Nitrogen-containing heterocyclic derivative for inhibiting WRN helicase, pharmaceutical composition containing same, and use thereof

Abstract

Provided in the present invention are a nitrogen-containing heterocyclic derivative for inhibiting WRN helicase, a pharmaceutical composition containing same, and the use thereof. Specifically, provided in the present invention is a compound represented by formula (I), or a stereoisomer thereof, an optical isomer thereof, a pharmaceutically acceptable salt thereof, a crystal form thereof, an isotope derivative thereof, a prodrug thereof, a metabolite thereof, a solvate thereof or a hydrate thereof. Further provided in the present invention are a composition containing the compound and the use thereof as a WRN helicase inhibitor in the preparation of a drug for treating WRN helicase-mediated diseases.

Description

A nitrogen-containing heterocyclic derivative that inhibits WRN helicase, a pharmaceutical composition containing the derivative, and their applications. Technical Field

[0001] This invention belongs to the field of medicinal chemistry, specifically relating to a nitrogen-containing heterocyclic derivative that inhibits WRN helicase, a pharmaceutical composition containing the derivative, and their applications. Background Technology

[0002] Werner syndrome helicase (WRN) is a helicase involved in DNA repair, telomere maintenance, and cell cycle checkpoint response, participating in various biological processes including DNA damage repair, telomere maintenance, autophagy, and genome maintenance. Microsatellite instability (MSI) is a phenomenon in tumor cells where new microsatellite alleles emerge due to impaired or defective mismatch repair. MSI can lead to further genomic disorder and mutation in tumor cells, thereby promoting the development of malignant tumors and is a recognized important oncogenic pathway. Recent studies have found that the growth of MSI malignant tumors is highly dependent on WRN helicase. Related results show that MSI-H cells undergo significant apoptosis after WRN silencing, while no apoptosis was observed in MSS (microsatellite stable) cells. With the discovery that WRN may be a novel synthetic lethal target for MSI malignant tumors, research on WRN inhibitors targeting MSI malignant tumors has attracted the interest of researchers and drug developers.

[0003] Therefore, there is an urgent need in this field for a small molecule inhibitor targeting WRN that can effectively treat tumors with MSI-H characteristics. Summary of the Invention

[0004] To address the above problems, the present invention provides a nitrogen-containing heterocyclic derivative that inhibits WRN helicase, a pharmaceutical composition containing the derivative, and its application.

[0005] In a first aspect, the present invention provides a compound of formula (I), its stereoisomer, its optical isomer, its pharmaceutically acceptable salt, its crystal form, its isotopic derivative, its prodrug, its metabolite, its solvate, or its hydrate.

[0006] in,

[0007] M, R, W, L, T and V are each independently selected from the following groups: C, CH and N, and M, R, W, L, T and V are not simultaneously C or CH;

[0008] U is C = O or N;

[0009] Y is C, CH, or N; Indicates a single bond or a double bond;

[0010] When Y is CH, Y is connected to the adjacent carbon atom by a single bond, and the CH is optionally replaced by -OH or a halogen;

[0011] When Y is C, Y is connected to adjacent atoms through double bonds;

[0012] When Y is N, Y is connected to adjacent atoms through single bonds;

[0013] y is 0, 1, 2, 3 or 4;

[0014] Each R3 is independently selected from the following groups: H, D, C 1-4 Alkyl, C 3-6 cycloalkyl, C 1-4 A haloalkyl group, or two R3 atoms on the same ring carbon atom and the carbon atom to which they are attached, together forming a 3- or 4-membered ring, wherein the 3- or 4-membered ring contains 0, 1, or 2 heteroatoms selected from N or O; or two R3 atoms on adjacent ring carbon atoms and the carbon atom to which they are attached, together forming a 3- or 6-membered ring, wherein the 3- or 6-membered ring is optionally separated by 1 or 2 R3 atoms. 3a replace;

[0015] R 3a Selected from the following groups: H, D, halogens, C 1-4 Alkyl, C 1-4 Hydroxyalkyl, C 1-4 Halogenated alkyl groups;

[0016] R4 is a 5-12 heteroaryl group; wherein the 5-12 heteroaryl group is optionally surrounded by 1, 2, 3 or 4 R groups. 4a replace;

[0017] Each R 4a Independently selected from the following groups: H, D, hydroxyl, halogen, cyano, C 1-4 Alkyl, C 1-4 Haloalkyl, C 3-6 cycloalkyl, C 3-6 Cycloalkoxy, C 1-4 Alkoxy, C 1-4 Deuterated alkyl, C 1-4 Halogenated alkoxy groups, or two R atoms on adjacent ring carbon atoms 4a Together with the carbon atoms to which they are attached, they form 4 to 6-membered rings, which contain 0, 1 or 2 heteroatoms selected from N or O;

[0018] X is CH or N; and when X is CH, the CH can be replaced by R5 or R6 (i.e., X is CR5 or CR6);

[0019] m and n are each independently 0, 1, 2 or 3;

[0020] R5 and R6 are each independently selected from the following groups: H, D, hydroxyl, halogen, cyano, C. 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Halogenated alkoxy groups, -SF3, -SF5, C 3-6 cycloalkyl, C 1-4 Alkylamino, C 1-4 alkenyl, C 2-6 alkynyl group, C 1-4 Haloalkenyl, C 2-6 Alkyne group; wherein the R5 is optionally surrounded by 1, 2, 3, 4 or 5 R groups. 5a Replace; the R6 is optionally replaced by 1, 2, 3, 4 or 5 Rs. 6a replace;

[0021] R 5a and R 6a Each is independently selected from the following groups: H, D, hydroxyl, halogen, amino, cyano, C. 1-4 Alkyl, C 1-4 Haloalkyl, C 3-6 cycloalkyl, C 3-6 Cycloalkoxy, C 1-4 Alkoxy, C 1-4 Deuterated alkyl, C 1-4 Halogenated alkoxy groups;

[0022] R1 is selected from the following group:

[0023] (1) 5-6 membered heterocyclic phenyl, 5-6 membered heterocyclic phenyl, 5-6 membered heterocyclic thiophene, 5-6 membered heterocyclic thiazolyl, 5-6 membered heterocyclic pyridyl, 5-6 membered heterocyclic pyridyl, 5-6 membered heterocyclic thiophene; wherein the above groups are optionally surrounded by 0, 1, 2, 3 or 4 R 1a Replace, each R 1a Same or different;

[0024] R1 is preferably structured as follows:

[0025] Among them, ring A is selected from the following group: 5-6 membered carbon rings, 5-6 membered heterocycles, and 5-6 membered heteroaromatic rings; ring A can be 0, 1, 2, 3, or 4 Rs. 1a Substitution; t is 0, 1, 2, 3, 4, or 5;

[0026] R 1a These are substituents on R1, each R 1a Independently selected from the following groups: H, D, halogen, cyano, amino, hydroxyl, C1-4 Alkyl, C 3-6 cycloalkyl, C 1-4 Deuterated alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 3-6 Cycloalkoxy, C 1-4 Halogenated alkoxy groups, C 3-6 alkynyl group, C 3-6 alkenyl, C 3-6 Haloalkenyl, C 1-4 alkylamine group, Or two R atoms on the same ring carbon atom 1a Together with the carbon atom it is attached to, they form a 3- or 4-membered ring, which contains 0, 1, or 2 heteroatoms selected from N or O, or two R atoms on adjacent ring atoms. 1a Together with the ring atoms it is attached to, it forms a 4- to 6-membered ring, which contains 0, 1, or 2 heteroatoms selected from N or O; wherein, R 1a Optionally divided by 0, 1, 2 or 3 R 1b Instead, the 4 to 6-membered rings are optionally replaced by 0, 1, or 2 R... 1c replace;

[0027] Each R 1b Independently selected from the following groups: H, D, hydroxyl, halogen, amino, cyano, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Halogenated alkoxy groups, C 1-4 Alkylamine group;

[0028] Each R 1c Independently selected from the following groups: H, D, halogen, hydroxyl, C 1-4 alkyl;

[0029] More preferably, R1 is In this ring, ring A is a tetrahydrofuran ring, a piperidine ring, or a pyrrole ring, and ring R... 1a The definitions of t are as described above;

[0030] More preferably, R1 has the following structure:

[0031] (2) 4-7 membered cycloalkenyl, 4-7 membered heterocyclic alkenyl; wherein the above groups are optionally surrounded by 0, 1, 2 or 3 R groups. 1d replace;

[0032] Each R 1d Independently selected from the following groups: H, D, halogen, cyano, amino, hydroxyl, C 1-4 Alkyl, C 3-6 cycloalkyl, C1-4 Deuterated alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 3-6 Cycloalkoxy, C 1-4 Halogenated alkoxy groups, C 3-6 alkynyl group, C 3-6 alkenyl, C 3-6 Haloalkenyl, C 1-4 Alkylamine group, or two R groups on the same carbon atom of a ring 1d Together with the carbon atoms they are attached to, they form 3- to 6-membered rings, which contain 0, 1, or 2 heteroatoms selected from N or O, or two R atoms on adjacent ring carbon atoms. 1d Together with the carbon atom it is attached to, it forms a 4- to 6-membered ring, wherein the 4- to 6-membered ring contains 0, 1, or 2 heteroatoms selected from N or O; wherein, the R 1d Optionally divided by 0, 1, 2 or 3 R 1e Replacement; the 3 to 6-membered rings described above are optionally replaced by 1 or 2 R... 1f Replace; the 4 to 6-membered rings described above are optionally replaced by 1 or 2 R... 1g replace;

[0033] Each R 1e Independently selected from the following groups: H, D, hydroxyl, halogen, amino, cyano, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Halogenated alkoxy groups, C 1-4 Alkylamine group;

[0034] Each R 1f and R 1g Independently selected from the following group: H, D, halogen, hydroxyl and C 1-4 alkyl;

[0035] R1 is preferably structured as follows:

[0036] (3) Substituted 5-6 membered heterocyclic groups; wherein at least one substituent is a halogenated C 2-4 Terminal olefins;

[0037] R1 is preferably structured as follows:

[0038] (4) 5-6-membered heteroaryl, phenyl; wherein the above groups are optionally surrounded by 1, 2, 3 or 4 R groups. 1h replace;

[0039] Each R 1h Independently selected from the following groups: H, D, hydroxyl, halogen, amino, cyano, C 1-4 Alkyl, C3-6 cycloalkyl, C 3-6 Cycloalkoxy, 3-6 oxetane, -O-3 to 6 oxetane, -C 0-4 Alkylene-NR 1m R 1n , or R on adjacent ring atoms 1h Together with the ring atoms it is attached to, they form C 4-6 Carbocyclic rings, 4-6 membered heterocyclic rings, or 5-6 membered heteroaromatic rings; wherein, each R 1h C 4-6 The carbocyclic ring, 4-6 membered heterocyclic ring, and 5-6 membered heteroaromatic ring are optionally surrounded by 1, 2, or 3 R groups. 1i replace;

[0040] Each R 1i Independently selected from the following groups: H, D, hydroxyl, halogen, amino, cyano, C 1-4 Alkyl, C 3-6 Cycloalkyl, 3-6 oxocycloalkyl, C 1-4 alkylamine group, C 1-4 Alkoxy groups; each R 1i Optionally by 1, 2 or 3 R 1j replace;

[0041] Each R 1j Independently selected from the following groups: H, D, hydroxyl, halogen, amino, cyano, C 1-4 Alkyl, C 3-6 Cycloalkyl, 3-6 oxocycloalkyl, C 1-4 alkylamine group, C 1-4 Alkoxy;

[0042] R 1m and R 1n Each is independently selected from the following groups: H, C 1-4 Alkyl, C 3-6 cycloalkyl, C 1-4 alkylamine group, C 1-4 alkoxy, or R 1m and R 1n Together they form a 4- to 8-membered heterocycle, wherein the 4- to 8-membered heterocycle contains one or two heteroatoms selected from N or O; the 4- to 8-membered heterocycle is optionally separated by 0, 1 or 2 R atoms. 1p replace;

[0043] Each R 1p Independently selected from the following groups: H, D, hydroxyl, halogen, amino, cyano, C 1-4 Alkyl, C 3-6 Cycloalkyl, 3-6 oxocycloalkyl, C 1-4 alkylamine group, C 1-4 alkoxy group, or two R atoms on the same ring atom1p Together with the ring atoms it is connected to, they form a 4- to 6-membered ring, or two R atoms on adjacent ring atoms. 1p Together with the ring atoms they are attached to, they form a 4- to 6-membered ring, which contains 0, 1, or 2 heteroatoms selected from N or O; the aforementioned 4- to 6-membered ring is optionally separated by 0, 1, or 2 R atoms. 1q replace;

[0044] Each R 1q Independently selected from the following groups: H, D, hydroxyl, halogen, amino, cyano, C 1-4 Alkyl, C 3-6 Cycloalkyl, 3-6 oxocycloalkyl, C 1-4 alkylamine group, C 1-4 Alkoxy;

[0045] Each R 1k The components are independently selected from the group consisting of 3-10 nitrogen-containing heterocycles; preferably 3-10 nitrogen-containing monocyclic rings, 3-10 nitrogen-containing fused rings, or 3-10 nitrogen-containing spirocyclic rings.

[0046] R1 is preferably structured as follows:

[0047] R2 is selected from the following group: C 1-4 Alkyl, C 3-6 cycloalkyl, C 1-4 Deuterated alkyl, C 1-4 Halogenated alkyl group; wherein the R2 is optionally surrounded by 1, 2 or 3 R groups. 2a replace;

[0048] Each R 2a Independently selected from the following groups: H, D, hydroxyl, halogen, cyano, C 1-4 Alkyl, C 1-4 Alkoxy, C 3-6 cycloalkyl, C 3-6 Cycloalkoxy, C 1-4 Alkylamino;

[0049] P is -CHR 11 or -NR 11 ;

[0050] R 11 Selected from the following groups: H, D, C 1-4 Alkyl, C 1-4 Deuterated alkyl, C 3-6 cycloalkyl, C 3-6 Cycloalkoxy, C 1-4 Alkylamine group, or R2, R 11 Together with the carbon atom it is attached to, it forms a 4- to 6-membered ring, wherein the 4- to 6-membered ring contains 0, 1, or 2 heteroatoms selected from N or O; wherein, the R11 The land can be optionally divided by 1, 2, 3, 4 or 5 Rs. 11a Instead, the 4 to 6-membered rings are optionally replaced by 0, 1, 2, or 3 R... 11b replace;;

[0051] Each R 11a Independently selected from the following groups: H, D, hydroxyl, halogen, cyano C 1-4 Alkyl groups; each R 11b Independently selected from the following groups: H, D, hydroxyl, halogen, amino, cyano, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Deuterated alkyl, C 1-4 Alkoxy, C 1-4 Halogenated alkoxy groups, C 1-4 alkylamine group, C 3-6 cycloalkyl, or two R atoms on adjacent ring atoms 11b The ring atoms connected to it together form a 5-membered ring, which contains 0, 1, or 2 heteroatoms selected from N or O, and is optionally bounded by 1, 2, 3, or 4 R atoms. 11c replace;

[0052] Each R 11c Independently selected from the following groups: H, D, hydroxyl, halogen, amino, cyano, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Deuterated alkyl, C 1-4 Alkoxy, C 1-4 Halogenated alkoxy groups, C 1-4 alkylamine group, C 3-6 Cycloalkyl, or two R atoms attached to the same carbon atom in a ring. 11c It can form a three-membered ring with the carbon atoms it is attached to;

[0053] The heteroaryl group is an aromatic cyclic group containing 1, 2, 3 or 4 heteroatoms selected from N, O or S on a cyclic skeleton; the heterocyclic group is a saturated or partially unsaturated cyclic group containing 1, 2, 3 or 4 heteroatoms selected from N, O or S and is non-aromatic, and is a monocyclic, fused, bridged or spirocyclic group.

[0054] In another preferred embodiment, the two R atoms on adjacent ring atoms 1a Together with the ring atoms it is connected to, they form 3 to 6-membered rings, in which the adjacent ring atoms are nitrogen atoms and carbon atoms, respectively.

[0055] In another preferred embodiment, when Y is CH, Y is connected to an adjacent carbon atom by a single bond, and the CH is optionally substituted with -OH or F.

[0056] In a preferred embodiment, the compound has the structure shown in formula (1a), (1b), (1c), (1d), or (1e):

[0057] Where o is 0, 1, 2, 3 or 4;

[0058] The definitions of Y, y, R3, R4, X, m, n, R5, and R6 are as described above;

[0059] When the compound has the structure shown in formula (1a), groups R1 and R 7b The definition is as follows:

[0060] When R 7b C 1-4 Alkyl, C 1-4 Halogenated alkyl or C 1-4 When hydroxyalkyl, R1 is selected from the following group:

[0061] (1) 5-6 membered heterocyclic phenyl, 5-6 membered heterocyclic phenyl, 5-6 membered heterocyclic thiophene, 5-6 membered heterocyclic thiazolyl, 5-6 membered heterocyclic pyridyl, 5-6 membered heterocyclic pyridyl, 5-6 membered heterocyclic thiophene; wherein the above groups are optionally surrounded by 0, 1, 2, 3 or 4 R 1a Replace, each R 1a Same or different;

[0062] R1 is preferably structured as follows:

[0063] Among them, ring A is selected from the following group: 5-6 membered carbon rings, 5-6 membered heterocycles, and 5-6 membered heteroaromatic rings; ring A can be 0, 1, 2, 3, or 4 Rs. 1a Substitution; t is 0, 1, 2, 3, 4, or 5;

[0064] R 1a These are substituents on R1, each R 1a Independently selected from the following groups: H, D, halogen, cyano, amino, hydroxyl, C 1-4 Alkyl, C 3-6 cycloalkyl, C 1-4 Deuterated alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 3-6 Cycloalkoxy, C 1-4 Halogenated alkoxy groups, C 3-6 alkynyl group, C 3-6 alkenyl, C 3-6 Haloalkenyl, C 1-4 alkylamine group, Or two R atoms on the same ring carbon atom1a Together with the carbon atom it is attached to, they form a 3- or 4-membered ring, which contains 0, 1, or 2 heteroatoms selected from N or O, or two R atoms on adjacent ring atoms. 1a Together with the ring atoms it is attached to, it forms a 3- to 6-membered ring, wherein the 3- to 6-membered ring contains 0, 1, or 2 heteroatoms selected from N or O; wherein, R 1a Optionally divided by 0, 1, 2 or 3 R 1b Instead, the 4 to 6-membered rings are optionally replaced by 0, 1, or 2 R... 1c replace;

[0065] Each R 1b Independently selected from the following groups: H, D, hydroxyl, halogen, amino, cyano, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Halogenated alkoxy groups, C 1-4 Alkylamine group;

[0066] Each R 1c Independently selected from the following groups: H, D, halogen, hydroxyl, C 1-4 alkyl;

[0067] More preferably, R1 is Among them, ring A is a tetrahydrofuran ring, a piperidine ring, and a pyrrole ring, and R... 1a The definitions of t are as described above;

[0068] More preferably, R1 has the following structure:

[0069] (2) 4-7-membered cycloalkenyl, 7-membered heterocyclic alkenyl; wherein the above groups are optionally surrounded by 1, 2, 3 or 4 R groups. 1d replace;;

[0070] Each R 1d Independently selected from the following groups: H, D, halogen, cyano, amino, hydroxyl, C 1-4 Alkyl, C 3-6 cycloalkyl, C 1-4 Deuterated alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 3-6 Cycloalkoxy, C 1-4 Halogenated alkoxy groups, C 3-6 alkynyl group, C 3-6 alkenyl, C 3-6 Haloalkenyl, C 1-4 Alkylamine group, or two R groups on the same carbon atom of a ring 1dTogether with the carbon atom it is attached to, it forms a 3- to 6-membered ring, which contains 0, 1, or 2 heteroatoms selected from N or O, or two R atoms on adjacent ring atoms. 1d Together with the carbon atom it is attached to, it forms a 4- to 6-membered ring, wherein the 4- to 6-membered ring contains 0, 1, or 2 heteroatoms selected from N or O; wherein, the R 1d Optionally divided by 0, 1, 2 or 3 R 1e Replacement; the 3 to 6-membered rings described above are optionally replaced by 1 or 2 R... 1f Replace; the 4 to 6-membered rings described above are optionally replaced by 1 or 2 R... 1g replace;

[0071] Each R 1e Independently selected from the following groups: H, D, hydroxyl, halogen, amino, cyano, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Halogenated alkoxy groups, C 1-4 Alkylamine group;

[0072] Each R 1f and R 1g Independently selected from the following group: H, D, halogen, hydroxyl and C 1-4 alkyl;

[0073] R1 is preferably structured as follows:

[0074] When R 7b It is cyano, C 1-4 alkylamine group, C 1-4 Deuterated alkyl, C 1-4 Halogenated alkoxy or C 1-4 alkoxy-substituted C 1-4 Alkyl groups, or two R atoms substituted on different ring atoms 7b When the atoms bonded to it together form a 3-membered carbon ring, R1 is defined as described above;

[0075] When the compound has the structure shown in formula (1b), the groups R1, R... 7b The definitions of K are as follows:

[0076] K is O, S, CH2, or NR 7b ;

[0077] Each R 7b Independently selected from the following groups: H, D, hydroxyl, halogen, amino, cyano, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Deuterated alkyl, C 1-4 Alkoxy, C1-4 Halogenated alkoxy groups, C 1-4 alkylamine group, C 3-6 Cycloalkyl, or substitution of two R atoms on different ring atoms 7b The atoms bonded to it together form a 3-membered carbon ring or a 5-membered heterocycle; the aforementioned 3-membered carbon ring or 5-membered heterocycle is optionally bounded by 1, 2, 3 or 4 R atoms. 7c replace;;

[0078] When K is NR 7b At that time, the R on the ring carbon atom 7b and R connected to N 7b A quinary ring can be formed; the quinary ring described above is optionally bounded by 1, 2, 3 or 4 R... 7c replace;

[0079] Each R 7c Independently selected from the following groups: H, D, hydroxyl, halogen, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Deuterated alkyl, C 1-4 Alkoxy, C 1-4 Halogenated alkoxy groups, C 3-6 Cycloalkyl; two R atoms attached to the same carbon atom in a ring. 7c It can form a three-membered ring with the carbon atoms it is attached to;

[0080] More specifically, compounds having the structure shown in formula (1b) can further form compounds having the structures shown in formula (1b-1), (1b-2), (1b-3), or (1b-4), and have the following characteristics:

[0081] Where o is 0, 1, 2 or 3;

[0082] Preferably, the compound having the structure shown in formula (1b-1), R 7b C is preferred 1-4 Alkyl or C 1-4 Deuterated alkyl groups;

[0083] More preferably, compounds having the structure shown in formula (1b-2) have R attached to the carbon or nitrogen atom. 7b They can be the same or different; R 7b C is preferred 1-4 Alkyl, C 1-4 Deuterated alkyl or C 3-6 cycloalkyl;

[0084] R1 is defined as described above;

[0085] R1 is selected from the following group:

[0086] (1) 5-6 membered heterocyclic phenyl, 5-6 membered heterocyclic phenyl, 5-6 membered heterocyclic thiophene, 5-6 membered heterocyclic thiazolyl, 5-6 membered heterocyclic pyridyl, 5-6 membered heterocyclic pyridyl, 5-6 membered heterocyclic thiophene; wherein the above groups are optionally surrounded by 0, 1, 2, 3 or 4 R 1a Replace, each R 1a Same or different;

[0087] R1 is preferably structured as follows:

[0088] Among them, ring A is selected from the following group: 5-6 membered carbon rings, 5-6 membered heterocycles, and 5-6 membered heteroaromatic rings; ring A can be 0, 1, 2, 3, or 4 Rs. 1a Substitution; t is 0, 1, 2, 3, 4, or 5;

[0089] R 1a These are substituents on R1, each R 1a Independently selected from the following groups: H, D, halogen, cyano, amino, hydroxyl, C 1-4 Alkyl, C 3-6 cycloalkyl, C 1-4 Deuterated alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 3-6 Cycloalkoxy, C 1-4 Halogenated alkoxy groups, C 3-6 alkynyl group, C 3-6 alkenyl, C 3-6 Haloalkenyl, C 1-4 alkylamine group, Or two R atoms on the same ring carbon atom 1a Together with the carbon atom it is attached to, they form a 3- or 4-membered ring, which contains 0, 1, or 2 heteroatoms selected from N or O, or two R atoms on adjacent ring atoms. 1a Together with the ring atoms it is attached to, it forms a 3- to 6-membered ring, wherein the 3- to 6-membered ring contains 0, 1, or 2 heteroatoms selected from N or O; wherein, R 1a Optionally divided by 0, 1, 2 or 3 R 1b Instead, the 4 to 6-membered rings are optionally replaced by 0, 1, or 2 R... 1c replace;

[0090] Each R 1b Independently selected from the following groups: H, D, hydroxyl, halogen, amino, cyano, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Halogenated alkoxy groups, C 1-4 Alkylamine group;

[0091] Each R1c Independently selected from the following groups: H, D, halogen, hydroxyl, C 1-4 alkyl;

[0092] More preferably, R1 is In this ring, ring A is a tetrahydrofuran ring, a piperidine ring, or a pyrrole ring, and ring R... 1a The definitions of t are as described above;

[0093] More preferably, R1 has the following structure:

[0094] (2) 4-7 membered cycloalkenyl, 4-7 membered heterocyclic alkenyl; wherein the above groups are optionally surrounded by 0, 1, 2 or 3 R groups. 1d replace;

[0095] Each R 1d Independently selected from the following groups: H, D, halogen, cyano, amino, hydroxyl, C 1-4 Alkyl, C 3-6 cycloalkyl, C 1-4 Deuterated alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 3-6 Cycloalkoxy, C 1-4 Halogenated alkoxy groups, C 3-6 alkynyl group, C 3-6 alkenyl, C 3-6 Haloalkenyl, C 1-4 Alkylamine group, or two R groups on the same carbon atom of a ring 1d Together with the carbon atoms they are attached to, they form 3- to 6-membered rings, which contain 0, 1, or 2 heteroatoms selected from N or O, or two R atoms on adjacent ring carbon atoms. 1d Together with the carbon atom it is attached to, it forms a 4- to 6-membered ring, wherein the 4- to 6-membered ring contains 0, 1, or 2 heteroatoms selected from N or O; wherein, the R 1d Optionally divided by 0, 1, 2 or 3 R 1e Replacement; the 3 to 6-membered rings described above are optionally replaced by 1 or 2 R... 1f Replace; the 4 to 6-membered rings described above are optionally replaced by 1 or 2 R... 1g replace;

[0096] Each R 1e Independently selected from the following groups: H, D, hydroxyl, halogen, amino, cyano, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Halogenated alkoxy groups, C 1-4 Alkylamine group;

[0097] Each R 1f and R1g Independently selected from the following group: H, D, halogen, hydroxyl and C 1-4 alkyl;

[0098] R1 is preferably structured as follows:

[0099] (3) Substituted 5-6 membered heterocyclic groups; wherein at least one substituent is a halogenated C 2-4 Terminal olefins;

[0100] R1 is preferably structured as follows:

[0101] (4) 5-6-membered heteroaryl, phenyl; wherein the above groups are optionally surrounded by 1, 2, 3 or 4 R groups. 1h replace;

[0102] Each R 1h Independently selected from the following groups: H, D, hydroxyl, halogen, amino, cyano, C 1-4 Alkyl, C 3-6 cycloalkyl, C 3-6 Cycloalkoxy, 3-6 oxetane, -O-3 to 6 oxetane, -C 0-4 Alkylene-NR 1m R 1n , or R on adjacent ring atoms 1h Together with the ring atoms it is attached to, they form C 4-6 Carbocyclic rings, 4-6 membered heterocyclic rings, or 5-6 membered heteroaromatic rings; wherein, each R 1h C 4-6 The carbocyclic ring, 4-6 membered heterocyclic ring, and 5-6 membered heteroaromatic ring are optionally surrounded by 1, 2, or 3 R groups. 1i replace;

[0103] Each R 1i Independently selected from the following groups: H, D, hydroxyl, halogen, amino, cyano, C 1-4 Alkyl, C 3-6 Cycloalkyl, 3-6 oxocycloalkyl, C 1-4 alkylamine group, C 1-4 Alkoxy groups; each R 1i Optionally by 1, 2 or 3 R 1j replace;

[0104] Each R 1j Independently selected from the following groups: H, D, hydroxyl, halogen, amino, cyano, C 1-4 Alkyl, C 3-6 Cycloalkyl, 3-6 oxocycloalkyl, C 1-4 alkylamine group, C 1-4 Alkoxy;

[0105] R 1m and R 1n Each is independently selected from the following groups: H, C 1-4 Alkyl, C 3-6 cycloalkyl, C 1-4 alkylamine group, C 1-4 alkoxy, or R 1m and R 1n Together they form a 4- to 8-membered heterocycle, wherein the 4- to 8-membered heterocycle contains one or two heteroatoms selected from N or O; the 4- to 8-membered heterocycle is optionally separated by 0, 1 or 2 R atoms. 1p replace;

[0106] Each R 1p Independently selected from the following groups: H, D, hydroxyl, halogen, amino, cyano, C 1-4 Alkyl, C 3-6 Cycloalkyl, 3-6 oxocycloalkyl, C 1-4 alkylamine group, C 1-4 alkoxy group, or two R atoms on the same ring atom 1p Together with the ring atoms it is connected to, they form a 4- to 6-membered ring, or two R atoms on adjacent ring atoms. 1p Together with the ring atoms they are attached to, they form a 4- to 6-membered ring, which contains 0, 1, or 2 heteroatoms selected from N or O; the aforementioned 4- to 6-membered ring is optionally separated by 0, 1, or 2 R atoms. 1q replace;

[0107] Each R 1q Independently selected from the following groups: H, D, hydroxyl, halogen, amino, cyano, C 1-4 Alkyl, C 3-6 Cycloalkyl, 3-6 oxocycloalkyl, C 1-4 alkylamine group, C 1-4 Alkoxy;

[0108] Each R 1k The components are independently selected from the group consisting of 3-10 nitrogen-containing heterocycles; preferably 3-10 nitrogen-containing monocyclic rings, 3-10 nitrogen-containing fused rings, or 3-10 nitrogen-containing spirocyclic rings.

[0109] R1 is preferably structured as follows:

[0110] When the compound has the structure shown in formula (1c), the groups R1, R2, and R8 are defined as follows:

[0111] R2 is selected from the following group: C 1-4 Alkyl, C 1-4 Deuterated alkyl, C 1-4Halogenated alkyl group; wherein, the R2 is optionally surrounded by 1, 2 or 3 R groups. 2a replace;

[0112] R 2a Selected from the following groups: H, D, hydroxyl, halogen, cyano, C 1-4 Alkyl, C 1-4 Alkoxy, C 3-6 cycloalkyl, C 3-6 Cycloalkoxy, C 1- 4-alkylamine group;

[0113] R8 is selected from the following groups: H, C 1-4 Alkyl, C 1-4 Deuterated alkyl, C 3-6 cycloalkyl, C 3-6 Cycloalkoxy, C 1-4 An alkylamine group, or R2, R8, together with the carbon atom it is attached to, forms a 5- to 6-membered ring, wherein the 5- to 6-membered ring contains 0, 1, or 2 heteroatoms selected from N or O; wherein the R8 is optionally surrounded by 1, 2, or 3 R atoms. 8a Instead, the 5- to 6-membered ring is optionally replaced by 1, 2, or 3 R... 8b replace;

[0114] Each R 8a Independently selected from the following groups: H, D, hydroxyl, halogen, cyano, C 1-4 alkyl;

[0115] Each R 8b Independently selected from the following groups: H, D, hydroxyl, halogen, amino, cyano, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Deuterated alkyl, C 1-4 Alkoxy, C 1-4 Halogenated alkoxy groups, C 1-4 Alkylamine group;

[0116] R1 is defined as described above;

[0117] When the compound has the structure shown in formula (1d), groups R1 and R 10b The definition is as follows:

[0118] J represents O, OCH2, -NR 10b CH2- or -NHR 10b -;

[0119] Each R 10b Independently selected from the following groups: H, D, hydroxyl, halogen, amino, cyano, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4Deuterated alkyl, C 1-4 Alkoxy, C 1-4 Halogenated alkoxy groups, C 1-4 Alkylamine group;

[0120] R1 is defined as described above;

[0121] When the compound has the structure shown in formula (1e), the groups R1, R2, and R9 are defined as follows:

[0122] R2 is selected from the following group: C 1-4 Alkyl, C 1-4 Deuterated alkyl, C 1-4 Halogenated alkyl group; wherein, the R2 is optionally surrounded by 1, 2 or 3 R groups. 2a replace;

[0123] R 2a Selected from the following groups: H, D, hydroxyl, halogen, cyano, C 1-4 Alkyl, C 1-4 Alkoxy, C 3-6 cycloalkyl, C 3-6 Cycloalkoxy, C 1- 4-alkylamine group;

[0124] R9 is selected from the following groups: H, D, C 1-4 Alkyl, C 1-4 Deuterated alkyl, C 3-6 cycloalkyl, C 3-6 Cycloalkoxy, C 1-4 An alkylamine group, or R2, R9, together with the carbon atom to which they are attached, forms a 4- to 6-membered ring, wherein the 4- to 6-membered ring contains 0 to 2 heteroatoms selected from N or O; wherein R9 is optionally surrounded by 1, 2, 3, 4, or 5 R atoms. 9a Instead, the 4 to 6-membered rings are optionally replaced by 0, 1, 2, or 3 R... 9b replace;

[0125] Each R 9a Independently selected from the following groups: H, D, hydroxyl, halogen, cyano, C 1-4 alkyl;

[0126] Each R 9b Independently selected from the following groups: H, D, hydroxyl, halogen, amino, cyano, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Deuterated alkyl, C 1-4 Alkoxy, C 1-4 Halogenated alkoxy groups, C 1-4 Alkylamine group;

[0127] R1 is defined as described above.

[0128] In another preferred embodiment, the compound having the structure shown in formula (1a) has the structure shown in formula (1a-1):

[0129] The definitions of R1, R3, R4, R5, R6, X, m, n, and y are as described above.

[0130] In another preferred example, o is 1, R 7b It is a methyl group.

[0131] In another preferred embodiment, the compound has the structure shown in formula (1b-5) or formula (1b-6):

[0132] In compounds having the structure shown in formula (1b-5), R 7b C 1-4 Alkyl or C 1-4 Deuterated alkyl groups;

[0133] In compounds having the structure shown in formulas (1b-6), R 7b and R 7b’ Each independently is C 1-4 Alkyl, C 1-4 Deuterated alkyl or C 3-6 cycloalkyl, or R 7b R 7b’ The atoms bonded to it together form a 3-membered carbon ring or a 5-membered heterocycle; the aforementioned 3-membered carbon ring or 5-membered heterocycle is optionally bounded by 1, 2, 3 or 4 R atoms. 7c replace;

[0134] 1* and 2* indicate that this area is chiral;

[0135] R1, R3, R4, R5, R6, R 7c The definitions of m, n, and y are as described above.

[0136] In another preferred embodiment, R 7b It is a methyl group.

[0137] In another preferred embodiment, 1* is the R configuration.

[0138] In another preferred embodiment, 2* is an S configuration.

[0139] In a preferred embodiment, R1 is preferably selected from the following group:

[0140] In a preferred embodiment, the Selected from the following group:

[0141] R4 is defined as described above.

[0142] In a preferred embodiment, the Selected from the following group:

[0143] In a preferred embodiment, R4 is controlled by 1, 2, or 3 R... 4a Substituted 5-7 membered heteroaryl groups; of which, R 4a Selected from the following groups: H, D, hydroxyl, halogen, C 1-4 Alkyl, C 1-4 Deuterated alkyl groups, or two R atoms on adjacent ring carbon atoms 4a Together with the carbon atoms to which they are attached, they form 4 to 6-membered rings, which contain 0, 1 or 2 heteroatoms selected from N or O;

[0144] Preferably, R4 is composed of 1, 2, or 3 R... 4a Substituted 5-7 nitrogen-containing heteroaryl groups; wherein, R 4a Selected from the following groups: H, D, hydroxyl, halogen, C 1-4 Alkyl, C 1-4 Deuterated alkyl groups, or two R atoms on adjacent ring carbon atoms 4a Together with the carbon atoms to which they are attached, they form 4 to 6-membered rings, which contain 0, 1 or 2 heteroatoms selected from N or O;

[0145] More preferably, R4 is selected from the group consisting of:

[0146] In a preferred embodiment, the compound is selected from the group consisting of:

[0147] A second aspect of the present invention provides a pharmaceutical composition comprising a compound as described in the first aspect of the present invention, its stereoisomer, its optical isomer, its pharmaceutically acceptable salt, its crystal form, its isotopic derivative, its prodrug, its metabolite, its solvate, or its hydrate; and a pharmaceutically acceptable carrier.

[0148] In another preferred embodiment, the pharmaceutical composition further comprises other therapeutic agents.

[0149] In another preferred embodiment, the other therapeutic agent is a chemotherapy drug, an immunotherapy drug, or a targeted drug.

[0150] In another preferred embodiment, the chemotherapeutic drugs are selected from: anastrozole, bicalutamide, bleomycin sulfate, busulfan, busulfan injection, capecitabine, N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytarabine, cytosine arabinoside, cytarabine liposome injection, dacarbazine, actinomycin, daunorubicin hydrochloride, daunorubicin citrate liposome injection, dexamethasone, docetaxel, doxorubicin hydrochloride, and etoposide. Fludarabine phosphate, 5-fluorouracil, flutamide, tezacitamine, gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan, L-asparaginase, leucovorin, melphalan, 6-mercaptopurine, methotrexate, mitoxantrone, gemtuzumab, paclitaxel, pentostatin, tamoxifen citrate, teniposide, 6-thioguanine, thiotepa, terazamine, topotecan hydrochloride for injection, vinblastine, vincristine, and vinorelbine; preferably 5-fluorouracil (5-FU) and irinotecan.

[0151] In another preferred embodiment, the immunotherapy drug is selected from: PD-1 inhibitors (including small molecule and antibody drugs), PD-L1 inhibitors (including small molecule and antibody drugs), CTLA-4 inhibitors, PD-1 / CTLA-4 bispecific antibodies, PD-1 / TIGIT bispecific antibodies, PD-L1 / 4-1BB bispecific antibodies, PD-1 / VEGF-A bispecific antibodies, and PD-1 / LAG-3 bispecific antibodies;

[0152] Among them, the PD-1 inhibitors are selected from: nivolumab, pembrolizumab, camrelizumab, toripalimab, sintilimab, tislelizumab, penaprilimab, cepalimumab, slulimab, dostarlimab and PDR001;

[0153] Among them, the PD-L1 inhibitors are selected from: durvalumab, atezolizumab, envorimab, and sugemalimab;

[0154] Among them, the CTLA-4 inhibitor was selected from ipilimumab;

[0155] Among them, the PD-1 / CTLA-4 bispecific antibody was selected from cantulimumab.

[0156] In another preferred embodiment, the targeted drug includes, but is not limited to, VEGFR inhibitors (lenvatinib), NTRK inhibitors (entrectinib), BRAFV600 inhibitors (dabrafenib), MEK inhibitors (trametinib), EGFR monoclonal antibodies (cetuximab), PARP inhibitors (olaparib, niraparib, fluzoparib, and pamiparib, etc.) and ATR inhibitors.

[0157] A third aspect of the present invention provides a method for preparing a pharmaceutical composition, comprising the steps of: mixing a pharmaceutically acceptable carrier with the compound of the present invention, its stereoisomer, its optical isomer, its pharmaceutically acceptable salt, its crystal form, its isotope derivative, its prodrug, its metabolite, its solvate, or its hydrate thereof, thereby forming a pharmaceutical composition as described in the second aspect of the present invention.

[0158] A fourth aspect of the present invention provides the use of a compound, its stereoisomer, its optical isomer, its pharmaceutically acceptable salt, its crystal form, its isotopic derivative, its prodrug, its metabolite, its solvate or hydrate thereof, or a pharmaceutical composition as described in the first aspect of the present invention, in the preparation of a medicament for treating WRN helicase-mediated diseases.

[0159] In another preferred embodiment, the disease is a tumor.

[0160] In another preferred embodiment, the disease is a microsatellite unstable tumor.

[0161] In another preferred embodiment, the tumor is characterized by high microsatellite instability (MSI-H) or by defective mismatch repair (dMMR) or “dMMR features”.

[0162] Among them, the microsatellite unstable tumors include, but are not limited to, uterine fibroids, endometrial cancer, colonic adenocarcinoma, gastric adenocarcinoma, rectal adenocarcinoma, adrenocortical carcinoma, uterine carcinosarcoma, cervical squamous cell carcinoma, cervical endometrial adenocarcinoma, esophageal cancer, breast cancer, renal clear cell carcinoma, and ovarian serous cyst carcinoma.

[0163] Tumors with "defective mismatch repair" (dMMR) or "dMMR features" include, but are not limited to, lung cancer, breast cancer, kidney cancer, colorectal cancer, ovarian cancer, prostate cancer, upper respiratory and digestive tract cancer, gastric cancer, endometrial cancer, liver cancer, pancreatic cancer, hematopoietic and lymphatic tissue cancer, skin cancer, thyroid cancer, pleural cancer, autonomic nervous system tumors, soft tissue tumors, rhabdomyosarcoma, melanoma, and other tumors.

[0164] In another preferred embodiment, the drug is administered orally, by injection, inhalation, or via a cavity.

[0165] In another preferred embodiment, the dosage form of the drug is selected from: tablets, capsules, dispersants, suspensions, granules, sprays, gels, sustained-release agents, oral liquids, pellets, and nanoformulations.

[0166] In a fifth aspect, the present invention provides a method for treating WRN helicase-mediated diseases, comprising the steps of administering to a subject requiring treatment an effective amount of a compound, stereoisomer thereof, optical isomer thereof, pharmaceutically acceptable salt thereof, crystal form thereof, isotopic derivative thereof, prodrug thereof, metabolite thereof, solvate thereof or hydrate thereof, or pharmaceutical composition as described in the second aspect.

[0167] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here.

[0168] Brief description of the attached figures

[0169] Figure 1 shows the crystal structure of the complex of compound 11-P2 and WRN protein.

[0170] Figure 2 shows the antitumor effect of the compound on xenografted colorectal cancer SW48 cells in nude mice. Detailed Implementation

[0171] Through extensive and in-depth research, the inventors discovered for the first time a nitrogen-containing heterocyclic derivative with a novel structure and good WRN helicase inhibitory activity. Based on this, the inventors completed this invention.

[0172] the term

[0173] Unless otherwise specified, the following terms used in this application (including the specification and claims) have the definitions given below.

[0174] "Alkyl" (alone or as part of other groups) refers to a monovalent straight-chain or branched saturated hydrocarbon group consisting only of carbon and hydrogen atoms. Among them, "C..." 1-4 "Alkyl" refers to an alkyl group containing 1, 2, 3, or 4 carbon atoms. Examples of alkyl groups include, but are not limited to: methyl, ethyl, propyl, isopropyl, isobutyl, sec-butyl, tert-butyl, pentyl, n-hexyl, octyl, dodecyl, etc.

[0175] "Terminal alkene" (alone or as part of other groups) refers to a hydrocarbon group consisting only of carbon and hydrogen atoms containing a carbon-carbon double bond, with the carbon-carbon double bond located at one end of the group. Specifically, "C..." 2-4 "Terminal olefin" refers to a terminal olefin group containing 2, 3, or 4 carbon atoms.

[0176] “C 1-4 "Deuterated alkyl" (alone or as part of other groups) refers to an alkyl group containing 1, 2, 3, or 4 carbon atoms that has been replaced by 1, 2, 3, 4, 5, 6, or 7 deuterium atoms. Examples of deuterated alkyl groups include, but are not limited to, CD3, CD2CD3, and CD2CD2CD3.

[0177] "Alkoxy" (alone or as part of other groups) refers to the -OR or -R'-OR group, where R is an alkyl group as defined herein, and R' is an alkylene group. C 1-4 Alkoxy groups refer to alkoxy groups containing 1, 2, 3, or 4 carbon atoms. Examples of alkoxy groups include, but are not limited to: methoxy, ethoxy, isopropoxy, tert-butoxy, -CH2O-CH3, -CH2CH2-O-CH3, -CH2-O-CH2CH3, etc.

[0178] "Halogen (halogenated)" (alone or as part of other groups) refers to fluorine, chlorine, bromine or iodine.

[0179] "Hydroxyalkyl" (alone or as part of other groups) refers to the group obtained by replacing one or more hydrogen atoms in an alkyl group as described above with hydroxyl groups.

[0180] "Halogenated alkyl" (alone or as part of other groups) refers to the group obtained by substituting one or more hydrogen atoms in an alkyl group as described above with the same or different halogens. Wherein, "C..." 1-4 "Halogenated alkyl" refers to an alkyl halogroup containing 1, 2, 3, or 4 carbon atoms. Examples of alkyl halogroups include, but are not limited to: -CH2Cl, -CH2CF3, -CH2CCl3, perfluoroalkyl (e.g., -CF3-, -CF2CF3), etc.

[0181] "Haloalkoxy" (alone or as part of other groups) refers to a group of formula -OR, where R is a haloalkyl group as defined herein. "C" 1-4 "Haloalkoxy" refers to an alkoxy group containing 1, 2, 3, or 4 carbon atoms that are substituted with multiple halogens. Examples of haloalkoxy groups include, but are not limited to, trifluoromethoxy, difluoromethoxy, and 2,2,2-trifluoroethoxy.

[0182] "Cycloalkyl" (alone or as part of other groups) refers to a monovalent saturated carbocyclic group consisting of only carbon and hydrogen atoms in a single or bicyclic ring. 3-6"Cycloalkyl" refers to a saturated carbocyclic group containing 3 to 6 carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. A cycloalkyl group may optionally be substituted by one or more substituents, wherein each substituent is independently a hydroxyl, alkyl, alkoxy, halogen, haloalkyl, amino, monoalkylamino, or dialkylamino group. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

[0183] "Cycloalkoxy" (alone or as part of other groups) refers to the formula -OR group, where R is a cycloalkyl group as defined herein. "C 3-6 "Cycloalkoxy" refers to a saturated carbon epoxy group containing 3 to 6 carbon atoms. Exemplary cycloalkyloxy groups include cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, etc.

[0184] A "heterocyclic group" (alone or as part of another group) refers to a fully saturated or partially unsaturated cyclic group (including, but not limited to, 3-7 membered monocyclic, 6-11 membered bicyclic, or 8-16 membered tricyclic systems), wherein at least one heteroatom selected from N, O, or S is present in a ring with at least one carbon atom. Each heterocycle containing a heteroatom has 1, 2, 3, or 4 heteroatoms selected from nitrogen, oxygen, or sulfur, wherein the nitrogen or sulfur atom may be oxidized or quaternized. Heterocyclic alkane(group) refers to a fully saturated heterocycle(group). Heterocyclic groups can be attached to any heteroatom or carbon residue in a ring or cyclic molecule. "3-6 membered heterocyclic group" refers to a group having 3, 4, 5, or 6 ring members. "3-4 membered heterocyclic group" refers to a group having 3 or 4 ring members. Typical monocyclic heterocyclic compounds include, but are not limited to: nitrogen-containing heterocyclic butyl groups, pyrrolyl groups, oxocyclic butyl groups, pyrazolinyl groups, imidazolinyl groups, imidazoalkyl groups, oxazolinyl groups, isoxazolinyl groups, thiazoalkyl groups, isothiazolinyl groups, tetrahydrofuranyl groups, piperidinyl groups, piperazinyl groups, 2-oxopiperidinyl groups, 2-oxopiperidinyl groups, 2-oxopiperylyl groups, hexahydroachenginyl groups, 4-piperidinoneyl groups, tetrahydropyranyl groups, morpholinyl groups, thiomorpholinyl groups, thiomorpholinyl sulfoxide groups, thiomorpholinyl sulfone groups, 1,3-dioxylyl groups, and tetrahydro-1,1-dioxothiophene groups, etc. Polycyclic heterocyclic groups include spirocyclic, fused-ring, and bridged-ring heterocyclic groups; the spirocyclic, fused-ring, and bridged-ring heterocyclic groups involved may optionally be connected to other groups by single bonds, or further cyclically linked to other cycloalkyl, heterocyclic, aryl, and heteroaryl groups by any two or more atoms on the ring.

[0185] "Oxycyclic alkyl" (alone or as part of other groups) refers to a fully saturated cyclic group (including, but not limited to, 3-7 membered monocyclic, 6-11 membered bicyclic, or 8-16 membered tricyclic systems) containing 1, 2, or 3 oxygen atoms in its ring atom. "3-6 membered oxycyclic alkyl" refers to a heterocyclic group having 3, 4, 5, or 6 ring members, at least one of which is an oxygen atom. Examples of 3-6 membered oxycyclic alkyl include, but are not limited to, oxycyclic butyl and oxycyclic hexyl.

[0186] "Azaheterocyclic alkyl" (alone or as part of other groups) refers to a fully saturated cyclic group containing one, two, or three nitrogen atoms in its ring (including, but not limited to, 3-7 membered monocyclic, 6-11 membered bicyclic, or 8-16 membered tricyclic systems). "3- to 6-membered azaheterocyclic alkyl" refers to a heterocyclic group having 3, 4, 5, or 6 ring members, at least one of which is a nitrogen atom. Examples of 3- to 6-membered azaheterocyclic alkyl include, but are not limited to, azaheterocyclic butyl and azaheterocyclic hexyl.

[0187] "Aryl" (alone or as part of other groups) refers to an aromatic cyclic hydrocarbon compound group having 1-5 rings, especially monocyclic and bicyclic groups. Any compound containing two or more aromatic rings (bicyclic, etc.) can have these rings linked by single bonds (e.g., biphenyl) or fused together (e.g., naphthalene, anthracene, etc.). Among these, "C..." 6-12 "Aryl" refers to an aromatic cyclic hydrocarbon compound group containing 6, 7, 8, 9, 10, 11, or 12 ring carbon atoms. Examples of aryl groups (especially monocyclic and bicyclic groups) include, but are not limited to, phenyl, biphenyl, or naphthyl. Aryl groups can be fused with heterocyclic groups through a single bond or any two adjacent ring carbon atoms, such as benzotetrahydrofuranyl, benzotetrahydropyranyl, and benzodioxaneyl. wait.

[0188] "Heteroaryl" (alone or as part of other groups) refers to an aromatic group whose skeleton has heteroatoms. "5-12 membered heterocyclic group" refers to a monocyclic, bicyclic, or tricyclic group with 5, 6, 7, 8, 9, 10, 11, or 12 ring atoms, containing at least one (e.g., 1, 2, or 3) ring heteroatoms selected from N, O, or S, with the remaining ring atoms being C aromatic rings. It should be clearly understood that the connection point of the heteroaryl group should be located on the aromatic ring. Preferably, the heteroaryl group has 5-8 ring atoms (5-8 members), more preferably 5-6 ring atoms (5-6 members). Examples of heteroaryl groups include, but are not limited to: imidazole, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, pyrazinyl, thiophene, furanyl, pyranyl, pyridinyl, pyrroleyl, pyrazolyl, pyrimidinyl, quinolinyl, isoquinolinyl, benzofuranyl, benzothiophene, benzothiaranyl, benzoimidazolyl, benzooxazolyl, benzooxadiazolyl, benzothiazolyl, benzothiadiazolyl, benzopyranyl, indolyl, isindolyl, triazolyl, triazinyl, quinoxolinyl, purine, quinazolinyl, quinazinyl, naphridinyl, pteridinyl, carbazole, azatriyl, diazatriyl, acridineyl, etc.

[0189] Cycloalkenyl groups (alone or as part of other groups) refer to groups whose skeletal portion contains a carbon-carbon double bond, either a carbon ring or a heterocycle; where "C" is the carbon ring. 4- 10 "Cycloalkenyl" refers to a monocyclic, bicyclic, or tricyclic group with 4, 5, 6, 7, 8, 9, or 10 ring atoms, containing at least one (e.g., 1, 2, or 3) carbon-carbon double bond; cycloalkenyl groups containing heteroatoms contain at least one (e.g., 1, 2, or 3) heteroatom selected from N, O, or S; C 4-7 Cycloalkenyl and 4-7 membered heterocyclic alkenyl groups refer to cycloalkenyl groups containing 4 to 7 ring atoms; it should be clearly understood that the cycloalkenyl group should be connected at a carbon-carbon double bond. In this invention, the alkyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups, unless otherwise specified, include substituted alkyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups, and the substituents are, for example (but not limited to): halogens, hydroxyl groups, cyano groups, alkyl groups, cycloalkyl groups, heterocyclic groups, aryl, and heteroaryl groups.

[0190] "Deuterated compounds" refer to compounds in which one or more hydrogen atoms (H) are replaced by deuterium atoms (D). 1-4 "Deuterated alkyl" refers to a compound obtained by replacing at least three hydrogen atoms (H) or more hydrogen atoms (H) with deuterium atoms (D), such as (but not limited to): deuterated methyl, deuterated ethyl, etc.

[0191] "3-12-membered heterocycles" refer to groups comprising a 3- to 12-membered non-aromatic ring system having a cyclic carbon atom and 1 to 5 cyclic heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon. In heterocycles containing one or more nitrogen atoms, the linking point may be a carbon or nitrogen atom, provided the valence allows. In some embodiments, 3- to 6-membered heterocycles are preferred, which are 3- to 6-membered non-aromatic ring systems having a cyclic carbon atom and 1 to 3 cyclic heteroatoms; in some embodiments, 4- to 6-membered heterocycles are preferred, which are 4- to 6-membered non-aromatic ring systems having a cyclic carbon atom and 1 to 3 cyclic heteroatoms; in some embodiments, 5- to 6-membered heterocycles are preferred, which are 5- to 6-membered non-aromatic ring systems having a cyclic carbon atom and 1 to 3 cyclic heteroatoms; exemplary 3-membered heterocycles containing one heteroatom include, but are not limited to: azirropropane, oxacyclopropane, and thiohexacyclopropane. Exemplary 4-membered heterocycles containing one heteroatom include, but are not limited to: azirrobutane, oxacyclobutane, and thiohexacyclobutane. Exemplary 5-membered heterocycles containing one heteroatom include, but are not limited to: tetrahydrofuran, dihydrofuran, tetrahydrothiophene, dihydrothiophene, pyrrole, dihydropyrrole, and pyrrole-2,5-dione. Exemplary 5-membered heterocycles containing two heteroatoms include, but are not limited to: dioxacyclopentane, oxothiocyclopentane, dithiocyclopentane, and oxazolidin-2-one. Exemplary 5-membered heterocycles containing three heteroatoms include, but are not limited to: triazoline, oxadiazoline, and thiazoline. Exemplary 6-membered heterocycles containing one heteroatom include, but are not limited to: piperidine, tetrahydrofuran, dihydropyridine, and thiohexane. Exemplary 6-membered heterocycles containing two heteroatoms include, but are not limited to: piperazine, morpholine, dithiohexane, and dioxane. Exemplary 6-membered heterocycles containing three heteroatoms include, but are not limited to: hexahydrotriazine. Exemplary 7-membered heterocycles containing one heterocycle include, but are not limited to: azirheptan, oxetane, and thionheptan. Exemplary 5-membered heterocycles fused to a C6 aryl ring include, but are not limited to: dihydroindole, isodihydroindole, dihydrobenzofuran, dihydrobenzothiophene, benzoxazolinone, etc. Exemplary 6-membered heterocycles fused to a C6 aryl ring include, but are not limited to: tetrahydroquinoline, tetrahydroisoquinoline, tetrahydrobenzopyran, and tetrahydropyranopyridine.

[0192] "5-6 membered heteroaromatic ring" refers to a 4n+2 aromatic ring system with a 5-6 membered monocyclic ring having a cyclic carbon atom and 1-4 cyclic heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur. In heteroaryl groups containing one or more nitrogen atoms, the linkage can be a carbon or nitrogen atom, provided the valence allows. Heteroaromatic rings also include ring systems in which the aforementioned heteroaromatic ring is fused with one or more cycloalkanes or heterocycles, and the linkage is on the heteroaryl ring, in which case the number of carbon atoms continues to represent the number of carbon atoms in the heteroaromatic ring system. In some embodiments, a 5-6 membered heteroaromatic ring is a 4n+2 aromatic ring system with a 5-6 membered monocyclic ring having a cyclic carbon atom and 1-4 cyclic heteroatoms. Exemplary 5-membered heteroaromatic rings containing one heteroatom include, but are not limited to: pyrrole, furan, and thiophene. Exemplary 5-membered heteroaromatic rings containing two heteroatoms include, but are not limited to: imidazole, pyrazole, oxazole, isoxazole, thiazole, and isothiazole. Exemplary 5-membered heteroaryl rings containing three heteroatoms include, but are not limited to: triazoles, oxadiazoles, and thiadiazoles. Exemplary 5-membered heteroaryl rings containing four heteroatoms include, but are not limited to: tetrazolium. Exemplary 6-membered heteroaryl rings containing one heteroatom include, but are not limited to: pyridine. Exemplary 6-membered heteroaryl rings containing two heteroatoms include, but are not limited to: pyridazines, pyrimidines, and pyrazines. Exemplary 6-membered heteroaryl rings containing three or four heteroatoms include, but are not limited to: triazines and tetraazines, respectively.

[0193] As used in this article, the term "multiple substitutions" refers to 2, 3, 4, 5, or 6 substitutions.

[0194] As used in this article, the term "one or more" refers to 1, 2, 3, 4, 5, or 6.

[0195] Active ingredients

[0196] As used herein, “compound of the present invention” means a compound of formula (I), and also includes its stereoisomers, its optical isomers, its pharmaceutically acceptable salts, its crystal forms, its isotopic derivatives, its prodrugs, its metabolites, its solvates or hydrates thereof.

[0197] Unless otherwise specified, the structural formulas described in this invention are intended to include all stereoisomers (such as cis-trans isomers, enantiomers, diastereomers, and conformational isomers): R and S configurations containing an asymmetric center, (Z) and (E) isomers of double bonds, cis-trans isomers of cycloalkanes, etc. Therefore, any single stereochemical isomer of the compounds of this invention, or a mixture of its enantiomers, diastereomers, or conformational isomers, is within the scope of this invention.

[0198] The compounds of this invention may contain cis-trans isomers, one or more chiral carbon atoms, and thus can produce cis-trans isomers, chiral isomers, enantiomers, diastereomers, and other combinations of stereoisomers. Cis-trans isomerism refers to the diastereomeric phenomenon in which different groups in a compound molecule are arranged differently in space due to a restrictive factor that limits free rotation. This restrictive factor is generally caused by non-rotating functional groups in the structure of organic compounds, such as C=C double bonds, C=N double bonds, C=S double bonds, N=N double bonds, heterocycles, or cycloalkanes. Organic molecules containing such isomers, such as alkenes, azo compounds, and alicyclic hydrocarbons, are considered cis-trans isomers. Cis refers to the same ligands being in adjacent positions, generally denoted by "cis" or "cis-"; trans refers to the same ligands being in diagonal positions, generally denoted by "trans" or "trans-". Each chiral carbon atom can be defined as (R)- or (S)- based on stereochemistry. This invention aims to include all possible isomers, their racemic and optically pure forms. The compounds of this invention can be prepared using racemic, cis-trans, chiral, diastereomer, or enantiomers as starting materials or intermediates. Optically active isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, such as crystallization and chiral chromatography.

[0199] Conventional techniques for preparing / separating individual optical isomers (i.e., cis-trans and chiral isomers) include chiral synthesis from suitable cis-trans precursors or optically pure precursors, or resolution of racemates (or racemates of salts or derivatives) using, for example, chiral high-performance liquid chromatography. See, for example, Gerald Gübitz and Martin G. Schmid (Eds.), Chiral Separations, Methods and Protocols, Methods in Molecular Biology, Vol. 243, 2004; AMStalcup, Chiral Separations, Annu. Rev. Anal. Chem. 3:341-63, 2010; Fumiss et al. (eds.), VOGEL'S ENCYCLOPEDIA OF PRACTICAL ORGANIC CHEMISTRY 5.sup. TH ED., Longman Scientific and Technical Ltd., Essex, 1991, 809-816; Heller, Acc. Chem. Res. 1990, 23, 128.

[0200] To design the synthesis of a specific stereoisomer of the compound of this invention, it can be prepared asymmetrically or derivatized with a chiral auxiliary. The resulting stereo mixture is then separated, and the chiral auxiliary is removed to obtain pure cis-trans monomers, chiral monomers, or mixed stereoisomers. If the molecule contains a cis-trans isomer center, it can be purified by column chromatography (normal-phase silica gel column or reverse-phase high-performance liquid chromatography) to obtain pure cis or trans products. Alternatively, if the molecule contains a basic functional group, such as an amino acid, or an acidic functional group, such as a carboxyl group, it can be formed with a suitable optically active acid or base to form a diastereomeric salt, which is then separated by conventional methods such as separation crystallization or chromatography to obtain pure enantiomers.

[0201] This invention also includes isotopically labeled compounds (i.e., isotopic derivatives), equivalent to the original compounds disclosed herein. However, it is common practice to see one or more atoms replaced by atoms with different atomic weights or mass numbers. Examples of isotopes in the isotopic derivatives of this invention include hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine isotopes, respectively as follows: 2 H, 3 H, 13 C 11 C 14 C 15 N、 18 O、 17 O、 31 P, 32 P, 35 S, 18 F and 36 Cl. Isotope derivatives of the compounds of this invention are all within the scope of protection of this invention. In this document, 3 H-labeled compounds and 14 C-labeled compounds are useful in tissue distribution experiments of drugs and substrates. Tritium (i.e., 3 H) and carbon-14 (i.e. 14 C) Labeled compounds are relatively easy to prepare and detect, making them the preferred choice among isotopes. Furthermore, heavier isotope substitutions, such as deuterium, are also possible. 2 H, due to its excellent metabolic stability, offers advantages in certain therapies, such as increasing half-life or reducing dosage in vivo, and therefore may be preferred in some cases. Isotopically labeled compounds can be prepared using general methods, by replacing the non-isotopic reagent with an readily available isotopically labeled reagent, according to the scheme disclosed in the examples.

[0202] As used herein, the term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0203] "Pharmaceutically acceptable acid addition salts" refer to salts formed with inorganic or organic acids that retain the bioavailability of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobroms, sulfates, nitrates, and phosphates; organic acid salts include, but are not limited to, formates, acetates, 2,2-dichloroacetate, trifluoroacetate, propionates, hexanoates, octanoates, decanoates, undecenoates, glycolates, gluconates, lactates, sebates, adipates, glutarate, malonates, oxalates, maleates, succinates, fumarates, tartrates, citrates, palmitates, stearates, oleates, cinnamates, laurates, malates, glutamates, pyroglutamates, aspartate, benzoates, methanesulfonates, benzenesulfonates, p-toluenesulfonates, alginates, ascorbic acid salts, salicylates, 4-aminosalicylic acid salts, and naphthalene disulfonates. These salts can be prepared using methods known in this field.

[0204] "Pharmaceutically acceptable base addition salts" refer to salts formed with inorganic or organic bases that retain the bioavailability of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Preferred inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, the following: primary amines, secondary amines, and tertiary amines; substituted amines, including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. These salts can be prepared by methods known in the art.

[0205] Metabolites of the compound represented by formula (I) and its pharmaceutically acceptable salts, as well as prodrugs that can be converted in vivo into the compound represented by formula (I) and its pharmaceutically acceptable salts, are also included within the scope of protection of this invention.

[0206] As used herein, the term "solvent" refers to a complex of a compound of formula (I) coordinated with a solvent molecule in a specific ratio.

[0207] As used herein, the term "hydrate" refers to a complex of the compound represented by formula (I) coordinated with water molecules in a specific ratio.

[0208] As described herein, the compounds of the present invention can be substituted with any number of substituents or functional groups to broaden their scope. Generally, the term "substitution" refers to replacing a hydrogen radical with a substituent of a specified structure. When multiple positions in a particular structure are substituted by multiple specific substituents, each position of the substituent can be the same or different. The term "substitution" as used herein includes all permissible organic group substitutions. In a broad sense, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic organic groups. As described herein, heteroatomic nitrogen may be supplemented with a hydrogen substituent or any permissible organic group described above to complete its valence state. Furthermore, the present invention is not intended to limit permissible substituted organic groups in any way. The present invention considers the combination of substituents and variable groups to be beneficial in the treatment of diseases in the form of stable compounds. The term "stable" here means having a stable compound that, when tested over a sufficiently long period, maintains sufficient integrity of the compound structure, preferably remaining effective for a sufficiently long period, and is used herein for the purposes described above.

[0209] Pharmaceutical Compositions and Administration

[0210] Because the compounds of the present invention can inhibit WRN helicase and are used to treat diseases such as tumors, the compounds of the present invention, their stereoisomers, their optical isomers, their pharmaceutically acceptable salts, their crystal forms, their isotopic derivatives, their prodrugs, their metabolites, their solvates or hydrates thereof, and pharmaceutical compositions containing the compounds of the present invention as the main active ingredient can be used to prevent and / or treat (stabilize, alleviate or cure) WRN helicase-related diseases, preferably tumors; more preferably, microsatellite unstable tumors; and even more preferably, tumors characterized by high microsatellite instability (MSI-H) or having "defective mismatch repair deficiency (dMMR)" or "dMMR characteristics".

[0211] Microsatellite unstable tumors are particularly prevalent in colorectal cancer, gastric cancer, and endometrial cancer, but are also found in adrenocortical carcinoma, uterine cancer, cervical cancer, esophageal cancer, breast cancer, kidney cancer, prostate cancer, and ovarian cancer. Microsatellite unstable tumors include, but are not limited to, uterine fibroids, endometrial cancer, colonic adenocarcinoma, gastric adenocarcinoma, rectal adenocarcinoma, adrenocortical carcinoma, uterine carcinosarcoma, cervical squamous cell carcinoma, cervical endometrial adenocarcinoma, esophageal cancer, breast cancer, renal clear cell carcinoma, and ovarian serous cystic carcinoma.

[0212] Tumors with “defective mismatch repair” (dMMR) or “dMMR features” include cancer-type mechanisms associated with recorded mutations or epigenetic silencing of MLH1, PMS2, MSH2, MSH3, MSH6, MLH3, and PMS1, microsatellite vulnerable sites, or other gene inactivation, including but not limited to lung cancer, breast cancer, kidney cancer, colorectal cancer, ovarian cancer, prostate cancer, upper respiratory and digestive tract cancers, gastric cancer, endometrial cancer, liver cancer, pancreatic cancer, hematopoietic and lymphoid tissue cancers, skin cancer, thyroid cancer, pleural cancer, autonomic nervous system tumors, soft tissue tumors, rhabdomyosarcoma, melanoma, and other tumors.

[0213] The pharmaceutical compositions of the present invention comprise the compound of the present invention within a safe and effective range and a pharmaceutically acceptable excipient or carrier. "Safe and effective range" refers to an amount of the compound sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, more preferably, 10-200 mg of the compound of the present invention per dose. Preferably, "one dose" is one capsule or tablet.

[0214] "Pharmaceutically acceptable carriers" refer to one or more compatible solid or liquid fillers or gelling substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with and with the compounds of the present invention without significantly reducing the efficacy of the compounds. Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as... Wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0215] There are no particular limitations on the administration of the compounds or pharmaceutical compositions of the present invention. Representative administration methods include, but are not limited to, oral administration and parenteral administration (intravenous, intramuscular, or subcutaneous).

[0216] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following components: (a) fillers or compatibilizers, such as starch, lactose, sucrose, glucose, mannitol, and silica; (b) binders, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; (c) humectants, such as glycerin; (d) disintegrants, such as agar, calcium carbonate, potato starch or cassava starch, alginate, certain complex silicates, and sodium carbonate; (e) slowing agents, such as paraffin; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, such as kaolin; and (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof. Buffers may also be included in capsules, tablets, and pills.

[0217] Solid dosage forms such as tablets, sugar pills, capsules, pellets, and granules can be prepared using coatings and shells, such as casings and other materials known in the art. They may contain opacifying agents, and the release of the active compound or compound in such compositions can be delayed in a portion of the digestive tract. Examples of encapsulating components that can be used are polymeric substances and waxes. If necessary, the active compound may also be formed into microcapsules with one or more of the excipients described above.

[0218] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active compound, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, e.g., ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures of these substances.

[0219] In addition to these inert diluents, the composition may also contain auxiliaries such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and fragrances.

[0220] In addition to the active compound, the suspension may contain suspending agents such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0221] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0222] The compounds of this invention can be administered alone or in combination with other pharmaceutically acceptable therapeutic agents. Drugs administered in combination with WRN inhibitors include, but are not limited to, chemotherapeutic agents, immunotherapeutic agents, and targeted therapies. When administered in combination, the pharmaceutical composition further includes one or more (two, three, four, or more) other pharmaceutically acceptable therapeutic agents. One or more (two, three, four, or more) of these other pharmaceutically acceptable therapeutic agents can simultaneously treat microsatellite instability (MSI) or DNA mismatch repair deficient (dMMR) tumors with the compounds of this invention.

[0223] Chemotherapy drugs

[0224] Chemotherapy drugs include, but are not limited to, 5-fluorouracil (5-FU), irinotecan, anastrozole, bicalutamide, bleomycin sulfate, busulfan, busulfan injection, capecitabine, N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytarabine, cytosine arabinoside, cytarabine liposome injection, dacarbazine, actinomycin, daunorubicin hydrochloride, daunorubicin citrate, liposome injection, and dexamethasone. Docetaxel, Doxorubicin Hydrochloride, Etoposide, Fludarabine Phosphate, 5-Fluorouracil, Flutamide, Tezacitamine, Gemcitabine, Hydroxyurea, Idarubicin, Ifosfamide, Irinotecan, L-Asparaginase, Leucovorin, Melphalan, 6-Mercaptopurine, Methotrexate, Mitoxantrone, Gelatumab, Paclitaxel, Pentostatin, Tamoxifen Citrate, Teniposide, 6-Thioguanine, Thiotepa, Tirazamine, Topotecan Hydrochloride for Injection, Vincristine, Vincristine, and Vinorelbine.

[0225] Immunotherapy drugs

[0226] Immunotherapy drugs include, but are not limited to, PD-1 inhibitors (including small molecule and antibody drugs), PD-L1 inhibitors (including small molecule and antibody drugs), CTLA-4 inhibitors, PD-1 / CTLA-4 bispecific antibodies, PD-1 / TIGIT bispecific antibodies, PD-L1 / 4-1BB bispecific antibodies, PD-1 / VEGF-A bispecific antibodies, PD-1 / LAG-3 bispecific antibodies, and other immunotherapy drugs.

[0227] Targeted drugs

[0228] Targeted therapies include, but are not limited to, VEGFR inhibitors (lenvatinib), NTRK inhibitors (entrectinib), BRAFV600 inhibitors (dabrafenib), MEK inhibitors (trametinib), EGFR monoclonal antibodies (cetuximab), PARP inhibitors (olaparib, niraparib, fluzoparib, and pamiparib, etc.) and ATR inhibitors.

[0229] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to the mammal (such as a human) requiring treatment. The dosage administered is the pharmaceutically considered effective dose. For a person weighing 60 kg, the daily dose is typically 1-2000 mg, preferably 20-500 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of the skill of a skilled physician.

[0230] The present invention is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods not specifically described in the following embodiments are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts. The compounds of the present invention have their structures determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). NMR shifts are in units of 10⁻¹⁰. -6 (ppm). The solvents used for NMR determination were deuterated dimethyl sulfoxide, deuterated chloroform, deuterated methanol, etc., and the internal standard was tetramethylsilane (TMS).

[0231] Prep HPLC separation conditions: Instrument: Agilent 1260 Prep HPLC; Preparative column: Agilent 10Prep–C18 250*21.2mm, Mobile phase A: 0.05% formic acid in H2O, Mobile phase B: acetonitrile, Flow rate: 30mL / min.

[0232] HPLC analysis conditions: Instrument: SHIMADZU LC-20AD; Column: Eclipse Plus C18, 3.5 μM, 4.6 x 150 mm, Column temperature: 40℃; Mobile phase A: 0.05% formic acid in H2O, Mobile phase B: acetonitrile, Flow rate: 1.0 mL / min. Gradient: 5 to 95% B in 9 mins; 95 to 95% B in 4 mins.

[0233] Analytical Method 1: ChiralCEL OJ, 100×3mm ID, 3μm; Mobile phase: A for CO2 and B for MeOH (0.1% DEA); Gradient: B 25%; Flow rate: 2.0mL / min; Column temperature: 35℃

[0234] Analytical Method 2: ChiralPAK IJ, 100×3mm ID, 3μm; Mobile phase: A for CO2 and B for MeOH; Gradient: B 40%; Flow rate: 2.0 mL / min; Column temperature: 35℃

[0235] Analytical Method 3: Welch Boltimate EXT-C18, 2.7 μm, 4.6*50 mm; Mobile Phase A: Water + 0.05% Formic Acid, Mobile Phase B: Acetonitrile + 0.05% Formic Acid; Detection Wavelength: 220 nm / 254 nm; Flow Rate: 1.0-1.5 mL / min; Column Temperature: 40℃; Gradient Elution Program: 10% B to 95% B from 0.1 min to 2.0 min and 95% B to 95% B from 2.0 min to 3.2 min

[0236] Analytical Method 4: Welch Boltimate EXT-C18, 2.7 μm, 4.6*50 mm; Mobile Phase A: Water + 0.05% Formic Acid, Mobile Phase B: Acetonitrile + 0.05% Formic Acid; Detection Wavelength: 220 nm / 254 nm; Flow Rate: 1.0-1.5 mL / min; Column Temperature: 40℃; Gradient Elution Program: 10% B to 95% B from 0.1 min to 3.9 min and 95% B to 95% B from 3.9 min to 5.9 min

[0237] Analytical Method 5: Welch Boltimate EXT-C18, 2.7 μm, 4.6*50 mm; Mobile Phase A: Water + 0.05% Formic Acid, Mobile Phase B: Acetonitrile + 0.05% Formic Acid; Detection Wavelength: 220 nm / 254 nm; Flow Rate: 1.0-1.5 mL / min; Column Temperature: 40℃; Gradient Elution Program: 5% B to 95% B from 0.1 min to 5.0 min and 95% B to 95% B from 5.0 min to 6.0 min.

[0238] Analysis of the co-crystal structure of WRN helicase domain and compound 11-P2

[0239] A plasmid containing the core helicase domain of human WRN (amino acids 500-946) and a six-histidine tag at the N-terminus was used for recombinant baculovirus generation. WRN protein expression was performed in virus-infected fall armyworm (sf9) cells. Cells were incubated with shaking at 130 rpm and 27°C, and infection success was detected 72 h post-infection. The cell pellet was collected by centrifugation at 5000 rpm for 15 minutes using a high-speed floor centrifuge (Sorvall Lynx 6000) at room temperature, and the cell pellet was rapidly frozen and stored at -80°C.

[0240] For protein purification, the cell pellet was dissolved in a solution containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 20 mM imidazole, 10% glycerol, 1 mM TCEP, and 250 U BeyoZonase. TM Cells were resuspended in a mixture of supernuclease (with 1 mM MgCl2) and EDTA-free cocktail protease inhibitors. Cells were lysed using a high-pressure homogenizer at 4°C and 400 MPa, followed by centrifugation at 12,000 rpm for 30 minutes at 4°C using a floor centrifuge, and the supernatant was collected. The supernatant was equilibrated using a pre-filled nickel affinity column in Buffer A (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 20 mM imidazole, 10% glycerol, 1 mM TCEP), and then the supernatant was solidified by passing it through a nickel column containing Ni. 2+ Ions form coordinate bonds with the imidazole groups on histidine residues, selectively binding to target proteins tagged with His. The column is washed with Buffer A, then washed with a linear gradient of 5%–15% Buffer B (50mM Tris-HCl pH 8.0 + 300mM NaCl + 300mM imidazole + 10% glycerol + 1mM TCEP), finally eluting the protein to 100% Buffer B. Fractional samples are collected, run on SDS-PAGE gels, and stained with Coomassie Brilliant Blue. The eluent containing WRN protein is treated with TEV protease to remove the His tag, then placed in a dialysis bag, sealed, and immersed in dialysis buffer (2L Buffer A) at 4°C for enzymatic digestion and dialyzing overnight. The next day, the protein that had broken through and eluted with Buffer A was collected by reverse nickel affinity column and concentrated to 2 mL. The purified WRN protein was then separated by Superdex 16 / 600 75 pg column. The concentrated protein was combined to 16.83 mg / mL, and the purified protein was aliquoted, rapidly frozen, and stored at -80 °C.

[0241] All crystallization experiments were performed at 18 °C. Crystals of the complex of compound 11-P2 and WRN protein were obtained by sitting-drop vapor-diffusion co-crystallization. WRN protein was mixed with compound 11-P2 (20 mM dissolved in DMSO) at a molar ratio of 1:8 at a concentration of 4.0 mg / mL in 25 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5% glycerol, and 0.5 mM TCEP, and incubated on ice for 1 h. An equal volume (1 μL) of the protein complex was mixed with the reservoir solution, and 96-well plates were incubated at 18 °C. After one week, single crystals were obtained in a solution of 20% PEG 3350, 0.1 M Sodium citrate / citric acid, and 0.2 M Sodium citrate tribasic. The crystals were cryogenically protected using a solution containing 10% glycerol and flash-frozen in liquid nitrogen for crystal data collection. Diffraction data were acquired at 100K and 0.97918 wavelength using a PIXEL DECTRIS EIGER X 16M detector on the BL18U1 beamline of the Shanghai Synchrotron Radiation Facility, and processed using XDS.

[0242] Conclusion: The crystal structure of the complex of compound 11-P2 and WRN protein (Figure 1) verifies the absolute configuration of the two chiral centers of compound 11-P2.

[0243] Intermediate 1a 2-bromo-6-(4-(tert-butoxycarbonyl)piperazin-1-yl)-7-methyl-5-oxo-5,7,8,9-tetrahydropyrrolo[1,2-c][1,2,4]triazolo[1,5-a]pyrimidine-9-carboxylic acid

[0244] Step 1: 4-(2-bromo-5-ethyl-7-oxo-4-((2-(trimethylsilyl)ethoxy)methyl)-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)piperazine-1-carboxylic acid tert-butyl ester 2a

[0245] Compound 3a (23.3 g, 54.7 mmol) was dissolved in tetrahydrofuran (500 mL), and sodium hydride (3.3 g, 82 mmol) was added in portions under ice bath conditions. After reacting for 1 hour, 2-(trimethylsilyl)ethoxymethyl chloride (13.7 g, 82 mmol) was added dropwise, and the reaction was continued at room temperature for 16 hours. After the reaction was complete, a saturated ammonium chloride solution (500 mL) was added, and the mixture was extracted with ethyl acetate. The organic phase was concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA = 3:1) to give compound 2a (13.0 g).

[0246] MS(ESI)m / z 501.1[M+H–t-Bu] + .

[0247] Step 2: 4-(2-bromo-7-oxo-5-(pent-4-en-2-yl)-4-((2-(trimethylsilyl)ethoxy)methyl)-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)piperazine-1-carboxylic acid tert-butyl ester 1a-1

[0248] Compound 2a (7.8 g, 14.0 mmol) was dissolved in tetrahydrofuran (80 mL), and bis(trimethylsilyl)amino potassium (16.8 mL, 16.8 mmol, 1 M in THF) was added dropwise at -78 °C. After stirring for 1 hour, allyl bromide (4.2 g, 35 mmol) was added dropwise. The reaction was then carried out at -78 °C for 2 hours, followed by a slow increase to -20 °C and a further 1 hour. The reaction was monitored by TLC until completion. A saturated ammonium chloride solution (100 mL) was added, and the mixture was washed three times with ethyl acetate (100 mL). The organic phase was concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA = 3:1) to give compound 1a-1 (6.0 g).

[0249] MS(ESI) m / z 597.2 [M+H] + .

[0250] Step 3: 4-(2-bromo-5-(1-(epoxyethylene-2-yl)prop-2-yl)-7-oxo-4-((2-(trimethylsilyl)ethoxy)methyl)-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)piperazine-1-carboxylic acid tert-butyl ester 1a-2

[0251] Compound 1a-1 (6.0 g, 10.0 mmol) was dissolved in dichloromethane (200 mL), and m-chloroperoxybenzoic acid (5.5 g, 27.1 mmol, 85%) was added. The reaction was carried out at room temperature for 16 hours. After the reaction was completed by TLC monitoring, the reaction solution was diluted with dichloromethane (200 mL), washed five times with saturated sodium bicarbonate (100 mL), and the organic phase was concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA = 2:1) to obtain compound 1a-2 (4.44 g).

[0252] MS(ESI) m / z 613.2 [M+H] + .

[0253] Step 4: 4-(2-bromo-9-(hydroxymethyl)-7-methyl-5-oxo-5,7,8,9-tetrahydropyrrolo[1,2-c][1,2,4]triazolo[1,5-a]pyrimidin-6-yl)piperazine-1-carboxylic acid tert-butyl ester 1a-3

[0254] Compound 1a-2 (4.44 g, 7.45 mmol) was dissolved in DMF (44 mL) and water (1.0 mL), and then potassium fluoride (2.16 g, 37.3 mmol) was added. The mixture was reacted at 60 °C for 16 hours. After the reaction was complete, saturated brine (150 mL) was added, and the mixture was extracted three times with ethyl acetate (100 mL). The organic phase was concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (DCM / MeOH = 10:1) to obtain compound 1a-3 (1.88 g).

[0255] MS(ESI)m / z 427.0[M+H–t-Bu] + .

[0256] Step 5: 2-Bromo-6-(4-(tert-butyloxycarbonyl)piperazin-1-yl)-7-methyl-5-oxo-5,7,8,9-tetrahydropyrrolo[1,2-c][1,2,4]triazolo[1,5-a]pyrimidine-9-carboxylic acid 1a

[0257] Compound 1a-3 (1.88 g, 3.9 mmol) was dissolved in dichloromethane (56 mL) and water (60 mL), followed by the addition of sodium bicarbonate (3.27 g, 39.0 mmol), 2,2,6,6-tetramethylpiperidine oxide (6.2 mg, 0.04 mmol), potassium bromide (46 mg, 0.39 mmol), and methyltrioctylammonium chloride (80 mg, 0.05 mmol). The mixture was placed in an ice bath, and an aqueous solution of sodium hypochlorite (721 mg, 9.7 mmol, 10% available chlorine) was added dropwise with rapid stirring. After the reaction was completed as monitored by TLC, the reaction solution was diluted with dichloromethane (200 mL), and the organic phase was washed three times with saturated sodium carbonate (50 mL). The aqueous phase was collected, and the pH was adjusted to 3-4 with dilute hydrochloric acid. The phase was then extracted three times with dichloromethane (100 mL), and the organic phase was concentrated under reduced pressure to obtain crude product 1a, which was used directly in the next step (0.6 g).

[0258] MS(ESI)m / z 441.0[M+H–t-Bu] + .

[0259] Intermediate 8a 4-(2-bromo-5-(ethyl-2,2,2-d3)-7-oxo-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)piperazine-1-carboxylic acid tert-butyl ester

[0260] Step 1: Synthesis of methyl 3-oxovalerate-5,5,5-d3 8a-2

[0261] Compound 8a-1 (1 g, 8.6 mmol) was dissolved in anhydrous tetrahydrofuran (15 mL). Sodium hydride (448 mg, 60% in oil, 11.2 mmol) was added in portions at 0 °C. The reaction was carried out at 0 °C for 10 min. The temperature was lowered to -20 °C, and n-BuLi (4.5 mL, 2.5 M in hexane, 11.2 mmol) was added dropwise. The mixture was stirred at -20 °C for 10 min. CD3I (1.6 g, 11.2 mmol) was added dropwise. After the addition was complete, the temperature was slowly raised to room temperature and the reaction was carried out for 4 h. The reaction was quenched by adding saturated ammonium chloride solution. The mixture was extracted three times with ethyl acetate. The organic phases were combined, dried, and concentrated under reduced pressure to give a yellow oily substance 8a-2 (530 mg).

[0262] MS(ESI) m / z 133.0 [M+H] + .

[0263] Step 2: Synthesis of methyl 2-bromo-3-oxovalerate-5,5,5-d3 (8a-3)

[0264] Compound 8a-2 (0.5 g, 3.8 mmol) was dissolved in anhydrous dichloromethane (7.5 mL), and NBS (736 mg, 4.1 mmol) and p-toluenesulfonic acid (65 mg, 0.37 mmol) were added sequentially at room temperature. After the addition was complete, the mixture was stirred at room temperature for 2 h, diluted with water, and extracted twice with dichloromethane. The organic phases were combined and concentrated under reduced pressure to give compound 8a-3 (630 mg), a yellow oil.

[0265] Step 3: Synthesis of 4-(1-methoxy-1,3-dioxolane-2-yl-5,5,5-d3)piperazine-1-carboxylic acid tert-butyl ester 8a-4

[0266] Piperazine-1-carboxylic acid tert-butyl ester (583 mg, 3.1 mmol) was dissolved in anhydrous acetonitrile (6 mL), potassium carbonate (983 mg, 7.1 mmol) was added at room temperature, and a solution of compound 8a-3 (600 mg, 2.8 mmol) in acetonitrile (10 mL) was added dropwise with stirring. After the addition was complete, the reaction was allowed to proceed at room temperature for 2 h, the reaction was quenched with water, and the mixture was extracted twice with ethyl acetate. The organic phases were combined and concentrated under reduced pressure to give compound 8a-4 (900 mg), a yellow oil.

[0267] MS(ESI) m / z 318.2 [M+H] + .

[0268] Step 4: Synthesis of tert-butyl piperazine-1-carboxylate (2-bromo-5-(ethyl-2,2,2-d3)-7-oxo-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)piperazine-1-carboxylate 8a

[0269] 3-Bromo-1H-1,2,4-triazol-5-amine (3.69 g, 22.6 mmol) was dissolved in anhydrous ethanol (37 mL). Compound 8a-4 (7.9 g, 24.9 mmol) and anhydrous phosphoric acid (2.44 g, 24.9 mmol) were added sequentially at room temperature. The mixture was heated in an oil bath to 90 °C and reacted for 18 h. The reaction was quenched with water, and the mixture was extracted three times with ethyl acetate. The organic phases were combined, concentrated under reduced pressure, and purified by column chromatography (PE:EtOAc = 100% to 0) to give compound 8a (2.1 g), a pale yellow solid.

[0270] MS(ESI) m / z 430.1,432.1[M+H] + .

[0271] Intermediate 11d-P2A 4-((7S,10R)-2-bromo-10-((2-chloro-4-(trifluoromethyl)phenyl)carbamoyl)-7-methyl-5-oxo-5,7,9,10-tetrahydro-[1,2,4]triazolo[5',1':2,3]pyrimidino[6,1-c][1,4]oxazin-6-yl)piperazine-1-carboxylic acid tert-butyl ester

[0272] Step 1: 4-(2-bromo-5-(1-((((R)-epoxy-2-yl)methoxy)ethyl)-7-oxo-4-((2-(trimethylsilyl)ethoxy)methyl)-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)piperazine-1-carboxylic acid tert-butyl ester 11a

[0273] Compound 2a (1.73 g, 3.0 mmol) was dissolved in DMF (17 mL), and sodium hydride (180 mg, 4.5 mmol) was added under ice bath conditions. After stirring for 20 minutes, glycidyl (R)-(+)-m-nitrobenzenesulfonic acid (940 mg, 3.6 mmol) was added, and the reaction was carried out at room temperature for 30 minutes. After the reaction was completed, saturated ammonium chloride aqueous solution (20 mL) was added, and the mixture was extracted with ethyl acetate. The organic phase was concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA = 5:1) to obtain compound 11a (1.06 g), which was used directly in the next step.

[0274] MS(ESI) m / z 629.2 [M+H] + .

[0275] Step 2: 4-(2-bromo-10-(hydroxymethyl)-7-methyl-5-oxo-5,7,9,10-tetrahydro-[1,2,4]triazolo[5',1':2,3]pyrimidine[6,1-c][1,4]oxazin-6-yl)piperazine-1-carboxylic acid tert-butyl ester 11b

[0276] Compound 11a was dissolved in DMF (10 mL) and water (1.0 mL), and potassium fluoride (490 mg, 8.44 mmol) was added. The mixture was reacted at 70 °C for 2 hours. After the reaction was complete, saturated brine was added, and the mixture was extracted with ethyl acetate. The organic phase was concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (DCM / MeOH = 10:1) to obtain compound 11b (370 mg).

[0277] MS(ESI) m / z 499.1 [M+H] + .

[0278] Step 3: 2-Bromo-6-(4-(tert-butyloxycarbonyl)piperazin-1-yl)-7-methyl-5-oxo-5,7,9,10-tetrahydro-[1,2,4]triazolo[5',1':2,3]pyrimido[6,1-c][1,4]oxazine-10-carboxylic acid 11c

[0279] Compound 11b (900 mg, 0.36 mmol) was dissolved in dichloromethane (7.0 mL) and water (7.0 mL), followed by the addition of sodium bicarbonate (395 mg, 3.66 mmol), 2,2,6,6-tetramethylpiperidine oxide (5 mg, 0.03 mmol), potassium bromide (4 mg, 0.03 mmol), and methyltrioctylammonium chloride (11 mg, 0.03 mmol). Sodium hypochlorite aqueous solution (1.83 mmol, 10% available chlorine) was added dropwise under ice bath conditions. After the reaction was complete, ice water (20 mL) and sodium sulfite solution (20 mL, 10% in water) were added, and the mixture was stirred for 30 minutes. The pH was adjusted to 3-4 with dilute hydrochloric acid, and the mixture was extracted with dichloromethane. The organic phase was concentrated under reduced pressure to obtain crude product 11c, which was used directly in the next step (1.1 g).

[0280] MS(ESI)m / z 413.0[M+H-Boc] + .

[0281] Step 4: 4-(2-bromo-10-((2-chloro-4-(trifluoromethyl)phenyl)carbamoyl)-7-methyl-5-oxo-5,7,9,10-tetrahydro-[1,2,4]triazolo[5',1':2,3]pyrimidine[6,1-c][1,4]oxazin-6-yl)piperazine-1-carboxylic acid tert-butyl ester 11d

[0282] Compound 11c (820 mg, 1.59 mmol) was dissolved in dichloromethane (20 mL), and (chloromethylene)dimethylammonium chloride (233 mg, 2.33 mmol) was added. After stirring at room temperature for 20 minutes, 2-chloro-4-(trifluoromethyl)aniline (360 mg, 1.84 mmol) and 2,6-dimethylpyridine (880 mg, 2.05 mmol) were added, and the reaction was carried out at room temperature for 1 hour. After the reaction was completed, saturated sodium bicarbonate aqueous solution was added, and the mixture was extracted with ethyl acetate. The organic phase was concentrated under reduced pressure to obtain the crude product. The crude product was separated by preparative HPLC to obtain two isomers, namely compounds 11d-P1 (294 mg) and 11d-P2 (287 mg).

[0283] 11d-P1: MS(ESI) m / z 688.0 [MH] - LC-MS:R t =5.35min

[0284] 1 H NMR (400MHz, DMSO-d6) δ10.46(brs,1H),7.95(d,J=8.00Hz,2H),7.69(d,J=8.38Hz,1H),5.48-5.33(m,2H),4.55(d d,J=12.94,3.56Hz,1H),4.37(d,J=12.88Hz,1H),4.05-3.77(m,2H),3.50-3.39(m,1H),3.26-3.14(m,1H),2.93(br s,2H),2.73-2.57(m,2H),1.61(d,J=6.75Hz,3H),1.43(s,9H).

[0285] 11d-P2: MS(ESI) m / z 688.1 [MH] - LC-MS:R t =5.46min

[0286] 1H NMR(400MHz,DMSO-d6)δ10.39(brs,1H),7.96(d,J=8.25Hz,2H),7.80-7.69(m,1 H),5.33(s,1H),5.25(q,J=6.42Hz,1H),4.40(dd,J=12.32,1.94Hz,1H),4.13(dd ,J=12.44,3.31Hz,1H),3.92(brs,2H),3.56-3.44(m,1H),3.20–3.15(m,J=9.94H z,1H),2.90(brs,2H),2.77-2.61(m,2H),1.60(d,J=6.38,3H)1.50-1.39(m,9H).

[0287] Step 5: 4-((7S,10R)-2-bromo-10-((2-chloro-4-(trifluoromethyl)phenyl)carbamoyl)-7-methyl-5-oxo-5,7,9,10-tetrahydro-[1,2,4]triazolo[5',1':2,3]pyrimidine[6,1-c][1,4]oxazin-6-yl)piperazine-1-carboxylic acid tert-butyl ester (11d-P2A) and 4-((7R,10S)-2-bromo-10-((2-chloro-4-(trifluoromethyl)phenyl)carbamoyl)-7-methyl-5-oxo-5,7,9,10-tetrahydro-[1,2,4]triazolo[5',1':2,3]pyrimidine[6,1-c][1,4]oxazin-6-yl)piperazine-1-carboxylic acid tert-butyl ester (11d-P2B).

[0288] Compound 11d-P2 (287 mg) was separated by SFC (column type: ChiralPak IJ, 250 x 40 mm ID, 10 μm; mobile phase A: carbon dioxide, mobile phase B: methanol; gradient: 40% methanol; flow rate: 130 mL / min; column temperature: 30 °C) to obtain compounds 11d-P2A (150 mg) and 11d-P2B (90 mg).

[0289] 11d-P2A:MS(ESI)m / z 688.1[MH] - SFC R t = 0.410 min (Analysis Method 2)

[0290] 11d-P2B:MS(ESI)m / z 688.1[MH] - SFC R t = 0.595 min (Analysis Method 2)

[0291] The compound 11-P2(SFC R) was synthesized using intermediate 11d-P2A. t= 5.587 min, analytical method 1) and the synthesis of compound 11-P2 (SFC R) obtained by SFC isolation using intermediate 11d-P2. t =5.584 min, analytical method 1), the SFC retention times of the two are consistent, indicating that the absolute configurations of intermediate 11d-P2A and compound 11-P2 are consistent.

[0292] Intermediate 13a 4,4,5,5-Tetramethyl-2-(5'H,7'H-spiro[cyclopropane-1,4'-thieno[2,3-c]pyran]-2'-yl)-1,3,2-dioxacyclopentaborane

[0293] Step 1 5'H,7'H-spiro[cyclopropane-1,4'-thieno[2,3-c]pyran]13a-2

[0294] The synthesis of compound 13a-1 is based on patent PCT / CN / 2022 / 083485. Compound 13a-1 (1.9 g, 12.33 mmol), paraformaldehyde (0.4 g, 13.33 mmol), and indium trichloride (0.13 g, 0.58 mmol) were dissolved in acetonitrile (20 mL), placed under nitrogen atmosphere, and reacted at 45 °C for 0.5 hours, then heated to 65 °C for another 0.5 hours, and finally heated to 85 °C for 2 hours. After cooling to room temperature, the mixture was diluted with water (20 mL), extracted with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the crude product was separated by column chromatography to obtain compound 13a-2 (0.8 g), an oily substance.

[0295] 1 H NMR (400MHz, CDCl3) δ7.11 (dd, J=5.1, 0.9Hz, 1H), 6.48 (d, J=5.1Hz, 1H), 4.92 (s, 2H), 3.70 (s, 2H), 0.99–0.94 (m, 2H), 0.93–0.87 (m, 2H).

[0296] Step 2: 4,4,5,5-Tetramethyl-2-(5'H,7'H-spiro[cyclopropane-1,4'-thieno[2,3-c]pyran]-2'-yl)-1,3,2-dioxacyclopentaborane 13a

[0297] Compound 13a-2 (0.8 g, 4.81 mmol) was dissolved in THF (10 mL). The reaction solution was cooled to -78 °C, and 2.5 M n-butyllithium hexane solution (1.9 mL, 5.0 mmol) was added dropwise. The reaction was allowed to proceed for 10 minutes, and the temperature was slowly raised to room temperature for 0.5 hours. The temperature was then lowered to -78 °C again, and isopropanol pinacol borate ester (0.98 g, 5.26 mmol) was added dropwise. The reaction was allowed to proceed for 10 minutes, and the reaction was quenched by adding water (20 mL) at low temperature. The mixture was extracted with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the crude product was separated by column chromatography to obtain compound 13a (0.5 g), a white solid.

[0298] MS(ESI) m / z 293.1 [M+H] +

[0299] Intermediate 15a 4,4,5,5-Tetramethyl-2-(4'H,6'H-spiro[cyclopropane-1,7'-thieno[3,2-c]pyran]-2'-yl)-1,3,2-dioxoborane

[0300] Step 1 4'H,6'H-spiro[cyclopropane-1,7'-thieno[3,2-c]pyran]15a-2

[0301] The synthesis of (1-(thiophen-2-yl)cyclopropyl)methanol (15a-1) is based on patent WO2022 / 206706. Compound (15a-1) (1.3 g, 8.44 mmol) was dissolved in dry acetonitrile (100 mL), followed by the sequential addition of paraformaldehyde (329 mg, 10.96 mmol) and indium trichloride (187 mg, 0.84 mmol). Under nitrogen protection, the mixture was heated to 70 °C for 2 h, cooled to room temperature, and concentrated under reduced pressure. The crude product was purified by column chromatography (petroleum ether) to give compound 15a-2 (600 mg), a colorless oil.

[0302] Step 2: 4,4,5,5-Tetramethyl-2-(4'H,6'H-spiro[cyclopropane-1,7'-thieno[3,2-c]pyran]-2'-yl)-1,3,2-dioxoborane 15a

[0303] Under nitrogen protection, compound 15a-2 (600 mg, 3.61 mmol) was dissolved in dry tetrahydrofuran (10 mL), cooled to -70 °C, and 2.5 M n-butyllithium hexane solution (1.52 mL, 3.8 mmol) was added dropwise. The mixture was allowed to return to room temperature for 1 h, and the reaction solution was cooled to -70 °C. 2-Isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborane (740 mg, 3.98 mmol) was added dropwise. The mixture was allowed to return to room temperature for 2 h, and the reaction was quenched by adding saturated ammonium chloride solution (30 mL). The mixture was extracted with ethyl acetate (30 mL x 3), the organic phases were combined, concentrated, and the crude product was purified by column chromatography (PE / EA = 3 / 2) to obtain compound 15a (520 mg), a yellow oil.

[0304] MS(ESI)m / z 293[M+H] + .

[0305] Intermediate 22a: 4,4,5,5-Tetramethyl-2-(2,3,6,7-tetrahydrooxetane-4-yl)-1,3,2-dioxaborane

[0306] Step 1: 1,3-Dioxoisoindoline-2-yl 2,3,6,7-tetrahydrooxetane-4-carboxylate 22a-2

[0307] The synthesis of compound 22a-1 was performed according to Angew Chem Int Ed Engl. 2013 Jun 3; 52(23):6072-5. Compound 22a-1 (500 mg, 3.52 mmol) was dissolved in DCM (15 mL), and N-hydroxyphthalimide (688 mg, 4.22 mmol), DMAP (42.7 mg, 0.35 mmol), and DCC (869 mg, 4.22 mmol) were added. The reaction was carried out at room temperature for 4 h. After the reaction was complete, water was slowly added to the reaction solution, and the mixture was extracted with ethyl acetate. The collected organic phase was washed once with saturated brine, dried over anhydrous sodium sulfate, concentrated, and the crude product was purified by flash separation (PE:EA = 5:1) to obtain compound 22a-2 (700 mg), a white solid.

[0308] 1 H NMR (400MHz, DMSO-d6) δ7.91 (dd, J=5.4, 3.1Hz, 2H), 7.80 (dd, J=5.5, 3.1Hz, 2H), 7.62 (t,J=6.1Hz,1H),3.82–3.72(m,4H),2.89–2.81(m,2H),2.63(dd,J=10.5,5.5Hz,2H).

[0309] Step 2: 4,4,5,5-Tetramethyl-2-(2,3,6,7-tetrahydrooxocycloheptane-4-yl)-1,3,2-dioxaborane 22a

[0310] Compound 22a-2 (320 mg, 1.11 mmol), pinacol diboronate (563 mg, 2.22 mmol), and tert-butyl isonicotinic acid (39.7 mg, 0.22 mmol) were dissolved in ethyl acetate (10 mL), purged three times with nitrogen, and reacted at 100 °C for 16 h. After the reaction was complete, the reaction solution was concentrated, and the crude product was purified by flash separation to obtain compound 22a (130 mg), a white solid.

[0311] 1 H NMR (400MHz, DMSO-d6) δ6.78 (t, J = 5.7Hz, 1H), 3.69–3.59 (m, 4H), 2.51–2.40 (m, 4H), 1.26 (s, 12H).

[0312] Intermediate 24a(2R)-4-(2-bromo-10-((2-chloro-4-(trifluoromethyl)phenyl)carbamoyl)-7-methyl-5-oxo-5,7,9,10-tetrahydro-[1,2,4]triazolo[5',1':2,3]pyrimidine[6,1-c][1,4]oxazin-6-yl)-2-methylpiperazine-1-carboxylic acid tert-butyl ester

[0313] The synthesis of compound 24a-1 is described in reference to patent WO202479623. The synthesis of compound 24a is described in reference 11d-P2A, where intermediate 24a-1 is used to replace compound 2b to obtain compound 24a, MS (ESI) m / z 704.1 [M+H]. +

[0314] Example 1. N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(4,7-dihydro-5H-thieno[2,3-c]pyran-2-yl)-6-(4-(5-hydroxy-6-methylpyrimidin-4-carbonyl)piperazin-1-yl)-7-methyl-5-oxo-5,7,8,9-tetrahydropyrrolo[1,2-c][1,2,4]triazolo[1,5-a]pyrimidin-9-carboxamide

[0315] Step 1: 4-(2-bromo-9-((2-chloro-4-(trifluoromethyl)phenyl)carbamoyl)-7-methyl-5-oxo-5,7,8,9-tetrahydropyrrolo[1,2-c][1,2,4]triazolo[1,5-a]pyrimidin-6-yl)piperazine-1-carboxylic acid tert-butyl ester 1b

[0316] The preparation of compound 1a was performed according to WO2024079623. Compound 1a (400 mg, 0.80 mmol) and 2-chloro-4-trifluoromethylaniline (173 mg, 0.88 mmol) were dissolved in 1,4-dioxane (10 mL), followed by the dropwise addition of triethylamine (640 mg, 6.4 mmol). 1-Propylphosphine anhydride (2.03 g, 3.2 mmol, 50% in EtOAc) was added, and the reaction was carried out at 50 °C for 2 hours. After the reaction was complete, the mixture was extracted three times with saturated sodium bicarbonate aqueous solution (20 mL) and ethyl acetate (20 mL). The organic phase was concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA = 3:1) to obtain compound 1b (290 mg).

[0317] MS(ESI) m / z 672.1 [MH] - .

[0318] Step 2: 4-(8-((2-chloro-4-(trifluoromethyl)phenyl)carbamoyl)-2-(4,7-dihydro-5H-thieno[2,3-c]pyran-2-yl)-6-methyl-4-oxo-3a,4,7,8-tetrahydro-6H-imidazo[4,5-e]indene-5-yl)piperazine-1-carboxylic acid tert-butyl ester 1d·

[0319] Compound 1b (290 mg, 0.42 mmol) was dissolved in 1,4-dioxane (10 mL) and water (2 mL), and intermediate 1c (138 mg, 0.50 mmol), potassium phosphate (267 mg, 1.26 mmol), and Pd(dppf)Cl2 (32 mg, 0.04 mmol) were added. The mixture was purged with nitrogen three times, and then reacted at 100 °C for 5 hours. After cooling, the reaction solution was diluted with ethyl acetate (30 mL), and washed three times with saturated brine (15 mL). The organic phase was concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (DCM / MeOH = 15:1) to obtain compound 1d (240 mg).

[0320] MS(ESI) m / z 732.2 [MH] - .

[0321] Step 3: N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(4,7-dihydro-5H-thieno[2,3-c]pyran-2-yl)-6-methyl-4-oxo-5-(piperazin-1-yl)-3a,4,7,8-tetrahydro-6H-imidazo[4,5-e] indene-8-carboxamide 1e·

[0322] Compound 1d (240 mg, 0.33 mmol) was dissolved in ethyl acetate (10 mL), and hydrogen chloride-ethanol (5 mL, 2 M HCl in EtOH) was added. The reaction was carried out at room temperature for 3 hours. After the reaction was completed by TLC monitoring, the reaction solution was concentrated under reduced pressure to obtain crude product 1e, which was used directly in the next step.

[0323] MS(ESI) m / z 634.1 [M+H] + .

[0324] Step 4: 1 g of 6-(4-(5-(benzyloxy)-6-methylpyrimidin-4-carbonyl)piperazin-1-yl)-N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(4,7-dihydro-5H-thieno[2,3-c]pyran-2-yl)-7-methyl-5-oxo-5,7,8,9-tetrahydropyrrolo[1,2-c][1,2,4]triazolo[1,5-a]pyrimidin-9-carboxamide

[0325] Compound 1e (63 mg, 0.1 mmol) and 5-(benzyloxy)-6-methylpyrimidine-4-carboxylic acid (1e) (36.6 mg, 0.15 mmol) were dissolved in N,N-dimethylformamide (3 mL), followed by the addition of triethylamine (30 mg, 0.3 mmol) and 1-propylphosphoric anhydride (127 mg, 0.2 mmol, 50% in EtOAc). The reaction was carried out at room temperature for 3 hours. After the reaction was completed, saturated sodium bicarbonate aqueous solution (20 mL) was added, and the mixture was extracted with ethyl acetate. The organic phase was concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (DCM / MeOH = 10:1) to give 1 g (60 mg) of compound.

[0326] MS(ESI) m / z 860.2 [M+H] + .

[0327] Step 5: N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(4,7-dihydro-5H-thieno[2,3-c]pyran-2-yl)-6-(4-(5-hydroxy-6-methylpyrimidin-4-carbonyl)piperazin-1-yl)-7-methyl-5-oxo-5,7,8,9-tetrahydropyrrolo[1,2-c][1,2,4]triazolo[1,5-a]pyrimidin-9-carboxamide 1·

[0328] 1 g (60 mg, 0.10 mmol) of compound was dissolved in 3 mL of TFA and reacted at 50 °C for 3 h. After the reaction was completed by TLC monitoring, the reaction solution was concentrated under reduced pressure to obtain the crude product. The crude product was purified by preparative HPLC to obtain compounds 1-P1 (3.1 mg) and 1-P2 (2.5 mg).

[0329] Compound 1-P1:

[0330] MS(ESI) m / z 770.2 [M+H] + 。

[0331] 1 H NMR(400 MHz, DMSO-d6) δ 10.51 (s, 1H), 8.55 (s, 1H), 7.98 - 7.95 (m, 2H), 7.75 (d, J = 8.76 Hz, 1H), 7.48 (s, 1H), 5.64 (dd, J = 8.63, 5.25 Hz, 1H), 5.32 (t, J = 4.63, 1H), 4.76 (s, 2H), 3.87 (t, J = 5.32, 2H), 3.78 - 3.69 (m, 1H), 2.90 (m, 4H), 2.75 - 2.52 (m, 4H), 2.44 (s, 3H), 2.04 - 1.94 (m, 3H), 1.54 - 1.40 (m, 3H).

[0332] HPLC: R t = 11.776 min

[0333] Compound 1-P2:

[0334] MS(ESI) m / z 770.1 [M+H] + 。

[0335] 1 H NMR(400 MHz, DMSO-d6) δ 10.52 (s, 1H), 8.55 (s, 1H), 7.99 - 7.94 (m, 2H), 7.76 (d, J = 9.01 Hz, 1H), 7.51 (s, 1H), 5.57 (d, J = 9.63 Hz, 1H), 5.33 (t, 1H), 4.77 (s, 2H), 3.88 (t, 2H), 3.79 - 3.68 (m, 1H), 3.06 - 2.95 (m, 4H), 2.74 - 2.65 (m, 4H), 2.62 - 2.52 (m, 2H), 2.44 (s, 3H), 2.07 - 1.92 (m, 3H), 1.51 - 1.43 (m, 3H).

[0336] HPLC: R t = 11.926 min

[0337] Example 2. N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(4,7-dihydro-5H-thieno[2,3-c]pyran-2-yl)-6-(4-(5-hydroxy-6-methylpyrimidin-4-carbonyl)piperazin-1-yl)-7-methyl-5-oxo-5,7,9,10-tetrahydro-[1,2,4]triazolo[5',1':2,3]pyrimidinyl[6,1-c][1,4]oxazine-10-carboxamide

[0338] Step 1: 4-(2-bromo-5-(1-hydroxyethyl)-7-oxo-4-((2-(trimethylsilyl)ethoxy)methyl)-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)piperazine-1-carboxylic acid tert-butyl ester 2b

[0339] Compound 2a was prepared according to patent WO2024079623. Compound 2a (268 mg, 0.48 mmol) was dissolved in THF (4 mL), and bis(trimethylsilylaminolithium) (1.2 mL, 1.2 mmol, 1 M in THF) was added dropwise at -78 °C. The mixture was stirred at this temperature for 1 hour, followed by the addition of 3-phenyl-2-benzenesulfonyl-1,2-oxazolidinium (188 mg, 0.72 mmol). The mixture was slowly brought to room temperature and reacted for 16 hours. The reaction was monitored by TLC until completion. The mixture was extracted three times with saturated ammonium chloride solution (10 mL) and ethyl acetate (10 mL). The organic phase was concentrated to obtain the crude product, which was purified by column chromatography (DCM / MeOH = 10:1) to obtain compound 2b (150 mg).

[0340] MS(ESI) m / z 573.1 [M+H] + .

[0341] Step 2: 4-(5-(1-(allyloxy)ethyl)-2-bromo-7-oxo-4-((2-(trimethylsilyl)ethoxy)methyl)-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)piperazine-1-carboxylic acid tert-butyl ester 2c·

[0342] Compound 2b (1.08 g, 1.88 mmol) was dissolved in dry tetrahydrofuran (10 mL) and dry dimethyl sulfoxide (1.0 mL). Sodium hydride (151 mg, 3.76 mmol) was added under ice bath conditions. After stirring for 1 hour, allyl iodine (1.0 g, 5.64 mmol) was added, and the reaction was allowed to proceed at room temperature for 3 hours. The reaction was monitored by TLC until completion. The reaction solution was diluted with saturated ammonium chloride (20 mL), and washed three times with ethyl acetate (15 mL). The organic phase was concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (DCM / MeOH = 10:1) to obtain compound 2c (1.3 g).

[0343] MS(ESI) m / z 613.2 [M+H] + .

[0344] Step 3: 4-(2-bromo-5-(1-(oxehero-2-ylmethoxy)ethyl)-7-oxo-4-((2-(trimethylsilyl)ethoxy)methyl)-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)piperazine-1-carboxylic acid tert-butyl ester 2d·

[0345] Compound 2c (1.1 g, 1.8 mmol) was dissolved in dichloromethane (11 mL), and m-chloroperoxybenzoic acid (1.1 g, 5.38 mmol, 85%) was added. The mixture was reacted at room temperature for 16 hours. After the reaction was completed by TLC monitoring, the reaction solution was diluted with dichloromethane (50 mL), washed five times with saturated sodium bicarbonate (10 mL), and the organic phase was concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA = 2:1) to obtain compound 2d (265 mg).

[0346] MS(ESI) m / z 629.1 [M+H] + .

[0347] Step 4: 4-(2-bromo-10-(hydroxymethyl)-7-methyl-5-oxo-5,7,9,10-tetrahydro-[1,2,4]triazolo[5',1':2,3]pyrimidino[6,1-c][1,4]oxazin-6-yl)piperazine-1-carboxylic acid tert-butyl ester 2e

[0348] Compound 2d (260 mg, 0.41 mmol) was dissolved in N,N-dimethylformamide (2.6 mL) and water (0.26 mL), and potassium fluoride (120 mg, 2.0 mmol) was added. The mixture was reacted at 70 °C for 3 hours. After the reaction was complete, saturated brine (10 mL) was added, and the mixture was extracted three times with ethyl acetate (10 mL). The organic phase was concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (DCM / MeOH = 10:1) to obtain compound 2e (150 mg).

[0349] MS(ESI) m / z 499.1 [M+H] + .

[0350] Step 5: 2-Bromo-6-(4-(tert-butyloxycarbonyl)piperazin-1-yl)-7-methyl-5-oxo-5,7,9,10-tetrahydro-[1,2,4]triazolo[5',1':2,3]pyrimido[6,1-c][1,4]oxazine-10-carboxylic acid 2f

[0351] Compound 2e (150 mg, 0.3 mmol) was dissolved in dichloromethane (4.5 mL) and water (4.5 mL), followed by the addition of sodium bicarbonate (250 mg, 3.0 mmol), 2,2,6,6-tetramethylpiperidine oxide (2 mg, 0.013 mmol), potassium bromide (2 mg, 0.017 mmol), and methyltrioctylammonium chloride (6 mg, 0.015 mmol). The mixture was placed in an ice bath, and an aqueous solution of sodium hypochlorite (555 mg, 0.75 mmol, 10% available chlorine) was added dropwise with rapid stirring. After the reaction was complete as monitored by TLC, ice water (10 mL) and sodium sulfite solution (10 mL, 10% in water) were added, and the mixture was stirred for 30 minutes. The pH was then adjusted to 3-4 with dilute hydrochloric acid. The mixture was extracted three times with dichloromethane (15 mL), and the organic phase was concentrated under reduced pressure to obtain crude product 2f, which was used directly in the next step (50 mg).

[0352] MS(ESI)m / z 413.0[M+H-Boc] + .

[0353] Step 6: 2g of 4-(2-bromo-10-((2-chloro-4-(trifluoromethyl)phenyl)carbamoyl)-7-methyl-5-oxo-5,7,9,10-tetrahydro-[1,2,4]triazolo[5',1':2,3]pyrimidino[6,1-c][1,4]oxazin-6-yl)piperazine-1-carboxylic acid tert-butyl ester

[0354] Compound 2f (75 mg, 0.146 mmol) and 2-chloro-4-(trifluoromethyl)aniline (34.2 mg, 0.175 mmol) were dissolved in dioxane (3 mL), followed by the addition of triethylamine (233 mg, 2.33 mmol). 1-Propylphosphine anhydride (737 mg, 1.16 mmol, 50% in EtOAc) was added, and the reaction was carried out at 70 °C for 2 hours. After the reaction was complete, saturated sodium bicarbonate (10 mL) was added, and the mixture was extracted three times with ethyl acetate (10 mL). The organic phase was concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (DCM / MeOH = 10:1) to give 2 g (37 mg) of compound.

[0355] MS(ESI) m / z 712.0 [M+Na] + .

[0356] Step 7: 4-(10-((2-chloro-4-(trifluoromethyl)phenyl)carbamoyl)-2-(4,7-dihydro-5H-thieno[2,3-c]pyran-2-yl)-7-methyl-5-oxo-5,7,9,10-tetrahydro-[1,2,4]triazolo[5',1':2,3]pyrimidino[6,1-c][1,4]oxazin-6-yl)piperazine-1-carboxylic acid tert-butyl ester 2h

[0357] 2 g (51 mg, 0.074 mmol) of compound was dissolved in 1,4-dioxane (2 mL) and water (0.4 mL), followed by the addition of 2-(5,7-dihydro-4H-thieno[2,3-c]pyran-2-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborhexacyclopentane (23 mg, 0.089 mmol), potassium phosphate (47 mg, 0.222 mmol), and Pd(dppf)Cl2 (8 mg, 0.01 mmol). The mixture was purged with nitrogen three times and then reacted at 100 °C for 5 hours. After cooling, the reaction solution was diluted with ethyl acetate (15 mL) and washed three times with saturated brine (10 mL). The organic phase was concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (DCM / MeOH = 15:1) to obtain compound 2 h (53 mg).

[0358] MS(ESI) m / z 650.1 [M+H-Boc] + .

[0359] Step 8: N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(4,7-dihydro-5H-thieno[2,3-c]pyran-2-yl)-7-methyl-5-oxo-6-(piperazin-1-yl)-5,7,9,10-tetrahydro-[1,2,4]triazolo[5',1':2,3]pyrimido[6,1-c][1,4]oxazine-10-carboxamide 2i

[0360] Compound 2h (53 mg, 0.07 mmol) was dissolved in dichloromethane (2 mL), and hydrogen chloride-ethanol (2 mL, 2 M HCl in EtOH) was added. The reaction was carried out at room temperature for 3 hours. After the reaction was completed by TLC monitoring, the reaction solution was concentrated under reduced pressure to obtain crude product 2i, which was directly used in the next step.

[0361] MS(ESI) m / z 650.1 [M+H] + .

[0362] Step 9: 6-(4-(5-(benzyloxy)-6-methylpyrimidin-4-carbonyl)piperazin-1-yl)-N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(4,7-dihydro-5H-thieno[2,3-c]pyran-2-yl)-7-methyl-5-oxo-5,7,9,10-tetrahydro-[1,2,4]triazolo[5',1':2,3]pyrimido[6,1-c][1,4]oxazine-10-carboxamide 2j

[0363] Crude product 2i (50 mg, 0.07 mmol) and 5-(benzyloxy)-6-methylpyrimidine-4-carboxylic acid (1f) (28 mg, 0.12 mmol) were dissolved in N,N-dimethylformamide (2 mL), and then triethylamine (23 mg, 0.23 mmol) was added dropwise. 1-Propylphosphine anhydride (96 mg, 0.15 mmol, 50% in EtOAc) was added, and the reaction was carried out at room temperature for 3 hours. After the reaction was complete, saturated sodium bicarbonate aqueous solution (10 mL) was added, and the mixture was extracted three times with ethyl acetate (10 mL). The organic phase was concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (DCM / MeOH = 10:1) to give compound 2j (38 mg).

[0364] MS(ESI) m / z 876.2 [M+H] + .

[0365] Step 10: N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(4,7-dihydro-5H-thieno[2,3-c]pyran-2-yl)-6-(4-(5-hydroxy-6-methylpyrimidin-4-carbonyl)piperazin-1-yl)-7-methyl-5-oxo-5,7,9,10-tetrahydro-[1,2,4]triazolo[5',1':2,3]pyrimido[6,1-c][1,4]oxazine-10-carboxamide 2

[0366] Compound 2j (38 mg, 0.10 mmol) was dissolved in trifluoroacetic acid (2 mL) and reacted at 50 °C for 3 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure to obtain the crude product. The crude product was purified by preparative HPLC to obtain compound 2 (9.3 mg). LC-MS analytical conditions: Column: Welch Boltimate EXT-C18 2.7 μm, 4.6 x 50 mm; Column temperature: 40 °C; Mobile phase A: 0.05% formic acid in H2O, Mobile phase B: 0.05% formic acid in CH3CN; Flow rate: 1.0 mL / min to 1.5 mL / min; Gradient: 5 to 95% B, 0–1.5 min; 95% B in 1.5–3.0 min.

[0367] LC-MS:R t =2.372min, MS(ESI)m / z 786.2[M+H] + .

[0368] 1H NMR (400MHz, CDCl3) δ11.78(s,1H),8.59(s,2H),8.51(d,J=8.8Hz,1H),7.67(s,1H),7.57(d,J=8.8Hz ,1H),7.55(s,1H),5.76-5.65(m,1H),5.37(q,J=6.4Hz,1H),5.24(t,J=4.2Hz,1H),4.86-4.76(m,1H) ,4.81(s,2H),4.64(dd,J=4.0,12.8Hz,1H),4.17(dd,J=4.4,12.4Hz,1H),4.00-3.88(m,3H),3.78-3. 65(m,1H),3.55-3.38(m,1H),3.09-2.99(m,1H),2.86-2.68(m,4H),2.57(s,3H),1.80(d,J=6.4Hz,3H)

[0369] Example 3 1-(2-(3,6-dihydro-2H-pyran-4-yl)-5-ethyl-6-(4-(5-hydroxy-6-methylpyrimidin-4-carbonyl)piperazin-1-yl)-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)-3-(4-(trifluoromethyl)phenyl)urea

[0370] Step 1: 4-(4-amino-2-bromo-5-ethyl-7-oxo-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)piperazine-1-carboxylic acid tert-butyl ester 3b

[0371] Compound 3a (3.6 g, 8.55 mmol) was dissolved in DMF (50 mL), and sodium hydride (684 mg, 17.1 mmol) was added at room temperature. After stirring at room temperature for 1 hour, diphenylphosphonohydroxylamine (4.0 g, 17.1 mmol) was added, and after reacting for 1 hour, DMF (50 mL) was added. The reaction mixture was then allowed to react for another 16 hours at room temperature. After the reaction was complete, saturated ammonium chloride solution (100 mL) was added, and the mixture was extracted three times with ethyl acetate (50 mL). The organic phase was concentrated to obtain the crude product, which was purified by column chromatography (DCM / MeOH = 10:1) to obtain compound 3b (450 mg).

[0372] MS(ESI) m / z 342.1 [M+H-Boc] + .

[0373] Step 2: 4-(2-bromo-5-ethyl-7-oxo-4-(3-(4-(trifluoromethyl)phenyl)ureo)-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)piperazine-1-carboxylic acid tert-butyl ester 3c

[0374] Compound 3b (135 mg, 0.3 mmol) was dissolved in tetrahydrofuran (2 mL), and sodium hydride (37 mg, 0.9 mmol) was added under ice bath conditions. After stirring for 1 hour, 1-isocyano-4-(trifluoromethyl)benzene (84 mg, 0.45 mmol) was added, and the reaction was carried out at room temperature for 16 hours. After the reaction was completed, the reaction solution was diluted with saturated ammonium chloride (10 mL), extracted with ethyl acetate, and the organic phase was concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (DCM / MeOH = 10:1) to obtain compound 3c (107 mg).

[0375] MS(ESI)m / z 573.1[M+H–t-Bu] + .

[0376] Step 3: 4-(2-(3,6-dihydro-2H-pyran-4-yl)-5-ethyl-7-oxo-4-(3-(4-(trifluoromethyl)phenyl)ureo)-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)piperazine-1-carboxylic acid tert-butyl ester 3d

[0377] Compound 3c (107 mg, 0.173 mmol) was dissolved in 1,4-dioxane (2 mL) and water (0.2 mL), and 3,6-dihydro-2H-pyran-4-boronic acid pinacol ester (44 mg, 0.208 mmol), potassium phosphate (110 mg, 0.519 mmol), and Pd(dppf)Cl2 (16 mg, 0.02 mmol) were added. The mixture was purged with nitrogen three times and reacted at 100 °C for 5 hours. After cooling, the reaction solution was diluted with ethyl acetate (15 mL), washed three times with saturated brine (10 mL), and the organic phase was concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (DCM / MeOH = 15:1) to obtain compound 3d (58 mg).

[0378] MS(ESI) m / z 631.2 [M–H] - .

[0379] Step 4: 1-(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)-3-(4-(trifluoromethyl)phenyl)urea 3e

[0380] Compound 3d (58 mg, 0.09 mmol) was dissolved in dichloromethane (2 mL), and hydrogen chloride-ethanol (2 mL, 2 M HCl in EtOH) was added. The reaction was carried out at room temperature for 3 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure to obtain crude product 3e, which was used directly in the next step.

[0381] MS(ESI) m / z 533.2 [M+H] + .

[0382] Step 5: 1-(6-(4-(5-(benzyloxy)-6-methylpyrimidin-4-carbonyl)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)-3-(4-(trifluoromethyl)phenyl)urea 3f

[0383] The crude product 3e (58 mg, 0.09 mmol) and compound 1f were dissolved in DMF (3 mL), followed by the addition of triethylamine (28 mg, 0.27 mmol) and 1-propylphosphoric anhydride (117 mg, 0.18 mmol, 50% in EtOAc). The reaction was carried out at room temperature for 4 hours. After the reaction was complete, saturated sodium bicarbonate aqueous solution (15 mL) was added, followed by extraction with ethyl acetate (15 mL). The organic phase was concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (DCM / MeOH = 10:1) to obtain compound 3f (22 mg).

[0384] MS(ESI) m / z 759.1 [M+H] + .

[0385] Step 6: 1-(2-(3,6-dihydro-2H-pyran-4-yl)-5-ethyl-6-(4-(5-hydroxy-6-methylpyrimidin-4-carbonyl)piperazin-1-yl)-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)-3-(4-(trifluoromethyl)phenyl)urea3

[0386] Compound 3f (38 mg, 0.10 mmol) was dissolved in trifluoroacetic acid (2 mL) and reacted at 50 °C for 3 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure to obtain the crude product. The crude product was purified by preparative HPLC to obtain compound 3 (1.1 mg).

[0387] MS(ESI) m / z 669.2 [M+H] + .

[0388] Example 4: N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(2-(3,6-dihydro-2H-pyran-4-yl)-6-ethyl-5-(4-(5-hydroxy-6-methylpyrimidin-4-carbonyl)piperazin-1-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)acetamide

[0389] Step 1: 2-Bromo-6-ethyl-[1,2,4]triazolo[1,5-a]pyrimidine-5,7(4H,6H)-dione 4b

[0390] 3-Bromo-1H-1,2,4-triazol-5-amine (4a) (5 g, 30.6 mmol), methyl 2-(methoxycarbonyl)butyrate (4.91 g, 30.6 mmol), and DIPEA (3.95 g, 30.6 mmol) were added to a sealed flask and reacted at 130 °C for 16 h. After the reaction was complete, the mixture was cooled to room temperature, diluted with water, extracted twice with ethyl acetate, and the pH of the aqueous phase was adjusted to 1. A solid precipitated out and was filtered to give compound 4b (4 g), a white solid.

[0391] MS(ESI) m / z 259.0 [M+H] + .

[0392] Step 2: 2-Bromo-5,7-dichloro-6-ethyl-[1,2,4]triazolo[1,5-a]pyrimidine 4c

[0393] Compound 4b (1.5 g, 5.79 mmol) was dissolved in POCl3 (10 mL) and reacted at 110 °C for 2 h. After the reaction was complete, the mixture was cooled to room temperature, and the reaction solution was slowly poured into ice water to quench it. The mixture was extracted with ethyl acetate, washed once with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give compound 4c (800 mg) as a white solid.

[0394] MS(ESI) m / z 294.9 [M+H] + .

[0395] Step 3: Ethyl 2-(2-bromo-5-chloro-6-ethyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)-3-oxohexanoate 4d

[0396] Diethyl malonate (518 mg, 3.24 mmol) was dissolved in DMF (10 mL), and NaH (129 mg, 3.24 mmol) was added at 0 °C. After reacting for 30 min at 0 °C, compound 4c (800 mg, 2.70 mmol) was added, and the reaction was carried out at 25 °C for 1 h. After the reaction was completed, the reaction solution was slowly quenched with water, extracted with ethyl acetate, washed once with saturated brine, dried over anhydrous sodium sulfate, concentrated, and the crude product was flash-separated to obtain compound 4d (1.1 g), a pale yellow solid.

[0397] MS(ESI) m / z 419.0 [M+H] +

[0398] Step 4: Ethyl 2-(2-bromo-5-chloro-6-ethyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)-3-oxohexanoate 4e

[0399] Compound 4d (1.1 g, 2.61 mmol) was dissolved in DMF (10 mL), and piperazine-1-carboxylic acid tert-butyl ester (584 mg, 3.14 mmol) and DIPEA (673 mg, 5.22 mmol) were added. The reaction was carried out at 60 °C for 2 h. After the reaction was completed, the reaction solution was diluted with water, extracted with ethyl acetate, the organic phase was washed once with saturated brine, dried over anhydrous sodium sulfate, concentrated, and the crude product was purified by flash separation to give compound 4e (1.2 g), a pale yellow solid.

[0400] MS(ESI) m / z 569.2 [M+H] + .

[0401] Step 5: 4-(2-bromo-7-(2-ethoxy-2-oxoethyl)-6-ethyl-[1,2,4]triazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylic acid tert-butyl ester 4f

[0402] Compound 4e (600 mg, 1.05 mmol) was dissolved in DMF (8 mL), and LiCl (88.2 mg, 2.10 mmol) and H₂O (37.8 mg, 2.10 mmol) were added. The mixture was reacted at 80 °C for 1 h. After the reaction was complete, the reaction solution was diluted with water, extracted with ethyl acetate, washed once with saturated brine, dried over anhydrous sodium sulfate, concentrated, and the crude product was purified by flash separation to obtain compound 4f (500 mg), a pale yellow solid.

[0403] MS(ESI) m / z 497.1 [M+H] +

[0404] 1H NMR (400MHz, DMSO-d6) δ4.27(s,2H),4.13(q,J=7.1Hz,2H),3.51(s,4H),3.36(s,4H),2.71(q,J=7.1Hz,2H),1.44(s,9H),1.17(q,J=7.2Hz,6H).

[0405] Step 6: 4g of 4-(2-(3,6-dihydro-2H-pyran-4-yl)-7-(2-ethoxy-2-oxoethyl)-6-ethyl-[1,2,4]triazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylic acid tert-butyl ester

[0406] Compound 4f (500 mg, 1.01 mmol), 2-(3,6-dihydro-2H-pyran-4-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborhexacyclopentane (315 mg, 1.51 mmol), Pd(dppf)Cl2 (73.1 mg, 0.10 mmol), and K2CO3 (347 mg, 2.52 mmol) were dissolved in 1,4-dioxane (10 mL) and reacted at 80 °C for 1 h under nitrogen protection. After the reaction was complete, the reaction solution was diluted with water, extracted with ethyl acetate, the organic phase was washed once with saturated brine, dried over anhydrous sodium sulfate, concentrated, and the crude product was purified by flash separation to give 4 g (420 mg) of compound as a pale yellow solid.

[0407] MS(ESI) m / z 501.2 [M+H] +

[0408] 1 H NMR(400MHz, DMSO-d6)δ6.85(s,1H),4.32–4.21(m,4H),4.12(q,J=7.1Hz,2H),3.82(t,J=5.4Hz ,2H),3.52(s,4H),3.31–3.24(m,4H),2.71(d,J=7.5Hz,2H),1.44(s,9H),1.16(t,J=7.0Hz,6H).

[0409] Step 7: 2-(5-(4-(tert-butyloxycarbonyl)piperazin-1-yl)-2-(3,6-dihydro-2H-pyran-4-yl)-6-ethyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)acetic acid for 4 hours

[0410] 4 g (360 mg, 0.72 mmol) of the compound was dissolved in MeOH (4 mL) and H₂O (1 mL), and LiOH (34.56 mg, 1.44 mmol) was added. The reaction was carried out at 25 °C for 1 h. After the reaction was completed, the pH of the reaction solution was adjusted to 3 with 1 M HCl, extracted with ethyl acetate, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give compound 4 h (320 mg), a pale yellow liquid.

[0411] MS(ESI) m / z: 473.2 [M+H] + .

[0412] Step 8: 4-(7-(2-((2-chloro-4-(trifluoromethyl)phenyl)amino)-2-oxoethyl)-2-(3,6-dihydro-2H-pyran-4-yl)-6-ethyl-[1,2,4]triazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylic acid tert-butyl ester 4i

[0413] Compound 4h (300 mg, 0.63 mmol) and 2-chloro-4-(trifluoromethyl)aniline (148 mg, 0.76 mmol) were dissolved in DMF (8 mL), and T3P (50% in EA, 601 mg, 0.94 mmol) and DPIEA (325 mg, 2.52 mmol) were added. The mixture was reacted at 25 °C for 1 h. After the reaction was complete, the reaction solution was diluted with water, extracted with ethyl acetate, and the collected organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and the crude product was purified by flash separation to give compound 4i (260 mg) as a pale yellow solid.

[0414] MS(ESI) m / z 650.2 [M+H] + .

[0415] Step 9: N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(2-(3,6-dihydro-2H-pyran-4-yl)-6-ethyl-5-(piperazin-1-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)acetamide 4j

[0416] Compound 4i (240 mg, 0.37 mmol) was dissolved in DCM (6 mL), and TFA (168 mg, 1.48 mmol) was added. The reaction was carried out at 25 °C for 1 h. After the reaction was completed, the pH of the reaction solution was adjusted to 8 with saturated NaHCO3 aqueous solution, extracted with ethyl acetate, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give compound 4j (180 mg), a pale yellow liquid.

[0417] MS(ESI) m / z 550.2 [M+H] + .

[0418] Step 10: 2-(5-(4-(5-(benzyloxy)-6-methylpyrimidin-4-carbonyl)piperazin-1-yl)-2-(3,6-dihydro-2H-pyran-4-yl)-6-ethyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)-N-(2-chloro-4-(trifluoromethyl)phenyl)acetamide 4k

[0419] Compounds 4j (170 mg, 0.31 mmol) and 1j (113 mg, 0.46 mmol) were dissolved in DMF (6 mL), and T3P (292 mg, 0.46 mmol) and DIPEA (160 mg, 1.24 mmol) were added. The mixture was reacted at 25 °C for 1 h. After the reaction was complete, water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic phase was washed once with saturated brine, dried over anhydrous sodium sulfate, concentrated, and the crude product was purified by flash separation to obtain compound 4k (160 mg), a pale yellow solid.

[0420] MS(ESI) m / z: 776.3 [M+H] + .

[0421] Step 11: N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(2-(3,6-dihydro-2H-pyran-4-yl)-6-ethyl-5-(4-(5-hydroxy-6-methylpyrimidin-4-carbonyl)piperazin-1-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)acetamide 4

[0422] Compound 4k (150 mg, 0.19 mmol) was dissolved in TFA (2 mL) and reacted at 50 °C for 1 h. After the reaction was complete, the pH of the reaction solution was adjusted to 8 with saturated NaHCO3 aqueous solution, extracted with ethyl acetate, the organic phase was washed once with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain the crude product. The crude product was purified by preparative high performance liquid chromatography to obtain compound 4 (24 mg), a white solid.

[0423] MS(ESI) m / z 686.2 [M+H] +

[0424] 1H NMR (400MHz, DMSO-d6) δ10.27(s,1H),8.50(s,1H),7.99(d,J=7.9Hz,1H),7.94(s,1H),7.71(d,J=8.3Hz,1H),6.85(s,1H),4 .48(s,2H),4.26(s,2H),3.81(d,J=25.3Hz,4H),3.46(d,J=23.7Hz,6H),2.74(s,2H),2.51(s,2H),2.42(s,3H),1.22(s,3H).

[0425] Example 5: N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(3,6-dihydro-2H-pyran-4-yl)-6-(4-(5-hydroxy-6-methylpyrimidin-4-carbonyl)piperazin-1-yl)-7-methyl-5-oxo-5,7,9,10-tetrahydro-[1,2,4]triazolo[5',1':2,3]pyrimido[6,1-c][1,4]oxazine-10-carboxamide

[0426] The synthesis of compound 5 was based on compound 2, but with 2-(5,7-dihydro-4H-pyran-4-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentane (1c) replaced by 2-(3,6-dihydro-4H-thieno[2,3-c]pyran-2-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentane, yielding compound 5, with an MS (ESI) m / z of 686.2 [M+H]. + .

[0427] Example 6: N-(2-chloro-4-(trifluoromethyl)phenyl)-6-(4-(5-hydroxy-6-methylpyrimidin-4-carbonyl)piperazin-1-yl)-7-methyl-2-(6-(methyl-d3)-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)-5-oxo-5,7,9,10-tetrahydro-[1,2,4]triazolo[5',1':2,3]pyrimido[6,1-c][1,4]oxazine-10-carboxamide

[0428] The synthesis of compound 6 was based on compound 2, but with 2-(5,7-dihydro-4H-thieno[2,3-c]pyridin-2-yl)boronic acid substituted for 2-(5,7-dihydro-4H-thieno[2,3-c]pyran-2-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentane (1c), yielding compound 6. MS (ESI) m / z 802.2 [M+H] + .

[0429] Example 7: N-(2-chloro-4-(trifluoromethyl)phenyl)-6-(4-(5-hydroxy-6-methylpyrimidin-4-carbonyl)piperazin-1-yl)-7-methyl-2-(6-(methyl-d3)-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)-5-oxo-5,7,8,9-tetrahydropyrrolo[1,2-c][1,2,4]triazolo[1,5-a]pyrimidin-9-carboxamide

[0430] Compound 7 was synthesized according to Example 1, by replacing 2-(5,7-dihydro-4H-thieno[2,3-c]pyridin-2-yl)boronic acid with (6-(methyl-d3)-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)boronic acid (1c) to obtain compound 7, MS (ESI) m / z 786.2 [M+H] + .

[0431] Example 8 N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(3,6-dihydro-2H-pyran-4-yl)-6-(4-(5-hydroxy-6-methylpyrimidin-4-carbonyl)piperazin-1-yl)-7-(methyl-d3)-5-oxo-5,7,8,9-tetrahydropyrrolo[1,2-c][1,2,4]triazolo[1,5-a]pyrimidin-9-carboxamide

[0432] Compound 8 was synthesized according to Example 1, with 4-(2-bromo-5-(ethyl-2,2,2-d3)-7-oxo-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)piperazine-1-carboxylic acid tert-butyl ester (8a) replaced by 4-(2-bromo-5-ethyl-7-oxo-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)piperazine- 1-Tertiaryl carboxylate (3a), substituted with 2-(5,7-dihydro-4H-pyran-4-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentane (1c) to give compound 8, MS (ESI) m / z 717.2 [M+H] + .

[0433] Example 9 N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(4,7-dihydro-5H-thieno[2,3-c]pyran-2-yl)-6-(4-(5-hydroxy-6-methylpyrimidin-4-carbonyl)piperazin-1-yl)-7-(methyl-d3)-5-oxo-5,7,8,9-tetrahydropyrrolo[1,2-c][1,2,4]triazolo[1,5-a]pyrimidin-9-carboxamide

[0434] The synthesis of compound 9 was performed according to Example 1, using compound 8g instead of compound 1b. MS (ESI) m / z 773.2 [M+H] + .

[0435] Example 10N-(2-chloro-4-(trifluoromethyl)phenyl)-6-(4-(5-hydroxy-6-methylpyrimidin-4-carbonyl)piperazin-1-yl)-7-(methyl-d3)-2-(6-(methyl-d3)-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)-5-oxo-5,7,8,9-tetrahydropyrrolo[1,2-c][1,2,4]triazolo[1,5-a]pyrimidin-9-carboxamide

[0436] Compound 10 was synthesized according to Example 1, by replacing compound 1b with compound 8g and 2-(5,7-dihydro-4H-thieno[2,3-c]pyridin-2-yl)boronic acid with (6-(methyl-d3)-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)boronic acid, yielding compound 10, with an MS (ESI) m / z of 789.2 [M+H]. + .

[0437] Example 11 (7S,10R)-N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(4,7-dihydro-5H-thieno[2,3-c]pyran-2-yl)-6-(4-(5-hydroxy-6-methylpyrimidin-4-carbonyl)piperazin-1-yl)-7-methyl-5-oxo-5,7,9,10-tetrahydro-[1,2,4]triazolo[5',1':2,3]pyrimido[6,1-c][1,4]oxazine-10-carboxamide (11-P 2) and (7R,10S)-N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(4,7-dihydro-5H-thieno[2,3-c]pyran-2-yl)-6-(4-(5-hydroxy-6-methylpyrimidin-4-carbonyl)piperazin-1-yl)-7-methyl-5-oxo-5,7,9,10-tetrahydro-[1,2,4]triazolo[5',1':2,3]pyrimido[6,1-c][1,4]oxazine-10-carboxamide (11-P1)

[0438] Method 1: Synthesis of compounds 11-P2 and 11-P1. Referring to Example 2, 2g of intermediate was replaced with intermediate 11d-P2. The final product was separated by SFC (column type: ChiralPak OJ, 250x 40mm ID, 10μm; mobile phase A: carbon dioxide, mobile phase B: MeOH (0.1% ammonia): acetonitrile = 7:3; flow rate: 130mL / min; column temperature: 30℃) to obtain compounds 11-P2 and 11-P1.

[0439] Compound 11-P2:

[0440] MS(ESI) m / z 786.2 [M+H] + SFC R t =2.662 min (analysis method 2), ee = 99.05%.

[0441] 1H NMR (400MHz, DMSO-d6) δ10.41(s,1H),8.58(s,1H),7.99(s,1H),7.93(t,J=8.0Hz,1H),7.80-7.7 2(m,1H),7.52(s,1H),5.42-5.37(m,1H),5.36-5.27(m,1H),4.77(s,2H),4.58-4.44(m,1H),4.43 -4.36(m,1H),4.24-4.13(m,1H),3.88(t,J=5.5Hz,2H),3.73-3.57(m,1H),3.54-3.43(m,1H),3. 26-3.16(m,2H),3.06-2.79(m,2H),2.75-2.63(m,3H),2.44(s,3H),1.64(dd,J=20.2,6.4Hz,3H).

[0442] Compound 11-P1:

[0443] MS(ESI) m / z 786.2 [M+H] + SFC R t =1.754min (Analysis Method 2), ee = 100%.

[0444] 1 H NMR (400MHz, DMSO-d6) δ10.42(s,1H),8.56(d,J=2.4Hz,1H),7.99(s,1H),7.92(t,J=8.0Hz,1H),7. 79-7.72(m,1H),7.52(s,1H),5.43-5.37(m,1H),5.36-5.25(m,1H),4.77(s,2H),4.58-4.44(m,1H), 4.43-4.35(m,1H),4.23-4.13(m,1H),3.88(t,J=5.4Hz,2H),3.72-3.59(m,1H),3.55-3.46(m,1H), 3.26-3.18(m,2H),3.06-2.81(m,2H),2.75-2.66(m,3H),2.44(s,3H),1.64(dd,J=20.2,6.4Hz,3H).

[0445] Example 12 (7S,10S)-N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(4,7-dihydro-5H-thieno[2,3-c]pyran-2-yl)-6-(4-(5-hydroxy-6-methylpyrimidin-4-carbonyl)piperazin-1-yl)-7,8-dimethyl-5-oxo-7,8,9,10-tetrahydro-5H-pyrazine[1,2-c][1,2,4]triazolo[1,5-a]pyrimidin-10-carboxamide (12-P1) A) and (7S,10R)-N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(4,7-dihydro-5H-thieno[2,3-c]pyran-2-yl)-6-(4-(5-hydroxy-6-methylpyrimidin-4-carbonyl)piperazin-1-yl)-7,8-dimethyl-5-oxo-7,8,9,10-tetrahydro-5H-pyrazine[1,2-c][1,2,4]triazolo[1,5-a]pyrimidin-10-carboxamide (12-P1B)

[0446] Step 1: 4-(2-bromo-5-(1-chloroethyl)-7-oxo-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)piperazine-1-carboxylic acid tert-butyl ester 12a

[0447] Compound 2b (4.0 g, 6.97 mmol) was dissolved in DCM (50 mL), and thionyl chloride (912 mg, 7.67 mmol) was added at 0 °C. The reaction was carried out at 0 °C for 30 minutes, quenched with saturated sodium bicarbonate aqueous solution, and extracted with ethyl acetate (100 mL × 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain compound 12a (3.0 g). The crude product was used directly for the next step.

[0448] MS(ESI) m / z 405.0 [M-56] + .

[0449] Step 2: 4-(2-bromo-5-(1-(methylamino)ethyl)-7-oxo-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)piperazine-1-carboxylic acid tert-butyl ester 12b

[0450] The crude compound 12a (4.2 g, 9.10 mmol) was dissolved in methanol (60 mL), and a methanol solution of methylamine (14.13 g, 91.0 mmol, 10% in MeOH) was added. The mixture was reacted at 25 °C for 18 h. After the reaction was completed, the crude product was concentrated and purified by column chromatography (DCM / MeOH = 7 / 3) to obtain compound 12b (1.8 g).

[0451] MS(ESI) m / z 456.0 [M+H]+

[0452] Step 3 involves 4-((7S,10R)-2-bromo-10-(hydroxymethyl)-7,8-dimethyl-5-oxo-7,8,9,10-tetrahydro-5H-pyrazino[1,2-c][1,2,4]triazolo[1,5-a]pyrimidin-6-yl)piperazine-1-carboxylic acid tert-butyl ester (12c-P1) and 4-((7R,10R)-2-bromo-10-(hydroxymethyl)-7,8-dimethyl-5-oxo-7,8,9,10-tetrahydro-5H-pyrazino[1,2-c][1,2,4]triazolo[1,5-a]pyrimidin-6-yl)piperazine-1-carboxylic acid tert-butyl ester (12c-P2).

[0453] Compound 12b (1.8 g, 3.94 mmol) was dissolved in DMF (18 mL), and (R)-(+)-m-nitrobenzenesulfonic acid glycidyl ester (1.33 g, 5.13 mmol) and cesium carbonate (2.57 g, 7.89 mmol) were added. The reaction was carried out at 25 °C for 18 h. After the reaction was completed, the mixture was diluted with water and extracted with ethyl acetate (30 mL × 3). The organic phases were combined, and the crude product was concentrated and purified by column chromatography (PE / EtOAc = 1 / 8) to give compounds 12c-P1 (300 mg) and 12c-P2 (340 mg).

[0454] MS(ESI) m / z 512.1 [M+H] +

[0455] Step 4 (6S,9R)-2-bromo-6-(4-(tert-butyloxycarbonyl)piperazin-1-yl)-7,8-dimethyl-5-oxo-7,8,9,10-tetrahydro-5H-pyrazino[1,2-c][1,2,4]triazolo[1,5-a]pyrimidine-10-carboxylic acid 12d-P1

[0456] Compound 12c-P1 (150 mg, 0.3 mmol) was dissolved in dichloromethane (7.0 mL), and Dess-Martin oxidant (380 mg, 0.9 mmol) was added in portions under ice bath conditions. After reacting for 1 h under ice bath conditions, the reaction was quenched by adding sodium sulfite solution. The mixture was extracted three times with dichloromethane, and the organic phase was concentrated under reduced pressure to obtain the crude product, which was directly used in the next step.

[0457] MS(ESI) m / z 454.0 [M–C4H7] + .

[0458] The crude product was dissolved in a mixed solvent of tetrahydrofuran (10 mL), tert-butanol (10 mL), and water (3 mL). NaH2PO4 (414 mg, 3.0 mmol) and 2-methyl-but-2-ene (2 mL) were added. NaClO2 (135 mg, 1.5 mmol) was added in portions at room temperature. The reaction was carried out at room temperature for 18 h. The crude product was concentrated under reduced pressure and purified by reverse preparative column chromatography to obtain compound 12d-P1 (50 mg).

[0459] MS(ESI) m / z 470.0 [M–C4H7] + .

[0460] Step 5: 4-((7S,10S)-2-bromo-10-((2-chloro-4-(trifluoromethyl)phenyl)carbamoyl)-7,8-dimethyl-5-oxo-7,8,9,10-tetrahydro-5H-pyrazinyl[1,2-c][1,2,4]triazolo[1,5-a]pyrimidin-6-yl)piperazine-1-carboxylic acid tert-butyl ester (12e-P1A) and 4-((7S,10R)-2-bromo-10-((2-chloro-4-(trifluoromethyl)phenyl)carbamoyl)-7,8-dimethyl-5-oxo-7,8,9,10-tetrahydro-5H-pyrazinyl[1,2-c][1,2,4]triazolo[1,5-a]pyrimidin-6-yl)piperazine-1-carboxylic acid tert-butyl ester (12e-P1B).

[0461] Compound 12d-P1 (53 mg, 0.1 mmol) was dissolved in dichloromethane (2 mL), and (chloromethylene)dimethylammonium chloride (26 mg, 0.2 mmol) was added. After stirring at room temperature for 20 minutes, 2,6-dimethylpyridine (53 mg, 0.5 mmol) and 2-chloro-4-(trifluoromethyl)aniline (23 mg, 0.12 mmol) were added. The mixture was reacted at room temperature for 1 hour, and saturated sodium bicarbonate (20 mL) was added. The mixture was extracted three times with dichloromethane, and the organic phase was concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography to give two isomers, 12e-P1A (21 mg) and 12e-P1B (20 mg).

[0462] 12e-P1A:MS(ESI)m / z 703.0[M+H] + .

[0463] 12e-P1B:MS(ESI)m / z 703.1[M+H] + .

[0464] Step 6: 4-((7S,10S)-10-((2-chloro-4-(trifluoromethyl)phenyl)carbamoyl)-2-(4,7-dihydro-5H-thieno[2,3-c]pyran-2-yl)-7,8-dimethyl-5-oxo-7,8,9,10-tetrahydro-5H-pyrazino[1,2-c][1,2,4]triazolo[1,5-a]pyrimidin-6-yl)piperazine-1-carboxylic acid tert-butyl ester (12f-P1A) ) and 4-((7S,10R)-10-((2-chloro-4-(trifluoromethyl)phenyl)carbamoyl)-2-(4,7-dihydro-5H-thieno[2,3-c]pyran-2-yl)-7,8-dimethyl-5-oxo-7,8,9,10-tetrahydro-5H-pyrazino[1,2-c][1,2,4]triazolo[1,5-a]pyrimidin-6-yl)piperazine-1-carboxylic acid tert-butyl ester (12f-P1B)

[0465] Compound 12e-P1A (16 mg, 0.023 mmol) was dissolved in 1,4-dioxane (2 mL) and water (0.4 mL), and intermediate 1c (7.3 mg, 0.026 mmol), sodium carbonate (7.2 mg, 0.069 mmol), and Pd(dppf)Cl2 (1.7 mg, 0.002 mmol) were added. The mixture was purged with nitrogen three times and reacted at 100 °C for 4 hours. After cooling, the reaction solution was diluted with ethyl acetate (25 mL), washed with saturated brine (5 mL), and the organic phase was concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography to obtain compound 12f-P1A (10 mg).

[0466] MS(ESI) m / z 763.2 [M+H] + .

[0467] Compound 12e-P1B (17 mg, 0.023 mmol) was dissolved in 1,4-dioxane (2 mL) and water (0.4 mL), and intermediate 1c (6.3 mg, 0.023 mmol), sodium carbonate (7.6 mg, 0.072 mmol), and Pd(dppf)Cl2 (1.8 mg, 0.002 mmol) were added. The mixture was purged with nitrogen three times and reacted at 100 °C for 3 hours. The crude product was concentrated under reduced pressure. The crude product was purified by column chromatography to give compound 12f-P1B (11 mg).

[0468] MS(ESI) m / z 763.2 [M+H] + .

[0469] Step 7 (7S,10S)-N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(4,7-dihydro-5H-thieno[2,3-c]pyran-2-yl)-7,8-dimethyl-5-oxo-6-(piperazin-1-yl)-7,8,9,10-tetrahydro-5H-pyrazino[1,2-c][1,2,4]triazolo[1,5-a]pyrimidine-10-carboxamide (12g-P1A) (7S,10R)-N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(4,7-dihydro-5H-thieno[2,3-c]pyran-2-yl)-7,8-dimethyl-5-oxo-6-(piperazin-1-yl)-7,8,9,10-tetrahydro-5H-pyrazino[1,2-c][1,2,4]triazolo[1,5-a]pyrimidine-10-carboxamide (12g-P1B)

[0470] Compound 12f-P1A (10 mg, 0.013 mmol) was dissolved in an ethanol solution of HCl (2 mL, 2.0 M) and reacted at room temperature for 3 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure to obtain crude product 12 g-P1A, which was used directly in the next step.

[0471] MS(ESI) m / z 663.1 [M+H] + LC-MS R t = 3.55 min (Analysis Method 5)

[0472] Compound 12f-P1B (11 mg, 0.013 mmol) was dissolved in an ethanol solution of HCl (2 mL, 2.0 M) and reacted at room temperature for 2 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure to obtain crude product 12 g-P1B, which was used directly in the next step.

[0473] MS(ESI) m / z 663.1 [M+H] + LC-MS R t = 3.36 min (Analysis Method 5)

[0474] Step 8 (7S,10S)-6-(4-(5-(benzyloxy)-6-methylpyrimidin-4-carbonyl)piperazin-1-yl)-N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(4,7-dihydro-5H-thieno[2,3-c]pyran-2-yl)-7,8-dimethyl-5-oxo-7,8,9,10-tetrahydro-5H-pyrazino[1,2-c][1,2,4]triazolo[1,5-a]pyrimidin-10-carboxamide (12h-P1A) (7S,10R)-6-(4-(5-(benzyloxy)-6-methylpyrimidin-4-carbonyl)piperazin-1-yl)-N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(4,7-dihydro-5H-thieno[2,3-c]pyran-2-yl)-7,8-dimethyl-5-oxo-7,8,9,10-tetrahydro-5H-pyrazino[1,2-c][1,2,4]triazolo[1,5-a]pyrimidin-10-carboxamide (12h-P1B)

[0475] The crude product 12 g-P1A and 5-(benzyloxy)-6-methylpyrimidine-4-carboxylic acid (3 mg, 0.012 mmol) were dissolved in tetrahydrofuran (2 mL), and DIPEA (23 mg, 0.18 mmol) and T3P (23 mg, 0.036 mmol, 50% in EtOAc) were added dropwise. The reaction was carried out at room temperature for 5 hours. After the reaction was completed, saturated sodium bicarbonate aqueous solution was added, and the mixture was extracted with ethyl acetate. The organic phase was concentrated under reduced pressure to obtain the crude product 12 h-P1A, which was directly used in the next reaction.

[0476] MS(ESI) m / z 889.2 [M+H] + LC-MS R t = 5.10 min (Analysis Method 5)

[0477] The crude product 12 g-P1B and 5-(benzyloxy)-6-methylpyrimidine-4-carboxylic acid (5 mg, 0.021 mmol) were dissolved in tetrahydrofuran (2 mL), and DIPEA (27 mg, 0.21 mmol) and T3P (27 mg, 0.042 mmol, 50% in EtOAc) were added dropwise. The reaction was carried out at room temperature for 3 hours. After the reaction was completed, saturated sodium bicarbonate aqueous solution was added, and the mixture was extracted with ethyl acetate. The organic phase was concentrated under reduced pressure to obtain the crude product 12 h-P1B, which was used directly in the next reaction.

[0478] MS(ESI) m / z 889.2 [M+H] + LC-MS R t = 5.08 min (Analysis Method 5)

[0479] Step 9 (7S,10S)-N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(4,7-dihydro-5H-thieno[2,3-c]pyran-2-yl)-6-(4-(5-hydroxy-6-methylpyrimidin-4-carbonyl)piperazin-1-yl)-7,8-dimethyl-5-oxo-7,8,9,10-tetrahydro-5H-pyrazine[1,2-c][1,2,4]triazolo[1,5-a]pyrimidin-10-carboxamide (12-P1A) (7S,10R)-N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(4,7-dihydro-5H-thieno[2,3-c]pyran-2-yl)-6-(4-(5-hydroxy-6-methylpyrimidin-4-carbonyl)piperazin-1-yl)-7,8-dimethyl-5-oxo-7,8,9,10-tetrahydro-5H-pyrazine[1,2-c][1,2,4]triazolo[1,5-a]pyrimidin-10-carboxamide (12-P1B)

[0480] The crude compound 12h-P1A obtained in the previous step was dissolved in trifluoroacetic acid (2 mL) and reacted at 50 °C for 3 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure to obtain the crude product. The crude product was purified by preparative HPLC to obtain compound 12-P1A (2.0 mg).

[0481] MS(ESI) m / z 799.2 [M+H] + HPLC R t =11.62min; SFC R t =0.85min (Analysis Method 2), SFC 100%ee.

[0482] The crude compound 12h-P1B obtained in the previous step was dissolved in trifluoroacetic acid (2 mL) and reacted at 50 °C for 3 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure to obtain the crude product, which was then purified by preparative HPLC to obtain compound 12-P1B (2.2 mg).

[0483] MS(ESI) m / z 799.2 [M+H] + HPLC R t =11.72min; SFC R t =0.89min (Analysis Method 2), SFC 100%ee.

[0484] Example 13 (7S,10R)-N-(2-chloro-4-(trifluoromethyl)phenyl)-6-(4-(5-hydroxy-6-methylpyrimidin-4-carbonyl)piperazin-1-yl)-7-methyl-5-oxo-2-(5'H,7'H-spiro[cyclopropane-1,4'-thieno[2,3-c]pyran]-2'-yl)-5,7,9,10-tetrahydro-[1,2,4]triazolo[5',1':2,3]pyrimido[6,1-c][1,4]oxazine-10-carboxamide

[0485] The synthesis of compound 13 was performed according to Example 2, with intermediate 2g replaced by intermediate 11d-P2A and intermediate 1c replaced by intermediate 13a, to obtain compound 13.

[0486] MS(ESI) m / z 812.1 [M+H] + SFC R t =3.374 min (Analysis Method 1); ee = 97.84%

[0487] 1 H NMR (400MHz, DMSO-d6) δ10.43(s,1H),8.58(s,1H),8.00(s,1H),7.93(t,J=7.8Hz,1H),7.80-7.72(m,1H ),7.22(s,1H),5.45-5.39(m,1H),5.38-5.27(m,1H),4.86(s,2H),4.57-4.45(m,1H),4.43-4.35(m,1H), 4.24-4.15(m,1H),3.69-3.60(m,1H),3.65(s,2H),3.56-3.45(m,1H),3.28-3.18(m,2H),3.06-2.82(m, 2H),2.74-2.64(m,1H),2.45(s,3H),1.64(dd,J=20.4,6.4Hz,3H),1.02-0.94(m,2H),0.90-0.83(m,2H).

[0488] Example 14 (7S,10R)-N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(3,6-dihydro-2H-pyran-4-yl)-6-(4-(5-hydroxy-6-methylpyrimidin-4-carbonyl)piperazin-1-yl)-7-methyl-5-oxo-5,7,9,10-tetrahydro-[1,2,4]triazolo[5',1':2,3]pyrimido[6,1-c][1,4]oxazine-10-carboxamide

[0489] The synthesis of compound 14 was performed according to Example 2, with intermediate 2g replaced by intermediate 11d-P2A; and intermediate 1c replaced by intermediate 2-(3,6-dihydro-2H-pyran-4-yl)-4,4,5,5-tetramethyl-1,3,2-dioxacyclopentaborane (14a) to obtain compound 14.

[0490] Chiral analysis conditions: ChiralCEL OJ, 100×3mm ID, 3μm; Mobile phase: A for CO2 and B for MeOH (0.1% DEA); Gradient: B 15%; Flow rate: 2.0mL / min; Column temperature: 35℃

[0491] MS(ESI) m / z 730.2 [M+H] + SFC R t =3.069min; ee=100%.

[0492] 1 H NMR (400MHz, DMSO-d6) δ10.40(s,1H),8.56(d,J=2.1Hz,1H),7.98(s,1H),7.93(t,J=7.9Hz,1H),7.78- 7.72(m,1H),6.82(s,1H),5.41-5.37(m,1H),5.31(dd,J=17.4,6.4Hz,1H),4.57-4.43(m,1H),4.42-4. 33(m,1H),4.28-4.22(m,2H),4.22-4.14(m,1H),3.87-3.75(m,2H),3.71-3.57(m,1H),3.55-3.42(m,2 H),3.27-3.14(m,4H),3.07-2.75(m,2H),2.74-2.63(m,1H),2.44(s,3H),1.63(dd,J=20.2,6.4Hz,3H).

[0493] Example 15 (7S,10R)-N-(2-chloro-4-(trifluoromethyl)phenyl)-6-(4-(5-hydroxy-6-methylpyrimidin-4-carbonyl)piperazin-1-yl)-7-methyl-5-oxo-2-(4'H,6'H-spiro[cyclopropane-1,7'-thieno[3,2-c]pyran]-2'-yl)-5,7,9,10-tetrahydro-[1,2,4]triazolo[5',1':2,3]pyrimido[6,1-c][1,4]oxazine-10-carboxamide

[0494] The synthesis of compound 15 was performed according to Example 2, in which intermediate 2g was replaced with intermediate 11d-P2A and intermediate 1c was replaced with intermediate 15a to obtain compound 15.

[0495] MS(ESI) m / z 812.1 [M+H] + SFC R t =5.554 min (Analysis Method 1), ee = 98.83%.

[0496] 1 H NMR (400MHz, DMSO-d6) δ10.40(s,1H),8.56(s,1H),8.00(s,1H),7.94(t,J=7.9Hz,1H),7.80-7.73(m,1H ),7.45(s,1H),5.41-5.36(m,1H),5.35-5.27(m,1H),4.73(s,2H),4.58-4.44(m,1H),4.43-4.35(m,1H), 4.23-4.11(m,1H),3.68(s,2H),3.67-3.59(m,1H),3.55-3.42(m,1H),3.25-3.13(m,2H),3.05-2.79(m, 2H),2.74-2.62(m,1H),2.44(s,3H),1.63(dd,J=20.3,6.4Hz,3H),1.10-1.05(m,2H),0.97-0.90(m,2H).

[0497] Example 16 (7S,10R)-N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(3-fluoro-2,6-dimethylpyridin-4-yl)-6-(4-(5-hydroxy-6-methylpyrimidin-4-carbonyl)piperazin-1-yl)-7-methyl-5-oxo-5,7,9,10-tetrahydro-[1,2,4]triazolo[5,1':2,3]pyrimidin[6,1-c][1,4]oxazine-10-carboxamide

[0498] The synthesis of compound 16 was performed according to Example 2, with intermediate 2g replaced by intermediate 11d-P2A; the synthesis of intermediate 3-fluoro-2,6-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxacyclopentaborane-2-yl)pyridine (16a) was performed according to patent WO2021224636A1. Replacing intermediate 1c with intermediate 16a yielded compound 16.

[0499] MS(ESI) m / z 771.2 [M+H] + SFC R t=0.773min (Analysis Method 1); ee = 98.59%.

[0500] 1 H NMR (400MHz, DMSO-d6) δ10.45(s,1H),10.25(s,1H),8.59(d,J=2.1Hz,1H),8.00(s,1H),7.94(t,J=8.2Hz, 1H),7.77(d,J=8.3Hz,1H),7.65(d,J=4.8Hz,1H),5.46(s,1H),5.36(dd,J=17.1,6.5Hz,1H),4.58–4.39(m, 2H),4.21(t,J=8.8Hz,1H),3.67(d,J=11.9Hz,1H),3.57–3.47(m,1H),3.24(s,2H),2.96(ddd,J=37.6,24.1 ,11.6Hz,2H),2.72(t,J=11.5Hz,1H),2.49(s,3H),2.48(s,3H),2.45(s,3H),1.66(dd,J=20.2,6.3Hz,3H).

[0501] Example 17 (7S,10R)-N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(6,7-dihydro-4H-thieno[3,2-c]pyran-2-yl)-6-(4-(5-hydroxy-6-methylpyrimidin-4-carbonyl)piperazin-1-yl)-7-methyl-5-oxo-5,7,9,10-tetrahydro-[1,2,4]triazolo[5',1':2,3]pyrimidin[6,1-c][1,4]oxazine-10-carboxamide

[0502] The synthesis of compound 17 was performed according to Example 2, with intermediate 2g replaced by intermediate 11d-P2A and intermediate 1c replaced by intermediate 17a, to obtain compound 20.

[0503] MS(ESI) m / z 786.1 [M+H] + SFC R t =5.208 min (Analysis Method 1); ee = 97.99%.

[0504] 1H NMR (400MHz, DMSO-d6) δ10.41(s,1H),8.58(d,J=2.2Hz,1H),7.99(s,1H),7.93(t,J=8.1Hz,1H),7. 76(d,J=8.5Hz,1H),7.46(s,1H),5.40(d,J=2.3Hz,1H),5.32(dd,J=17.4,6.5Hz,1H),4.64(s,2H),4 .58–4.36(m,2H),4.18(t,J=9.3Hz,1H),3.90(t,J=5.5Hz,2H),3.66(d,J=10.1Hz,1H),3.55–3.45(m ,1H),3.22(s,2H),3.05–2.82(m,4H),2.74–2.64(m,1H),2.44(s,3H),1.64(dd,J=20.2,6.3Hz,3H).

[0505] Example 18 (7S,10R)-N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(3-fluoro-4-(2-hydroxypropyl-2-yl)phenyl)-6-(4-(5-hydroxy-6-methylpyrimidin-4-carbonyl)piperazin-1-yl)-7-methyl-5-oxo-5,7,9,10-tetrahydro-[1,2,4]triazolo[5',1':2,3]pyrimido[6,1-c][1,4]oxazine-10-carboxamide

[0506] Step 1: 4-((7S,10R)-10-((2-chloro-4-(trifluoromethyl)phenyl)carbamoyl)-2-(3-fluoro-4-(2-hydroxypropyl-2-yl)phenyl)-7-methyl-5-oxo-5,7,9,10-tetrahydro-[1,2,4]triazolo[5',1':2,3]pyrimidino[6,1-c][1,4]oxazin-6-yl)piperazine-1-carboxylic acid tert-butyl ester 18b

[0507] The synthesis of intermediate 18a was performed according to WO2011145035A1. Compound 18a (15 mg, 57.4 μmol) was dissolved in 1,4-dioxane (3 mL) and water (0.3 mL), followed by compound 11d-P2A (36 mg, 52 μmol), potassium phosphate (22 mg, 104 μmol), and Pd(dppf)Cl2 (7 mg, 10 μmol). The mixture was purged with nitrogen three times and then reacted at 90 °C for 5 hours. After cooling, the reaction solution was diluted with ethyl acetate (20 mL) and washed three times with saturated brine (10 mL). The organic phase was concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (DCM / MeOH = 15:1) to obtain compound 18b (37 mg).

[0508] MS(ESI) m / z 763.1 [M+H] + .

[0509] Step 2 (7S,10R)-N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(3-fluoro-4-(2-hydroxypropyl-2-yl)phenyl)-7-methyl-5-oxo-6-(piperazin-1-yl)-5,7,9,10-tetrahydro-[1,2,4]triazolo[5',1':2,3]pyrimidino[6,1-c][1,4]oxazine-10-carboxamide18c

[0510] Compound 18b (37 mg, 0.11 mmol) was dissolved in dichloromethane (2 mL), and hydrogen chloride-ethanol (2 mL, 2 M HCl in EtOH) was added. The reaction was carried out at room temperature for 3 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure to obtain crude product 18c, which was used directly in the next step.

[0511] MS(ESI) m / z 664.2 [M+H] + .

[0512] Step 3 (7S,10R)-N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(3-fluoro-4-(2-hydroxypropyl-2-yl)phenyl)-6-(4-(5-hydroxy-6-methylpyrimidin-4-carbonyl)piperazin-1-yl)-7-methyl-5-oxo-5,7,9,10-tetrahydro-[1,2,4]triazolo[5',1':2,3]pyrimido[6,1-c][1,4]oxazine-10-carboxamide 18

[0513] Crude product 18c (34 mg, 51.3 μmol) and 5-hydroxy-6-methylpyrimidine-4-carboxylic acid (11.5 mg, 61.6 μmol) were dissolved in acetonitrile (2 mL), followed by dropwise addition of N-methylimidazole (14.7 mg, 180 μmol) and TCFH (17.2 mg, 61.6 μmol). The reaction was carried out at room temperature for 3 hours. After the reaction was completed, saturated sodium bicarbonate aqueous solution (10 mL) was added, followed by extraction with ethyl acetate (10 mL). The organic phase was concentrated under reduced pressure to obtain the crude product, which was purified by preparative HPLC to obtain compound 18 (5.3 mg).

[0514] MS(ESI) m / z 800.2 [M+H] + SFC R t =1.754 min (Analysis Method 1); ee = 99.71%.

[0515] 1H NMR(400MHz,DMSO-d6)δ10.48(brs.,1H),8.46(brs.,1H),8.00(s,1H),7.96-7.84(m,2H),7.83-7.63(m,3H),5.50-5.37(m,3H),4.61-4.35(m .,2H),4.21(brs.,1H),3.75-3.60(m,2H),3.05-2.60(m,4H),2.41(s,3 H),2.02-1.95(m,1H),1.73-1.58(m,2H),1.51(s,6H),1.45(brs.,1H).

[0516] Example 19 (7S,10R)-N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(2,3-dihydrobenzofuran-5-yl)-6-(4-(5-hydroxy-6-methylpyrimidin-4-carbonyl)piperazin-1-yl)-7-methyl-5-oxo-5,7,9,10-tetrahydro-[1,2,4]triazolo[5',1':2,3]pyrimidin[6,1-c][1,4]oxazine-10-carboxamide

[0517] The synthesis of compound 19 was performed according to Example 2, by replacing intermediate 2g with intermediate 11d-P2A and intermediate 1c with intermediate 19a, to obtain compound 19.

[0518] MS(ESI) m / z 766.2 [M+H] + SFC R t = 9.462 min (Analysis Method 1); ee = 99.34%.

[0519] 1 H NMR(400MHz,DMSO-d6)δ10.48(brs,1H),8.48(s,1H),8.34(s,1H),8.01-7.90(m,3H),7.86 (d,J=8.3Hz,1H),7.75(d,J=8.0Hz,1H),6.88(d,J=8.4Hz,1H),5.45(s,1H),5.39-5.25(m, 1H),4.66-4.45(m,3H),4.44-4.34(m,1H),4.27-4.14(m,1H),3.73-3.60(m,1H),3.29-3.2 3(m,4H),3.06-2.80(m,3H),2.77-2.61(m,1H),2.42(s,3H),1.65(dd,J=6.3,19.6Hz,3H).

[0520] Example 20 (7S,10R)-N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(1-cyclopropyl-1,2,3,6-tetrahydropyridin-4-yl)-6-(4-(5-hydroxy-6-methylpyrimidin-4-carbonyl)piperazin-1-yl)-7-methyl-5-oxo-5,7,9,10-tetrahydro-[1,2,4]triazolo[5',1':2,3]pyrimidin[6,1-c][1,4]oxazine-10-carboxamide

[0521] The synthesis of compound 20 was performed according to Example 2, in which intermediate 2g was replaced with intermediate 11d-P2A and intermediate 1c was replaced with intermediate 20a to obtain compound 20.

[0522] MS(ESI) m / z 769.2 [M+H] + SFC R t =0.884min (Analysis Method 1); ee = 98.20%.

[0523] 1 H NMR (400MHz, DMSO-d6) δ10.39(s,1H),8.58(d,J=2.0Hz,1H),8.14(s,1H),7.94(dd,J=19.5,11.3Hz,2H),7.75(d,J= 7.7Hz,1H),6.76(s,1H),5.40–5.24(m,2H),4.50(dd,J=21.2,12.7Hz,1H),4.38(d,J=13.3Hz,1H),4.18(t,J=9.2Hz ,1H),3.65(d,J=10.4Hz,1H),3.47(d,J=10.9Hz,1H),3.25(t,J=12.5Hz,7H),3.05–2.82(m,2H),2.82–2.75(m,2H), 2.67(dd,J=13.4,11.5Hz,1H),2.44(s,3H),1.62(dd,J=20.2,6.3Hz,3H),0.51–0.40(m,2H),0.36(d,J=2.8Hz,2H).

[0524] Example 21 (7S,10R)-N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(2-(difluoromethyl)pyridin-4-yl)-6-(4-(5-hydroxy-6-methylpyrimidin-4-carbonyl)piperazin-1-yl)-7-methyl-5-oxo-5,7,9,10-tetrahydro-[1,2,4]triazolo[5',1':2,3]pyrimidin[6,1-c][1,4]oxazine-10-carboxamide

[0525] The synthesis of compound 21 was carried out in reference to Example 2, by replacing intermediate 2g with intermediate 11d-P2A and intermediate 1c with intermediate 21a, to obtain compound 21.

[0526] MS(ESI) m / z 775.2 [M+H] + SFC R t =0.745min (Analysis Method 1); ee = 97.83%.

[0527] 1 H NMR (400MHz, DMSO-d6) δ10.48(s,1H),8.88(d,J=5.0Hz,1H),8.57(d,J=2.9Hz,1H),8.25(s,1H),8.19(d,J=4.9Hz, 1H),8.00(s,1H),7.92(d,J=7.9Hz,1H),7.75(d,J=8.6Hz,1H),7.09(t,J=54.8Hz,1H),5.49(d,J=2.4Hz,1H),5.43 –5.30(m,1H),4.61–4.39(m,2H),4.21(t,J=8.9Hz,1H),3.74–3.61(m,1H),3.49(d,J=15.3Hz,1H),3.27–3.19(m,2 H), 2.96 (ddd, J=37.9, 24.1, 11.8Hz, 2H), 2.71 (dd, J=21.6, 8.1Hz, 1H), 2.44 (s, 3H), 1.66 (dd, J=20.1, 6.4Hz, 3H).

[0528] Example 22 (7S,10R)-N-(2-chloro-4-(trifluoromethyl)phenyl)-6-(4-(5-hydroxy-6-methylpyrimidin-4-carbonyl)piperazin-1-yl)-7-methyl-5-oxo-2-(2,3,6,7-tetrahydrooxetane-4-yl)-5,7,9,10-tetrahydro-[1,2,4]triazolo[5',1':2,3]pyrimidin[6,1-c][1,4]oxazine-10-carboxamide

[0529] The synthesis of compound 22 was performed according to Example 2, with intermediate 2g replaced by intermediate 11d-P2A and intermediate 1c replaced by intermediate 22a, to obtain compound 22.

[0530] MS(ESI)m / z 744.2[M+H]+; SFC R t =0.910min (Analysis Method 1); ee = 96.23%.

[0531] 1 H NMR (400MHz, DMSO-d6) δ10.40(s,1H),8.56(d,J=2.1Hz,1H),7.95(dd,J=18.1,10.1Hz,2 H),7.76(d,J=8.7Hz,1H),7.11(t,J=6.1Hz,1H),5.42-5.24(m,2H),4.57-4.44(m,1H),4 .38(d,J=13.1Hz,1H),4.19(t,J=8.6Hz,1H),3.70-3.60(m,5H),3.48(s,1H),3.22(s,4H ),3.03-2.80(m,4H),2.67(t,J=11.0Hz,1H),2.44(s,3H),1.62(dd,J=20.2,6.3Hz,3H).

[0532] Example 23 (7S,10R)-N-(2-chloro-4-(trifluoromethyl)phenyl)-6-(4-(5-hydroxy-6-methylpyrimidin-4-carbonyl)piperazin-1-yl)-7-methyl-5-oxo-2-(5-(2,2-trifluoroethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl)-5,7,9,10-tetrahydro-[1,2,4]triazolo[5',1':2,3]pyrimidinyl[6,1-c][1,4]oxazine-10-carboxamide

[0533] The synthesis of compound 23 was performed according to Example 2, with intermediate 2g replaced by intermediate 11d-P2A and intermediate 1c replaced by intermediate 23a, to obtain compound 23.

[0534] MS(ESI) m / z 867.2 [M+H] + SFC R t =5.073 min (Analysis Method 1); ee = 97.74%.

[0535] 1H NMR(400MHz,DMSO-d6)δ10.44(brs,1H),8.51-8.30(m,1H),8.12-7.85(m,2 H),7.76(d,J=7.6Hz,1H),7.46(s,1H),5.40(s,1H),5.37-5.24(m,1H),4.59 -4.34(m,2H),4.24-4.10(m,1H),3.82-3.71(m,2H),3.70-3.61(m,3H),3.04 -2.78(m,8H),2.74-2.63(m,2H),2.41(s,3H),1.64(dd,J=6.3,19.4Hz,3H).

[0536] Example 24 Synthesis of compounds 24-P2A and 24-P2B

[0537] Step 1 (2R)-4-(10-((2-chloro-4-(trifluoromethyl)phenyl)carbamoyl)-2-(4,7-dihydro-5H-thieno[2,3-c]pyran-2-yl)-7-methyl-5-oxo-5,7,9,10-tetrahydro-[1,2,4]triazolo[5',1':2,3]pyrimido[6,1-c][1,4]oxazin-6-yl)-2-methylpiperazine-1-carboxylate 24b

[0538] Compound 24a (1 g, 1.41 mmol), compound 23a (738 mg, 2.12 mmol), Pd(dppf)Cl2 (103 mg, 141 μmol), and K2CO3 (486 mg, 3.52 mmol) were dissolved in 1,4-dioxane (20 mL) and H2O (4 mL) under nitrogen protection and reacted at 80 °C for 12 h. After the reaction was complete, the reaction solution was diluted with water, extracted with ethyl acetate, the organic phase was washed once with saturated brine, dried over anhydrous sodium sulfate, concentrated, and the crude product was purified by flash separation (PE:EA = 1:2) to give compound 24b (700 mg), a white solid.

[0539] MS(ESI) m / z 845.2 [M+H] +

[0540] Step 2: N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(4,7-dihydro-5H-thieno[2,3-c]pyran-2-yl)-7-methyl-6-((R)-3-methylpiperazin-1-yl)-5-oxo-5,7,9,10-tetrahydro-[1,2,4]triazolo[5',1':2,3]pyrimidine[6,1-c][1,4]oxazine-10-carboxamide 24c

[0541] Compound 24b (700 mg, 828 μmol) was dissolved in HCl / EtOH (2 M, 10 mL) and reacted at room temperature for 1 h. After the reaction was complete, the pH of the reaction solution was adjusted to 8 with saturated NaHCO3 aqueous solution, extracted with ethyl acetate, and the collected organic phase was washed once with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give compound 24c (580 mg), a pale yellow liquid.

[0542] MS(ESI) m / z 745.2 [M+H] +

[0543] Step 3: 6-((R)-4-(5-(benzyloxy)-6-methylpyrimidin-4-carbonyl)-3-methylpiperazin-1-yl)-N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(4,7-dihydro-5H-thieno[2,3-c]pyran-2-yl)-7-methyl-5-oxo-5,7,9,10-tetrahydro-[1,2,4]triazolo[5',1':2,3]pyrimido[6,1-c][1,4]oxazine-10-carboxamide 24d

[0544] Compound 24c (560 mg, 751 μmol) and compound 1f (366 mg, 1.50 mmol) were dissolved in DMF (10 mL), and HATU (855 mg, 2.25 mmol) and DIEA (774 mg, 6.01 mmol) were added. The mixture was reacted at room temperature for 2 h. After the reaction was complete, water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic phase was washed once with saturated brine, dried over anhydrous sodium sulfate, concentrated, and the crude product was purified by flash separation (PE:EA = 5:95) to give compound 24d (500 mg), a white solid.

[0545] MS(ESI) m / z 971.2 [M+H] +

[0546] The fourth step involves the synthesis of compounds 24-P2A and 24-P2B.

[0547] Compound 24d (500 mg, 514 μmol) was dissolved in TFA (10 mL) and reacted at 50 °C for 2 h. After the reaction was complete, the pH of the reaction solution was adjusted to 8 with saturated NaHCO3 aqueous solution, extracted with ethyl acetate, and the collected organic phase was washed once with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain the crude product. The crude product was first separated by reverse-phase preparative analysis to obtain two peaks, namely compounds 24-P1 (20 mg) and 24-P2 (300 mg).

[0548] Compound 24-P1: LC-MS retention time 2.260 min (analytical method 3), MS (ESI) m / z 881.1 [M+H] + .

[0549] Compound 24-P2: LC-MS retention time 2.332 min (analytical method 3), MS (ESI) m / z 881.1 [M+H] + .

[0550] Compound 24-P2 was further separated by SFC to obtain compounds 24-P2A (153.9 mg) and 24-P2B (81.1 mg), which were white solids.

[0551] Chiral preparation conditions: IC, 10μm, 30*250mm; Mobile phase A: acetonitrile + 0.2% formic acid, Mobile phase B: ethanol + 0.2% formic acid; Flow rate: 25mL / min; Column temperature: room temperature; Isocratic elution program: Mobile phase A: Mobile phase B = 85:15 (V / V).

[0552] Chiral analysis conditions: IC, 5μm, 4.6mm*250mm; Mobile phase A: acetonitrile + 0.2% formic acid, Mobile phase B: ethanol + 0.2% formic acid; Flow rate: 1.0mL / min; Column temperature: 30℃; Isocratic elution program: Mobile phase A: Mobile phase B = 85:15 (V / V).

[0553] Compound 24-P2A: MS (ESI) m / z 881.1 [M+H] + SFC 100% ee. Retention time 5.423 min

[0554] 1 H NMR (400MHz, DMSO-d6) δ10.45(s,1H),8.56(d,J=9.4Hz,1H),8.00(s,1H),7.94(t,J=8.3Hz,1H),7.77(d, J=8.2Hz,1H),7.46(d,J=2.0Hz,1H),5.58–5.45(m,1H),5.42(s,1H),4.49(dd,J=97.0,83.7Hz,2H),4.27– 4.15(m,1H),3.91(t,J=8.7Hz,1H),3.77(s,2H),3.55–3.37(m,3H),3.30–3.26(m,2H),2.98(d,J=5.5Hz, 2H),2.90–2.60(m,4H),2.45(d,J=6.9Hz,3H),1.61(dd,J=19.2,6.4Hz,3H),1.41(dd,J=14.0,6.8Hz,3H).

[0555] Compound 24-P2B: MS (ESI) m / z 881.1 [M+H] + SFC 99.44% ee. Retention time 7.202 min

[0556] 1 H NMR (400MHz, DMSO-d6) δ10.42(s,1H),8.56(d,J=3.0Hz,1H),7.95(dd,J=21.1,12.1Hz,2H),7.77(d,J=2.5H z,1H),7.46(s,1H),5.41(d,J=2.8Hz,1H),5.25–5.11(m,1H),4.50(dd,J=82.7,72.1Hz,2H),4.19(t,J=9.7H z,1H),3.79–3.62(m,3H),3.58–3.49(m,1H),3.39(d,J=10.3Hz,3H),3.16(d,J=13.1Hz,1H),2.98(d,J=5.5H z,2H),2.90–2.62(m,4H),2.44(d,J=2.3Hz,3H),1.68(dd,J=20.2,6.4Hz,3H),1.37(dd,J=10.1,6.8Hz,3H).

[0557] Example 25 Synthesis of compounds 25-P2A and 25-P2B

[0558] Step 1 (2S,6S)-4-(2-bromo-5-(1-(epoxy-2-ylmethoxy)ethyl)-7-oxo-4-((2-(trimethylsilyl)ethoxy)methyl)-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-2,6-dimethylpiperazine-1-carboxylic acid tert-butyl ester 25b

[0559] Compound 25a (4.5 g, 7.48 mmol) and (R)-(+)-m-nitrobenzenesulfonic acid glycidyl ester (2.13 g, 8.23 ​​mmol) were dissolved in DMF (200 mL). t-BuOK (14.9 mL, 1 M, 14.9 mmol) was slowly added at 0 °C, and the reaction was carried out at 0 °C for 1 h. After the reaction was completed, water was slowly added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic phase was washed once with saturated brine, dried over anhydrous sodium sulfate, concentrated, and the crude product was purified by flash separation (PE:EA = 1:1) to obtain compound 25b (2 g), a pale yellow oil.

[0560] MS(ESI) m / z 659.2 [M+H] +

[0561] Step 2: (2S,6S)-4-(2-bromo-10-(hydroxymethyl)-7-methyl-5-oxo-5,7,9,10-tetrahydro-[1,2,4]triazolo[5',1':2,3]pyrimidino[6,1-c][1,4]oxazin-6-yl)-2,6-dimethylpiperazine-1-tert-butyl carboxylate 25c

[0562] Compound 25b (2 g, 3.04 mmol) was dissolved in DMF (30 mL) and H2O (3 mL), and KF (1.05 g, 18.2 mmol) was added. The reaction was carried out at 70 °C for 12 h. After the reaction was completed, the mixture was cooled to room temperature, and the reaction solution was quenched slowly with water. The mixture was extracted three times with ethyl acetate, and the collected organic phase was washed once with saturated brine, dried over anhydrous sodium sulfate, concentrated, and the crude product was purified by flash separation (PE:EA = 1:5) to obtain compound 25c (1.4 g), a pale yellow oil.

[0563] MS(ESI) m / z 529.1 [M+H] +

[0564] Step 3 (2S,6S)-4-(2-bromo-10-formyl-7-methyl-5-oxo-5,7,9,10-tetrahydro-[1,2,4]triazolo[5',1':2,3]pyrimidino[6,1-c][1,4]oxazin-6-yl)-2,6-dimethylpiperazine-1-carboxylic acid tert-butyl ester 25d

[0565] Compound 25c (1.1 g, 2.08 mmol) was dissolved in DCM (15 mL), and DMP (1.76 g, 4.16 mmol) was added. The reaction was carried out at room temperature for 1 h. After the reaction was complete, water was added to the reaction solution, and the mixture was extracted with DCM. The collected organic phase was washed once with saturated brine, dried over anhydrous sodium sulfate, concentrated, and the crude product was purified by flash separation (DCM:MeOH = 20:1) to give compound 25d (700 mg), a pale yellow oil.

[0566] MS(ESI) m / z 527.1 [M+H] +

[0567] Step 4: 2-Bromo-6-((3S,5S)-4-(tert-Butoxycarbonyl)-3,5-dimethylpiperazin-1-yl)-7-methyl-5-oxo-5,7,9,10-tetrahydro-[1,2,4]triazolo[5',1':2,3]pyrimido[6,1-c][1,4]oxazine-10-carboxylic acid 25e

[0568] Compound 25d (700 mg, 1.33 mmol) was dissolved in THF (7 mL), t-BuOH (7 mL), and H2O (3.5 mL). 2-Methylbut-2-ene (1.86 g, 26.6 mmol) and NaH2PO4 (1.59 g, 13.3 mmol) were added. NaClO2 (359 mg, 3.99 mmol) was slowly added while stirring at 0 °C, and the reaction was carried out at 0 °C for 1 h. After the reaction was complete, the mixture was extracted with ethyl acetate, washed once with saturated brine, dried over anhydrous sodium sulfate, concentrated, and the crude product was purified by flash separation (DCM:MeOH = 20:1) to give compound 25e (380 mg), a pale yellow oil.

[0569] MS(ESI) m / z 541.1 [M+H] +

[0570] Step 5 (2S,6S)-4-(2-bromo-10-(2-chloro-4-(trifluoromethyl)phenyl)carbamoyl)-7-methyl-5-oxo-5,7,9,10-tetrahydro-[1,2,4]triazolo[5',1':2,3]pyrimidino[6,1-c][1,4]oxazin-6-yl)-2,6-dimethylpiperazine-1-carboxylic acid tert-butyl ester 25f

[0571] Compound 25e (380 mg, 702 μmol) and 2-chloro-4-(trifluoromethyl)aniline (165 mg, 842 μmol) were dissolved in DMF (30 mL), and T3P (2.67 g, 4.21 mmol) and TEA (850 mg, 8.42 mmol) were added. The mixture was reacted at 70 °C for 2 h. After the reaction was complete, water was slowly added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic phase was washed once with saturated brine, dried over anhydrous sodium sulfate, concentrated, and the crude product was purified by flash separation (PE:EA = 1:1) to give compound 25f (200 mg), a white solid.

[0572] MS(ESI) m / z 720.2 [M+H] +

[0573] Step 6: 25g of (2S,6S)-4-(10-((2-chloro-4-(trifluoromethyl)phenyl)carbamoyl)-2-(4,7-dihydro-5H-thieno[2,3-c]pyran-2-yl)-7-methyl-5-oxo-5,7,9,10-tetrahydro-[1,2,4]triazolo[5',1':2,3]pyrimidino[6,1-c][1,4]oxazin-6-yl)-2,6-dimethylpiperazine-1-carboxylic acid tert-butyl ester

[0574] Compound 25f (200 mg, 278 μmol), compound 1c (111 mg, 417 μmol), Pd(dppf)Cl2 (20.4 mg, 27.8 μmol), and K2CO3 (96.1 mg, 696 μmol) were dissolved in 1,4-dioxane (5 mL) and H2O (1 mL), and the reaction was carried out at 80 °C for 12 h under nitrogen protection. After the reaction was completed, water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The collected organic phase was washed once with saturated brine, dried over anhydrous sodium sulfate, concentrated, and the crude product was purified by flash separation (PE:EA = 1:3) to obtain compound 25 g (120 mg) as a white solid.

[0575] MS(ESI) m / z 778.2 [M+H] +

[0576] Step 7: N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(4,7-dihydro-5H-thieno[2,3-c]pyran-2-yl)-6-((3S,5S)-3,5-dimethylpiperazin-1-yl)-7-methyl-5-oxo-5,7,9,10-tetrahydro-[1,2,4]triazolo[5',1':2,3]pyrimido[6,1-c][1,4]oxazine-10-carboxamide 25h

[0577] 25 g (120 mg, 154 μmol) of the compound was dissolved in HCl / EtOH (2 M, 3 mL) and reacted at room temperature for 1 h. After the reaction was complete, the pH of the reaction solution was adjusted to 8 with saturated NaHCO3 aqueous solution, extracted with ethyl acetate, the collected organic phase was washed once with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give compound 25 h (100 mg), a pale yellow liquid.

[0578] MS(ESI) m / z 678.2 [M+H] +

[0579] Step 8: 6-((3S,5S)-4-(5-(benzyloxy)-6-methylpyrimidin-4-carbonyl)-3,5-dimethylpiperazin-1-yl)-N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(4,7-dihydro-5H-thieno[2,3-c]pyran-2-yl)-7-methyl-5-oxo-5,7,9,10-tetrahydro-[1,2,4]triazolo[5',1':2,3]pyrimido[6,1-c][1,4]oxazine-10-carboxamide 25i

[0580] Compounds 25h (100 mg, 147 μmol) and 1f (71.7 mg, 294 μmol) were dissolved in DMF (2 mL), and HATU (167 mg, 441 μmol) and DIEA (150 mg, 1.17 mmol) were added. The mixture was reacted at room temperature for 2 h. After the reaction was complete, water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic phase was washed once with saturated brine, dried over anhydrous sodium sulfate, concentrated, and the crude product was purified by flash separation (PE:EA = 5:95) to give compound 25i (80 mg) as a white solid.

[0581] MS(ESI) m / z 904.2 [M+H] +

[0582] Step 9: Synthesis of compounds 25-P2A and 25-P2B

[0583] Compound 25i (70 mg, 77.4 μmol) was dissolved in TFA (2 mL) and reacted at 50 °C for 2 h. After the reaction was complete, the reaction solution was concentrated under reduced pressure, and saturated NaHCO3 aqueous solution was added to adjust the pH to 8. The solution was extracted with DCM, the organic phase was washed once with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain the crude product. The crude product was then separated by Prep HPLC, yielding two peaks: compound 25-P1 (3 mg) and compound 25-P2 (30 mg).

[0584] Compound 25-P1: MS (ESI) m / z 814.1 [M+H] + LC-MS retention time: 2.152 min (Analysis Method 3)

[0585] Compound 25-P2: MS (ESI) m / z 814.1 [M+H] + LC-MS retention time: 2.237 min (Analysis Method 3)

[0586] Compound 25-P2 was purified by chiral separation to give compounds 25-P2A (16.1 mg) and 25-P2B (3.2 mg), which were white solids.

[0587] Chiral preparation conditions: IC, 10μm, 30*250mm; Mobile phase A: acetonitrile + 0.2% formic acid, Mobile phase B: ethanol + 0.2% formic acid; Flow rate: 25mL / min; Column temperature: room temperature; Isocratic elution program: Mobile phase A: Mobile phase B = 70:30 (V / V).

[0588] Chiral analysis conditions: IC, 5μm, 4.6mm*250mm; Mobile phase A: acetonitrile + 0.2% formic acid, Mobile phase B: ethanol + 0.2% formic acid; Flow rate: 1.0mL / min; Column temperature: 30℃; Isocratic elution program: Mobile phase A: Mobile phase B = 70:30 (V / V).

[0589] Compound 25-P2A: MS (ESI) m / z 814.1 [M+H] + SFC 100%ee. Retention time 4.815 min.

[0590] 1 H NMR (400MHz, DMSO-d6) δ10.42(s,1H),8.56(s,1H),7.97(dd,J=17.6,5.0Hz,2H),7.76(dd,J=8.6,1.6Hz ,1H),7.51(s,1H),5.42(t,J=2.8Hz,1H),5.15(q,J=6.4Hz,1H),4.77(s,2H),4.47–4.35(m,1H),4.19(dd ,J=12.3,3.3Hz,1H),3.89(dd,J=16.2,10.8Hz,3H),3.64(d,J=8.7Hz,1H),3.50(s,1H),3.30–3.23(m,1 H),2.92(d,J=9.4Hz,1H),2.77–2.57(m,3H),2.44(s,3H),1.66(d,J=6.4Hz,3H),1.34(t,J=14.1Hz,6H).

[0591] Compound 25-P2B: MS (ESI) m / z 814.1 [M+H] + SFC 99.18% ee. Retention time 6.424 min

[0592] 1H NMR(400MHz,DMSO-d6)δ10.47(s,1H),8.33(s,1H),8.03–7.89(m,2H),7.76(d,J=8.6Hz,1H),7 .49(d,J=9.8Hz,1H),5.35(dd,J=29.5,9.9Hz,2H),4.77(s,2H),4.42(d,J=11.1Hz,1H),4.17(d d,J=38.7,29.8Hz,3H),3.88(t,J=5.4Hz,2H),3.49(d,J=11.2Hz,1H),3.14–2.98(m,2H),2.68( dd,J=9.0,7.2Hz,3H),2.36(dd,J=22.9,21.1Hz,3H),1.58(d,J=5.9Hz,3H),1.52–1.31(m,6H).

[0593] Example 26 Preparation of compounds 26-P2A and 26-P2B

[0594] The synthesis of compound 26 was performed according to Example 24, with compound 26a replacing compound 24a. Compound 26 was separated by Prep HPLC to yield two peaks: compound 26-P1 (24 mg) and 26-P2 (140 mg).

[0595] Compound 25-P1: MS (ESI) m / z 800.1 [M+H] + LC-MS retention time: 2.079 min (Analysis Method 3)

[0596] Compound 25-P2: MS (ESI) m / z 800.1 [M+H] + LC-MS retention time: 2.142 min (Analysis Method 3)

[0597] Compound 26-P2 was isolated by chiral preparation to yield compounds 26-P2A and 26-P2B. The chiral preparation and chiral analysis conditions were the same as in Example 25.

[0598] Compound 26-P2A: MS (ESI) m / z 800.1 [M+H] + SFC 100%ee. Retention time 6.174 min.

[0599] 1H NMR (400MHz, DMSO-d6) δ10.42(s,1H),8.57(d,J=2.5Hz,1H),7.95(dd,J=20.1,11.1Hz,2H),7.82–7.71(m, 1H),7.52(s,1H),5.41(d,J=2.9Hz,1H),5.18(dq,J=19.4,6.3Hz,1H),4.77(s,2H),4.41(dd,J=10.0,6.6H z,1H),4.26–4.13(m,1H),3.88(t,J=5.5Hz,2H),3.77–3.47(m,3H),3.44–3.35(m,1H),3.15(t,J=11.1Hz, 2H),2.91–2.53(m,4H),2.45(d,J=2.2Hz,3H),1.68(dd,J=20.0,6.4Hz,3H),1.37(dd,J=10.6,6.8Hz,3H).

[0600] Compound 26-P2B: MS (ESI) m / z 800.1 [M+H] + SFC 99.14%ee. Retention time 8.129 min.

[0601] 1 H NMR (400MHz, DMSO-d6) δ10.47(s,1H),8.55(d,J=10.6Hz,1H),7.95(dd,J=18.8,10.5Hz,2H),7.76(d ,J=8.5Hz,1H),7.51(d,J=1.8Hz,1H),5.48(ddd,J=23.7,14.8,4.3Hz,2H),4.77(s,2H),4.39(dd,J= 12.9,7.5Hz,1H),4.28–4.16(m,1H),4.00–3.70(m,4H),3.49(d,J=15.2Hz,1H),3.24–3.10(m,2H),2 .92–2.59(m,4H),2.44(d,J=7.0Hz,3H),1.61(dd,J=19.0,6.4Hz,3H),1.41(dd,J=13.2,6.8Hz,3H).

[0602] Example 27 Preparation of compounds 27-P2A and 27-P2B

[0603] The synthesis of compound 27 was performed according to Example 24, with compound 23a replaced by compound 15a. Compound 27 was separated by Prep HPLC to obtain two peaks, namely compound 27-P1 (28 mg) and 27-P2 (280 mg).

[0604] Compound 27-P1: MS (ESI) m / z 826.1 [M+H] + LC-MS retention time: 4.26 min (Analysis Method 4)

[0605] Compound 27-P2: MS (ESI) m / z 826.1 [M+H] + LC-MS retention time: 4.29 min (Analysis Method 4)

[0606] Compound 27-P2 was isolated by chiral preparation to yield compounds 27-P2A (69.1 mg) and 27-P2B (61.9 mg).

[0607] Chiral preparation conditions: IC, 10μm, 30*250mm; Mobile phase A: acetonitrile + 0.2% formic acid, Mobile phase B: ethanol + 0.2% formic acid; Flow rate: 25mL / min; Column temperature: room temperature; Isocratic elution program: Mobile phase A: Mobile phase B = 80:20 (V / V).

[0608] Chiral analysis conditions: IC, 5μm, 4.6mm*250mm; Mobile phase A: acetonitrile + 0.2% formic acid, Mobile phase B: ethanol + 0.2% formic acid; Flow rate: 1.0mL / min; Column temperature: 30℃; Isocratic elution program: Mobile phase A: Mobile phase B = 80:20 (V / V).

[0609] Compound 27-P2A: MS (ESI) m / z 826.1 [M+H] + SFC 100%ee. Retention time 7.676 min.

[0610] 1H NMR(400MHz,DMSO-d6)δ10.43(s,2H),8.54(s,0.5H),8.51(s,0.5H),8.03-7.90(m,2H),7.79-7.72(m,1H),7.44(s,1H),5.55-5.43(m,1H),5.42 -5.36(m,1H),4.85-4.75(m,0.5H),4.72(s,2H),4.44-4.33(m,1.5H),4 .25-4.15(m,1H),3.95-3.85(m,1H),3.78-3.69(m,1H),3.67(s,2H),3.5 4-3.43(m,1H),3.40-3.31(m,1H),2.90-2.85(m,0.5H),2.78-2.72(m,0 .5H),2.70-2.65(m,0.5H),2.64-2.58(m,0.5H),2.45(s,1.5H),2.43(s, 1.5H),1.63(d,J=6.4Hz,1.5H),1.58(d,J=6.4Hz,1.5H),1.41(d,J=6.8H z,1.5H),1.38(d,J=6.8Hz,1.5H),1.10-1.03(m,2H),0.96-0.89(m,2H).

[0611] Compound 27-P2B: MS (ESI) m / z 826.1 [M+H] + SFC 99.44%ee. Retention time 8.776min.

[0612] 1H NMR (400MHz, DMSO-d6) δ10.44(s,1H),8.52(s,0.5H),8.50(s,0.5H),8.05-7.92(m,2H),7.82-7.75(m,1H),7.47(s,1H),5.44-5.38(m,1 H),5.22(q,J=6.4Hz,0.5H),5.18(q,J=6.4Hz,0.5H),4.83-4.75(m,0.5H),4.75(s,2H),4.47-4.39(m,1.5H),4.26-4.16(m,1H),3.77-3. 1 .72(d,J=6.4Hz,1.5H),1.67(d,J=6.4Hz,1.5H),1.40(d,J=6.8Hz,1.5H),1.38(d,J=6.8Hz,1.5H),1.13-1.06(m,2H),0.99-0.92(m,2H).

[0613] Example 28 (7S,10R)-2-(benzothiophene-2-yl)-N-(2-chloro-4-(trifluoromethyl)phenyl)-6-(4-(5-hydroxy-6-methylpyrimidin-4-carbonyl)piperazin-1-yl)-7-methyl-5-oxo-5,7,9,10-tetrahydro-[1,2,4]triazolo[5',1':2,3]pyrimido[6,1-c][1,4]oxazine-10-carboxamide

[0614] Compound 28 was synthesized according to Example 22, except that compound 22a was replaced with benzothiophene-2-ylboronic acid. The chiral analysis conditions were the same as in Example 25.

[0615] MS(ESI) m / z 780.1 [M+H] + SFC 100%ee. Retention time 5.999min.

[0616] 1H NMR(400MHz,DMSO-d6)δ10.46(s,1H),10.25(s,1H),8.61(s,1H),8.14(s,1H),8.10–7.94( m,4H),7.79(s,1H),7.53–7.41(m,2H),5.48(s,1H),5.36(dd,J=17.3,6.4Hz,1H),4.64–4. 40(m,2H),4.23(d,J=8.8Hz,1H),3.69(d,J=10.1Hz,1H),3.58–3.40(m,2H),3.32–3.19(m, 1H),3.12–2.85(m,2H),2.71(d,J=17.1Hz,1H),2.47(s,3H),1.67(dd,J=20.1,6.2Hz,3H).

[0617] The synthesis of control compound 1 is based on patent WO2022249060A1.

[0618] Test Example 1: Test of the compound's activity against WRN(517-1238) enzyme

[0619] This experiment investigated the inhibitory effect of the compound on the activity of WRN(517-1238) enzyme by examining its effect on the activity of WRN(517-1238) enzyme in vitro.

[0620] Experimental reagents and consumables

[0621] The compound was serially diluted using DMSO in a 384PP Plate compound dilution plate. 0.15 μL of the compound was transferred to a 384 reaction microplate (Corning 4514) using an Echo, ensuring a final DMSO concentration of 1%. 5 μL of WRN (517-1238) enzyme solution (containing 0.2 mM ATP) was added to each well of the 384 reaction microplate, and incubated at 25°C for 4 hours. Wells containing 1% DMSO and enzyme served as high-value controls, and wells containing the same concentration of DMSO and buffer served as low-value controls. 5 μL of a mixture of double-stranded DNA and capture DNA was added to each well, briefly centrifuged, and then 5 μL of ATP solution was added. The plate was incubated at 25°C for 30 minutes (final concentration: 5 nM WRN, 100 nM double-stranded DNA, 1000 nM capture DNA, 4.07 mM ATP). Fluorescence signal values ​​were read on a BMG (CLARIO Star Plusacu) microplate reader (excitation wavelength: 620 nm, emission wavelength: 685 nm). The inhibition percentage of the compound-treated wells was normalized between the high-value and low-value controls (inhibition rate % = (signal value)). 高值对照 -Signal value化合物处理 ) / (signal value) 高值对照 -Signal value 低值对照 (*100). Then, fit the four-parameter IC using XLfit 5.5.0. 50 The curve was analyzed.

[0622] Table 1. Inhibition results of the tested compounds on WRN(517-1238)_FI

[0623] As can be seen from the table above, the compounds of the present invention have a good inhibitory effect on WRN(517-1238)_FI.

[0624] Test Example 2: Test of the inhibitory activity of the compound on the proliferation of SW48 cells

[0625] Prepare complete culture medium (L-15 + 10% FBS + 1% P / S) to revive SW48 cells (ATCC), passage them two times, centrifuge to collect and count the cells in the logarithmic growth phase, resuspend the cells to an appropriate concentration, and seed the cell suspension into 96-well plates, adding 100 μL of cell suspension to each well, with a seeding density of 5000 cells / well. Place the 96-well plates in a CO2 incubator (0% CO2) overnight, then aspirate 50 μL of the supernatant culture medium and add 40 μL of fresh culture medium. The test compound was prepared as a stock solution using DMSO. Nine concentration gradients were obtained by stepwise 4-fold dilution with DMSO, starting at a maximum concentration of 2.5 mM. The test compound was diluted 25-fold with culture medium. 10 μL of each solution was added to a 96-well cell plate containing 90 μL of cells. Cell-free culture medium (containing 0.4% DMSO) was added to the Min control wells, and 10 μL of a DMSO-cell culture medium mixture (final DMSO concentration 0.4%) was added to the Max control wells. The plates were incubated at 37°C, 0% relative humidity (≥90%) for 120 h. 50 μL / well CellTiter Glo was added to terminate the reaction. The plates were incubated at room temperature in the dark for 10 min, gently shaken, and then detected using Envision. The fluorescence value (RLU) of each well was read, and the cell inhibition rate (%) was calculated using the formula: Cell Growth Inhibition Rate (%) = (1 - As / Ac) × 100. Where As: RLU sample (cells + CTG + test compound) - RLU Min (cell-free culture medium), Ac: RLU normal growth cell control (cells + CTG + DMSO) - RLU Min (cell-free culture medium). Enter the inhibition rate Inh% (Y) corresponding to each concentration (X) in Excel, and use Graphpad Prism 8 software to fit the formula Y = Bottom + (Top - Bottom) / (1 + (IC)) using the built-in four-parameter fitting formula. 50The half-maximal inhibitory concentration (IC50) of each compound was calculated using the formula ( / X)*HillSlope). 50 value.

[0626] Table 2. Results of the inhibitory effects of the tested compounds on the proliferation of SW48 cells.

[0627] Experimental results show that, compared with control compound 1, the compounds of the present invention have a good inhibitory effect on the proliferation of SW48 cells. In particular, after introducing a thiophene ring structure to replace the original dihydropyran fragment based on the structure of compound 14, the resulting compounds (such as 11-P2, 15, 23 and 24-P2B) showed a significant improvement in the inhibitory activity against the proliferation of SW48 cells.

[0628] Test Example 3: Beagle Dog Pharmacokinetic Study

[0629] The test animals used in this study were male beagle dogs (9-11 kg, Yizheng Anlimao Biotechnology Co., Ltd.). Three dogs were assigned to each oral administration group and three to each intravenous administration group. The solvent for both oral and intravenous administration was 5% DMSO + 10% Solutol + 85% Saline. The clear solution of the test compound was administered to the beagle dogs via tail vein injection or gavage (the dogs were fasted overnight before each administration and fed 4 hours after administration; water was not restricted throughout the experiment). For intravenous administration, 1 mL of blood was collected from the forelimb vein at 0 h (before administration) and 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h after administration. The blood was anticoagulated with EDTA-2K, and the mixture was thoroughly vortexed and centrifuged at 6000 rpm for 3 minutes at 4°C. For oral administration via gavage, 1 mL of blood was collected from the forelimb vein at 0 h (before administration) and 0.083, 0.25, 1, 2, 4, 6, 8, and 24 h after administration. The blood was anticoagulated with EDTA-2K, and the mixture was thoroughly vortexed and centrifuged at 6000 rpm for 3 minutes. Blood drug concentrations were determined by LC-MS / MS. Relevant pharmacokinetic parameters were calculated using the non-compartmental linear logarithmic trapezoidal method with Phoenix WinNonlin 8.2.0 pharmacokinetic software.

[0630] Table 3. Results of the beagle dog pharmacokinetic studies of the tested compounds.

[0631] Experimental results show that, compared with control compound 1, the compound of the present invention exhibits lower clearance, longer half-life and higher exposure, and has better pharmacokinetic properties.

[0632] Test Example 4: In vivo efficacy test of SW48 xenograft BALB / c nude mice

[0633] The experimental animals used in this study were female Balb / c nude mice (6-8 weeks old, weighing 20-22g, provided by Zhejiang Vital River Laboratory Animal Technology Co., Ltd.). Animals were housed in SPF-grade animal facilities in individually air-conditioned cages, with no more than 5 mice per cage. The SW48 cells used in this study were purchased from ATCC (Cat#: CCL-231, Lot. No: 70026601). Cell culture was performed using Leibovitz's L-15 + 10% FBS + 1% P / S at 37℃ with 5% CO2. Cells were harvested and seeded when the cell saturation reached 80%–90% and the required number was achieved. 0.2 mL (2 × 10⁶ cells) of SW48 cells (with matrix gel, volume ratio 1:1) were subcutaneously seeded into the right posterior back of each mouse. Drug administration began when the average tumor volume reached 100-150 mm³. During the drug administration period, body weight and tumor volume were measured twice weekly, and clinical symptoms were observed and recorded daily throughout the experiment. Experimental data are expressed as mean ± SEM. One-way ANOVA was used for comparisons between groups. p < 0.05 was considered statistically significant, and p < 0.01 was considered highly statistically significant (IBM SPSS Statistics 19.0). The results are shown in Figure 2.

[0634] The results showed that compounds 11-P2, 24-P2B, and 23 of the present invention exhibited tumor growth inhibition effects comparable to those of control compound 1 (10 mg / kg) at a dose of 5 mg / kg. At the same dose (10 mg / kg), compound 23 showed significantly better antitumor effects than control compound 1. The compounds of the present invention exhibited low toxicity in mice (body weight loss in mice did not exceed 5% in any dose group). In summary, compared to control compound 1, the compounds of the present invention significantly improved tumor inhibition effects in vivo.

[0635] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A compound of formula (1b), its stereoisomers, its optical isomers, its pharmaceutically acceptable salts, its crystal form, its isotopic derivatives, its prodrugs, its metabolites, its solvates, or its hydrates. in, Y is C, CH, or N; Indicates a single bond or a double bond; When Y is CH, Y is connected to the adjacent carbon atom by a single bond, and the CH is optionally replaced by -OH or a halogen; When Y is C, Y is connected to adjacent atoms through double bonds; When Y is N, Y is connected to adjacent atoms through single bonds; o and y are each independently 0, 1, 2, 3 or 4; Each R3 is independently selected from the following groups: H, D, C 1-4 Alkyl, C 3-6 cycloalkyl, C 1-4 A haloalkyl group, or two R3 atoms on the same ring carbon atom and the carbon atom to which they are attached, together forming a 3- or 4-membered ring, wherein the 3- or 4-membered ring contains 0, 1, or 2 heteroatoms selected from N or O; or two R3 atoms on adjacent ring carbon atoms and the carbon atom to which they are attached, together forming a 3- or 6-membered ring, wherein the 3- or 6-membered ring is optionally separated by 1 or 2 R3 atoms. 3a replace; R 3a Selected from the following groups: H, D, halogens, C 1-4 Alkyl, C 1-4 Hydroxyalkyl, C 1-4 Halogenated alkyl groups; R4 is a 5-12 heteroaryl group; wherein the 5-12 heteroaryl group is optionally surrounded by 1, 2, 3 or 4 R groups. 4a replace; Each R 4a Independently selected from the following groups: H, D, hydroxyl, halogen, cyano, C 1-4 Alkyl, C 1-4 Haloalkyl, C 3-6 cycloalkyl, C 3-6 Cycloalkoxy, C 1-4 Alkoxy, C 1-4 Deuterated alkyl, C 1-4 Halogenated alkoxy groups, or two R atoms on adjacent ring carbon atoms 4a Together with the carbon atoms to which they are attached, they form 4 to 6-membered rings, which contain 0, 1 or 2 heteroatoms selected from N or O; X is CH or N; and when X is CH, the CH can be replaced by R5 or R6 (i.e., X is CR5 or CR6); m and n are each independently 0, 1, 2 or 3; R5 and R6 are each independently selected from the following groups: H, D, hydroxyl, halogen, cyano, C. 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Halogenated alkoxy groups, -SF3, -SF5, C 3-6 cycloalkyl, C 1-4 Alkylamino, C 1-4 alkenyl, C 2-6 alkynyl group, C 1-4 Haloalkenyl, C 2-6 Alkyne group; wherein the R5 is optionally surrounded by 1, 2, 3, 4 or 5 R groups. 5a Replace; the R6 is optionally replaced by 1, 2, 3, 4 or 5 Rs. 6a replace; R 5a and R 6a Each is independently selected from the following groups: H, D, hydroxyl, halogen, amino, cyano, C. 1-4 Alkyl, C 1-4 Haloalkyl, C 3-6 cycloalkyl, C 3-6 Cycloalkoxy, C 1-4 Alkoxy, C 1-4 Deuterated alkyl, C 1-4 Halogenated alkoxy groups; K is O, S, or NR 7b ; Each R 7b Independently selected from the following groups: H, D, hydroxyl, halogen, amino, cyano, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Deuterated alkyl, C 1-4 Alkoxy, C 1-4 Halogenated alkoxy groups, C 1-4 alkylamine group, C 3-6 Cycloalkyl, or substitution of two R atoms on different ring atoms 7b The atoms bonded to it together form a 3-membered carbon ring or a 5-membered heterocycle; the aforementioned 3-membered carbon ring or 5-membered heterocycle is optionally bounded by 1, 2, 3 or 4 R atoms. 7c replace; When K is NR 7b At that time, the R on the ring carbon atom 7b and R connected to N 7b They can collectively form a five-membered heterocycle; the five-membered heterocycle described above is optionally divided by 1, 2, 3 or 4 R... 7c replace; Each R 7c Independently selected from the following groups: H, D, hydroxyl, halogen, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Deuterated alkyl, C 1-4 Alkoxy, C 1-4 Halogenated alkoxy groups, C 3-6 Cycloalkyl; or two R atoms attached to the same carbon atom in a ring. 7c It can form a ternary carbon ring together with the carbon atom it is attached to; R1 is selected from the following group: t can be 0, 1, 2, 3, 4, or 5; R 1a These are substituents on R1, each R 1a Independently selected from the following groups: H, D, halogen, cyano, amino, hydroxyl, C 1-4 Alkyl, C 3-6 cycloalkyl, C 1-4 Deuterated alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 3-6 Cycloalkoxy, C 1-4 Halogenated alkoxy groups, C 3-6 alkynyl group, C 3-6 alkenyl, C 3-6 Haloalkenyl, C 1-4 Alkylamine group, Or two R atoms on the same ring carbon atom 1a Together with the carbon atom it is attached to, they form a 3- or 4-membered ring, which contains 0, 1, or 2 heteroatoms selected from N or O, or two R atoms on adjacent ring atoms. 1a Together with the ring atoms it is attached to, it forms a 3- to 6-membered ring, wherein the 3- to 6-membered ring contains 0, 1, or 2 heteroatoms selected from N or O; wherein, R 1a Optionally divided by 0, 1, 2 or 3 R 1b Instead, the 4 to 6-membered rings are optionally replaced by 0, 1, or 2 R... 1c replace; Each R 1b Independently selected from the following groups: H, D, hydroxyl, halogen, amino, cyano, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Halogenated alkoxy groups, C 1-4 Alkylamine group; Each R 1c Independently selected from the following groups: H, D, halogen, hydroxyl, C 1-4 alkyl.

2. The compound, its stereoisomer, its optical isomer, its pharmaceutically acceptable salt, its crystal form, its isotopic derivative, its prodrug, its metabolite, its solvate, or its hydrate as claimed in claim 1, characterized in that, The compound has the structure shown in formula (1b-1), formula (1b-2), (1b-3), (1b-4) or formula (1a-1): Where o is 0, 1, 2 or 3; R1, R3, R4, R5, R6, R 7b R 7c The definitions of X, m, n, and y are as described in claim 1; Preferably, in compounds having the structure shown in formula (1b-1), R 7b C 1-4 Alkyl or C 1-4 Deuterated alkyl groups; More preferably, in compounds having the structure shown in formula (1b-2), R attached to the carbon or nitrogen atom 7b Same or different, R 7b C 1-4 Alkyl, C 1-4 Deuterated alkyl or C 3-6 Cycloalkyl.

3. The compound, its stereoisomer, its optical isomer, its pharmaceutically acceptable salt, its crystal form, its isotopic derivative, its prodrug, its metabolite, its solvate, or its hydrate as claimed in claim 1, characterized in that, The compound has the structure shown in formula (1b-5) or formula (1b-6): In compounds having the structure shown in formula (1b-5), R 7b C 1-4 Alkyl or C 1-4 Deuterated alkyl groups; In compounds having the structure shown in formulas (1b-6), R 7b and R 7b’ Each independently is C 1-4 Alkyl, C 1-4 Deuterated alkyl or C 3-6 cycloalkyl, or R 7b R 7b’ The atoms bonded to it together form a 3-membered carbon ring or a 5-membered heterocycle; the aforementioned 3-membered carbon ring or 5-membered heterocycle is optionally bounded by 1, 2, 3 or 4 R atoms. 7c replace; 1* and 2* indicate that this area is chiral; R1, R3, R4, R5, R6, R 7c The definitions of m, n, and y are as described in claim 1.

4. The compound, its stereoisomer, its optical isomer, its pharmaceutically acceptable salt, its crystal form, its isotopic derivative, its prodrug, its metabolite, its solvate, or its hydrate as claimed in claim 1, characterized in that, R1 is selected from the following group:

5. The compound, its stereoisomer, its optical isomer, its pharmaceutically acceptable salt, its crystal form, its isotopic derivative, its prodrug, its metabolite, its solvate, or its hydrate as claimed in claim 1, characterized in that, The Selected from the following group: R4 is defined as described in claim 1.

6. The compound, its stereoisomer, its optical isomer, its pharmaceutically acceptable salt, its crystal form, its isotopic derivative, its prodrug, its metabolite, its solvate, or its hydrate as claimed in claim 1, characterized in that, The Selected from the following group:

7. The compound, its stereoisomer, its optical isomer, its pharmaceutically acceptable salt, its crystal form, its isotopic derivative, its prodrug, its metabolite, its solvate, or its hydrate as claimed in claim 1, characterized in that, R4 is defined by 1, 2, or 3 Rs. 4a Substituted 5-7 membered heteroaryl groups; of which, R 4a Selected from the following groups: H, D, hydroxyl, halogen, C 1-4 Alkyl, C 1-4 Deuterated alkyl groups, or two R atoms on adjacent ring carbon atoms 4a Together with the carbon atoms to which they are attached, they form 4 to 6-membered rings, which contain 0, 1 or 2 heteroatoms selected from N or O; Preferably, R4 is composed of 1, 2, or 3 R... 4a Substituted 5-7 nitrogen-containing heteroaryl groups; wherein, R 4a Selected from the following groups: H, D, hydroxyl, halogen, C 1-4 Alkyl, C 1-4 Deuterated alkyl groups, or two R atoms on adjacent ring carbon atoms 4a Together with the carbon atoms to which they are attached, they form 4 to 6-membered rings, which contain 0, 1 or 2 heteroatoms selected from N or O; More preferably, R4 is selected from the group consisting of:

8. The compound of claim 1, its stereoisomers, its optical isomers, its pharmaceutically acceptable salts, its crystal forms, its isotopic derivatives, its prodrugs, its metabolites, its solvates, or its hydrates, characterized in that, The compounds are selected from the following group:

9. A pharmaceutical composition comprising the compound of any one of claims 1-8, its stereoisomer, its optical isomer, its pharmaceutically acceptable salt, its crystal form, its isotopic derivative, its prodrug, its metabolite, its solvate, or its hydrate; and a pharmaceutically acceptable carrier.

10. Use of a compound, stereoisomer, optical isomer, pharmaceutically acceptable salt, crystal form, isotopic derivative, prodrug, metabolite, solvate or hydrate thereof, or pharmaceutical composition as described in claim 9 in the preparation of a medicament for treating WRN helicase-mediated diseases. Preferably, the disease is a tumor; more preferably, the disease is a microsatellite instability tumor; even more preferably, the tumor is a tumor characterized by high microsatellite instability (MSI-H) or having defective mismatch repair (dMMR) or "dMMR features"; in, The microsatellite unstable tumors include, but are not limited to, uterine fibroids, endometrial cancer, colonic adenocarcinoma, gastric adenocarcinoma, rectal adenocarcinoma, adrenocortical carcinoma, uterine carcinosarcoma, cervical squamous cell carcinoma, cervical endometrial adenocarcinoma, esophageal cancer, breast cancer, renal clear cell carcinoma, and ovarian serous cyst carcinoma. Tumors with "defective mismatch repair (dMMR)" or "dMMR features" include, but are not limited to, lung cancer, breast cancer, kidney cancer, colorectal cancer, ovarian cancer, prostate cancer, upper respiratory and digestive tract cancer, gastric cancer, endometrial cancer, liver cancer, pancreatic cancer, hematopoietic and lymphatic tissue cancer, skin cancer, thyroid cancer, pleural cancer, autonomic nervous system tumors, soft tissue tumors, rhabdomyosarcoma, melanoma, and other tumors.

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