Oxo-six-membered heterocyclic thiazole compound and application thereof

Abstract

The invention relates to an oxo-six-membered heterocyclic thiazole compound shown in a formula I or pharmaceutically acceptable salts thereof, a pharmaceutical composition containing the oxo-six-membered heterocyclic thiazole compound and the pharmaceutically acceptable salts thereof, and application of the oxo-six-membered heterocyclic thiazole compound and the pharmaceutically acceptable salts thereof in preparation of drugs for preventing or treating DNA polymerase theta mediated diseases. The compound with the structure as shown in the formula I has good inhibitory activity and good patent medicine property.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to an oxo six-membered heterocyclic fused thiazole compound, a pharmaceutically acceptable salt thereof, a pharmaceutical composition containing the compound, and the use of the compound as a DNA polymerase theta inhibitor in the preparation of a medicine for preventing or treating a disease mediated by DNA polymerase theta. BACKGROUND

[0002] When the cells in the eukaryotic organism are subjected to continuous oxidative stress, ionizing radiation or endogenous or exogenous damage such as chemotherapy drugs, DNA damage breaks occur, including DNA single-strand break (SSB) and DNA double-strand break (DSB), and then the DNA damage repair response (DDR) in the body cells for maintaining the stability of the genome is activated to identify and repair the DNA damage. Among the above two kinds of DNA damage, DNA double-strand break (DSB) is a very serious damage, and improper repair will lead to cell apoptosis or genomic rearrangement.

[0003] The repair pathway of DNA double-strand break (DSB) includes at least three repair pathways, i.e. homologous recombination (HR), non-homologous end joining (NHEJ) and microhomology-mediated end joining (MMEJ). Among them, homologous recombination (HR) which is active only in S and G2 phases of the cell cycle is the only precise DSB repair pathway; non-homologous end joining (NHEJ) which is active mainly in G1 phase is the main DSB repair pathway in mammalian cells; in addition, microhomology-mediated end joining (MMEJ) involving DNA polymerase theta (POLQ) is part of a more extensive end joining mechanism, and this pathway is also known as POLQ-mediated end joining (TMEJ), when HR and NHEJ are damaged, the substrate is transferred to the MMEJ pathway, such as cells lacking BRCA1 / 2, Ku and Lig4 which depend on MMEJ for survival.

[0004] In the DNA damage repair of cells, POLQ plays a key role as a key protein in the MMEJ repair pathway. POLQ is highly expressed in various tumor cells, such as in lung cancer, colon cancer, breast cancer and gastric cancer tumor tissues resected from patients, and is associated with poor prognosis. Inhibition of the function of POLQ will cause the tumor cells to undergo apoptosis due to increased replication stress. At the same time, POLQ has a synthetic lethal effect with HR defects (such as BRCA1 / 2 deletion), and a POLQ inhibitor can inhibit the proliferation of tumor cells with HR defects, especially the proliferation of tumor cells resistant to PARP inhibitors, so that the POLQ inhibitor can make the tumor cells again sensitive to PARP inhibitors.

[0005] In summary, POLQ is a potential target for tumor treatment, and searching for POLQ inhibitors that are suitable for drug development while meeting certain activity requirements will provide new strategies and better options for targeted treatment of tumors, especially HR-deficient tumors. SUMMARY

[0006] The present application aims to provide an oxo-hexa-heterocyclic thiazole compound having good inhibitory effect on DNA polymerase theta, which is suitable for drug development, a pharmaceutically acceptable salt thereof, a pharmaceutical composition and use thereof.

[0007] The purpose of the present application is achieved by the following technical solutions:

[0008] In one aspect, the present application provides a compound as shown in formula I and a pharmaceutically acceptable salt thereof:

[0009]

[0010] Wherein:

[0011] Ring A is selected from C6-C 12 aryl, 5-10 membered heteroaryl, which aryl or heteroaryl is unsubstituted or substituted with 1-5 substituents each independently selected from halogen, cyano, =O, C1-C6alkyl, C1-C6haloalkyl, C3-C9cycloalkyl, -OR b ;

[0012] R b is selected from H, C1-C6alkyl, C1-C6haloalkyl, C3-C9cycloalkyl;

[0013] Y is selected from N or C, when Y is N, R1 is absent;

[0014] Z is selected from N or CR a ;

[0015] R a is selected from H, C1-C6alkyl;

[0016] R1 is selected from H, C1-C6alkyl, wherein said C1-C6alkyl is unsubstituted or optionally substituted with halogen, C1-C6alkoxy, -C(O)NR c R d ;

[0017] R c , R d are each independently selected from H, C1-C6alkyl;

[0018] R2 is selected from H, halogen, cyano, C1-C6alkyl, C1-C6haloalkyl, C1-C6alkoxy;

[0019] R3is selected from C6-C 12 aryl, 5-14 membered heteroaryl, C3-C9cycloalkyl, 5-14 membered heterocyclyl, which are unsubstituted or substituted by 1-5 substituents each independently selected from the group consisting of cyano, nitro, halogen, =0, -C(O)NR c’ R d’ , -OR e , -C(O)R f , -ORg, C1-C6alkyl, C6-C 12 aryl, 5-14 membered heteroaryl, C3-C9cycloalkyl, 5-14 membered heterocyclyl, which are unsubstituted or substituted by 1-5 substituents each independently selected from the group consisting of cyano, nitro, halogen, =0, -C(O)NR 12 aryl, 5-14 membered heteroaryl, C3-C9cycloalkyl, 5-14 membered heterocyclyl, which are unsubstituted or substituted by 1-5 substituents each independently selected from the group consisting of cyano, nitro, halogen, =0, -C(O)NR

[0020] R c’ , R d’ each independently selected from H, C1-C6alkyl;

[0021] R e selected from H, C1-C6alkyl;

[0022] R f selected from H, C1-C6alkyl;

[0023] R g selected from H, C1-C6alkyl, which is unsubstituted or optionally substituted by C1-C6alkoxy, C6-C 12 aryl, 5-14 membered heteroaryl, C3-C9cycloalkyl, 5-14 membered heterocyclyl.

[0024] In certain preferred embodiments of the present application, the compounds as illustrated in formula I and pharmaceutically acceptable salts thereof, wherein:

[0025] Ring A is selected from phenyl, 5-6 membered heteroaryl containing at least one N, which phenyl or heteroaryl is unsubstituted or substituted by 1-5 substituents each independently selected from halogen, cyano, =0, C1-C3alkyl, C1-C3haloalkyl, C3-C6cycloalkyl, -OR b ;

[0026] R b selected from H, C1-C3alkyl, C1-C3haloalkyl, C3-C6cycloalkyl;

[0027] Y is selected from N or C, when Y is N, R1is absent;

[0028] Z is selected from N or CR a ;

[0029] R a is selected from H, C1-C3 alkyl;

[0030] R1is selected from H, C1-C3 alkyl, wherein the C1-C3 alkyl is unsubstituted or optionally substituted with halogen, C1-C3 alkoxy, -C(O)NR c R d ;

[0031] R c , R d are each independently selected from H, C1-C3 alkyl;

[0032] R2is selected from H, halogen, cyano, C1-C3 alkyl, C1-C3 haloalkyl, C1-C3 alkoxy;

[0033] R3is selected from C6-C 10 aryl, 5-10 membered heteroaryl containing 1, 2, 3, 4 heteroatoms selected from O, N, S, C3-C6 cycloalkyl, 5-10 membered heterocyclyl containing 1, 2, 3, 4 heteroatoms selected from O, N, S, which are unsubstituted or substituted with 1 -5 substituents each independently selected from cyano, nitro, halogen, =0, -C(O)NR c’ R d’ , -N(R e )-C(O)R f , -ORg, C1-C3 alkyl, C3-C6 cycloalkyl, C6-C 10 aryl, 5-9 membered heteroaryl, which C1-C3 alkyl, C3-C6 cycloalkyl, C6-C 10 aryl, 5-9 membered heteroaryl are optionally substituted with halogen, cyano, hydroxy, C1-C3 alkyl, C1-C3 alkoxy;

[0034] R c’ , R d’ are each independently selected from H, C1-C3 alkyl;

[0035] R e is selected from H, C1-C3 alkyl;

[0036] R f is selected from H, C1-C3 alkyl;

[0037] R g is selected from H, C1-C3 alkyl, which alkyl is unsubstituted or optionally substituted with C1-C3 alkoxy, C6-C 10C6cycloalkyl, 5-10 membered heteroaryl containing 1, 2, 3, 4 heteroatoms selected from O, N, S, C3-C6cycloalkyl, 5-10 membered heterocyclyl containing 1, 2, 3, 4 heteroatoms selected from O, N, S, unsubstituted or substituted with 1 -5 substituents each independently selected from cyano, nitro, halogen, =0, -C(O)NR

[0038] In certain preferred embodiments of the application, the compound as shown in formula I has the structure of formula la:

[0039]

[0040] wherein,

[0041] X is selected from N or CH;

[0042] Y is selected from N or C, R1is absent when Y is N;

[0043] Z is selected from N or CR a ;

[0044] R a is selected from H, C1-C3alkyl;

[0045] R1is selected from H, C1-C3alkyl, wherein said C1-C3alkyl is unsubstituted or optionally substituted with halogen, C1-C3alkoxy, C(O)NR c R d ;

[0046] R c , R d are each independently selected from H, C1-C3alkyl;

[0047] R2is selected from H, halogen, cyano, C1-C3alkyl, C1-C3haloalkyl, C1-C3alkoxy;

[0048] R3is selected from C6-C 10 C6cycloalkyl, 5-10 membered heteroaryl containing 1, 2, 3, 4 heteroatoms selected from O, N, S, C3-C6cycloalkyl, 5-10 membered heterocyclyl containing 1, 2, 3, 4 heteroatoms selected from O, N, S, unsubstituted or substituted with 1 -5 substituents each independently selected from cyano, nitro, halogen, =0, -C(O)NR c’ R d’ , -N(R e )-C(O)R f , -ORg, C1-C3alkyl, C3-C6cycloalkyl, C6-C 10 aryl, 5-9 membered heteroaryl, said C1-C3alkyl, C3-C6cycloalkyl, C6-C 10aryl, 5-9 membered heteroaryl, optionally substituted with halogen, cyano, hydroxy, C1-C3alkyl, C1-C3alkoxy;

[0049] R4is selected from hydrogen, halogen, cyano, =0, C1-C3alkyl, C1-C3alkoxy, C1-C3haloalkyl, C3-C6cycloalkyl;

[0050] R5is selected from C1-C3alkyl, C1-C3haloalkyl, C3-C6cycloalkyl;

[0051] R6is selected from H, halogen, C1-C3alkyl;

[0052] R c’ , R d’ are each independently selected from H, C1-C3alkyl;

[0053] R e is selected from H, C1-C3alkyl;

[0054] R f is selected from H, C1-C3alkyl;

[0055] R g is selected from H, C1-C3alkyl, said alkyl being unsubstituted or optionally substituted with C1-C3alkoxy, C6-C10aryl, 5-10 membered heteroaryl, C3-C6cycloalkyl, 5-10 membered heterocyclyl; 10 aryl, 5-9 membered heteroaryl, optionally substituted with halogen, cyano, hydroxy, C1-C3alkyl, C1-C3alkoxy;

[0056] In certain preferred embodiments of the application, the compounds as described by formula I have the structure of formula la:

[0057]

[0058] wherein,

[0059] X is selected from N or CH;

[0060] Y is selected from N or C, when Y is N, R1is absent;

[0061] Z is selected from N or CR a ;

[0062] R a is selected from H, methyl, ethyl, propyl, isopropyl;

[0063] R1is selected from H, methyl, ethyl, propyl, isopropyl, wherein said alkyl is unsubstituted or optionally substituted with F, Cl, Br, methoxy, ethoxy, propoxy, -C(O)NR c Rd ;

[0064] R c , R d each independently is selected from H, methyl, ethyl, propyl, isopropyl;

[0065] R2is selected from H, F, Cl, Br, cyano, methyl, ethyl, propyl, isopropyl, CF3, CF2H, CFH2, methoxy, ethoxy, propoxy;

[0066] R3is selected from phenyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, triazolyl, furazanyl, furanyl, thienyl, thiazolyl, isothiazolyl, oxadiazolyl, naphthyl, quinolinyl, isoquinolinyl, indolyl, benzopyrazolyl, benzimidazolyl, benzoxazolyl, benzothiazolyl, benzoisoxazolyl, benzoisothiazolyl, pyrimido-oxazolyl, pyrimido-thiazolyl, pyrimido-imidazolyl, pyridazino-imidazolyl, pyrazino-imidazolyl, indigo, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, oxolanyl, dioxolanyl, morpholinyl, piperidinyl, piperazinyl, 1,4-benzodioxanyl, 2,3-dihydrobenzofuranyl, benzomorpholinyl, 1,2,3,4-tetrahydroquinolinyl, unsubstituted or substituted with 1 to 5 substituents each independently selected from cyano, nitro, F, Cl, Br, =0, -N(R e )-C(O)R f , -ORg, methyl, ethyl, propyl, isopropyl, cyclopropyl, phenyl, isoxazolyl, said alkyl, phenyl, isoxazolyl optionally substituted with F, Cl, Br, cyano, hydroxy, methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, propoxy;

[0067] R4is selected from H, F, Cl, Br, cyano, =0, methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, propoxy, CF3, CF2H, CFH2, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl;

[0068] R5is selected from methyl, ethyl, propyl, isopropyl, CF3, CF2H, CFH2, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl;

[0069] R6is selected from H, F, Cl, Br, methyl, ethyl, propyl, isopropyl;

[0070] R e is selected from H, methyl, ethyl, propyl, isopropyl;

[0071] R f is selected from H, methyl, ethyl, propyl, isopropyl;

[0072] Rg selected from H, methyl, ethyl, propyl, isopropyl, said alkyl is unsubstituted or optionally substituted with methoxy, ethoxy, propoxy, phenyl, naphthyl, pyridyl, oxazolyl, imidazolyl, pyrazolyl, triazolyl, oxolanyl, dioxanyl, morpholinyl, piperidinyl, piperazinyl.

[0073] In certain preferred embodiments of the application, the compounds as described by formula I have the structure of formula la:

[0074]

[0075] wherein,

[0076] X is selected from N or CH;

[0077] Y is selected from N, R1 is absent, Z is selected from N;

[0078] or Y is selected from N, R1 is absent, Z is selected from CH;

[0079] or Y is selected from C, Z is selected from N;

[0080] R1 is selected from H, methyl, ethyl, propyl, isopropyl, wherein said alkyl is unsubstituted or optionally substituted with F, Cl, Br, methoxy, ethoxy, propoxy, -C(O)NR c R d ;

[0081] R c , R d are each independently selected from H, methyl, ethyl, propyl, isopropyl;

[0082] R2 is selected from H, F, Cl, Br, cyano, methyl, ethyl, propyl, isopropyl, CF3, CF2H, CFH2, methoxy, ethoxy, propoxy;

[0083] R3 is selected from

[0084] R4 is selected from H, F, Cl, Br, cyano, =O, methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, propoxy, CF3, CF2H, CFH2, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl;

[0085] R5 is selected from methyl, ethyl, propyl, isopropyl, CF3, CF2H, CFH2, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl;

[0086] R6 is selected from H, F, Cl, Br, methyl, ethyl, propyl, isopropyl.

[0087] In certain preferred embodiments of the application, the compound according to Formula I has the structure of Formula Ib:

[0088]

[0089] wherein,

[0090] X is selected from N or C, and when X is N, R7 is absent;

[0091] Y is selected from N or C, and when Y is N, R1 is absent;

[0092] Z is selected from N or CR a ;

[0093] R a is selected from H, C1-C3 alkyl;

[0094] R7 is selected from hydrogen, halogen;

[0095] R1 is selected from H, C1-C3 alkyl, wherein said C1-C3 alkyl is unsubstituted or optionally substituted with halogen, C1-C3 alkoxy, C(O)NR c R d ;

[0096] R c , R d are each independently selected from H, C1-C3 alkyl;

[0097] R2 is selected from H, halogen, cyano, C1-C3 alkyl, C1-C3 haloalkyl, C1-C3 alkoxy;

[0098] R3 is selected from C6-C 10 aryl, said C6-C 10 aryl being unsubstituted or substituted with 1-5 substituents each independently selected from nitro, halogen, =O, -C(O)NR c’ R d’ , -N(R e )-C(O)R f , -ORg, C1-C3 alkyl, C3-C6 cycloalkyl, C6-C 10 aryl, 5-9 membered heteroaryl, said C1-C3 alkyl, C3-C6 cycloalkyl, C6-C 10 aryl, 5-9 membered heteroaryl being optionally substituted with halogen, cyano, hydroxy, C1-C3 alkyl, C1-C3 alkoxy;

[0099] R4 is selected from hydrogen, halogen, cyano, =O, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, C3-C6 cycloalkyl;

[0100] R5is selected from C1-C3alkyl, C1-C3haloalkyl, C3-C6cycloalkyl;

[0101] R6is selected from H, halogen, C1-C3alkyl;

[0102] R c’ , R d’ are each independently selected from H, C1-C3alkyl;

[0103] R e is selected from H, C1-C3alkyl;

[0104] R f is selected from H, C1-C3alkyl;

[0105] R g is selected from H, C1-C3alkyl, which alkyl is unsubstituted or optionally substituted by C1-C3alkoxy, C6-C10aryl, 5-10 membered heteroaryl containing 1, 2, 3 heteroatoms selected from O, N, S, C3-C6cycloalkyl, 5-10 membered heterocyclyl containing 1, 2, 3 heteroatoms selected from O, N, S. 10 C6-C10aryl, 5-10 membered heteroaryl containing 1, 2, 3 heteroatoms selected from O, N, S, C3-C6cycloalkyl, 5-10 membered heterocyclyl containing 1, 2, 3 heteroatoms selected from O, N, S.

[0106] In certain preferred embodiments of the application, the compounds as described by formula I have the structure of formula Ib:

[0107]

[0108] wherein,

[0109] X is selected from N or C, R7is absent when X is N;

[0110] Y is selected from N or C, R1is absent when Y is N;

[0111] Z is selected from N or CR a ;

[0112] R a is selected from H, C1-C3alkyl;

[0113] R7is selected from hydrogen, halogen;

[0114] R1is selected from H, C1-C3alkyl, wherein said C1-C3alkyl is unsubstituted or optionally substituted by halogen;

[0115] R2is selected from H, halogen, C1-C3alkyl, C1-C3haloalkyl;

[0116] R3is selected from C6-C 10 aryl, which C6-C 10aryl unsubstituted or substituted with 1 to 5 substituents each independently selected from halogen, C1-C3alkyl, phenyl, pyridyl, said alkyl, phenyl, pyridyl optionally substituted with halogen, C1-C3alkyl;

[0117] R4is selected from hydrogen, halogen, C1-C3alkyl, C1-C3haloalkyl;

[0118] R5is selected from C1-C3alkyl, C1-C3haloalkyl;

[0119] R6is selected from H, halogen, C1-C3alkyl.

[0120] In certain preferred embodiments of the present application, the compound as described by formula I has the structure of formula Ib:

[0121]

[0122] wherein,

[0123] X is selected from N or C, R7is absent when X is N;

[0124] Y is selected from N or C, R1is absent when Y is N;

[0125] Z is selected from N;

[0126] R7is selected from H, F, Cl, Br;

[0127] R1is selected from H, methyl, ethyl, propyl, isopropyl, wherein said alkyl is unsubstituted or optionally substituted with F, Cl, Br;

[0128] R2is selected from H, F, Cl, Br, methyl, ethyl, propyl, isopropyl;

[0129] R3is selected from phenyl, said phenyl unsubstituted or substituted with 1 to 5 substituents each independently selected from methyl, ethyl, propyl, isopropyl, phenyl, pyridyl, said alkyl, phenyl, pyridyl optionally substituted with F, Cl, Br, methyl, ethyl, propyl, isopropyl;

[0130] R4is selected from H, F, Cl, Br, methyl, ethyl, propyl, isopropyl;

[0131] R5is selected from methyl, ethyl, propyl, isopropyl, CF3, CF2H, CFH2;

[0132] R6is selected from H, F, Cl, Br, methyl, ethyl, propyl, isopropyl.

[0133] In certain preferred embodiments of the present application, the compound as described by formula I has the structure of formula Ib:

[0134]

[0135] wherein,

[0136] X is selected from N or C, and when X is N, R7 is absent;

[0137] Y is selected from C, and Z is selected from N;

[0138] R7 is selected from H, F;

[0139] R1 is selected from methyl;

[0140] R2 is selected from H;

[0141] R3 is selected from

[0142] R4 is selected from Cl;

[0143] R5 is selected from methyl;

[0144] R6 is selected from H, F.

[0145] In certain preferred embodiments of the present application, the compound according to Formula I is any one of the following:

[0146]

[0147]

[0148]

[0149] The compounds according to Formula I described herein can exist in stereoisomeric or tautomeric forms, depending on the type of substituents. The present application contemplates both such isomers and mixtures thereof.

[0150] In another aspect, the present application provides a pharmaceutical composition comprising a compound according to Formula I described above, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0151] In another aspect, the present application provides the use of a compound according to Formula I described above, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, for the manufacture of a medicament for preventing or treating a DNA polymerase theta mediated disease.

[0152] In certain preferred embodiments of the present application, the DNA polymerase theta mediated disease is cancer.

[0153] In certain preferred embodiments of the present application, the cancer is lung cancer, breast cancer, HR-deficient ovarian cancer, gastric cancer, prostate cancer, pancreatic cancer, or colon cancer.

[0154] Unless otherwise indicated herein, the terms used in the specification and claims have the following meanings.

[0155] The present application relates to the case where each substituent can be the same or different when the compound is substituted by a plurality of substituents.

[0156] The elements carbon, hydrogen, oxygen, sulfur, nitrogen or halogen involved in the groups and compounds described in the present application include their isotopic cases, and the elements carbon, hydrogen, oxygen, sulfur or nitrogen involved in the groups and compounds described in the present application are optionally further replaced by one or more of their corresponding isotopes, wherein the isotopes of carbon include 12 C, 13 C, 14 C, the isotopes of hydrogen include protium (H), deuterium (D, also called heavy hydrogen), tritium (T, also called super-heavy hydrogen), the isotopes of oxygen include 16 O, 17 O and 18 O, the isotopes of sulfur include 32 S, 33 S, 34 S and 36 S, the isotopes of nitrogen include 14 N and 15 N, the isotopes of fluorine 19 F, the isotopes of chlorine include 35 Cl and 37 Cl, the isotopes of bromine include 79 Br and 81 Br.

[0157] The term "optionally", "optional" or "optionally" means that the event or circumstance described subsequently can but need not occur, including the case where the event or circumstance occurs or does not occur. For example, "aryl optionally substituted with alkyl" means that alkyl can but need not be present, and the description includes the case where aryl is substituted with alkyl and the case where aryl is not substituted with alkyl.

[0158] "Substituted or unsubstituted" refers to the case where the group can be substituted or not substituted, and if it is not indicated in the present application that the group can be substituted, it means that the group is in the unsubstituted case.

[0159] "Substituted" refers to the case where one or more hydrogen atoms in the group are replaced by other groups, and if the group is replaced by hydrogen atoms, the group formed is the same as the group replaced by hydrogen atoms. The case where the group is substituted, for example, amino, C 1-4 alkyl, C 1-4 alkoxy, C 3-6 carbocyclic ring, 3- to 6-membered heterocyclic ring optionally further substituted by 0, 1, 2, 3 or 4 groups selected from H, F, Cl, Br, I, hydroxy, cyano, amino, C 1-4 alkyl or C 1-4Substitution of the alkyl group with substituents forms groups including, but not limited to, methyl, chloromethyl, trichloromethyl, hydroxymethyl, -CH2OCH3, -CH2SH, -CH2CH2CN, -CH2NH2, -NHOH, -NHCH3, -OCH2Cl, -OCH2OCH2CH3, -OCH2CH2NH2, -OCH2CH2SH, -OCH2CH2OH, 1-hydroxycyclopropyl, 2-hydroxycyclopropyl, 2-aminocyclopropyl, 4-methylfuryl, 2-hydroxyphenyl, 4-aminophenyl, phenyl.

[0160] "Alkyl" refers to saturated aliphatic hydrocarbon groups, both straight- chain and branched, containing one to twenty carbon atoms. Preferred are alkyl groups containing one to ten carbon atoms, non-limiting examples of which include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, n-pentyl, n-nonyl, and various branched isomers thereof; more preferred are lower alkyl groups containing one to four carbon atoms, non-limiting examples of which include methyl, ethyl, propyl, i-propyl, n-butyl, i-butyl, or t-butyl, and the like. Alkyl groups can be substituted or unsubstituted, and when substituted, the substituents are preferably one to five, independently selected from F, Cl, Br, I, =0, alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, thiol, hydroxyl, nitro, cyano, amino, alkylacylamino, cycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthiol, hydroxyalkyl, carboxyl, carboxylate, heterocycloalkylthiol.

[0161] "Cycloalkyl" refers to a fully saturated carbon ring, which exists in the form of a monocyclic, fused ring, bridged ring, or spirocyclic ring, and the like. Unless otherwise indicated, the carbon ring is typically a 3- to 10-membered ring. The term "C3-C10cycloalkyl" can be understood to mean a saturated monocyclic, fused ring, bridged ring, or spirocyclic ring having 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. Specific examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, norbornyl (bicyclo[2.2.1]heptyl), bicyclo[2.2.2]octyl, adamantyl, spiro[4.5]decyl, and the like. The term "C3-C6cycloalkyl" can be understood to mean a saturated monocyclic or bicyclic hydrocarbon ring having 3, 4, 5, or 6 carbon atoms, specific examples of which include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, and the like. 10 "Cycloalkyl" refers to a fully saturated carbon ring, which exists in the form of a monocyclic, fused ring, bridged ring, or spirocyclic ring, and the like. Unless otherwise indicated, the carbon ring is typically a 3- to 10-membered ring. The term "C3-C10cycloalkyl" can be understood to mean a saturated monocyclic, fused ring, bridged ring, or spirocyclic ring having 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. Specific examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, norbornyl (bicyclo[2.2.1]heptyl), bicyclo[2.2.2]octyl, adamantyl, spiro[4.5]decyl, and the like. The term "C3-C6cycloalkyl" can be understood to mean a saturated monocyclic or bicyclic hydrocarbon ring having 3, 4, 5, or 6 carbon atoms, specific examples of which include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, and the like. 10 "Cycloalkyl" refers to a fully saturated carbon ring, which exists in the form of a monocyclic, fused ring, bridged ring, or spirocyclic ring, and the like. Unless otherwise indicated, the carbon ring is typically a 3- to 10-membered ring. The term "C3-C10cycloalkyl" can be understood to mean a saturated monocyclic, fused ring, bridged ring, or spirocyclic ring having 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. Specific examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, norbornyl (bicyclo[2.2.1]heptyl), bicyclo[2.2.2]octyl, adamantyl, spiro[4.5]decyl, and the like. The term "C3-C6cycloalkyl" can be understood to mean a saturated monocyclic or bicyclic hydrocarbon ring having 3, 4, 5, or 6 carbon atoms, specific examples of which include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, and the like.

[0162] "Alkoxy" refers to -O-alkyl, wherein alkyl is as defined herein above. Alkoxy groups can be substituted or unsubstituted, non-limiting examples of which include methoxy, ethoxy, propoxy, isopropoxy, butoxy, t-butoxy, pentoxy, or hexoxy, preferably having 1 to 12 members. When substituted, the substituents are preferably 1 to 5, independently selected from F, Cl, Br, I, =0, alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, thiol, hydroxyl, nitro, cyano, amino, alkylacylamino, cycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthiol, hydroxyalkyl, carboxyl, carboxylate, or heterocycloalkylthiol.

[0163] "Heterocyclyl" refers to a monocyclic, bicyclic, spiro, or bridged ring radical whose ring atoms contain one, two, three, four, or five heteroatoms or heteroatom groups (i.e., groups of atoms that contain heteroatoms), including, but not limited to, N, O, S, P, B, -S(=O)2-, -S(=O)-, -P(=O)2-, -P(=O)-, -NH-, -S(=O)(=NH)-, -C(=O)NH-, or -NHC(=O)NH-, and the like. The term "3-18 membered heterocyclyl" refers to a heterocyclyl radical having a number of ring atoms from 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18, and having one, two, three, four, or five ring atoms independently selected from the heteroatoms or heteroatom groups described above. The term "4-12 membered heterocyclyl" refers to a heterocyclyl radical having a number of ring atoms from 4, 5, 6, 7, 8, 9, 10, 11, or 12, and having one, two, three, four, or five ring atoms independently selected from the heteroatoms or heteroatom groups described above. The term "4-10 membered heterocyclyl" refers to a heterocyclyl radical having a number of ring atoms from 4, 5, 6, 7, 8, 9, or 10, and having one, two, three, four, or five ring atoms independently selected from the heteroatoms or heteroatom groups described above, where specific examples of 4-membered heterocyclyl groups include, but are not limited to, azetidinyl, thietanyl, or oxetanyl; specific examples of 5-membered heterocyclyl groups include, but are not limited to, tetrahydrofuranyl, dioxolyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl, 4,5-dihydrooxazolyl, or 2,5-dihydro-lH-pyrrolyl; specific examples of 6-membered heterocyclyl groups include, but are not limited to, tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl, trithianyl, tetrahydropyridinyl, or 4H-[l,3,4]thiadiazinyl; specific examples of 7-membered heterocyclyl groups include, but are not limited to, diazepanyl. Specific examples of 8-membered heterocyclyl groups include, but are not limited to, hexahydrocyclopenta[c]pyrrol-2(lH)-yl. Specific examples of 9-membered heterocyclyl groups include, but are not limited to, 2,3-dihydrobenzofuranyl. Specific examples of 10-membered heterocyclyl groups include, but are not limited to, 1,4-benzodioxanyl, benzomorpholinyl, 1,2,3,4-tetrahydroquinolinyl. The heterocyclyl group can also be a bicyclic group, where specific examples of 5,5 membered bicyclic groups include, but are not limited to, hexahydrocyclopenta[c]pyrrol-2(lH)-yl; specific examples of 5,6 membered bicyclic groups include, but are not limited to, hexahydropyrrolo[l,2-a]pyrazin-2(lH)-yl, 5,6,7,8-tetrahydro-[l,2,4]triazolo[4,3-a]pyrazinyl, or 5,6,7,8-tetrahydroimidazo[l,5-a]pyrazinyl. Optionally, the heterocyclyl group can be a benzo-fused ring of the above 4-7 membered heterocyclyl groups, specific examples include, but are not limited to, dihydroisoquinolinyl, benzo[d][l,3]dioxolyl, and the like."4-10 membered heterocyclyl" can include the range of "5-10 membered heterocyclyl", "4-7 membered heterocyclyl", "5-6 membered heterocyclyl", "6-8 membered heterocyclyl", "4-10 membered heterocycloalkyl", "5-10 membered heterocycloalkyl", "4-7 membered heterocycloalkyl", "5-6 membered heterocycloalkyl", "6-8 membered heterocycloalkyl", and the like. "4-7 membered heterocyclyl" can further include the range of "4-6 membered heterocyclyl", "5-6 membered heterocyclyl", "4-7 membered heterocycloalkyl", "4-6 membered heterocycloalkyl", "5-6 membered heterocycloalkyl", and the like.

[0164] "Aryl" refers to an all-carbon monocyclic or fused ring poly cyclic, aromatic ring moiety, having a conjugated pi-electron system. Aryl groups can have 6-20 carbon atoms, 6-14 carbon atoms, or 6-10 carbon atoms. The term "C6-C 14 Aryl" can be understood as aryl having 6 to 14 carbon atoms. In particular a ring having 6 carbon atoms ("C6-aryl"), such as, for example, a phenyl group; or a ring having 9 carbon atoms ("C9-aryl"), such as, for example, an indane or indenyl group; or a ring having 10 carbon atoms ("C 10 Aryl") such as, for example, a tetrahydronaphthyl, dihydronaphthyl or naphthyl group; or a ring having 13 carbon atoms ("C 13 Aryl") such as, for example, a fluorenyl group; or a ring having 14 carbon atoms ("C 14 Aryl") such as, for example, a chrysenyl group. The term "C6-C 10 Aryl" can be understood as aryl having 6 to 10 carbon atoms. In particular a ring having 6 carbon atoms ("C6-aryl"), such as, for example, a phenyl group; or a ring having 9 carbon atoms ("C9-aryl"), such as, for example, an indane or indenyl group; or a ring having 10 carbon atoms ("C 10 Aryl") such as, for example, a tetrahydronaphthyl, dihydronaphthyl or naphthyl group.

[0165] "Heteroaryl" means a monocyclic or fused polycyclic ring system having aromaticity, which contains at least one ring atom selected from N, O, S, the remaining ring atoms being C. The term "5-10 membered heteroaryl" is understood to include monocyclic or bicyclic aromatic ring systems having 5, 6, 7, 8, 9 or 10 ring atoms, in particular 5 or 6 or 9 or 10 ring atoms, and which contain 1, 2, 3, 4 or 5, preferably 1, 2 or 3 heteroatoms independently selected from N, O and S. In particular, the heteroaryl group is selected from thienyl, furanyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl or thiadiazolyl and the like as well as their benzo derivatives, such as, for example, benzofuranyl, benzothienyl, benzothiazolyl, benzoxazolyl, benzisoxazolyl, benzimidazolyl, benzotriazolyl, indazolyl, indolyl or isoindolyl and the like; or pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl or triazinyl and the like as well as their benzo derivatives, such as, for example, quinolinyl, quinazolinyl or isoquinolinyl and the like; or azocinyl, indolizinyl, purinyl and the like as well as their benzo derivatives; or cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, pteridinyl, carbazolyl, acridinyl, phenoxazinyl, phenothiazinyl or phenoxazinyl and the like. The term "5-6 membered heteroaryl" means an aromatic ring system having 5 or 6 ring atoms, and which contains 1, 2 or 3, preferably 1-2 heteroatoms independently selected from N, O and S. The term "9-10 membered heteroaryl" means an aromatic ring system having 9 or 10 ring atoms, and which contains 1, 2 or 3, preferably 1-2 heteroatoms independently selected from N, O and S. "6 membered heteroaryl" means an aromatic ring system having 6 ring atoms, and which contains 1, 2 or 3, preferably 1-2 heteroatoms independently selected from N, O and S.

[0166] "=0" is the usual custom in the art and means an oxygen atom connected by a double bond, such as the double-bonded oxygen atom in a carbonyl group, which is connected to a carbon atom.

[0167] "stereoisomers" means isomers that differ in the configuration of a stereocenter, i.e., an atom that is attached to four non-identical groups or atoms. Stereoisomers include enantiomers (mirror image isomers) and diastereomers (isomers that are not mirror image isomers).

[0168] "pharmaceutically acceptable" means relatively non-toxic, safe, and suitable for use with patients.

[0169] "Pharmaceutically acceptable salts" refer to salts obtained by reacting a compound with a pharmaceutically acceptable acid or base. When a compound contains a relatively acidic functional group, a base addition salt can be obtained by contacting the compound with a sufficient amount of a pharmaceutically acceptable base in a suitable inert solvent. Pharmaceutically acceptable base addition salts include, but are not limited to, sodium salts, potassium salts, calcium salts, aluminum salts, magnesium salts, bismuth salts, and ammonium salts. When a compound contains a relatively basic functional group, an acid addition salt can be obtained by contacting the compound with a sufficient amount of a pharmaceutically acceptable acid in a suitable inert solvent. Pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochloride salts, sulfate salts, trifluoroacetate salts, formate salts, and methanesulfonate salts.

[0170] "Pharmaceutical composition" means a mixture of one or more compounds of this application or their salts with pharmaceutically acceptable excipients. The purpose of a pharmaceutical composition is to facilitate the administration of the compounds of this application to an organism.

[0171] "Pharmaceutical acceptable excipients" refer to those excipients that do not cause significant irritation to the organism and do not impair the biological activity and properties of the active compound. Suitable excipients are well known to those skilled in the art, such as carbohydrates, water-soluble and / or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, etc. Detailed Implementation

[0172] The technical solution of the present invention will be described in detail below through embodiments, but the scope of protection of the present invention includes, but is not limited to, these embodiments. This application has been described in detail herein, and specific embodiments thereof have also been disclosed. It will be apparent to those skilled in the art that various changes and improvements can be made to the specific embodiments of this application without departing from the spirit and scope of this application.

[0173] The structure of the compound was determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). NMR shifts (δ) were expressed in 10⁻¹⁰. -6 The unit (ppm) is given. The solvents used for NMR determination are deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), etc., and the internal standard is tetramethylsilane (TMS).

[0174] Thin-layer chromatography uses GF254 silica gel plates with a thickness of 0.9 mm to 1 mm. Column chromatography typically uses 100–200 mesh or 200–300 mesh silica gel as the support.

[0175] The known starting materials of this invention can be synthesized using or according to methods known in the art, or obtained by purchasing commercially available materials.

[0176] "Nitrogen protection" refers to connecting a nitrogen balloon with a volume of approximately 1L to the reaction flask.

[0177] Unless otherwise specified, the reaction temperature in the examples is room temperature, and the room temperature is 20-30°C.

[0178] Unless otherwise specified, the ratio indicated by the mixed solvent represents the volume mixing ratio. For example, "petroleum ether: ethyl acetate = 1:2" indicates that the volume ratio of petroleum ether and ethyl acetate is 1:2.

[0179] Intermediate I-1: 5-(5-bromo-2-methoxyphenyl)pyridazine-4-carboxylic acid

[0180]

[0181] Into a 100 mL flask, compound I-1-1 (5.5 g, 44.32 mmol), N,N-dimethylformamide (60 mL), dimethylamine hydrochloride (7.23 g, 88.66 mmol), triethylamine (16.1 g, 159.12 mmol) and HATU (20.22 g, 53.18 mmol) were added successively, and the reaction solution was stirred at room temperature for 24 hours. After concentration under reduced pressure, the residue was separated by silica gel column chromatography to obtain intermediate I-1-2 (6.69 g, yield 99.9%, the product had a large amount of triethylamine residue, and no further purification was needed before being used in the next step).

[0182] Into a 500 mL flask, intermediate I-1-2 (6.69 g, 44.25 mmol), tetrahydrofuran (100 mL) were added successively under nitrogen protection, and the reaction solution was cooled to 0°C. A 2-methoxyphenyl magnesium bromide tetrahydrofuran solution (1 N, 44 mL) was added dropwise. After the addition was completed, the reaction solution was stirred at room temperature for 15 hours. The solvent was removed by concentration under reduced pressure, and ethyl acetate (300 mL), acetyl iodobenzene (24.2 g, 75.13 mmol) were added. The reaction solution was continuously stirred at room temperature for 3 hours. The reaction solution was washed successively with saturated Na2S2O3 aqueous solution (100 mL*2), water (50 mL), and saturated brine (50 mL), and dried over anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure, and separated by silica gel column chromatography to obtain intermediate I-1-3 (2.45 g, yield 16.5%).

[0183] Into a 100 mL flask, intermediate I-1-3 (2.0 g, 5.95 mmol), ethanol (20 mL), water (20 mL), and potassium hydroxide (2.2 g, 39.21 mmol) were added successively. The reaction solution was heated to 100°C, and reacted for 24 hours. After being cooled to room temperature, the pH was adjusted to 2 with 4N hydrochloric acid aqueous solution. Ethanol was removed by concentration, and a solid was precipitated. After filtration, the solid was washed with water (20 mL), and dried to obtain intermediate I-1 (1.67 g, yield 90.8%).

[0184]

[0185] Into a 250 mL jar flask, 3-bromoisatnic acid methyl ester (I-2-1, 2.12 g, 9.81 mmol), 1,4-dioxane (60 mL), water (12 mL), 2-fluoro-6-methoxybenzoic acid (2 g, 11.77 mmol), Pd(dppf)Cl2(717 mg, 0.98 mmol), potassium carbonate (2.71 g, 19.61 mmol) were added successively, and the reaction solution was heated to 90 °C for 2 h under nitrogen protection. The solvent was removed by concentration under reduced pressure, water (50 mL) was added, and the mixture was extracted with ethyl acetate (100 mL*3). The organic phase was combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was separated by silica gel column chromatography to obtain intermediate I-2-2 (2.16 g, yield 84.2%).

[0186] Into a 100 mL jar flask, intermediate I-2-2 (650 mg, 2.49 mmol) and 5 mL of methanol solution were added, and sodium hydroxide aqueous solution (398 mg of sodium hydroxide dissolved in 5 mL of water) was added dropwise to the mixed solution under stirring. The mixture was reacted at 40 °C for 3 h. The methanol was removed by concentration under reduced pressure, and the mixture was extracted with ethyl acetate (40 mL). The organic phase was discarded, and the aqueous phase was adjusted to pH 4-5 with dilute hydrochloric acid (2N). The mixture was extracted with ethyl acetate (30 mL*5), and the organic phase was combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain intermediate I-2 (320 mg, yield 52.0%). 1 H NMR (400 MHz, DMSO-d6) δ 13.25 (s, 1H), 8.71 (d, J = 5.0 Hz, 1H), 8.55 (dd, J = 1.4, 0.7 Hz, 1H), 7.77 (dd, J = 5.0, 0.8 Hz, 1H), 7.45-7.36 (m, 1H), 6.99-6.86 (m, 2H), 3.70 (s, 3H).

[0187] Intermediate I-3: 4-(5-cyano-2-methoxyphenyl)-6-methylnicotinic acid

[0188]

[0189] Into a 100 mL jar flask, 4-chloro-6-methyl nicotinic acid methyl ester (I-3-1, 2 g, 10.77 mmol), 1,4-dioxane (30 mL), 5-cyano-2-methoxyphenyl boronic acid (2 g, 11.30 mmol), Pd(dppf)Cl2(400 mg, 0.55 mmol), potassium carbonate (4.46 g, 32.27 mmol) and water (6 mL) were added successively; nitrogen protection, 90 °C for 2 h; the solvent was removed by concentration under reduced pressure, water (50 mL) was added, and the organic phase was extracted with ethyl acetate (100 mL*3), the organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure; the residue was separated by silica gel column chromatography to obtain intermediate I-3-2 (2.8 g, yield 92.0%).

[0190] Into a 100 mL jar flask, intermediate I-3-2 (2.8 g, 9.92 mmol) and 15 mL of methanol solution were added, and sodium hydroxide aqueous solution (1.58 g of sodium hydroxide dissolved in 15 mL of water) was added dropwise under stirring; the reaction system was heated to 40 °C, and reacted for 3 h; methanol was removed by concentration under reduced pressure, extracted with ethyl acetate (40 mL), and the organic phase was discarded; the aqueous phase was adjusted to pH 4-5 with dilute hydrochloric acid (2N), extracted with ethyl acetate (30 mL*5), the organic phase was combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain intermediate I-3 (1.5 g, yield 56.4%). 1 H NMR (400 MHz, DMSO-d6) δ 12.84 (s, 1H), 8.84 (s, 1H), 7.89 (dd, J = 8.6, 2.2 Hz, 1H), 7.74 (d, J = 2.1 Hz, 1H), 7.30 (s, 1H), 7.24 (d, J = 8.7 Hz, 1H), 3.76 (s, 3H), 2.56 (s, 3H).

[0191] Intermediate I-4: 2'-chloro-5'-methoxy-6-methyl-[4,4'-bipyridine]-3-carboxylic acid

[0192]

[0193] Into a 250 mL flask, add 4-bromo-6-methylnicotinic acid methyl ester (I-4-1, 2.05 g, 8.91 mmol), (2-chloro-5-methoxypyridin-4-yl)boronic acid (2.0 g, 10.67 mmol), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (651 mg, 0.89 mmol), potassium carbonate (2.46 g, 17.80 mmol), dioxane (60 mL), water (12 mL), and protect with nitrogen. Heat the reaction system to 90 °C and react for 2 hours. Remove the solvent by concentration under reduced pressure. Add water (50 mL) and extract with ethyl acetate (100 mL*3). Combine the organic phases and wash with water (50 mL) and saturated sodium chloride aqueous solution (50 mL) successively. Dry over anhydrous sodium sulfate, concentrate under reduced pressure, and separate the residue by silica gel column chromatography to obtain intermediate I-4-2 (2.3 g, yield 88.2%).

[0194] Into a 100 mL flask, add intermediate I-4-2 (500 mg, 1.71 mmol) and 5 mL of methanol. Under stirring, add sodium hydroxide aqueous solution (273 mg of sodium hydroxide dissolved in 5 mL of water) dropwise. React at 40 °C for 3 hours. Remove methanol by concentration under reduced pressure. Extract with ethyl acetate (40 mL) and discard the organic phase. Adjust the pH of the aqueous phase to 4-5 with dilute hydrochloric acid (2N) and extract with ethyl acetate (30 mL*5). Combine the organic phases, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain intermediate I-4 (390 mg, yield: 81.9%). 1 H NMR (400 MHz, DMSO-d6) δ 13.05 (s, 1H), 8.89 (s, 1H), 8.21 (s, 1H), 7.44 (s, 1H), 7.30 (s, 1H), 3.78 (s, 3H), 2.56 (s, 3H).

[0195] Intermediate I-5~I-6

[0196] Intermediate I-5 or I-6 is prepared in a similar manner as intermediate I-4, using 2-fluoro-6-methoxybenzeneboronic acid or 5-chloro-2-methoxybenzeneboronic acid as the starting material.

[0197]

[0198]

[0199] Intermediate I-7: 5'-Methoxy-2',6-dimethyl-[4,4'-bipyridin]-3-carboxylic acid

[0200]

[0201] Into a 100 mL vial, was placed compound I-4-2 (1.0 g, 3.42 mmol), ethylene glycol dimethyl ether (30 mL), trimethyl cyclo-trioxonium (1.29 g, 10.28 mmol), potassium carbonate (1.18 g, 8.54 mmol), Pd(dppf)Cl2(250 mg, 0.34 mmol), the reaction system was heated to 100 °C under nitrogen protection and stirred for 2 h; the solvent was removed by concentration under reduced pressure, water (30 mL) was added, and the mixture was extracted with dichloromethane (50 mL*3), the organic phases were combined, washed with water (30 mL) and saturated brine (30 mL), and concentrated under reduced pressure. The residue was separated by silica gel column chromatography to obtain intermediate I-7-1 (713 mg, yield: 76.7%).

[0202] Intermediate I-7-1 (300 mg, 1.1 mmol) was dissolved in anhydrous methanol (6 mL), and sodium hydroxide aqueous solution (132 mg of sodium hydroxide dissolved in 1.5 mL of water) was added dropwise under stirring. The reaction system was heated to 45 °C and stirred for 2 h; most of the methanol was removed by concentration under reduced pressure, and the residue was adjusted to pH 3 with dilute hydrochloric acid (2N), and the solvent was removed by concentration under reduced pressure; 15 mL of dichloromethane / methanol solution (volume ratio 10:1) was added to the residue, stirred for 10 min, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent; 15 mL of ethyl acetate was added to the residue, stirred for 10 min, filtered, and the filter cake was collected and dried to obtain intermediate I-7 (250 mg, yield: 87.9%). 1 H NMR (400 MHz, DMSO-d6) δ 13.34 (s, 1H), 8.97 (s, 1H), 8.49 (s, 1H), 7.78 (s, 1H), 7.35 (s, 1H), 3.86 (s, 3H), 2.69 (s, 3H), 2.60 (s, 3H).

[0203] Intermediate I-8: 2'-cyclopropyl-5'-methoxy-6-methyl-[4,4'-bipyridine]-3-carboxylic acid

[0204]

[0205] Intermediate I-4-2 (2.0 g, 6.83 mmol) and cyclopropylboronic acid (5.85 g, 68.1 mmol) were dissolved in ethylene glycol dimethyl ether (40 mL), potassium carbonate (1.89 g, 13.67 mmol), Pd(dppf)Cl2(500 mg, 0.68 mmol) were added, the reaction system was protected by nitrogen, heated to 100 °C and stirred for 3 h; the solvent was removed by concentration under reduced pressure, water (50 mL) was added, dichloromethane was extracted (100 mL*3), the organic phase was combined, washed with water (50 mL), saturated brine (50 mL), concentrated under reduced pressure, and the residue was separated by silica gel column chromatography to obtain intermediate I-8-1 (1.38 g, yield 67.7%).

[0206] Intermediate I-8-1 (300 mg, 1 mmol) was dissolved in anhydrous methanol (6 mL), and an aqueous solution of sodium hydroxide (132 mg of sodium hydroxide dissolved in 1.5 mL of water) was added dropwise under stirring conditions, the reaction system was heated to 45 °C and stirred for 2 h; most of the methanol was removed by concentration under reduced pressure, the residue was adjusted to pH 3 with dilute hydrochloric acid (2N), and the solvent was removed by concentration under reduced pressure; the residue was added to 15 mL of dichloromethane / methanol solution (volume ratio 10:1), stirred for 10 min, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent; the residue was added to 15 mL of ethyl acetate, stirred for 10 min, filtered, and the filter cake was collected and dried to obtain intermediate I-8 (270 mg, yield 94.4%). 1 HNMR (400 MHz, DMSO-d6) δ 8.97 (s, 1H), 8.36 (s, 1H), 7.51 (s, 1H), 7.44 (s, 1H), 3.84 (s, 3H), 2.62 (s, 3H), 2.49-2.40 (m, 1H), 1.28-1.19 (m, 2H), 1.19-1.10 (m, 2H). Intermediate I-9:

[0207]

[0208] Compound I-9-1 (4 g, 24.91 mmol) was dissolved in 80 mL of anhydrous tetrahydrofuran, the reaction system was cooled to below -70 °C under nitrogen protection, LDA (2M in THF, 25 mL) was added dropwise, after the addition was completed, the reaction was continued at -70 °C for 1 hour, triisopropyl borate (9.4 g, 49.98 mmol) was added, and the reaction was continued at -70 °C for 2 hours. 30 mL of saturated aqueous ammonium chloride solution was added to the reaction solution, hydrochloric acid (2N) was used to adjust the pH to 2-3, and ethyl acetate was extracted (100 mL*3), the organic phase was combined, washed with water (30 mL), saturated brine (30 mL), and concentrated under reduced pressure to obtain compound I-9-2 (4.2 g, yield: 82.49%).

[0209] Into a 100 mL of a flask, 4-bromo-6-methyl nicotinic acid methyl ester (2.5 g, 10.87 mmol), (3-chloro-2-fluoro-6-methoxyphenyl)boronic acid (I-9-2, 3.2 g, 15.66 mmol), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (795 mg, 1.09 mmol), potassium carbonate (3 g, 21.71 mmol), dioxane (25 mL), water (5 mL), and nitrogen protection were added sequentially, the reaction system was heated to 90 °C, and the reaction was performed for 3 h; the solvent was removed by concentration under reduced pressure, water (40 mL) was added, extraction was performed with ethyl acetate (50 mL*3), the organic phases were combined, washed with water (50 mL) and saturated sodium chloride aqueous solution (50 mL) sequentially, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was separated by silica gel column chromatography to obtain intermediate I-9-3 (2.7 g, yield: 80.22%).

[0210] Into a 100 mL of a flask, 4-bromo-6-methyl nicotinic acid methyl ester (2.5 g, 10.87 mmol), (3-chloro-2-fluoro-6-methoxyphenyl)boronic acid (I-9-2, 3.2 g, 15.66 mmol), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (795 mg, 1.09 mmol), potassium carbonate (3 g, 21.71 mmol), dioxane (25 mL), water (5 mL), and nitrogen protection were added sequentially, the reaction system was heated to 90 °C, and the reaction was performed for 3 h; the solvent was removed by concentration under reduced pressure, water (40 mL) was added, extraction was performed with ethyl acetate (50 mL*3), the organic phases were combined, washed with water (50 mL) and saturated sodium chloride aqueous solution (50 mL) sequentially, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was separated by silica gel column chromatography to obtain intermediate I-9-3 (2.7 g, yield: 80.22%). 1 H NMR (400 MHz, CDCl3) δ 9.18 (s, 1H), 7.38 (t, J = 8.6 Hz, 1H), 7.15 (s, 1H), 6.70 (d, J = 8.9 Hz, 1H), 3.73 (s, 3H), 2.67 (s, 3H).

[0211] Intermediate I-10:

[0212]

[0213] Into a 100 mL of a flask, 4-bromo-6-methyl nicotinic acid methyl ester (2.5 g, 10.87 mmol), (3-chloro-2-fluoro-6-methoxyphenyl)boronic acid (I-9-2, 3.2 g, 15.66 mmol), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (795 mg, 1.09 mmol), potassium carbonate (3 g, 21.71 mmol), dioxane (25 mL), water (5 mL), and nitrogen protection were added sequentially, the reaction system was heated to 90 °C, and the reaction was performed for 3 h; the solvent was removed by concentration under reduced pressure, water (40 mL) was added, extraction was performed with ethyl acetate (50 mL*3), the organic phases were combined, washed with water (50 mL) and saturated sodium chloride aqueous solution (50 mL) sequentially, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was separated by silica gel column chromatography to obtain intermediate I-9-3 (2.7 g, yield: 80.22%).

[0214] Into a 100 mL flask, was added intermediate I-10-2 (2.1 g, 8.77 mmol), 6-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)nicotinate methyl ester (2.8 g, 10.10 mmol), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (641 mg, 0.88 mmol), potassium carbonate (2.42 g, 17.51 mmol), dioxane (30 mL), water (6 mL), and the reaction system was heated to 85 °C for 3 h under nitrogen protection. The solvent was removed by concentration under reduced pressure, water (50 mL) was added, and the organic phase was extracted with ethyl acetate (100 mL*3). The combined organic phase was washed with water (50 mL), saturated sodium chloride aqueous solution (50 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was separated by silica gel column chromatography to give intermediate I-10-3 (1.8 g, yield: 66.27%).

[0215] Into a 100 mL flask, was added intermediate I-10-3 (420 mg, 1.36 mmol) and 10 mL of methanol solution, and sodium hydroxide aqueous solution (217 mg of sodium hydroxide dissolved in 10 mL of water) was added dropwise under stirring. The reaction was carried out at 40 °C for 3 h. The methanol was removed by concentration under reduced pressure, and the organic phase was extracted with dichloromethane (10 mL*3). The organic phase was discarded, and the aqueous phase was adjusted to pH 4-5 with dilute hydrochloric acid (2N). A solid was precipitated, and the filtrate was collected by suction filtration, washed with water, and dried to give compound I-10 (300 mg, yield: 74.82%). 1 H NMR (400 MHz, DMSO-d6) δ 8.87 (s, 1H), 7.53 (d, J = 8.4 Hz, 1H), 7.46 (s, 1H), 7.26 (d, J = 11.5 Hz, 1H), 3.71 (s, 3H), 2.60 (s, 3H).

[0216] Intermediate II-1: 2-(ethylsulfonyl)-6-(4-chlorophenyl)-thiazolo[4,5-d]pyrimidin-7(6H)-one

[0217]

[0218] Into a 100 mL flask, was added intermediate I-10-2 (2.1 g, 8.77 mmol), 6-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)nicotinate methyl ester (2.8 g, 10.10 mmol), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (641 mg, 0.88 mmol), potassium carbonate (2.42 g, 17.51 mmol), dioxane (30 mL), water (6 mL), and the reaction system was heated to 85 °C for 3 h under nitrogen protection. The solvent was removed by concentration under reduced pressure, water (50 mL) was added, and the organic phase was extracted with ethyl acetate (100 mL*3). The combined organic phase was washed with water (50 mL), saturated sodium chloride aqueous solution (50 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was separated by silica gel column chromatography to give intermediate I-10-3 (1.8 g, yield: 66.27%).

[0219] Into a 100 mL flask, add intermediate II-1-1 (5 g, 39.2 mmol) and 20 mL of dichloromethane solution, add triethylamine (5 g, 49.4 mmol), cool the system to 0 °C, and drop the chloroacetyl chloride solution (3.75 mL, 47.1 mmol) into the mixed solution under stirring, and stir at room temperature for 12 hours; add water (50 mL) into the reaction solution, extract with dichloromethane (100 mL*3), combine the organic phase, wash with saturated brine (50 mL), dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain intermediate II-1-2 (5.44 g, yield 68.1%).

[0220] Dissolve intermediate II-1-2 (2.04 g, 10 mmol) in 20 mL of acetone, add the aqueous solution of compound II-1-3 (1.96 g of compound II-1-3 dissolved in 20 mL of water), add aqueous sodium hydroxide solution (0.4 g of sodium hydroxide dissolved in 5 mL of water), stir at room temperature for 2 hours, and then heat to 80 °C for 0.5 hours; cool to room temperature; drop iodomethane (1.56 g, 10 mmol) into the reaction solution, and stir at room temperature for 12 hours; pour the reaction solution into cold water (100 mL), filter, wash with water (30 mL), collect the filter cake, and dry to obtain intermediate II-1-4 (2.2 g, yield 70.1%).

[0221] Heat compound II-1-4 (2.2 g, 7.01 mmol) in formic acid (10 mL) to reflux for 2 hours, cool to room temperature, and precipitate yellow solid; add water (50 mL), precipitate solid, filter, wash with water (30 mL), collect the filter cake, and dry to obtain compound II-1-5 (2.16 g, yield 95.2%).

[0222] Into a 50 mL flask, add 10 mL of ethanol solution, add II-1-5 (100 mg, 0.31 mmol) and Na2WO4 (23 mg, 0.08 mmol), add 1 mL of H2O2 under stirring, react at room temperature for 1 hour, and then heat to 50 °C for 1 hour; cool to room temperature, pour the reaction solution into 20 mL of ice water, precipitate solid, filter, wash the filter cake with 5 mL of water, collect the filter cake, and dry to obtain intermediate II-1 (50 mg, yield 45.5%). 1 HNMR (400 MHz, CDCl3) δ 8.33 (s, 1H), 7.63-7.53 (m, 2H), 7.42-7.34 (m, 2H), 3.61 (q, J = 7.4 Hz, 2H), 1.47 (t, J = 7.4 Hz, 3H).

[0223] Intermediate II-2 to II-17

[0224] Intermediate II-2 to II-17 were prepared in a similar manner to that of Intermediate II-1 using p-cyanylaniline, p-methoxyaniline, aniline, 2-methylaniline, 4-cyanomethylaniline, 1-naphthylamine, 1-methyl-1H-pyrazol-4-amine, p-bromoaniline, 4-chloro-2-methylaniline, 2,3-dihydrobenzo[b][1,4]dioxan-5-amine, 4-chloro-2,6-dimethylaniline, 4-chloro-2,6-dicyclopropylaniline, 2,6-dimethylaniline, 2,3-dimethylaniline, 2-chloro-6-methylaniline, 4-cyano-2,6-dimethylaniline as the starting material

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231] Intermediate II-18: 2-(ethylsulfonyl)-6-(4-(2-methoxyethoxy)phenyl)thiazolo[4,5- d]pyrimidin-7(6H)-one

[0232]

[0233] Compound II-18-1 was prepared in a similar manner to that of Intermediate II-1-5 using 4-(2-methoxyethoxy)aniline as the starting material.

[0234] Compound II-18-1 (525 mg, 1.44 mmol) was dissolved in 10 ml of dichloromethane, and m-chloroperoxybenzoic acid (879 mg, 5.09 mmol) was added with stirring, and the reaction was allowed to proceed at room temperature for 5 hours. Upon completion of the reaction, which was confirmed by TLC, the reaction mixture was filtered, and the filtrate was washed with an aqueous sodium thiosulfate solution (30 mL x 2), a saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure, and the resulting product was separated by thin layer chromatography to obtain Compound II-18 (340 mg, yield 59.5%). 1 H NMR (400 MHz, CDC13) δ 8.34 (s, 1H), 7.36-7.30 (m, 2H), 7.13-7.07 (m, 2H), 4.27-4.16 (m, 2H), 3.83-3.77 (m, 2H), 3.61 (q, J = 7.5 Hz, 2H), 3.47 (s, 3H), 1.47 (t, J = 7.4 Hz, 3H).

[0235] Intermediate II-19: 2-(ethylsulfonyl)-5-methyl-6-(1-naphthyl)thiazolo[4,5-d]pyrimidin-7(6H)-one

[0236]

[0237] Compound II-19-1 was prepared in a similar manner as that of Intermediate II-1-4, using 1-naphthylamine as the starting material.

[0238] Compound II-19-1 (1.2 g) was weighed into a sealed tube with 10 mL of triethyl orthoacetate, and 0.3 mL of hydrochloric acid was added dropwise with stirring. The reaction was carried out at 140 °C for 3 hours. TLC detection showed that the reaction was completed, and the reaction solution was concentrated under reduced pressure. Compound II-19-2 (770 mg, yield 60.1%) was obtained by thin layer chromatography.

[0239] Compound II-19-2 (300 mg, 0.85 mmol) was dissolved in 10 mL of dichloromethane, and m-chloroperbenzoic acid (517 mg, 2.99 mmol) was added with stirring. The reaction was carried out at room temperature for 5 hours. TLC detection showed that the reaction was completed, and the filtrate was washed with an aqueous solution of sodium thiosulfate (30 mL*2), a saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Compound II-19 (180 mg, yield 55.0%) was obtained by thin layer chromatography. 1 H NMR (400 MHz, CDCl3) δ 8.08 (d, J = 8.7 Hz, 1H), 8.00-7.95 (m, 1H), 7.91 (dd, J = 8.0, 1.5 Hz, 1H), 7.93-7.89 (m, 1H), 7.67-7.58 (m, 2H), 7.31 (dd, J = 8.6, 2.1 Hz, 1H), 3.62 (q, J = 7.4 Hz, 2H), 2.39 (s, 3H), 1.48 (t, J = 7.4 Hz, 3H).

[0240] Intermediate II-20 to II-35

[0241] Intermediates II-20 to II-35 were prepared in a similar manner as that of Intermediate II-1, using the corresponding arylamine as the starting material

[0242]

[0243]

[0244]

[0245]

[0246]

[0247] Intermediate III-1: 2-amino-6-(4-cyanophenyl)-thiazolo[4,5-d]pyrimidin-7(6H)-one

[0248]

[0249] Compound II-2 (614 mg, 1.77 mmol) was weighed into a 30 mL sealed tube, and a solution of ammonia in dioxane (0.6 N, 5 mL) was added. The reaction was stirred at 65 °C for 5 h, cooled to room temperature, filtered, washed with water (20 mL), and the filter cake was dried to give compound III-1 (405 mg, 84.8% yield). 1 H NMR (400 MHz, DMSO-d6) δ 8.37 - 8.33 (m, 3H), 8.10 - 7.97 (m, 2H), 7.83 - 7.65 (m, 2H).

[0250] Intermediate III-2

[0251] Intermediate III-2, III-12, and III-13 were synthesized in a similar manner as Intermediate III-1

[0252]

[0253]

[0254] Intermediate III-3: 5-phenyl-4,5,6,7-tetrahydro-thiazolo[5,4-c]pyridin-2-amine

[0255]

[0256] To a 100 ml tomato bottle, 10 ml ethanol solution was added, and III-3-1 (200 mg, 1.14 mmol) and diethylamine (167 mg, 2.28 mmol) were added, stirred for 30 min, and sulfur (40 mg, 1.25 mmol) was added. The reaction system was cooled to 0 °C, and a solution of cyanamide in ethanol (58 mg, 1.37 mmol, 2 ml of ethanol) was added dropwise at this temperature. After the dropwise addition was completed, the reaction was warmed to 70 °C and reacted for 6 h. The reaction was monitored by TLC, and after the reaction was completed, the organic phase was concentrated under reduced pressure, extracted with ethyl acetate (30 mL*3), and the combined organic phase was washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was separated by silica gel column chromatography to give intermediate III-3 (80 mg, yield: 30.30%).

[0257] Intermediate III-4: 2-amino-7-phenyl-5H-[l,3,4]thiadiazolo[3,2- a]pyrimidin-5-one

[0258]

[0259] To a solution of compound III-4-1 (6.4 g, 33.29 mmol) in polyphosphoric acid (20 mL) was added compound 2-bromo-5-aminothiadiazole (5.0 g, 27.77 mmol) and heated to 100 °C for 8 hours. The reaction was monitored by TLC. After cooling to room temperature, the reaction mixture was diluted with water and extracted with ethyl acetate (60 mL*3). The organic phase was combined and washed with saturated brine (50 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel to give compound III-4-2 (723 mg, yield: 9.13%).

[0260] A solution of compound III-4-2 (300 mg, 0.97 mmol), 2,4-dimethoxybenzylamine (200 mg, 1.20 mmol), DIEA (250 mg, 1.93 mmol) in DMF (5 mL) was heated to 100 °C for 3 hours. The reaction was monitored by TLC. After cooling to room temperature, the solid was precipitated by water, filtered and dried to give compound III-4-3 (320 mg, yield: 83.33%).

[0261] A solution of 320 mg of compound III-4-3 in dichloromethane (5 mL) was stirred at room temperature for 24 hours. The reaction was monitored by TLC. After filtration, the solid was dried to give intermediate III-4 (150 mg, yield: 75.76%).

[0262] Intermediate III-5: 2-amino-6-(4-(pyridin-4-yl)phenyl)thiazolo[4,5-d]pyrimidin-7(6H)- one

[0263]

[0264] Intermediate III-5-1 was synthesized in a similar manner as intermediate III-1 using intermediate II-9 as the starting material.

[0265] Compound III-5-1 (400 mg, 1.24 mmol) was dissolved in 20 mL of acetonitrile, and then Boc2O (324 mg, 1.48 mmol), DMAP (15 mg, 0.12 mmol), and TEA (250 mg, 2.47 mmol) were added in sequence. The reaction was carried out at room temperature for 10 hours, and TLC was used to monitor the completion of the reaction. The reaction solution was concentrated under reduced pressure, and the residue was extracted with dichloromethane (20 mL*3). The combined organic phases were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was separated by silica gel column chromatography to obtain compound III-5-2 (400 mg, yield: 76.35%).

[0266] A 100 mL tomato bottle was sequentially added with III-5-2 (300 mg, 0.71 mmol), 1,4-dioxane (15 mL), water (4 mL), pyridin-4-ylboronic acid (113 mg, 0.92 mmol), Pd(dppf)Cl2(52 mg, 0.07 mmol), and potassium carbonate (196 mg, 1.42 mmol). The reaction solution was heated to 90°C under nitrogen protection for 3 hours. The solvent was removed by reduced pressure concentration, water (3 mL) was added, and extraction was performed with ethyl acetate (15 mL*3). The combined organic phases were washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was separated by silica gel column chromatography to obtain intermediate III-5-3 (200 mg, yield: 66.95%).

[0267] A dichloromethane solution (6 mL) of 200 mg of compound III-5-3 and trifluoroacetic acid (3 mL) was stirred at room temperature for 24 hours. TLC was used to monitor the completion of the reaction. Saturated aqueous sodium bicarbonate solution (15 mL) was added to the reaction solution, and a solid was precipitated. Filtration and drying of the solid yielded intermediate III-5 (127 mg, yield: 83.28%). 1 H NMR (400 MHz, DMSO-d6) δ 8.83 (d, J = 6.3 Hz, 2H), 8.37 (s, 1H), 8.34 (s, 2H), 8.11-8.03 (m, 4H), 7.71 (d, J = 8.5 Hz, 2H).

[0268] Intermediate III-6 to III-11 were synthesized in a similar manner as intermediate III-5, using the corresponding arylamine as the starting material

[0269]

[0270]

[0271]

[0272] Intermediate IV-1: 5-(5-bromo-2-methoxyphenyl)pyridazine-4-carboxamide

[0273]

[0274] Compound I-1 (400 mg, 1.29 mmol) was weighed into a 50 mL vial, to which was added 4 mL of dichloromethane and 1 mL of methanol solution, and a solution of TMSCHN2 in n-hexane (2 N, 0.8 mL, 1.6 mmol) was added dropwise with stirring, and the reaction was allowed to proceed at room temperature for 3 hours; the solution was concentrated under reduced pressure, water (20 mL) was added, and the organic phase was extracted with ethyl acetate (20 mL*3), the organic phases were combined, washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, and the solution was concentrated under reduced pressure; the residue was separated by silica gel column chromatography to obtain intermediate IV-1-1 (220 mg, yield 52.6%).

[0275] Compound IV-1-1 (220 mg, 0.68 mmol) was weighed into a 30 mL sealed tube, 5 mL of 7M ammonium methanol solution was added, and the reaction was allowed to proceed at 65°C for 4 hours; the reaction was cooled to room temperature, filtered, washed with water (5 mL), and the filter cake was collected and dried to obtain intermediate IV-1 (120 mg, yield 57.2%). 1 HNMR (400 MHz, DMSO-d6) δ 9.26 (s, 2H), 8.02 (s, 1H), 7.73 (s, 1H), 7.62 (dd, J = 8.8, 2.5 Hz, 1H), 7.54 (d, J = 2.6 Hz, 1H), 7.10 (d, J = 8.9 Hz, 1H), 3.72 (s, 3H).

[0276] Intermediate IV-2: 4-(5-cyano-2-methoxyphenyl)-6-methylnicotinamide

[0277]

[0278] A 100 mL vial was charged with compound I-3 (1.34 g, 4.99 mmol), DMF (10 mL), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 1.43 g, 7.46 mmol) and 1-hydroxybenzotriazole (HOBt, 1.35 g, 9.99 mmol) were added at 0°C with stirring for 5 min; ammonium chloride (1.6 g, 29.9 mmol) was added to the reaction vial, and stirring was continued for 5 min; finally, N,N-diisopropylethylamine (DIPEA, 2.58 g, 19.96 mmol) was added dropwise to the reaction solution, which was allowed to react at room temperature for 8 hours; 50 mL of water was added to the reaction solution, which was stirred for 30 min, filtered, washed with water (20 mL), and the filter cake was collected and dried to obtain intermediate IV-2 (700 mg, yield 52.4%). 1HNMR (400 MHz, DMSO-d6) δ 8.58 (s, 1H), 7.86 (dd, J = 8.6, 2.2 Hz, 1H), 7.72-7.62 (m, 2H), 7.27 (s, 1H), 7.25-7.20 (m, 2H), 3.76 (s, 3H), 2.52 (s, 3H).

[0279] Intermediate IV-3

[0280] Intermediate IV-3 and IV-4 were synthesized from intermediate I-4, I-6 by similar procedures as described for intermediate IV-2

[0281]

[0282] Example 1: 5-(5-bromo-2-methoxyphenyl)-N-(6-phenyl-7-oxo-6,7-dihydrothiazolo[4,5- d]pyrimidin-2-yl)pyridazine-4-carboxamide (Compound 1)

[0283]

[0284] Intermediate II-4 (25 mg, 0.07 mmol) was dissolved in 5 mL of tetrahydrofuran, intermediate IV-1 (48 mg, 0.15 mmol) and NaH (6 mg, 0.15 mmol) were added with stirring, the reaction solution was heated to 65 °C for 30 min; cooled to room temperature, 5 mL of ethanol was added to quench the reaction, the solvent was removed by reduced pressure concentration, the residue was separated by thin layer chromatography to obtain compound 1 (12 mg, yield 14.4%). 1 H NMR (400 MHz, DMSO-d6) δ 13.69 (s, 1H), 9.60-9.45 (m, 2H), 8.51 (s, 1H), 7.81-7.50 (m, 7H), 7.04 (d, J = 8.9 Hz, 1H), 3.51 (s, 3H).

[0285] Example 2: 4-(5-cyano-2-methoxyphenyl)-N-(6-(4-cyanophenyl)-7-oxo-6,7- dihydrothiazolo[4,5-d]pyrimidin-2-yl)-6-methylnicotinamide (Compound 2)

[0286]

[0287] Intermediate I-3 (40 mg, 0.15 mmol) was dissolved in 5 mL of dichloromethane and placed in an ice water bath at 0 °C, and N,N'-dicyclohexylcarbodiimide (DCC, 102 mg, 0.49 mmol) was added with stirring, and reacted at 0 °C for 1 hour; a solution of intermediate III-1 (48 mg, 0.18 mmol) and 4-dimethylaminopyridine (DMAP, 9 mg, 0.07 mmol) in dichloromethane (5 mL) was added to the reaction flask, and reacted at room temperature for 12 hours; the reaction solution was filtered, the filtrate was collected, and the filtrate was washed sequentially with a dilute hydrochloric acid solution (1 N, 5 mL), a saturated aqueous sodium bicarbonate solution (5 mL), and a saturated aqueous sodium chloride solution (5 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure, and the residue was separated by thin layer chromatography to obtain compound 2 (19 mg, yield 24.5%). 1 HNMR (400 MHz, DMSO-d6) δ 13.40 (s, 1H), 8.81 (s, 1H), 8.54 (s, 1H), 8.12-8.04 (m, 2H), 7.95-7.90 (m, 2H), 7.83-7.77 (m, 2H), 7.45 (s, 1H), 7.22-7.14 (m, 1H), 3.57 (s, 3H), 2.60 (s, 3H).

[0288] Examples 3 to 8, 45 to 46, 49, 51, 53 to 59

[0289] The following compounds were prepared in a similar manner to compound 2.

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298] Examples 9 to 42, 47 to 48

[0299] The following compounds were prepared in a similar manner to compound 1.

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311]

[0312]

[0313]

[0314] Example 43: 5-(5-bromo-2-methoxyphenyl)-N-(5-phenyl-4,5,6,7-tetrahydrothiazo[5,4-c]pyridin-2-yl)pyridazin-4-carboxamide (Compound 43)

[0315]

[0316] Intermediate I-1 (78 mg, 0.25 mmol) was dissolved in 5 mL of LMF, and HOBt (114 mg, 0.84 mmol) and EDCI (97 mg, 0.51 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 5 min. Intermediate III-3 (95 mg, 0.41 mmol) was added to the reaction mixture, and the reaction was carried out at 70 °C for 12 h. After the reaction was completed by TLC, the mixture was extracted with ethyl acetate (15 mL * 3), and the organic phases were combined. The organic phases were washed with saturated brine (15 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was separated by thin-layer chromatography to give compound 43 (22 mg, yield: 16.79%). 1H NMR(400MHz,DMSO-d6)δ12.76(s,1H),9.42(s,1H),9.40(s,1H),7.72–7.58(m,2H),7.28–7.18(m,2H),7.0 8–6.96(m,3H),6.77(t,J=7.2Hz,1H),4.40(s,2H),3.66(t,J=5.7Hz,2H),3.52(s,3H),2.75-2.71(m,2H).

[0317] Example 44: 5-(5-bromo-2-methoxyphenyl)-N-(5-oxo-7-phenyl-5H-[1,3,4]thiadiazo[3,2-c]pyrimidin-2-yl)pyridazine-4-carboxamide (Compound 44)

[0318]

[0319] Intermediate I-1 (25 mg, 0.09 mmol) was dissolved in 5 mL of LMF, and HOBt (33 mg, 0.24 mmol) and EDCI (29 mg, 0.15 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 5 min. Intermediate III-4 (29 mg, 0.12 mmol) was added to the reaction mixture, and the reaction was carried out at 60 °C for 16 h. After the reaction was completed by TLC, the mixture was extracted with ethyl acetate (15 mL * 3), and the organic phases were combined. The organic phases were washed with saturated brine (15 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was separated by thin-layer chromatography to give compound 44 (11 mg, yield: 25.41%). 1 H NMR(400MHz,DMSO-d6)δ9.51(s,1H),9.31(s,1H),8.12–8.01(m,2H),7.67–7. 58(m,2H),7.52-7.46(m,3H),7.05(d,J=8.7Hz,1H),6.84(s,1H),3.60(s,3H).

[0320] Example 50: 4'-(2-(2'-chloro-5'-methoxy-6-methyl-[4,4'-bipyridine]-3-carboxamido)-7-oxothiazo[4,5-d]pyrimidin-6(7H)-yl)-2,3'-dimethyl-[1,1'-biphenyl]-4-carboxylic acid (Compound 50)

[0321]

[0322] Compound 49 (760 mg, 1.14 mmol) was dissolved in methanol (10 mL). Under stirring, an aqueous solution of sodium hydroxide (182 mg sodium hydroxide dissolved in 10 mL of water) was added dropwise. The reaction system was heated to 40 °C and stirred for 4 h. Most of the methanol was removed by concentration under reduced pressure. The mixture was extracted with ethyl acetate (5 mL * 3) and the organic phase was discarded. The pH of the aqueous phase was adjusted to 4-5 with dilute hydrochloric acid (2 N). A solid precipitated out. The solid was filtered, the filter cake was washed with water, and dried to give compound 50 (312 mg, yield: 41.93%). 1 H NMR (400MHz, DMSO-d6) δ13.58(s,1H),8.92(s,1H),8.52(s,1H),8.20(s,1H),7.92(s,1H),7.85(d,J=7.9Hz,1H),7.63(s,1H) ,7.56(s,1H),7.53(d,J=8.1Hz,1H),7.48(s,1H),7.40(t,J=7.9Hz,2H),3.64(s,3H),2.64(s,3H),2.36(s,3H),2.17(s,3H).

[0323] Example 52: 4'-(2-(2'-chloro-5'-methoxy-6-methyl-[4,4'-bipyridine]-3-carboxamido)-7-oxothiazo[4,5-d]pyrimidin-6(7H)-yl)-[1,1'-biphenyl]-4-carboxylic acid ester (Compound 52)

[0324]

[0325] Compound 52 (130 mg, yield: 66.46%) was prepared from compound 51 (200 mg, 0.31 mmol) using the same synthetic method as compound 50. 1 H NMR (400MHz, DMSO-d6) δ13.59(s,1H),8.96(s,1H),8.58(s,1H),8.21(s,1H),8.06(d,J=8.3Hz,2H),7.93( d,J=8.5Hz,2H),7.89(d,J=8.4Hz,2H),7.68(d,J=8.5Hz,2H),7.65-7.62(m,2H),3.62(s,3H),2.67(s,3H).

[0326] Comparative Example A: 4-(5-cyano-2-methoxyphenyl)-N-(5-(4-chlorophenyl)thiazo[5,4-b]pyridin-2-yl)-6-methylnicotinamide (Comparative Compound A)

[0327]

[0328] Compound A-1 (1 g, 4.35 mmol) was dissolved in 20 mL of acetonitrile, and triethylamine (570 mg, 5.63 mmol) and DMAP (53 mg, 0.43 mmol) were added. Boc2O (1.23 g, 5.64 mmol) was added with stirring. The reaction solution was heated to 50 °C and reacted for 2 hours. A solid precipitated during the reaction. The mixture was cooled to room temperature, water (20 mL) was added, and the mixture was filtered. The mixture was washed with water (10 mL), and the filter cake was collected and dried to give intermediate A-2 (1.2 g, yield 83.6%).

[0329] Compound A-2 (500 mg, 1.51 mmol), 1,4-dioxane (15 mL), p-chlorophenylboronic acid (308 mg, 1.97 mmol), Pd(dppf)Cl2 (110 mg, 0.15 mmol), potassium carbonate (628 mg, 4.54 mmol), and water (3 mL) were added sequentially to a 100 mL flask. The mixture was reacted at 90 °C for 2 h under nitrogen protection. After cooling to room temperature, the reaction solution was filtered through diatomaceous earth, concentrated under reduced pressure, and the residue was slurried with ethyl acetate (5 mL) to give intermediate A-3 (500 mg, yield 91.3%).

[0330] Weigh 500 mg of compound A-3 and dissolve it in 10 mL of dichloromethane. Add 2 mL of trifluoroacetic acid dropwise while stirring at room temperature and react at room temperature for 3 hours. Add saturated sodium bicarbonate aqueous solution (5 mL) to the reaction solution and extract with ethyl acetate (30 mL * 3). Combine the organic phases, wash the organic phase with saturated brine (10 mL), dry with anhydrous sodium sulfate, concentrate under reduced pressure, and slurry the residue with dichloromethane (5 mL) to obtain intermediate A-4 (260 mg, yield 71.9%).

[0331] Compound A-4 (32 mg, 0.12 mmol) and intermediate I-3 (30 mg, 0.11 mmol) were dissolved in 5 mL of acetonitrile. N-methylimidazole (NMI, 50 mg, 0.61 mmol) was added, followed by an acetonitrile solution (2 mL) of tetramethylchlorourea hexafluorophosphate (TCFH, 31 mg, 0.11 mmol) under stirring. The mixture was stirred at room temperature for 8 hours. The reaction solution was concentrated under reduced pressure, water (10 mL) was added, and the mixture was extracted with ethyl acetate (20 mL x 3). The organic phases were combined. The organic phases were washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was separated by thin-layer chromatography to obtain the control compound A (14 mg, yield 22.4%). 1H NMR(400MHz,DMSO-d6)δ13.02(s,1H),8.80(s,1H),8.23-8.08(m,4H),7.96-7.88(m ,2H),7.61-7.54(m,2H),7.44(s,1H),7.21-7.16(m,1H),3.60(s,3H),2.60(s,3H).

[0332] Comparative Example B: 4-(5-cyano-2-methoxyphenyl)-N-(6-(4-chlorophenyl)thiazo[4,5-b]pyrazin-2-yl)-6-methylnicotinamide (Comparative Compound B)

[0333]

[0334] Compound B-1 (3 g, 12.98 mmol) was dissolved in 40 mL of dichloromethane, and triethylamine (2.63 g, 25.99 mmol) and DMAP (159 mg, 1.30 mmol) were added. Boc2O (3.12 g, 14.29 mmol) was added under stirring. The reaction solution was reacted at room temperature for 2 hours, during which solid precipitated. The mixture was filtered, and the filter cake was washed with dichloromethane (5 mL) and water (10 mL). The filter cake was collected and dried to give intermediate B-2 (3.9 g, yield 90.91%).

[0335] Compound B-2 (3.9 g, 11.78 mmol), 1,4-dioxane (50 mL), p-chlorophenylboronic acid (3.5 g, 22.38 mmol), Pd(dppf)Cl2 (861 mg, 1.17 mmol), potassium carbonate (3.25 g, 23.52 mmol), and water (10 mL) were added sequentially to a 100 mL flask. The mixture was reacted at 90 °C for 2 h under nitrogen protection. After cooling to room temperature, the reaction solution was filtered through diatomaceous earth, concentrated under reduced pressure, and the residue was purified by column chromatography to give intermediate B-3 (3.6 g, yield 84.31%).

[0336] 3.6 g of compound B-3 was weighed and dissolved in 35 mL of dichloromethane. 7 mL of trifluoroacetic acid was added dropwise while stirring at room temperature, and the reaction was carried out at room temperature for 6 hours. Saturated sodium bicarbonate aqueous solution (15 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (50 mL * 3). The organic phases were combined, washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was slurried with dichloromethane (10 mL) to give intermediate B-4 (2.2 g, yield 84.61%).

[0337] Compound B-4 (150 mg, 0.57 mmol) and intermediate I-3 (200 mg, 0.75 mmol) were dissolved in 5 mL of DMF. HATU (283.5 mg, 0.75 mmol) was added, followed by DIPEA (192 mg, 1.49 mmol) under stirring. The mixture was stirred at 60 °C for 12 hours. The reaction solution was concentrated under reduced pressure, and water (10 mL) was added. The mixture was extracted with ethyl acetate (20 mL x 3), and the organic phases were combined. The organic phases were washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was separated by thin-layer chromatography to obtain the control compound B (62 mg, yield 16.23%). 1 H NMR(400MHz,DMSO-d6)δ13.40(s,1H),9.26(s,1H),8.83(s,1H),8.25-8.17(m,2H),7.96- 7.89(m,2H),7.66-7.57(m,2H),7.46(s,1H),7.23-7.15(m,1H),3.61(s,3H),2.61(s,3H).

[0338] Comparative Example C: 4-(5-cyano-2-methoxyphenyl)-N-(6-(4-chlorophenyl)thiazo[4,5-c]pyridin-2-yl)-6-methylnicotinamide (Comparative Compound C)

[0339]

[0340] Compound C-1 (3.2 g, 19.63 mmol) was dissolved in 30 ml of acetone. Benzoyl isothiocyanate (6.36 g, 38.97 mmol) was added to the mixture. The reaction mixture was then reacted at room temperature for 3 hours. After the reaction was completed, the mixture was filtered, the filter cake was washed with acetone, and dried to give intermediate C-2 (6.3 g, yield: 98.43%).

[0341] Compound C-2 (6.3 g, 19.31 mmol) was dissolved in an aqueous solution of sodium hydroxide (2.35 g, 57.93 mmol). The reaction solution was reacted at 100 °C for 40 minutes. After the reaction was completed, the mixture was filtered, the filter cake was washed with water and ethanol, and dried to obtain intermediate C-3 (3.5 g, yield: 81.78%).

[0342] Compound C-3 (3.5 g, 15.76 mmol) was dissolved in 10 ml of DMF, and 60% NaH (1.26 g, 31.52 mmol) was added in portions with stirring. The reaction solution was reacted at 80 °C for 20 minutes under nitrogen protection. After the reaction was completed, saturated ammonium chloride aqueous solution was slowly added, filtered, and the filter cake was washed with water and ethanol and dried to obtain intermediate C-4 (2.4 g, yield: 82.19%).

[0343] Compound C-4 (1 g, 5.38 mmol) was dissolved in 20 mL of dichloromethane, and triethylamine (1.09 g, 10.77 mmol) and DMAP (66 mg, 0.54 mmol) were added. Boc2O (1.29 g, 5.91 mmol) was added under stirring. The reaction solution was reacted at room temperature for 12 hours, filtered, washed with water (10 mL), and the filter cake was collected and dried to give intermediate C-5 (1.1 g, yield 71.89%).

[0344] Compound C-5 (1.1 g, 3.85 mmol), 1,4-dioxane (30 mL), p-chlorophenylboronic acid (903 mg, 5.77 mmol), Pd(dppf)Cl2 (280 mg, 0.38 mmol), cesium carbonate (2.51 g, 7.70 mmol), and water (6 mL) were added sequentially to a 100 mL flask. The mixture was reacted at 90 °C for 2 h under nitrogen protection. After cooling to room temperature, the reaction solution was filtered through diatomaceous earth, concentrated under reduced pressure, and the residue was purified by column chromatography to give compound C-6 (680 mg, yield 48.92%).

[0345] 680 mg of compound C-6 was weighed and dissolved in 10 mL of dichloromethane. 5 mL of trifluoroacetic acid was added dropwise while stirring at room temperature, and the reaction was carried out at room temperature for 3 hours. Saturated sodium bicarbonate aqueous solution (10 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (30 mL * 3). The organic phases were combined, washed with saturated brine (15 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was slurried with dichloromethane (5 mL) to give intermediate C-7 (450 mg, yield 91.46%).

[0346] Compound C-7 (150 mg, 0.57 mmol) and intermediate I-3 (230 mg, 0.86 mmol) were dissolved in 5 mL of DMF. HATU (327 mg, 0.86 mmol) was added, followed by DIPEA (222 mg, 1.72 mmol) under stirring. The mixture was stirred at 65 °C for 12 hours. The reaction solution was concentrated under reduced pressure, and water (10 mL) was added. The mixture was extracted with ethyl acetate (20 mL x 3), and the organic phases were combined. The organic phases were washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was separated by thin-layer chromatography to obtain the control compound C (51 mg, yield 17.46%). 1H NMR(400MHz,DMSO-d6)δ13.14(s,1H),9.10(s,1H),8.81(s,1H),8.68(s,1H),8.18–8.12(m,2H),7 .95–7.88(m,2H),7.60–7.53(m,2H),7.45(s,1H),7.18(d,J=9.2Hz,1H),3.58(s,3H),2.61(s,3H).

[0347] Example of results:

[0348] 1. POLQ enzyme activity test

[0349] Experimental procedure:

[0350] The N-terminus of the POLQ protein contains a helicase domain with ATPase activity, which can hydrolyze ATP into ADP. The ATPase activity of the POLQ protein can be analyzed experimentally to reflect the inhibitory effect of small molecule compounds on the protein. Specific detection methods are as follows:

[0351] (1) Prepare the Assay buffer;

[0352] (2) Preparation of compound concentration gradient: The test compound was gradient diluted in EP tubes to prepare three solutions of different concentrations, and the solutions were tested in duplicate. The concentrations of the test compound were 1000 nM, 100 nM, 10 nM, or 100 nM, 10 nM, 1 nM, or 10 nM, 1 nM, 0.1 nM. 0.1 μL was transferred to a 384-well plate by Echo.

[0353] (3) Add 5 μL of POLQ enzyme solution prepared with Assay buffer to a 384-well plate, centrifuge at 1000 rpm for 1 min, and incubate at 25°C for 10 min.

[0354] (4) Add 5 μL of ATP & ssDNA solution prepared with Assay buffer to a 384-well plate, mix well and incubate at 25°C for 60 min.

[0355] (5) Add 5 μL of romegaADP-Glo ​​Kinase assay reaction buffer, centrifuge at 1000 rpm for 1 min, and incubate at 25 °C for 40 min;

[0356] (6) Add 10 μL of PromegaADP-Glo ​​Kinase assay detection buffer, centrifuge at 1000 rpm for 1 min, incubate at 25 °C for 40 min, and measure the autoluminescence value;

[0357] Data Analysis:

[0358] Inhibition rate calculation formula:

[0359] %Inhibition = (luminescence value) 阳性孔 -Luminescence value 实验孔 ) / (luminous value) 阳性孔 -Luminescence value 阴性孔 )×100%

[0360] Luminous value 实验孔 This is the reading from the compound sample well; luminescence value. 阳性孔 The reading represents the amount of light emitted from the compound-free suppression aperture; luminescence value. 阴性孔 This represents the reading of the enzyme-free active well.

[0361] The activity data of the compounds are shown in the table below:

[0362]

[0363]

[0364]

[0365] 2. POLQ cell viability test

[0366] The inhibitory activity of the inhibitors in vitro was detected using the Cell Counting-Lite 2.0 Luminescent Cell Viability Assay reagent. The specific detection method is as follows:

[0367] (1) Cultivate DLD-1BRCA2 - / - Cell lines were digested with trypsin, centrifuged at 1200 rpm for 3 min and the cells were collected. After cell counting, the cells were seeded into 96-well white culture plates at an appropriate seeding ratio and cultured at 37°C and 5% CO2.

[0368] (2) After the cells adhered, the compound was dissolved in DMSO and serially diluted 10-fold with cell culture medium. The original culture medium in the cell plate was discarded and replaced with drug-added culture medium to make the final concentration of the compound 1000 nM, 100 nM, 10 nM, or 100 nM, 10 nM, 1 nM. Three concentrations were set for each compound, and two replicates were made for each concentration. A DMSO control group and a blank culture medium control group were set up. The cell culture plate was cultured at 37°C and 5% CO2.

[0369] (3) On the 4th and 7th days after drug administration, the medium was replaced with fresh drug-treated medium and cultured for another 10 days.

[0370] (4) After incubation, add Cell Counting-Lite 2.0 Luminescent Cell ViabilityAssay reagent to the wells of the cell culture plate, mix well for 2 min, and incubate at room temperature for 30 min;

[0371] (5) Perform chemiluminescence detection using an enzyme-linked immunosorbent assay (ELISA) reader and process the data:

[0372] Inhibition rate % = (Mean value of DMSO control group - Mean value of experimental group) * 100% / (Mean value of DMSO control group - Mean value of blank culture medium control group)

[0373] (The control group's test results showed a CV% of less than 20% and a Z' value of greater than 0.5.)

[0374] The cell proliferation inhibitory activity data of the compounds are shown in the table below:

[0375]

[0376]

[0377]

[0378] 3. Human liver microsomal metabolic stability test

[0379] Experimental procedure:

[0380] Each incubation system had a total volume of 150 μL and included 0.1 M PBS buffer (pH 7.4); an NADPH regeneration system (1 mM NADP, 10 mM glucose-6-phosphate, 1 U / mL glucose-6-phosphate dehydrogenase, 5 mM MgCl2 solution); and an appropriate concentration of the analyte; with a final concentration of human liver microsomes of 0.2 mg / mL. The incubation system was run at 37°C, with each sample in triplicate. A sample without the NADPH generation system served as a negative control. After a 5-minute pre-incubation, the NADPH regeneration system was added to initiate the reaction. Incubation times were 0 min, 10 min, 20 min, 35 min, and 50 min. At each time point, 200 μL of ice-cold acetonitrile was added to the corresponding well to terminate the reaction. The wells were then sealed and stored at -80°C. All sealed samples were added to 100 μL of acetonitrile containing internal standard and shaken for 5 min. After centrifugation at 4000 rpm for 25 min, 100 μL of the supernatant was transferred to a sample vial for LC-MS / MS analysis. The mass spectrometric response As of the original compound and the mass spectrometric response Ai of the internal standard were detected in each sample.

[0381] Data Analysis:

[0382] Calculate the clearance rate and recovery rate: Calculate the As / Ai value using the compound mass spectrometry response As and the internal standard mass spectrometry response Ai, and calculate the remaining percentage; Remaining percentage = (As / Ai) at different time points / (As / Ai) at 0 min * 100;

[0383] Logarithmically transform the remaining proportions at different times, plot a semi-logarithmic curve over time, and perform linear fitting; the slope is -k. e (Elimination rate constant), elimination half-life t 1 / 2 =0.693 / k e Liver microsomal intrinsic clearance rate CL int =k e / C protein .

[0384] The results of the liver microsomal stability test of the compound are shown in the table below:

[0385] Compound No. T 1 / 2 (min)]]> CL int (μL·min -1 ·mg -1 )]]> Recovery (%) Compound 4 1047 3.3 97.59 Compound 7 455 7.6 99.83 Compound 8 854 4.1 96.55 Compound 9 44 79.2 97.71 Compound 10 604 5.7 98.72 Compound 14 1151 3.0 92.86 Compound 16 1038 3.3 96.69 Compound 18 487 7.1 96.67 Compound 22 81 43.0 89.28 Compound 26 406 8.5 98.74 Compound 28 276 12.5 95.72 Compound 29 246 14.1 96.64 Compound 30 177 19.5 97.23 Compound 31 137 25.3 91.63 Compound 32 282 12.3 86.17 Compound 33 181 19.2 96.53 Compound 36 164 21.2 90.96 Compound 39 1227 2.8 101.58 Compound 40 211 16.5 82.13 Compound 45 330 10.5 93.83 Compound 46 180 19.2 98.67 Compound 47 155 22.3 98.98 Compound 49 - - 11.84 Compound 50 611 5.7 100.94 Compound 53 133 26.0 86.73 Compound 54 94 36.9 88.88 Comparative Compound A 160 21.6 88.40 Comparative Compound B 303 11.4 91.66 Comparative Compound C 293 11.8 92.08

[0386] 4. Caco-2 Permeability Test

[0387] Experimental Methods: Caco-2 cells were seeded at a specific density in 12-well Transwell plates and cultured for 21 days. The formation of a complete cell monolayer in each well was observed under an inverted microscope, and the TEER value of each well was measured to ensure successful model construction. The test compound was dissolved in DMSO and diluted with buffer to a final concentration of 5 μM (final DMSO concentration less than 0.1%). The cells were incubated in drug-containing buffer for 60 min, and the drug concentration on the receiver side of each well was detected by LC-MS / MS.

[0388] Data processing: Calculate the P of the compound based on the above results. app Value and R E The value is calculated using the following formula:

[0389]

[0390] R E =P app (BL→AP) / P app (AP→BL)

[0391] V R The volume of the solution in the receiving chamber is represented by dC / dt; A represents the area of ​​the membrane; C0 represents the initial concentration of the drug in the supply solution; and dC / dt represents the drug concentration obtained in the receiving chamber per unit time.

[0392] The results of the Caco-2 permeability test of the compound are shown in the table below:

[0393]

[0394]

[0395] 5. Rat pharmacokinetic test

[0396] Experimental animals: Healthy male SPF-grade SD rats, weighing approximately 220g. Provided by Spiford (Beijing) Biotechnology Co., Ltd.

[0397] Experimental design: Rats were administered 5 mg / kg via gavage and 2.5 mg / kg via intravenous injection. Three healthy male SD rats were used in each group. Rats were fasted for 12 hours before administration but allowed free access to water. Two hours after administration, rats were allowed free access to food and water throughout the experiment. Blood samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours post-administration in the gavage group; and at 0.033, 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours post-administration in the intravenous administration group.

[0398] The concentration of the compounds of the present invention in biological samples was determined by LC-MS / MS, and plasma concentration analysis and pharmacokinetic parameters were calculated.

[0399] The results of the rat pharmacokinetic studies of the compound are shown in the table below:

[0400]

Claims

1. A compound of formula I and a pharmaceutically acceptable salt thereof: in: Ring A is selected from C6-C 12 Aryl, 5-10 heteroaryl, wherein the aryl or heteroaryl group is unsubstituted or substituted by 1-5 substituents, each independently selected from the following: halogen, cyano, =O, C1-C6 alkyl, C1-C6 haloalkyl, C3-C9 cycloalkyl, -OR b ; R b Selected from H, C1-C6 alkyl, C1-C6 haloalkyl, and C3-C9 cycloalkyl; Y is selected from N or C. When Y is N, R1 does not exist. Z is selected from N or CR. a ; R a Selected from H, C1-C6 alkyl groups; R1 is selected from H, C1-C6 alkyl, wherein the C1-C6 alkyl is unsubstituted or optionally substituted with the following groups: halogen, C1-C6 alkoxy, -C(O)NR c R d ; R c R d Each is independently selected from H and C1-C6 alkyl groups; R2 is selected from H, halogen, cyano, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 alkoxy. R3 is selected from C6-C 12 Aryl, 5-14 membered heteroaryl, C3-C9 cycloalkyl, 5-14 membered heterocyclic, wherein the above groups are unsubstituted or substituted by 1-5 substituents independently selected from the following: cyano, nitro, halogen, =O, -C(O)NR c’ R d’ -N(R) e )-C(O)R f -ORg, C1-C6 alkyl, C6-C 12 aryl, 5-14 membered heteroaryl, C3-C9 cycloalkyl, 5-14 membered heterocyclic, wherein the C1-C6 alkyl, C6-C 12 The aryl, 5-14-membered heteroaryl, C3-C9 cycloalkyl, and 5-14-membered heterocyclic groups may optionally be substituted with the following groups: halogen, cyano, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy; R c’ R d’ Each is independently selected from H and C1-C6 alkyl groups; R e Selected from H, C1-C6 alkyl groups; R f Selected from H, C1-C6 alkyl groups; R g Selected from H, C1-C6 alkyl groups, wherein the alkyl group is unsubstituted or optionally substituted with the following groups: C1-C6 alkoxy, C6-C 12 Aryl, 5-14 membered heteroaryl, C3-C9 cycloalkyl, 5-14 membered heterocyclic.

2. The compound of claim 1 and its pharmaceutically acceptable salt, wherein: Ring A is selected from phenyl, or a 5-6 membered heteroaryl group containing at least one nitrogen atom, wherein the phenyl or heteroaryl group is unsubstituted or substituted by 1-5 substituents, each independently selected from the following: halogen, cyano, =O, C1-C3 alkyl, C1-C3 haloalkyl, C3-C6 cycloalkyl, -OR b ; R b Selected from H, C1-C3 alkyl, C1-C3 haloalkyl, and C3-C6 cycloalkyl; Y is selected from N or C. When Y is N, R1 does not exist. Z is selected from N or CR. a ; R a Selected from H, C1-C3 alkyl groups; R1 is selected from H, C1-C3 alkyl, wherein the C1-C3 alkyl is unsubstituted or optionally substituted with the following groups: halogen, C1-C3 alkoxy, -C(O)NR c R d ; R c R d Each is independently selected from H and C1-C3 alkyl groups; R2 is selected from H, halogen, cyano, C1-C3 alkyl, C1-C3 haloalkyl, and C1-C3 alkoxy. R3 is selected from C6-C 10 Aryl, 5-10 membered heteroaryl containing 1, 2, 3, or 4 heteroatoms selected from O, N, and S, C3-C6 cycloalkyl, 5-10 membered heterocyclic group containing 1, 2, 3, or 4 heteroatoms selected from O, N, and S, wherein the above groups are unsubstituted or substituted by 1-5 substituents each independently selected from the following: cyano, nitro, halogen, =O, -C(O)NR c’ R d’ -N(R) e )-C(O)R f -ORg, C1-C3 alkyl, C3-C6 cycloalkyl, C6-C 10 aryl, 5-9 membered heteroaryl, wherein the C1-C3 alkyl, C3-C6 cycloalkyl, C6-C 10 The aryl and 5-9 membered heteroaryl groups may optionally be substituted with the following groups: halogen, cyano, hydroxyl, C1-C3 alkyl, C1-C3 alkoxy; R c’ R d’ Each is independently selected from H and C1-C3 alkyl groups; R e Selected from H, C1-C3 alkyl groups; R f Selected from H, C1-C3 alkyl groups; R g Selected from H, C1-C3 alkyl groups, wherein the alkyl group is unsubstituted or optionally substituted with the following groups: C1-C3 alkoxy, C6-C 10 Aryl, 5-10 membered heteroaryl containing 1, 2, or 3 heteroatoms selected from O, N, or S, C3-C6 cycloalkyl, 5-10 membered heterocyclic group containing 1, 2, or 3 heteroatoms selected from O, N, or S.

3. The compound of claim 1 and its pharmaceutically acceptable salt, wherein the compound has formula Ia: in, X is selected from N or CH; Y is selected from N or C. When Y is N, R1 does not exist. Z is selected from N or CR. a ; R a Selected from H, C1-C3 alkyl groups; R1 is selected from H, C1-C3 alkyl, wherein the C1-C3 alkyl is unsubstituted or optionally substituted with the following groups: halogen, C1-C3 alkoxy, C(O)NR. c R d ; R c R d Each is independently selected from H and C1-C3 alkyl groups; R2 is selected from H, halogen, cyano, C1-C3 alkyl, C1-C3 haloalkyl, and C1-C3 alkoxy. R3 is selected from C6-C 10 Aryl, 5-10 membered heteroaryl containing 1, 2, 3, or 4 heteroatoms selected from O, N, and S, C3-C6 cycloalkyl, 5-10 membered heterocyclic group containing 1, 2, 3, or 4 heteroatoms selected from O, N, and S, wherein the above groups are unsubstituted or substituted by 1-5 substituents each independently selected from the following: cyano, nitro, halogen, =O, -C(O)NR c’ R d’ -N(R) e )-C(O)R f -ORg, C1-C3 alkyl, C3-C6 cycloalkyl, C6-C 10 aryl, 5-9 membered heteroaryl, wherein the C1-C3 alkyl, C3-C6 cycloalkyl, C6-C 10 The aryl and 5-9 membered heteroaryl groups may optionally be substituted with the following groups: halogen, cyano, hydroxyl, C1-C3 alkyl, C1-C3 alkoxy; R4 is selected from hydrogen, halogen, cyano, =O, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, and C3-C6 cycloalkyl. R5 is selected from C1-C3 alkyl, C1-C3 haloalkyl, and C3-C6 cycloalkyl; R6 is selected from H, halogens, and C1-C3 alkyl groups; R c’ R d’ Each is independently selected from H and C1-C3 alkyl groups; R e Selected from H, C1-C3 alkyl groups; R f Selected from H, C1-C3 alkyl groups; R g Selected from H, C1-C3 alkyl groups, wherein the alkyl group is unsubstituted or optionally substituted with the following groups: C1-C3 alkoxy, C6-C 10 Aryl, 5-10 membered heteroaryl containing 1, 2, or 3 heteroatoms selected from O, N, or S, C3-C6 cycloalkyl, 5-10 membered heterocyclic group containing 1, 2, or 3 heteroatoms selected from O, N, or S.

4. The compound of claim 1 and its pharmaceutically acceptable salt, wherein the compound has formula Ia: in, X is selected from N or CH; Y is selected from N or C. When Y is N, R1 does not exist. Z is selected from N or CR. a ; R a Selected from H, methyl, ethyl, propyl, and isopropyl; R1 is selected from H, methyl, ethyl, propyl, isopropyl, wherein the alkyl group is unsubstituted or optionally substituted with the following groups: F, Cl, Br, methoxy, ethoxy, propoxy, -C(O)NR. c R d ; R c R d Each is independently selected from H, methyl, ethyl, propyl, and isopropyl; R2 is selected from H, F, Cl, Br, cyano, methyl, ethyl, propyl, isopropyl, CF3, CF2H, CFH2, methoxy, ethoxy, propoxy; R3 is selected from phenyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrazolyl, imidazoleyl, oxazolyl, isoxazolyl, triazolyl, furazolyl, furanyl, thiophenyl, thiazolyl, isothiazolyl, oxadiazolyl, naphthyl, quinolinyl, isoquinolinyl, indolyl, benzopyrazolyl, benzimidazolyl, benzoxazolyl, benzoxazolyl, benzoxisoxazolyl, benziisothiazolyl, pyrimidinoxazolyl, pyrimidinthiazolyl, pyrimidinimidazolyl, pyridinyl The following groups are included: azizimidazolyl, pyrazinzimidazolyl, indigo, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, oxacyclopentyl, dioxacyclohexyl, morpholinyl, piperidinyl, piperazinyl, 1,4-benzodioxane, 2,3-dihydrobenzofuranyl, benzomorpholinyl, 1,2,3,4-tetrahydroquinolinyl, wherein the above groups are unsubstituted or substituted by 1-5 substituents independently selected from the following: cyano, nitro, F, Cl, Br, =O, -N(R) e )-C(O)R f -ORg, methyl, ethyl, propyl, isopropyl, cyclopropyl, phenyl, isoxazolyl, wherein the alkyl, phenyl, isoxazolyl groups are optionally substituted with the following groups: F, Cl, Br, cyano, hydroxyl, methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, propoxy; R4 is selected from H, F, Cl, Br, cyano, =O, methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, propoxy, CF3, CF2H, CFH2, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. R5 is selected from methyl, ethyl, propyl, isopropyl, CF3, CF2H, CFH2, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. R6 is selected from H, F, Cl, Br, methyl, ethyl, propyl, and isopropyl. R e Selected from H, methyl, ethyl, propyl, and isopropyl; R f Selected from H, methyl, ethyl, propyl, and isopropyl; R g The alkyl group is selected from H, methyl, ethyl, propyl, and isopropyl, and the alkyl group is unsubstituted or optionally substituted with the following groups: methoxy, ethoxy, propoxy, phenyl, naphthyl, pyridinyl, oxazolyl, imidazolyl, pyrazolyl, triazolyl, oxacyclopentyl, dioxacyclohexyl, morpholinyl, piperidinyl, and piperazine.

5. The compound of claim 1 and its pharmaceutically acceptable salt, wherein the compound has formula Ia: in, X is selected from N or CH; Y is selected from N, R1 does not exist, and Z is selected from N; Alternatively, Y can be selected from N, R1 does not exist, and Z can be selected from CH; Alternatively, Y can be selected from C, and Z from N; R1 is selected from H, methyl, ethyl, propyl, isopropyl, wherein the alkyl group is unsubstituted or optionally substituted with the following groups: F, Cl, Br, methoxy, ethoxy, propoxy, -C(O)NR. c R d ; R c R d Each is independently selected from H, methyl, ethyl, propyl, and isopropyl; R2 is selected from H, F, Cl, Br, cyano, methyl, ethyl, propyl, isopropyl, CF3, CF2H, CFH2, methoxy, ethoxy, propoxy; R3 is selected from R4 is selected from H, F, Cl, Br, cyano, =O, methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, propoxy, CF3, CF2H, CFH2, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. R5 is selected from methyl, ethyl, propyl, isopropyl, CF3, CF2H, CFH2, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. R6 is selected from H, F, Cl, Br, methyl, ethyl, propyl, and isopropyl.

6. The compound of claim 1 and its pharmaceutically acceptable salt, wherein the compound has the formula Ib: in, X is selected from N or C. When X is N, R7 does not exist. Y is selected from N or C. When Y is N, R1 does not exist. Z is selected from N or CR. a ; R a Selected from H, C1-C3 alkyl groups; R7 is selected from hydrogen and halogens; R1 is selected from H, C1-C3 alkyl, wherein the C1-C3 alkyl is unsubstituted or optionally substituted with the following groups: halogen, C1-C3 alkoxy, C(O)NR. c R d ; R c R d Each is independently selected from H and C1-C3 alkyl groups; R2 is selected from H, halogen, cyano, C1-C3 alkyl, C1-C3 haloalkyl, and C1-C3 alkoxy. R3 is selected from C6-C 10 Aryl, the C6-C 10 The aryl group is unsubstituted or substituted by 1 to 5 substituents, each independently selected from the following: nitro, halogen, =O, -C(O)NR. c’ R d’ -N(R) e )-C(O)R f -ORg, C1-C3 alkyl, C3-C6 cycloalkyl, C6-C 10 aryl, 5-9 membered heteroaryl, wherein the C1-C3 alkyl, C3-C6 cycloalkyl, C6-C 10 The aryl and 5-9 membered heteroaryl groups may optionally be substituted with the following groups: halogen, cyano, hydroxyl, C1-C3 alkyl, C1-C3 alkoxy; R4 is selected from hydrogen, halogen, cyano, =O, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, and C3-C6 cycloalkyl. R5 is selected from C1-C3 alkyl, C1-C3 haloalkyl, and C3-C6 cycloalkyl; R6 is selected from H, halogens, and C1-C3 alkyl groups; R c’ R d’ Each is independently selected from H and C1-C3 alkyl groups; R e Selected from H, C1-C3 alkyl groups; R f Selected from H, C1-C3 alkyl groups; R g Selected from H, C1-C3 alkyl groups, wherein the alkyl group is unsubstituted or optionally substituted with the following groups: C1-C3 alkoxy, C6-C 10 Aryl, 5-10 membered heteroaryl containing 1, 2, or 3 heteroatoms selected from O, N, or S, C3-C6 cycloalkyl, 5-10 membered heterocyclic group containing 1, 2, or 3 heteroatoms selected from O, N, or S.

7. The compound of claim 1 and its pharmaceutically acceptable salt, wherein the compound has formula Ib: in, X is selected from N or C. When X is N, R7 does not exist. Y is selected from N or C. When Y is N, R1 does not exist. Z is selected from N or CR. a ; R a Selected from H, C1-C3 alkyl groups; R7 is selected from hydrogen and halogens; R1 is selected from H, C1-C3 alkyl, wherein the C1-C3 alkyl is unsubstituted or optionally substituted with the following groups: halogen; R2 is selected from H, halogens, C1-C3 alkyl groups, and C1-C3 haloalkyl groups; R3 is selected from C6-C 10 Aryl, the C6-C 10 The aryl group is unsubstituted or substituted by 1 to 5 substituents, each independently selected from the following: halogen, C1-C3 alkyl, phenyl, pyridyl, wherein the C1-C3 alkyl, phenyl, pyridyl are optionally substituted by the following groups: halogen, C1-C3 alkyl; R4 is selected from hydrogen, halogen, C1-C3 alkyl, and C1-C3 haloalkyl; R5 is selected from C1-C3 alkyl and C1-C3 haloalkyl; R6 is selected from H, halogens, and C1-C3 alkyl groups.

8. The compound of claim 1 and its pharmaceutically acceptable salt, wherein the compound has the formula Ib: in, X is selected from N or C. When X is N, R7 does not exist. Y is selected from N or C. When Y is N, R1 does not exist. Z is selected from N; R7 is selected from H, F, Cl, and Br; R1 is selected from H, methyl, ethyl, propyl, isopropyl, wherein the alkyl group is unsubstituted or optionally substituted with the following groups: F, Cl, Br; R2 is selected from H, F, Cl, Br, methyl, ethyl, propyl, and isopropyl. R3 is selected from phenyl, wherein the phenyl is unsubstituted or substituted by 1 to 5 substituents each independently selected from the following: methyl, ethyl, propyl, isopropyl, phenyl, pyridyl, wherein the alkyl, phenyl, pyridyl is optionally substituted by the following groups: F, Cl, Br, methyl, ethyl, propyl, isopropyl; R4 is selected from H, F, Cl, Br, methyl, ethyl, propyl, and isopropyl. R5 is selected from methyl, ethyl, propyl, isopropyl, CF3, CF2H, CFH2; R6 is selected from H, F, Cl, Br, methyl, ethyl, propyl, and isopropyl.

9. The compound of claim 1 and its pharmaceutically acceptable salt, wherein the compound has the formula Ib: in, X is selected from N or C. When X is N, R7 does not exist. Y is selected from C, and Z is selected from N; R7 is selected from H and F; R1 is selected from methyl; R2 is selected from H; R3 is selected from R4 is selected from Cl; R5 is selected from methyl; R6 is selected from H and F.

10. The compound according to claim 1 and its pharmaceutically acceptable salt, wherein, The structure of the compound is selected from:

11. A pharmaceutical composition comprising the compound of any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

12. Use of any compound of claims 1 to 10 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 11, in the preparation of a medicament for the prevention or treatment of DNA polymerase θ-mediated diseases; wherein the DNA polymerase θ-mediated diseases are preferably cancers; wherein the cancers are preferably lung cancer, breast cancer, HR-deficient ovarian cancer, gastric cancer, prostate cancer, pancreatic cancer, or colon cancer.

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