Cyano-containing heterocyclic compound and use thereof

Abstract

Disclosed in the present invention are a nitrogen-containing heterocyclic compound as shown in a formula (I), or a stereoisomer, a deuterated substance, a solvate, a pharmaceutically acceptable salt, or a cocrystal thereof and a pharmaceutical composition containing same, and the use thereof in the preparation of a drug for treating / preventing diseases mediated by PRMT5·MTA. Each group in formula (I) is as defined in the description.

Description

A cyano-containing heterocyclic compound and its use Technical Field

[0001] The present invention relates to an inhibitor of PRMT5·MTA complex and use thereof in preparing an inhibitor for treating tumors. Background Art

[0002] Protein arginine methyltransferases (PRMTs) catalyze protein arginine methylation, a key post-translational modification. PRMTs, using S-adenosylmethionine (SAM) as a methyl donor, methylate the nitrogen atom of the arginine side chain of proteins. The nine identified human PRMTs (PRMT1–9) are further divided into types I, II, and III based on their arginine methylation status. Type I PRMTs, including PRMT1, PRMT2, PRMT3, PRMT4, PRMT6, and PRMT8, catalyze the formation of monomethylarginine (MMA) and asymmetric dimethylarginine (aDMA); type II PRMTs, including PRMT5 and PRMT9, catalyze the formation of MMA and symmetric dimethylarginine (sDMA); and type III PRMTs, including PRMT7, catalyze the formation of MMA. PRMT5 belongs to type II PRMT, which transfers methyl group from SAM to the guanidine nitrogen of arginine residues to generate MMA and sDMA, thereby regulating the expression of target genes in this epigenetic manner or regulating key signaling molecules through post-translational methylation modification pathways.

[0003] Homozygous deletions of p16 / CDKN2a are common in cancer. These mutations often involve co-deletions of adjacent genes, including the gene encoding methylthioadenosine phosphorylase (MTAP). It is estimated that approximately 15% of human cancers have homozygous deletions of the MTAP gene (e.g., see Firestone & Schramm (2017) J. Am. Chem Soc. 139(39):13754-13760. doi:10.1021 / jacs.7b05803. Epub 2017 Sep 20).

[0004] In MTAP-deficient cells, methylthioadenosine (MTA) overaccumulates and forms a complex with PRMT5, PRMT5·MTA, partially inhibiting PRMT5 enzymatic activity and increasing the sensitivity of cell proliferation to PRMT5 deficiency or inactivity. Consequently, MTAP deficiency reduces PRMT5 methylation activity, rendering cells selectively dependent on PRMT5 activity. In MTAP-deficient cancers, the synergistic inhibition of PRMT5 activity with MTA could provide therapeutic benefits for a variety of cancers.

[0005] Therefore, it is necessary to develop new MTA-synergistic PRMT5 inhibitors that can inhibit PRMT5 activity under elevated MTA concentrations, especially in MTAP-deficient cells.

[0006] Summary of the Invention

[0007] The present invention provides a compound of formula (I), formula (I-1), or formula (I-2), and its stereoisomers, deuterated forms, solvates, or pharmaceutically acceptable salts. The compound has good activity, high selectivity, excellent pharmacokinetic properties, excellent physicochemical properties, ease of preparation, high bioavailability, and low toxic and side effects.

[0008] The compound of formula (I), formula (I-1), formula (I-2), its stereoisomer, deuterated substance, solvate, or pharmaceutically acceptable salt,

[0009] in,

[0010] D is selected from D1, D2, D3, and D4;

[0011] In some embodiments, D is selected from the following structures:

[0012] Each R5 is independently selected from H, D, OH, C 1-4 Alkyl, CN, halogen, C 1-4 Alkoxy, C 2-4 Alkenyl, C 2-4 Alkynyl, 5-6 membered heteroaryl containing 1-3 heteroatoms selected from N, O, S, 4-7 membered heterocycloalkyl containing 1-3 heteroatoms selected from N, O, S or C 3-6 Cycloalkyl, wherein the alkyl, alkoxy, alkenyl, alkynyl, heteroaryl or cycloalkyl is optionally substituted by 1-3 groups selected from halogen, D, CN, OH, C 1-4 Alkyl, C 1-4 In some embodiments, each R5 is independently H, D, C 1-4 Alkyl, CN, halogen, C2-4 Alkenyl, C 2-4 Alkynyl, 4-7 membered heterocycloalkyl containing 1-3 heteroatoms selected from N, O, S or C 3-6 Cycloalkyl, wherein the alkyl, alkoxy, alkenyl, alkynyl, heterocycloalkyl or cycloalkyl is optionally substituted by 1-3 groups selected from halogen, D, CN, OH, C 1-2 Alkyl, C 1-2 In some embodiments, each R5 is independently H, D, methyl, ethyl, propyl, isopropyl, CN, F, Cl, vinyl, propenyl, ethynyl, propynyl, azetidinyl, azepanyl, azepanyl, oxetanyl, oxolanyl, oxetanyl, morpholinyl, piperazinyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl; In some embodiments, each R5 is independently H, D, methyl, CN, F, vinyl, azetidinyl, cyclopropyl; In some embodiments, each R5 is independently H, D, methyl, F;

[0013] R7 is independently H, D, C 1-4 Alkyl, halogenated C 1-4 Alkyl, deuterated C 1-4 Alkyl, C 2-4 Alkenyl, C 2-4 Alkynyl, C 3-6 Cycloalkyl or 4-7 membered heterocycloalkyl containing 1-3 heteroatoms selected from N, O, S; in some embodiments, R7 is independently C 1-3 Alkyl, halogenated C 1-4 Alkyl, deuterated C 1-3 Alkyl; in some embodiments, R7 is independently methyl, ethyl, propyl, isopropyl, -CH2F, -CHF2, -CF3, -CH2CH2F, -CH2CHF2, -CH2CF3, -CHFCH2F, -CHFCHF2, -CHFCF3, -CF2CH2F, -CF2CHF2, -CF2CF3, -CH2D, -CHD2, -CD3, -CH2CH2D, -CH2CHD2, -CH2CD3, -CHDCH2D, -CHDCHD2, -CHDCD3, -CD2CH2D, -CD2CHD2, -CD2CD3;

[0014] Each R 8a 、R 8b 、R 8c Each independently selected from H, D, amino, C 1-6 Alkoxy, OH, CN, halogen, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkyl, 4-7 membered heterocycloalkyl containing 1-3 heteroatoms selected from N, O, S, C3-8 Cycloalkyl, -COC 1-4 Alkyl, -N(R7')2, -C(=O)N(R7')2, -NR7'C(=O)-R7', -C(=O)-R7', -O-(CH2) r -R8'、-SC 1-6 Alkyl, -S(O)-C 1-6 Alkyl, -S(O)2-C 1-6 Alkyl, -S-(CH2) r -R8'、-NR7'-(CH2) r -R8' or -(CH2) r -R8', the alkyl, alkenyl, alkynyl, heterocycloalkyl, cycloalkyl are optionally substituted by 1-5 groups selected from R 8” Substituted; in some embodiments, each R 8a 、R 8b 、R 8c Each independently selected from amino, C 1-6 Alkoxy, OH, CN, halogen, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkyl, 4-7 membered heterocycloalkyl containing 1-3 heteroatoms selected from N, O, S, C 3-8 Cycloalkyl, -O-(CH2) r -R8', the alkyl, alkenyl, alkynyl, heterocycloalkyl, cycloalkyl are optionally substituted by 1-5 groups selected from R 8” replace;

[0015] In some embodiments, R 8a Selected from C 2-6 Alkenyl, C 2-6 Alkynyl, 4-7 membered heterocycloalkyl containing 1-3 heteroatoms selected from N, O, S, C 3-8 Cycloalkyl, -OC 3-8 Cycloalkyl, the alkenyl, alkynyl, cycloalkyl, heterocycloalkyl are optionally substituted by 1-5 groups selected from R 8” Substituted; in some embodiments, R 8a Selected from Cyclopropyl, cyclobutyl, -O-cyclopropyl, -O-cyclobutyl, vinyl, the Cyclopropyl and cyclobutyl are further optionally substituted by 1-3 groups selected from methyl, F, Cl, CN, In some embodiments, R 8a Selected from Cyclopropyl, cyclobutyl, -O-cyclopropyl, -O-cyclobutyl, vinyl, the Cyclopropyl and cyclobutyl are further optionally substituted by 1-3 groups selected from methyl, F, Cl, CN, In some embodiments, R 8a Selected from -O-cyclopropyl, vinyl,

[0016] R 8b Selected from C 1-4 Alkyl, halogen or C 2-4 Alkynyl; in some embodiments, R 8b is selected from F, Cl, methyl, ethynyl or propynyl; in some embodiments, R 8b is selected from Cl, methyl, ethynyl or propynyl; in some embodiments, R 8b Selected from halogen or C 2- 4 alkynyl; in some embodiments, R 8b is selected from F, Cl, ethynyl or propynyl; in some embodiments, R 8b Selected from Cl, ethynyl or propynyl;

[0017] R 8c is selected from halogen; in some embodiments, R 8c is selected from F, Cl; in some embodiments, R 8c Selected from F;

[0018] Alternatively, R 8b With R 8c Together form a 4-6 membered carbocyclic ring, said carbocyclic ring is optionally further substituted by 1-3 groups selected from F, Cl, C 1-4 Alkyl, halogenated C 1-4 Alkyl, deuterated C 1-4 In some embodiments, R 8b With R 8c Together they form a 5- or 6-membered carbocyclic ring, which is optionally further substituted with 1-2 substituents selected from F, Cl, methyl, ethyl, propyl, isopropyl, tert-butyl, -CH2F, -CHF2, -CF3, -CH2CH2F, -CH2CHF2, -CH2CF3, -CHFCH2F, -CHFCHF2, -CHFCF3, -CF2CH2F, -CF2CHF2, -CF2CF3, -CH2D, -CHD2, -CD3, -CH2CH2D, -CH2CHD2, -CH2CD3, -CHDCH2D, -CHDCHD2, -CHDCD3, -CD2CH2D, -CD2CHD2, -CD2CD3;

[0019] Each R7' is independently H, D, amino, hydroxyl, C 1-6 Alkyl, halogenated C 1-6Alkyl, deuterated C 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkoxy, C 3-6 Cycloalkyl, C 3-12 Heterocycloalkyl, -NHC 1-6 Alkyl, -N(C 1-6 Alkyl)2, or deuterated C 1-6 Alkoxy, the cycloalkyl, heterocycloalkyl is optionally substituted by 1 to 3 groups selected from deuterium, halogen, cyano, amino, hydroxyl, C 1-6 Alkyl, halogenated C 1-6 Alkyl, deuterated C 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkoxy or deuterated C 1-6 In some embodiments, each R7' is independently H, D, amino, hydroxy, C 1-4 Alkyl, halogenated C 1-4 Alkyl, deuterated C 1-4 Alkyl, C 1-4 Alkoxy, halogenated C 1-4 Alkoxy, C 3-6 Cycloalkyl, C 3-12 Heterocycloalkyl, -NHC 1-4 Alkyl, -N(C 1-4 Alkyl)2, or deuterated C 1-4 Alkoxy, the cycloalkyl, heterocycloalkyl is optionally substituted by 1 to 3 groups selected from deuterium, halogen, cyano, amino, hydroxyl, C 1-4 Alkyl, halogenated C 1-4 Alkyl, deuterated C 1-4 Alkyl, C 1-4 Alkoxy, halogenated C 1-4 Alkoxy or deuterated C 1-4 In some embodiments, each R7' is independently H, D, amino, hydroxy, C 1-4 Alkyl, halogenated C 1-2 Alkyl, deuterated C 1-2 Alkyl, C 1-2 Alkoxy, halogenated C 1-2 Alkoxy, C 3-6 Cycloalkyl, C 3-12 Heterocycloalkyl, -NHC 1-2 Alkyl, -N(C 1-2 Alkyl)2, or deuterated C 1-2 Alkoxy, the cycloalkyl, heterocycloalkyl is optionally substituted by 1 to 3 groups selected from deuterium, halogen, cyano, amino, hydroxyl, C 1-2 Alkyl, halogenated C 1-2 Alkyl, deuterated C 1-2 Alkyl, C1-2 Alkoxy, halogenated C 1-2 Alkoxy or deuterated C 1-2 In some embodiments, each R7' is independently H, D, amino, hydroxy, C 1-4 In some embodiments, each R7' is independently H, C 1-4 Alkyl or halogenated C 1-4 In some embodiments, each R7' is independently H or C 1-4 Alkyl; in some embodiments, each R7' is independently H;

[0020] Each R8' is independently H, D, C 1-6 Alkyl, C 1-4 Alkoxy, C 3-8 Cycloalkyl, 4-12 membered heterocyclic ring containing 1-3 heteroatoms selected from N, O, S, wherein the alkyl and alkoxy groups are optionally substituted by 1-3 heteroatoms selected from halogen, =O, D, OH, NH2, CN, C 1-6 Alkyl, halogenated C 1-6 Alkyl, deuterated C 1-6 Alkyl, -C(O)C 1-4 Alkyl, -C(O)NHC 1-4 Alkyl, -C(O)NH2, -NHC(O)C 1-4 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkoxy and deuterated C 1-6 The cycloalkyl or heterocyclic ring is optionally substituted by 1-5 groups selected from R 8” In some embodiments, each R8' is independently H, C 1-4 Alkoxy, C 3-6 Cycloalkyl, phenyl, 4-7 membered heterocycloalkyl containing 1-3 heteroatoms selected from N, O, S, or 5-6 membered heteroaryl containing 1-3 heteroatoms selected from N, O, S, wherein the cycloalkyl, phenyl, heterocycloalkyl or heteroaryl is optionally substituted by 1-3 halogen, =O, D, OH, NH2, CN, C 1-4 Alkyl, halogenated C 1-4 Alkyl, and C 1-4 In some embodiments, each R8' is independently H, C 1-4 Alkoxy, C 3-6 Cycloalkyl, phenyl, 4-7 membered heterocycloalkyl containing 1-3 heteroatoms selected from N, O, S, or 5-6 membered heteroaryl containing 1-3 heteroatoms selected from N, O, S, wherein the cycloalkyl, phenyl, heterocycloalkyl or heteroaryl is optionally substituted by 1-3 halogen, =O, D, OH, NH2, CN, C 1-4 Alkyl, halogenated C1-4 Alkyl, and C 1-4 In some embodiments, R8' is independently C 1-4 Alkoxy, cyclopropyl, cyclobutyl, cyclopentyl, phenyl, tetrahydropyrrolyl, azetidinyl, piperidinyl, piperazinyl, morpholinyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, Thiphenyl, thiazolyl, pyrrolyl, imidazolyl, oxazolyl, pyridinyl, pyrazinyl, pyrimidinyl or pyridazinyl, optionally substituted by 1-3 groups selected from F, Cl, Br, I, =O, D, OH, NH2, CN, C 1-4 Alkyl, halogenated C 1-2 Alkyl, and C 1-2 Alkoxy group substitution;

[0021] R 8” Selected from halogen, D, OH, NH2, CN, C 1-6 Alkyl, C 1-6 Alkoxy, The alkyl or alkoxy group is optionally further substituted by 1 to 5 groups selected from halogen, deuterium, cyano, amino, hydroxyl, C 1-6 Alkyl or C 1-6 In some embodiments, R 8” Selected from halogen, D, OH, NH2, CN, C 1-4 alkyl, and C 1-4 Alkoxy, the alkyl or alkoxy group is optionally further substituted by 1 to 3 groups selected from halogen, deuterium, cyano, amino, hydroxyl, C 1-2 Alkyl or C 1-2 In some embodiments, R 8” Selected from deuterium, F, Cl, methyl, ethyl, propyl, methoxy, ethoxy, or propoxy, wherein the methyl, ethyl, propyl, methoxy, ethoxy or propoxy group is optionally further substituted with 1 to 3 groups selected from F, Cl, deuterium, hydroxyl, methyl, ethyl, propyl, methoxy, ethoxy or propoxy; in some embodiments, R 8” Selected from halogen, D, OH, NH2, CN, C 1-6 Alkyl, C 1-6 Alkoxy, The alkyl or alkoxy group is optionally further substituted by 1 to 5 groups selected from halogen, deuterium, cyano, amino, hydroxyl, C 1-6 Alkyl or C 1-6 In some embodiments, R 8”Selected from halogen, D, OH, NH2, CN, C 1-4 alkyl, and C 1-4 Alkoxy, the alkyl or alkoxy group is optionally further substituted by 1 to 3 groups selected from halogen, deuterium, cyano, amino, hydroxyl, C 1-2 Alkyl or C 1-2 In some embodiments, R 8” Selected from deuterium, F, Cl, methyl, ethyl, propyl, methoxy, ethoxy, or propoxy, wherein the methyl, ethyl, propyl, methoxy, ethoxy or propoxy group is optionally further substituted with 1 to 3 groups selected from F, Cl, deuterium, hydroxy, methyl, ethyl, propyl, methoxy, ethoxy or propoxy;

[0022] Each r is independently 0, 1, 2, or 3; in some embodiments, each r is independently 0, 1, or 2;

[0023] In some specific embodiments, the compound of formula (I) of the present invention is not selected from the following structures:

[0024] As a more specific first technical solution of the present invention, it relates to a compound represented by formula (I), its stereoisomers, deuterated substances, solvates, or pharmaceutically acceptable salts,

[0025] in,

[0026] D is selected from D1, D2, D3, and D4;

[0027] Each R5 is independently selected from H, D, OH, C 1-4 Alkyl, CN, halogen, C 1-4 Alkoxy, C 2-4 Alkenyl, C 2-4 Alkynyl, 5-6 membered heteroaryl containing 1-3 heteroatoms selected from N, O, S, 4-7 membered heterocycloalkyl containing 1-3 heteroatoms selected from N, O, S or C 3-6 Cycloalkyl, wherein the alkyl, alkoxy, alkenyl, alkynyl, heteroaryl or cycloalkyl is optionally substituted by 1-3 groups selected from halogen, D, CN, OH, C 1-4 Alkyl, C 1-4 Alkoxy and NH2 group substitution;

[0028] R7 is independently H, D, C 1-4 Alkyl, halogenated C 1-4 Alkyl, deuterated C 1-4 Alkyl, C 2-4 Alkenyl, C2-4 Alkynyl, C 3-6 Cycloalkyl or 4-7 membered heterocycloalkyl containing 1-3 heteroatoms selected from N, O, and S;

[0029] Each R 8a 、R 8b 、R 8c Each independently selected from H, D, amino, C 1-6 Alkoxy, OH, CN, halogen, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkyl, 4-7 membered heterocycloalkyl containing 1-3 heteroatoms selected from N, O, S, C 3-8 Cycloalkyl, -COC 1-4 Alkyl, -N(R7')2, -C(=O)N(R7')2, -NR7'C(=O)-R7', -C(=O)-R7', -O-(CH2) r -R8'、-SC 1-6 Alkyl, -S(O)-C 1-6 Alkyl, -S(O)2-C 1-6 Alkyl, -S-(CH2) r -R8'、-NR7'-(CH2) r -R8' or -(CH2) r -R8', the alkyl, alkenyl, alkynyl, heterocycloalkyl, cycloalkyl are optionally substituted by 1-5 groups selected from R 8” replace;

[0030] Alternatively, R 8b With R 8c Together form a 4-6 membered carbocyclic ring, said carbocyclic ring is optionally further substituted by 1-3 groups selected from F, Cl, C 1-4 Alkyl, halogenated C 1-4 Alkyl, deuterated C 1-4 Substitution of alkyl groups;

[0031] Each R7' is independently H, D, amino, hydroxyl, C 1-6 Alkyl, halogenated C 1-6 Alkyl, deuterated C 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkoxy, C 3-6 Cycloalkyl, C 3-12 Heterocycloalkyl, -NHC 1-6 Alkyl, -N(C 1-6 Alkyl)2, or deuterated C 1-6 Alkoxy, the cycloalkyl, heterocycloalkyl is optionally substituted by 1 to 3 groups selected from deuterium, halogen, cyano, amino, hydroxyl, C 1-6 Alkyl, halogenated C1-6 Alkyl, deuterated C 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkoxy or deuterated C 1-6 Alkoxy group substitution;

[0032] Each R8' is independently H, D, C 1-6 Alkyl, C 1-4 Alkoxy, C 3-8 Cycloalkyl, 4-12 membered heterocyclic ring containing 1-3 heteroatoms selected from N, O, S, wherein the alkyl and alkoxy groups are optionally substituted by 1-3 heteroatoms selected from halogen, =O, D, OH, NH2, CN, C 1-6 Alkyl, halogenated C 1-6 Alkyl, deuterated C 1-6 Alkyl, -C(O)C 1-4 Alkyl, -C(O)NHC 1-4 Alkyl, -C(O)NH2, -NHC(O)C 1-4 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkoxy and deuterated C 1-6 The cycloalkyl or heterocyclic ring is optionally substituted by 1-5 groups selected from R 8” replace;

[0033] R 8” Selected from halogen, D, OH, NH2, CN, C 1-6 Alkyl, C 1-6 Alkoxy, The alkyl or alkoxy group is optionally further substituted by 1 to 5 groups selected from halogen, deuterium, cyano, amino, hydroxyl, C 1-6 Alkyl or C 1-6 In some embodiments, R 8” Selected from halogen, D, OH, NH2, CN, C 1-6 Alkyl, C 1-6 Alkoxy, The alkyl or alkoxy group is optionally further substituted by 1 to 5 groups selected from halogen, deuterium, cyano, amino, hydroxyl, C 1-6 Alkyl or C 1-6 Alkoxy group substitution;

[0034] Each r is independently 0, 1, 2 or 3.

[0035] As a more specific second technical solution of the present invention, a compound represented by formula (I) is provided, and its stereoisomers, deuterated substances, solvates, or pharmaceutically acceptable salts have the following structures (I-1) and (I-2):

[0036] The definitions of other groups are consistent with any of the above technical solutions.

[0037] Furthermore, in some specific embodiments, the compounds represented by formula (I-1) and (I-2), their stereoisomers, deuterated forms, solvates, or pharmaceutically acceptable salts, have the following structures: (I-1A), (I-1B), (I-2A), (I-2B):

[0038] The definitions of other groups are consistent with any of the above technical solutions.

[0039] As a more specific third technical solution of the present invention, the compounds of formula (I), formula (I-1), formula (I-2), formula (I-1A), (I-1B), (I-2A), (I-2B), their stereoisomers, deuterated substances, solvates, or pharmaceutically acceptable salts, wherein D is selected from

[0040] The definitions of other groups are consistent with any of the above technical solutions.

[0041] As a more specific fourth technical solution of the present invention, the compounds of formula (I), formula (I-1), formula (I-2), formula (I-1A), (I-1B), (I-2A), (I-2B), their stereoisomers, deuterated substances, solvates, or pharmaceutically acceptable salts, wherein

[0042] R 8a Selected from C 2-6 Alkenyl, C 2-6 Alkynyl, 4-7 membered heterocycloalkyl containing 1-3 heteroatoms selected from N, O, S, C 3-8 Cycloalkyl, -OC 3-8 Cycloalkyl, the alkenyl, alkynyl, cycloalkyl, heterocycloalkyl are optionally substituted by 1-5 groups selected from R 8” replace;

[0043] In some embodiments, R 8a Selected from Cyclopropyl, cyclobutyl, -O-cyclopropyl, -O-cyclobutyl, vinyl, the Cyclopropyl and cyclobutyl are further optionally substituted by 1-3 groups selected from methyl, F, Cl, CN, The group substitution;

[0044] In some embodiments, R 8a Selected from Cyclopropyl, cyclobutyl, -O-cyclopropyl, -O-cyclobutyl, vinyl, the Cyclopropyl and cyclobutyl are further optionally substituted by 1-3 groups selected from methyl, F, Cl, CN, The group substitution;

[0045] In some embodiments, R 8a Selected from -O-cyclopropyl, vinyl,

[0046] The definitions of other groups are consistent with any of the above technical solutions.

[0047] As a more specific fifth technical solution of the present invention, the compounds of formula (I), formula (I-1), formula (I-2), formula (I-1A), (I-1B), (I-2A), (I-2B) and their stereoisomers, deuterated substances, solvates, or pharmaceutically acceptable salts, wherein

[0048] R 8b is selected from F, Cl, methyl, ethynyl or propynyl; in some embodiments, R 8b is selected from Cl, methyl, ethynyl or propynyl; in some embodiments, R 8b is selected from F, Cl, ethynyl or propynyl; in some embodiments, R 8b is selected from Cl, ethynyl or propynyl; or

[0049] R 8b With R 8c Together they form a 5- or 6-membered carbocyclic ring, which is optionally further substituted by 1-2 substituents selected from F, Cl, methyl, ethyl, propyl, isopropyl, tert-butyl, -CH2F, -CHF2, -CF3, -CH2CH2F, -CH2CHF2, -CH2CF3, -CHFCH2F, -CHFCHF2, -CHFCF3, -CF2CH2F, -CF2CHF2, -CF2CF3, -CH2D, -CHD2, -CD3, -CH2CH2D, -CH2CHD2, -CH2CD3, -CHDCH2D, -CHDCHD2, -CHDCD3, -CD2CH2D, -CD2CHD2, -CD2CD3.

[0050] The definitions of other groups are consistent with any of the above technical solutions.

[0051] As a more specific sixth technical solution of the present invention, the compounds of formula (I), formula (I-1), formula (I-2), formula (I-1A), (I-1B), (I-2A), (I-2B) and their stereoisomers, deuterated substances, solvates, or pharmaceutically acceptable salts, wherein

[0052] Each R7' is independently H, C 1-4 Alkyl or halogenated C 1-4alkyl;

[0053] Each R8' is independently H, C 1-4 Alkoxy, C 3-6 Cycloalkyl, phenyl, 4-7 membered heterocycloalkyl containing 1-3 heteroatoms selected from N, O, S, or 5-6 membered heteroaryl containing 1-3 heteroatoms selected from N, O, S, wherein the cycloalkyl, phenyl, heterocycloalkyl or heteroaryl is optionally substituted by 1-3 halogen, =O, D, OH, NH2, CN, C 1-4 Alkyl, halogenated C 1-4 Alkyl, and C 1-4 Alkoxy group substitution;

[0054] Each r is independently 0, 1, or 2;

[0055] The definitions of other groups are consistent with any of the above technical solutions.

[0056] As a more specific seventh technical solution of the present invention, the compound of the present invention, its stereoisomer, deuterated substance, solvate, or pharmaceutically acceptable salt, the compound of formula (I) is not selected from the following structures:

[0057] As a more specific eighth technical solution of the present invention, the compounds represented by Formula (I), Formula (I-1), (I-2), Formula (I-1A), (I-1B), (I-2A), (I-2B) of the present invention, their stereoisomers, deuterated forms, solvates, or pharmaceutically acceptable salts, wherein:

[0058] Ring D is selected from

[0059] R 8a Selected from C 2-6 Alkenyl, C 2-6 Alkynyl, 4-7 membered heterocycloalkyl containing 1-3 heteroatoms selected from N, O, S, C 3-8 Cycloalkyl, -OC 3-8 Cycloalkyl, the alkenyl, alkynyl, cycloalkyl, heterocycloalkyl are optionally substituted by 1-5 groups selected from R 8” Substituted; preferably R 8a Selected from Cyclopropyl, cyclobutyl, -O-cyclopropyl, -O-cyclobutyl, vinyl, the Cyclopropyl and cyclobutyl are further optionally substituted by 1-3 groups selected from methyl, F, Cl, CN, More preferably, R 8a Selected from -O-cyclopropyl, vinyl,

[0060] R8b is selected from F, Cl, methyl, ethynyl or propynyl; preferably R 8b is selected from Cl, methyl, ethynyl or propynyl.

[0061] As a more specific ninth technical solution of the present invention, the compound of the present invention, its stereoisomer, deuterated substance, solvate, or pharmaceutically acceptable salt is selected from the structure of Table A below.

[0062] Table A

[0063] As a more specific tenth technical solution of the present invention, a pharmaceutical composition is provided, which contains the compound described in any one of the first to ninth technical solutions, its stereoisomers, deuterated substances, solvates, or pharmaceutically acceptable salts, and pharmaceutically acceptable carriers and / or excipients.

[0064] As a more specific eleventh technical solution of the present invention, a use is provided, namely, use of the compound described in any one of the first to third technical solutions, its stereoisomer, deuterated form, solvate, or pharmaceutically acceptable salt, or the composition described in the tenth technical solution in the preparation of a medicament for treating / preventing PRMT5·MTA-mediated diseases.

[0065] Furthermore, the PRMT5·MTA-mediated disease is selected from liver cancer, breast cancer, skin cancer, bladder cancer, pancreatic cancer or head and neck cancer.

[0066] The present invention also provides a composition or pharmaceutical formulation comprising a compound according to any of the foregoing embodiments, a stereoisomer, deuterated form, solvate, or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and / or excipient. The pharmaceutical composition may be in unit dosage form (a unit dosage is also referred to as a "dose strength").

[0067] Furthermore, the composition or pharmaceutical preparation of the present invention contains 1-1500 mg of the compound described in any one of the aforementioned schemes, its stereoisomers, deuterated substances, solvates, or pharmaceutically acceptable salts, and a pharmaceutically acceptable carrier and / or excipient.

[0068] The present invention also provides the use of a compound according to any of the preceding embodiments, or a stereoisomer, deuterated form, solvate, or pharmaceutically acceptable salt thereof, in the preparation of a medicament for treating or preventing a PRMT5·MTA-mediated disease. Furthermore, the PRMT5·MTA-mediated disease is liver cancer, breast cancer, skin cancer, bladder cancer, pancreatic cancer, or head and neck cancer.

[0069] The present invention also provides a method for treating a disease in a mammal, comprising administering to a subject a therapeutically effective amount of a compound of any of the foregoing schemes, a stereoisomer, deuterated form, solvate, or pharmaceutically acceptable salt thereof, wherein the disease is preferably liver cancer, breast cancer, skin cancer, bladder cancer, pancreatic cancer, or head and neck cancer, and wherein the therapeutically effective amount is preferably 1-1500 mg. In some embodiments, the mammal of the present invention includes a human.

[0070] As used herein, an "effective amount" or "therapeutically effective amount" refers to the administration of a sufficient amount of a compound disclosed herein to alleviate, to some extent, one or more symptoms of the disease or condition being treated. In some embodiments, the result is a reduction and / or alleviation of the signs, symptoms, or causes of the disease, or any other desired alteration of a biological system. For example, an "effective amount" for therapeutic uses is the amount of a composition comprising a compound disclosed herein required to provide a clinically significant reduction in disease symptoms. Examples of therapeutically effective amounts include, but are not limited to, 1-1500 mg, 1-1400 mg, 1-1300 mg, 1-1200 mg, 1-1000 mg, 1-900 mg, 1-800 mg, 1-700 mg, 1-600 mg, 1-500 mg, 1-400 mg, 1-300 mg, 1-250 mg, 1-200 mg, 1-150 mg, 1-125 mg, 1-100 mg, 1-80 mg, 1-60 mg, 1-50 mg, 1-40 mg, 1-25 mg, 1- 20mg, 5-1500mg, 5-1000mg, 5-900mg, 5-800mg, 5-700mg, 5-600mg, 5-500mg, 5-400mg, 5-300mg, 5-250mg, 5-200mg, 5 -150mg, 5-125mg, 5-100mg, 5-90mg, 5-70mg, 5-80mg, 5-60mg, 5-50mg, 5-40mg, 5-30mg, 5-25mg, 5-20mg, 10-1500mg, 10-1000mg, 10-900mg, 10-800mg, 10-700mg, 10-600mg, 10-500mg, 10-450mg, 10-400mg, 10-300mg, 10-250mg, 10-20 0mg, 10-150mg, 10-125mg, 10-100mg, 10-90mg, 10-80mg, 10-70mg, 10-60mg, 10-50mg, 10-40mg, 10-30mg, 10-20mg; 2 0-1500mg, 20-1000mg, 20-900mg, 20-800mg, 20-700mg, 20-600mg, 20-500mg, 20-400mg, 20-350mg, 20-300mg, 20-25 0mg, 20-200mg, 20-150mg, 20-125mg, 20-100mg, 20-90mg, 20-80mg, 20-70mg, 20-60mg, 20-50mg, 20-40mg, 20-30mg;50-1500mg, 50-1000mg, 50-900mg, 50-800mg, 50-700mg, 50-600mg, 50-500mg, 50-400mg, 50-300mg, 50-250mg, 50-200mg, 50-150mg, 50-125mg, 5 0-100mg; 100-1500mg, 100-1000mg, 100-900mg, 100-800mg, 100-700mg, 100-600mg, 100-500mg, 100-400mg, 100-300mg, 100-250mg, 100-200mg;

[0071] In some embodiments, the pharmaceutical composition or formulation of the present invention contains the above-mentioned therapeutically effective amount of the compound of the present invention or its stereoisomer, deuterated substance, solvate, or pharmaceutically acceptable salt;

[0072] The present invention relates to a pharmaceutical composition or pharmaceutical preparation comprising a therapeutically effective amount of a compound of the present invention, or a stereoisomer, deuterated form, solvate, or pharmaceutically acceptable salt thereof, and a carrier and / or excipient. The pharmaceutical composition may be in the form of a unit dosage form (the amount of the active ingredient in a unit dosage form is also referred to as the "drug strength"). In some embodiments, the pharmaceutical composition includes but is not limited to 1 mg, 1.25 mg, 2.5 mg, 5 mg, 10 mg, 12.5 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 110 mg, 120 mg, 125 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg g, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, 1500 mg of a compound of the present invention or a stereoisomer, deuterated form, solvate, or pharmaceutically acceptable salt thereof.

[0073] A method for treating a disease in a mammal, comprising administering to a subject a therapeutically effective amount of a compound of the present invention, a stereoisomer, deuterated form, solvate, or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and / or excipient, wherein the therapeutically effective amount is preferably 1-1500 mg, and the disease is preferably liver cancer, breast cancer, skin cancer, bladder cancer, pancreatic cancer, or head and neck cancer.

[0074] A method for treating a disease in a mammal, comprising administering a compound of the present invention, a stereoisomer, deuterated form, solvate, or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and / or excipient to a subject at a daily dose of 1-1500 mg / day. The daily dose may be a single dose or divided doses. In some embodiments, the daily dose includes but is not limited to 10-1500 mg / day, 20-1500 mg / day, 25-1500 mg / day, 50-1500 mg / day, 75-1500 mg / day, 100-1500 mg / day, 200-1500 mg / day, 10-1000 mg / day, 20-1000 mg / day, 25-1000 mg / day. , 50-1000 mg / day, 75-1000 mg / day, 100-1000 mg / day, 200-1000 mg / day, 25-800 mg / day, 50-800 mg / day, 100-800 mg / day, 200-800 mg / day, 25-400 mg / day, 50-400 mg / day, 100-400 mg / day, 200-400 mg / day, in some embodiments, daily doses include but are not limited to 1 mg / day, 5 mg / day, 10 mg / day, 20 mg / day, 25 mg / day, 50 mg / day, 75 mg / day, 100 mg / day, 125 mg / day, 150 mg / day, 200 mg / day, 400 mg / day, 600 mg / day day, 800 mg / day, 1000 mg / day, 1200 mg / day, 1400 mg / day, 1500 mg / day.

[0075] The present invention relates to a kit, which may include a composition in single-dose or multi-dose form, wherein the kit contains a compound of the present invention or a stereoisomer, deuterated substance, solvate, or pharmaceutically acceptable salt thereof, and the amount of the compound of the present invention or its stereoisomer, deuterated substance, solvate, or pharmaceutically acceptable salt is the same as that in the above-mentioned pharmaceutical composition.

[0076] The amount of the compound of the present invention or its stereoisomer, deuterated form, solvate or pharmaceutically acceptable salt in the present invention is in each case calculated based on the free base form.

[0077] "Preparation specifications" refers to the weight of the main drug contained in each vial, tablet or other unit preparation.

[0078] Synthesis route

[0079] Those skilled in the art can prepare the compounds of the present invention by combining known organic synthesis techniques, using commercially available chemicals and / or compounds described in the chemical literature as starting materials. "Commercially available chemicals" are obtained from reputable commercial sources, including suppliers such as Titan Technology, Anage Chemical, Shanghai Demo, Chengdu Kelon Chemical, Shaoyuan Chemical Technology, Nanjing Yaoshi, WuXi AppTec, and J&K Technology.

[0080] the term

[0081] Unless otherwise specified in the present invention, the terms of the present invention have the following meanings:

[0082] The carbon, hydrogen, oxygen, sulfur, nitrogen or halogen involved in the groups and compounds of the present invention include their isotopes, and the carbon, hydrogen, oxygen, sulfur, nitrogen or halogen involved in the groups and compounds of the present invention are optionally further replaced by one or more of their corresponding isotopes, wherein the isotopes of carbon include 12 C. 13 C and 14 C, hydrogen isotopes include protium (H), deuterium (deuterium, also known as heavy hydrogen), tritium (T, also known as super tritium), oxygen isotopes include 16 O. 17 O and 18 O, sulfur isotopes include 32 S. 33 S. 34 S and 36 S, nitrogen isotopes include 14 N and 15 N, an isotope of fluorine 19 F, chlorine isotopes include 35 Cl and 37 Isotopes of Cl, bromine include 79 Br and 81 Br.

[0083] "Halogen" herein refers to F, Cl, Br, I, or isotopes thereof.

[0084] "Halo" or "halogen-substituted" refers to substitution with one or more halogens selected from F, Cl, Br, I, or isotopes thereof. The upper limit of the number of halogen substituents is equal to the sum of the number of hydrogen atoms that can be replaced by the substituted group. Unless otherwise specified, the number of halogen substituents is any integer between 1 and the upper limit. When the number of halogen substituents is greater than 1, they may be the same or different halogens. Typical examples include 1-5 halogen substitutions, 1-3 halogen substitutions, 1-2 halogen substitutions, and 1 halogen substitution.

[0085] "Deuterium" refers to the isotope of hydrogen (H) and is synonymous with "D".

[0086] "Deuterated" or "deuterated compound" refers to a situation where a hydrogen atom on an alkyl, cycloalkyl, alkylene, aryl, heteroaryl, thiol, heterocycloalkyl, alkenyl, alkynyl or other group is replaced by at least one deuterium atom. The upper limit of the number of deuterated groups is equal to the sum of the number of replaceable hydrogen atoms in the substituted group. Unless otherwise specified, the number of deuterated groups is any integer between 1 and the upper limit, for example, 1-20 deuterium atoms, 1-10 deuterium atoms, 1-6 deuterium atoms, 1-3 deuterium atoms, 1-2 deuterium atoms or 1 deuterium atom.

[0087] “C x-y " group refers to a group containing x to y carbon atoms, such as "C 1-6 "Alkyl" refers to an alkyl group containing 1 to 6 carbon atoms.

[0088] "Alkyl" refers to a monovalent straight-chain or branched saturated aliphatic hydrocarbon group. Typically, it is an alkyl group having 1 to 20 carbon atoms, or an alkyl group having 1 to 8 carbon atoms, or an alkyl group having 1 to 6 carbon atoms, or an alkyl group having 1 to 4 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, neobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, and the like. The alkyl group may be further substituted with a substituent.

[0089] "Alkylene" refers to a divalent straight-chain or branched saturated alkyl group. Examples of alkylene groups include, but are not limited to, methylene, ethylene, and the like.

[0090] "Haloalkyl" refers to a situation where one or more hydrogen atoms in an alkyl group are replaced by one or more halogen atoms (such as fluorine, chlorine, bromine, iodine, or their isotopes). The upper limit of the number of halogen substituents is equal to the sum of the number of replaceable hydrogen atoms in the alkyl group. Unless otherwise specified, the number of halogen substituents is any integer between 1 and the upper limit. Typically, an alkyl group is substituted with 1-5 halogens, or 1-3 halogens, or 1-2 halogens, or 1 halogen. When the number of halogen substituents is greater than 1, they can be the same or different halogens. Specific examples include, but are not limited to, -CF3, -CH2Cl, -CH2CF3, -CCl2, CF3, etc.

[0091] "Alkoxy" or "alkyloxy" refers to an -O-alkyl group. For example, -OC 1-8 Alkyl, -OC 1-6 Alkyl, -OC 1-4 Alkyl or -OC 1-2Specific non-limiting examples include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, n-hexyloxy, cyclopropyloxy, and cyclobutyloxy, and the like; the alkoxy groups may be optionally substituted with substituents.

[0092] "Haloalkoxy" refers to an -O-haloalkyl group. For example, -O-haloC 1-8 Alkyl, -O-halogenated C 1-6 Alkyl, -O-halogenated C 1-4 Alkyl or -O-halogenated C 1-2 Alkyl; the upper limit of the number of halogen substituents is equal to the sum of the number of hydrogen atoms that can be substituted by the substituted group. Unless otherwise specified, the number of halogen substituents is any integer between 1 and the upper limit, preferably 1-5 halogen substitutions, 1-3 halogen substitutions, 1-2 halogen substitutions, or 1 halogen substitution; when the number of halogen substituents is greater than 1, they may be the same or different halogen substitutions; non-limiting examples include monofluoromethoxy, difluoromethoxy, trifluoromethoxy, difluoroethyloxy, and the like.

[0093] "Alkenyl" refers to a straight or branched chain hydrocarbon group containing at least one carbon-carbon double bond (C=C), typically containing 2 to 18 carbon atoms, such as 2 to 8 carbon atoms, further such as 2 to 6 carbon atoms, and further such as 2 to 4 carbon atoms, examples of which include but are not limited to vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-methyl-1-butenyl, 2-methyl-1-butenyl, 2-methyl-2-butenyl, 2-methyl-3-butenyl, 2-methyl-4-butenyl, 2-methyl-5-butenyl, 2-methyl-6-butenyl, 2-methyl-7-butenyl, 2-methyl-8-butenyl, 2-methyl-9-butenyl, 2-methyl-1-butenyl, 2-methyl-2-butenyl, 2-methyl-3 ... -methyl-3-butenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-methyl-1-pentenyl, 2-methyl-1-pentenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 1-octenyl, 3-octenyl, 1-nonenyl, 3-nonenyl, 1-decenyl, 4-decenyl, 1,3-butadiene, 1,3-pentadiene, 1,4-pentadiene and 1,4-hexadiene, etc.; the alkenyl group may be optionally further substituted with a substituent.

[0094] "Alkynyl" refers to a straight-chain or branched hydrocarbon group containing at least one carbon-carbon triple bond (C≡C), typically containing 2 to 18 carbon atoms, further containing 2 to 8 carbon atoms, further containing 2 to 6 carbon atoms, and further containing 2 to 4 carbon atoms. Examples include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, butynyl, 2-butynyl, 3-butynyl, 1-methyl-2-propynyl, 4-pentynyl, 3-pentynyl, 1-methyl-2-butynyl, 2-hexynyl, 3-hexynyl, 2-heptynyl, 3-heptynyl, 4-heptynyl, 3-octynyl, 3-nonynyl, and 4-decynyl. The alkynyl group may be optionally substituted with a substituent.

[0095] "Cycloalkyl" refers to a saturated or partially unsaturated, non-aromatic carbocyclic hydrocarbon group containing no ring heteroatoms. Cycloalkyl can be monocyclic, bicyclic or polycyclic. Bicyclic or polycyclic rings can be cyclic, spirocyclic, bridged or a combination thereof. Bicyclic or polycyclic rings can include one or more aromatic rings, but the ring system as a whole does not have aromaticity. The attachment site can be on the aromatic ring or on the non-aromatic ring. Usually, the cycloalkyl group contains 3 to 20 carbon atoms, further contains 3-8 carbon atoms, and further contains 3-6 carbon atoms; when it is a monocyclic cycloalkyl group, it contains 3-15 carbon atoms, or 3-10 carbon atoms, or 3-8 carbon atoms, or 3-6 carbon atoms; when it is a bicyclic or polycyclic cycloalkyl group, it contains 5-12 carbon atoms, or 5-11 carbon atoms, or 6-10 carbon atoms; non-limiting examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, butenyl, cyclopentenyl, cyclohexenyl, The cycloalkyl group may be optionally substituted with a substituent.

[0096] "Aryl" refers to a carbocyclic ring having aromaticity and not containing heteroatoms, including monocyclic aryl and condensed ring aryl. It generally contains 6 to 14 carbon atoms, and further contains 6 to 10 carbon atoms. Non-limiting examples include phenyl, naphthyl, anthracenyl, and phenanthrenyl. Aryl can optionally be substituted with a substituent.

[0097] "Carbocycle" or "carbocyclyl" refers to a saturated, partially unsaturated, or aromatic carbocycle, including aryl and cycloalkyl. Carbocycles can be monocyclic, bicyclic, or polycyclic, including bridged, fused, and spirocyclic rings and combinations thereof. Carbocycles typically have 3 to 12 carbon atoms, or 3-10 carbon atoms, or 3-6 carbon atoms. In non-limiting examples, monocyclic carbocycles include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, or phenyl, and bicyclic bridged rings include etc., double ring and ring include etc., bicyclic spiro ring includes The carbocyclic ring may be optionally substituted with a substituent.

[0098] "Heterocycloalkyl" refers to a saturated or partially unsaturated non-aromatic carbocyclic ring containing 1, 2, 3, or 4 heteroatoms selected from N, S, and O. Heterocycloalkyl can be monocyclic, bicyclic, or polycyclic. Bicyclic or polycyclic rings can be bridged, fused, spirocyclic, or a combination thereof. Bicyclic or polycyclic rings can include one or more aromatic or heteroaromatic rings, but the ring system as a whole is not aromatic. The attachment point can be on the aromatic ring or on the non-aromatic ring. Heterocycloalkyl groups are usually 3 to 20-membered rings. When they are monocyclic heterocycloalkyl groups, they are usually 3 to 15-membered rings, or 3-10-membered rings, or 3-8-membered rings, or 3-6-membered rings; when they are bicyclic or polycyclic heterocycloalkyl groups, they are usually 5-12-membered rings, or 5-11-membered rings, or 6-9-membered rings. The heteroatoms N and S include their oxidation states. Non-limiting examples of heterocycloalkyl groups include azetidinyl, morpholinyl, piperazinyl, piperidinyl, tetrahydropyranyl, oxetanyl, pyranyl, azolidinyl, azolinyl, oxolanyl, oxekinyl, and the like. Heterocycloalkyl groups may be optionally substituted with substituents.

[0099] "Heteroaromatic ring" or "heteroaryl" unless otherwise specified refers to a ring containing 1 to 4 heteroatoms selected from N, O or S and their oxidation states and having aromatic properties, which may be monocyclic, bicyclic or polycyclic, and the bicyclic or polycyclic rings may be bridged, parallel, spirocyclic or combinations thereof; when bicyclic or polycyclic, it may be a fusion of a heteroaryl and an aryl, or a fusion of a heteroaryl and a heteroaryl, wherein either the heteroaryl or the aryl may be a connection site. Non-limiting examples include furanyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, indolyl, purinyl, The heteroaryl group may be optionally substituted by a substituent.

[0100] "Heterocycle" or "heterocyclyl" refers to a saturated or unsaturated, aromatic or non-aromatic ring containing 1 to 4 heteroatoms selected from N, O, or S and their oxidation states, and includes heteroaryl and heterocycloalkyl. Heterocycles include monocyclic heterocycles, bicyclic bridged heterocycles, bicyclic heterocycles, and bicyclic spiro heterocycles, or combinations thereof. Heterocycles are typically 3- to 12-membered heterocycles, 5- to 12-membered heterocycles, or 5- to 7-membered heterocycles. The heterocyclic group may be attached to a heteroatom or a carbon atom, and non-limiting examples include oxirane, aziridine, oxetanyl, azetidinyl, 1,3-dioxolanyl, 1,4-dioxolanyl, 1,3-dioxanyl, piperazinyl, azepanyl, pyridinyl, furanyl, thienyl, pyranyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, pyrazolyl, pyridazinyl, imidazolyl, piperidinyl, piperidinyl, morpholinyl, thiomorpholinyl, 1,3-dithianyl , dihydrofuranyl, dihydropyranyl, dithiolanyl, tetrahydrofuranyl, tetrahydropyrrolyl, tetrahydroimidazolyl, oxazolyl, dihydrooxazolyl, tetrahydrooxazolyl, tetrahydrothiazolyl, tetrahydropyranyl, benzimidazolyl, benzopyridinyl, pyrrolopyridinyl, benzodihydrofuranyl, azabicyclo[3.2.1]octyl, azabicyclo[5.2.0]nonyl, oxatricyclo[5.3.1.1]dodecyl, azaadamantyl and oxaspiro[3.3]heptanyl, The heterocyclic ring may be optionally substituted with a substituent.

[0101] "Spiro" refers to a polycyclic group that shares a carbon atom (called a spiro atom) between the rings, which may contain 0 or more double bonds or triple bonds, and may contain 0 to 5 heteroatoms selected from N, O, S, P, Si and their oxidation states. Typically, the spiro ring is a 6- to 14-membered ring, or a 6- to 12-membered ring, or a 6- to 10-membered ring. Typically, the spiro ring is a trispirotri (representing a three-membered ring spirotricycle), a trispirotetra, a trispiropenta, a trispirohexa, a tetraspirotetra, a tetraspiropenta, a tetraspirohexa, a pentaspiropenta or a pentaspirohexa. Non-limiting examples of spiro rings include The spiro ring may be optionally substituted with a substituent.

[0102] "Parallel ring" or "condensed ring" refers to a polycyclic group in which the rings share two adjacent ring atoms and a chemical bond, and may contain one or more double bonds or triple bonds, and the rings may contain 0 to 5 heteroatoms selected from N, S, O, P, Si and their oxidation states. Usually, the cyclic ring is a 5- to 20-membered ring, or a 5- to 14-membered ring, or a 5- to 12-membered ring, or a 5- to 10-membered ring. Usually, the cyclic ring is a three- to four-membered ring (representing a cyclic ring formed by a three-membered ring and a four-membered ring. According to the IUPC naming rules, it is possible to have a three-membered ring as the basic ring or a four-membered ring as the basic ring. The same applies below), a three- to five-ring ring, a three- to six-ring ring, a four- to four-ring ring, a four- to five-ring ring, a four- to six-ring ring, a five- to five-ring ring, a five- to six-ring ring, and a six- to six-ring ring. Non-limiting examples of cyclic rings include purine, quinoline, isoquinoline, benzopyran, benzofuran, benzothiophene, The cyclic ring may be aromatic or non-aromatic and may be optionally substituted with a substituent.

[0103] "Bridged ring" means two rings share two non-adjacent ring atoms and may contain one or more double or triple bonds. A bridged ring may contain 0 to 5 heteroatoms selected from N, S, O, P, Si, and oxidation states thereof. Typically, a bridged ring has 5 to 20, or 5 to 14, or 5 to 12, or 5 to 10 ring atoms.

[0104] "Substitution" or "substituent" unless otherwise specified refers to any substitution at a position permitted by chemical theory, and the number of substituents complies with the chemical bond rules. Exemplary substituents include but are not limited to: C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-8 Heteroalkyl, C 5-12 Aryl, 5-12 membered heteroaryl, hydroxyl, C 1-6 Alkoxy, C 5-12 Aryloxy, thiol, C 1-6 Alkylthio, cyano, halogen, C 1-6 Alkylthiocarbonyl, C 1-6 Alkylcarbamoyl, N-carbamoyl, nitro, silyl, sulfinyl, sulfonyl, sulfoxide, halo 1-6 Alkyl, halogenated C 1-6 Alkoxy, amino, phosphonic acid, -CO2(C 1-6 alkyl), -OC(=O)(C 1-6 alkyl), -OCO2(C 1-6 alkyl), -C(=O)NH2, -C(=O)N(C 1- 6-alkyl)2,-OC(=O)NH(C 1-6 alkyl), -NHC(=O)(C 1-6 alkyl), -N(C 1-6alkyl)C(=O)(C 1-6 Alkyl), -NHCO2(C 1-6 alkyl), -NHC(=O)N(C 1-6 alkyl)2,-HC(=O)NH(C 1-6 alkyl), -NHC(=O)NH2, -NHSO2(C 1-6 alkyl), -SO2N(C 1-6 Alkyl)2,-SO2NH(C 1-6 alkyl), -SO2NH2, -SO2C 1-6 Alkyl, etc.

[0105] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and the description includes instances where the event or circumstance occurs and instances where it does not. For example, "alkyl optionally substituted with F" means that the alkyl group may but need not be substituted with F, and the description includes instances where the alkyl group is substituted with F and instances where the alkyl group is not substituted with F.

[0106] "Pharmaceutically acceptable salts" refer to salts of the compounds of the present invention that retain the biological effectiveness and properties of the free acids or free bases, and the free acids are reacted with non-toxic inorganic or organic bases, or the free bases are reacted with non-toxic inorganic or organic acids.

[0107] A "pharmaceutical composition" refers to a mixture of one or more compounds described herein, or stereoisomers, deuterated forms, solvates, pharmaceutically acceptable salts, or cocrystals thereof, with other ingredients, wherein the other ingredients include physiologically / pharmaceutically acceptable carriers and / or excipients.

[0108] "Carrier" refers to a system that does not cause significant irritation to the organism and does not eliminate the biological activity and properties of the administered compound, and can change the way the drug enters the human body and its distribution in the body, control the release rate of the drug and deliver the drug to the target organ. Non-limiting examples include microcapsules and microspheres, nanoparticles, liposomes, etc.

[0109] "Excipient" refers to a substance that is not itself a therapeutic agent but serves as a diluent, excipient, binder, and / or vehicle that is added to a pharmaceutical composition to improve its handling or storage properties or to allow or facilitate the formation of a compound or pharmaceutical composition into a unit dosage form for administration. As known to those skilled in the art, pharmaceutical excipients can serve a variety of functions and can be described as wetting agents, buffers, suspending agents, lubricants, emulsifiers, disintegrants, absorbents, preservatives, surfactants, colorants, flavoring agents, and sweeteners. Examples of pharmaceutical excipients include, but are not limited to: (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, cellulose acetate, hydroxypropyl methylcellulose, hydroxypropyl cellulose, microcrystalline cellulose, and cross-linked carboxymethylcellulose (e.g., cross-linked sodium carboxymethylcellulose); (4) tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn starch, and maltodextrin. oil and soybean oil; (10) glycols such as propylene glycol; (11) polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) pH buffer solutions; (21) polyesters, polycarbonates and / or polyanhydrides; and (22) other non-toxic compatible substances used in pharmaceutical preparations.

[0110] "Stereoisomers" refer to isomers resulting from different spatial arrangements of atoms in a molecule, including cis-trans isomers, enantiomers, and conformational isomers.

[0111] The compounds of the present invention also include their tautomers. For example, when the present invention describes a compound on the left side in which the pyrimidine ring is substituted with OH, it also includes the tautomer compound on the right side.

[0112] "Solvate" refers to a substance formed by a compound of the present invention or a salt thereof and a stoichiometric or non-stoichiometric amount of a solvent bound to the compound or salt thereof by non-covalent forces between the molecules. When the solvent is water, the solvate is a hydrate.

[0113] A "cocrystal" is a crystal formed by the active pharmaceutical ingredient (API) and cocrystal former (CCF) bound together by hydrogen bonds or other non-covalent bonds. Both the API and CCF are solid in their pure form at room temperature, and the components exist in a fixed stoichiometric ratio. A cocrystal is a multi-component crystal, encompassing both binary cocrystals formed between two neutral solids and multi-component cocrystals formed between a neutral solid and a salt or solvate. DETAILED DESCRIPTION

[0114] The present invention will be described in detail below through examples. Where specific conditions are not specified in the examples, the experimental methods are carried out according to conventional conditions. The examples are provided to better illustrate the present invention, but it should not be understood that the present invention is limited to the examples. Any non-essential improvements and adjustments made by those skilled in the art to the embodiments based on the above invention are still within the scope of protection of the present invention.

[0115] Test Method

[0116] The structures of the compounds were determined by nuclear magnetic resonance (NMR) or / and mass spectrometry (MS). NMR shifts (δ) were expressed in 10 -6 The unit of (ppm) is given. NMR measurements were performed using a Bruker Avance III 400 and Bruker Avance 300 NMR spectrometer. The solvents used were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD). The internal standard was tetramethylsilane (TMS).

[0117] MS was determined using (Agilent 6120B (ESI) and Agilent 6120B (APCI));

[0118] HPLC determination was performed using an Agilent 1260DAD high pressure liquid chromatograph (Zorbax SB-C 18 100 × 4.6 mm, 3.5 μM);

[0119] Thin layer chromatography silica gel plates used were Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates. The specifications of the silica gel plates used for thin layer chromatography (TLC) were 0.15 mm to 0.20 mm, and the specifications used for thin layer chromatography separation and purification products were 0.4 mm to 0.5 mm.

[0120] Column chromatography generally uses Yantai Huanghai silica gel 200-300 mesh silica gel as the carrier.

[0121] Example 1

[0122] Synthesis of intermediate 1

[0123] Step 1: Dissolve 4-chloro-2,5-difluorobenzonitrile (Compound 1a, CAS No.: 135748-35-5) (200g, 1.15mol) and N-bromosuccinimide (411g, 2.31mol) in dichloroethane (1.5L), and then add toluene-4-sulfonic acid (39.6g, 230mmol) and palladium (II) acetate (19g, 115mmol). Stir at 77°C for 48h under nitrogen protection. Cool, add dichloromethane, wash, dry, and concentrate to obtain a crude product, which is separated by column chromatography (petroleum ether: ethyl acetate (v / v) = 10:1) to obtain compound 1b (210g crude product, containing compound 1a, 1 H NMR analysis showed: 1b:1a=2.3:1).

[0124] Step 2: 1b (50 g, 199 mmol) and 1-methyl-5-(tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (62 g, 299 mmol) were added to a flask, followed by diethylene glycol dimethyl ether (800 mL), followed by rubidium fluoride (38 g, 365 mmol) and bis(triphenylphosphine)palladium(II) chloride (14 g, 19.9 mmol). The mixture was stirred magnetically at 90°C under nitrogen for 16 hours. After cooling, ethyl acetate and water were added, and the mixture was extracted with ethyl acetate. The organic phases were combined and concentrated to obtain the crude product, which was separated by column chromatography (petroleum ether:ethyl acetate (v / v) = 10:1) to obtain the product. Compound 1c (30 g, yield: 60%) was obtained by recrystallization (petroleum ether:ethyl acetate (v / v) = 10:1).

[0125] LC-MS (ESI): m / z = 254.0 [M+H] + .

[0126] Step 3: 1c (5 g, 19.71 mol) was dissolved in acetonitrile (80 mL), and N-iodosuccinimide (8.87 g, 39.42 mol) and trifluoroacetic acid (1.12 g, 9.86 mol) were added. The mixture was stirred at 50°C for 5 h. LCMS showed that the reaction was complete. The product was concentrated to give a crude product, which was separated by column chromatography (petroleum ether:ethyl acetate (v / v) = 5:1) to give compound 1d (6 g, yield: 80%).

[0127] LC-MS (ESI): m / z = 379.9 [M+H] + .

[0128] Step 4: Dissolve compound 1d (900 mg, 2.37 mmol) in tetrahydrofuran (15 mL), add methyl 1-hydroxy-1-cyclopropanecarboxylate (116 mg, 3.32 mmol), cool the system to 0°C, add sodium hydride (133 mg, 3.32 mmol) with stirring, and slowly warm to room temperature with stirring for 2 hours. Slowly add water to quench the reaction, extract with ethyl acetate (15 mL x 3), collect the organic phase, wash with saturated brine, dry, and concentrate to obtain compound 1e (1 g crude product), which is used directly in the next reaction.

[0129] LC-MS (ESI): m / z = 476.0 [M+H] + .

[0130] Step 5: Compound 1e (1 g, 2.1 mmol) was dissolved in a mixture of tetrahydrofuran (10 mL), methanol (10 mL), and water (6 mL). Lithium hydroxide monohydrate (264 mg, 6.3 mmol) was added and stirred at room temperature for 3 hours. The mixture was concentrated directly, and dilute hydrochloric acid was added to the resulting residue to precipitate a solid. The solid was filtered and the filter cake was washed with water. The filter cake was collected and dried to obtain compound 1f (800 mg, yield: 82.4%).

[0131] LC-MS (ESI): m / z = 462.0 [M+H] + .

[0132] Step 6: Compound 1f (800 mg, 1.73 mmol) was dissolved in N,N-dimethylformamide (10 mL). Ammonium chloride (741 mg, 13.86 mmol), N,N-diisopropylethylamine (894 mg, 6.92 mmol), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (658 mg, 1.73 mmol) were added and stirred at room temperature overnight. Ethyl acetate and water were added for separation and extraction. The organic phase was collected, dried, and concentrated to obtain compound 1g (700 mg crude product), which was used directly in the next reaction.

[0133] LC-MS (ESI): m / z = 461.2 [M+H] + .

[0134] Step 7: Dissolve compound 1g (700 mg, 1.52 mmol) in dichloromethane (10 mL), add triethylamine (465 mg, 4.56 mmol) and trifluoroacetic anhydride (479 mg, 2.28 mmol), and stir at room temperature for 3 hours. Extraction was performed with water and dichloromethane. The organic phase was collected, dried, and concentrated. The resulting residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate (v / v) = 4:1) to afford compound 1h (580 mg, yield: 83%).

[0135] LC-MS (ESI): m / z = 443.0 [M+H] + .

[0136] Step 8: Compound 1h (500 mg, 1.13 mmol) was dissolved in dioxane (12 mL), and triethylamine (692 mg, 6.79 mmol), pinacol borane (723 mg, 5.65 mmol), and tetrakis(triphenylphosphine)palladium (127 mg, 0.11 mmol) were added. The atmosphere was purged with nitrogen three times, and the mixture was stirred at 90°C for 16 h. LCMS indicated completion of the reaction, and the mixture was concentrated directly. Water (20 mL) was added to the resulting product, and the mixture was extracted with ethyl acetate. The organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The resulting residue was separated by column chromatography (petroleum ether:ethyl acetate (v / v) = 5:1) to afford Intermediate 1 (350 mg, yield: 70%).

[0137] 1 H NMR (400MHz, DMSO-d6) δ8.06(d,1H),7.77(s,1H),3.76(s,3H),1.93-1.81(m,2H),1.74–1.53(m,2H),1.15(d,12H); LC-MS(ESI): m / z=443.1[M+H] + .

[0138] Synthesis of compounds 1 and 2:

[0139] Step 1: Compound 3-amino-5-bromo-2-methylbenzoic acid methyl ester (1A) (100 g, 410 mmol) and acetonitrile (1.1 L) were added to a 3 L single-necked flask, followed by triethylamine (110 mL, 820 mmol), di-tert-dicarbonate (119 g, 533 mmol) and 4-dimethylaminopyridine (5.0 g, 41 mmol), and the mixture was reacted at 50°C for 2 hours. After the reaction, the reaction solution was cooled and concentrated under reduced pressure to remove the organic solvent. 1 L of water was then added, and the mixture was extracted with ethyl acetate (1 L x 3). The organic phases were combined, washed with saturated brine (1 L), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain compound 1B (123 g, yield: 87%).

[0140] 1 H NMR (400MHz, DMSO-d6) δ8.88(s,1H),7.74(t,1H),7.63(d,1H),3.84(s,3H),2.27(s,3H),1.48(s,9H).

[0141] Step 2: Compound 1B (123 g, 358 mmol) and lithium hydroxide monohydrate (45 g, 1.07 mol) were added to a 3 L single-necked flask, followed by the addition of THF (800 mL) and water (250 mL). The mixture was allowed to react at room temperature for 18 hours. After the reaction, the organic solvent was removed under reduced pressure, and the mixture was diluted with water (300 mL). The pH was then adjusted to 2-3 with 2 mol / L hydrochloric acid, and the aqueous phase was extracted with ethyl acetate (1 L x 3). The organic phases were combined, washed with saturated brine (1 L), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain compound 1C (102 g, yield: 86%).

[0142] 1 H NMR (400MHz, DMSO-d6) δ8.82(s,1H),7.65(d,1H),7.57(d,1H),2.27(s,3H),1.47(s,9H).

[0143] Step 3: Compound 1C (102 g, 308.9 mmol) was added to a 3 L three-necked flask and dissolved in DMF (1.0 L). DIPEA (102 mL, 620 mmol) and HATU (153 g, 403 mmol) were added at room temperature and stirred at room temperature for 30 min. Finally, dimethylhydroxylamine hydrochloride (45.6 g, 465 mmol) was added and allowed to react at room temperature for 2 h. After completion of the reaction, the reaction solution was poured into 2 L of water and extracted three times with ethyl acetate (1 L x 3). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated. The resulting residue was purified by column chromatography (PE:EA (v:v) = 3:1) to obtain compound 1D (82 g, yield: 71%).

[0144] 1 H NMR (400MHz, DMSO-d6) δ8.78(s,1H),7.63(s,1H),7.24(d,1H),3.43(s,3H),3.26(s,3H),2.04(s,3H),1.47(s,9H).

[0145] Step 4: Compound 1D (82 g, 220 mmol) was placed in a 3 L three-necked flask and dissolved in THF (800 mL). Methylmagnesium bromide (220 mL, 660 mmol) was added at 0°C and the mixture was slowly warmed to room temperature for 16 hours. After completion of the reaction, saturated ammonium chloride solution (1 L) was added to the reaction system, stirred for 10 minutes, and then extracted with EA (500 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated. The resulting residue was purified by silica gel column chromatography (PE:EA (v:v) = 3:1) to afford compound 1E (70 g, yield: 97%).

[0146] 1H NMR (400MHz, DMSO-d6) δ8.84(s,1H),7.67(d,1H),7.62(d,1H),2.53(s,3H),2.13(s,3H),1.47(s,9H).

[0147] Step 5: Compound 1E (53 g, 160 mmol) was added to a 3L three-necked flask and dissolved with tert-butyl alcohol (500 mL). Water (500 mL) was then added, followed by anhydrous potassium carbonate (44 g, 320 mmol). The mixture was heated to 70°C and potassium permanganate (179 g, 1.13 mol) was added to the reaction system in six batches at 30-min intervals. After the addition was complete, the reaction was continued at 70°C for 1 hour. The dark purple color of potassium permanganate disappeared, indicating that the reaction was complete. The reaction solution was cooled to room temperature and filtered through celite. The filtrate was collected and the pH was subsequently adjusted to 2-3 with 2 mol / L hydrochloric acid. The mixture was then extracted with a mixed solvent of EA:THF = 10:1 (700 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated to give compound 1F (39 g, yield: 62%).

[0148] LC-MS (ESI): m / z = 388.1 [M+H] + .

[0149] Step 6: Compound 1F (39 g, 100 mmol) was placed in a 1 L single-necked flask and dissolved in ethanol (200 mL). Hydrazine hydrate (7.24 mL, 120 mmol) was then added and reacted at 75°C for 15 hours. The precipitated solid was cooled to room temperature, filtered, and the filter cake was washed with ethanol (100 mL). The filter cake was collected and dried to obtain compound 1G (17 g, 44% yield).

[0150] 1 H NMR (400MHz, DMSO-d6) δ12.00(s,1H),8.63(d,1H),8.05(d,1H),1.50(s,9H).

[0151] Step 7: Compound 1G (17 g, 44.2 mmol) was placed in a 1 L single-necked flask, and methanol (150 mL) was added and mixed thoroughly. Hydrochloric acid methanol solution (4 mol / L, 240 mL) was then added and reacted at 70°C for 2 hours. After completion of the reaction, the organic solvent was removed by concentration under reduced pressure. The residue was added with water (200 mL), and the pH was adjusted to 8 with sodium bicarbonate. The mixture was filtered, and the filter cake was washed with water (100 mL) and dried to obtain compound 1H (13 g, yield: 98%).

[0152] 1H NMR (400MHz, DMSO-d6) δ12.87(s,1H),7.58(s,2H),7.13(s,1H),3.87(s,3H); LC-MS(ESI):m / z=298.0[M+H] + .

[0153] Step 8: Compound 1H (13 g, 43.6 mmol) and p-toluenesulfonic acid (33 g, 174.4 mmol) were placed in a 3L three-necked flask, followed by acetonitrile (2.0 L) and nitrogen substitution three times. The mixture was mixed at 0°C. Sodium nitrite (7.5 g, 109 mmol) was then dissolved in water (20 mL) and added dropwise to the reaction mixture. The reaction was maintained at this temperature for 1 hour. Finally, potassium iodide (21.7 g, 130.8 mmol) was dissolved in water (20 mL) and added dropwise to the reaction mixture. The mixture was warmed to room temperature and reacted for 2 hours. After completion of the reaction, saturated sodium thiosulfate solution (200 mL) was added to the mixture, stirred for 10 minutes, and then the organic phase was removed by concentration under reduced pressure. The mixture was filtered, and the filter cake was washed with clean water (300 mL). The filter cake was collected and dried to obtain compound 1I (8.2 g, yield: 45.98%).

[0154] 1 H NMR (400MHz, DMSO-d6) δ13.24(s,1H),8.73(d,1H),8.61(d,1H),3.90(s,3H); LC-MS(ESI): m / z=409.1[M+H] + .

[0155] Step 9: Compound 1I (8.2 g, 20 mmol) was placed in a 1 L single-necked flask, and THF (240 mL) and EtOH (240 mL) were added to dissolve the mixture. Anhydrous calcium chloride (2.5 g, 2.4 mmol) was added at 0°C, and sodium borohydride (1.47 g, 40 mmol) was then added to the reaction system in four batches at 10-min intervals. The mixture was allowed to react for 10 min after the addition was complete. Saturated ammonium chloride solution (100 mL) was added to the system, and the mixture was stirred at room temperature for 10 min. The mixture was filtered, and the filter cake was washed with water (100 mL). The filter cake was collected and dried. The resulting solid was mixed with methanol (200 mL), stirred for three hours, filtered, and the filter cake was collected and dried to obtain compound 1J (4.8 g, yield: 62.84%).

[0156] 1 H NMR (400MHz, DMSO-d6) δ12.61(s,1H),8.55(d,1H),8.28(d,1H),5.55(t,1H),4.61(d,2H); LC-MS(ESI): m / z=381.1[M+H] + .

[0157] Step 10: Compound 1J (4.6 g, 12.1 mmol) was added to a 100 mL single-necked flask and mixed with DCM:THF (1:1) (100 mL). Sulfur oxychloride (5 mL) was then added, followed by DMF (1 mL). The mixture was allowed to react at room temperature for 16 hours. After completion, the organic solvent was removed by concentration under reduced pressure. The residue was slurried with petroleum ether (100 mL) for 20 minutes and then filtered. The filter cake was washed with petroleum ether (30 mL), collected, and dried to afford compound 1K (4.4 g, yield: 91.67%).

[0158] 1 H NMR (400MHz, DMSO-d6) δ12.86(s,1H),8.60(d,1H),8.27(d,1H),5.01(s,2H).

[0159] Step 11: Compound 1K (4.4 g, 11.1 mmol) was added to a 100 mL single-necked flask and dissolved in DMF (50 mL). Potassium phthalimide (2.05 g, 11.1 mmol) was then added and allowed to react at room temperature for 4 hours. After completion of the reaction, the reaction solution was poured into water (500 mL), stirred for 10 minutes, and then filtered. The filter cake was washed with water (100 mL), collected, and dried to obtain compound 1L (5.0 g, yield: 89.28%).

[0160] 1 H NMR (400MHz, DMSO-d6) δ12.54(s,1H),8.61(d,1H),8.40(d,1H),7.96–7.94(m,2H),7.90–7.88(m,2H),5.12(s,2H); LC-MS(ESI): m / z=510.3[M+H] + .

[0161] Step 12: Compound 1L (5.0 g, 9.8 mmol) was added to a reaction flask and mixed with anhydrous ethanol (25 mL). Hydrazine hydrate (3 mL, 39.2 mmol) was then added and reacted at 80°C for 1 hour. After the reaction was completed, the mixture was cooled to room temperature and filtered. The filter cake was washed once with anhydrous ethanol (25 mL) and once with water (50 mL). The filter cake was collected and dried to obtain compound 1M (3.5 g, yield: 94.59%).

[0162] LC-MS (ESI): m / z = 382.0 [M+H] + .

[0163] Step 13: Compound 1M (3.5 g, 9.2 mmol) and triethylamine (3.8 mL, 27.6 mmol) were added to a reaction flask and dissolved in DCM (60 mL). Di-tert-butyl dicarbonate (4.0 g, 18.4 mmol) was then added and allowed to react at room temperature for 3 hours. After completion of the reaction, the mixture was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (PE:EA (v:v) = 2:1 to 1:2) to obtain compound 1N (2.0 g, yield: 45.24%).

[0164] 1 H NMR (400MHz, DMSO-d6) δ12.64(s,1H),8.56(d,1H),8.23(s,1H),7.40(s,1H),4.35(d,2H),1.40(s,9H); LC-MS(ESI): m / z=480.3[M+H] + .

[0165] Step 14: Compound 1N (2.0 g, 4.16 mmol), Pd(PPh3)2Cl2 (292 mg, 0.42 mmol), and tetraethylammonium chloride (1.40 g, 8.32 mmol) were added to a reaction flask, dissolved with DMF (60 mL), and replaced with nitrogen three times. Tributyl vinyl tin (1.32 g, 4.16 mmol) was then added, and the mixture was reacted at 100°C for 2 hours. After the reaction, the reaction solution was poured into water (200 mL) and extracted with ethyl acetate (100 mL × 3). The organic phases were combined, washed once with saturated brine, and dried over anhydrous sodium sulfate. The residue was concentrated and purified by silica gel column chromatography (PE:EA (v:v) = 2:1 to 0:1) to obtain compound 1O (1.0 g, yield: 63.29%).

[0166] 1 H NMR (400MHz, DMSO-d6) δ12.57(s,1H),8.15(s,1H),8.13–8.07(m,1H),8.06(d,1H),7.36–7. 30(m,1H),5.83(d,1H),5.45(d,1H),4.38(t,2H),1.40(s,9H); LC-MS(ESI):m / z=382.1[M+H] + .

[0167] Step 15: Compound 1O (1.0 g, 2.63 mmol), intermediate 1 (1.16 g, 2.63 mmol), Pd(dppf)Cl2 (190 mg, 0.26 mmol), and potassium carbonate (1.09 g, 7.89 mmol) were added to a reaction flask. 1,4-dioxane and water (v:v = 6:1, 70 mL) were then added. The atmosphere was purged with nitrogen three times and the reaction was carried out at 85°C for 6 hours. After completion of the reaction, the mixture was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (PE:EA (v:v) = 4:1 to 1:1) to obtain compound 1P (370 mg, yield: 23%).

[0168] LC-MS (ESI): m / z = 616.40 [M+H] + .

[0169] Step 16: Compound 1P (370 mg, 0.60 mmol) was separated by SFC to obtain P1 (SFC analysis retention time 2.115 min, designated as compound 1P-1. SFC chiral analysis method: instrument: SHIMADZU LC-30AD; chromatographic column: Chiralcel Cellulose-2 column; mobile phase: A for CO2; B for 0.05% DEA in ETOH; gradient: B 5-40%; flow rate: 3 mL / min; column pressure: 100 bar; column temperature: 35°C; wavelength: 220 nm) and P2 (retention time 2.329 min, designated as compound 1P-2). SFC separation method: Instrument: Waters 150SFC, Column: Chiralcel Cellulose-2 column, Mobile phase: A for CO2; B for 0.1% NH3·H2O in MeOH, Gradient: B 35%, Flow rate: 120 mL / min, Column pressure: 100 bar; Column temperature: 25°C; Absorption wavelength: 220 nm, Cycle time: 3 min. Sample preparation: The compound was dissolved in acetonitrile at a concentration of 10 mg / mL; Injection: 2 mL per injection; Workup: After separation, the mixture was concentrated by rotary evaporation at 35°C, and the solvent was then dried in a lyophilizer at -80°C to obtain compounds 1P-1 (150 mg, 40.54%) and 1P-2 (140 mg, 37.83%).

[0170] Step 17: Dissolve compound 1P-1 (150 mg, 0.24 mmol) in dichloromethane (8 mL), then add trifluoroacetic acid (2 mL) and stir at room temperature for 2 hours. Concentrate the reaction mixture and dilute with dichloromethane. Then, add sodium bicarbonate solution to make the mixture alkaline. Extract the mixture with dichloromethane, collect the organic phase, dry it, and concentrate it. The resulting residue is purified by silica gel column chromatography (dichloromethane:methanol (v / v) = 10:1) to obtain compound 1 (70 mg, yield: 56.6%).

[0171] 1 H NMR(400MHz,DMSO-d6)δ12.33(s,1H),8.39(s,1H),8.18(d,1H),8.15–8.08(m,1H),7.68(d,2H),5.44(d,1H) ,5.32(d,1H),3.82(s,3H),3.74(s,2H),1.90-1.85(m,2H),1.71-1.59(m,2H); LC-MS(ESI):m / z=516.2[M+H] + .

[0172] Compound 1P-2 (140 mg, 0.23 mmol) was dissolved in dichloromethane (8 mL), followed by the addition of trifluoroacetic acid (2 mL) and stirring at room temperature for 2 hours. The reaction system was concentrated and diluted with dichloromethane, followed by the addition of sodium bicarbonate solution to adjust the solution to alkalinity. The separated liquids were extracted with dichloromethane, and the organic phase was collected, dried, and concentrated. The resulting residue was purified by silica gel column chromatography (dichloromethane:methanol (v / v) = 10:1) to afford compound 2 (50 mg, 42.7%).

[0173] 1 H NMR(400MHz,DMSO-d6)δ12.33(s,1H),8.39(s,1H),8.18(d,1H),8.15–8.09(m,1H),7.68(d,2H),5.44(d,1H) ,5.31(d,1H),3.82(s,3H),3.75(s,2H),1.89-1.85(m,2H),1.72–1.58(m,2H); LC-MS(ESI):m / z=516.2[M+H] + .

[0174] Synthesis of intermediate 2:

[0175] Compound 3a (10 g, 59.09 mmol) was dissolved in dichloromethane (60 mL), and trifluoroacetic acid (30.59 g, 268.37 mmol) was slowly added. The mixture was reacted at room temperature for 3 hours. After the reaction was completed, the product was directly dried to obtain intermediate 2 (4 g crude product), which was used directly in the next reaction.

[0176] LC-MS (ESI): m / z = 70.3 [M+H] + .

[0177] Synthesis of intermediate 3:

[0178] Compound 1O (4 g, 10.52 mmol), potassium acetate (2.58 g, 26.30 mmol), bis(pentanoyldiboron) (5.34 g, 21.04 mmol), and xphos Pd g2 (0.83 g, 1.05 mmol) were dissolved in 1,4-dioxane (50 mL) and replaced with nitrogen three times. The mixture was reacted at 100°C for 3 hours. After completion of the reaction, the mixture was filtered, the filtrate was dried, and the mixture was slurried to obtain intermediate 3 (3.8 g crude product), which was used directly in the next reaction.

[0179] LC-MS (ESI): m / z = 428.2 [M+H] + .

[0180] Example 3 and Example 4

[0181] Synthesis of compound 3 and compound 4:

[0182] Step 1: Compound 1d (5 g, 13.17 mmol), intermediate 2 (3.64 g, 52.68 mmol), and ethyldiisopropylamine (6.81 g, 52.68 mmol) were dissolved in acetonitrile (40 mL) and reacted at 50°C for 4 hours. After completion of the reaction, the product was concentrated and the resulting residue was purified by column chromatography (petroleum ether:ethyl acetate (v / v) = 10:1) to afford compound 3D (5.5 g, 97% yield).

[0183] LC-MS (ESI): m / z = 429.1 [M+H] + .

[0184] Step 2: Compound 3D (0.5 g, 1.31 mmol), intermediate 3 (0.84 g, 1.97 mmol), 1,1-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (0.096 g, 0.13 mmol), and potassium carbonate (0.45 g, 3.28 mmol) were dissolved in 1,4-dioxane (8 mL) and water (2 mL). The atmosphere was purged with nitrogen three times and the mixture was reacted at 90°C for 5 hours. After completion of the reaction, the reaction solution was directly concentrated, and the resulting residue was purified by column chromatography (dichloromethane:ethyl acetate (v / v) = 4:1) to afford compound 3E (0.2 g, yield: 25%).

[0185] LC-MS (ESI): m / z = 602.3 [M+H] + .

[0186] Step 3: Compound 3E (0.9 g, 1.49 mmol), tributyl(prop-1-ynyl)stannane (1.96 g, 5.96 mmol), and Xphos Pd G2 (0.23 g, 0.30 mmol) were dissolved in 1,4-dioxane (25 mL). The atmosphere was purged with nitrogen three times and the mixture was reacted at 100°C overnight. The reaction solution was concentrated, and the resulting residue was purified by column chromatography (dichloromethane:ethyl acetate (v / v) = 4:1) to afford compound 3F (0.38 g, 42% yield).

[0187] LC-MS (ESI): m / z = 606.3 [M+H] + .

[0188] Step 4: Compound 3F (300 mg, 0.50 mmol) was separated by SFC to give P1 (SFC retention time: 1.560 min, designated as compound 3F-1. SFC chiral analysis method: instrument: SHIMADZU LC-30AD; chromatographic column: Chiral OX column; mobile phase: A for CO2; B for 0.05% DEA in MeOH and acetonitrile; gradient: B 40%; flow rate: 3 mL / min; column pressure: 100 bar; column temperature: 35°C; wavelength: 220 nm) and P2 (SFC retention time: 2.130 min, designated as compound 3F-2). SFC separation method: Instrument: Waters 150SFC, Column: Chiral OX column, Mobile phase: A for CO2; B for 0.1% NH3·H2O in MeOH and acetonitrile, Gradient: B 60%, Flow rate: 80 mL / min, Column pressure: 100 bar; Column temperature: 25°C; Absorption wavelength: 220 nm, Cycle time: 4.5 min. Sample preparation: The compound was dissolved in acetonitrile at a concentration of 10 mg / mL; Injection: 3 mL per injection; Workup: After separation, the mixture was concentrated by rotary evaporation at 35°C, and the solvent was dried in a lyophilizer at -80°C to obtain compounds 3F-1 (45 mg, 15%) and 3F-2 (53 mg, 17%).

[0189] Step 5: Dissolve compound 3F-1 (45 mg, 0.074 mmol) in dichloromethane (4 mL), then add trifluoroacetic acid (1 mL) and stir at room temperature for 2 hours. The reaction mixture was concentrated and diluted with dichloromethane, followed by sodium bicarbonate solution to make the solution alkaline. Extract the solution with dichloromethane, collect the organic phase, dry it, and concentrate it. The resulting residue was purified by preparative HPLC. Method: Waters 2767 preparative HPLC; Column: SunFire@Prep C18 (19 mm × 150 mm); Mobile phase composition: Mobile phase A: acetonitrile; Mobile phase B: water (containing 1 / 1000th of ammonium acetate); Gradient: 20% to 70% acetonitrile. Compound 3 (13 mg, yield: 35%) was obtained.

[0190] 1H NMR (400MHz, DMSO-d6) δ12.34(s,1H),8.35(s,1H),8.19-8.08(m,1H),7.76(d,1H),7.64(d,1H),6.99(d,1H),5.46–5. 28(m,2H),5.09-5.03(m,2H),4.78–4.65(m,4H),3.81(s,2H),3.77(s,3H),2.13(s,3H); LC-MS(ESI):m / z=506.6[M+H] + .

[0191] Compound 3F-2 (53 mg, 0.088 mmol) was dissolved in dichloromethane (4 mL), followed by the addition of trifluoroacetic acid (1 mL) and stirring at room temperature for 2 hours. The reaction system was concentrated and diluted with dichloromethane, followed by the addition of sodium bicarbonate solution to make the solution alkaline. The separated liquids were extracted with dichloromethane, the organic phase was collected, dried, and concentrated, and the resulting residue was purified by preparative HPLC. Method: Waters 2767 preparative HPLC; Column: SunFire@Prep C18 (19 mm × 150 mm); Mobile phase composition: Mobile phase A: acetonitrile; Mobile phase B: Water (containing 1 / 1000th of ammonium acetate); Gradient: 20% to 70% acetonitrile. Compound 4 (13 mg, yield: 29%) was obtained.

[0192] 1 H NMR (400MHz, DMSO-d6) δ12.33(s,1H),8.35(s,1H),8.19-8.07(m,1H),7.76(d,1H),7.65(d,1H),6.99(d,1H),5.49–5. 22(m,2H),5.14–4.99(m,2H),4.78–4.65(m,4H),3.80(s,2H),3.77(s,3H),2.13(s,3H); LC-MS(ESI):m / z=506.6[M+H] + .

[0193] Example 5

[0194] Step 1: Compound 1c (10 g, 39.43 mmol) and [1,3-bis(2,6-diisopropylphenyl)imidazol-2-ylidene](3-chloropyridine)palladium dichloride (2.69 g, 3.94 mmol) were dissolved in anhydrous cyclopentyl methyl ether (50 mL). Under nitrogen, trimethylaluminum (18 mL, a 2 mol / L solution in toluene) was slowly added. The temperature was raised to 80°C and the reaction was allowed to react for 4 hours. After completion of the reaction, the mixture was cooled and concentrated. The resulting residue was purified by column chromatography (ethyl acetate:petroleum ether (v:v) = 0-40%) to afford compound 5A (6.2 g, yield: 67.4%).

[0195] LC-MS (ESI): m / z = 234.1 [M+H] + .

[0196] Step 2: Compound 5A (0.50 g, 2.14 mmol), trifluoroacetic acid (49 mg, 0.43 mmol), and N-bromosuccinimide (0.50 g, 2.78 mmol) were dissolved in acetonitrile (5 mL) and reacted at 60°C for 8 hours. The mixture was concentrated directly, and the resulting residue was separated by column chromatography (ethyl acetate:petroleum ether (v:v) = 0-20%) to afford compound 5B (0.63 g, yield: 94.8%).

[0197] LC-MS(ESI):m / z=312.0,314.0[M+H] + .

[0198] Step 3: Compound 5B (1.00 g, 3.20 mmol), 3-methyleneazetidine (2.21 g, 32.00 mmol), triethylamine (3.24 g, 32.00 mmol), and potassium carbonate (1.33 g, 9.60 mmol) were dissolved in N,N-dimethylformamide (5 mL) and reacted at 60°C overnight. After the reaction, water was added to the system, and the mixture was extracted with ethyl acetate. The organic phase was collected, dried, and concentrated. The resulting residue was separated by column chromatography (ethyl acetate:petroleum ether (v:v) = 0-20%) to afford compound 5C (0.75 g, yield: 64.8%).

[0199] LC-MS(ESI):m / z=361.1,363.1[M+H] + .

[0200] Step 4: Compound 5C (400 mg, 1.11 mmol), intermediate 3 (711.45 mg, 1.67 mmol) and sodium bicarbonate (0.23 g, 2.78 mmol) were added sequentially to 1,4-dioxane (15 mL) solvent, followed by addition of distilled water (5 mL). After nitrogen substitution, chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II) (87 mg, 0.11 mmol) was added. After nitrogen substitution three times, the temperature was raised to 60°C and the reaction was allowed to proceed overnight. After completion of the reaction, the mixture was cooled to room temperature and filtered. The filtrate was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by reverse-phase column chromatography (acetonitrile:water (v:v) = 0-80%) to give compound 5D (58 mg, yield: 9.00%).

[0201] LC-MS (ESI): m / z = 582.2 [M+H] + .

[0202] Step 5: Compound 5D (8.00 mg, 0.01 mmol) was dissolved in dichloromethane (3 mL), and trifluoroacetic acid (1 mL) was added. The mixture was reacted at room temperature for 3 hours. After the reaction was complete, the reaction solution was concentrated to dryness under reduced pressure. Acetonitrile (10 mL) and water (10 mL) were added to the residue, and the mixture was lyophilized in a freeze dryer at -85°C to obtain compound 5 (3 mg, yield: 45.30%).

[0203] 1 H NMR(400MHz,DMSO-d6)δ12.76(s,1H),8.41(s,1H),8.40-8.34(m,2H),8.13-8.04(m,1H),7.95(s,1H),7.70–7.61(m,1H),6.92 –6.71(m,1H),5.39–4.93(m,4H),3.76-3.74(m,4H),3.62-3.58(m,2H),2.89(s,3H),2.73(s,3H); LC-MS(ESI):m / z=482.2[M+H] + .

[0204] Example 6 and Example 7

[0205] Step 1: Compound 5D (80 mg, 0.14 mmol) was subjected to SFC separation to obtain P1 (SFC analysis retention time: 0.882 min, set as compound 6A-1) and P2 (SFC analysis retention time: 1.082 min, set as compound 6A-2). SFC chiral analysis method: instrument: Waters UPC2 analytical SFC (SFC-H); chromatographic column: ChiralPak IH, 150×4.6 mm ID, 3 μm; mobile phase: A for CO2 and B for Ethanol (0.05% DEA); gradient: B 5-40%; flow rate: 2.5 mL / min; column pressure: 100 bar; column temperature: 35°C; wavelength: 220 nm). SFC chiral separation method: Instrument: WATERS 150 preparative SFC (SFC-26); Column: ChiralPak IH, 250×30 mm ID, 10 μm; Mobile phase: A for CO2 and B for Ethanol (0.1% NH3·H2O); Gradient: B 30%; Flow rate: 120 mL / min; Column pressure: 100 bar; Column temperature: 38°C; Wavelength: 220 nm; Cycle time: ~6 min. Sample preparation: Dissolve the compound in methanol and dichloromethane (20 mL); Injection: 2.5 mL per injection; Workup: After separation, concentrate on a rotary evaporator at 35°C to yield compounds 6A-1 (32 mg) and 6A-2 (33 mg).

[0206] Step 2: Dissolve compound 6A-1 (32.00 mg, 0.05 mmol) in dichloromethane (3 mL), add trifluoroacetic acid (1 mL), and react at room temperature for 3 hours. After the reaction is complete, the reaction solution is concentrated under reduced pressure. The resulting residue is purified by preparative HPLC to obtain compound 6 (5 mg, yield: 18%).

[0207] 1 H NMR (400MHz, DMSO-d6) δ12.32-12.31(m,1H),8.36-8.35(m,1H),8.18-8.11(m,1H),7.74-7.69(m,2H),6.89-6.87(m,1H),5.42-5.28(m,2H),5 .09-5.06(m,2H),4.80–4.61(m,4H),3.80-2.77(m,2H),3.76-3.70(m,3 H),2.34-2.33(m,3H),1.90-1.89(m,2H); LC-MS(ESI):m / z=482.2[M+H] + .

[0208] Compound 6A-2 (33.00 mg, 0.05 mmol) was dissolved in dichloromethane (3 mL), and trifluoroacetic acid (1 mL) was added. The mixture was reacted at room temperature for 3 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The residue was purified by preparative HPLC to give compound 7 (7 mg, yield: 25%).

[0209] 1 H NMR (400MHz, DMSO-d6) δ12.62-12.61(m,1H),8.40-8.39(m,1H),8.15-8.08(m,1H),7.78-7.57(m,2H),6.90-6.88(m,1H),5.34–5.18(m,2H),5 .14–5.05(m,2H),4.76-4.65(m,4H),4.15-4.14(m,2H),3.77-3.76(m,3 H),2.34-2.33(m,3H),1.92-1.91(m,2H); LC-MS(ESI):m / z=482.2[M+H] + .

[0210] Biological test evaluation

[0211] 1. PRMT5 and PRMT5·MTA binding assay

[0212] 1.1 PRMT5 enzyme activity test method

[0213] Prepare 1× assay buffer (10 mM Tris, 1 mM DTT, 0.01% BSA, 0.01% Tween-20, adjusted to pH 8.0). Dissolve the test compound in DMSO to a 10 mM stock solution and dilute to a 100-fold final concentration. Add 100 nL of compound to each well in the treatment group, and 100 nL of DMSO to the blank and negative control wells. Then, add 5 μL of PRMT5 / MEP50 (BPS, Cat#31921) enzyme solution (final concentration 1 nM) to the treatment and negative control wells, and 5 μL of 1× assay buffer to the blank wells. Incubate at room temperature for 15 minutes. Add 5 μL of a mixture of H4(1-21)S1ac (0.035 μM; GL Biochem, customized) and SAM (0.434 μM; Sigma, Cat#A7007) to each well and incubate at room temperature for 1 hour. Prepare 1× Epigenetics buffer (PerkinElmer, Cat#AL008F) to dilute the acceptor and donor beads. Add 15 μL of acceptor beads (final concentration 10 μg / mL; PerkinElmer, Cat#AL150C) and donor beads (final concentration 10 μg / mL; PerkinElmer, Cat#AS106M) and incubate at room temperature for 1 hour in the dark. Detection was performed using the Alpha module of an Enspire microplate reader. The resulting data were used to calculate the inhibition rate in Excel: Inhibition rate (%) = (maximum negative control - detection signal value) / (maximum negative control - minimum blank control) × 100. IC values ​​were fitted using XL-Fit. 50 value.

[0214] 1.2 PRMT5·MTA enzyme activity assay

[0215] Prepare 1× assay buffer (10 mM Tris, 1 mM DTT, 0.01% BSA, 0.01% Tween-20, adjusted to pH 8.0). Dissolve the test compound in DMSO to a 10 mM stock solution and dilute to a 100-fold final concentration. Add 100 nL of compound to each well in the treatment group, and 100 nL of DMSO to the blank and negative control wells. Then, add 5 μL of a solution of PRMT5 / MEP50 (BPS, Cat#31921) (final concentration 1 nM) and MTA (final concentration 1 μM) to the treatment and negative control wells, and 5 μL of 1× assay buffer to the blank wells. Incubate at room temperature for 15 minutes. Add 5 μL of a mixture of H4(1-21)S1ac (0.035 μM; GL Biochem, customized) and SAM (0.434 μM; Sigma, Cat#A7007) to each well and incubate at room temperature for 1 hour. Prepare 1× Epigenetics buffer (PerkinElmer, Cat# AL008F) to dilute the acceptor and donor beads. Add 15 μL of acceptor beads (final concentration 10 μg / mL; PerkinElmer, #AL150C) and donor beads (final concentration 10 μg / mL; PerkinElmer, Cat# AS106M) and incubate at room temperature for 1 hour in the dark. Detection is then performed using the Alpha module of an Enspire microplate reader.

[0216] The obtained data were used to calculate the inhibition rate in Excel: inhibition rate (%) = (maximum negative control - detection signal value) / (maximum negative control - minimum blank control) × 100, and IC was fitted using XL-Fit. 50 value.

[0217] The compounds of the present invention have an IC<100 nM for PRMT5·MTA 50 IC values ​​of some compounds 50 The values ​​are shown in Table 1, where AA represents IC 50 <5nM, A represents 5nM≤IC 50 <100nM, B represents 100nM≤IC 50 <500nM, C represents 500nM≤IC 50 <1000nM, D represents 1000nM≤IC 50 .

[0218] Table 1 Inhibitory activity of compounds against PRMT5·MTA

[0219] Conclusion: The compounds of the present invention, such as the compounds in the examples, have significant inhibitory activity against PRMT5·MTA, and some preferred compounds have an IC of less than 1 nM.50 At the same time, in the absence of MTA, the inhibitory activity against PRMT5 is relatively weak, that is, the compounds of the present invention have selective inhibition against PRMT5·MTA. For example, the IC50 value of compound 3 against PRMT5·MTA is 1.15 nM, and the IC50 value against PRMT5 is 845 nM. The selectivity of compound 7 is greater than 10 times, showing excellent selectivity.

[0220] 2. MIAPACA-2 cell proliferation inhibition experiment

[0221] MIAPACA-2 cells were cultured in DMEM (ATCC, Cat#30-2002; supplemented with 10% FBS and 1% double-antibody) until confluence reached approximately 85% before plating. The cells were discarded, rinsed with 1× PBS, and then digested with trypsin (Gibco, Cat#15400-054). When the cells became round and began to detach, culture medium was added to terminate digestion. The cells were pipetted off and transferred to a sterile centrifuge tube. Centrifuged at 1000 rpm for 3 minutes, the supernatant was removed, and the supernatant discarded. Culture medium was added to the centrifuge tube to resuspend the cells and count them. Based on the count results, the cell suspension was adjusted to the appropriate concentration and poured into a sample reservoir. The suspension was added to the first 96-well cell plate (Corning, Cat#3903). Ten Day 0 wells were also plated in the second 96-well cell plate. The compounds were dissolved in DMSO to 10 mM and stored for later use. During the experiment, the compound stock solution was serially diluted 5-fold. 24 hours after plating, add the diluted compound to the first 96-well plate, set up a DMSO vehicle control group, and continue incubation at 37°C, 5% CO2 for 5 days. After 5 days of incubation, slowly and carefully aspirate the supernatant from the first 96-well plate using a multichannel pipette and discard it. Add TrypLE (Gibco, Cat#12604-013) to each well and place in a 37°C incubator for 5 minutes until the cells are completely detached. Then, add culture medium to terminate the digestion and pipette evenly. Take a third 96-well plate (Corning, Cat#3903), add DMEM medium and the digested cell suspension to each well, and mix thoroughly with a pipette. Add the diluted compound and set up a DMSO vehicle control group. Continue incubation at 37°C, 5% CO2 for 5 days. 24 hours after plating, CELL VIABILITY reagent (Vazyme, Cat#DD1101-03) was added to each well of the second 96-well cell plate. CELL VIABILITY reagent was added to each well of the first 96-well cell plate on the 5th day after administration. CELL VIABILITY reagent was added to each well of the third 96-well cell plate on the 10th day after the first administration. The cells were incubated at room temperature for 10 minutes. After the incubation, the cells were gently shaken 5 times, and then the chemiluminescence readings were measured on a microplate reader. The cell proliferation inhibition rate was calculated according to the following formula: [1-(25×T test - T0 / 25×T control - T0)]×100. The GI of the compound for inhibiting cell proliferation was then calculated using the DoseResp function using Origin 9.2 software. 50 value.

[0222] Effect of the compounds of the present invention on the GI function of MIAPACA-2 cells 50 Less than 1000nM, some compounds GI 50 Less than 100nM, some compounds GI 50Less than 50nM. The test results of some embodiments are shown in Table 2, where A < 50nM, 50nM ≤ B < 100nM, 100nM ≤ C < 200nM, and 200nM ≤ D < 500nM:

[0223] Table 2 Antiproliferation activity of compounds on MIAPACA-2 cells

[0224] Conclusion: The compounds of the present invention, such as the compounds in the examples, have good inhibitory activity against MIAPACA-2 cells.

[0225] 3. HCT-116 cell proliferation inhibition experiment

[0226] HCT-116 cells were cultured in RPMI 1640 medium (ATCC, Cat#30-2001; supplemented with 10% FBS and 1% double-antibody) until the confluence reached approximately 85% before plating. The medium was discarded, and the cells were washed with 1× PBS and then digested with trypsin (Gibco, Cat#15400-054). When the cells became round and began to detach, the medium was added to terminate the digestion. The cells were pipetted off and transferred to a sterile centrifuge tube and centrifuged at 1000 rpm for 3 minutes. After centrifugation, the cells were removed and the supernatant discarded. The centrifuge tube was resuspended in medium and counted. According to the counting results, the cell suspension was adjusted to the appropriate concentration and poured into the sample tank. It was added to the first 96-well cell plate (Corning, Cat#3903). At the same time, 10 Day 0 wells were plated in the second 96-well cell plate. The compound was dissolved in DMSO to 10 mM and stored for later use. During the experiment, the compound stock solution was diluted 5-fold in sequence. 24 hours after plating, add the diluted compound to the first 96-well plate, set up a DMSO vehicle control group, and continue incubation at 37°C, 5% CO2 for 5 days. After 5 days of incubation, slowly and carefully aspirate the supernatant from the first 96-well plate using a multichannel pipette and discard. Add TrypLE (Gibco, Cat#12604-013) to each well and place in a 37°C incubator for 5 minutes until the cells are completely detached. Then, add culture medium to terminate digestion and pipette evenly. Take a third 96-well plate (Corning, Cat#3903), add RPMI-1640 medium and the digested cell suspension to each well, and mix thoroughly with a pipette. Add the diluted compound and set up a DMSO vehicle control group. Continue incubation at 37°C, 5% CO2 for 5 days. 24 hours after plating, CELL VIABILITY reagent (Vazyme, Cat# DD1101-03) was added to each well of the second 96-well cell plate. CELL VIABILITY reagent was added to each well of the first 96-well cell plate on the 5th day after administration. CELL VIABILITY reagent was added to each well of the third 96-well cell plate on the 10th day after the first administration. The cells were incubated at room temperature for 10 minutes. After the incubation, the cells were gently shaken 5 times, and then the chemiluminescence readings were measured using a microplate reader. The cell proliferation inhibition rate was calculated according to the following formula: [1-(25×T test-T0 / 25×T control-T0)]×100. The GI of the compound for inhibiting cell proliferation was then calculated using the DoseResp function using Origin 9.2 software. 50 value.

[0227] Table 3 Inhibitory activity of compounds on HCT-116 cell proliferation

[0228] The compounds of the present invention have a GI of greater than 100 nM against HCT-116 cells.50 Values, GI of some compounds 50 The values ​​are shown in Table 3, where AA represents GI 50 <5nM, A represents 5nM≤GI 50 <100nM, B represents 100nM≤GI 50 <500nM, C represents 500nM≤GI 50 <1000nM, D represents 1000nM≤GI 50 .

[0229] Conclusion: The compounds of the present invention, such as the compounds in the examples, have weak inhibitory activity against HCT-116 cells. For example, the GI50 of compound 4 for inhibiting HCT-116 cell proliferation reached 1084 nM, while that of compound 7 was 758 nM.

[0230] 4. HCT-116 MTAP - / - Cell proliferation inhibition assay

[0231] HCT-116MTAP - / -Cells were cultured in RPMI 1640 medium (ATCC, Cat#30-2001; supplemented with 10% FBS and 1% double antibody) until the confluence reached about 85% before plating. The culture medium was discarded, and the cells were washed with 1×PBS and then digested with trypsin (Gibco, Cat#15400-054). When the cells became round and began to fall off, the culture medium was added to terminate the digestion. The cells were blown off and transferred to a sterile centrifuge tube and centrifuged at 1000 rpm for 3 minutes. After centrifugation, the cells were removed and the supernatant was discarded. The centrifuge tube was added with culture medium to resuspend the cells and counted. According to the counting results, the cell suspension was adjusted to the appropriate concentration and poured into the sample tank. It was added to the first 96-well cell plate (Corning, Cat#3903). At the same time, 10 Day 0 wells were plated in the second 96-well cell plate. The compound was dissolved in DMSO to 10 mM and stored for later use. During the experiment, the compound stock solution was diluted 5-fold in sequence. 24 hours after plating, add the diluted compound to the first 96-well plate, set up a DMSO vehicle control group, and continue incubation at 37°C, 5% CO2 for 5 days. After 5 days of incubation, slowly and carefully aspirate the supernatant from the first 96-well plate using a multichannel pipette and discard. Add TrypLE (Gibco, Cat#12604-013) to each well and place in a 37°C incubator for 5 minutes until the cells are completely detached. Then, add culture medium to terminate digestion and pipette evenly. Take a third 96-well plate (Corning, Cat#3903), add RPMI-1640 medium and the original cell suspension that has been evenly digested to each well, and mix thoroughly with a pipette. Add the diluted compound, set up a DMSO vehicle control group, and continue incubation at 37°C, 5% CO2 for 5 days. 24 hours after plating, CELL VIABILITY reagent (Vazyme, Cat# DD1101-03) was added to each well of the second 96-well cell plate. CELL VIABILITY reagent was added to each well of the first 96-well cell plate on the 5th day after administration. CELL VIABILITY reagent was added to each well of the third 96-well cell plate on the 10th day after the first administration. The cells were incubated at room temperature for 10 minutes. After the incubation, the cells were gently shaken 5 times, and then the chemiluminescence readings were measured using a microplate reader. The cell proliferation inhibition rate was calculated according to the following formula: [1-(25×T test-T0 / 25×T control-T0)]×100. The GI of the compound for inhibiting cell proliferation was then calculated using the DoseResp function using Origin 9.2 software. 50 value.

[0232] Table 4 Compounds for HCT-116MTAP - / - Cell proliferation inhibitory activity

[0233] The compounds of the present invention have an effect on HCT-116MTAP- / - Cells have a GI of less than 100 nM 50 Values, GI of some compounds 50 The values ​​are shown in Table 4, where AA represents GI 50 <5nM, A represents 5nM≤GI 50 <100nM, B represents 100nM≤GI 50 <500nM, C represents 500nM≤GI 50 <1000nM, D represents 1000nM≤GI 50 .

[0234] Conclusion: The compounds of the present invention, such as the compounds in the examples, have an effect on HCT-116MTAP. - / - Strong cell inhibitory activity.

[0235] 5. Cell sDMA detection test

[0236] 5.1. Detection of sDMA in MIAPACA-2 cells

[0237] MIAPACA-2 cells were cultured in DMEM (ATCC, Cat#30-2002; supplemented with 10% FBS and 1% double-antibody) until confluence reached approximately 85% before plating. The cells were discarded, rinsed with 1× PBS, and then digested with trypsin (Gibco, Cat#15400-054). When the cells became round and began to detach, culture medium was added to terminate digestion. The cells were pipetted off and transferred to a sterile centrifuge tube. Centrifuged at 1000 rpm for 3 minutes. After centrifugation, the supernatant was removed and discarded. Culture medium was added to the centrifuge tube to resuspend the cells and count them. Based on the count results, the cell suspension was adjusted to the appropriate concentration and poured into the sample well. 100 μL was added to each well of a 96-well plate. The compound was dissolved in DMSO to 10 mM and stored until ready for use. During the experiment, the compound was serially diluted 5-fold in culture medium. 100 μL of the diluted compound was added to the 96-well plate with the cells. A DMSO vehicle control group was also set up. The cells were cultured at 37°C and 5% CO2 for 3 days. After 3 days, each well was washed twice with cold PBS. Protease and phosphatase inhibitor cocktail (CST, Cat#5872S) and RIPA lysis buffer were mixed in the required volume and added to a 96-well plate at 50 μL / well. The plates were placed on ice for 10 minutes, with repeated pipetting until the cells were completely lysed. The plates were then centrifuged at 2500 rpm at 4°C for 35 minutes, and the supernatant protein samples were collected. Protein detection was performed using a BCA kit (Beyotime, Cat#P0011). Based on the assay results, the protein concentration of the lysate was adjusted to the desired concentration using PBS carbonate-bicarbonate buffer, pH 9.6 (Sigma, Cat#C3041). 100 μL was then added to each well of a black high-binding 96-well plate (Nunc Maxisorp, Cat#437111) and incubated at room temperature for 2 hours. The lysate was removed and the plate was washed four times with PBST (Cell Signaling, Cat#9809S). Blocking was performed with 5% BSA blocking buffer (solarbio, Cat#SW3015) for 1 hour, followed by washing four times with PBST. 100 μL of either sDMA antibody (CST, Cat#13222S) or SMD3 antibody (Abgent, Cat#AP12451A) was added to each well and shaken overnight at 4°C. After removing the antibody, the plate was washed four times with PBST. 100 μL of rabbit secondary antibody (Cell Signaling Technology, Cat#7074) was added to each well and incubated at room temperature for 1 hour. After removing the secondary antibody, the plate was washed four times with PBST. 100 μL of Immobilon Forte Western HRP Substrate (Millipore, Cat#WBLUF0500) was added to each well and incubated with shaking for 15 minutes before detection using chemiluminescence in a BMG microplate reader.After data normalization (SDMA reading / SMD3 reading), Graphpad Prism 8.3.0 software was used to plot the data and calculate the IC. 50 value.

[0238] 5.2. SDMA Detection in HCT-116 Cells

[0239] HCT-116 cells were cultured in RPMI 1640 medium (ATCC, Cat#30-2001, supplemented with 10% FBS and 1% double-antibody) until the confluence reached approximately 85% before plating. The culture medium was discarded, and the cells were rinsed with 1×PBS and then digested with trypsin (Gibco, Cat#15400-054). When the cells became round and began to fall off, the culture medium was added to terminate the digestion. The cells were pipetted down and transferred to a sterile centrifuge tube and centrifuged at 1000 rpm for 3 minutes. After centrifugation, the cells were removed and the supernatant discarded. The centrifuge tube was resuspended in culture medium and counted. According to the counting results, the cell suspension was adjusted to the appropriate concentration and poured into the sample tank. 100 μL was added to each well of the 96-well plate. The compound was dissolved in DMSO to 10 mM and stored for later use. During the experiment, the compound was diluted 5-fold in the culture medium. Add 100 μL of diluted compound to a 96-well plate containing cells. A DMSO vehicle control group was also established and cultured at 37°C, 5% CO2 for 3 days. After 3 days, each well was washed twice with cold PBS. Protease and phosphatase inhibitor cocktail (CST, Cat#5872S) and RIPA lysis buffer were mixed to the required volume and added 50 μL / well to the 96-well plate. The plate was incubated on ice for 10 minutes, with repeated pipetting until the cells were completely lysed. The supernatant was then centrifuged at 2500 rpm at 4°C for 35 minutes, and the supernatant protein sample was collected. Protein detection was performed using a BCA kit (Beyotime, Cat#P0011). Based on the assay results, the protein concentration of the lysate was adjusted to the desired concentration using PBS carbonate-bicarbonate buffer, pH 9.6 (Sigma, Cat#C3041). 100 μL was then added to each well of a black high-binding 96-well plate (Nunc Maxisorp, Cat#437111) and incubated at room temperature for 2 hours. The lysate was removed and the plate was washed four times with PBST (Cell Signaling, Cat#9809S). Blocking was performed with 5% BSA blocking buffer (solarbio, Cat#SW3015) for 1 hour, followed by washing four times with PBST. 100 μL of either sDMA antibody (CST, Cat#13222S) or SMD3 antibody (Abgent, Cat#AP12451A) was added to each well and shaken overnight at 4°C. After removing the antibody, the plate was washed four times with PBST. 100 μL of rabbit secondary antibody (Cell Signaling Technology, Cat#7074) was added to each well and incubated at room temperature for 1 hour. After removing the secondary antibody, the plate was washed four times with PBST. 100 μL of Immobilon Forte Western HRP substrate (Millipore, Cat#WBLUF0500) was added to each well and incubated with shaking for 15 minutes before detection using chemiluminescence on a BMG microplate reader.After data normalization (SDMA reading / SMD3 reading), Graphpad Prism 8.3.0 software was used to plot the data and calculate the IC. 50 value.

[0240] Table 5.1 Inhibitory activity of compounds on SDMA production in HCT-116 cells

[0241] The compounds of the present invention have an IC greater than 100 nM for SDMA production in HCT-116 cells. 50 IC values ​​of some compounds 50 The values ​​are shown in Table 5.1, where AA represents IC 50 <5nM, A represents 5nM≤IC 50 <100nM, B represents 100nM≤IC 50 <500nM, C represents 500nM≤IC 50 <1000nM, D represents 1000nM≤IC 50 .

[0242] Conclusion: The compounds of the present invention, such as the compounds in the examples, have weak inhibitory activity on SDMA production in HCT-116 cells.

[0243] 5.3 HCT-116MTAP - / - SDMA detection in cells

[0244] HCT-116MTAP- / - cells were cultured with RPMI 1640 medium (ATCC, #30-2001, supplemented with 10% FBS and 1% double antibody) until the confluence reached about 85% and then plated. Discard the culture medium, rinse with 1×PBS, and then add trypsin (Gibco, Cat#15400-054) for digestion. When the cells become round and begin to fall off, add culture medium to terminate digestion, blow down the cells and transfer them to a sterile centrifuge tube, centrifuge at 1000rpm for 3 minutes, remove them after centrifugation, and discard the supernatant. Add culture medium to the centrifuge tube to resuspend the cells and count them. According to the counting results, adjust the cell suspension to the appropriate concentration and pour it into the sample tank. Add 100 μL to each well of the 96-well plate. Dissolve the compound to 10mM with DMSO and save it for later use. During the experiment, dilute the compound in a 5-fold gradient with culture medium. Add 100 μL of diluted compound to a 96-well plate containing cells. A DMSO vehicle control group was also established and cultured at 37°C, 5% CO2 for 3 days. After 3 days, each well was washed twice with cold PBS. Protease and phosphatase inhibitor cocktail (CST, Cat#5872S) and RIPA lysis buffer were mixed to the required volume and added 50 μL / well to the 96-well plate. The plate was incubated on ice for 10 minutes, with repeated pipetting until the cells were completely lysed. The supernatant was then centrifuged at 2500 rpm at 4°C for 35 minutes, and the supernatant protein sample was collected. Protein detection was performed using a BCA kit (Beyotime, Cat#P0011). Based on the assay results, the protein concentration of the lysate was adjusted to the desired concentration using PBS carbonate-bicarbonate buffer, pH 9.6 (Sigma, Cat#C3041). 100 μL was then added to each well of a black high-binding 96-well plate (Nunc Maxisorp, Cat#437111) and incubated at room temperature for 2 hours. The lysate was removed and the plate was washed four times with PBST (Cell Signaling, Cat#9809S). Blocking was performed with 5% BSA blocking buffer (solarbio, Cat#SW3015) for 1 hour, followed by washing four times with PBST. 100 μL of either sDMA antibody (CST, Cat#13222S) or SMD3 antibody (Abgent, Cat#AP12451A) was added to each well and shaken overnight at 4°C. After removing the antibody, the plate was washed four times with PBST. 100 μL of rabbit secondary antibody (Cell Signaling Technology, Cat#7074) was added to each well and incubated at room temperature for 1 hour. After removing the secondary antibody, the plate was washed four times with PBST. 100 μL of Immobilon Forte Western HRP substrate (Millipore, Cat#WBLUF0500) was added to each well and incubated with shaking for 15 minutes before detection using chemiluminescence on a BMG microplate reader.After data normalization (SDMA reading / SMD3 reading), Graphpad Prism 8.3.0 software was used to plot the data and calculate the IC. 50 value.

[0245] Table 5.2 Compounds for HCT-116MTAP - / - Inhibitory activity of SDMA production in cells

[0246] The compounds of the present invention have an effect on HCT-116MTAP - / - Cells produce SDMA with an IC of less than 100 nM 50 IC values ​​of some compounds 50 The values ​​are shown in Table 5.2, where AA represents IC 50 <5nM, A represents 5nM≤IC 50 <100nM, B represents 100nM≤IC 50 <500nM, C represents 500nM≤IC 50 <1000nM, D represents 1000nM≤IC 50 .

[0247] Conclusion: The compounds of the present invention, such as the compounds in the examples, have an effect on HCT-116MTAP. - / - The cell production of SDMA has a strong inhibitory activity. For example, compound 7 has a strong inhibitory effect on HCT-116MTAP. - / - Cells generate SDMA IC 50 The value is 1.23nM.

[0248] 6. hERG potassium channel effect test

[0249] Experimental platform: electrophysiology manual patch clamp system

[0250] Cell line: Chinese hamster ovary (CHO) cell line stably expressing hERG potassium channel

[0251] Experimental methods: CHO (Chinese Hamster Ovary) cells stably expressing hERG potassium channels were used to record hERG potassium channel currents using the whole-cell patch clamp technique at room temperature. Glass microelectrodes were pulled from glass electrode blanks (BF150-86-10, Sutter) using a puller. The tip resistance after perfusing the electrode liquid was about 2-5 MΩ. The glass microelectrode was inserted into the amplifier probe to connect to the patch clamp amplifier. The clamping voltage and data recording were controlled and recorded by a computer using pClamp 10 software, with a sampling frequency of 10 kHz and a filter frequency of 2 kHz. After obtaining the whole-cell recording, the cell was clamped at -80 mV to induce the hERG potassium current (I hERG) was administered with a 2-second depolarization step from -80 mV to +20 mV, followed by repolarization to -50 mV for 1 second before returning to -80 mV. This voltage stimulus was administered every 10 seconds, and administration began after confirming that the hERG potassium current was stable (for at least 1 minute). Compounds were administered for at least 1 minute at each test concentration, and at least two cells (n ≥ 2) were tested for each concentration.

[0252] Data processing: Data analysis was performed using pClamp 10, GraphPad Prism 5, and Excel software. The degree of inhibition of hERG potassium current (peak hERG tail current induced at -50 mV) by different compound concentrations was calculated using the following formula: Inhibition% = [1–(I / Io)] × 100%

[0253] Wherein, Inhibition% represents the inhibition percentage of the compound on hERG potassium current, and I and Io represent the amplitude of hERG potassium current before and after drug addition, respectively.

[0254] Compound IC 50 Calculated using GraphPad Prism 5 software by fitting the following equation: Y = Bottom + (Top-Bottom) / (1 + 10^((LogIC50-X)*HillSlope))

[0255] Where X is the Log value of the test sample concentration, Y is the inhibition percentage at the corresponding concentration, and Bottom and Top are the minimum and maximum inhibition percentages, respectively.

[0256] Conclusion: The compounds of the present invention, such as the compounds in the examples, do not inhibit the hERG potassium ion channel.

[0257] 7. CYP450 enzyme inhibition test

[0258] The purpose of this study was to evaluate the effects of test substances on the activities of five isoenzymes of cytochrome P450 (CYP) in human liver microsomes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) using an in vitro test system. Specific probe substrates for the CYP450 isoenzymes were incubated with human liver microsomes and varying concentrations of the test substances. Reduced nicotinamide adenine dinucleotide phosphate (NADPH) was added to initiate the reaction. After the reaction, the samples were processed and the metabolites produced by the specific substrates were quantitatively detected by liquid chromatography-tandem mass spectrometry (LC-MS / MS). Changes in CYP enzyme activity were measured, and the IC values ​​were calculated. 50 The inhibitory potential of the test substance on each CYP enzyme isoform was evaluated.

[0259] Conclusion: The compounds of the present invention, such as the compounds in the examples, do not inhibit the activity of CYP enzymes.

[0260] 8. Pharmacokinetic test in mice

[0261] 8.1. Experimental Animals: Male ICR mice, 22-25 g, 6 mice per compound, purchased from Chengdu Dashuo Experimental Animal Co., Ltd.

[0262] 8.2 Experimental Design: On the day of the experiment, ICR mice were randomly divided into groups according to body weight. They were fasted (but not water) for 12–14 h before dosing and fed 4 h after dosing. Dosing was performed according to Table 6.

[0263] Table 6 Dosage information Note: Intravenous administration solvent: 5% DMA + 5% Solutol + 90% Saline; Oral administration solvent: 0.5% MC (DMA: dimethylacetamide; Solutol: polyethylene glycol-15-hydroxystearate; Saline: normal saline; MC: methylcellulose solution)

[0264] Before and after drug administration, 0.06 mL of blood was collected via the orbital cavity under isoflurane anesthesia. The blood was placed in an EDTAK2 centrifuge tube and centrifuged at 5000 rpm at 4°C for 10 minutes to collect plasma. Blood was collected at 0, 5, 15, 30 minutes, 1, 2, 4, 7, and 24 hours for both the intravenous and oral gavage groups. All samples were stored at -80°C prior to analysis and quantitative analysis was performed using LC-MS / MS.

[0265] Table 7 Pharmacokinetic parameters of test compounds in mouse plasma

[0266] -: Not applicable. MRTX1719 Structure:

[0267] Conclusion: The compounds of the present invention, such as the compounds in the examples, have good pharmacokinetic characteristics in mice.

[0268] 9. Pharmacokinetic test in rats

[0269] 9.1. Experimental Animals: Male SD rats, approximately 220 g, 6-8 weeks old, 6 rats per compound, purchased from Chengdu Dashuo Experimental Animal Co., Ltd.

[0270] 9.2 Experimental Design: On the day of the experiment, SD rats were randomly divided into groups according to body weight. They were fasted (but not water) for 12–14 hours prior to dosing and fed 4 hours after dosing. Dosing was performed according to Table 8.

[0271] Table 8 Dosage information Note: Intravenous administration solvent: 5% DMA + 5% Solutol + 90% Saline; Oral administration solvent: 0.5% MC (DMA: dimethylacetamide; Solutol: polyethylene glycol-15-hydroxystearate; Saline: normal saline; MC: methylcellulose solution)

[0272] Before and after drug administration, 0.15 mL of blood was collected via the orbital cavity under isoflurane anesthesia. The blood was placed in an EDTAK2 centrifuge tube and centrifuged at 5000 rpm at 4°C for 10 minutes to collect plasma. Blood was collected from both the intravenous and oral gavage groups at 0, 5, 15, 30 minutes, and 1, 2, 4, 6, 8, and 24 hours. All samples were stored at -80°C prior to analysis and quantitative analysis was performed using LC-MS / MS.

[0273] Conclusion: The compounds of the present invention, such as the compounds in the examples, have good pharmacokinetic characteristics in rats.

[0274] 10. Beagle dog pharmacokinetic test

[0275] 10.1. Experimental Animals: Male beagle dogs, weighing approximately 8-11 kg, 6 per compound, purchased from Beijing Mas Biotechnology Co., Ltd.

[0276] 10.2 Test Methods: On the day of the test, (6 × number of test compounds n) beagle dogs were randomly divided into groups based on body weight. They were fasted (but not watered) for 12-14 hours prior to dosing and fed 4 hours after dosing. Dosing was performed according to Table 9.

[0277] Table 9 Dosage information Note: Intravenous administration solvent: 5% DMA + 5% Solutol + 90% Saline; Oral administration solvent: 0.5% MC (DMA: dimethylacetamide; Solutol: polyethylene glycol-15-hydroxystearate; Saline: normal saline; MC: methylcellulose solution)

[0278] Before and after dosing, 1 mL of blood was collected from the jugular vein or limb vein into an EDTAK2 centrifuge tube. The samples were centrifuged at 5000 rpm at 4°C for 10 minutes, and plasma was collected. Blood was collected from both the intravenous and oral administration groups at the following time points: 0, 5, 15, 30 minutes, and 1, 2, 4, 6, 8, 10, 12, 24, 48, and 72 hours. All samples were stored at -80°C prior to analysis and quantitative analysis was performed using LC-MS / MS.

[0279] Conclusion: The compounds of the present invention, such as the compounds in the examples, have good pharmacokinetic characteristics in beagle dogs.

[0280] 11. Monkey pharmacokinetic test

[0281] 11.1. Experimental Animals: Male cynomolgus monkeys, 3-5 kg, 3-6 years old, 4 per compound. Purchased from Suzhou Xishan Biotechnology Co., Ltd.

[0282] 11.2. Test Methods: On the day of the test, (4 × the number of test compounds) monkeys were randomly divided into groups based on body weight. One day before dosing, they were fasted but not watered for 14-18 hours. Food was allowed 4 hours after dosing. Dosing was performed according to Table 10.

[0283] Table 10 Dosage information Note: Intravenous administration solvent: 5% DMA + 5% Solutol + 90% Saline; Oral administration solvent: 0.5% MC

[0284] Before and after dosing, 1.0 mL of blood was collected from a limb vein and placed in an EDTAK2 centrifuge tube. The samples were centrifuged at 5000 rpm at 4°C for 10 minutes, and plasma was collected. Blood was collected from both the intravenous and oral administration groups at the following time points: 0, 5 minutes, 15 minutes, 30 minutes, and 1, 2, 4, 6, 8, 10, 12, and 24 hours. All samples were stored at -80°C prior to analysis and quantitative analysis was performed using LC-MS / MS.

[0285] Conclusion: The compounds of the present invention, such as the compounds in the examples, have good pharmacokinetic characteristics in monkeys.

[0286] 12. Liver microsome stability test

[0287] In this study, liver microsomes from five species, including humans, dogs, rats, and mice, were used as in vitro models to evaluate the metabolic stability of the test substances.

[0288] At 37°C, 1 μM of the test substance was incubated with microsomal proteins and coenzyme NADPH. The reaction was terminated by adding ice-cold acetonitrile containing an internal standard after a certain time (5, 10, 20, 30, 60 min). The concentration of the test substance in the sample was detected by LC-MS / MS. The T value was calculated based on the ln value of the drug residual rate in the incubation system and the incubation time. 1 / 2 , and further calculated the liver microsomal intrinsic clearance CL int(mic) and hepatic intrinsic clearance CL int(Liver) .

[0289] Conclusion: The compounds of the present invention, such as the compounds in the examples, showed good metabolic stability in the liver microsome stability test.

[0290] 13. Caco-2 permeability test

[0291] The experiment used Caco-2 cell monolayers in 96-well Transwell plates, incubated in triplicate. Transport buffer (HBSS, 10 mM HEPES, pH 7.4 ± 0.05) containing the compound of the invention (2 μM) or the control compounds digoxin (10 μM), nadolol (2 μM), and metoprolol (2 μM) was added to the apical or basolateral wells of the cell monolayer. Transport buffer containing DMSO was added to the corresponding receiving wells. After incubation at 37 ± 1°C for 2 hours, the cell plate was removed and appropriate samples were taken from the apical and basolateral wells to a new 96-well plate. Proteins were then precipitated by adding acetonitrile containing an internal standard. Samples were analyzed using LC-MS / MS to determine the concentrations of the compound of the invention and the control compound. The concentration data were used to calculate the apparent permeability coefficients for transport from the apical to basolateral side of the cell monolayer and from the basolateral to apical side, thereby calculating the efflux rate. The integrity of the cell monolayer after 2 hours of incubation was assessed by leakage of Lucifer Yellow.

[0292] Conclusion: The compounds of the present invention, such as the compounds in the examples, showed good permeability in the Caco-2 permeability test.

[0293] 14. In vivo efficacy experiments

[0294] HCT-116MTAP- / - cells were placed in RPMI-1640 medium and cultured in an incubator at 37°C containing 5% CO2. The cells were passaged every 2 to 3 days. When the number reached the requirement, the cells were collected, counted, and the cell concentration was adjusted to an appropriate concentration with PBS. The cell concentration was diluted to 2.5×107 cells / ml with Matrigel at a ratio of 1:1. 5×106 cells / 200μL / mouse (purchased from Weitong Lihua Experimental Animal Technology Co., Ltd., Balb / c nude, female) were inoculated into the axillary area of ​​the right forelimb. When the tumor grew to 80-100mm 3 (Inoculation Days 5-7) Groups were sorted by tumor size and dosed (the day of group dosing was designated Day 0). Tumors and animal body weights were measured three times per week during dosing. After the experiment, multi-point PK analysis and tumor samples were collected, and tumor weights were recorded and photographed.

[0295] The experimental data were expressed as mean ± standard error (SEM). The data were statistically analyzed using one-way ANOVA plus Dunnett's multiple comparison test in Graphpad Prism. P < 0.05 was considered to be significantly different.

[0296] Conclusion: The compounds of the present invention, such as Example compounds 5 and 7, have good tumor inhibition effects in the mouse CDX (HCT-116MTAP- / - cell) model.

Claims

1. A compound represented by formula (I), its stereoisomer, deuterated substance, solvate, or pharmaceutically acceptable salt, in, D is selected from D1, D2, D3, and D4; Each R5 is independently selected from H, D, OH, C 1-4 Alkyl, CN, halogen, C 1-4 Alkoxy, C 2-4 Alkenyl, C 2-4 Alkynyl, 5-6 membered heteroaryl containing 1-3 heteroatoms selected from N, O, S, 4-7 membered heterocycloalkyl containing 1-3 heteroatoms selected from N, O, S or C 3-6 Cycloalkyl, wherein the alkyl, alkoxy, alkenyl, alkynyl, heteroaryl or cycloalkyl is optionally substituted by 1-3 selected from halogen, D, CN, OH, C 1-4 Alkyl, C 1-4 Alkoxy and NH2 group substitution; R7 is independently H, D, C 1-4 Alkyl, halogenated C 1-4 Alkyl, deuterated C 1-4 Alkyl, C 2-4 Alkenyl, C 2-4 Alkynyl, C 3-6 Cycloalkyl or 4-7 membered heterocycloalkyl containing 1-3 heteroatoms selected from N, O, S; Each R 8a , R 8b , R 8c Each independently selected from H, D, amino, C 1-6 Alkoxy, OH, CN, halogen, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkyl, 4-7 membered heterocycloalkyl containing 1-3 heteroatoms selected from N, O, S, C 3-8 Cycloalkyl, -COC 1-4 Alkyl, -N(R7')2, -C(=O)N(R7')2, -NR7'C(=O)-R7', -C(=O)-R7', -O-(CH2) r -R8', -SC 1-6 Alkyl, -S(O)-C 1-6 Alkyl, -S(O)2-C 1-6 Alkyl, -S-(CH2) r -R8', -NR7'-(CH2) r -R8' or -(CH2) r -R8', the alkyl, alkenyl, alkynyl, heterocycloalkyl, cycloalkyl are optionally substituted by 1-5 groups selected from R 8” replace; Alternatively, R 8b With R 8c Together they form a 4-6 membered carbocyclic ring, which is optionally further substituted by 1-3 members selected from F, Cl, C 1-4 Alkyl, halogenated C 1-4 Alkyl, deuterated C 1-4 Substitution of alkyl groups; Each R7' is independently H, D, amino, hydroxyl, C 1-6 Alkyl, halogenated C 1-6 Alkyl, deuterated C 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkoxy, C 3-6 Cycloalkyl, C 3-12 Heterocycloalkyl, -NHC 1-6 Alkyl, -N(C 1-6 Alkyl)2, or deuterated C 1-6 Alkoxy, the cycloalkyl, heterocycloalkyl is optionally substituted by 1 to 3 groups selected from deuterium, halogen, cyano, amino, hydroxyl, C 1-6 Alkyl, halogenated C 1-6 Alkyl, deuterated C 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkoxy or deuterated C 1-6 Alkoxy group substitution; Each R8' is independently H, D, C 1-6 Alkyl, C 1-4 Alkoxy, C 3-8 Cycloalkyl, 4-12 membered heterocyclic ring containing 1-3 heteroatoms selected from N, O, S, wherein the alkyl and alkoxy groups are optionally substituted by 1-3 halogen, =O, D, OH, NH2, CN, C 1-6 Alkyl, halogenated C 1-6 Alkyl, deuterated C 1-6 Alkyl, -C(O)C 1-4 Alkyl, -C(O)NHC 1-4 Alkyl, -C(O)NH2, -NHC(O)C 1-4 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkoxy and deuterated C 1-6 The cycloalkyl or heterocyclic ring is optionally substituted by 1-5 groups selected from R8"; R 8” Selected from halogen, D, OH, NH2, CN, C 1-6 Alkyl, C 1-6 Alkoxy, The alkyl or alkoxy group may be further substituted by 1 to 5 groups selected from halogen, deuterium, cyano, amino, hydroxyl, C 1-6 Alkyl or C 1-6 Alkoxy group substitution; Each r is independently 0, 1, 2 or 3.

2. The compound of formula (I) according to claim 1, wherein the stereoisomer, deuterated substance, solvate, or pharmaceutically acceptable salt thereof has the following structure:

3. The compound according to claim 1, its stereoisomer, deuterated substance, solvate, or pharmaceutically acceptable salt, wherein D is selected from 4. The compound according to claim 1, its stereoisomer, deuterated substance, solvate, or pharmaceutically acceptable salt, wherein: R 8a Selected from C 2-6 Alkenyl, C 2-6 Alkynyl, 4-7 membered heterocycloalkyl containing 1-3 heteroatoms selected from N, O, S, C 3-8 Cycloalkyl, -OC 3-8 Cycloalkyl, the alkenyl, alkynyl, cycloalkyl, heterocycloalkyl is optionally substituted by 1-5 selected from R8"; Preferably, R 8a Selected from Cyclopropyl, cyclobutyl, -O-cyclopropyl, -O-cyclobutyl, vinyl, the The cyclopropyl and cyclobutyl groups are further optionally substituted by 1 to 3 groups selected from methyl, F, Cl, CN, The group substitution; More preferably, R 8a Selected from -O-cyclopropyl, vinyl, 5. The compound according to claim 1, its stereoisomer, deuterated substance, solvate, or pharmaceutically acceptable salt, wherein: R 8b is selected from F, Cl, methyl, ethynyl or propynyl; Preferably, R 8b is selected from Cl, methyl, ethynyl or propynyl; or R 8b With R 8c Together they form a 5- or 6-membered carbocyclic ring, which is optionally further substituted by 1-2 substituents selected from F, Cl, methyl, ethyl, propyl, isopropyl, tert-butyl, -CH2F, -CHF2, -CF3, -CH2CH2F, -CH2CHF2, -CH2CF3, -CHFCH2F, -CHFCHF2, -CHFCF3, -CF2CH2F, -CF2CHF2, -CF2CF3, -CH2D, -CHD2, -CD3, -CH2CH2D, -CH2CHD2, -CH2CD3, -CHDCH2D, -CHDCHD2, -CHDCD3, -CD2CH2D, -CD2CHD2, -CD2CD3.

6. The compound according to claim 1, its stereoisomer, deuterated substance, solvate, or pharmaceutically acceptable salt, wherein: Each R7' is independently H, C 1-4 Alkyl or halogenated C 1-4 alkyl; Each R8' is independently H, C 1-4 Alkoxy, C 3-6 Cycloalkyl, phenyl, 4-7 membered heterocycloalkyl containing 1-3 heteroatoms selected from N, O, S, or 5-6 membered heteroaryl containing 1-3 heteroatoms selected from N, O, S, wherein the cycloalkyl, phenyl, heterocycloalkyl or heteroaryl is optionally substituted by 1-3 halogen, =O, D, OH, NH2, CN, C 1-4 Alkyl, halogenated C 1-4 Alkyl, and C 1-4 Alkoxy group substitution; Each r is independently 0, 1 or 2.

7. The compound according to claim 1, its stereoisomer, deuterated substance, solvate, or pharmaceutically acceptable salt, wherein: The compound of formula (I) is not selected from the following structures:

8. The compound according to claim 1, its stereoisomer, deuterated form, solvate, or pharmaceutically acceptable salt, wherein the compound is selected from the structure in Table A.

9. A pharmaceutical composition comprising the compound according to any one of claims 1 to 8, or a stereoisomer, deuterated substance, solvate, or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and / or excipient.

10. The pharmaceutical composition or pharmaceutical preparation according to claim 9, comprising 1-1500 mg of a compound selected from any one of claims 1-8 or a stereoisomer, deuterated substance, solvate, cocrystal or pharmaceutically acceptable salt thereof and a carrier and / or excipient.

11. Use of the compound according to any one of claims 1 to 8, its stereoisomer, deuterated form, solvate, or pharmaceutically acceptable salt, or the composition according to claim 9 or 10 in the preparation of a medicament for treating / preventing a PRMT5·MTA-mediated disease.

12. The use according to claim 11, wherein the PRMT5·MTA-mediated disease is selected from liver cancer, breast cancer, skin cancer, bladder cancer, pancreatic cancer or head and neck cancer.

13. A method for treating a disease in a mammal, the method comprising administering to a subject a therapeutically effective amount of a compound according to any one of claims 1 to 8 or a stereoisomer, deuterated form, solvate, prodrug, metabolite, pharmaceutically acceptable salt or cocrystal thereof, the therapeutically effective amount preferably being 1-1500 mg, and the disease preferably being liver cancer, breast cancer, skin cancer, bladder cancer, pancreatic cancer or head and neck cancer.

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