperature for 2 hours. LCMS confirmed the reaction was complete. The mixture was extracted with ethyl acetate (20 mL x 2), the organic phase discarded, and 1N dilute hydrochloric acid was added to the aqueous phase to adjust the pH to 3. Reverse phase reaction was performed to obtain A67-3 (38.00 mg, yield: 16%). ESI-MS m / z = 226.30 [M+H] + .

[0360] Step 4: Synthesis of Compound A67

[0361] In a 50 mL single-necked bottle, 5-hydroxy-2-morpholin-4-ylpyrimidine-4-carboxylic acid (24.8 mg, 0.11 mmol) was suspended in DCM (5 mL), and 1-chloro-N,N,2-trimethylpropenamine (20.0 mg, 0.15 mmol) was added. After reacting at room temperature for 1.5 hours, a mixed solution of 2-(2-(3,6-dihydro-2H-pyran-4-yl)-5-ethyl-7-oxo-6-(piperazin-1-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)-N-(2-methyl-4-(trifluoromethyl)phenyl)acetamide (54 mg, 0.10 mmol) and DIEA (47 mg, 0.37 mmol) in DCM (5 mL) was added. The reaction was continued at room temperature for 1 hour. The reaction was completed after LCMS monitoring. 20 mL of water and dichloromethane (20 mL) were added to the reaction solution. x2) was extracted, the organic phase was dried by rotary evaporation, and the product A67 (15.8 mg, yield: 23%) was obtained by reverse phase preparation. ESI-MS m / z = 753.15 [M+H] + .

[0362] Example 73 and Example 74 Synthesis of Compound A73 and Compound A74

[0363] 2-(6-(4-(1-cyclopropyl-4-hydroxy-1H-pyrazole-3-carbonyl)piperazin-1-yl)-2-(3,6-dihydro-2H-pyrazol-4-yl)-5-ethyl-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)-N-(2-methyl-4-(trifluoromethyl)phenyl)acetamide and 2-(6-(4-(1-cyclopropyl-4-hydroxy-1H-pyrazole-5-carbonyl)piperazin-1-yl)-2-(3,6-dihydro-2H-pyrazol-4-yl)-5-ethyl-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)-N-(2-methyl-4-(trifluoromethyl)phenyl)acetamide

[0364] Step 1: Synthesis of Compounds A73-1 and A74-1

[0365] In a 50 mL single-necked bottle, 4-formyl-1H-pyrazole-3-carboxylic acid ethyl ester (500.0 mg, 2.97 mmol), Na2CO3 (630.0 mg, 5.94 mmol) and cyclopropylboronic acid (639.0 mg, 7.44 mmol) and DCE (13 mL) were added pyridine (1.18 g, 14.87 mmol) and Cu(OAc)2 (810.0 mg, 4.46 mmol), and the mixture was heated at 70 ° C in air overnight. The mixture was cooled, quenched with saturated NaHCO3 aqueous solution (13 mL) and extracted with ethyl acetate (20 mL x 2). The organic phases were combined and dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography PE:EtOAc=90:10-70:30 to provide a mixture of A73-1, A74-1 (258.0 mg, yield: 42%), ESI-MS m / z=208.22 [M+H] + .

[0366] Step 2: Synthesis of Compounds A73-2 and A74-2

[0367] In a 10 mL single-necked bottle, m-CPBA (316.1 mg, 1.84 mmol) was added to a solution of A73-1 and A74-1 (258.0 mg, 1.23 mmol) in chloroform (4.0 mL) and stirred at room temperature overnight. The reaction was quenched with aqueous NaHCO 3 at 0° C. and extracted with EtOAc. The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography (PE:EtOAc=90:10-70:30) to provide a mixture of A73-2 and A74-2 (200 mg, yield: 83%), ESI-MS m / z=196.21 [M+H] + .

[0368] Step 3: Synthesis of Compounds A73-3 and A74-3

[0369] In a 50 mL single-necked vial, A73-2 and A74-2 (200 mg, 1.02 mmol) and NaOH (122.4 mg, 3.06 mmol) were added to THF (3.0 mL), H2O (1.0 mL), and MeOH (1.0 mL) and reacted at room temperature for 2 h. The THF was removed by distillation under reduced pressure, and the mixture was dissolved in 2 mL of water and extracted with EtOAc (2 x 5 mL). The organic phase was discarded, and the aqueous phase was adjusted to pH 3 (1N HCl) and extracted with EtOAc (3 x 10 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated to afford a mixture of A73-3 and A74-3 as a yellow solid (120 mg, 70% yield). ESI-MS m / z = 168.15 [M+H] + .

[0370] Step 4: Synthesis of compounds A73 and A74

[0371] In a 10 mL single-necked vial, substrates A73-2, A74-2 (40.0 mg, 0.24 mmol), and B9 (129.8 mg, 0.24 mmol) were added to DMF (2 mL). HOBt (64.29 mg, 0.48 mmol) and EDCI (136.8 mg, 0.71 mmol) were then added sequentially. Finally, DIPEA (153.7 mg, 1.19 mmol) was added dropwise. The reaction was allowed to react at 80°C for 1 h. After cooling, the reaction mixture was poured into water (10 mL) and extracted with ethyl acetate (50 mL). The organic phase was dried, evaporated under reduced pressure, and lyophilized to afford A73 (7.3 mg) as a white solid. ESI-MS m / z = 696.28 [M+H] + , A74 (11.2mg), ESI-MS m / z=696.28[M+H] + .

[0372] Example 78 Synthesis of Compound A78

[0373] N-(7-chlorobenzofuran-4-yl)-2-(2-(3,6-dihydro-2H-pyran-4-yl)-5-ethyl-6-(4-(3-hydroxy-2-pyridinyl)piperazin-1-yl)-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)acetamide

[0374] Step 1: Synthesis of compound A78-1

[0375] In a 50 mL single-necked bottle, 4-bromo-7-chlorobenzofuran (200 mg, 0.86 mmol), benzophenone imine (164 mg, 0.91 mmol), Pd2dba3 (95 mg, 0.1 mmol), BINAP (129 mg, 0.21 mmol), and sodium tert-butoxide (208 mg, 2.16 mmol) were dissolved in toluene (8 mL). The atmosphere in the reaction system was replaced with nitrogen three times. Under nitrogen protection, the reaction was incubated at 110°C for 2 hours. LCMS monitored the reaction to be nearly complete. The mixture was cooled to room temperature and concentrated. The residue was dissolved in dichloromethane (30 mL) and washed sequentially with tap water (30 mL) and saturated brine (30 mL). The organic layer was concentrated and purified by flash column chromatography to obtain A78-1 (245 mg, 86% yield).

[0376] Step 2: Synthesis of compound A78-2

[0377] In a 50 mL single-necked vial, compound A78-1 (245 mg, 0.74 mmol) was dissolved in DCM (20 mL), followed by the addition of 4 M HCl in 1,4-dioxane (1 mL). The mixture was stirred at room temperature for 1 h. LCMS confirmed the reaction was complete. The reaction solution was concentrated to dryness, and the residue was dissolved in THF (10 mL), sonicated, and filtered. The filter cake was rinsed with THF (2 mL) and dried under reduced pressure to afford A78-2 (44 mg, 36% yield).

[0378] Step 3: Synthesis of compound A78-3

[0379] In a 50 mL single-necked vial, compound B5 (80 mg, 0.16 mmol), A78-2 (44 mg, 0.26 mmol), HATU (124 mg, 0.33 mmol), and DMAP (20 mg, 0.16 mmol) were dissolved in anhydrous DCM (8 mL) and anhydrous DMF (0.8 mL). DIEA (170 mg, 1.31 mmol) was then added. After addition, the mixture was stirred at room temperature for 5 min and then at 35°C for 16 h. LCMS monitoring was used. The reaction solution was diluted with dichloromethane (30 mL) and washed sequentially with tap water (30 mL x 3) and saturated brine (30 mL). The organic layer was concentrated, and the residue was separated and purified by PLC using a developing solvent of DCM / MeOH = 20 / 1 to afford A78-3 (63 mg, 60% yield).

[0380] Step 4: Synthesis of compound A78-4

[0381] In a 50 mL single-necked vial, compound A78-3 (63 mg, 0.10 mmol) was dissolved in DCM (5 mL) and MeOH (2 mL). A 4M HCl solution in 1,4-dioxane (2 mL) was added. After addition, the mixture was stirred at room temperature for 1 h. LCMS confirmed the reaction was complete. The reaction solution was concentrated to dryness, and the residue was directly carried to the next step to afford A78-4 (53 mg, 99% yield).

[0382] Step 5: Synthesis of Compound A78

[0383] In a 10 mL single-necked vial, compound A78-4 (53 mg, 0.10 mmol), 3-hydroxypyridine-2-carboxylic acid (18 mg, 0.13 mmol), HATU (75 mg, 0.20 mmol), and DMAP (12 mg, 0.10 mmol) were dissolved in anhydrous DCM (2 mL) and anhydrous DMF (0.2 mL). DIEA (127 mg, 0.99 mmol) was then added. After addition, the mixture was stirred at room temperature for 10 min and then at 35°C for 16 h. LCMS monitoring was used. The reaction solution was diluted with dichloromethane (20 mL), washed sequentially with tap water (30 mL) and saturated brine (30 mL). The organic layer was concentrated, and the residue was purified by prep-HPLC to afford product A78 (17 mg, 25% yield). ESI-MS m / z = 659.21 [M+H] + .

[0384] Example 81 Synthesis of Compound A81

[0385] 5-Ethyl-6-(4-(3-hydroxypyridinoyl)piperazin-1-yl)-4-(2-((2-methyl-4-(trifluoromethyl)phenyl)amino)-2-oxoethyl)-N-(oxetan-3-yl)-7-oxo-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidine-2-carboxamide

[0386] Step 1: Synthesis of compound A81-1

[0387] To a 25 mL single-necked flask, intermediate B2 (3.50 g, 11.13 mmol), methanol (10.00 mL), and a 4 M HCl solution in 1,4-dioxane (11 mL, 44.53 mmol) were added and allowed to react at room temperature for 2 h. The reaction was complete as monitored by LCMS. The mixture was then dried to give compound A81-1 (2.39 g, yield: 100.00%). ESI-MS m / z = 215.13 [M+H] + .

[0388] Step 2: Synthesis of compound A81-2

[0389] A81-1 (2.50 g, 11.67 mmol), dichloromethane (20.00 mL), and N,N-diisopropylethylamine (9.64 mL, 58.34 mmol) were added to a 25 mL single-necked flask. Benzyl chloroformate (1.97 mL, 14.00 mmol) was added at room temperature and allowed to react for 2 h. The mixture was diluted with dichloromethane, washed with water, dilute phosphoric acid, and sodium chloride solution. The organic phase was dried and subjected to column chromatography to afford compound A81-2 (3.2 g, 78% yield). ESI-MS m / z = 349.17 [M+H] + .

[0390] Step 3: Synthesis of compound A81-3

[0391] A81-2 (3.20 g, 9.19 mmol), methyl 5-amino-1H-1,2,4-triazole-3-carboxylate (1.44 g, 10.10 mmol), p-toluenesulfonic acid (2.37 g, 13.78 mmol), and N-methylpyrrolidone (30.00 mL) were added to a 100 mL glass bottle and reacted at 100°C for 2 h. The reaction was complete and the system was directly purified by reverse phase preparation to obtain compound A81-3 (740 mg, yield: 18.00%). ESI-MS m / z = 441.18 [M+H] + .

[0392] Step 4: Synthesis of compound A81-4

[0393] A81-3 (180.00 mg, 0.41 mmol), intermediate 7 (168.25 mg, 0.49 mmol), 1,4-dioxane (4.00 mL), and N,N-diisopropylethylamine (0.20 mL, 1.23 mmol) were added to a 10 mL glass bottle and reacted at 80°C for 16 h. The reaction was complete as monitored by LCMS. The mixture was diluted with EtOAc, washed with water, and then washed with sodium chloride solution. The organic phase was dried, spin-dried, and reversed to obtain compound A81-4 (730 mg, yield: 66.00%). ESI-MS m / z = 656.24 [M+H] + .

[0394] Step 5: Synthesis of Compound A81-5

[0395] A81-4 (730.00 mg, 1.11 mmol) and MeOH (5.00 mL) were added to a 50 mL single-necked bottle, followed by 5% Pd / C (592.44 mg, 5.57 mmol). The reaction was allowed to react at room temperature for 2 h. The reaction was monitored by LCMS. The mixture was filtered and dried to give compound A81-5 (560 mg, yield: 96.00%). ESI-MS m / z = 522.20 [M+H] + .

[0396] Step 6: Synthesis of Compound A81-6

[0397] A81-5 (560.00 mg, 1.07 mmol), 3-hydroxy-2-pyridinecarboxylic acid (179.26 mg, 1.29 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (411.69 mg, 2.15 mmol), 1-hydroxybenzotriazole (290.18 mg, 2.15 mmol), N,N-dimethylformamide (10.0 mL), and N,N-diisopropylethylamine (0.89 mL, 5.37 mmol) were added to a 25 mL single-necked bottle and reacted overnight at 40°C. LCMS monitored the reaction to be complete. The mixture was diluted with EtOAc, washed with water, and then washed with chlorinated solution. The organic phase was dried and spin-dried to give compound A81-6 (600 mg, 86% yield). ESI-MS m / z = 643.22 [M+H] + .

[0398] Step 7: Synthesis of Compound A81-7

[0399] A81-6 (600.00 mg, 0.93 mmol), lithium hydroxide monohydrate (156.71 mg, 3.73 mmol), tetrahydrofuran (5.00 mL), and water (5.00 mL) were added to a 25 mL single-necked bottle and reacted at room temperature for 2 h. The reaction was complete by LCMS. The product was purified by reverse phase chromatography to give compound A81-7 (254 mg, yield: 43%). ESI-MS m / z = 629.20 [M+H] + .

[0400] Step 8: Synthesis of Compound A81

[0401] In a 25 mL glass vial, A81-7 (50.00 mg, 0.08 mmol) was dissolved in N,N-dimethylformamide (1.0 mL). N,N-diisopropylethylamine (0.07 mL, 0.40 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (60.46 mg, 0.16 mmol), and 3-oxetanamine were added and reacted at 80°C for 16 h. The reaction solution was directly purified by reverse phase preparative reaction to obtain compound A81 (8.8 mg, yield: 12.00%). ESI-MS m / z = 684.24 [M+H] + .

[0402] Example 84 Synthesis of Compound A84

[0403] 2-(5-ethyl-6-(4-(3-hydroxypyridinoyl)piperazin-1-yl)-2-(1-(2-methoxyethyl)-1H-pyrazol-4-yl)-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)-N-(2-methyl-4-(trifluoromethyl)phenyl)acetamide

[0404] Step 1: Synthesis of compound A84-1

[0405] In a 100 mL single-necked bottle, tert-butyl 4-[8-bromo-4-ethyl-5-[[2-methyl-4-(trifluoromethyl)phenyl]carbamoylmethyl]-2-oxo-1,5,7,9-tetraazabicyclo[4.3.0]nona-3,6,8-trien-3-yl]piperazine-1-carboxylate (200 mg, 0.31 mmol) was dissolved in ethyl acetate (10 mL). A 4.0 M solution of hydrogen chloride in 1,4-dioxane (4.0 mL) was added and the mixture was allowed to react at room temperature for 2 hours. The reaction was complete as monitored by LCMS and the product A84-1 was obtained by spin drying (160 mg, 95% yield). ESI-MS m / z = 543.12 [M+H] + .

[0406] Step 2: Synthesis of compound A84-2

[0407] In a 50 mL single-necked vial, 3-hydroxypyridinic acid (52 mg, 0.37 mmol) was suspended in DCM (5 mL). 1-Chloro-N,N,2-trimethylpropenamine (62 mg, 0.46 mmol) was added and allowed to react at room temperature for 1.5 hours. A mixture of A84-1 (168 mg, 0.31 mmol) and DIEA (80 mg, 0.62 mmol) in DCM (5 mL) was then added. The reaction was continued at room temperature for 1 hour. LCMS monitored the reaction completion. 20 mL of water was added to the reaction solution, and the mixture was extracted with dichloromethane (20 mL x 2). The organic phase was dried by spin drying and purified by column chromatography using DCM / MeOH = 90 / 10 to afford product A84-2 (164 mg, 80% yield). ESI-MS m / z = 664.15 [M+H] + .

[0408] Step 3: Synthesis of compound A84

[0409] In a 100 mL single-necked bottle, (1-(2-methoxyethyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (57 mg, 0.23 mmol), A84-2 (60 mg, 0.09 mmol), XPhos Pd G3 (15 mg, 0.02 mmol), and K3PO4 (76 mg, 0.36 mmol) were added 1,4-dioxane (5 mL) and water (1 mL). After nitrogen replacement, the temperature was raised to 100°C and the reaction was allowed to react for 2 hours. The reaction was detected by liquid chromatography-mass spectrometry. The reaction was complete and extracted with ethyl acetate (50 mL x 2). The organic phase was spin-dried and the product A84 (19.3 mg, 30% yield) was obtained by reverse phase chromatography. ESI-MS m / z = 709.23 [M+H] + .

[0410] Example 142 Synthesis of Compound A142

[0411] N-(4-chloronaphthyl)-2-(5-ethyl-6-(4-(5-hydroxy-6-methylpyrimidine-4-carbonyl)piperazin-1-yl)-2-(1-methyl-1H-pyrazol-4-yl)-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)acetamide

[0412] Step 1: Synthesis of compound A142-1:

[0413] In a 50 mL single-necked vial, intermediate B10 (120 mg, 0.25 mmol), 4-chloronaphthalene-1-amine (44 mg, 0.25 mmol), pyridine (0.06 mL, 0.74 mmol), and DCM (5 mL) were mixed thoroughly. Phosphine oxychloride (0.03 mL, 0.30 mmol) was added under ice-cooling and the mixture was incubated for 20 min. The reaction was complete as monitored by LCMS, and the product was directly dried and used in the next step. ESI-MS m / z = 646.26 [M+H] + .

[0414] Step 2: Synthesis of compound A142-2:

[0415] In a 50 mL single-necked vial, the reactant A142-1 from the previous step was added to a 4.0 M solution of hydrogen chloride in 1,4-dioxane (5 mL) and allowed to react at room temperature for 2 h. The reaction was monitored by LCMS and the mixture was directly dried and used in the next reaction. ESI-MS m / z = 546.21 [M+H] + .

[0416] Step 3: Synthesis of compound A142:

[0417] The synthesis method of compound A142 was prepared by referring to the synthesis method of compound A81-6. ESI-MS m / z=682.23[M+H] + .

[0418] Example 143 Synthesis of Compound A143

[0419] N-(4-chloronaphth-1-yl)-2-(2-(3,6-dihydro-2H-pyran-4-yl)-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)acetamide

[0420] Step 1: Synthesis of compound A143:

[0421] In a 10 mL single-necked vial, compound A64-2 (40 mg, 0.07 mmol), 5-hydroxy-6-methylpyrimidine-4-carboxylic acid (11 mg, 0.07 mmol), EDCI (21 mg, 0.11 mmol), HOBt (15 mg, 0.11 mmol), DIEA (0.06 mL, 0.36 mmol), and DMF (2 mL) were mixed and allowed to react at 70°C for 30 min. The reaction was monitored for completion by LCMS, and the product was directly prepared by Prep-HPLC and lyophilized to afford compound A143 (10.5 mg, 21% yield). ESI-MS m / z = 684.24 [M+H] + .

[0422] Example 186 Synthesis of Compound A186

[0423] 2-(2-(3,6-dihydro-2H-pyran-4-yl)-5-ethyl-6-(4-(3-hydroxy-2-pyridinyl)piperazin-1-yl)-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)-N-(3-(dimethylamino)-4-(trifluoromethyl)phenyl)acetamide

[0424] Step 1: Synthesis of compound A186-1

[0425] In a 50 mL single-necked vial, 2-fluoro-4-nitrotrifluoromethylbenzene (300 mg, 1.43 mmol) was dissolved in a 2M solution of dimethylamine in tetrahydrofuran (12 mL) and stirred at 50°C for 4 days. The reaction mixture was concentrated to dryness using LCMS monitoring, and the residue was directly used in the next step to obtain A186-1 (378 mg). ESI-MS m / z = 235.06 [M+H] + .

[0426] Step 2: Synthesis of compound A186-2

[0427] In a 50 mL single-necked vial, compound A186-1 (200 mg, 0.85 mmol) was dissolved in THF (20 mL) and MeOH (5 mL). 10% palladium on carbon (60 mg) was then added. The mixture was purged with hydrogen three times and allowed to react under a hydrogen balloon. The mixture was stirred at room temperature for 1.5 h. LCMS monitoring indicated the reaction was complete. The reaction solution was concentrated to dryness to afford A186-2 (174 mg, 99% yield), which was used directly in the next step. ESI-MS m / z = 205.09 [M+H] + .

[0428] Step 3: Synthesis of compound A186-3

[0429] In a 50 mL single-necked vial, compound B5 (80 mg, 0.16 mmol), A186-2 (100 mg, 0.49 mmol), HATU (124 mg, 0.33 mmol), and DMAP (20 mg, 0.16 mmol) were dissolved in anhydrous DCM (8 mL) and anhydrous DMF (0.8 mL). DIEA (170 mg, 1.31 mmol) was then added. The mixture was stirred at room temperature for 5 min and then at 35°C for 16 h. LCMS monitoring was used. The reaction solution was diluted with dichloromethane (30 mL) and washed sequentially with tap water (30 mL x 3) and saturated brine (30 mL). The organic layer was concentrated, and the residue was separated and purified by PLC using a developing solvent of DCM / MeOH = 20 / 1 to afford A186-3 (62 mg, 56% yield). ESI-MS m / z = 675.32 [M+H] + .

[0430] Step 4: Synthesis of compound A186-4

[0431] In a 50 mL single-necked vial, compound A186-3 (62 mg, 0.09 mmol) was dissolved in DCM (5 mL) and MeOH (2 mL). A 4M solution of hydrogen chloride in 1,4-dioxane (2 mL) was then added. The mixture was stirred at room temperature for 40 min. LCMS confirmed the reaction was complete. The reaction solution was concentrated to dryness, and the residue was directly used in the next step to obtain A186-4 (52 mg, 98% yield). ESI-MS m / z = 575.26 [M+H] + .

[0432] Step 5: Synthesis of compound A186

[0433] In a 10 mL single-necked vial, compound A186-4 (52 mg, 0.09 mmol), 3-hydroxypyridine-2-carboxylic acid (17 mg, 0.12 mmol), HATU (70 mg, 0.18 mmol), and DMAP (11 mg, 0.09 mmol) were dissolved in anhydrous DCM (2 mL) and anhydrous DMF (0.2 mL). DIEA (119 mg, 0.92 mmol) was then added. After addition, the mixture was stirred at room temperature for 10 min and then at 35°C for 16 h. LCMS monitoring was used. The reaction solution was diluted with dichloromethane (20 mL), washed sequentially with tap water (30 mL) and saturated brine (30 mL). The organic layer was concentrated, and the residue was purified by prep-HPLC to afford product A186 (10 mg, 15% yield). ESI-MS m / z = 696.44 [M+H] + .

[0434] Example 190 Synthesis of Compound A190

[0435] 2-(2-(3,6-dihydro-2H-pyran-4-yl)-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-ethyl-4-(trifluoromethyl)phenyl)acetamide

[0436] Step 1: Synthesis of compound A190-1

[0437] In a 25 mL single-necked bottle, 4-amino-3-iodobenzotrifluoride (500 mg, 1.74 mmol), trimethylethynylsilane (342.1 mg, 3.48 mmol), bistriphenylphosphine palladium dichloride (122.2 mg, 0.17 mmol), and cuprous iodide (66.3 mg, 0.35 mmol) were dissolved in diethylamine (5.0 mL). The atmosphere in the reaction system was replaced with nitrogen three times. The reaction was carried out at 25°C under a nitrogen balloon for 12 hours. The mixture was diluted with dichloromethane (30 mL) and washed sequentially with tap water (30 mL) and saturated brine (30 mL). The organic layer was concentrated and purified by flash column chromatography to obtain A190-1 (200 mg, 0.77 mmol, 45% yield). ESI-MS m / z = 257.33 [M+H] + .

[0438] Step 2: Synthesis of compound A190-2

[0439] In a 25 mL single-necked vial, substrate B5 (200 mg, 0.41 mmol) and A190-1 (105.3 mg, 0.41 mmol) were added to DCM (2 mL). Pyridine (161.9 mg, 2.05 mmol) and POCl (125.5 mg, 0.82 mmol) were added sequentially in an ice bath. The mixture was allowed to react at 25°C for 1 hour. The reaction mixture was poured into ice water (10 mL) and extracted with DCM (30 mL x 2). The organic phase was dried over anhydrous sodium sulfate and evaporated under reduced pressure to afford A190-2 (200 mg, 0.27 mmol, 67% yield) as a yellow solid. ESI-MS m / z = 727.86 [M+H] + .

[0440] Step 3: Synthesis of compound A190-3

[0441] In a 25 mL single-necked vial, substrate A190-2 (200 mg, 0.27 mmol) was added to DCM (5 mL). Trifluoroacetic acid (307.8 mg, 2.7 mmol) was added dropwise at room temperature. The mixture was stirred at room temperature for 1 hour. Evaporation under reduced pressure afforded A190-3 (140 mg, 0.22 mmol, 82% yield) as a yellow solid. ESI-MS m / z = 627.74 [M+H] + .

[0442] Step 4: Synthesis of compound A190-4

[0443] In a 25 mL single-necked vial, substrate A190-3 (140 mg, 0.22 mmol) and 5-hydroxy-6-methylpyrimidine-4-carboxylic acid (48.1 mg, 0.31 mmol) were added to DMF (2 mL). HOBt (60.2 mg, 0.45 mmol) and EDCI (128.2 mg, 0.67 mmol) were then added dropwise. Finally, DIPEA (144.1 mg, 1.11 mmol) was added dropwise. The mixture was reacted at 80°C for 1 hour. After cooling, the reaction mixture was poured into water (10 mL) and extracted with ethyl acetate (50 mL). The organic phase was dried, evaporated under reduced pressure, and purified by prep-HPLC using DMF (3 mL). Freeze-dried, the resulting white solid A190-4 (100 mg, 0.13 mmol, 59% yield) was obtained. ESI-MS m / z = 763.85 [M+H] + .

[0444] Step 5: Synthesis of compound A190

[0445] In a 50 mL single-necked vial, substrate A190-4 (100 mg, 0.13 mmol) was added to methanol (5 mL) and potassium fluoride (114.1 mg, 1.96 mmol) was added. The mixture was reacted at 25°C for 1 hour. The reaction mixture was poured into water (10 mL) and extracted with ethyl acetate (50 mL). The organic phase was dried, evaporated under reduced pressure, dissolved in DMF (2 mL), purified by prep-HPLC, and lyophilized to obtain A190 (26.1 mg, 0.036 mmol, yield: 28%) as a white solid. ESI-MS m / z = 691.67 [M+H] + .

[0446] Example 192 Synthesis of Compound A192

[0447] 2-(2-(3,6-dihydro-2H-pyran-4-yl)-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-(4-methylbenzofuran-7-yl)acetamide

[0448] Step 1: Synthesis of compound A192-1

[0449] In a 250 mL single-necked flask, 7-bromo-4-methylbenzofuran (4.70 g, 22.27 mmol), benzophenone imine (4.44 g, 24.50 mmol), Pd2(dba)3 (1.63 g, 1.78 mmol), BINAP (2.22 g, 3.56 mmol), sodium tert-butoxide (5.35 g, 55.67 mmol), and toluene (40 mL) were added. The atmosphere in the reaction system was replaced with nitrogen three times. Under a nitrogen balloon, the reaction was carried out in an external bath at 110°C for 2 hours. LCMS monitored the reaction to be essentially complete. The mixture was cooled to room temperature and concentrated. The residue was dissolved in dichloromethane (100 mL), washed sequentially with tap water (100 mL) and saturated brine (100 mL). The organic layer was concentrated and purified by flash column chromatography to obtain A192-1 (7.92 g).

[0450] Step 2: Synthesis of compound A192-2

[0451] In a 1000 mL single-necked flask, compound A192-1 (7.92 g, 25.44 mmol) was dissolved in DCM (100 mL) and methanol (100 mL). The mixture was cooled to below 5°C in an ice-water bath. A 4 M solution of hydrogen chloride in 1,4-dioxane (100 mL) and a 1 M aqueous solution of hydrochloric acid (100 mL) were added. After addition, the mixture was transferred to room temperature and stirred for 2 hours. LCMS monitoring indicated that the reaction was complete. The reaction mixture was allowed to stand and the layers were separated. The aqueous layer was washed with DCM (100 mL x 2). The DCM layer was removed and the pH of the aqueous layer was adjusted to 8 with saturated aqueous potassium carbonate solution. The mixture was extracted with EtOAc (100 mL x 3). The organic phases were combined and washed with tap water (100 mL x 2). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated to dryness to a reddish-brown solid, A192-2 (2.72 g, 83% yield over two steps).

[0452] Step 3: Synthesis of compound A192-3

[0453] In a 250 mL single-necked bottle, compound intermediate B5 (3.00 g, 6.14 mmol) and intermediate A192-2 (1.08 g, 7.37 mmol) were dissolved in anhydrous DCM (60 mL). Pyridine (3.95 mL, 49.13 mmol) was slowly added dropwise in an ice-water bath below 5°C. The mixture was stirred at this temperature for 5 min, and then POCl3 (1.14 mL, 12.28 mmol) was slowly added dropwise. After the addition was complete, the mixture was stirred at this temperature for 30 minutes, then transferred to room temperature and stirred for 1 hour. LCMS monitored the reaction completion. The reaction solution was slowly added with 150 mL of tap water, 30 mL of 1 M dilute hydrochloric acid, and 50 mL of saturated brine to quench the reaction. The liquid was separated, and the aqueous phase was extracted with DCM (30 mL). The organic phases were combined and washed with saturated brine (60 mL). The organic layer was concentrated and purified by flash column chromatography to obtain A192-3 (1.97 g, 52% yield).

[0454] Step 4: Synthesis of compound A192-4

[0455] In a 100 mL single-necked vial, compound A192-3 (1.60 g, 2.59 mmol) was dissolved in DCM (15 mL) and MeOH (10 mL). A 4 M solution of hydrogen chloride in 1,4-dioxane (15 mL) was added and the mixture was stirred at room temperature for 2 hours. The reaction was complete as monitored by LCMS. The reaction solution was concentrated to dryness, and the residue was added with 30 mL of EtOAc. The mixture was sonicated for 0.5 hours and filtered. The filter cake was rinsed with EtOAc and dried under reduced pressure to afford A194-4 (1.27 g, 95% yield).

[0456] Step 5: Synthesis of compound A192

[0457] In a 50 mL single-necked vial, compound A192-4 (300 mg, 0.58 mmol), 5-hydroxy-6-methylpyrimidine-4-carboxylic acid (125 mg, 0.81 mmol), HOBt (157 mg, 1.16 mmol), and EDCI (333 mg, 1.74 mmol) were dissolved in anhydrous DMF (6 mL). DIEA (375 mg, 2.90 mmol) was then added. After addition, the mixture was stirred at 80°C for 40 minutes. The reaction mixture was filtered and purified directly by prep-HPLC using LCMS monitoring to afford product A192 (163 mg, 40.87% yield). ESI-MS m / z = 654.27 [M+H] + .

[0458] Compound A192: 1H NMR (500MHz, DMSO-d6) δ10.56(s,1H),8.57(s,1H),8.05(d,J=2.2Hz,1H),7.58(d,J=7.9Hz,1H),7.0 6(d,J=2.2Hz,1H),6.98(d,J=8.0Hz,1H),6.88-6.77(m,1H),5.28(s,2H),4.52(d,J=12.6Hz,1H),4.2 5(q,J=2.8Hz,2H),3.80(t,J=5.5Hz,2H),3.49(t,J=11.4Hz,3H),3.29-3.19(m,2H),2.98(d,J=11.2 Hz,3H),2.82(d,J=11.1Hz,1H),2.64(d,J=11.8Hz,1H),2.44(d,J=1.8Hz,6H),1.20(t,J=7.5Hz,3H).

[0459] Example 193 Synthesis of Compound A193

[0460] 2-(2-(3,6-dihydro-2H-pyran-4-yl)-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-(7-methylbenzofuran-4-yl)acetamide

[0461] Step 1: Synthesis of compound A193

[0462] In a 25 mL single-necked vial, compound A59-2 (54 mg, 0.10 mmol), 5-hydroxy-6-methylpyrimidine-4-carboxylic acid (19 mg, 0.15 mmol), HATU (79 mg, 0.21 mmol), and DMAP (13 mg, 0.10 mmol) were dissolved in anhydrous DCM (2 mL) and anhydrous DMF (0.2 mL). DIEA (135 mg, 1.04 mmol) was then added. After addition, the mixture was stirred at room temperature for 10 minutes and then at 35°C for 16 hours. The reaction mixture was filtered directly under LCMS monitoring, and the filtrate was purified by prep-HPLC to afford product A193 (11 mg, 15% yield). ESI-MS m / z = 654.32 [M+H] + .

[0463] Example 217 Synthesis of Compound A217

[0464] 2-(2-(3,6-dihydro-2H-pyran-4-yl)-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-(4-(trifluoromethyl)naphthalen-1-yl)acetamide

[0465] Step 1: Synthesis of compound A217-1

[0466] 1-Naphthylamine (15.00 g, 104.76 mmol) and N,N-dimethylformamide dimethyl acetal (75.00 mL) were added to a 250 mL single-necked bottle and reacted at 100°C for 16 hours. The reaction was complete by LCMS. The system was directly purified by reverse phase purification to obtain compound A217-1 (16 g, yield: 77%). ESI-MS m / z = 199.12 [M+H] + .

[0467] Step 2: Synthesis of compound A217-2

[0468] In a 250 mL single-necked flask, A217-1 (10 g, 50.44 mmol) was dissolved in DMF (100.00 mL). NIS (14.75 g, 65.57 mmol) was added and stirred at room temperature for 1 hour. After the reaction, ethyl acetate and water were added, the mixture was separated, and the solvent was removed by rotary evaporation. Silica gel was added and the sample was purified, with the eluent changing from pure PE to pure EtOAc. The sample was concentrated to obtain compound A217-2 (11.5 g, yield: 67%). ESI-MS m / z = 325.01 [M+H] + .

[0469] Step 3: Synthesis of compound A217-3

[0470] A217-2 (500.00 mg, 1.54 mmol), N,N-dimethylformamide (5.00 mL), cuprous chloride (45.81 mg, 0.46 mmol), and methyl fluorosulfonyldifluoroacetate (888.95 mg, 4.63 mmol) were added to a 25 mL single-necked flask and reacted at 100°C for 3 hours. The reaction solution was poured into ice water to precipitate a solid, which was filtered. The product was extracted with EtOAc in the aqueous phase, and the organic phase was dried and spin-dried to give compound A217-3 (180 mg, 43% yield). ESI-MS m / z = 267.10 [M+H] + .

[0471] Step 4: Synthesis of compound A217-4

[0472] A217-3 (4.30 g, 16.15 mmol), ethanol (25.00 mL), and hydrazine hydrate (40.00 mL) were added to a 250 mL single-necked bottle and heated to 80°C for 2 hours. After the reaction was complete, the system was directly purified by reverse phase purification and dried to give compound A217-4 (1.5 g, yield: 41%). ESI-MS m / z = 212.06 [M+H] + .

[0473] Step 5: Synthesis of compound A217-5

[0474] To a 100 mL single-necked flask, intermediate B5 (1.50 g, 3.07 mmol), A217-4 (0.59 g, 2.79 mmol), dichloromethane (10.00 mL), and pyridine (1.12 mL, 13.96 mmol) were added. Phosphorus oxychloride (0.52 mL, 5.58 mmol) was added in an ice bath and allowed to react at room temperature for 1 hour. LCMS confirmed the reaction was complete. The reaction was quenched with water and extracted with DCM. The organic phase was dried and spin-dried to give compound A217-5 (2.0 g, 98% yield). ESI-MS m / z = 682.29 [M+H] + .

[0475] Step 6: Synthesis of compound A217-6

[0476] A217-5 (2.0 g, 4.11 mmol), dichloromethane (30.00 mL), methanol (30.00 mL), and 4.0 M hydrogen chloride in dioxane (20.00 mL) were added to a 250 mL single-necked bottle and reacted at room temperature for 1 hour. The mixture was concentrated and spin-dried to dryness. EtOAc (60.00 mL) was added, the mixture was slurried, and filtered to obtain 2.4 g of a solid (88% pure). Methanol was added to slurry to obtain compound A217-6 (1.2 g, yield: 60%). ESI-MS m / z = 582.24 [M+H] + .

[0477] Step 7: Synthesis of Compound A217

[0478] A217-6 (1.20 g, 2.06 mmol), 5-hydroxy-6-methylpyrimidine-4-carboxylic acid (0.38 g, 2.48 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.79 g, 4.13 mmol), 1-hydroxybenzotriazole (0.56 g, 4.13 mmol), and N,N-dimethylformamide (12.00 mL) were added to a 50 mL single-necked vial. N,N-diisopropylethylamine (1.71 mL, 10.32 mmol) was added dropwise. The reaction was allowed to proceed at room temperature for 1 hour. The reaction was complete. The system was then directly reversed and lyophilized to give compound A217 (861 mg, 56% yield). ESI-MS m / z = 718.26 [M+H] + .

[0479] Compound A217: 1 H NMR (500MHz, DMSO-d6) δ10.71(s,1H),10.23(s,1H),8.58(s,1H),8.42(d,J=8.6Hz,1H),8.18-8.09(m,1H),8. 00(d,J=8.1Hz,1H),7.87(d,J=8.0Hz,1H),7.81(ddd,J=8.4,6.9,1.3Hz,1H),7.78-7.68(m,1H),6.89(s,1H),5 .37(s,2H),4.53(d,J=12.5Hz,1H),4.27(q,J=2.9Hz,2H),3.82(t,J=5.5Hz,2H),3.50(t,J=11.9Hz,3H),3.26 (d,J=25.2Hz,2H),3.13-2.95(m,3H),2.83(d,J=11.3Hz,1H),2.66(s,1H),2.44(s,3H),1.23(t,J=7.4Hz,3H).

[0480] Example 226 Synthesis of Compound A226

[0481] N-(4-chlorobenzofuran-7-yl)-2-(2-(3,6-dihydro-2H-pyran-4-yl)-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)acetamide

[0482] Step 1: Synthesis of compound A226-1

[0483] In a 50 mL single-necked flask, substrate B5 (200 mg, 0.41 mmol) and 4-chloro-1-benzofuran-7-amine (137.2 mg, 0.82 mmol) were added to DCM (10 mL). The condensing agent HOBt (165.9 mg, 1.23 mmol) and PyBOP (639.1 mg, 1.23 mmol) were then added sequentially. Finally, DIPEA (476.2 mg, 3.68 mmol) was added dropwise. The mixture was reacted at 25°C for 2 hours. The reaction mixture was poured into ice water (10 mL), extracted three times with DCM (10 mL), dried over anhydrous sodium sulfate, and filtered to give A226-1 (246.0 mg, 94% yield) as a yellow solid. ESI-MS m / z = 638.12 [M+H] + .

[0484] Step 2: Synthesis of compound A226-2

[0485] In a 25 mL single-necked vial, substrate A226-1 (220 mg, 0.34 mmol) was added to DCM (5 mL). Trifluoroacetic acid (387.6 mg, 3.4 mmol) was added dropwise at room temperature. The mixture was stirred at room temperature for 1 hour. Evaporation under reduced pressure afforded A226-2 (160.0 mg, 86% yield) as a yellow solid. ESI-MS m / z = 538.01 [M+H] + .

[0486] Step 3: Synthesis of compound A226

[0487] In a 10 mL single-necked vial, substrate A226-2 (100 mg, 0.19 mmol) and 5-hydroxy-6-methylpyrimidine-4-carboxylic acid (28.6 mg, 0.19 mmol) were added to DMF (2 mL). HOBt (50.1 mg, 0.37 mmol) and EDCI (106.9 mg, 0.56 mmol) were then added dropwise. Finally, DIPEA (120.1 mg, 0.78 mmol) was added dropwise. The mixture was reacted at 80°C for 1 hour. After cooling, the reaction mixture was poured into water (10 mL) and extracted with ethyl acetate (50 mL). The organic phase was dried, evaporated under reduced pressure, and dissolved in DMF (3 mL) for preparation. A226 (13.1 mg, 10% yield) was obtained by lyophilization as a white solid. ESI-MS m / z = 674.12 [M+H] + .

[0488] Compound A226: 1H NMR(500MHz,DMSO-d6)δ10.81(s,1H),8.53(s,1H),8.22(d,J=2.3Hz,1H),7.78(d,J=8.5Hz,1H), 7.29(d,J=8.5Hz,1H),7.07(d,J=2.3Hz,1H),6.87-6.76(m,1H),5.31(s,2H),4.52(d,J=12.6Hz,1 H),4.24(q,J=2.9Hz,2H),3.80(t,J=5.5Hz,2H),3.57-3.45(m,3H),3.29-3.21(m,2H),2.99(d,J =13.1Hz,3H),2.82(d,J=11.5Hz,1H),2.64(d,J=10.5Hz,1H),2.43(s,3H),1.19(t,J=7.4Hz,3H).

[0489] Example 231 Synthesis of Compound A231

[0490] 2-(2-(3,6-dihydro-2H-pyran-4-yl)-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-(4-(trifluoromethyl)benzofuran-7-yl)acetamide

[0491] Step 1: Synthesis of compound A231-1

[0492] To a 250 mL single-necked flask, 2-bromo-5-(trifluoromethyl)phenol (10.00 g, 41.49 mmol), toluene (100 mL), 2,2-diethoxyethanol (7.24 g, 53.94 mmol), and cyanomethylenetri-n-butylphosphine (20 g, 82.99 mmol) were added. Under nitrogen, the mixture was reacted at 90°C for 3 hours. The reaction progress was monitored by TLC. After completion, the reaction was concentrated under reduced pressure, and the residue was purified by flash column chromatography to obtain A231-1 (12.90 g, 87% yield).

[0493] Step 2: Synthesis of compound A231-2

[0494] To a 100 mL single-necked flask, add compound A231-1 (2.0 g, 5.6 mmol), toluene (30 mL), and polyphosphoric acid (4.0 g). Incubate at 100°C for 4 hours, monitoring the reaction progress with LCMS. Upon completion, cool the reaction, remove the supernatant, and use it directly in the next step. Use 600 mg (2.26 mmol) of the product in the next step.

[0495] Step 3: Synthesis of compound A231-3

[0496] To a 100 mL single-necked flask, the reaction mixture from the previous step (A231-2) was added with benzophenone imine (410 mg, 2.26 mmol), Pd2(dba)3 (207 mg, 0.23 mmol), BINAP (282 mg, 0.45 mmol), and sodium tert-butoxide (652.7 mg, 6.79 mmol). The air in the reaction flask was replaced with nitrogen three times. Under nitrogen protection, the reaction was carried out at 100°C for 3 hours. The progress of the reaction of A231-2-1 was monitored by LCMS. After completion of the reaction, the mixture was concentrated under reduced pressure. The residue was added with EtOAc (60 mL) and water (80 mL), shaken, and the layers separated. The organic phase was concentrated under reduced pressure, and the residue was added with 20 mL of methanol. The mixture was sonicated and filtered. The filtrate was used directly in the next step.

[0497] To a 100 mL single-necked flask, the filtrate from the previous step was added a 4 M solution of hydrogen chloride in 1,4-dioxane (6 mL). Deprotection was monitored by LCMS. After the reaction was complete, water (60 mL), EtOAc (35 mL), and saturated aqueous potassium carbonate (15 mL) were added and the mixture was shaken to separate the layers. The mixture was extracted with EtOAc (20 mL), and the combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was subjected to reverse phase preparative separation to afford A231-3 (85 mg). ESI-MS m / z = 202.04 [M+H] + .

[0498] Step 4: Synthesis of compound A231-4

[0499] In a 25 mL single-necked vial, compound intermediate B5 (247.7 mg, 0.51 mmol) and A231-3 (85 mg, 0.42 mmol) were dissolved in anhydrous DCM (5 mL). Pyridine (200 mg, 2.54 mmol) and phosphorus oxychloride (194 mg, 1.27 mmol) were added and the mixture was allowed to warm to room temperature for 30 minutes. The reaction was monitored for completion by LCMS. DCM (20 mL) and water (30 mL) were added to the system, and the mixture was shaken. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain A231-4 (270 mg, 95% yield).

[0500] Step 5: Synthesis of compound A231-5

[0501] In a 25 mL single-necked vial, substrate A231-4 (270 mg, 0.40 mmol) was added to DCM (6 mL). Trifluoroacetic acid (2 mL) was added at room temperature and the reaction was stirred at room temperature for 1 hour. After LCMS monitoring, the system was added with DCM (30 mL), water (30 mL), and saturated aqueous potassium carbonate (15 mL). The mixture was shaken and separated. The aqueous phase was extracted with DCM (15 mL). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was added with EtOAc:PE = 1:10 (10 mL), sonicated, and filtered to afford A231-5 (100 mg, 44% yield). ESI-MS m / z = 672.30 [M+H] + .

[0502] Step 6: Synthesis of Compound A231

[0503] In a 10 mL single-necked vial, compound A231-5 (22 mg, 0.04 mmol), 5-hydroxy-6-methylpyrimidine-4-carboxylic acid (8.9 mg, 0.06 mmol), HOBt (10.4 mg, 0.08 mmol), and EDCI (14.8 mg, 0.08 mmol) were dissolved in anhydrous DMF (2 mL). DIEA (19.9 mg, 0.15 mmol) was then added. After addition, the mixture was reacted at 80°C and monitored by LCMS. After completion of the reaction, the reaction solution was filtered and directly purified by prep-HPLC to afford product A231 (11.2 mg, 41%). ESI-MS m / z = 708.30 [M+H] + .

[0504] Compound A231: 1 H NMR (500MHz, DMSO-d6) δ11.10(s,1H),10.25(s,1H),8.59(s,1H),8.37(d,J=2.2Hz,1H),8.06(d,J=8.4 Hz,1H),7.61(d,J=8.5Hz,1H),7.14(p,J=1.6Hz,1H),6.81(td,J=3.0,1.5Hz,1H),5.37(s,2H),4.59-4 .48(m,1H),4.23(q,J=2.9Hz,2H),3.79(t,J=5.4Hz,2H),3.58-3.46(m,4H),3.26(td,J=13.1,12.7,3. 2Hz,1H),3.08-2.93(m,3H),2.83(d,J=11.1Hz,1H),2.72-2.60(m,1H),2.45(s,3H),1.25-1.16(m,3H).

[0505] Example 232 Synthesis of Compound A232

[0506] 2-(2-(3,6-dihydro-2H-pyran-4-yl)-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-(7-(trifluoromethyl)benzofuran-4-yl)acetamide

[0507] Step 1: Synthesis of intermediate A232-1

[0508] In a 50 mL single-necked bottle, 2-bromo-5-trifluoromethylphenol (1.0 g, 4.15 mmol) was added to toluene (10.0 mL), followed by 2,2-diethoxyethanol (0.67 g, 4.98 mmol). Cyanomethylenetri-n-butylphosphine (4.01 g, 16.60 mmol) was then added dropwise. The reaction was allowed to proceed at 100°C for 3 hours. The mixture was cooled to room temperature, concentrated under reduced pressure, and purified by column chromatography using PE / EtOAc = 1:20 to afford Intermediate A232-1 (1.4 g, 94% yield).

[0509] Step 2: Synthesis of intermediate A232-2

[0510] In a 50 mL single-necked bottle, intermediate A232-1 (700 mg, 1.96 mmol) was added to chlorobenzene (10.0 mL) and Amberlyst 15 ion exchange resin (272.4 mg, 1.96 mmol). The mixture was reacted at 120°C for 5 hours. The mixture was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure to afford intermediate A232-2 (500 mg, 96% yield).

[0511] Step 3: Synthesis of intermediate A232-3

[0512] In a 50 mL single-necked flask, intermediate A232-2 (500 mg, 1.89 mmol), tert-butyl carbamate (265.2 mg, 2.26 mmol), Pd2(dba)3 (172.8 mg, 0.19 mmol), Xantphos (109.1 mg, 0.19 mmol), and Cs2CO3 (1.54 g, 4.72 mmol) were added sequentially to 1,4-dioxane (10.0 mL) and reacted at 100°C for 3 hours. The reaction mixture was added with water, saturated sodium bicarbonate, and EtOAc (3 x 10 mL). The mixture was washed with saturated brine and dried over anhydrous sodium sulfate. The organic phases were combined, concentrated under reduced pressure, and purified by column chromatography using a PE / EtOAc ratio of 1:5 to afford intermediate A232-3 (100 mg, 18% yield). ESI-MS m / z = 302.27 [M+H].

[0513] Step 4: Synthesis of intermediate A232-4

[0514] In a 50 mL single-necked flask, compound A232-3 (100 mg, 0.33 mmol) was added to the reaction flask. DCM (5 mL) was added, and trifluoroacetic acid (376.3 mg, 3.3 mmol) was added dropwise at room temperature. The mixture was stirred at room temperature for 1 hour. After the reaction was complete, stirring was stopped. The reaction liquid was rotary evaporated to remove the solvent to obtain crude product A232-4 (50 mg, 0.25 mmol). ESI-MS m / z = 202.15 [M+H] + .

[0515] Step 5: Synthesis of compound A232-5

[0516] In a 10 mL single-necked vial, substrate B5 (100 mg, 0.20 mmol) and intermediate A232-4 (41.1 mg, 0.20 mmol) were added to DCM (2 mL). Pyridine (80.9 mg, 1.02 mmol) and POCl (62.7 mg, 0.41 mmol) were added sequentially under ice-cooling, and the mixture was reacted at 25°C for 1 hour. The reaction mixture was poured into ice water (10 mL), extracted with DCM (10 mL x 2), dried over anhydrous sodium sulfate, and evaporated under reduced pressure to afford A232-5 (80.0 mg, 58% yield) as a yellow solid. ESI-MS m / z = 671.68 [M+H] + .

[0517] Step 6: Synthesis of compound A232-6

[0518] In a 25 mL single-necked vial, substrate A232-5 (80 mg, 0.12 mmol) was added to DCM (5 mL). Trifluoroacetic acid (136.8 mg, 1.20 mmol) was added dropwise at room temperature. The mixture was stirred at room temperature for 1 hour. Evaporation under reduced pressure afforded A232-6 (45 mg, 65% yield) as a yellow solid. ESI-MS m / z = 571.56 [M+H] + .

[0519] Step 7: Synthesis of Compound A232

[0520] In a 10 mL single-necked vial, substrate A232-6 (45 mg, 0.08 mmol) and 5-hydroxy-6-methylpyrimidine-4-carboxylic acid (12.4 mg, 0.08 mmol) were added to DMF (2 mL). HOBt (32.6 mg, 0.24 mmol) and EDCI (46.1 mg, 0.24 mmol) were then added sequentially. Finally, DIPEA (51.6 mg, 0.40 mmol) was added dropwise. The mixture was reacted at 80°C for 1 hour. After cooling, the reaction mixture was poured into water (10 mL) and extracted with ethyl acetate (50 mL). The organic phase was dried, evaporated under reduced pressure, and dissolved in DMF (3 mL) for preparation. Freeze-dried, A232 (7.8 mg, 0.01 mmol, 14% yield) was obtained as a white solid. ESI-MS m / z = 707.67 [M+H] + .

[0521] Example 254 Synthesis of Compound A231

[0522] 2-(2-(3,6-dihydro-2H-pyran-4-yl)-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-(4-(trifluoromethyl)benzofuran-7-yl)acetamide

[0523] Step 1: Synthesis of compound A231-6

[0524] 4,6-Dichloro-5-methoxypyrimidine (36.0 g, 201.1 mmol) and tetrahydrofuran (200.00 mL) were added to a 1000 mL three-necked flask. A 3.0 M methylmagnesium chloride THF solution (100 mL, 300 mmol) was slowly added dropwise under an ice bath. The temperature was naturally raised to room temperature and the reaction was allowed to proceed overnight. After completion of the reaction, saturated aqueous ammonium chloride solution was added to quench the reaction. The mixture was extracted with ethyl acetate and washed with brine. The organic phase was dried and spin-dried. The mixture was separated by column chromatography with PE / EA = 1 / 10 to obtain compound A231-6 (28 g, yield: 88%). ESI-MS m / z = 159.2 [M+H] + .

[0525] Step 2: Synthesis of compound A231-7

[0526] Compound A231-6 was dissolved in methanol (150.00 mL), and triethylamine (52.59 mL, 378.34 mmol) and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane complex (15.45 g, 18.92 mmol) were added. The mixture was reacted at 60°C for 3 days under a carbon monoxide atmosphere. The reaction was complete by liquid chromatography-mass spectrometry. The filtrate was filtered, dried, and column chromatography was performed using a PE / EA ratio of 1:10 to obtain compound A231-7 (29 g, 84% yield). ESI-MS m / z = 183.1 [M+H] + .

[0527] Step 3: Synthesis of compound A231-8

[0528] Compound A231-7 (18.22 g, 100 mmol) was dissolved in a mixed solvent of methanol (50.00 mL) and water (10.00 mL). Sodium hydroxide (4.40 g, 110 mmol) was added and the mixture was allowed to react at room temperature for 1 hour. The reaction was complete after liquid chromatography-mass spectrometry. Methanol was removed by rotary evaporation and then extracted with ethyl acetate. The aqueous phase was separated and 6N hydrochloric acid was added to adjust the pH to 1. A large amount of off-white solid precipitated. The solid was filtered and dried to give compound A231-8 (14.2 g, yield: 84%). ESI-MS m / z = 169.2 [M+H] + .

[0529] Step 4: Synthesis of compound A231-9

[0530] Compound A231-8 (9.03 g, 53.7 mmol) was dissolved in N,N-dimethylformamide (50.00 mL), and EDCI (12.35 g, 64.4 mmol) and HOBt (8.71 g, 64.43 mmol) were added. The mixture was stirred at room temperature for 10 minutes, and then tert-butyl piperazine-1-carboxylate (10.00 g, 53.7 mmol) was added. After stirring for 5 minutes, N,N-diisopropylethylamine (26.62 mL, 161 mmol) was added. After reacting for 1 hour, the reaction was completed by liquid chromatography-mass spectrometry. The reaction was quenched with water and extracted with ethyl acetate. The organic phase was dried and purified by column chromatography with EA / PE = 1 / 1 to obtain compound A231-9 (14.10 g, yield: 78.1%), ESI-MS m / z = 281.1 [M+H-56]. + .

[0531] Step 5: Synthesis of compound A231-10

[0532] Compound A231-9 (10.0 g, 29.7 mmol) was dissolved in methanol (30.00 mL) and a 4.0 M hydrogen chloride solution in dioxane (10.00 mL) was added. The mixture was allowed to react at room temperature for 2 hours. After liquid chromatography-mass spectrometry, the reaction was complete. Most of the solvent was removed by rotary evaporation, and ethyl acetate was added. The mixture was filtered, and the filter cake was washed with ethyl acetate. The solid was then dried to give compound A231-10 (8.0 g, yield: 99%). ESI-MS m / z = 237.3 [M+H] + .

[0533] Step 6: Synthesis of Compound A231-11

[0534] Method 1: Methyl 3-oxopentanoate (13.00 g, 99.9 mmol) was dissolved in DCM / dichloromethane (130.00 mL). N-chlorosuccinimide (14.67 g, 109.9 mmol) and p-toluenesulfonic acid (1.72 g, 10.0 mmol) were added under ice bath and reacted at room temperature overnight. TLC showed that a slightly less polar spot was generated. The mixture was filtered, the filter cake was washed with dichloromethane, the filtrate was washed twice with water, and then washed with saturated sodium chloride aqueous solution. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was dried to give compound A231-11 (15.00 g, 91.2%).

[0535] Method 2: Methyl 3-oxopentanoate (5.20 g, 40.0 mmol) was dissolved in dichloromethane (50.00 mL). Methanesulfonyl chloride (5.93 g, 44.0 mmol) was added dropwise under ice-bath and the mixture was allowed to warm naturally. The reaction mixture was allowed to react for 6 hours. TLC showed that the starting material disappeared. The reaction mixture was poured into ice-water for extraction. The organic phase was separated and washed twice with water, dried over anhydrous sodium sulfate, filtered, and the filtrate was dried to give compound A231-11 (6.20 g, 94.3%).

[0536] Step 7: Synthesis of Compound A231-12

[0537] Compound A231-10 (5.76 g, 21.1 mmol) was dissolved in acetonitrile (60.00 mL), and compound A231-11 (5.21 g, 31.68 mmol) and N,N-diisopropylethylamine (17.5 mL, 105.6 mmol) were added. The temperature was raised to 60°C and the reaction was allowed to proceed overnight. The reaction was detected by liquid chromatography-mass spectrometry. The product was dried and passed through a column with EA / PE = 1 / 1 to obtain compound A231-12 (5.70 g, yield: 74.1%). ESI-MS m / z = 365.20 [M+H] + .

[0538] Step 8: Synthesis of Compound A231-13

[0539] Compound A231-12 (0.82 g, 5.01 mmol) and 5-bromo-2H-1,2,4-triazole-3-amine (0.82 g, 5.01 mmol) were cyclized in an appropriate solvent by an acid-catalyzed method to give compound A231-13 (1.51 g, yield: 75.8%), ESI-MS m / z = 477.3 / 479.3 [M+H] + .

[0540] Step 9: Synthesis of Compound A231-14

[0541] Compound A231-3 (6.70 g, 28.44 mmol) was dissolved in dichloromethane (67.00 mL), and triethylamine (13.84 mL, 99.54 mmol) was added. Chloroacetyl chloride (4.82 g, 42.66 mmol) was added dropwise in an ice bath. The reaction was allowed to react overnight. Ice water was added to the reaction system to quench the reaction. After separation, the organic phase was dried and evaporated to dryness. After purification by column chromatography, compound A231-14 (7.2 g, yield: 91%) was obtained.

[0542] Step 10: Synthesis of compound A231-15

[0543] Compound A231-13 (1.50 g, 3.14 mmol), A231-14 (0.96 g, 3.46 mmol), N,N-diisopropylethylamine (1.56 mL, 9.43 mmol) and potassium iodide (0.52 g, 3.14 mmol) were added to acetonitrile (20.00 mL). The system was heated to 70°C and reacted overnight. The reaction was detected by liquid chromatography-mass spectrometry. The mixture was filtered, dried, and passed through a column. The mixture was purified by DCM / MeOH = 10 / 1 to obtain compound A231-15 (1.83 g, yield: 81%). ESI-MS m / z = 718.2 / 720.2 [M+H] + .

[0544] Step 11: Synthesis of Compound A231-16

[0545] In a 100 mL single-necked bottle, compound A231-15 (1.20 g, 1.67 mmol), 3,6-dihydro-2H-pyran-4-boronic acid pinacol ester (0.53 g, 2.51 mmol), methanesulfonic acid (2-dicyclohexylphosphino-2',4',6'-tri-isopropyl-1,1'-biphenyl)(2'-amino-1,1'-biphenyl-2-yl) palladium (II) (0.14 g, 0.17 mmol) were added. l), K3PO4 (1.06 g, 5.01 mmol), 1,4-dioxane (20.00 mL) and water (5.00 mL), heated to 100°C under nitrogen protection for 3 hours. After completion of the reaction, HPLC-MS / MS was performed, and the mixture was extracted with ethyl acetate. The organic phase was separated and dried, and separated by column chromatography with DCM / MeOH = 10 / 1 to obtain compound A231-16 (780 mg, yield: 64.7%). ESI-MS m / z = 722.3 [M+H] + .

[0546] Step 12: Synthesis of Compound A231

[0547] Compound A231-16 (50.00 mg, 0.07 mmol) was dissolved in DMF / N,N-dimethylformamide (2.00 mL), and sodium methylthiolate (14.57 mg, 0.21 mmol) was added. The temperature was raised to 100°C and the reaction was allowed to react for 2 hours. Liquid chromatography-mass spectrometry showed that the starting material had disappeared. The reaction solution was filtered and reverse phase was used to obtain compound A231 (16 mg, yield: 32%). ESI-MS m / z = 708.2 [M+H] +.

[0548] Example 255 Synthesis of Compound A231

[0549] 2-(2-(3,6-dihydro-2H-pyran-4-yl)-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-(4-(trifluoromethyl)benzofuran-7-yl)acetamide

[0550] Step 1: Synthesis of compound A231-17

[0551] Benzyl-1-piperazine carbonate (6.01 g, 27.27 mmol) was dissolved in acetonitrile (60 mL), and methyl 2-bromo-3-oxopentanoate (5.00 g, 23.92 mmol) and potassium carbonate (6.94 g, 50.23 mmol) were added, respectively. The mixture was allowed to react overnight at room temperature. After completion of the reaction, the mixture was filtered and the filter cake was washed with ethyl acetate. The filtrate was concentrated under reduced pressure and separated by column chromatography using PE / EA = 80 / 20 to give compound A231-17 (4.58 g, yield: 55%). ESI-MS m / z = 349.1 [M+H] + .

[0552] Step 2: Synthesis of compound A231-18

[0553] In a 100 mL single-necked bottle, compound 5-bromo-2H-1,2,4-triazole-3-amine (0.48 g, 2.96 mmol), A231-18 (1.03 g, 2.96 mmol), 1,4-dioxane (10.00 mL) and trifluoroacetic acid (5.00 mL) were added respectively. The reaction was carried out at 100°C overnight and concentrated under reduced pressure. The crude product was diluted with water and the pH was adjusted to about 8 with aqueous sodium bicarbonate solution. The product was extracted twice with DCM, the mixture was combined, dried, concentrated under reduced pressure, and separated by column chromatography with PE / EA = 1 / 4 as the eluent to give compound A231-18 (0.45 g, yield: 33%). ESI-MS m / z = 462 / 464 [M+H] + .

[0554] Step 3: Synthesis of compound A231-19

[0555] In a 100 mL single-necked bottle, compound A231-14 (415 mg, 1.27 mmol), compound A231-18 (390.00 mg, 0.85 mmol), acetonitrile (10.00 mL), and N,N-diisopropylethylamine (1.40 mL, 8.45 mmol) were added, respectively, and the mixture was reacted at 80°C overnight. The mixture was concentrated under reduced pressure and separated by column chromatography with PE / EA = 1 / 3 as the eluent to obtain compound A231-19 (385.00 mg, 64.83%). ESI-MS m / z = 703.4 [M+H] + .

[0556] Step 4: Synthesis of compound A231-20

[0557] Compound A231-19 (375.00 mg, 0.53 mmol), 3,6-dihydro-2H-pyran-4-boronic acid pinacol ester (224.28 mg, 1.07 mmol), potassium phosphate (566.54 mg, 2.67 mmol), methanesulfonic acid (2-dicyclohexylphosphino-2',4',6'-tri-isopropyl-1,1'-biphenyl) (2'-amino-1, 1'-biphenyl-2-yl)palladium(II) (90.40 mg, 0.11 mmol), 1,4-dioxane (10.0 mL) and water (5.0 mL) were reacted at 80°C under nitrogen for 3 hours. The reaction solution was filtered, concentrated under reduced pressure, and purified by column chromatography with DCM / MeOH = 2 / 1 as the eluent (105.00 mg, 34.4%) to give compound A231-20 (53.00 mg, 14.07%), ESI-MS m / z = 706.7 [M+H] + .

[0558] Step 5: Synthesis of compound A231-21

[0559] Compound A231-20 (21.0 mg, 0.03 mmol) was dissolved in DCM (2 mL). 1.0 M boron trichloride dichloromethane solution (0.6 mL) was added under ice-cooling and the mixture was kept warm for 1 hour. After completion of the reaction, the mixture was quenched with ice water, extracted with DCM, washed with water, and dried. The organic phase was spin-dried to give compound A231-21 (16.0 mg, 85%). ESI-MS m / z = 572.6 [M+H] + .

[0560] Step 6: Synthesis of Compound A231

[0561] Compound A231-22 (90.00 mg, 0.16 mmol), 5-hydroxy-6-methylpyrimidine-4-carboxylic acid (60.69 mg, 0.39 mmol), EDCI (90.58 mg, 0.47 mmol), HOBt (63.84 mg, 0.47 mmol), DMF (5.00 mL) and DIEA (0.26 mL, 1.57 mmol) were added sequentially to a 50 mL single-necked bottle and reacted at 80°C for 1 hour. The reaction was quenched by adding water and the compound A231 (27.20 mg, 23.18%) was prepared by reverse phase reaction. ESI-MS m / z = 708.3 [M+H] + .

[0562] Example 256 Synthesis of Compound A231

[0563] 2-(2-(3,6-dihydro-2H-pyran-4-yl)-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-(4-(trifluoromethyl)benzofuran-7-yl)acetamide

[0564] Step 1: Synthesis of compound A231-22

[0565] Compound A231-13 (500.00 mg, 1.05 mmol), tert-butyl bromoacetate (408.64 mg, 2.10 mmol), potassium carbonate (579.10 mg, 4.19 mmol), and acetonitrile (20.00 mL) were added sequentially to a 100 mL single-necked vial. The mixture was heated to 75°C and reacted for 16 hours. The reaction was complete by liquid chromatography-mass spectrometry. The filtrate was filtered and dried, and then passed through a column. Elution with DCM:MeOH = 10:1 afforded A231-22 (500.00 mg, 80.7%). ESI-MS m / z = 591.30 [M+H] + .

[0566] Step 2: Synthesis of compound A231-23

[0567] Compound A231-22 (1.20 g, 2.03 mmol), 3,6-dihydro-2H-pyran-4-boronic acid pinacol ester (0.85 g, 4.06 mmol), methanesulfonic acid (2-dicyclohexylphosphino-2',4',6'-tri-isopropyl-1,1'-biphenyl)(2'-amino-1,1'-biphenyl-2-yl) palladium (II) (0.17 g, 0.20 mmol), potassium phosphate (1.29 g, 6.09 mmol), 1,4-dioxane (40.00 mL) and water (8.00 mL) were added sequentially to a 100 mL single-necked bottle. The reaction system was heated to 100 ° C for 6 hours. After the reaction was completed, the product was spin-dried and passed through a column. DCM:MeOH = 10:1 was used as the eluent to obtain A231-23 (600.00 mg, 79%). ESI-MS m / z=595.40[M+H] + .

[0568] Step 3: Synthesis of compound A231-24

[0569] Compound A231-23 (200.00 mg, 0.34 mmol) was dissolved in N-methylpyrrolidone (2.00 mL), and pyridine hydrochloride (1.17 g, 10.09 mmol) was added. The temperature was raised to 126°C and the reaction was allowed to proceed for 18 hours. The reaction was completed by liquid chromatography-mass spectrometry. The reaction solution was filtered and A231-24 (88 mg, 50%) was prepared by reverse phase chromatography. ESI-MS m / z = 525.20 [M+H] + .

[0570] Step 4: Synthesis of compound A231

[0571] Compound A231-24 (52 mg, 0.10 mmol), compound A231-3 (20 mg, 0.1 mmol), EDCI (38.5 mg, 0.2 mmol), HOBt (27 mg, 0.2 mmol), DMF (5.00 mL) and DIEA (52 mg, 0.4 mmol) were added to a 50 mL single-necked bottle in sequence. The mixture was reacted at 80°C for 1 hour. The reaction was quenched by adding water and the compound A231 (37 mg, 52%) was prepared by reverse phase reaction. ESI-MS m / z = 708.30 [M+H] + .

[0572] Example 260 Synthesis of Compound A260

[0573] 2-(2-(dimethylamino)-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-(4-(trifluoromethyl)benzofuran-7-yl)acetamide

[0574] Step 1: Synthesis of compound A260-1

[0575] Compound B3 (4.26 g, 10 mmol), compound A231-14 (3.05 g, 11 mmol), acetonitrile (35 mL), and N,N-diisopropylethylamine (5.20 mL, 30 mmol) were added to a 100 mL single-necked bottle, and the mixture was reacted at 80°C overnight. After monitoring the completion of the reaction, the mixture was concentrated under reduced pressure and separated by column chromatography with PE / EA = 1 / 3 as the eluent to obtain compound A260-1 (5.52 g, 82.58%). ESI-MS m / z = 668.2 [M+H] + .

[0576] Step 2: Synthesis of compound A260-2

[0577] Dimethylamine hydrochloride (2.46 g, 30.22 mmol), A260-1 (1.01 g, 1.51 mmol), DMF (10.00 mL), and DIEA (10.24 mL, 61.95 mmol) were added sequentially to a 50 mL single-necked flask. The temperature was raised to 120°C for reaction. After monitoring the completion of the reaction, water was added to quench the reaction. The mixture was extracted with EA, washed twice with brine, concentrated under reduced pressure, and purified by column chromatography to obtain compound A260-2 (0.92 g, 96.25%). ESI-MS m / z = 633.3 [M+H] + .

[0578] Step 3: Synthesis of compound A260-3

[0579] A260-2 (0.92 g, 1.45 mmol), DCM / dichloromethane (10.00 mL), and HCl / hydrochloric acid (4M in dioxane) (10.91 mL, 4.00 mol / L, 43.63 mmol) were added sequentially to a 50 mL single-necked bottle. The reaction was allowed to react at room temperature. After monitoring the completion of the reaction, the product was concentrated under reduced pressure. The crude product A260-3 was directly used in the next step. ESI-MS m / z = 533.3 [M+H] + .

[0580] Step 4: Synthesis of compound A260

[0581] A260-3 (750.00 mg, 1.41 mmol), 5-hydroxy-6-methylpyrimidine-4-carboxylic acid (651.20 mg, 4.23 mmol), EDCI (809.97 mg, 4.23 mmol), HOBt (380.61 mg, 2.82 mmol), DIEA (2.33 mL, 14.08 mmol), and DMF (15.00 mL) were added sequentially to a 50 mL single-necked bottle. The reaction was stirred at 60°C for 1 h. After monitoring the reaction completion, the product was purified by reverse phase chromatography to obtain compound A260 (433.4 mg, 46.02%). ESI-MS m / z = 669.6 [M+H] + .

[0582] Example 264 Synthesis of Compound A264

[0583] 2-(2-(1-cyclopropyl-1H-pyrazol-4-yl)-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-(4-(trifluoromethyl)benzofuran-7-yl)acetamide

[0584] Step 1: Synthesis of compound A264-2

[0585] In a 100 mL single-necked bottle, A260-1 (1.08 g, 2 mmol), A264-1 (536 mg, 5 mmol), XPhos Pd G3 PdCl2(dppf)CH2Cl2 (163 mg, 0.2 mmol), and K2CO3 (829 mg, 6 mmol) were added to 1,4-dioxane (10.00 mL) and water (2.00 mL). After nitrogen replacement, the temperature was raised to 100°C and the reaction was allowed to react for 3 hours. LCMS confirmed the reaction was complete. Water (20 mL) was added to the reaction solution, and the product was extracted with ethyl acetate (50 mL x 2). The organic phase was dried by spin drying and then purified by column chromatography using a PE / EA ratio of 2 / 1 to afford the product A264-2 (1.04 g, 75% yield). ESI-MS m / z = 696.3 [M+H] + .

[0586] Step 2: Synthesis of compound A264-3

[0587] A264-2 (1.02 g, 1.47 mmol), DCM / dichloromethane (10.00 mL), and HCl / hydrochloric acid (4M in dioxane) (10.91 mL, 4.00 mol / L, 43.63 mmol) were added sequentially to a 50 mL single-necked bottle. The reaction was allowed to react at room temperature. After monitoring the reaction completion, the product was concentrated under reduced pressure. The crude product A264-3 was directly used in the next step. ESI-MS m / z = 595.3 [M+H] + .

[0588] Step 3: Synthesis of compound A264

[0589] A264-3 (840.00 mg, 1.41 mmol), 5-hydroxy-6-methylpyrimidine-4-carboxylic acid (651.20 mg, 4.23 mmol), EDCI (809.97 mg, 4.23 mmol), HOBt (380.61 mg, 2.82 mmol), DIEA (2.33 mL, 14.08 mmol), and DMF (15.00 mL) were added sequentially to a 50 mL single-necked bottle. The reaction was stirred at 60°C for 1 h. After monitoring the reaction completion, the product was purified by reverse phase chromatography to obtain compound A260 (533 mg, 51.66%). ESI-MS m / z = 732.3 [M+H] + .

[0590] Other example compounds in Table 2 were prepared by similar methods to those used to synthesize the above-mentioned final products:

[0591] Table 2

[0592] Comparative Compound A

[0593] The above compound was prepared according to Example 42 of WO2022249060.

[0594] Pharmacological tests

[0595] Example A: In vitro enzymatic activity detection of WRN ATPase

[0596] The experiment used the ATPase kit ADP-Glo TM Kinase Assay (Promage, V9102) was used to detect the amount of ADP generated and to evaluate the inhibitory activity of the compounds against WRN ATPase. The reaction buffer consisted of 20 mM Tris-HCl [pH 8.0], 50 mM NaCl, 1 mM EDTA, 3 mM MgCl2, 0.1 mg / mL BSA, and 1 mM DTT. 8uL WRN protein solution and 4uL of serially diluted test compound solution were added to a 384-well reaction plate and incubated at 25°C for 15 min. Then, 8uL of ssDNA and ATP mixed solution were added to make the final concentrations of each component of the 20uL reaction system 1nM WRN protein, 20nM ssDNA, 1uM ATP, and the final concentrations of the test compound were 1000, 333.33, 111.11, 37.04, 12.35, 4.12, 1.37, 0.46, 0.15, 0.05, and 0nM, respectively. The final DMSO concentration was 1%. The reaction was incubated at 25°C for 60 min. 10uL ADP-Glo ​​Reagent solution was added to each well of the reaction plate and incubated at 25°C for 40 min. Finally, ADP-Glo ​​Detection reagent was added and incubated at 25°C in the dark for 40 min. Luminescence luminescence values ​​(RLU values) were read on Envision (Perkin Elmer) and raw data were collected.

[0597] Convert RLU values ​​to Inhibition % values:

[0598] The inhibition percentage of each well (%inh) = 100*(maximum value - measured value) / (maximum value - Blank)

[0599] ("Maximum" is from the 1% DMSO control wells, "Blank" is from the blank control wells, and "Measured" is from the compound reaction wells).

[0600] GraphPad Prism software was used for curve fitting and IC 50 The results are shown in Table 3.

[0601] Table 3

[0602] Example B: MSI-H cell proliferation inhibition experiment

[0603] HCT-116 (MSI-H) and SW48 (MSI-H) cells were plated at a density of 1000 cells / well and 1500 cells / well in a white 96-well plate, respectively. After stabilization for 24 h in a cell culture incubator, the test compounds were added to the 96-well plate at final concentrations of 20,000, 6666.67, 2222.22, 740.74, 246.91, 82.30, 27.43, 9.14, 3.05, and 0 nM (DMSO final concentration was 0.1%). The cell plates were cultured in a 37°C, 5% CO2 cell culture incubator for 7 days. After that, 50 μL of Cell-titer GLO working solution was added to each well, mixed by vortexing, and incubated at room temperature for 10 min. Luminescence luminescence (RLU) values ​​were read on an Envision (Perkin Elmer) and converted to percent inhibition. The percent inhibition of cell proliferation was calculated according to the following formula:

[0604] Inhibition percentage = (maximum value - measured value) / (maximum value - Blank) × 100

[0605] ("Maximum value" is from the 0.1% DMSO control well, "Blank" is from the blank control well, and "Measured value" is from the compound reaction well).

[0606] GraphPad Prism software was used for curve fitting and IC 50 The results are shown in Table 4.

[0607] Table 4 “-” means not detected.

[0608] Example C: Western Blot Experiment

[0609] For cell or tissue samples, add 1× Lysis Buffer and incubate at 100°C for 5 minutes to fully denature the protein sample. After denaturation, determine the protein concentration using the BCA protein quantification assay and adjust all samples to the same concentration. Before loading the sample, add 4× LDS Lysis Buffer, incubate at 100°C for 5 minutes, and centrifuge at 1000 rpm for one minute before loading.

[0610] Prepared protein samples were added to the wells of a 4-12% precast gel and transferred by wet transfer at 80V for 20 minutes, followed by 120V for 90 minutes. Following transfer, the membrane was blocked with 1×TBST containing 2.5% BSA at room temperature for 1 hour, followed by incubation with primary antibody at 4°C overnight. The next day, the membrane was washed three times with 1×TBST at room temperature on a shaker for 10 minutes at high speed, then developed by adding secondary antibody dilution and shaking slowly on a shaker at room temperature for 1 hour.

[0611] The western blot results of tumor tissue and cells are shown in Figures 1 and 2, respectively.

[0612] The experiments showed that compared with the control compound A, the compound of the present invention had a more significant inhibitory effect on WRN protein activity, and the changes in downstream biomarkers γH2AX and p21 were also more obvious.

[0613] Example D: Plasma Protein Binding Assay

[0614] Plasma protein binding was determined as follows.

[0615] 1) Preparation of 10 mM PBS buffer (pH 7.3 ± 0.1)

[0616] Take a bag of PBS powder purchased from Solebol (product number: P1010) and dissolve it in 2 L of ultrapure water, and dissolve it by ultrasonication.

[0617] 2) Preparation of plasma

[0618] Frozen plasma was thawed immediately at room temperature.

[0619] Centrifuge the plasma at 3,220 x g for 10 minutes to remove clots and collect the supernatant into a fresh tube. Check and record the pH of the plasma. This test can be performed with whole plasma or 50% (or 10%) plasma-PBS.

[0620] Note: a) Use only plasma that has been thawed no more than twice upon arrival. b) Use only plasma within the pH range of 7 to 8.

[0621] 3) Preparation of working solution

[0622] Working solutions of the test compound and the control compound ketoconazole were prepared at a concentration of 200 μM using a dilution of ACN:MeOH:H 2 O=1:1:1.

[0623] 4) Preparation of incubation system mother solution

[0624] Take 3 μL of the working solution and add it to 597 μL of blank whole plasma or 50% (or 10%) plasma-PBS, mix well, and obtain a 1 μM incubation stock solution (final DMSO concentration ≤ 0.5%). Then, take 50 μL of this stock solution and add it to an equal volume of blank PBS, mix well, and use it as the zero-time sample for recovery. Separately, take 50 μL of blank whole plasma or 50% (or 10%) plasma-PBS and add it to an equal volume of PBS, mix well, and use it as a double blank sample.

[0625] 5) Add plate and incubate

[0626] Select the appropriate well on a RED disposable incubation plate and add 200 μL of the above stock solution to the red-circled side of the insert tube in the well (plasma side). Add 400 μL of PBS to the other side (buffer side). Seal the incubation plate with sealing film and place it in a 37°C constant temperature shaker with reciprocating shaking at 150 rpm for 4 hours.

[0627] 6) Sample collection and processing

[0628] After incubation, remove 50 μL of sample from each of the plasma and buffer sides and transfer them to preselected wells on the sample collection plate. Then, add 50 μL of blank buffer to the plasma side and 50 μL of blank whole plasma or 50% (or 10%) plasma-PBS to the buffer side, and mix thoroughly. Add 300 μL of stop solution containing the internal standard to the sample collection plate (including the double blank and the recovery time zero sample) and shake at 600 rpm for 10 minutes at room temperature to mix thoroughly. Centrifuge the precipitated collection plate at 3,200 × g for 12 minutes at 4°C. Add 50 μL of the supernatant to ultrapure water pre-mixed with the appropriate ratio and shake at 600 rpm for 10 minutes at room temperature to mix thoroughly. Place the plate in the LC-MS / MS autosampler for injection.

[0629] 7) Data processing

[0630] The peak areas of the analyte and the internal standard were measured on the plasma side and the buffer side, respectively, and the plasma protein binding rate was calculated using the peak area ratio of the analyte and the internal standard.

[0631] The free fraction is calculated as follows:

[0632] When the matrix used is whole plasma,

[0633] Protein binding rate (Bound%) = PAR buffer / PAR plasma × 100

[0634] Free fraction (fu) = 1-PAR buffer / PAR plasma

[0635] Note: PAR buffer represents the ratio of analyte to internal standard on the buffer side;

[0636] PAR plasma represents the ratio of the plasma analyte to the internal standard.

[0637] When the matrix used is 50% (or 10%) plasma-PBS,

[0638] fu100%=fu50% / (2-fu50%); fu100%=fu10% / (10-9×fu10%)

[0639] The recovery rate calculation formula is as follows

[0640] % recovery = (PAR buffer × 0.4 + PAR plasma × 0.2) / PAR t = 0 × 0.2 × 100

[0641] Note: PAR t=0 represents the ratio of analyte to internal standard in the time zero sample used for recovery.

[0642] Example E: Pharmacokinetics in mice

[0643] Mouse PK studies were conducted using female ICR mice (20-30 g). Compounds were prepared and prepared immediately prior to use. Blood was collected from the mouse orbital venous plexus via intravenous injection or oral gavage. At each time point, 100 μL of whole blood was collected into a K2-EDTA tube. The whole blood was centrifuged at 4000 rpm for 10 minutes, and approximately 40 μL of the supernatant was collected to obtain plasma samples.

[0644] After intravenous administration of 2 mg / kg, plasma samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 7, and 24 hours. After oral administration of 10 mg / kg, plasma samples were collected at 0.25, 0.5, 1, 2, 4, 7, and 24 hours. Plasma samples were stored at -80°C until further use. After protein precipitation with acetonitrile, the supernatant of these plasma samples was collected and mixed with water in a 1:2 ratio. 10 μL of the supernatant was used for LC-MS / MS analysis.

[0645] The pharmacokinetic results of single intravenous and oral administration in mice are shown in Tables 5 and 6, respectively.

[0646] Table 5

[0647] Table 6

[0648] The experiments showed that the compound of the present invention exhibited low clearance and high exposure in mice compared with the comparative compound A, showing excellent pharmacokinetic properties.

[0649] Example F: Canine Pharmacokinetic Study

[0650] Beagle PK studies were conducted using male beagle dogs (9-12 kg). Compound dosing solutions were prepared immediately prior to use. Oral administration was performed by gavage. Blood was collected via the jugular vein. At each time point, 200 μL of whole blood was collected into a K2-EDTA tube. The whole blood was centrifuged at 4000 rpm for 10 minutes, and approximately 100 μL of the supernatant was collected to obtain plasma samples.

[0651] The oral gavage dose was 5 mg / kg, and the administration volume was 5 mL / kg. Plasma samples were collected before dosing and at 0.25, 0.5, 1, 2, 4, 7, 10, 24, 48, and 72 hours after dosing. The plasma samples were stored at -80°C until analysis. 30 μL of the plasma sample was added to 200 μL of acetonitrile containing an internal standard to precipitate protein. The supernatant was then centrifuged and mixed with water in a 1:2 ratio. 10 μL of the supernatant was then analyzed by LC-MS / MS. The pharmacokinetic results of oral administration are shown in Table 7.

[0652] Table 7

[0653] Example G: In vivo efficacy experiment in SW48 xenograft tumor model

[0654] 200uL of 5×10 6 A PBS solution of SW48 (MSI-H) cells and matrigel were inoculated subcutaneously into Balb / c nude mice at a ratio of 1:1 to construct a xenograft tumor model. Tumor growth was monitored regularly after cell inoculation. When the tumor volume reached approximately 110 mm 2 At 40 mg / kg, the animals were randomly divided into a blank group, a control group, and a test compound group (n=6). The animals were orally administered with a dose of 40 mg / kg, and the tumor size was measured twice a week. The animals were observed daily for any obvious signs of adverse reactions.

[0655] The tumor growth inhibition percentage (TGI%) and body weight change percentage (BWC%) were calculated according to the following formula:

[0656] Tumor growth inhibition percentage = 100-[(tumor volume treated -tumor volume treated at strat) / (Δtumor volume Control -Tumor volume treated )×100]

[0657] Percentage change in body weight = (body weight treated -Body weight treated at strat) / Body weight treated at strat×100%.

[0658] Statistical analysis of tumor and body weight change data was performed using GraphPad Prism, and the results are presented as mean ± SEM as a measure of efficacy. The experimental results are shown in Figures 3 and 4.

[0659] Experiments show that compared with the comparative compound A, the compound of the present invention can significantly reduce tumor volume and has better in vivo efficacy; the weight loss is less, indicating better safety.

[0660] Although the present invention has been fully described through its embodiments, it is noteworthy that various changes and modifications are obvious to those skilled in the art. Such changes and modifications should be included in the scope of the appended claims of the present invention.

Claims

1. A compound as shown in formula I, or its stereoisomer, tautomer, solvate, pharmaceutically acceptable salt or deuterated substance: Indicates whether the bond on the ring is a single bond or a double bond; represents a single bond or a double bond; Z and M are independently selected from CR x , CR x R x NR x , N, O or S; Q, W, J, T, K, G are independently selected from C, CR x or N; X is a bond or C 1-4 A divalent saturated or unsaturated straight or branched hydrocarbon group, wherein one, two or three methylene groups in the hydrocarbon group may be optionally and independently replaced by: -CH(R e )-、-C(R e )2-、C 3-5 Cycloalkylene, -N(R e )-、-N(R e )C(=O)-、-C(=O)N(R e )-、-N(R e )C(=S)-、-C(=S)N(R e )-、-N(R e )S(=O)2-、-S(=O)2N(R e )-, -O-, -C(=O)-, -C(=S)-, -OC(=O)-, -C(=O)O-, -S-, -S(=O)- or -S(=O)2-; Ring A is C 6-14 Aryl, C 3-14 Carbocyclyl, 5-14 membered heteroaryl or 3-14 membered heterocyclyl; R1 is selected from halogen, -CN, -OR e 、-NR e R f 、-SR e 、-C(O)R e 、-N(R e )C(O)R f 、-C(O)NR e R f 、-N(R e )C(O)OR f 、-OC(O)NR e R f 、-N(R e )C(O)NR f R j 、-S(O)R e 、-S(O)2R e 、-S(O)(=NRe)R f 、-S(O)NR e R f 、-S(O)2NR e R f 、-N(R e )S(O)R f 、-N(R e )S(O)2R f 、-C(O)OR e 、-OC(O)R f 、-OC(O)OR j , C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, -C 0-6 Alkylene-C 3-14 Carbocyclic group, -C 0-6 Alkylene-C 6-14 Aryl, -C 0-6 Alkylene-3-14 membered heterocyclic group or -C 0-6 Alkylene-5-14 membered heteroaryl or in, The C 1- 6 alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, -C 0-6 Alkylene-C 3-14 Carbocyclic group, -C 0-6 Alkylene-C 6-14 Aryl, -C 0-6 Alkylene-3-14 membered heterocyclic group, -C 0-6 Alkylene-5-14 membered heteroaryl, R e , R f , R j , R 1’ and R 1” Optionally replaced by one or more hydrogen, deuterium, halogen, CN, oxo, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, -OR h 、-NR h R i , substituted or unsubstituted -C 0-6 Alkylene-C 3- 14 Carbocyclic, substituted or unsubstituted -C 0-6 Alkylene-3-14 membered heterocyclic group, -C(O)OR h 、-C(O)NR h R i or R h replace; R 1’ and R 1” are independently selected from C 1-6 Alkyl and C 1-6 haloalkyl; or R 1’ and R 1” Together with the atoms to which it is attached, it forms C 3-6 carbocyclic group, 3-7 membered heterocyclic group; wherein the C 3-6 Carbocyclic group, 3-7 membered heterocyclic group are optionally substituted by one or more R h replace; Each R x Same or different, R x , R2 is selected from hydrogen, deuterium, halogen, CN, OH, NH2, -COOH, -NH(C 1-6 Alkyl), -N(C 1-6 Alkyl)2, oxo, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, -NHCO-C 1-6 Alkyl, -NHCO-C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, -OC 3-14 Cycloalkyl, -O-3-14 membered heterocyclic group; wherein the OH, NH2, -COOH, -NH(C 1-6 Alkyl), -N(C 1-6 Alkyl)2, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, -NHCO-C 1-6 Alkyl, -NHCO-C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, -OC 3- 14 Cycloalkyl, -O-3-14 membered heterocyclic group is optionally substituted with one or more hydrogen, deuterium, halogen, CN, OH, NH2, -COOH, oxo, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy, -NH(C 1-6 Alkyl), -N(C 1-6 Alkyl)2, -COO(C 1- 6 alkyl), -CONH(C 1-6 Alkyl) or -S(O)2-C 1-6 Alkyl substitution; Ring B is C 6-14 Aryl, C 3-14 Carbocyclyl, 5-14 membered heteroaryl or 3-14 membered heterocyclyl; Each R4 is the same or different and is independently selected from hydrogen, deuterium, halogen, CN, OH, NH2, -COOH, -NH(C 1-6 Alkyl), -N(C 1-6 Alkyl)2, oxo, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, -NHCO-C 1-6 Alkyl, -NHCO-C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, -OC 3-14 Cycloalkyl or -O-3-14 membered heterocyclic group; wherein the OH, NH2, -COOH, -NH(C 1-6 Alkyl), -N(C 1-6 Alkyl)2, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, -NHCO-C 1-6 Alkyl, -NHCO-C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, -OC 3- 14 Cycloalkyl and -O-3-14 membered heterocyclyl are optionally substituted with one or more hydrogen, deuterium, halogen, CN, OH, NH2, -CF3, -COOH, oxo, C 1- 6 alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy, -NH(C 1-6 Alkyl), -N(C 1-6 Alkyl)2, -COO(C 1-6 alkyl), -CONH(C 1-6 Alkyl) or -S(O)2-C 1-6 Alkyl substitution; L is a key, C 1-3 Alkylene, C 2-4 Alkenylene, C 2-4 Alkynylidene, C 1-4 Heteroalkylene, -OC 1-6 Alkylene-, -N(R e )C(O)-、-C(O)N(R e )-、 -C(O)-, -S(O)-, -S(O)2-, -N(R e )S(=O)2-、-S(=O)2N(R e )-or Among them, the C 1-3 Alkylene, C 2-4 Alkenylene, C 2-4 Alkynylidene, C 1-4 Heteroalkylene and -OC 1-6 Alkylene - optionally substituted with one or more R h replace; Ring C is C 6-14 Aryl, C 3-14 Carbocyclyl, 5-14 membered heteroaryl or 3-14 membered heterocyclyl; Each R3, R5 is the same or different and is independently selected from H, deuterium, halogen, -CN, oxo, thio, -OR e 、-NR e R f , -BR e R f 、-SR e 、-C(O)R e 、-N(R e )C(O)R f 、-C(O)NR e R f 、-N(R e )C(O)OR f 、-OC(O)NR e R f 、-N(R e )C(O)NR f R j 、-S(O)R e 、-S(O)2R e 、-S(O)(=NRe)R f 、-S(O)NR e R f 、-S(O)2NR e R f 、-N(R e )S(O)R f 、-N(R e )S(O)2R f 、-C(O)OR e 、-OC(O)R f 、-OC(O)OR j , C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, -C 0-6 Alkylene-C 3-14 Carbocyclic group, -C 0-6 Alkylene-C 6-14 Aryl, -C 0-6 Alkylene-3-14 membered heterocyclic group or -C 0-6 Alkylene-5-14 membered heteroaryl; wherein the C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, -C 0- 6-Alkylene-C 3-14 Carbocyclic group, -C 0-6 Alkylene-C 6-14 Aryl, -C 0-6 Alkylene-3-14 membered heterocyclic group, -C 0-6 Alkylene-5-14 membered heteroaryl, R e , R f and R j Optionally replaced by one or more hydrogen, deuterium, halogen, CN, oxo, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, -OR h 、-NR h R i , substituted or unsubstituted -C 0-6 Alkylene-C 3-14 Carbocyclic, substituted or unsubstituted -C 0-6 Alkylene-3-14 membered heterocyclic group, -C(O)OR h 、-OC(O)R h 、-NC(O)R h R i 、-C(O)NR h R i or R h replace; R e , R f , R h , R i , R j are independently selected from hydrogen, deuterium, halogen, CN, OH, NH2, -COOH, -NH(C 1-6 Alkyl), -N(C 1-6 Alkyl)2, oxo, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, -OC 3-14 Cycloalkyl, -O-3-14 membered heterocyclic group, -S-3-14 membered heterocyclic group, -SC 3-14 Cycloalkyl, -SC 1-6 Alkyl, -CONH2, -CONH(C 1-6 Alkyl), -CON(C 1-6 Alkyl)2, -CONH(C 2-6 -CON(C 2-6 alkenyl)2, -CONH(C 2-6 Alkynyl), -CON(C 2-6 Alkynyl)2, -S(O)-C 1-6 Alkyl, -S(O)2-C 1-6 Alkyl, -S(O)-C 2-6 Alkenyl, -S(O)2-C 2-6 Alkenyl, -S(O)-C 2-6 Alkynyl, -S(O)2-C 2-6 Alkynyl, -S(O)-C 3-14 Cycloalkyl, -S(O)2-3-14 membered heterocyclic group, -S(O)NH2, -S(O)NHC 1-6 Alkyl, -S(O)N(C 1-6 Alkyl)2, -CHO, -C(O)-C 1-6 Alkyl, -C(O)-C 2-6 Alkenyl, -C 0-6 Alkylene-C 6-14 Aryl, -C 0-6 Alkylene-5-14 membered heteroaryl, -C 0-6 Alkylene-C 3-14 Carbocyclic group, -C 0-6 Alkylene-3-14 membered heterocyclic group, -CO-C 6-14 Aryl, -CO-5-14 membered heteroaryl, -CO-C 3-14 Carbocyclic group, -CO-3-14 membered heterocyclic group, -NHCO-C 6-14 Aryl, -NHCO-5-14 membered heteroaryl, -NHCO-C 3-14 carbocyclic group or -NHCO-3-14 membered heterocyclic group; wherein the -NH(C 1-6 Alkyl), -N(C 1-6 Alkyl)2, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, -OC 3-14 Cycloalkyl, -O-3-14 membered heterocyclic group, -S-3-14 membered heterocyclic group, -SC 3-14 Cycloalkyl, -SC 1-6 Alkyl, -CONH(C 1-6 Alkyl), -CON(C 1-6 Alkyl)2, -CONH(C 2-6 -CON(C 2-6 alkenyl)2, -CONH(C 2-6 Alkynyl), -CON(C 2-6 Alkynyl)2, -S(O)-C 1-6 Alkyl, -S(O)2-C 1-6 Alkyl, -S(O)-C 2-6 Alkenyl, -S(O)2-C 2-6 Alkenyl, -S(O)-C 2-6 Alkynyl, -S(O)2-C 2-6 Alkynyl, -S(O)-C 3-14 Cycloalkyl, -S(O)2-3-14 membered heterocyclic group, -S(O)NHC 1-6 Alkyl, -S(O)N(C 1- 6 alkyl)2, -C(O)-C 1-6 Alkyl, -C(O)-C 2-6 Alkenyl, -C 0-6 Alkylene-C 6-14 Aryl, -C 0-6 Alkylene-5-14 membered heteroaryl, -C 0-6 Alkylene-C 3-14 Cycloalkyl, -C 0-6 Alkylene-3-14 membered heterocyclic group, -CO-C 6-14 Aryl, -CO-5-14 membered heteroaryl, -CO-C 3-14 Carbocyclic group, -CO-3-14 membered heterocyclic group, -NHCO-C 6-14 Aryl, -NHCO-5-14 membered heteroaryl, -NHCO-C 3-14 The carbocyclyl and -NHCO-3-14 membered heterocyclyl are optionally substituted with one or more hydrogen, deuterium, halogen, CN, OH, NH2, -COOH, oxo, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2- 6 alkynyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy, -NH(C 1-6 Alkyl), -N(C 1-6 Alkyl)2, -COO(C 1-6 alkyl), -CONH(C 1-6 Alkyl), -CON(C 1-6 Alkyl)2, -S(O)-C 1-6 Alkyl, -S(O)2-C 1-6 Alkyl, -C 0-6 Alkylene-NHC(=O)C 1-6 Alkyl, -C 0-6 Alkylene-NHC(=O)C 1- 6-haloalkyl, -C 0-6 Alkylene-NHC(=O)C 2-6 Alkenyl, -CON(C 1-6 alkyl)2, substituted or unsubstituted -C 0-6 Alkylene-C 3-14 Carbocyclyl, substituted or substituted -C 0-6 Alkylene-3-14 membered heterocyclic group substitution; m is 0, 1, 2, 3 or 4; n is 0, 1, 2, 3 or 4; q is 0, 1, 2, 3 or 4.

2. The compound according to claim 1, or its stereoisomer, tautomer, solvate, pharmaceutically acceptable salt or deuterated form, characterized in that ,, Indicates whether the bonds on the ring are single or double bonds.

3. The compound according to claim 1 or 2, or its stereoisomer, tautomer, solvate, pharmaceutically acceptable salt or deuterated substance, characterized in that: Z is selected from N; K is selected from N; M is selected from N; G is selected from C; T is selected from C or N; Q is selected from C; W is selected from C; J is selected from N.

4. The compound according to any one of claims 1 to 3, or its stereoisomer, tautomer, solvate, pharmaceutically acceptable salt or deuterated substance, characterized in that: Ring A is phenyl, naphthyl, 5-6 membered monocyclic heteroaryl, 5-6 membered monocyclic heterocyclyl, 9-10 membered bicyclic heterocyclyl or 9-10 membered bicyclic heteroaryl.

5. The compound according to any one of claims 1 to 4, or its stereoisomer, tautomer, solvate, pharmaceutically acceptable salt or deuterated substance, characterized in that: Ring A is phenyl, naphthyl, 5-membered heteroaryl, 6-membered heteroaryl, 10-membered bicyclic heterocyclyl or 10-membered bicyclic heteroaryl.

6. The compound according to any one of claims 1 to 5, or its stereoisomer, tautomer, solvate, pharmaceutically acceptable salt or deuterated substance, characterized in that: for wherein r is selected from 0, 1, 2, 3 or 4; p is selected from 0, 1, 2, 3 or 4; r+p=m.

7. The compound according to any one of claims 1 to 6, or its stereoisomer, tautomer, solvate, pharmaceutically acceptable salt or deuterated substance, characterized in that: R3 is H, halogen, CN, oxo, thio, NH2, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, -COOH, -C(O)NH2, -NHCOOC 1-6 Alkyl, -NHCOC 1-6 Alkyl, -S(O)2-C 1-6 Alkyl, C 3-6 Cycloalkyl, -OC 3- 6-membered cycloalkyl, 3-7-membered heterocyclic group, phenyl or 5-6-membered heteroaryl; wherein the NH2, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, -COOH, -C(O)NH2, -NHCOOC 1-6 Alkyl, -NHCOC 1-6 Alkyl, -S(O)2-C 1-6 Alkyl, C 3-6 Cycloalkyl, -OC 3-6 Cycloalkyl, 3-7 membered heterocyclyl, phenyl and 5-6 membered heteroaryl are optionally substituted with one or more halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkoxy, C 3-6 The cycloalkyl group or the 3-6-membered heterocyclic group is substituted.

8. The compound according to any one of claims 1 to 7, or its stereoisomer, tautomer, solvate, pharmaceutically acceptable salt or deuterated substance, characterized in that: R3 is H, F, Cl, Br, CN, NH2, oxo, thio, CH3, -CF3, -CHF2, -CH2F, -C2F5, 9. The compound according to any one of claims 1 to 8, or a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt or deuterated substance thereof, characterized in that: X is a key, 10. The compound according to any one of claims 1 to 9, or a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt or deuterated substance thereof, characterized in that: for 11. The compound according to any one of claims 1 to 10, or a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt or deuterated substance thereof, characterized in that: R1 is halogen, -NH2, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, -C(O)-C 1-6 Alkyl, -C(O)OC 1-6 Alkyl, -C(O)NH2, -C(O)NH-C 1-6 Alkyl, -C(O)N(C 1-6 alkyl)2, 5-6 membered heteroaryl, 5-6 membered heterocyclyl, 8-9 membered heteroaryl, 7-9 membered heterocyclyl, -NH-3-6 membered heterocyclyl, -C(O)-3-6 membered heterocyclyl, -C(O)NH-3-6 membered heterocyclyl, -5-6 membered heterocyclyl-C 1-3 Alkyl or Wherein, the -NH2, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, -C(O)-C 1-6 Alkyl, -C(O)OC 1-6 Alkyl, -C(O)NH2, -C(O)NH-C 1-6 Alkyl, -C(O)N(C 1-6 alkyl)2, 5-6 membered heteroaryl, 5-6 membered heterocyclyl, 8-9 membered heteroaryl, 7-9 membered heterocyclyl, -NH-3-6 membered heterocyclyl, -C(O)-3-6 membered heterocyclyl, -C(O)NH-3-6 membered heterocyclyl and -5-6 membered heterocyclyl-C 1-3 The alkyl group is optionally substituted with one or more halogen, substituted or unsubstituted C 1-6 Alkyl, substituted or unsubstituted C 1-6 Alkoxy, substituted or unsubstituted C 3-6 Cycloalkyl, substituted or unsubstituted 3-6 membered heterocyclic group or -S(O)2-C 1-6 Alkyl substitution.

12. The compound according to any one of claims 1 to 11, or a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt or deuterated substance thereof, characterized in that: R1 is Br, NH2, 13. The compound according to any one of claims 1 to 12, or a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt or deuterated substance thereof, characterized in that: R2 is H, CN, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl or C 1-6 Haloalkoxy; wherein the C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 The haloalkoxy group is optionally substituted with one or more halogen, CN, C 1-6 Alkoxy, C 1-6 Halogenated alkoxy, C 1-6 Haloalkyl or -S(=O)2C 1-6 Alkyl substitution.

14. The compound according to any one of claims 1 to 13, or a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt or deuterated substance thereof, characterized in that: R2 is H, CN, CH2CH3, 15. The compound according to any one of claims 1 to 14, or a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt or deuterated substance thereof, characterized in that: Ring B is phenyl, a 4-6 membered monocyclic heterocyclic group, a 5-6 membered heteroaryl group or a 7-8 membered bicyclic heterocyclic group.

16. The compound according to any one of claims 1 to 15, or a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt or deuterated substance thereof, characterized in that: for wherein j is selected from 0, 1, 2, 3 or 4; k is selected from 0, 1, 2, 3 or 4; j+k=q.

17. The compound according to any one of claims 1 to 16, or a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt or deuterated substance thereof, characterized in that: R4 is H, halogen or C 1-6 alkyl.

18. The compound according to any one of claims 1 to 17, or a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt or deuterated substance thereof, characterized in that: R4 is H, Cl or Br.

19. The compound according to any one of claims 1 to 18, or a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt or deuterated substance thereof, characterized in that: L is a key, C 1-3 Alkylene, C 2-4 Alkenylene, C 2-4 Alkyne, -C(O)NH-, -NHC(O)-, -C(O)-, -OC 1-6 Alkylene- or 20. The compound according to any one of claims 1 to 19, or a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt or deuterated substance thereof, characterized in that: L is a bond, CH2, 21. The compound according to any one of claims 1 to 20, or a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt or deuterated substance thereof, characterized in that: Ring C is phenyl, 5-membered heteroaryl, 6-membered heteroaryl, 5-membered heterocyclyl or 6-membered heterocyclyl.

22. The compound according to any one of claims 1 to 21, or a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt or deuterated substance thereof, characterized in that: R5 is H, deuterium, halogen, CN, oxo, -OH, -NH2, -N(C 1-6 Alkyl)2, -COOH, boric acid, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, -C(O)OC 1-6 Alkyl, phenyl, 3-10 membered heterocyclic group, C 3-6 Carbocyclic group, 5-6 membered heteroaryl, -SC 1-6 Alkyl, -S(O)-C 1-6 Alkyl or -S(O)2-C 1-6 Alkyl; wherein the -OH, -NH2, -N(C 1-6 Alkyl)2, -COOH, boric acid, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, -C(O)OC 1-6 Alkyl, phenyl, 3-10 membered heterocyclic group, C 3-6 Carbocyclic group, 5-6 membered heteroaryl, -SC 1-6 Alkyl, -S(O)-C 1-6 Alkyl and -S(O)2-C 1-6 The alkyl group is optionally substituted with one or more OH, halogen, C 1-6 Alkyl, C 1- 6 alkoxy, C 1-6 Haloalkyl, C 1-6 Haloalkoxy, 3-6 membered heterocyclic group, -S(O)-C 1-6 Alkyl or -S(O)2-C 1-6 Alkyl substitution.

23. The compound according to any one of claims 1 to 22, or a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt or deuterated substance thereof, characterized in that: H、F、Cl、OH、CH3、 -C≡CH、CN、NH2、COOH、 24. The compound according to any one of claims 1 to 23, or a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt or deuterated substance thereof, characterized in that: for 25. The compound according to any one of claims 1 to 24, or a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt or deuterated substance thereof, characterized in that: for 26. The compound according to any one of claims 1 to 25, or a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt or deuterated substance thereof, characterized in that: The compound of formula I is selected from the compounds in Table 1.

27. A method for preparing the compound of formula (Z), characterized in that: The steps include: in, R 100 is a hydroxyl protecting group; preferably, R 100 is selected from tert-butyldimethylsilyl, methyl, benzyl, p-methoxybenzyl, methoxymethyl, and the other groups are as defined in formula I of any one of claims 1 to 26.

28. A method for preparing the compound of formula Z, characterized in that: The steps include: The groups therein are as defined in formula I according to any one of claims 1 to 26.

29. A method for preparing the compound of formula Z, characterized in that: The steps include: in, R 100 is a hydroxyl protecting group; preferably, R 100 is selected from tert-butyldimethylsilyl, methyl, benzyl, p-methoxybenzyl, methoxymethyl, and the other groups are as defined in formula I of any one of claims 1 to 26.

30. An intermediate compound, the intermediate compound is selected from the compounds shown below: in, Y is selected from halogen; preferably, Y is selected from chlorine or bromine; R 100 is a hydroxyl protecting group; preferably, R 100 Selected from tert-butyldimethylsilyl, methyl, benzyl, p-methoxybenzyl, methoxymethyl; R1, R2, R3, R4, Ring A, m and q are as defined in formula I according to any one of claims 1-26.

31. An intermediate compound, wherein the intermediate compound is selected from the following compounds:

32. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises a therapeutically effective amount of at least one compound according to any one of claims 1 to 26, or a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt or deuterated substance thereof and at least one pharmaceutically acceptable excipient.

33. Use of the compound according to any one of claims 1 to 26, or its stereoisomer, tautomer, solvate, pharmaceutically acceptable salt or deuterated substance, or the pharmaceutical composition according to claim 32 in the preparation of an anti-MSI-H / dMMR tumor drug.

34. The use according to claim 33, characterized in that The MSI-H / dMMR tumor is a cancer with high microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR), selected from prostate cancer, endometrial cancer, colon adenocarcinoma, gastric adenocarcinoma, rectal adenocarcinoma, adrenal cortical carcinoma, uterine carcinosarcoma, cervical squamous cell carcinoma, endocervical adenocarcinoma, esophageal cancer, breast cancer, renal clear cell carcinoma and ovarian serous cystadenocarcinoma.

35. A method for regulating WRN activity in a subject, characterized in that The method comprises administering to a subject a therapeutically effective amount of a compound according to any one of claims 1 to 26, or a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt or deuterated substance thereof, or a pharmaceutical composition according to claim 32.

36. A method for inhibiting WRN in a subject, characterized in that The method comprises administering to a subject a therapeutically effective amount of a compound according to any one of claims 1 to 26, or a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt or deuterated substance thereof, or a pharmaceutical composition according to claim 32.

37. A method of treating a disease by inhibiting WRN in a subject, characterized in that The method comprises administering to a subject a therapeutically effective amount of the compound of any one of claims 1 to 26, or a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt or deuterated substance thereof, or the pharmaceutical composition of claim 32.

38. The method according to claim 37, characterized in that wherein the compound is a WRN inhibitor.

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