Preparation method of benzoxazole compounds and their application in the medical field

Abstract

The present invention provides a benzoxazole small molecule compound, which is an inhibitor of the Zeste gene enhancer homolog 2 (EZH2) and can be used to prevent and / or treat EZH2-mediated related diseases, such as malignant tumors.

Description

Technical Field

[0001] The present invention relates to the field of medicine and relates to a small molecule benzoxazole compound, a method for preparing the compound, a pharmaceutical composition containing the compound, and its use in medicine. The present invention discloses the compound as an inhibitor of enhancer of zeste homolog 2 (EZH2), useful for preventing and / or treating EZH2-related diseases, such as malignant tumors. Background Art

[0002] Malignant tumors are diseases that seriously threaten human health. The occurrence and development of tumors is a multifactorial and multi-stage evolutionary process involving mutations in multiple genes and epigenetic changes. Epigenetics refers to a genetic phenomenon in which the expression level and function of genes change, resulting in heritable phenotypes, without changing the DNA sequence of the gene. Polycomb group protein (PcG) is an important protein factor involved in the epigenetic negative regulation of chromatin genes. The PcG family includes two multimeric complexes: polycomb repressive complex 1 (PRC1) and polycomb repressive complex 2 (PRC2). Enhancer of Zeste homolog 2 (EZH2) is a core member of the polycomb group protein (PcG) family. EZH2 is a catalytic subunit of the PRC2 protein complex and plays a central role in its function. EZH2 contains a highly conserved SET domain and possesses histone methyltransferase (HMT) activity. It catalyzes the trimethylation of lysine 27 (H3K27me3) on histone H3, triggering the aggregation of PCR1 complex components at specific gene loci, leading to the silencing of downstream target genes. These target genes are involved in the regulation of various fundamental biological processes, such as apoptosis, cell cycle regulation, cell aging, and differentiation. Current studies have shown that EZH2 is highly expressed in various tumor tissues and is closely associated with tumor malignant progression, invasiveness, and metastatic potential.

[0003] Overexpression of EZH2 is often associated with advanced cancer progression and poor prognosis in human cancers, such as prostate cancer, breast cancer, bladder cancer, lung cancer, colorectal cancer, and lymphoma. Mutations or deletions of EZH2 are associated with tumors such as diffuse large B-cell lymphoma, follicular lymphoma, myelodysplasia, and myeloproliferative disorders. EZH2 Y641 and A677 mutations have been shown to increase the activity of the encoded protein, leading to elevated H3K27me3 levels and promoting the proliferation of lymphoma cells.

[0004] In summary, EZH2, as an epigenetic enzyme, is involved in the occurrence and development of tumors, and EZH2 inhibitors have good application prospects in the pharmaceutical industry as drugs.

[0005] Disclosed EZH2 selective inhibitors include WO2012005805, WO2012050532, WO2012118812, WO2015143424A2, WO2016102493A1, WO2017084494A1, and WO2018045971A1, etc. Currently, a series of EZH2 inhibitor patents have been disclosed, but new EZH2 inhibitors are still needed to meet market demand.

[0006] The present invention redesigns and synthesizes a class of EZH2 inhibitors. Experimental studies have shown that the compounds have high selectivity for the EZH2 target and can exhibit excellent pharmacodynamic effects in in vivo animal experiments. Summary of the Invention

[0007] The object of the present invention is to provide a compound represented by general formula (I), and its tautomers, enantiomers, diastereomers, racemates and pharmaceutically acceptable salts.

[0008]

[0009] R 0 -C 1-6 Alkyl, -C 1-3 Alkylene-NR a R b 、-T 0 or -C 1-3 Alkylene-T 0 ;

[0010] R a and R b are independently hydrogen, -C 1-3 Alkyl, -C 1-4 Alkylene-OH, -T 0 、-C 1-3 Alkylene-T 0 、-(CH2) n -CF3, -(CH2) n -CHF2, -(CH2) n -CH2F, -C(O)-C 1-3 Alkyl, -C(O)-C 2-4 Alkenyl, -C(O)-(CH2) n -CF3, -C(O)-(CH2) n -CHF2, -C(O)-(CH2) n -CH2F, -C(O)-T 0 、-C(O)-C 1-3 Alkylene-T 0 、-C 2-4Alkylene -OCH3 or -C 2-6 Alkylene -CH3, where C 2-6 The alkylene group is optionally interrupted by oxygen atoms and / or optionally by one or more C 1-3 Alkyl substitution, R a and R b More preferably, methyl, ethyl, propyl, -C 1-4 Alkylene-OH, -C 2-4 Alkylene -OCH3 or -C 2-6 Alkylene -CH3, where C 2-6 The alkylene groups are optionally interrupted by oxygen atoms;

[0011] or R a and R b Together with the nitrogen atom to which they are attached, they form an unsubstituted or substituted 4-6 membered heterocycloalkyl group, wherein the methylene group on the 4-6 membered heterocycloalkyl group is unsubstituted or substituted with one or two T groups, and the 4-6 membered heterocycloalkyl group is a heterocycloalkyl group having one nitrogen heteroatom, a heterocycloalkyl group having two nitrogen heteroatoms, or a heterocycloalkyl group having one nitrogen heteroatom and one oxygen heteroatom. When the 4-6 membered heterocycloalkyl group is a heterocycloalkyl group having two nitrogen heteroatoms, the secondary nitrogen on the ring is unsubstituted or substituted with T';

[0012] -NR a R b Can be further specified as

[0013] T is halogen, -C 1-4 Alkyl, -C 1-3 Alkyl-substituted-C 2-4 Alkyl or -NR c R d , T may further preferably be fluorine, -C 1-4 Alkyl, -C 1-2 Alkyl-substituted-C 2-3 Alkyl or -NR c R d ;

[0014] When a methylene group on the 4-6 membered heterocycloalkane is simultaneously substituted by two T groups, T is fluorine, methyl or ethyl;

[0015] R c and R d are independently hydrogen, -C 1-3 Alkyl, -C 1-4 Alkylene-OH, -C 1-4 Alkylene -OCH3 or -C 2-6 Alkylene -CH3, where C 2-6The alkylene group is optionally interrupted by oxygen atoms and / or optionally by one or more C 1-3 Alkyl substitution;

[0016] T' is -C 1-4 Alkyl, -C 1-3 Alkyl-substituted-C 2-4 Alkyl, -C 1-4 Alkylene-OH, -C 3-6 Cycloalkyl, 4-6 membered heterocycloalkyl, -(CH2) n -CF3, -(CH2) n -CHF2, -(CH2) n -CH2F, -C(O)-C 1-3 Alkyl, -C(O)-C 2-4 Alkenyl, -C(O)-(CH2) n -CF3, -C(O)-(CH2) n -CHF2, -C(O)-(CH2) n -CH2F, tert-butyloxycarbonyl, -S(O)2-C 1-3 Alkyl, -S(O)2-(CH2) n -CF3, -S(O)2-(CH2) n -CHF2;

[0017] T 0 Has not been replaced or replaced by T 1 Replaced by -C 3-8 Cycloalkyl, 4-6 membered heterocycloalkyl, phenyl or 5-6 membered heteroaryl, when T 0 When the heteroatom is a 4-6 membered heterocycloalkyl group or a 5-6 membered heteroaryl group, the nitrogen atom is not substituted or replaced by T 2 Replace, T 0 It can be further specified as

[0018] T 1 Halogen, -C 1-6 Alkyl, -C 1-3 Alkoxy, -C 1-3 Alkyl-substituted-C 1-6 Alkyl or -NR c R d ;T 1 It may further be preferentially fluorine, -C 1-3 Alkyl, -C 1-3 Alkoxy, -C 1-2 Alkyl-substituted-C 2-3 Alkyl or -NR c R d ;

[0019] T 2 -C 1-4 Alkyl, -C 1-3 Alkyl-substituted-C 2-4 Alkyl, -C 1-4 Alkylene-OH, -C 3-6 Cycloalkyl, 4-6 membered heterocycloalkyl, -(CH2) n -CF3, -(CH2) n -CHF2, -(CH2) n -CH2F, -C(O)-C 1-3 Alkyl, -C(O)-C 2-4 Alkenyl, -C(O)-(CH2) n -CF3, -C(O)-(CH2) n -CHF2, -C(O)-(CH2) n -CH2F, tert-butyloxycarbonyl, -S(O)2-C 1-3 Alkyl, -S(O)2-(CH2) n -CF3, -S(O)2-(CH2) n -CHF2;

[0020] n is 0, 1, 2, 3 or 4;

[0021] R 3 For hydrogen, -C 1-4 Alkyl or substituted -C 1-4 Alkyl, wherein the substituted -C 1-4 The alkyl group is optionally substituted with one or more of the following substituents: hydroxy, carboxyl or -C(O)OR ’ , R 3 Preferably methyl or ethyl;

[0022] R ’ -C 1-6 Alkyl, -C 2-6 Alkenyl, -C 2-6 Alkynyl, -C 3-8 Cycloalkyl or -C 4-10 heterocycloalkyl;

[0023] R 4 and R 5 Separately, C 1-6 Alkyl, preferably methyl or ethyl;

[0024] R 5a -C 1-6 Alkyl or -C 1-6 Alkoxy, preferably methyl, ethyl, methoxy or ethoxy;

[0025] R6 -C 1-6 Alkyl, five to six membered cycloalkyl, five to six membered heterocycloalkyl or a bicyclic ring with 8 to 10 carbon atoms, wherein the five to six membered heterocycloalkyl, the heteroatom is selected from nitrogen, sulfur or oxygen, the bicyclic ring is fused, any one ring in the bicyclic ring is saturated, unsaturated or aromatic, the cycloalkyl, heterocycloalkyl or bicyclic ring with 8 to 10 carbon atoms is not substituted or is replaced by one or more R 6a Group substitution, R 6a Halogen, hydroxyl, -C 1-3 Alkyl, -C 1-3 Alkoxy, 3-6 membered cycloalkyl, 4-6 membered heterocyclic group, -NR h R k 、-C(O)-C 1-3 Alkyl, -C(O)-C 3-6 Cycloalkyl, -S(O)2-C 1-3 Alkyl, -S(O)2-C 3-6 Cycloalkyl;

[0026] When R 6 When it is a sulfur heterocyclic group containing one sulfur atom, the sulfur heteroatom is not oxidized or is oxidized by two oxygen groups to form a sulfone group;

[0027] When R 6 When it is a nitrogen heterocyclic group containing one nitrogen atom, the nitrogen atom is not substituted or replaced by R 6b Replacement, R 6b -C 1-4 Alkyl, -C 1-3 Alkyl-substituted-C 2-4 Alkyl, -C 1-4 Alkylene-OH, -C 3-8 Cycloalkyl, 4-6 membered heterocycloalkyl, -C(O)-C 1-3 Alkyl, -C(O)-C 3-6 Cycloalkyl, -S(O)2-C 1-3 Alkyl, -S(O)2-C 3-6 Cycloalkyl, wherein the 4-6 membered heterocycloalkyl, the heteroatom is selected from nitrogen or oxygen;

[0028] R 6 Preferably, methyl, ethyl, propyl,

[0029] R h and R k are independently hydrogen, -C 1-3 Alkyl, -C 1-4 Alkylene-OH, -T 0 、-C 1-3 Alkylene-T0 、-(CH2) n -CF3, -(CH2) n -CHF2, -(CH2) n -CH2F, -C(O)-C 1-3 Alkyl, -C(O)-C 2-4 Alkenyl, -C(O)-(CH2) n -CF3, -C(O)-(CH2) n -CHF2, -C(O)-(CH2) n -CH2F, -C(O)-T 0 、-C(O)-C 1-3 Alkylene-T 0 、-C 2-4 Alkylene -OCH3 or -C 2-6 Alkylene -CH3, where C 2-6 The alkylene group is optionally interrupted by oxygen atoms and / or optionally by one or more C 1-3 Alkyl substitution,

[0030] R h and R k Or together with the nitrogen atom to which they are attached, they form a 4-6 membered heterocycloalkyl group which is unsubstituted or substituted by one or two T groups, wherein T is halogen, -C 1-4 Alkyl, -C 1-3 Alkyl-substituted-C 2-4 Alkyl or -NR c R d , R c and R d are independently hydrogen, -C 1-3 Alkyl, -C 1-4 Alkylene-OH, -C 2-4 Alkylene -OCH3 or -C 2-6 Alkylene -CH3, where C 2-6 The alkylene group is optionally interrupted by oxygen atoms and / or optionally by one or more C 1-3 Alkyl substitution.

[0031] R h and R k Preferably, hydrogen, -C 1-3 Alkyl, -C 2-3 Alkylene -OCH3 or -C 2-6 Alkylene -CH3, wherein

[0032] C 2-6 The alkylene group is optionally interrupted by oxygen atoms and / or optionally by one or more C 1-3 Alkyl substitution.

[0033] The synthesis process of the compound provided by the present invention is:

[0034] The compounds represented by the general formula of the present invention can be synthesized according to a variety of reaction schemes. Those skilled in the art can easily design reaction schemes for other compounds using some of the preparation methods provided in the examples herein.

[0035] The present invention relates to a method for preparing a compound represented by general formula (I) or a pharmaceutically acceptable salt thereof, wherein when R 0 -C 1-3 Alkylene-NR a R b , m is 1, 2 or 3, the preparation method of the compound represented by general formula (II) or its pharmaceutically acceptable salt comprises the following steps:

[0036]

[0037] The compound of the general formula (II-1) reacts with di-tert-butyl dicarbonate under alkaline conditions to obtain a compound of the general formula (II-2); the compound of the general formula (II-2) is reduced to a compound of the general formula (II-3) under palladium-carbon reducing agent conditions; the compound of the general formula (II-3) undergoes bromination reaction under alkaline conditions to obtain a compound of the general formula (II-4), the reagent for the alkaline conditions provided under the conditions is preferably calcium carbonate, and the brominating agent is preferably benzyltrimethylammonium tribromide; the compound of the general formula (II-4) reacts with chloroacetyl chloride under alkaline conditions to obtain a compound of the general formula (II-5), the reagent for the alkaline conditions provided under the conditions is preferably triethylamine; the compound of the general formula (II-5) reacts with the corresponding amine (R a R b NH) undergoes a substitution reaction to obtain a compound of the general formula (II-6), wherein the alkaline reagent provided under the conditions is preferably cesium carbonate; the compound of the general formula (II-6) undergoes a ring-closing reaction under the conditions of heating, alkalinity, and the presence of a catalyst to obtain a compound of the general formula (II-7), wherein the alkaline reagent provided under the conditions is preferably cesium carbonate, and the catalyst is preferably cuprous iodide and 1,10-phenanthroline; the compound of the general formula (II-7) undergoes a ring-closing reaction under the conditions of heating, alkalinity, and the presence of a catalyst with the corresponding amine (R 6-NH2) undergoes a substitution reaction to obtain a compound of the general formula (II-8), the reagent for the alkaline conditions provided under the conditions is preferably cesium carbonate, and the catalyst is preferably tris(dibenzylideneacetone)dipalladium and 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene; the compound of the general formula (II-8) undergoes a reductive amination reaction with the corresponding ketone in the presence of a reducing agent (preferably sodium acetate borohydride) to obtain a compound of the general formula (II-9); the compound of the general formula (II-9) undergoes a tert-butyl removal under acidic conditions to obtain a compound of the general formula (II-10), the reagent for the acidic conditions provided under the conditions is preferably trifluoroacetic acid; the compound of the general formula (II-10) undergoes an acylation reaction with the compound of the general formula (II-11) to obtain a compound of the general formula (I).

[0038] When R 0 -C 1-6 Alkyl, -T 0 or -C 1-3 Alkylene-T 0 , a method for preparing a compound represented by general formula (I) or a pharmaceutically acceptable salt thereof, comprising the following steps:

[0039]

[0040] The compound of formula (II-4) is reacted with acyl chloride (R 0 COCl) to obtain a compound of the general formula (IV-1), wherein the alkaline reagent provided under the conditions is preferably triethylamine; the compound of the general formula (IV-1) undergoes a ring-closing reaction under the conditions of heating, alkalinity, and the presence of a catalyst to obtain a compound of the general formula (IV-2), wherein the alkaline reagent provided under the conditions is preferably cesium carbonate, and the catalyst is preferably cuprous iodide and 1,10-phenanthroline; the compound of the general formula (IV-2) is reacted with the corresponding amine (R 6 -NH2) undergoes a substitution reaction to obtain a compound of the general formula (IV-3), the reagent for the alkaline conditions provided under the conditions is preferably cesium carbonate, and the catalyst is preferably tris(dibenzylideneacetone)dipalladium and 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene; the compound of the general formula (IV-3) undergoes a reductive amination reaction with the corresponding aldehyde or ketone in the presence of a reducing agent (preferably sodium acetate borohydride) to obtain a compound of the general formula (IV-4); the compound of the general formula (IV-4) undergoes a tert-butyl removal under acidic conditions to obtain a compound of the general formula (IV-5), the reagent for the acidic conditions provided under the conditions is preferably trifluoroacetic acid; the compound of the general formula (IV-5) undergoes an acylation reaction with a compound of the general formula (II-11) to obtain a compound of the general formula (IV).

[0041] According to the compound represented by general formula (I) of the present invention and its pharmaceutically acceptable salt, wherein the compound is specifically:

[0042] N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-5-methyl-2-(morpholinomethyl)benzo[d]oxazole-4-carboxamide;

[0043] N-(4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-5-methyl-2-(tetrahydro-2H-pyran-4-yl)benzo[d]oxazole-4-carboxamide;

[0044] 2-cyclopropyl-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-5-methylbenzoxazole-4-carboxamide;

[0045] N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-5-methyl-2-(1-methyl-1H-pyrazol-3-yl)benzo[d]oxazole-4-carboxamide;

[0046] N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2,5-dimethylbenzo[d]oxazole-4-carboxamide;

[0047] N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-(4-methoxyphenyl)-5-methylbenzo[d]oxazole-4-carboxamide;

[0048] N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-5-methyl-2-(pyridin-4-yl)benzo[d]oxazole-4-carboxamide;

[0049] N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(methyl(tetrahydro-2H-pyran-4-yl)amino)-5-methyl-2-(morpholinomethyl)benzo[d]oxazole-4-carboxamide;

[0050] N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-5-methyl-2-((dimethylamino)methyl)benzo[d]oxazole-4-carboxamide;

[0051] N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-5-methyl-2-(piperidin-1-ylmethyl)benzo[d]oxazole-4-carboxamide;

[0052] N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-5-methyl-2-((4-methylpiperazin-1-yl)methyl)benzo[d]oxazole-4-carboxamide;

[0053] N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-5-methyl-2-((4-isopropylpiperazin-1-yl)methyl)benzo[d]oxazole-4-carboxamide;

[0054] N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-5-methyl-2-(((4-dimethylamino)piperidin-1-yl)methyl)benzo[d]oxazole-4-carboxamide;

[0055] N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-5-methyl-2-(morpholinomethyl)benzo[d]oxazole-4-carboxamide;

[0056] N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(1-methylpiperidin-4-yl)amino)-5-methyl-2-(dimethylaminomethyl)benzo[d]oxazole-4-carboxamide;

[0057] N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(tetrahydro-2H-thiopyran-4-yl)amino)-5-methyl-2-(dimethylaminomethyl)benzo[d]oxazole-4-carboxamide;

[0058] 2-(azetidin-1-ylmethyl)-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-5-methylbenzo[d]oxazole-4-carboxamide;

[0059] N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-5-methyl-2-(pyrrolidin-1-ylmethyl)benzo[d]oxazole-4-carboxamide;

[0060] N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(tetrahydro-2H-thiopyran-4-yl)amino)-5-methyl-2-((4,4-difluoropiperidin-1-yl)methyl)benzo[d]oxazole-4-carboxamide;

[0061] N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(4-dimethylaminocyclohexyl)amino)-5-methyl-2-((dimethylamino)methyl)benzo[d]oxazole-4-carboxamide;

[0062] N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(1-acetylpiperidin-4-yl)amino)-5-methyl-2-(morpholinomethyl)benzo[d]oxazole-4-carboxamide;

[0063] N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(1-methylsulfonylpiperidin-4-yl)amino)-5-methyl-2-(morpholinomethyl)benzo[d]oxazole-4-carboxamide;

[0064] N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(1-methylpyrrolidin-3-yl)amino)-5-methyl-2-(dimethylamino)methyl)benzo[d]oxazole-4-carboxamide;

[0065] 6-((2,3-dihydro-1H-inden-2-yl)(ethyl)amino)-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-(dimethylamino)methyl)-5-methyl-benzo[d]oxazole-4-carboxamide;

[0066] N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-5-methyl-2-(2-morpholinoethyl)benzo[d]oxazole-4-carboxamide;

[0067] N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-5-methyl-2-(morpholinomethyl)benzo[d]oxazole-4-carboxamide;

[0068] 2-(Dimethylaminomethyl)-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-methylbenzo[d]oxazole-4-carboxamide;

[0069] N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-(((2-methoxyethyl)(methyl)amino)methyl)-5-methylbenzo[d]oxazole-4-carboxamide;

[0070] N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-5-methyl-2-(1-methylpiperidin-4-yl)benzo[d]oxazole-4-carboxamide;

[0071] N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-5-methyl-2-(4-methoxycyclohexyl)benzo[d]oxazole-4-carboxamide.

[0072] The term "substituted" as used in the present invention means that any one or more hydrogen atoms on a designated atom are replaced by a substituent selected from a designated group, and the result of the substitution is a stable compound. When the substituent is an oxo group or a keto group (i.e., =O), two hydrogen atoms on the atom are replaced, and the keto substituent does not exist on the aromatic ring.

[0073] The pharmaceutically acceptable salts described in the present invention refer to inorganic base salts, such as sodium salts, potassium salts, calcium salts, magnesium salts, zinc salts, ammonium salts, quaternary ammonium salts or aluminum salts; organic base salts, such as lysine salts, arginine salts, diethylamine salts, triethylamine salts, ethanolamine salts, trimethylamine salts, dicyclohexylamine salts, choline salts, dibenzylamine salts, piperidine salts and other pharmaceutically acceptable organic amine salts.

[0074] When the compound of the present invention contains at least one nitrogen atom capable of forming a salt, the compound can be converted into a corresponding salt by reacting with a corresponding organic acid or inorganic acid in an organic solvent such as acetonitrile or tetrahydrofuran. Typical organic acids include oxalic acid, tartaric acid, maleic acid, succinic acid, methanesulfonic acid, benzoic acid, benzenesulfonic acid, toluenesulfonic acid, sulfamic acid, citric acid, glutamic acid, pyroglutamic acid, aspartic acid, glucuronic acid, naphthalenesulfonic acid, glutaric acid, acetic acid, trifluoroacetic acid, malic acid, fumaric acid, salicylic acid, 4-aminosalicylic acid, lactic acid, palmitate, stearic acid, lauric acid, cinnamic acid, alginic acid, and ascorbate. Typical inorganic acids include nitric acid, hydrochloric acid, sulfuric acid, and phosphoric acid.

[0075] When there are one or more asymmetric carbon atoms in the compounds of the present invention, they can exist in the form of optically pure enantiomers, pure diastereomers, mixtures of enantiomers, mixtures of diastereomers, racemic mixtures of enantiomers, racemates or racemate mixtures. All possible isomers, stereoisomers and mixtures thereof of compounds of formula (II) are also within the scope of the present invention.

[0076] The present invention also provides a pharmaceutical composition comprising at least one compound as described above and optionally one or more pharmaceutically acceptable carriers and / or diluents.

[0077] The pharmaceutical compositions provided by the present invention can be prepared in any form, such as granules, powders, tablets, coated tablets, capsules, pills, syrups, drops, solutions, suspensions and emulsions, or sustained-release preparations of the active ingredients, wherein examples of capsules include hard or soft gelatin capsules, and granules and powders can be non-effervescent or effervescent forms.

[0078] The pharmaceutical composition of the present invention may further include one or more pharmaceutically or physiologically acceptable carriers, which are suitably formulated for ease of administration. For example, the pharmaceutically or physiologically acceptable carrier may be saline, autoclaved water, Ringer's solution, buffered saline, glucose, maltodextrin, glycerol, ethanol, and mixtures thereof. The pharmaceutical composition of the present invention may also include pharmaceutically or physiologically acceptable additives, such as diluents, lubricants, binders, glidants, disintegrants, sweeteners, flavoring agents, wetting agents, dispersants, surfactants, solvents, coating agents, foaming agents, or fragrances.

[0079] Examples of diluents that may be used include, but are not limited to, lactose, sucrose, starch, kaolin, salt, mannitol, and dicalcium phosphate; examples of lubricants include, but are not limited to, talc, starch, magnesium or calcium stearate, lycopodium, and stearic acid; examples of binders include, but are not limited to, microcrystalline cellulose, tragacanth gum, glucose solution, acacia mucilage, gelatin solution, sucrose, and starch paste; examples of glidants include, but are not limited to, colloidal silicon dioxide; examples of disintegrants include, but are not limited to, cross-linked sodium carboxymethylcellulose, sodium starch glycolate, alginic acid, corn starch, maize starch, and malt starch. Potato starch, bentonite, methylcellulose, agar and carboxymethylcellulose; examples of sweeteners include, but are not limited to, sucrose, lactose, mannitol and artificial sweeteners such as sodium cyclamate and saccharin, and any number of spray-dried flavors; examples of flavors include, but are not limited to, natural flavors extracted from plants, such as fruits, and flavorful compounds such as, but not limited to, mint and methyl salicylate; examples of wetting agents include, but are not limited to, propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylene lauryl ether.

[0080] The pharmaceutical composition of the present invention can be administered by various routes according to conventional methods, including oral, intravenous, intraarterial, intraperitoneal, intrathoracic, transdermal, nasal, inhalation, rectal, ocular and subcutaneous administration.

[0081] The pharmaceutically acceptable carrier optionally added to the pharmaceutical composition of the present invention is: one or more of water, alcohol, honey, mannitol, sorbitol, dextrin, lactose, caramel, gelatin, calcium sulfate, magnesium stearate, talc, kaolin, glycerol, Tween, agar, calcium carbonate, calcium bicarbonate, surfactants, cyclodextrin and its derivatives, phospholipids, phosphates, starches and its derivatives, silicon derivatives, celluloses and its derivatives, pyrrolidones, polyethylene glycols, acrylic resins, phthalates, acrylic acid copolymers, and trimellitic acid esters.

[0082] Pharmacological experiments have verified that the compounds or pharmaceutical compositions provided by the present invention can treat tumors, myeloproliferative diseases or autoimmune diseases through EZH2. The tumors are lymphomas, melanomas, gliomas, gastrointestinal stromal tumors, prostate cancer, breast cancer, ovarian cancer, bladder cancer, lung cancer, rectal cancer, skin cancer, epithelial cell cancer, nasopharyngeal cancer, bone cancer, esophageal cancer or leukemia. The autoimmune diseases are inflammatory bowel disease, autoimmune encephalomyelitis or multiple sclerosis.

[0083] The general dosage range of the compounds provided by the present invention is about 0.001 mg / kg to 1000 mg / kg per day, preferably about 0.01 mg / kg to 100 mg / kg, and more preferably about 0.1 to 20 mg / kg. The dosage range of the pharmaceutical composition is calculated based on the amount of the above-mentioned compound contained therein. DETAILED DESCRIPTION

[0084] The present invention is further described below with reference to the following examples, but these examples are not intended to limit the scope of the present invention.

[0085] Example 1 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-5-methyl-2-(morpholinomethyl)benzo[d]oxazole-4-carboxamide

[0086]

[0087] Step 1: tert-Butyl 2-methyl-6-nitrobenzoate 1b

[0088] Compound 2-methyl-6-nitrobenzoic acid 1a (4.0 g, 22.1 mmol) was dissolved in tetrahydrofuran (40 mL), and di-tert-butyl dicarbonate (12 g, 55.2 mmol) and 4-dimethylaminopyridine (2.7 g, 22.1 mmol) were added. The mixture was stirred at room temperature overnight. Water and ethyl acetate were added for extraction. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain the title compound 1b (1.8 g, 7.6 mmol) in a 34% yield.

[0089] Step 2: tert-Butyl 2-amino-6-methylbenzoate 1c

[0090] Compound 1b (1.8 g, 7.6 mmol) was dissolved in methanol (20 mL), and palladium carbon (0.9 g) was added. The mixture was stirred at room temperature for 2 h under hydrogen protection. TLC showed that the reaction was complete. The mixture was filtered and concentrated to give the title compound 1c (1.5 g, 7.2 mmol) in a yield of 95%.

[0091] Step 3 tert-Butyl 2-amino-3,5-dibromo-6-methylbenzoate 1d

[0092] Compound 1c (1.5 g, 7.25 mmol) was dissolved in dichloromethane (30 mL), and benzyltrimethylammonium tribromide (7.0 g, 18 mmol) and calcium carbonate (1.9 g, 18 mmol) were added. The mixture was stirred at room temperature overnight. After completion of the reaction, the mixture was filtered, extracted with water and ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to afford the title compound 1d (2.4 g, 6.6 mmol) in a 91% yield.

[0093] Step 4: tert-Butyl 3,5-dibromo-2-(2-chloroacetylamino)-6-methylbenzoate 1e

[0094] Compound 1d (1.7 g, 4.66 mmol) was dissolved in dichloromethane (20 mL), and triethylamine (1.4 g, 14 mmol) was added, followed by the slow dropwise addition of chloroacetyl chloride (1.6 g, 14 mmol). The mixture was stirred at room temperature for 4 h. After completion of the reaction, the mixture was extracted with water and ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to afford the title compound 1e (1.6 g, 3.6 mmol) in a 78% yield.

[0095] Step 5: tert-Butyl 3,5-dibromo-2-methyl-6-(2-morpholinoacetamido)benzoate 1f

[0096] Compound 1e (0.8 g, 1.8 mmol) was dissolved in N,N-dimethylformamide (10 mL), and morpholine (0.32 g, 3.6 mmol) and cesium carbonate (0.6 g, 1.8 mmol) were added. The mixture was stirred at room temperature for 2 h. After completion of the reaction, water and ethyl acetate were added for extraction, and the mixture was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain the title compound 1f (0.6 g, 1.2 mmol) in a yield of 67%.

[0097] Step 6: tert-Butyl 6-bromo-5-methyl-2-(morpholinomethyl)benzo[d]oxazole-4-carboxylate 1 g

[0098] Compound 1f (550 mg, 1.12 mmol) and cuprous iodide (107 mg, 0.56 mmol) were dissolved in ethylene glycol dimethyl ether (10 mL). 1,10-Phenanthroline (162 mg, 0.44 mmol) and cesium carbonate (730 mg, 2.24 mmol) were added, and the mixture was heated at 110°C under nitrogen and stirred for 3 h. Ethyl acetate and water were added, and the mixture was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain 1 g (300 mg, 0.73 mmol) of the title compound in a 65% yield.

[0099] 1 H NMR (400MHz, DMSO) δ8.19 (s, 1H), 3.83 (s, 2H), 3.56–3.53 (t, J = 4.4Hz, 4H), 2.51 - 2.38 (m, 4H), 2.38 (s, 3H), 1.54 (s, 9H).

[0100] Step 7: tert-Butyl 5-methyl-2-(morpholinomethyl)-6-((tetrahydro-2H-pyran-4-yl)amino)benzo[d]oxazole-4-carboxylate 1h

[0101] Compound 1g (300 mg, 0.73 mmol) and tetrahydro-2H-pyran-4-amine (147 mg, 1.46 mmol) were dissolved in toluene (10 mL). Anhydrous cesium carbonate (476 mg, 1.46 mmol), tris(dibenzylideneacetone)dipalladium (67 mg, 0.073 mmol), and 4,5-bis(diphenylphosphino-9,9-dimethylxanthene) (43 mg, 0.073 mmol) were added. The mixture was heated at 100°C and stirred for 3 h under nitrogen. After cooling, the mixture was spin-dried and extracted three times with dichloromethane. The mixture was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by plate chromatography to obtain the title compound 1h (150 mg, 0.35 mmol) in a yield of 47.6%.

[0102] MSm / z(ESI):432[M+H] + .

[0103] Step 8: tert-Butyl 6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-5-methyl-2-(morpholinomethyl)benzo[d]oxazole-4-carboxylate 1i

[0104] Compound 1h (70 mg, 0.16 mmol) was dissolved in 1,2-dichloroethane (5.0 mL), and acetic acid (58 mg, 0.96 mmol) and acetaldehyde (22 mg, 0.48 mmol) were added. The reaction was stirred at room temperature for 30 minutes, and then sodium acetate borohydride (103 mg, 0.48 mmol) was added. After stirring at room temperature for 2 hours, the reaction was completed. The reaction solution was quenched with water, extracted three times with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by plate chromatography to obtain the title compound 1i (50 mg, 0.11 mmol) in a yield of 67%.

[0105] Step 9: 6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-5-methyl-2-(morpholinomethyl)benzo[d]oxazole-4-carboxylic acid 1j

[0106] Compound 1i (50 mg, 0.11 mmol) was dissolved in dichloromethane (2.0 mL), and trifluoroacetic acid (1.0 ml) was added. The reaction was stirred at room temperature for 2 h, and the reaction was complete. The reaction solution was washed with water, extracted three times with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by plate chromatography to obtain the title compound 1j (40 mg, 0.10 mmol) in a 91% yield.

[0107] Step 10: N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-5-methyl-2-(morpholinomethyl)benzo[d]oxazole-4-carboxamide

[0108] Compound 1j (40 mg, 0.10 mmol) and 3-(aminomethyl)-4,6-dimethyl-1H-pyridin-2-one hydrochloride (22 mg, 0.12 mmol) were dissolved in N,N-dimethylformamide (5.0 mL). 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (38 mg, 0.2 mmol), 1-hydroxybenzotriazole (14 mg, 0.1 mmol), and triethylamine (50 mg, 0.5 mmol) were added and stirred at room temperature for 2 h. The reaction was complete. The reaction solution was washed with water, extracted three times with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by plate chromatography to obtain the title compound 1 (20 mg, 0.037 mmol) in a yield of 38%.

[0109] 1H NMR(400MHz,DMSO)δ11.42(s,1H),8.24(s,1H),7.53(s,1H),5.83(s,1H),4.32 (d,J=4.8Hz,1H)3.80-3.76(d,J=16Hz,4H),3.55-3.52(t,J=4.4Hz,4H),3.21-3 .16(t,J=11.2Hz,2H),3.02-2.99(t,J=7.6Hz,2H),2.91(m,1H),2.21(s,6H),2. 06(s,3H),1.61-1.58(d,J=12Hz,2H),1.44(m,2H),0.77-0.73(t,J=7.2Hz,3H).

[0110] MSm / z(ESI):538[M+H] + .

[0111] Example 2 N-(4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-5-methyl-2-(tetrahydro-2H-pyran-4-yl)benzo[d]oxazole-4-carboxamide

[0112]

[0113] Step 1: tert-Butyl 3,5-dibromo-2-methyl-6-(tetrahydro-2H-pyran-4-carboxamido)benzoate 5a

[0114] Compound 1d (530 mg, 1.452 mmol) was dissolved in dichloromethane (20 mL), and triethylamine (147 mg, 1.452 mmol) was added. Tetrahydro-2H-pyran-4-carbonyl chloride (644 mg, 4.356 mmol) was then slowly added dropwise. The mixture was stirred at room temperature for 4 h. After completion of the reaction, the mixture was extracted with water and ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to afford the title compound 5a (480 mg, 1.01 mmol) in a 69% yield.

[0115] Step 2 tert-Butyl 6-bromo-5-methyl-2-(tetrahydro-2H-pyran-4-yl)benzo[d]oxazole-4-carboxylate 5b

[0116] Compound 5a (480 mg, 1.01 mmol) and cuprous iodide (190 mg, 1 mmol) were dissolved in ethylene glycol dimethyl ether (10 mL). 1,10-Phenanthroline (180 mg, 1 mmol) and cesium carbonate (652 mg, 2 mmol) were added, and the mixture was heated at 110°C and stirred for 3 h under nitrogen. Ethyl acetate and water were added, and the mixture was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain the title compound 5b (260 mg, 0.658 mmol) in a 65% yield.

[0117] Step 3 tert-Butyl 5-methyl-2-(tetrahydro-2H-pyran-4-yl)-6-((tetrahydro-2H-pyran-4-yl)amino)benzo[d]oxazole-4-carboxylate 5c

[0118] Compound 5b (260 mg, 0.658 mmol) and tetrahydro-2H-pyran-4-amine (133 mg, 1.316 mmol) were dissolved in toluene (10 mL). Anhydrous cesium carbonate (429 mg, 1.316 mmol), tris(dibenzylideneacetone)dipalladium (60 mg, 0.0658 mmol), and 4,5-bis(diphenylphosphino-9,9-dimethylxanthene) (76 mg, 0.1316 mmol) were added. The mixture was heated at 100°C and stirred for 3 h under nitrogen. After cooling, the mixture was spin-dried and extracted three times with dichloromethane. The mixture was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by plate chromatography to obtain the title compound 5c (130 mg, 0.3125 mmol) in a 48% yield.

[0119] MSm / z(ESI):417[M+H] + .

[0120] Step 4: tert-Butyl 6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-5-methyl-2-(tetrahydro-2H-pyran-4-yl)benzo[d]oxazole-4-carboxylate 5d

[0121] Compound 5c (130 mg, 0.3125 mmol) was dissolved in 1,2-dichloroethane (5.0 mL), and acetic acid (19 mg, 0.3125 mmol) and acetaldehyde (28 mg, 0.625 mmol) were added. The reaction was stirred at room temperature for 30 minutes, and then sodium acetate borohydride (133 mg, 0.625 mmol) was added. After stirring at room temperature for 2 hours, the reaction was completed. The reaction solution was quenched with water, extracted three times with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by plate chromatography to obtain the title compound 5d (90 mg, 0.202 mmol) in a yield of 64.6%.

[0122] Step 5: 6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-5-methyl-2-(tetrahydro-2H-pyran-4-yl)benzo[d]oxazole-4-carboxylic acid 5e

[0123] Compound 5d (90 mg, 0.202 mmol) was dissolved in dichloromethane (2.0 mL), and trifluoroacetic acid (1.0 ml) was added. The reaction was stirred at room temperature for 2 h, and the reaction was completed. The reaction solution was washed with water, extracted three times with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by plate chromatography to obtain the title compound 5e (77 mg, 0.2 mmol) in a yield of 99%. Step 6: N-(4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-5-methyl-2-(tetrahydro-2H-pyran-4-yl)benzo[d]oxazole-4-carboxamide 5

[0124] Compound 5e (77 mg, 0.2 mmol) and 3-(aminomethyl)-4,6-dimethyl-1H-pyridin-2-one hydrochloride (87 mg, 0.46 mmol) were dissolved in N,N-dimethylformamide (5.0 mL). 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (88 mg, 0.46 mmol), 1-hydroxybenzotriazole (63 mg, 0.46 mmol), and triethylamine (187 mg, 1.85 mmol) were added and stirred at room temperature for 2 h. The reaction was complete. The reaction solution was washed with water, extracted three times with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by plate chromatography to obtain the title compound 5 (38 mg, 0.072 mmol) in a yield of 36%.

[0125] 1 H NMR (400MHz, DMSO-d6) δ11.45(s,1H),8.27(t,J=5.1Hz,1H),7.52(s,1H),5.84(s,1H),4.32( d,J=5.0Hz,2H),3.87(d,J=11.1Hz,2H),3.78(d,J=11.3Hz,2H),3.52–3.40(m,2H),3.28–3.13 (m,3H),3.06–2.85(m,3H),2.24(d,J=5.4Hz,5H),2.07(s,3H),1.95(d,J=13.0Hz,2H),1.77( dd,J=12.8,8.6Hz,2H),1.60(d,J=12.4Hz,2H),1.44(d,J=11.1Hz,2H),0.74(t,J=6.9Hz,3H).

[0126] MSm / z(ESI):523[M+H] + .

[0127] Example 3 2-Cyclopropyl-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-5-methylbenzoxazole-4-carboxamide

[0128]

[0129] A similar synthesis method to that of Example 2 was used, except that tetrahydro-2H-pyran-4-carbonyl chloride was replaced by cyclopropylcarbonyl chloride to obtain the title product 6 in a yield of 33%.

[0130] 1 H NMR (400MHz, DMSO-d6) δ11.46(s,1H),7.44(s,1H),5.84(s,1H),4.31(d,J=5.1 Hz,2H),3.30(s,1H),3.18(td,J=13.5,12.6,2.9Hz,2H),2.99(q,J=6.9Hz,2H), 2.23(d,J=5.9Hz,6H),2.07(s,3H),1.59(d,J=12.5Hz,2H),1.48–1.36(m,1H), 1.11(dt,J=8.2,3.1Hz,2H), 1.06(dt,J=5.3,2.9Hz,2H), 0.73(t,J=6.9Hz,3H).

[0131] MSm / z(ESI):479[M+H] + .

[0132] Example 4 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-5-methyl-2-(1-methyl-1H-pyrazol-3-yl)benzo[d]oxazole-4-carboxamide

[0133]

[0134] A similar synthesis method to that of Example 2 was used, except that tetrahydro-2H-pyran-4-carbonyl chloride was replaced with 1-methyl-1H-pyrazol-3-ylcarbonyl chloride to obtain the title product 7 in a yield of 13%.

[0135] 1H NMR (400MHz, DMSO-d6) δ11.47(s,1H),8.37(t,J=5.0Hz,1H),7.91(d,J=2.3Hz,1H),7 .57(s,1H),6.88(d,J=2.3Hz,1H),5.85(s,1H),4.35(d,J=4.9Hz,2H),3.95(s,3H),3. 84–3.71(m,2H),3.20(t,J=11.4Hz,2H),3.09–2.88(m,3H),2.25(d,J=4.0Hz,6H),2.0 7(s,3H),1.62(d,J=12.4Hz,2H),1.48(dt,J=12.0,5.9Hz,2H),0.77(t,J=6.9Hz,3H).

[0136] MS m / z (ESI): 519 [M+H] +.

[0137] Example 5 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2,5-dimethylbenzo[d]oxazole-4-carboxamide 8

[0138]

[0139] A similar synthesis method to that of Example 2 was used, except that tetrahydro-2H-pyran-4-carbonyl chloride was replaced by acetyl chloride to obtain the title product 8 in a yield of 40%.

[0140] 1 H NMR (400MHz, Chloroform-d) δ11.16(s,1H),7.98(s,1H),7.26(s,1H),5.94(s,1H),4.63(d,J=5.8Hz,2H),3.92(d,J=11.4Hz,2H),3.28(td,J=11.2,3.7 Hz,2H),3.06(d,J=7.2Hz,2H),2.94(s,1H),2.54(s,3H),2.47(s,2H),2.44 (s,3H),2.22–2.17(m,3H),1.66(s,2H),1.23(s,3H),0.84(t,J=7.0Hz,3H).

[0141] MSm / z(ESI):453.1[M+H] +

[0142] Example 6 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-(4-methoxyphenyl)-5-methylbenzo[d]oxazole-4-carboxamide 9

[0143]

[0144] A similar synthesis method to that of Example 2 was used, except that tetrahydro-2H-pyran-4-carbonyl chloride was replaced with 4-methoxybenzoyl chloride to obtain the title product 9 in a yield of 9%.

[0145] MSm / z(ESI):545[M+H] +

[0146] Example 7 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-5-methyl-2-(pyridin-4-yl)benzo[d]oxazole-4-carboxamide 10

[0147]

[0148] A similar synthesis method to that of Example 2 was used, except that tetrahydro-2H-pyran-4-carbonyl chloride was replaced with pyridine-4-carbonyl chloride to obtain the title product 10 in 8% yield.

[0149] MSm / z(ESI):516[M+H] +

[0150] Example 8 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(methyl(tetrahydro-2H-pyran-4-yl)amino)-5-methyl-2-(morpholinomethyl)benzo[d]oxazole-4-carboxamide

[0151]

[0152] Step 1: tert-Butyl 5-methyl-6-(methyl(tetrahydro-2H-pyran-4-yl)amino)-2-(morpholinomethyl)benzo[d]oxazole-4-carboxylate 14a

[0153] Compound 1h (70 mg, 0.16 mmol) was dissolved in 1,2-dichloroethane (5.0 mL), and acetic acid (58 mg, 0.96 mmol) and paraformaldehyde (31 mg, 0.98 mmol) were added. The reaction was stirred at room temperature for 30 minutes, and then sodium acetate borohydride (103 mg, 0.48 mmol) was added. After stirring at room temperature for 2 hours, the reaction was completed. The reaction solution was quenched with water, extracted three times with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by plate chromatography to obtain the title compound 14a (80 mg, 0.16 mmol) in a 100% yield.

[0154] Step 2: 5-Methyl-6-(methyl(tetrahydro-2H-pyran-4-yl)amino)-2-(morpholinomethyl)benzo[d]oxazole-4-carboxylic acid 14b

[0155] Compound 14a (80 mg, 0.16 mmol) was dissolved in dichloromethane (2.0 mL), and trifluoroacetic acid (1.0 ml) was added. The reaction was stirred at room temperature for 2 h, and the reaction was complete. The reaction solution was washed with water, extracted three times with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by plate chromatography to obtain the title compound 14b (54 mg, 0.14 mmol) in an 88% yield.

[0156] Step 3 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(methyl(tetrahydro-2H-pyran-4-yl)amino)-5-methyl-2-(morpholinomethyl)benzo[d]oxazole-4-carboxamide 14

[0157] Compound 14b (54 mg, 0.14 mmol) and 3-(aminomethyl)-4,6-dimethyl-1H-pyridin-2-one hydrochloride (39 mg, 0.21 mmol) were dissolved in N,N-dimethylformamide (5.0 mL). 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (54 mg, 0.28 mmol), 1-hydroxybenzotriazole (19 mg, 0.14 mmol), and triethylamine (43 mg, 0.42 mmol) were added and stirred at room temperature for 2 h. The reaction was completed. The reaction solution was washed with water, extracted three times with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by plate chromatography to obtain the title compound 14 (33.2 mg, 0.063 mmol) in a yield of 45%.

[0158] 1H NMR (400MHz, DMSO-d6) δ11.43 (s, 1H), 8.23 ​​(t, J = 5.0Hz, 1H), 7.49 (s, 1H), 5. 83(s,2H),4.31(d,J=5.1Hz,2H),3.80(d,J=11.2Hz,2H),3.76(s,2H),3.54(t ,J=4.6Hz,4H),3.26–3.16(m,2H),2.93(dd,J=10.5,5.6Hz,1H),2.56(s,3H), 2.47(d,J=1.9Hz,4H),2.22(s,6H),2.07(d,J=0.9Hz,3H),1.60–1.48(m,4H).

[0159] MS m / z (ESI): 524.3 [M+H] +.

[0160] Example 9 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-5-methyl-2-((dimethylamino)methyl)benzo[d]oxazole-4-carboxamide

[0161]

[0162] A similar synthesis method to that of Example 1 was used, except that morpholine was replaced by dimethylamine, to obtain the title product 15 in a yield of 22%.

[0163] MSm / z(ESI):496[M+H] + .

[0164] Example 10 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-5-methyl-2-(piperidin-1-ylmethyl)benzo[d]oxazole-4-carboxamide

[0165]

[0166] A similar synthesis method to Example 1 was used, except that morpholine was replaced by piperidine, to obtain the title product 30 in a yield of 10%.

[0167] MSm / z(ESI):536.3[M+H] + .

[0168] Example 11 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-5-methyl-2-((4-methylpiperazin-1-yl)methyl)benzo[d]oxazole-4-carboxamide

[0169]

[0170] A similar synthesis method to that of Example 1 was used, except that morpholine was replaced by 1-methylpiperazine, to obtain the title product 31 in a yield of 15%.

[0171] MSm / z(ESI):551.3[M+H] + .

[0172] Example 12 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-5-methyl-2-((4-isopropylpiperazin-1-yl)methyl)benzo[d]oxazole-4-carboxamide

[0173]

[0174] A similar synthesis method to Example 1 was used, except that morpholine was replaced by 1-isopropylpiperazine, to obtain the title product 31 in a yield of 15%.

[0175] MSm / z(ESI):579.3[M+H] + .

[0176] Example 13 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-5-methyl-2-(((4-dimethylamino)piperidin-1-yl)methyl)benzo[d]oxazole-4-carboxamide

[0177]

[0178] A similar synthesis method to that of Example 1 was used, except that morpholine was replaced by 4-dimethylaminopiperidine, to obtain the title product 33 in a yield of 26%.

[0179] MSm / z(ESI):579.3[M+H] + .

[0180] Example 14 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-5-methyl-2-(morpholinomethyl)benzo[d]oxazole-4-carboxamide

[0181]

[0182] A similar synthesis method to that of Example 1 was used, except that acetaldehyde was replaced by paraformaldehyde, to obtain the title product 34 in a yield of 13%.

[0183] MSm / z(ESI):524.2[M+H] + .

[0184] Example 15 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(1-methylpiperidin-4-yl)amino)-5-methyl-2-(dimethylaminomethyl)benzo[d]oxazole-4-carboxamide

[0185]

[0186] A similar synthesis method to that of Example 1 was used, except that morpholine was replaced by dimethylamine and tetrahydropyrone was replaced by N-methyl-4-piperidone to obtain the title product 35 in a yield of 9%.

[0187] MSm / z(ESI):509[M+H] + .

[0188] Example 16 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(tetrahydro-2H-thiopyran-4-yl)amino)-5-methyl-2-(dimethylaminomethyl)benzo[d]oxazole-4-carboxamide

[0189]

[0190] A similar synthesis method to that of Example 1 was used, except that morpholine was replaced by dimethylamine and tetrahydropyrone was replaced by tetrahydrothiopyranone, to obtain the title product 36 in an 11% yield.

[0191] MS m / z (ESI): 512 [M+H] +.

[0192] Example 17 2-(azetidin-1-ylmethyl)-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-5-methylbenzo[d]oxazole-4-carboxamide

[0193]

[0194] A similar synthesis method to Example 1 was used, except that morpholine was replaced by azetidine, to obtain the title product 37 in a yield of 26%.

[0195] MS m / z (ESI): 508 [M+H] +.

[0196] Example 18 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-5-methyl-2-(pyrrolidin-1-ylmethyl)benzo[d]oxazole-4-carboxamide

[0197]

[0198] A similar synthesis method to Example 1 was used, except that morpholine was replaced by tetrahydropyrrole, to obtain the title product 38 in 8% yield.

[0199] MS m / z (ESI): 522.3 [M+H] +.

[0200] Example 19 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(tetrahydro-2H-thiopyran-4-yl)amino)-5-methyl-2-((4,4-difluoropiperidin-1-yl)methyl)benzo[d]oxazole-4-carboxamide

[0201]

[0202] A similar synthesis method to that of Example 1 was used, except that morpholine was replaced by tetrahydropyrrole, to obtain the title product 39 in a yield of 35%.

[0203] MS m / z (ESI): 572.3 [M+H] +.

[0204] Example 20 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(4-dimethylaminocyclohexyl)amino)-5-methyl-2-((dimethylamino)methyl)benzo[d]oxazole-4-carboxamide

[0205]

[0206] A similar synthesis method to that of Example 1 was used, except that morpholine was replaced by dimethylamine and tetrahydropyrone was replaced by 4-dimethylaminocyclohexanone to obtain the title product 40 in an 18% yield.

[0207] MS m / z (ESI): 537.3 [M+H] +.

[0208] Example 21 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(1-acetylpiperidin-4-yl)amino)-5-methyl-2-(morpholinomethyl)benzo[d]oxazole-4-carboxamide

[0209]

[0210] A similar synthesis method to that of Example 1 was used, except that tetrahydropyrone was replaced with 1-acetylpiperidin-4-one to obtain the title product 41 in a yield of 17%.

[0211] MS m / z (ESI): 579.3 [M+H] +.

[0212] Example 22 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(1-methylsulfonylpiperidin-4-yl)amino)-5-methyl-2-(morpholinomethyl)benzo[d]oxazole-4-carboxamide

[0213]

[0214] A similar synthesis method to that of Example 1 was used to prepare the title product 42 in a yield of 35%, except that tetrahydropyrone was replaced by 1-methylsulfonylpiperidin-4-one.

[0215] MS m / z (ESI): 615 [M+H] +.

[0216] Example 23 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(1-methylpyrrolidin-3-yl)amino)-5-methyl-2-(dimethylamino)methyl)benzo[d]oxazole-4-carboxamide

[0217]

[0218] A similar synthesis method to that of Example 1 was used, except that morpholine was replaced by dimethylamine and tetrahydropyrone was replaced by 1-methyl-3-pyrrolidone, to obtain the title product 43 in a yield of 29%.

[0219] MS m / z (ESI): 495 [M+H] +.

[0220] Example 24 6-((2,3-dihydro-1H-inden-2-yl)(ethyl)amino)-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-(dimethylamino)methyl)-5-methyl-benzo[d]oxazole-4-carboxamide

[0221]

[0222] A similar synthesis method to that of Example 1 was used, except that morpholine was replaced by dimethylamine and tetrahydropyrone was replaced by 2-indanone, to obtain the title product 44 in an 18% yield.

[0223] MS m / z (ESI): 528 [M+H] +.

[0224] Example 25 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-5-methyl-2-(2-morpholinoethyl)benzo[d]oxazole-4-carboxamide

[0225]

[0226] A similar synthesis method to that of Example 1 was used, except that chloroacetyl chloride was replaced with 3-chloropropionyl chloride to obtain the title product 45 in a yield of 38%.

[0227] MS m / z (ESI): 552.3 [M+H] +.

[0228] Example 26 N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-5-methyl-2-(morpholinomethyl)benzo[d]oxazole-4-carboxamide

[0229]

[0230] A synthetic method similar to that of Example 1 was used to obtain the title product 46 in 10% yield by replacing 3-(aminomethyl)-4,6-dimethyl-1H-pyridin-2-one hydrochloride with 3-(aminomethyl)-4-methoxy-6-methyl-1H-pyridin-2-one hydrochloride.

[0231] MS m / z (ESI): 554.3 [M+H] +.

[0232] Example 27 2-(Dimethylaminomethyl)-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-methylbenzo[d]oxazole-4-carboxamide

[0233]

[0234] A synthetic method similar to that of Example 1 was used, except that morpholine was replaced by dimethylamine, and 3-(aminomethyl)-4,6-dimethyl-1H-pyridin-2-one hydrochloride was replaced by 3-(aminomethyl)-4-methoxy-6-methyl-1H-pyridin-2-one hydrochloride to obtain the title product 47 in a yield of 29%.

[0235] MS m / z (ESI): 512 [M+H] +.

[0236] Example 28 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-(((2-methoxyethyl)(methyl)amino)methyl)-5-methylbenzo[d]oxazole-4-carboxamide

[0237]

[0238] A similar synthesis method to that of Example 1 was used, except that morpholine was replaced by 2-methoxy-N-methylethylamine, to obtain the title product 48 in 18% yield.

[0239] MS m / z (ESI): 540.3 [M+H] +.

[0240] Example 29 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-5-methyl-2-(1-methylpiperidin-4-yl)benzo[d]oxazole-4-carboxamide

[0241]

[0242] A similar synthesis method to Example 2 was used to prepare the title product 79 in 20% yield by replacing tetrahydro-2H-pyran-4-carbonyl chloride with 1-methylpiperidine-4-carbonyl chloride.

[0243] MS m / z (ESI): 536.2 [M+H] +.

[0244] Example 30 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-5-methyl-2-(4-methoxycyclohexyl)benzo[d]oxazole-4-carboxamide

[0245]

[0246] A similar synthesis method to that of Example 2 was used, except that tetrahydro-2H-pyran-4-carbonyl chloride was replaced with 4-methoxycyclohexanoyl chloride to obtain the title product 80 in an 11% yield.

[0247] MS m / z (ESI): 551 [M+H] +.

[0248] Biological experiments

[0249] Experiment 1: Determination of the activity of the compounds of the present invention on wild-type Polycomb Repressive Complex 2 (PRC2) (Drosophila enhancer of zeste homolog 2 (EZH2) wild-type)

[0250] 1. Detection method: Isotope method (Radiometric assay)

[0251] 2. Experimental steps

[0252] The test compound and 10 μl of wild-type EZH2 were added to each well and incubated at room temperature for 15 minutes. 3 H]-labeled methyl donor S-adenosylmethionine (SAM) was reacted at room temperature for 1 hour. The reaction was terminated by the addition of cold SAM. 25 μl of the reaction solution was transferred to a scintillation plate and incubated at room temperature for 1 hour. The plate was washed three times with deionized water and 0.1% Tween 20. The results were read using a PerkinElmer liquid scintillation counter, and the half-maximal inhibitory concentration (IC50) of the compound was calculated using GraphPad Prism 5 software.

[0253] 3. Experimental Results

[0254] The results showed that the IC50 of the positive drug EPZ-6438 in inhibiting wild-type EZH2 was 5.1 nanomoles per liter (nM). The example compounds all had strong inhibitory effects on wild-type EZH2. The results are shown in Table 1.

[0255] Experiment 2: Determination of the activity of the compounds of the present invention on mutant PRC2 complex (EZH2 mutant)

[0256] 1. Detection Method Homogeneous Time-Resolved Fluorescence (HTRF)

[0257] 2. Experimental steps

[0258] PRC2 complex (EZH2 Y641F / A677G / Y641N), H3(1-50)me1 substrate, methyl donor SAM, and compound were added to each well. The total reaction volume was 10 μl and incubated at room temperature in the dark for 4 hours. 5 μl of EU-labeled H3K27me3 antibody and 5 μl of anti-H3K27me3 were added to each well.

[0259] 3. Experimental Results

[0260] The results showed that the IC50 of the positive drug EPZ-6438 against mutant Y641F EZH2 was 4.6 nM, the IC50 against A677GEZH2 was 2.91 nM, and the IC50 against Y641N EZH2 was 2.24 nM. The example compounds all had strong inhibitory effects on mutant EZH2. The results are shown in Table 1.

[0261] Table 1. Inhibitory activity of compounds against PRC2 complex

[0262]

[0263]

[0264] Experiment 3: Analysis of the Effect of the Compounds of the Invention on the Proliferation of Diffuse Large B-Cell Lymphoma Cells (WSU-DLCL2 Cells)

[0265] 1. Experimental steps

[0266] When diffuse large B-cell lymphoma cells (WSU-DLCL2 cell line) are growing well, harvest and count them. Adjust the cell concentration to 100,000 cells / ml. Seed the cells at this concentration into a 96-well plate, 100 μl / well (10,000 cells per well). Prepare a compound plate with a 500x final concentration by serially diluting the compound in dimethyl sulfoxide (DMSO). Pipette 1.2 μl of the 500x concentration of compound into 200 μl of culture medium and mix thoroughly by pipetting to create a 3x compound intermediate plate. Pipette 50 μl of the 3x concentration of compound and add it to the cell plate according to the designated arrangement. Incubate the cell plate in a CO2 incubator for 4 days. Remove the cell plate, mix thoroughly, and aspirate a volume of the cell suspension. Stain with Calcein AM and count the cells in each well using Acumen. Based on the number of cells tested, 10,000 cells were re-seeded in a 96-well plate. Compounds were added as above. The cells were cultured in a CO2 incubator for 3 days (Day 7). The cell plate was removed again, mixed, a certain volume of cell suspension was aspirated, and the cells were stained with Calcein AM. The number of cells in each well was counted using Acumen. The cells were cultured for another 4 days (Day 11). The cell plate was removed for the third time, mixed, a certain volume of cell suspension was aspirated, and the cells were stained with Calcein AM. The number of cells in each well was counted using Acumen. Based on the number of cells tested, 10,000 cells were re-seeded in a 96-well plate. Compounds were added as above. The cells were cultured in a CO2 incubator for 3 days (Day 7). The cells were cultured for another 3 days (Day 14), and step 5 was repeated to obtain the final count result. The data from day 14 was processed to obtain the corresponding half-maximal inhibitory concentration IC50.

[0267] 2. Experimental Results and Conclusions

[0268] The results showed that the IC50 of EPZ-6438 on WSU-DLCL2 proliferation inhibition was 67.23 nM. The compounds in the examples all had a strong inhibitory effect on WSU-DLCL2 proliferation, and their inhibitory activity was superior to that of EPZ6438. The results are shown in Table 2.

[0269] Experiment 4: Analysis of the Effect of the Compounds of the Invention on the Proliferation of Human Diffuse Large Cell Lymphoma B Lymphocytes (Pfeiffer Cells)

[0270] 1. Experimental steps

[0271] When human diffuse large cell lymphoma B lymphocytes (Pfeiffer) are growing well, harvest and count the cells. Adjust the cell concentration to 100,000 cells / mL. Inoculate the cells at the above concentration into a 96-well plate at 100 μl / well (10,000 cells per well). Serially dilute the compound in DMSO to prepare a 500x final concentration compound plate. Pipette 1.2 μl of the 500x compound into 200 μl of culture medium and mix thoroughly by pipetting to create a 3x compound intermediate plate. Pipette 50 μl of the 3x compound into the cell plate according to the designated arrangement. Incubate the cell plate in a CO2 incubator for 4 days. Remove the cell plate, mix thoroughly, aspirate a volume of the cell suspension, stain with Calcein AM, and count the number of cells in each well using Acumen. Based on the number of cells detected, re-seed 10,000 cells into the 96-well plate. Add the compound as above. Place in a carbon dioxide incubator and culture for 3 days (day 7). Take out the cell plate again, mix well, aspirate a certain volume of cell suspension, stain with Calcein AM, and count the number of cells in each well using Acumen. Continue to culture the cells for 4 days (day 11), take out the cell plate for the third time, mix well, aspirate a certain volume of cell suspension, stain with Calcein AM, and count the number of cells in each well using Acumen. 6. Based on the number of cells detected, re-inoculate 10,000 cells in a 96-well plate. Add the compound as above. Place in a carbon dioxide incubator and culture for 3 days (day 7). Continue to culture the cells for 3 days (day 14), repeat step 5, and obtain the final count result. Process the data from day 14 to obtain the corresponding half-maximal inhibitory concentration IC50.

[0272] 2. Experimental Results and Conclusions

[0273] The results showed that the IC50 of EPZ-6438 in inhibiting Pfeiffer cell proliferation was 27.14 nM. The example compounds all had strong inhibitory effects on Pfeiffer cell proliferation, and their inhibitory activities were superior to those of EPZ6438. The results are shown in Table 2.

[0274] Table 2. Inhibitory effects of compounds on the proliferation of EZH2-sensitive cells

[0275]

[0276]

[0277] Experiment 5: Pharmacokinetics of the compounds of the present invention in rats

[0278] 1. Experimental Purpose

[0279] SD rats were used to determine the drug concentrations in plasma at different times after oral administration of the compounds of Example 2, Example 6, and Example 25 by liquid chromatography-mass spectrometry (LS / MS / MS) and to calculate the relevant pharmacokinetic parameters to evaluate the pharmacokinetic properties of the compounds of the present invention in rats.

[0280] 2. Medication

[0281] The specific administration was carried out according to the experimental plan, and the details are shown in Table 3.

[0282] Table 3. Pharmacokinetic study protocol in rats

[0283]

[0284] 3. Blood Collection Time and Sample Processing

[0285] Blood was collected through the eye socket before administration and 10 minutes, 20 minutes, 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, and 24 hours after administration. 0.2 ml of blood was collected each time and placed in a potassium ethylenediaminetetraacetic acid (EDTA-2K) test tube. The blood was centrifuged at 12,000 revolutions per minute (rpm) for 5 minutes to collect plasma, which was then stored in a -20°C refrigerator for later use.

[0286] 4. Sample testing and data analysis

[0287] Liquid chromatography-mass spectrometry (LC / MS / MS) was used to determine the content of the compound in mouse plasma. The pharmacokinetic parameters of the compound after administration were calculated using the non-compartmental model of WinNonLin5.3 software.

[0288] 5. Test Results

[0289] The compounds of Example 2, Example 6, and Example 25 all have good metabolic characteristics and good bioavailability. Experiment 6: Acute toxicity test of the compounds of the present invention

[0290] 1. Experimental Purpose and Methods

[0291] The purpose of this experiment is to test the toxic effects of compounds in mice.

[0292] Mice were given a single dose of compound 11 at different doses and observed for 14 days. Animal mortality, toxic reactions, weight changes, diet, appearance, behavior, etc. were recorded. At the end point, the animals were dissected and their organs were removed for histopathological examination.

[0293] 2. Experimental Results and Conclusions

[0294] The compound of the present invention has a median lethal dose (LD50) of >1000 mg / kg, demonstrating good safety. Compared to the control group, the mice in the treated group showed no weight or behavioral abnormalities within 14 days of administration, indicating that the compound of the present invention exhibited no significant toxicity.

[0295] Experiment 7: Pharmacological efficacy of the compounds of Examples 5, 15, and 26 of the present invention in a mouse transplant tumor model of human diffuse large B-cell lymphoma cells (WSU-DLCL2 cells)

[0296] 1. Experimental steps

[0297] Under sterile conditions, logarithmically growing diffuse large B-cell lymphoma cells (WSU-DLCL2 cell line) were digested and mixed with Matrigel before being implanted subcutaneously on the right side of the back of CB17 / SCID mice. Each mouse was inoculated with 1 x 107 cells in a volume of 100 μl. After inoculation, the mice were randomly divided into five groups of six mice each, balanced by tumor size, for in vivo efficacy testing. The positive control group received EPZ-6438, and the negative control group received an equal amount of vehicle. The detailed design is shown in Table 4.

[0298] Table 4. In vivo efficacy experiments of compounds

[0299]

[0300]

[0301] 2. Experimental Results and Conclusions

[0302] EPZ-6438 only inhibited tumor growth by 52% at a dose of 150 mg / kg. The compounds of Examples 5, 15, and 26, at a dose of 150 mg / kg, inhibited tumor growth by 56%, 63%, and 65%, respectively. This indicates that the compounds of Examples 5, 15, and 26 of the present invention have a stronger tumor growth inhibitory effect than EPZ-6438 in a diffuse large B-cell lymphoma cell (WSU-DLCL2 cell line) xenograft model. Experiment 8: Pharmacological efficacy of the compound of Example 20 of the present invention in a mouse xenograft tumor model of human diffuse large B-cell lymphoma cells (WSU-DLCL2 cells).

[0303] 1. Experimental steps

[0304] Under sterile conditions, logarithmically growing human diffuse large B-cell lymphoma cells (WSU-DLCL2) were digested and mixed with Matrigel before being implanted subcutaneously on the right side of the back of CB17 / SCID mice. Each mouse was inoculated with 1 x 107 cells in a volume of 100 μl. After inoculation, the mice were randomly divided into five groups of six mice each, balanced by tumor size, for in vivo efficacy testing. The positive control group received EPZ-6438, and the negative control group received an equal amount of vehicle. The detailed design is shown in Table 5.

[0305] Table 5. In vivo efficacy experiments of compounds

[0306]

[0307] 2. Experimental Results and Conclusions

[0308] The tumor growth inhibition rates of EPZ-6438 at dosage concentrations of 125 mg / kg and 250 mg / kg were 47% and 72%, respectively. The tumor growth inhibition rates of the compound of Example 20 at dosage concentrations of 125 mg / kg, 250 mg / kg, and 500 mg / kg were 56%, 85%, and 91%, respectively. This indicates that the compound of Example 20 of the present invention has a stronger tumor growth inhibitory effect than EPZ-6438 in the human diffuse large B-cell lymphoma cell (WSU-DLCL2 cell line) xenograft model.

[0309] Experiment 9: Pharmacological efficacy of the compound of Example 23 of the present invention in a human diffuse large cell lymphoma B lymphocyte (Pfeiffer cell) mouse transplant tumor model

[0310] 1. Experimental steps

[0311] Under sterile conditions, logarithmically growing human diffuse large cell lymphoma B lymphocytes (Pfeiffer cells) were digested and mixed with Matrigel before being subcutaneously transplanted into the right side of the back of beige SCID mice. Each mouse was inoculated with 1 x 107 cells in a volume of 100 μl. After inoculation, the mice were randomly divided into five groups of six mice each, balanced by tumor size, for in vivo efficacy testing. The positive control group received EPZ-6438, and the negative control group received an equal amount of vehicle. The detailed design is shown in Table 6.

[0312] Table 6. In vivo efficacy experiments of compounds

[0313]

[0314] 2. Experimental Results and Conclusions

[0315] The tumor growth inhibition rates of EPZ-6438 at dosage concentrations of 50 mg / kg and 100 mg / kg were 53% and 81%, respectively. The tumor growth inhibition rates of the compound of Example 23 at dosage concentrations of 50 mg / kg and 100 mg / kg were 78% and 92%, respectively. This indicates that the compound of Example 23 of the present invention has a stronger tumor growth inhibition effect than EPZ-6438 in the human diffuse large cell lymphoma B lymphocyte (Pfeiffer cell) xenograft model.

[0316] Experiment 10: Pharmacological efficacy of the compound of Example 18 of the present invention in a mouse transplant tumor model of human renal cancer cell line (G401 cells)

[0317] 1. Experimental steps

[0318] Under sterile conditions, human renal carcinoma cells (G401) in logarithmic growth phase were digested and mixed with Matrigel before being implanted subcutaneously on the right side of the back of severe combined immunodeficient (SCID) mice. Each mouse was inoculated with 1 x 107 cells in a volume of 100 μl. After inoculation, the mice were randomly divided into five groups of six mice each, balanced by tumor size, for in vivo efficacy testing. The positive control group received EPZ-6438, and the negative control group received an equal amount of vehicle. The detailed design is shown in Table 7.

[0319] Table 7. In vivo efficacy experiments of compounds

[0320]

[0321]

[0322] 2. Experimental Results and Conclusions

[0323] The tumor growth inhibition rates of EPZ-6438 at dosage concentrations of 125 mg / kg and 250 mg / kg were 41% and 66%, respectively. The tumor growth inhibition rates of the compound of Example 18 at dosage concentrations of 125 mg / kg and 250 mg / kg were 50% and 71%, respectively. This indicates that the compound of Example 18 of the present invention has a stronger tumor growth inhibition effect than EPZ-6438 in the human renal cancer cell line (G401 cell) xenograft model.

[0324] Experiment 11: Pharmacological efficacy of the compound of Example 18 of the present invention in a mouse transplant tumor model of human gastric cancer cells (NCI-N87 cells)

[0325] 1. Experimental steps

[0326] Under sterile conditions, logarithmically growing human gastric cancer cells (NCI-N87 cells) were digested and mixed with Matrigel before being implanted subcutaneously on the right side of the back of severe combined immunodeficient (SCID) mice. Each mouse was inoculated with 1 x 107 cells in a volume of 100 μl. After inoculation, the mice were randomly divided into five groups of six mice each, balanced by tumor size, for in vivo efficacy testing. The positive control group received EPZ-6438, and the negative control group received an equal amount of vehicle. The detailed design is shown in Table 8.

[0327] Table 8. In vivo efficacy experiments of compounds

[0328]

[0329] 2. Experimental Results and Conclusions

[0330] The tumor growth inhibition rates of EPZ-6438 at dosage concentrations of 125 mg / kg and 250 mg / kg were 42% and 63%, respectively. The tumor growth inhibition rates of the compound of Example 18 at dosage concentrations of 125 mg / kg and 250 mg / kg were 51% and 68%, respectively. This indicates that the compound of Example 18 of the present invention has a stronger tumor growth inhibition effect than EPZ-6438 in the human gastric cancer cell (NCI-N87 cell) xenograft model.

Claims

1. A compound represented by general formula (I) or a pharmaceutically acceptable salt thereof, in, R 0 -C 1-6 Alkyl, -C 1-3 Alkylene-NR a R b or -T 0 ; R a and R b Each independently is -C 1-3 Alkyl, -C 2-4 Alkylene -OCH3, or R a and R b Together with the nitrogen atom to which they are attached, they form an unsubstituted or substituted 4-6 membered heterocycloalkyl group, wherein the substituted group means that the methylene group on the 4-6 membered heterocycloalkyl group is substituted by one or two T groups, and the 4-6 membered heterocycloalkyl group is a heterocycloalkyl group having one nitrogen as a heteroatom, a heterocycloalkyl group having two nitrogen as heteroatoms, or a heterocycloalkyl group having one nitrogen and one oxygen as a heteroatom. When the 4-6 membered heterocycloalkyl group is a heterocycloalkyl group having two nitrogen as heteroatoms, the secondary nitrogen on the ring is unsubstituted or substituted by T'; T is halogen or -C 1-4 alkyl; T' is -C 1-4 alkyl; T 0 Has not been replaced or replaced by T 1 Replaced by -C 3-8 Cycloalkyl, 4-6 membered heterocycloalkyl, phenyl or 5-6 membered heteroaryl, when T 0 When the heteroatom is a 4-6 membered heterocycloalkyl or a 5-6 membered heteroaryl, if the heteroatom is a nitrogen atom, the nitrogen atom is not substituted or is replaced by T 2 replace; T 1 -C 1-6 Alkyl, -C 1-3 alkoxy; T 2 -C 1-4 alkyl; R 3 -C 1-4 alkyl; R 4 and R 5 Each independently is -C 1-6 alkyl; R 5a -C 1-6 Alkyl or -C 1-6 alkoxy; R 6 is a five- to six-membered cycloalkyl, a five- to six-membered heterocycloalkyl, or a bicyclic ring having 8 to 10 carbon atoms, wherein the heteroatom in the five- to six-membered heterocycloalkyl is a nitrogen, sulfur, or oxygen atom, the bicyclic rings are fused together, any one ring in the bicyclic ring is saturated, unsaturated, or aromatic, the cycloalkyl, heterocycloalkyl, or bicyclic ring having 8 to 10 carbon atoms is unsubstituted or replaced by one or more R 6a Group substitution, R 6a -C 1-3 Alkyl, -NR h R k 、-C(O)-C 1-3 Alkyl, -S(O)2-C 1-3 alkyl; When R 6 When it is a nitrogen heterocyclic group containing one nitrogen atom, the nitrogen atom is not substituted or replaced by R 6b Replacement, R 6b -C 1-4 Alkyl, -C(O)-C 1-3 Alkyl, -S(O)2-C 1-3 alkyl; R h and R k Each independently is -C 1-3 alkyl.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that R 0 -C 1-3 Alkyl, -C 1-3 Alkylene-NR a R b or -T 0 ; R a and R b are independently methyl, ethyl, propyl or -C 2-4 Alkylene -OCH3, or R a and R b Together with the nitrogen atom to which they are attached, they form an unsubstituted or substituted 4-6 membered heterocycloalkyl group, wherein the substituted group refers to a methylene group on the 4-6 membered heterocycloalkyl group being replaced by one or two fluorine groups, and the 4-6 membered heterocycloalkyl group is a heterocycloalkyl group having one nitrogen atom, a heterocycloalkyl group having two nitrogen atoms, or a heterocycloalkyl group having one nitrogen atom and one oxygen atom. When the 4-6 membered heterocycloalkyl group is a heterocycloalkyl group having two nitrogen atoms, the secondary nitrogen on the ring is unsubstituted or replaced by T', and T' is -C 1-4 alkyl; T 0 Has not been replaced or replaced by T 1 Replaced by -C 3-6 Cycloalkyl, 4-6 membered heterocycloalkyl, phenyl or 5-6 membered heteroaryl, wherein the heteroatoms of the 4-6 membered heterocycloalkyl are 1-2 nitrogen or oxygen atoms, and the heteroatoms of the 5-6 membered heteroaryl are 1-2 nitrogen atoms; T 1 -C 1-3 Alkyl, -C 1-3 alkoxy; R 3 、R 4 and R 5 Separately, C 1-3 alkyl; R 5a -C 1-2 Alkyl or -C 1-2 alkoxy; R 6b -C 1-3 Alkyl, -C(O)-C 1-3 Alkyl, -S(O)2-C 1-3 alkyl; R h and R k Each independently is -C 1-3 alkyl.

3. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that T 0 for T 1 -C 1-3 Alkoxy, T 2 -C 1-4 alkyl; -NR a R b for R a is methyl, ethyl or propyl; R 6 for 4. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that The compound is:

5. The method for preparing the compound represented by general formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, characterized in that: When R 0 -C 1-3 Alkylene-NR a R b , when m is 1 or 2, the preparation method comprises the following steps: Step (1): reacting the compound of formula (II-1) with di-tert-butyl dicarbonate under alkaline conditions to obtain a compound of formula (II-2); Step (2): The compound of formula (II-2) is reduced to a compound of formula (II-3) under palladium-carbon reducing agent conditions; Step (3): The compound of formula (II-3) undergoes bromination reaction under alkaline conditions to obtain a compound of formula (II-4); Step (4): reacting the compound of formula (II-4) with chloroacetyl chloride or chloropropionyl chloride under alkaline conditions to obtain a compound of formula (II-5); Step (5): The compound of formula (II-5) is reacted with the corresponding amine R under alkaline conditions. a R b NH undergoes a substitution reaction to obtain a compound of formula (II-6); Step (6): The compound of formula (II-6) undergoes a ring-closure reaction under heating, alkalinity, and the presence of a catalyst to obtain a compound of formula (II-7); Step (7): The compound of formula (II-7) is reacted with the corresponding amine R under heating, alkalinity and the presence of a catalyst. 6 -NH2 undergoes a substitution reaction to obtain a compound of formula (II-8); Step (8): The compound of formula (II-8) undergoes a reductive amination reaction with the corresponding ketone or aldehyde in the presence of a reducing agent to obtain a compound of formula (II-9); Step (9): removing the tert-butyl group from the compound of formula (II-9) under acidic conditions to obtain a compound of formula (II-10); Step (10): Acylation reaction is carried out between the compound of formula (II-10) and the compound of formula (II-11) to obtain a compound of formula (I); When R 0 -C 1-6 Alkyl or -T 0 , the preparation method comprises the following steps: Step (a): The compound of formula (II-4) is reacted with an acyl chloride R under alkaline conditions. 0 COCl reacts to obtain a compound of formula (IV-1); Step (b): The compound of formula (IV-1) undergoes a ring-closure reaction under heating, alkalinity, and the presence of a catalyst to obtain a compound of formula (IV-2); Step (c): The compound of formula (IV-2) is reacted with the corresponding amine R under heating, alkalinity and the presence of a catalyst. 6 -NH2 undergoes a substitution reaction to obtain a compound of formula (IV-3); Step (d): The compound of formula (IV-3) undergoes a reductive amination reaction with the corresponding aldehyde or ketone in the presence of a reducing agent to obtain a compound of formula (IV-4); Step (e): removing the tert-butyl group from the compound of formula (IV-4) under acidic conditions to obtain a compound of formula (IV-5); Step (f): The compound of general formula (IV-5) undergoes acylation reaction with the compound of general formula (II-11) to obtain the compound of general formula (IV).

6. The preparation method according to claim 5, characterized in that In step (3), the reagent providing the alkaline condition is calcium carbonate, and the brominated brominating agent is benzyltrimethylammonium tribromide.

7. The preparation method according to claim 5, characterized in that In step (4), the reagent providing the alkaline condition is triethylamine.

8. The preparation method according to claim 5, characterized in that In step (5), the reagent providing the alkaline condition is cesium carbonate.

9. The preparation method according to claim 5, characterized in that In step (6), the reagent providing the alkaline condition is cesium carbonate, and the catalyst is cuprous iodide and 1,10-phenanthroline.

10. The preparation method according to claim 5, characterized in that In step (7), the reagent providing the alkaline condition is cesium carbonate, and the catalyst is tris(dibenzylideneacetone)dipalladium and 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene.

11. The preparation method according to claim 5, characterized in that In step (8), the reducing agent is sodium acetate borohydride.

12. The preparation method according to claim 5, characterized in that In step (9), the reagent providing the acidic condition is trifluoroacetic acid.

13. The preparation method according to claim 5, characterized in that In step (a), the reagent providing the alkaline condition is triethylamine.

14. The preparation method according to claim 5, characterized in that In step (b), the reagent providing the alkaline condition is cesium carbonate, and the catalyst is cuprous iodide and 1,10-phenanthroline.

15. The preparation method according to claim 5, characterized in that In step (c), the reagent providing the alkaline condition is cesium carbonate, and the catalyst is tris(dibenzylideneacetone)dipalladium and 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene.

16. The preparation method according to claim 5, characterized in that In step (d), the reducing agent is sodium acetate borohydride.

17. The preparation method according to claim 5, characterized in that In step (e), the reagent providing the acidic condition is trifluoroacetic acid.

18. A pharmaceutical composition, characterized in that The invention comprises a therapeutically effective amount of the compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, and optionally one or more pharmaceutically acceptable carriers and / or diluents.

19. Use of the compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 18, in the preparation of a medicament for preventing and / or treating EZH2-mediated related diseases.

20. The use according to claim 19, characterized in that The EZH2-mediated related disease is tumor.

21. The use according to claim 20, characterized in that The tumor is renal cancer, gastric cancer or lymphoma.

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