Disclosure of Invention
The invention aims to: the first object of the invention is to provide a protein kinase inhibitor and a derivative thereof, the second object is to provide a preparation method of the protein kinase inhibitor and the derivative thereof, the third object is to provide a pharmaceutical composition containing the protein kinase inhibitor and the derivative thereof, and the fourth object is to provide the application of the protein kinase inhibitor and the derivative thereof in preparing medicines for treating and/or preventing hyperproliferative diseases such as cancers, virus-induced infectious diseases and/or cardiovascular diseases.
The technical scheme is as follows: the protein kinase inhibitor and the derivative thereof have the structure shown in the formula (I), wherein the derivative is an isomer, a diastereoisomer, an enantiomer, a tautomer, a solvate, a salt of the solvate, a pharmaceutically acceptable salt or a mixture of the isomers and the diastereoisomer, the enantiomer, the tautomer, the solvate and the salt of the solvate of the compound:
wherein:
v, W, X, Y or Z is CR4 Or a nitrogen atom;
R1 is thatOr->
R2 Is hydrogen atom, hydroxy, C1 -C6 Alkyl, C1 -C6 Alkylamino, C1 -C6 Alkoxy or Het1 Substituents, wherein C1 -C6 Alkyl, C1 -C6 Alkylamino, C1 -C6 Alkoxy, het1 Substituted by one or more halogen, hydroxy, amino, hetero atoms, C1 -C6 Alkyl, C1 -C6 Haloalkyl, C1 -C6 Alkylamino, C1 -C6 Alkoxy or Het1 Substitution; het1 Substituents are morpholinyl, morpholinylalkyl, morpholinylalkoxy, morpholinylalkylamino, piperazinyl, piperazinylalkyl, piperazinylalkylamino, homopiperazinyl, homopiperazinylalkyl, homopiperazinylalkoxy, homopiperazinylalkylamino, piperidinyl, piperidinylalkyl, piperidinylalkoxy, piperidinylalkylamino, tetrakisA hydropyrrolyl, tetrahydropyrrolylalkyl, tetrahydropyrrolylalkoxy, tetrahydropyrrolylalkylamino, tetrahydrofuranyl, tetrahydrofuranalkyl, tetrahydrofuranalkoxy, tetrahydrofuranalkylamino, tetrahydropyranyl, tetrahydropyranoxy or tetrahydropyranyl group;
R3 is hydrogen atom, halogen, C1 -C6 Alkyl or cyano;
R4 is hydrogen atom, halogen, C1 -C6 Alkyl, C1 -C6 Alkoxy or cyano;
R5 hydrogen atom, halogen, nitro, C substituted in any position on the aromatic ring1 -C6 Alkyl, C1 -C6 Alkoxy, C1 -C6 Alkylamino or phenoxy, wherein C1 -C6 Alkyl, C1 -C6 Alkoxy or C1 -C6 Alkylamino is substituted with 1-3 halogens, C1 -C6 Alkyl, C3 -C7 Cycloalkyl, heterocyclyl, C1 -C6 Alkenyl, C1 -C6 Alkynyl, at least 1 halogen-substituted phenyl, at least 1 halogen-substituted heteroaryl or Het1 Substitution;
R6 or R is7 Is one or more hydrogen atoms, halogen, C1 -C3 Alkyl, C1 -C3 Alkoxy or C1 -C3 An alkylamino group;
q is an oxygen atom or an imino group.
Preferably, the protein kinase inhibitor and its derivative structure are as follows:
v is a nitrogen atom;
R2 is hydrogen atom, C1 -C6 Alkyl, C1 -C6 Alkylamino, C1 -C6 Alkoxy or Het1 Substituents, wherein C1 -C6 Alkyl, C1 -C6 Alkylamino, C1 -C6 Alkoxy, het1 Substituted by one or more fluorine atoms, hetero atoms, C1 -C6 Alkyl, C1 -C6 Haloalkyl, C1 -C6 Alkylamino, C1 -C6 Alkoxy or Het1 Substitution; het1 Substituents are morpholinyl, morpholinylalkyl, morpholinylalkoxy, morpholinylalkylamino, piperazinyl, piperazinylalkyl, piperazinylalkoxy, piperazinylalkylamino, homopiperazinyl, homopiperazinylalkyl, homopiperazinylalkoxy, homopiperazinylalkylamino, piperidinyl, piperidinylalkyl, piperidinylalkoxy, piperidinylalkylamino, tetrahydropyrrolyl, tetrahydropyrrolylalkyl, tetrahydropyrrolylalkoxy, tetrahydropyrrolylalkylamino, tetrahydrofuranyl, tetrahydrofuranalkyl, tetrahydrofuranalkoxy, tetrahydrofuranylamino, tetrahydropyranyl, tetrahydropyranoxy or tetrahydropyran alkylamino;
R3 is a hydrogen atom, a fluorine atom, a chlorine atom, a cyano group or C1 -C6 An alkyl group;
R4 is hydrogen atom, fluorine atom, C1 -C3 Alkyl or C1 -C3 An alkoxy group;
R5 hydrogen atom substituted at any position on aromatic ring, fluorine atom, nitro group, C1 -C6 Alkyl, C1 -C6 Alkoxy, C1 -C6 Alkylamino, phenoxy, wherein C1 -C6 Alkyl, C1 -C6 Alkoxy or C1 -C6 Alkylamino is substituted with 1-3 fluorine atoms, C1 -C6 Alkyl, C3 -C7 Cycloalkyl, C1 -C6 Alkenyl, C1 -C6 Alkynyl, phenyl substituted with 1-2 fluorine atoms, heteroaryl substituted with 1-2 fluorine atoms, piperazinyl or morpholinyl;
R6 or R is7 Is one or more hydrogen atoms, fluorine atoms, C1 -C3 Alkyl, C1 -C3 Alkoxy or C1 -C3 An alkylamino group.
Preferably, the protein kinase inhibitor and derivatives thereof have the structure of formula (II):
wherein:
R1 is thatOr->
R2 Is hydrogen atom, methyl, morpholin-4-yl, piperazinyl, 4-methylpiperazinyl, 4-ethylpiperazinyl, 4- (2-methoxyethyl) piperazinyl, homopiperazinyl, N-methylpiperazinyl, thiomorpholin-4-yl, piperidinyl, 4- (N, N-dimethylpiperazinyl, (N-methylpiperazin-4-yl) piperidinyl, 3-methylaminopyrrolidinyl, (morpholin-4-yl) methyl, (piperazin-1-yl) methyl, (4-methylpiperazin-1-yl) methyl, (N-methylpiperazin-1-yl) methyl, (tetrahydropyran-4-yl) amino, (tetrahydrofuran-3-yl) amino, (N-methylpiperidin-4-yl) amino, 2- (dimethylamino) ethylamino, 2-methoxyethylamino, 3-methoxypropylamino, 2, 5-diazabicyclo [2.2.1]Heptyl-2-yl, 4-methylpiperazin-3-one, (S) -3-methylpiperazin, (R) -3-methylpiperazin, (8-azabicyclo [ 3.2.1)]Octan-3-yl) amino, (R) -2-methylpiperazinyl, (S) -2-methylpiperazinyl, pyrrolidinyl, (2-methoxyethyl) amino, (3-methoxypropyl) amino, (tetrahydropyran-4-yl) amino, (tetrahydrofuran-3-yl) amino, N-dimethylamino, azido or 4, 4-difluoropiperidinyl;
R3 is a hydrogen atom, a fluorine atom, a chlorine atom, a methyl group or a cyano group;
R4 is a hydrogen atom, a fluorine atom, a methyl group or a methoxy group;
R5 is hydrogen atom, fluorine atom, nitro group, methyl group, trifluoromethyl group, ethyl group, methoxy group, difluoromethoxy group, trifluoromethoxy group, methylamino group, dimethylamino group, ethoxy group, butoxy group, isopropoxy group, isobutoxy group, 1-cyclopropylmethoxy group, 1-cyclopentylmethoxy group, 1-cyclohexylmethoxy group, phenoxy group, benzyloxy group, 1-phenylethoxy group, 4-fluoro groupBenzyloxy, 2, 4-difluorobenzyloxy, benzylamino, 3- (4-methylpiperazin-1-yl) propoxy, 3-morpholin-1-ylpropoxy, but-2-en-1-yloxy, but-2-yn-1-yloxy, 1- (pyridin-4-yl) methoxy;
R6 、R7 is one or more hydrogen atoms, fluorine atoms, C1 -C3 Alkyl, methoxy or methylamino;
q is an oxygen atom or an imino group.
Further preferably, the protein kinase inhibitor and its derivative structure:
R1 is phenyl, 2-methoxyphenyl, 4-fluoro-2-methoxyphenyl, 2-ethoxyphenyl, 4-fluoro-2-ethoxyphenyl, 2-benzyloxyphenyl, 4-fluoro-2-benzyloxyphenyl, 3-isopropyl-2-methyl-2H-indazol-5-yl, 7-fluoro-3-isopropyl-2-methyl-2H-indazol-5-yl, 1-isopropyl-2-methyl-1H-benzo [ d ]]Imidazol-6-yl, 4-fluoro-1-isopropyl-2-methyl-1H-benzo [ d ]]Imidazol-6-yl, 1-methyl-1H-indol-3-yl, 1-methyl-1H-benzo [ d ]][1,2,3]Triazol-6-yl or 1-isopropyl-1H-benzo [ d ]][1,2,3]Triazol-6-yl;
R2 is hydrogen atom, morpholin-4-yl, piperazinyl, piperidinyl, 4-methylpiperazinyl, homopiperazinyl, N-methylpiperazinyl, 4-ethylpiperazinyl, (piperidin-4-yl) amino, (1-methylpiperidin-4-yl) amino, 2, 5-diazabicyclo [2.2.1]Heptyl-2-yl, (2-aminoethyl) amino, (2- (methylamino) ethyl) amino, (2- (dimethylamino) ethyl) (methyl) amino, (3-methoxyethyl) amino, (3-methoxypropyl) amino, 4-methylpiperazin-2-one, 4-methylpiperazin-3-one, (S) -3-methylpiperazin-yl, (R) -2-methylpiperazin-yl, (S) -2-methylpiperazin-yl, (8-azabicyclo [ 3.2.1)]Octane-3-yl) amino, pyrrolidinyl, (2-methoxyethyl) amino, (3-methoxypropyl) amino, (tetrahydropyran-4-yl) amino, (tetrahydrofuran-3-yl) amino, N-dimethylamino, azido or 4, 4-difluoropiperidinyl.
Preferably, the protein kinase inhibitor is any one of the following compounds:
4- (4-methylpiperazin-1-yl) -N- (4-phenylpyrimidin-2-yl) quinazolin-7-amine (I-1),
n- (4- (2-methoxyphenyl) pyrimidin-2-yl) -4- (4-methylpiperazin-1-yl) quinazolin-7-amine (I-2),
n- (4- (4-fluoro-2-methoxyphenyl) pyrimidin-2-yl) -4- (4-methylpiperazin-1-yl) quinazolin-7-amine (I-3),
n- (5-fluoro-4- (4-fluoro-2-methoxyphenyl) pyrimidin-2-yl) -4- (4-methylpiperazin-1-yl) quinazolin-7-amine (I-4),
n- (4- (2-ethoxyphenyl) pyrimidin-2-yl) -4- (4-methylpiperazin-1-yl) quinazolin-7-amine (I-5),
n- (4- (2-ethoxy-4-fluorophenyl) pyrimidin-2-yl) -4- (4-methylpiperazin-1-yl) quinazolin-7-amine (I-6),
n- (4- (2- (benzyloxy) phenyl) pyrimidin-2-yl) -4- (4-methylpiperazin-1-yl) quinazolin-7-amine (I-7),
n- (4- (2- (benzyloxy) -4-fluorophenyl) pyrimidin-2-yl) -4- (4-methylpiperazin-1-yl) quinazolin-7-amine (I-8),
n- (4- (3-isopropyl-2-methyl-2H-indazol-5-yl) pyrimidin-2-yl) quinazolin-7-amine (I-9),
n- (4- (3-isopropyl-2-methyl-2H-indazol-5-yl) pyrimidin-2-yl) -4-morpholinylquinazolin-7-amine (I-10),
n- (4- (3-isopropyl-2-methyl-2H-indazol-5-yl) pyrimidin-2-yl) -4- (piperazin-1-yl) quinazolin-7-amine (I-11),
n- (4- (3-isopropyl-2-methyl-2H-indazol-5-yl) pyrimidin-2-yl) -4- (4-methylpiperazin-1-yl) quinazolin-7-amine (I-12),
4- (4-ethylpiperazin-1-yl) -N- (4- (3-isopropyl-2-methyl-2H-indazol-5-yl) pyrimidin-2-yl) quinazolin-7-amine (I-13),
4- (7- ((4- (3-isopropyl-2-methyl-2H-indazol-5-yl) pyrimidin-2-yl) amino) quinazolin-4-yl) -1-methylpiperazin-2-one (I-14),
1- (7- ((4- (3-isopropyl-2-methyl-2H-indazol-5-yl) pyrimidin-2-yl) amino) quinazolin-4-yl) -4-methylpiperazin-2-one (I-15),
(S) -N- (4- (3-isopropyl-2-methyl-2H-indazol-5-yl) pyrimidin-2-yl) -4- (3-methylpiperazin-1-yl) quinazolin-7-amine (I-16),
(R) -N- (4- (3-isopropyl-2-methyl-2H-indazol-5-yl) pyrimidin-2-yl) -4- (3-methylpiperazin-1-yl) quinazolin-7-amine (I-17),
(R) -N- (4- (3-isopropyl-2-methyl-2H-indazol-5-yl) pyrimidin-2-yl) -4- (2-methylpiperazin-1-yl) quinazolin-7-amine (I-18),
(S) -N- (4- (3-isopropyl-2-methyl-2H-indazol-5-yl) pyrimidin-2-yl) -4- (2-methylpiperazin-1-yl) quinazolin-7-amine (I-19),
4- (1, 4-homopiperazin-1-yl) -N- (4- (3-isopropyl-2-methyl-2H-indazol-5-yl) pyrimidin-2-yl) quinazolin-7-amine (I-20),
n- (4- (3-isopropyl-2-methyl-2H-indazol-5-yl) pyrimidin-2-yl) -4- (4-methyl-1, 4-homopiperazin-1-yl) quinazolin-7-amine (I-21),
4- (2, 5-diazabicyclo [2.2.1] hept-2-yl) -N- (4- (3-isopropyl-2-methyl-2H-indazol-5-yl) pyrimidin-2-yl) quinazolin-7-amine (I-22),
N4 - (8-azabicyclo [ 3.2.1)]Octane-3-yl) -N7 - (4- (3-isopropyl-2-methyl-2H-indazol-5-yl) pyrimidin-2-yl) quinazoline-4, 7-diamine (I-23),
N7 - (4- (3-isopropyl-2-methyl-2H-indazol-5-yl) pyrimidin-2-yl) -N4 - (piperidin-4-yl) quinazoline-4, 7-diamine (I-24),
N7 - (4- (3-isopropyl-2-methyl-2H-indazol-5-yl) pyrimidin-2-yl) -N4 - (1-methylpiperidin-4-yl) quinazoline-4, 7-diamine (I-25),
N4 - (2- (dimethylamino) ethyl) -N7 - (4- (3-isopropyl-2-methyl-2H-indazol-5-yl) pyrimidin-2-yl) quinazoline-4, 7-diamine (I-26),
N4 - (2-aminoethyl) -N7 - (4- (3-isopropyl-2-methyl-2H-indazol-5-yl) pyrimidin-2-yl) quinazoline-4, 7-diamine (I-27),
N4 - (2- (dimethylamino) ethyl) -N7 - (4- (3-isopropyl-2-methyl-2H-indazol-5-yl) pyrimidin-2-yl) -N4 Methyl quinazoline-4, 7-diamine (I-28),
N7 - (4- (3-isopropyl-2-methyl-2H-indazol-5-yl) pyrimidin-2-yl) -N4 - (2- (methylamino) ethyl) quinazolinesThe 4, 7-diamine (I-29),
N7 - (4- (3-isopropyl-2-methyl-2H-indazol-5-yl) pyrimidin-2-yl) -N4 - (2-methoxyethyl) quinazoline-4, 7-diamine (I-30),
N7 - (4- (3-isopropyl-2-methyl-2H-indazol-5-yl) pyrimidin-2-yl) -N4 - (3-methoxypropyl) quinazoline-4, 7-diamine (I-31),
5- (7- (((4- (3-isopropyl-2-methyl-2H-indazol-5-yl) pyrimidin-2-yl) amino) quinazolin-4-yl) -2, 5-diazabicyclo [2.2.1] heptane-2-carboxylic acid ester (I-32),
n- (4- (1-methyl-1H-indol-3-yl) pyrimidin-2-yl) -4- (4-methylpiperazin-1-yl) quinazolin-7-amine (I-33),
n- (4- (4-fluoro-1-isopropyl-2-methyl-1H-benzo [ d ] imidazol-6-yl) pyrimidin-2-yl) -4- (4-methylpiperazin-1-yl) quinazolin-7-amine (I-34),
n- (5-fluoro-4- (4-fluoro-1-isopropyl-2-methyl-1H-benzo [ d ] imidazol-6-yl) pyrimidin-2-yl) -4- (4-methylpiperazin-1-yl) quinazolin-7-amine (I-35),
N7 - (4- (3-isopropyl-2-methyl-2H-indazol-5-yl) pyrimidin-2-yl) -N4 - (tetrahydrofuran-3-yl) quinazolin-4, 7-diamine (I-36),
N7 - (4- (3-isopropyl-2-methyl-2H-indazol-5-yl) pyrimidin-2-yl) -N4 - (tetrahydro-2H-pyran-4-yl) quinazoline-4, 7-diamine (I-37),
N7 - (4- (3-isopropyl-2-methyl-2H-indazol-5-yl) pyrimidin-2-yl) -N4 ,N4 Dimethylquinazoline-4, 7-diamine (I-38),
n- (4- (3-isopropyl-2-methyl-2H-indazol-5-yl) pyrimidin-2-yl) -2-methyl-4- (4-methylpiperazin-1-yl) quinazolin-7-amine (I-39),
4- (azido-1-yl) -N- (4- (3-isopropyl-2-methyl-2H-indazol-5-yl) pyrimidin-2-yl) quinazolin-7-amine (I-40),
n- (4- (3-isopropyl-2-methyl-2H-indazol-5-yl) pyrimidin-2-yl) -4- (piperidin-1-yl) quinazolin-7-amine (I-41),
n- (4- (3-isopropyl-2-methyl-2H-indazol-5-yl) pyrimidin-2-yl) -4- (pyrrolidin-1-yl) quinazolin-7-amine (I-42),
1- (4- (7- ((4- (3-isopropyl-2-methyl-2H-indazol-5-yl) pyrimidin-2-yl) amino) quinazolin-4-yl) piperazin-1-yl) ethanone (I-43),
4- (4, 4-difluoropiperidin-1-yl) -N- (4- (3-isopropyl-2-methyl-2H-indazol-5-yl) pyrimidin-2-yl) quinazolin-7-amine (I-44),
n- (4- (1-isopropyl-2-methyl-1H-benzo [ d ] imidazol-6-yl) pyrimidin-2-yl) -4- (4-methylpiperazin-1-yl) quinazolin-7-amine (I-45),
n- (4- (1-isopropyl-1H-indol-3-yl) pyrimidin-2-yl) -4- (4-methylpiperazin-1-yl) quinazolin-7-amine (I-46),
N4 - (2-methoxyphenyl) -N2 - (4- (4-methylpiperazin-1-yl) quinazolin-7-yl) pyrimidine-2, 4-diamine (I-47),
n- (4- (2-methoxyphenoxy) pyrimidin-2-yl) -4- (4-methylpiperazin-1-yl) quinazolin-7-amine (I-48),
n- (4- (1-isopropyl-1H-benzo [ d ] [1,2,3] triazol-6-yl) pyrimidin-2-yl) -4- (4-methylpiperazin-1-yl) quinazolin-7-amine (I-49),
n- (4- (1-methyl-1H-benzo [ d ] [1,2,3] triazol-6-yl) pyrimidin-2-yl) -4- (4-methylpiperazin-1-yl) quinazolin-7-amine (I-50).
Preferably, the pharmaceutically acceptable salt is a salt of the protein kinase inhibitor with an acid or a base, the acid being hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid or mandelic acid, the base being an inorganic base containing a basic metal cation, alkaline earth metal cation or ammonium cation salt.
The preparation method of the protein kinase inhibitor and the derivative thereof is characterized by comprising the following steps:
the bromo-compound A0 is firstly converted into the pinacol borate A1 and then the coupling reaction is carried out to obtain the compound A, or the bromo-compound A0 is directly coupled to obtain the compound A; the compound B1 is alkylated to introduce R2 Obtaining a compound B; the compound A and the compound B are obtained through alkylation reactionTo obtain the compound (I);
therein, W, V, X, Y, Z, R1 、R2 、R3 Is as defined in any one of claims 1 to 4;
adding the corresponding acid or alkali solution into the solution of the compound (I) prepared by the method, and removing the solvent under reduced pressure after complete salification to obtain the pharmaceutically acceptable salt of the protein kinase inhibitor.
The pharmaceutical composition of the invention comprises the protein kinase inhibitor and/or a derivative thereof and a pharmaceutically acceptable carrier.
The protein kinase inhibitor and the derivatives thereof can be added with pharmaceutically acceptable carriers to prepare common medicinal preparations such as tablets, capsules, syrup, suspending agents and injection, and can be added with common medicinal auxiliary materials such as spice, sweetener, liquid or solid filler or diluent and the like.
The protein kinase inhibitor and the application of the derivative thereof in preparing medicines for treating and/or preventing hyperproliferative diseases, virus-induced infectious diseases and/or cardiovascular diseases.
Preferably, the hyperproliferative disease is lung cancer, prostate cancer, cervical cancer, colorectal cancer, melanoma, ovarian cancer, breast cancer, renal cancer, a tumor of the nervous system, lymphoma or leukemia.
The protein kinase inhibitor and the derivative thereof can be prepared into medicines for treating and/or preventing acute myelogenous leukemia, chronic myelogenous leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, multiple myeloma, diffuse large B-cell lymphoma, mantle cell lymphoma, burkitt's lymphoma (Burkitt's lymphoma), follicular lymphoma, breast cancer, non-small cell lung cancer, melanoma, renal cancer, ovarian cancer, prostate cancer, colon cancer or central nervous system tumor.
The beneficial effects are that: compared with the prior art, the invention has the following remarkable advantages:
(1) The protein kinase inhibitor, the derivative and the pharmaceutical composition thereof can effectively inhibit CDK9 kinase activity, and CDK9 kinase inhibits IC50 Values of no more than 1. Mu.M, most preferably no more than 0.1. Mu.M, can reach nanomolar concentration levels; can also inhibit proliferation of MV4-11 tumor cells, and inhibit proliferation of MV4-11 tumor cells50 Values of most not more than 1. Mu.M, most preferably not more than 0.1. Mu.M; and has inhibiting effect on various tumor cells, and various tumor cells inhibit IC50 Values of no more than 1 mu M are adopted, and the nanomolar concentration level can be optimally achieved; the application is wide, and the medicine can be prepared into medicines for treating and/or preventing hyperproliferative diseases, virus-induced infectious diseases and/or cardiovascular diseases;
(2) The medicine has wide application, and can be used for treating and/or preventing hyperproliferative diseases, virus-induced infectious diseases and/or cardiovascular diseases; the medicine can exert drug effect at enzyme level and cell level, and has excellent effect and IC50 The value is optimal and can reach the nanomolar concentration level;
(3) The preparation method of the compound is simple and convenient and is easy to operate.
Detailed Description
The technical scheme of the invention is further described below by referring to examples.
Reagent and material:
4, 7-dichloropquinazolin, N-methylpiperazine, 4-phenylpyrimidin-2-amine, 4, 5-bis-diphenylphosphine-9, 9-dimethylxanthene, (2-methoxyphenyl) boric acid, [1,1' -bis (diphenylphosphino) ferrocene ] palladium dichloride is derived from Shanghai Biget medical science and technology Co, shanghai Haohong biomedical science and technology Co;
CDK9/Cyclin T1 was derived from the American Reaction Biology Corp (Malvern PA) company and MV4-11 tumor cell line was derived from Nanjing An Nakang Biotech Co.
Instrument:
1 h NMR was measured using a BRUKER AVANCE-300 nuclear magnetic resonance apparatus (Brucker, switzerland) with TMS as the internal standard and displacement value (δ) in ppm; low resolution mass spectrometry was determined using an expression compact fourier transform mass spectrometer.
Example 1: synthesis of 4- (4-methylpiperazin-1-yl) -N- (4-phenylpyrimidin-2-yl) quinazolin-7-amine (Compound I-1)
(1) Synthesis of 7-chloro-4- (4-methylpiperazin-1-yl) quinazoline (Compound B-1)
In a 50mL single-necked flask, 4, 7-dichloroquinazoline (199mg, 1.00 mmol) was added, 10mL of dichloromethane was added for dissolution, a solution of N-methylpiperazine (110 mg,1.10 mmol) and triethylamine (111 mg,1.10 mmol) in dichloromethane was slowly added, the reaction was stirred at room temperature for about 12h after the dropwise addition was completed, after the TLC monitoring was completed, the reaction solution was concentrated, and the residue was separated and purified by silica gel column chromatography (dichloromethane: methanol=100:1) to obtain 234mg of pale yellow solid in 89% yield; ESI-MS m/z 263[ M+H ]]+ 。
(2) Synthesis of 4- (4-methylpiperazin-1-yl) -N- (4-phenylpyrimidin-2-yl) quinazolin-7-amine (Compound I-1)
To a 25mL two-necked flask was added 4-phenylpyrimidine-2-amine (34 mg,0.20 mmol), compound B-1 (53 mg,0.20 mmol), cesium carbonate (130 mg,0.40 mmol), palladium acetate (4.5 mg,0.02 mmol) and 4, 5-bis-diphenylphosphine-9, 9-dimethylxanthene (11.5 mg,0.02 mmol), anhydrous toluene (5-10 mL) were dissolved, degassed with nitrogen, reacted under nitrogen protection at 100℃for 2-6 h by heating, the reaction solution was filtered and concentrated, and the residue was separated and purified by silica gel column chromatography (dichloromethane: methanol=100:1) to give 28mg of pale yellow solid in 35% yield;1 H NMR(300MHz,DMSO-d6 )δ10.32(s,1H),8.51(d,J＝5.7Hz,1H),8.35(s,1H),8.23(dd,J＝8.5,0.4Hz,1H),7.95–8.06(m,2H),7.78(dd,J＝2.2,0.6Hz,1H),7.56–7.62(m,1H),7.55–7.38(m,2H),7.35(dd,J＝8.4,2.3Hz,1H),7.16(d,J＝5.6Hz,1H),3.66–3.73(m,4H),2.50–2.59(m,4H),2.26(s,3H).ESI-MS m/z:398[M+H]+ 。
example 2: synthesis of N- (4- (2-methoxyphenyl) pyrimidin-2-yl) -4- (4-methylpiperazin-1-yl) quinazolin-7-amine (compound I-2)
(1) Synthesis of 4- (2-methoxyphenyl) pyrimidin-2-amine (compound A-1)
To a 25mL eggplant-shaped bottle was added (2-methoxyphenyl) boronic acid (502 mg,3.3 mmol), 4-chloropyrimidin-2-amine (390 mg,3.0 mmol) and [1,1' -bis (diphenylphosphino) ferrocene]Palladium dichloride (110 mg,0.15 mmol), sodium carbonate (636 mg,6.0 mmol), 1, 4-dioxane 12mL and purified water 2mL, degassing with nitrogen, heating at 100 ℃ for 6h under nitrogen protection, extracting the reaction liquid with ethyl acetate, concentrating, purifying by column chromatography (ethyl acetate: petroleum ether=1:1), and recrystallizing to obtain light yellow powder 450mg with a yield of 75%; ESI-MS m/z 202[ M+H ]]+ 。
(2) Synthesis of N- (4- (2-methoxyphenyl) pyrimidin-2-yl) -4- (4-methylpiperazin-1-yl) quinazolin-7-amine (compound I-2)
Starting with compound A-1 (40 mg,0.20 mmol) and compound B-1 (53 mg,0.20 mmol), according to the method of example 1, 39mg of a white solid was obtained in 45% yield;1 H NMR(300MHz,DMSO-d6)δ10.25(s,1H),8.47(d,J＝5.6Hz,1H),8.35(s,1H),8.24(dd,J＝8.5,0.5Hz,1H),7.74(dd,J＝2.3,0.5Hz,1H),7.55(dd,J＝7.8,1.3Hz,1H),7.44(d,J＝5.7Hz,1H),7.40–7.29(m,2H),7.14(td,J＝7.6,1.3Hz,1H),7.00(dd,J＝7.6,1.4Hz,1H),3.87(s,3H),3.63–3.70(m,4H),2.47–2.57(m,4H),2.37(s,3H).ESI-MS m/z:428[M+H]+ 。
by operating in a similar manner to examples 1-2, the following compounds were prepared:
example 3: inhibition of CDK9 kinase activity by Compounds
The synthesized compounds were tested for their inhibitory activity on CDK9 by HotSpotSM kinase method/Fluorescence Resonance Energy Transfer (FRET) method by the American Reaction Biology Corp. (Malvern PA), for example CDK9/Cyclin T1.
The specific operation method comprises the following steps: CDK9/Cyclin T1 was diluted to the appropriate concentration with kinase diluent for use. The kinase reaction mixture contains CDK9/Cyclin T1, peptide substrate, HEPES (pH 7.5), BRIJ-35, and MgCl2 And EDTA. CDK9 phospho-peptide substrate was used as a 100% phosphorylation control, and no ATP was added as a 0% phosphorylation control. After 1h of reaction at room temperature, development Reagent A was added to the reaction system in moderate dilution. The reaction was continued at room temperature for 1h, sto was addedp Reagent stopped the reaction. The excitation light wavelength was 400nm, and fluorescence intensities at 445nm (coumarin) and 520nm (fluorescein) were detected. Test compound inhibition (n=2) was calculated as per the formula, IC50 The results of the analysis are shown in Table 1, which are obtained by plotting the percent inhibition and the logarithmic concentration values.
Inhibition of CDK9 kinase Activity by Compounds of Table 1
In the table, "A" represents IC50 The value is less than 0.1. Mu.M, and "B" represents IC50 The value is between 0.1. Mu.M and 1. Mu.M, and "C" represents IC50 The value is greater than 1. Mu.M.
As shown in Table 1, all compounds have inhibitory effect on CDK9 kinase activity, wherein compounds I-1, I-2, I-5, I-7-I-10, I-18-I-19, I-27, I-32 and I-38-I-40 inhibit CDK9 kinase IC50 IC with CDK9 kinase inhibition by the remaining compounds having a value of no more than 1. Mu.M50 Values of no more than 0.1. Mu.M can be achieved at nanomolar concentration levels.
Example 4: antiproliferative effect of compounds on tumor cells
Experimental principle: the MTT method for measuring the inhibition of leukemia cell line MV4-11 tumor cell line by MTT method and in vitro test of anti-tumor proliferation activity is a method for detecting survival and growth of cells, wherein the detection principle is that NADP related dehydrogenase (succinate dehydrogenase) in mitochondria of living cells can reduce exogenous MTT into insoluble blue purple crystalline Formazan (Formazan) and deposit in cells, and dead cells have no function. Purple crystal formazan in cells is dissolved by using dimethyl sulfoxide (DMSO) or triple solution (10% SDS-5% isobutanol-0.01 mol/L HCl), and the light absorption value (OD value) at 570nm wavelength is detected by an ELISA detector, so that the living cell quantity can be indirectly reflected.
The specific operation method comprises the following steps: inoculating tumor cells in logarithmic growth phase into 96-well culture plate according to certain cell amount, culturing for 24 hr, adding test compound (directly after suspending cell plate), and culturing at 37deg.C with 5% CO2 After culturing for 48h or 72h, adding MTT, culturing for 4h, dissolving in DMSO, crystallizing, measuring OD value at 570nm wavelength by ELISA, and calculating inhibition rate and IC of the compound50 Values, analytical results are shown in tables 2 and 3.
Antiproliferative effect of compounds of Table 2 on MV4-11 tumor cells
In the table, "A" represents IC50 The value is less than 0.1. Mu.M, and "B" represents IC50 The value is between 0.1. Mu.M and 1. Mu.M, and "C" represents IC50 The value is greater than 1. Mu.M.
TABLE 3 inhibition of various tumor cell lines by Compounds I-12
As shown in Table 2, all of the compounds had inhibitory effect on MV4-11 tumor cells, wherein compounds I-3 to I-4, I-6, I-9, I-13, I-21, I-32 and I-38 inhibited the IC of MV4-11 tumor cells50 IC with values not exceeding 1 mu M for inhibiting MV4-11 tumor cells by compounds I-10-I-12, I-14-I-20, I-22-I-31, I-33-I-37 and I-39-I-4650 Values of no more than 0.1. Mu.M can be achieved at nanomolar concentration levels.
As shown in Table 3, compound I-12 has inhibitory effects on various solid organ cancers, including, but not limited to, various hematological malignancies, such as acute myelogenous leukemia, chronic myelogenous leukemia, lymphocytic leukemia, multiple myeloma, diffuse large B-cell lymphoma, mantle cell lymphoma, burkitt's lymphoma, follicular lymphoma, and solid tumors such as breast cancer, non-small cell lung cancer, melanoma, renal cancer, ovarian cancer, prostate cancer, colon cancer, and central nervous system tumors.
In summary, the protein kinase inhibitor, the derivatives and the pharmaceutical composition thereof can be prepared into anticancer drugs, and can also be prepared into drugs for treating other diseases, including heart, virology, inflammation and pain related diseases.