CN106478700B - Boron-substituted aniline protein kinase inhibitor - Google Patents

Abstract

The invention discloses a compound which can regulate the activity of protein kinase and is used for treating or preventing diseases related to the protein kinase. Specifically, the invention relates to a boron-substituted aniline protein kinase inhibitor, belongs to a compound for regulating the activity of Anaplastic Lymphoma Kinase (ALK), and provides a preparation method of the compound and pharmaceutical application of the compound in treating or preventing ALK-related diseases.

Description

Boron-substituted aniline protein kinase inhibitor

Technical Field

The present invention relates to compounds that modulate the activity of protein kinases and are useful in the treatment or prevention of diseases associated with protein kinases. Specifically, the invention relates to a boron-substituted aniline protein kinase inhibitor, belongs to a compound for regulating the activity of Anaplastic Lymphoma Kinase (ALK), and provides a preparation method of the compound and pharmaceutical application of the compound in treating or preventing ALK-related diseases.

Background

Malignant tumors are a common and frequently occurring disease that seriously threatens human health and are characterized by abnormal proliferation of cells or variant cells. The proliferation, apoptosis, metastasis and the like of tumor cells are closely related to the abnormality of a certain link in a series of signal transduction pathways inside and outside the cells. Among these signaling pathways, an important class of molecules is protein kinases, and abnormalities of protein kinases are closely related to the occurrence, development and prognosis of tumors, and also are the main causes of a series of other human diseases related to inflammation or proliferative responses; the development of drugs targeting protein kinases is a main means for treating related diseases, and many drugs are approved to be on the market, and the drugs have the characteristics of clear target, definite curative effect and high safety, so the drugs are more and more accepted and supported by clinical medical practice.

Anaplastic Lymphoma Kinase (ALK) is an important member of the protein kinase family, and prior studies have shown that overexpression, mutations and fusion proteins of ALK are directly associated with a variety of tumors, including but not limited to neuroblastoma, Anaplastic Large Cell Lymphoma (ALCL), non-small cell lung cancer (NSCLC), and Inflammatory Myofibroblastoma (IMT), among others. First-generation medicines of Crizotinib (Crizotinib) and second-generation medicine of Ceritinib (Ceritinib) aiming at the ALK fusion gene are respectively marketed in 2011 and 2014, and remarkable progression-free survival and objective effective rate are obtained when the Crizotinib and Ceritinib are used for treating ALK positive lung cancer patients, so that the clinical value of the target spot is confirmed. Despite the remarkable drug effect, due to the heterogeneous characteristics of the tumor and the adaptation of tumor cells to the environmental stress, more and more research reports show that the continuous development of tumor resistance and diseases is almost the inevitable fate of patients of the type; in addition, the serious adverse reactions of the existing medicines, such as high incidence rate of adverse reactions in the digestive tract, hepatotoxicity, prolongation of QT interval and the like, also limit the application of the medicines. In view of the above, there are significant social benefits and values in developing new compounds having excellent ALK inhibitory activity and safety and in developing them to the market to cope with the above problems.

Disclosure of Invention

The invention aims to provide a boron-substituted aniline protein kinase inhibitor with a novel structure, and a series of compounds with antitumor activity are synthesized and screened through group substitution modification.

In order to realize the purpose, the invention adopts the following technical scheme:

a boron-substituted aniline protein kinase inhibitor is a compound with the following structural general formula I and pharmaceutically acceptable salts thereof:

wherein R is₁Is selected from

Q is-O-, -S-or-NR₁₃-；R₂、R₃、R₄、R₅、R₆Each independently selected from hydrogen, halogen, C_1-6Alkyl, halo C_1-6Alkyl radical, C_1-6Alkoxy, halo C_1-6Alkoxy radical, C_2-6Alkenyl radical, C_2-6Alkynyl, cyano or amino; r₇Selected from hydroxy or C_1-6An alkoxy group; r₀、R₈、R₉、R₁₃Each independently selected from hydrogen and C_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6One or more of alkynyl, acyl, acylamino, sulfo, sulfonamido, hydroxyl, aryl and heterocyclic radical; r₁₀Selected from hydrogen, C_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Alkynyl, cyano, aryl, heterocyclyl, - (CH)₂)_wOR₁₁、 -(CH₂)_wNR₁₁R₁₂、-CO₂R₁₁、-CONR₁₁R₁₂One or more of the above; r₁₁、R₁₂Each independently selected from hydrogen and C_1-6Alkyl, halo C_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Alkynyl, aryl or heterocyclyl; heterocyclyl is a 3-12 membered heterocyclic ring selected from the group consisting of the heteroatoms N, O; and n is selected from any integer value of 1-3, L, p is selected from any integer value of 0-6, k is selected from any integer value of 2-6, and m and w are independently selected from any integer value of 0-3.

Preferably, in the general structural formula I, R₁Is selected from

Q is-O-, -S-or-NR₁₃-；R₂、R₃、R₄、R₅、R₆Each independently selected from hydrogen, halogen, C_1-6Alkyl, halo C_1-6Alkyl radical, C_1-6Alkoxy, halo C_1-6Alkoxy, cyano or amino; r₇Selected from hydroxy or C_1-6An alkoxy group; r₀、R₈、R₉、R₁₃Each independently selected from hydrogen and C_1-6Alkyl, acyl, amido, sulfo, sulfonamido, hydroxyl, phenyl, or heterocyclyl; r₁₀Selected from hydrogen, C_1-6Alkyl, cyano, phenyl, heterocyclyl, - (CH)₂)_wOR₁₁、-(CH₂)_wNR₁₁R₁₂、-CO₂R₁₁or-CONR₁₁R₁₂；R₁₁、R₁₂Each independently selected from hydrogen and C_1-6Alkyl, halo C_1-6Alkyl, phenyl or heterocyclyl; heterocyclyl is a 3-6 membered heterocyclic ring selected from the group consisting of the heteroatoms N, O; and n is selected from any integer value of 1-3, L, p is selected from any integer value of 0-6, k is selected from any integer value of 2-6, and m and w are independently selected from any integer value of 0-3.

A boron-substituted aniline protein kinase inhibitor also comprises a compound with the following structural general formula II and pharmaceutically acceptable salts thereof:

wherein R is₁Is selected from

Q is-O-, -S-or-NR₁₃-；R₂、R₃、R₄、R₅、R₆Each independently selected from hydrogen, halogen, C_1-6Alkyl, halo C_1-6Alkyl radical, C_1-6Alkoxy, halo C_1-6Alkoxy radical, C_2-6Alkenyl radical、C_2-6Alkynyl, cyano or amino; r₇、R₁₄Each independently selected from hydroxy, C_1-6Alkoxy or R₇、R₁₄The cycloalkoxy group formed; r₀、R₈、R₉、R₁₃Each independently selected from hydrogen and C_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6One or more of alkynyl, acyl, acylamino, sulfo, sulfonamido, hydroxyl, aryl and heterocyclic radical; r₁₀Selected from hydrogen, C_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Alkynyl, cyano, aryl, heterocyclyl, - (CH)₂)_wOR₁₁、-(CH₂)_wNR₁₁R₁₂、-CO₂R₁₁、-CONR₁₁R₁₂One or more of the above; r₁₁、R₁₂Each independently selected from hydrogen and C_1-6Alkyl, halo C_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Alkynyl, aryl or heterocyclyl; heterocyclyl is a 3-12 membered heterocyclic ring selected from the group consisting of the heteroatoms N, O; n is selected from any integer value of 0-3, L, p is selected from any integer value of 0-6, k is selected from any integer value of 2-6, and m and w are independently selected from any integer value of 0-3.

Preferably, in the general structural formula II, R₁Is selected from

Q is-O-, -S-or-NR₁₃-；R₂、R₃、R₄、R₅、R₆Each independently selected from hydrogen, halogen, C_1-6Alkyl, halo C_1-6Alkyl radical, C_1-6Alkoxy, halo C_1-6Alkoxy, cyano or amino; r₇、R₁₄Each independently selected from hydroxy, C_1-6Alkoxy or R₇、R₁₄Form aCycloalkoxy of (a); r₀、R₈、R₉、R₁₃Each independently selected from hydrogen and C_1-6Alkyl, acyl, amido, sulfo, sulfonamido, hydroxyl, phenyl, or heterocyclyl; r₁₀Selected from hydrogen, C_1-6Alkyl, cyano, phenyl, heterocyclyl, - (CH)₂)_wOR₁₁、-(CH₂)_wNR₁₁R₁₂、-CO₂R₁₁or-CONR₁₁R₁₂；R₁₁、R₁₂Each independently selected from hydrogen and C_1-6Alkyl, halo C_1-6Alkyl, phenyl or heterocyclyl; heterocyclyl is a 3-6 membered heterocyclic ring selected from the group consisting of the heteroatoms N, O; n is selected from any integer value of 0-3, L, p is selected from any integer value of 0-6, k is selected from any integer value of 2-6, and m and w are independently selected from any integer value of 0-3.

Preferably, aryl is phenyl, naphthyl or anthracenyl; the heterocyclic radical is morpholinyl, piperidyl, pyranyl, furyl, pyridyl or pyrimidyl.

Preferably, the halogen is one or more of fluorine, chlorine, bromine and iodine.

A boron-substituted aniline protein kinase inhibitor is selected from the following characteristic compounds with the numbers of REX-C1-REX-C23:

REX-C1: 7- ((5-chloro-2- ((2-isopropoxy-5-methyl-4- (4-piperidinyl) phenyl) amine) 4-pyrimidinyl) amine) benzo [ c ] [1,2] oxo 1(3H) boronic acid;

REX-C2: 4- ((5-chloro-2- ((2-isopropoxy-5-methyl-4- (4-piperidinyl) phenyl) amine) 4-pyrimidinyl) amine) benzo [ c ] [1,2] oxo 1(3H) boronic acid;

REX-C3: 7- ((5-trifluoromethoxy-2- ((2-isopropoxy-5-methyl-4- (4-piperidinyl) phenyl) amine) 4-pyrimidinyl) amine) benzo [ c ] [1,2] oxo 1(3H) boronic acid;

REX-C4: 7- ((5-chloro-2- ((2-trifluoromethoxy-5-methyl-4- (4-piperidinyl) phenyl) amine) 4-pyrimidinyl) amine) benzo [ c ] [1,2] oxo 1(3H) boronic acid;

REX-C5: 7- ((5-chloro-2- ((2-isopropoxy-5-chloro-4- (4-piperidinyl) phenyl) amine) 4-pyrimidinyl) amine) benzo [ c ] [1,2] oxo 1(3H) boronic acid;

REX-C6: 7- ((5-fluoro-2- ((2-isopropoxy-5-fluoro-4- (4-piperidinyl) phenyl) amine) 4-pyrimidinyl) amine) benzo [ c ] [1,2] oxo 1(3H) boronic acid;

REX-C7: 7- ((5-chloro-2- ((2-isopropoxy-5-methyl-4- (4-methylpiperidinyl) phenyl) amine) 4-pyrimidinyl) amine) benzo [ c ] [1,2] oxo 1(3H) boronic acid;

REX-C8: 7- ((5-chloro-2- ((2-isopropoxy-5-methyl-4- (1- (4-tetrahydropyranyl) -4-piperidinyl) phenyl) amine) 4-pyrimidinyl) amine) benzo [ c ] [1,2] oxo 1(3H) boronic acid;

REX-C9: 7- ((5-chloro-2- ((2-isopropoxy-5-methyl-4- (1- (morpholinylmethyl) cyclopropyl) phenyl) amine) 4-pyrimidinyl) amine) benzo [ c ] [1,2] oxo 1(3H) boronic acid;

REX-C10: (2- ((5-chloro-2- ((2-isopropoxy-5-methyl-4- (4-piperidinyl) phenyl) amine) 4-pyrimidinyl) amine) phenyl) boronic acid;

REX-C11: (2- ((5-chloro-2- ((2-isopropoxy-5-methyl-4- (4-piperidinyl) phenyl) amine) 4-pyrimidinyl) amine) benzyl) boronic acid;

REX-C12: 8- ((5-chloro-2- ((2-isopropoxy-5-methyl-4- (4-piperidinyl) phenyl) amino) -4-pyrimidinyl) amine) -3, 4-dihydro-benzo [ c ] [1,2] oxo 1(3H) boronic acid;

REX-C13: 7- ((5-chloro-2- ((2-isopropoxy-5-methyl-4- (4- (1-methylpiperidinyl)) phenyl) amine) 4-pyrimidinyl) amine) benzo [ c ] [1,2] oxo 1(3H) boronic acid;

REX-C14: 7- ((5-chloro-2- ((2-isopropoxy-5-methyl-4- (4- (1-acetamidopiperidinyl)) phenyl) amine) 4-pyrimidinyl) amine) benzo [ c ] [1,2] oxo 1(3H) boronic acid;

REX-C15: 7- ((5-chloro-2- ((2-isopropoxy-5-methyl-4- (4- (1-ethanolpiperidyl)) phenyl) amine) 4-pyrimidinyl) amine) benzo [ c ] [1,2] oxo 1(3H) boronic acid;

REX-C16: 7- ((5-chloro-2- ((2-isopropoxy-4- (4-piperidinyl) phenyl) amine) 4-pyrimidinyl) amine) benzo [ c ] [1,2] oxo 1(3H) boronic acid;

REX-C17: 7- ((5-methyl-2- ((2-isopropoxy-5-methyl-4- (4- (1-methylpiperidinyl)) phenyl) amine) 4-pyrimidinyl) amine) benzo [ c ] [1,2] oxo 1(3H) boronic acid;

REX-C18: 7- ((5-trifluoromethyl-2- ((2-isopropoxy-5-methyl-4- (4- (1-methylpiperidinyl)) phenyl) amine) 4-pyrimidinyl) amine) benzo [ c ] [1,2] oxo 1(3H) boronic acid;

REX-C19: 5-chloro-N²- (2-isopropoxy-5-methyl-4- (4-piperidinyl) phenyl) -N⁴- (2- ((4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) methyl) phenyl) -2, 4-diaminopyrimidine;

REX-C20: (2- ((5-chloro-2- ((4- (1- (ethoxymethyl) cyclopropyl) -2-isopropoxy-5-methylphenyl) amino) 4-pyrimidinyl) amine) benzyl) boronic acid;

REX-C21: 7- ((5-chloro-2- ((2-isopropoxy-5-methyl-4- (1- (4-tetrahydropyranyl) -4-piperidinyl) phenyl) amine) 4-pyrimidinyl) amine) benzyl) boronic acid;

REX-C22: 5-chloro-N²- (2-isopropoxy-5-methyl-4- (4-piperidinyl) phenyl) -N⁴- (4- ((4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) methyl) phenyl) -2, 4-diaminopyrimidine;

REX-C23: (4- ((5-chloro-2- ((4- (1- (ethoxymethyl) cyclopropyl) -2-isopropoxy-5-methylphenyl) amino) 4-pyrimidinyl) amine) benzyl) boronic acid.

The structural formula of the compounds numbered REX-C1-REX-C23 is shown as follows:

the invention also provides a synthesis method of the compound shown as the general formula I and the general formula II, and the general reaction route is as follows:

general formula I:

general formula II:

based on the general reaction route, the method comprises the following synthetic schemes:

(1) synthesis scheme 1: synthesis of Compounds 1-3

Step 1: dissolving the compound 1-1 in an organic solvent, slowly adding a catalyst, replacing nitrogen, introducing hydrogen, and heating for reacting for N hours; after the reaction is finished, filtering to remove the redundant catalyst, drying the organic solvent, decompressing and drying to obtain the compound 1-2.

Step 2: dissolving the compound 1-3 in an organic solvent, slowly adding sodium hydride and 2,4, 5-trichloropyrimidine, replacing with nitrogen, and heating for reaction for N hours; after the reaction is finished, adding a small amount of ice water to destroy redundant sodium hydride, adding an extracting agent, extracting, drying, decompressing and spin-drying to obtain the compound 1-3.

In the synthesis scheme 1, the organic solvent is selected from one or more of N, N-dimethylformamide, tetrahydrofuran, methanol and dioxane; the extractant is selected from one or more of pure water, dichloromethane and ethyl acetate; the catalyst is selected from one or more of palladium carbon, tetrakis (triphenylphosphine) palladium and platinum dioxide.

In the synthesis scheme 1, the heating reaction temperature is 60-120 ℃, preferably 60 ℃ and 120 ℃; the reaction time is 10 to 14 hours, preferably 10 hours and 14 hours.

(2) Synthesis scheme 2: synthesis of Compounds 2-3

Step 1: will be provided with

(i.e., Compound 2-1) and

dissolving the mixture in a mixed solution of an organic solvent and water which are prepared in proportion, slowly adding potassium phosphate and a catalyst, replacing nitrogen, and heating for reaction for N hours; and after the reaction is finished, adding an extracting agent, extracting, drying, decompressing and spin-drying to obtain the compound 2-2.

Step 2: dissolving the compound 2-2 in an organic solvent, adding a catalyst, replacing nitrogen, introducing hydrogen, and heating for reacting for N hours; and after the reaction is finished, filtering under reduced pressure to remove redundant catalyst, and performing reduced pressure and spin drying to obtain the compound 2-3.

In the synthesis scheme 2, the organic solvent is selected from one or more of N, N-dimethylformamide, methanol, isopropanol and dioxane; the extractant is selected from one or more of pure water, dichloromethane and ethyl acetate; the catalyst is selected from one or more of palladium carbon, tetrakis (triphenylphosphine) palladium and platinum dioxide.

In the synthesis scheme 2, the heating reaction temperature is 100-120 ℃, and preferably 100 ℃; the reaction time is 12 to 16 hours, preferably 12 hours and 16 hours.

(3) Synthesis scheme 3: synthesis of target Compound

Step 1: dissolving the compounds 1-4 prepared in the synthesis scheme 1 and the compounds 2-3 prepared in the synthesis scheme 2 in an organic solvent, continuously adding cesium carbonate and a catalyst, and carrying out nitrogen replacement and microwave heating reaction for N hours; after the reaction is finished, filtering under reduced pressure to remove redundant catalyst, and performing column chromatography to obtain the target compound (i.e. the compound of the general formula I or the general formula II).

In the synthesis scheme 3, the organic solvent is selected from one or more of N, N-dimethylformamide, tetrahydrofuran, methanol and dioxane; the catalyst is selected from one or more of palladium carbon, palladium tetrakis (triphenylphosphine) palladium, platinum dioxide, palladium acetate and 4, 5-bis (diphenylphosphino) -9, 9-dimethyl xanthene.

In the synthesis scheme 3, the reaction time is 0.5 to 1 hour, preferably 0.5 hour.

In schemes 1,2 and 3, R₁Is selected from

Q is-O-, -S-or-NR₁₃-；R₂、R₃、R₄、R₅、R₆Each independently selected from hydrogen, halogen, C_1-6Alkyl, halo C_1-6Alkyl radical, C_1-6Alkoxy, halo C_1-6Alkoxy radical, C_2-6Alkenyl radical, C_2-6Alkynyl, cyano or amino; r₇Selected from hydrogen, C_1-6One or more of alkyl, amido, hydroxyl, aryl and heterocyclic radical; r₀、R₈、R₉、R₁₃Each independently selected from hydrogen and C_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6One or more of alkynyl, acyl, acylamino, sulfo, sulfonamido, hydroxyl, aryl and heterocyclic radical; r₁₀Selected from hydrogen, halogen, C_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Alkynyl, cyano, aryl, heterocyclyl, - (CH)₂)_wOR₁₁、-(CH₂)_wNR₁₁R₁₂、-CO₂R₁₁、-CONR₁₁R₁₂One or more of the above; r₁₁、R₁₂Each independently selected from hydrogen and C_1-6Alkyl, halo C_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Alkynyl, aryl or heterocyclyl; heterocyclyl is a 3-12 membered heterocyclic ring selected from the group consisting of the heteroatoms N, O; and n is selected from any integer value of 1-6, L, p is selected from any integer value of 0-6, k is selected from any integer value of 2-6, and m and w are independently selected from any integer value of 0-3.

The term "compound" as used herein includes all stereoisomers, geometric isomers, tautomers and isotopes.

The "compounds" of the present invention may be asymmetric, e.g., having one or more stereoisomers. Unless otherwise indicated, all stereoisomers include, for example, enantiomers and diastereomers. The compounds of the invention containing asymmetric carbon atoms can be isolated in optically active pure form or in racemic form. The optically active pure form can be resolved from a racemic mixture or synthesized by using chiral starting materials or chiral reagents.

The "compound" of the present invention also includes tautomeric forms. Tautomeric forms result from the exchange of one single bond with an adjacent double bond and the concomitant migration of one proton.

The invention also includes all isotopic atoms, whether in the intermediate or final compound. Isotopic atoms include those having the same atomic number but different mass numbers. For example, isotopes of hydrogen include deuterium and tritium.

Compounds containing the foregoing general structure, the terms used herein have the following meanings:

the term "halogen" denotes fluorine, chlorine, bromine or iodine, preferably fluorine, chlorine or bromine.

The term "cyano" refers to — CN.

The term "hydroxy" refers to-OH.

The term "alkyl" denotes a straight or branched chain saturated hydrocarbon group consisting of carbon and hydrogen atoms, such as C_1-20Alkyl, preferably C_1-6Alkyl groups such as methyl, ethyl, propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl, sec-butyl or tert-butyl), pentyl (including n-pentyl, isopentyl, neopentyl), n-hexyl, 2-methylhexyl and the like. The alkyl group may be unsubstituted or substituted with one or more substituents including, but not limited to, alkyl, alkoxy, cyano, hydroxy, carbonyl, carboxy, aryl, heteroaryl, amino, halo, sulfonyl, sulfinyl, phosphoryl.

The term "amino" refers to the group-NH₂-NH (alkyl) and-N (alkyl)₂Alkyl has the meaning as previously described. The structural form of-NH (alkyl) is

Specific examples include, but are not limited to-NHCH₃、-NHCH(CH₃)₂、-NHC₂H₅Etc.; -N (alkyl)₂In the structural form of

Specific examples include, but are not limited to, -N (CH)₃)₂、-N(CH₃)C₂H₅And the like.

The term "aryl" refers to an all-carbon monocyclic or fused ring having a fully conjugated pi-electron system, typically having 6 to 14 carbon atoms, preferably having 6 to 12 carbon atoms, and most preferably having 6 carbon atoms. Aryl groups may be unsubstituted or substituted with one or more substituents including, but not limited to, alkyl, alkoxy, cyano, hydroxy, carbonyl, carboxy, aryl, aralkyl, amino, halo, sulfonyl, sulfinyl, phosphoryl. Examples of unsubstituted aryl groups include, but are not limited to, phenyl, naphthyl, and anthracenyl.

The term "heterocyclyl" refers to a monocyclic or fused ring having from 3 to 12 (an integer) ring atoms, of which 1,2 or 3 ring atoms are selected from one or more of N, O, the remaining ring atoms being C, and having a fully conjugated pi-electron system. The heterocyclyl group may be unsubstituted or substituted with one or more substituents including, but not limited to, alkyl, alkoxy, cyano, hydroxy, carbonyl, carboxy, aryl, aralkyl, amino, halo, sulfonyl, sulfinyl, phosphoryl. Examples of unsubstituted heterocyclyl groups include, but are not limited to, pyrrolyl, indolyl, pyrrolidinyl, imidazolyl, pyrazolyl, tetrazolyl, pyridyl, quinolyl, isoquinolyl, piperidyl, pyrimidinyl, pyrazinyl, piperazinyl, furyl, pyranyl, morpholinyl.

The general formula of the invention is shown as the structure I,

the invention also provides a pharmaceutical composition, which comprises the compound or the pharmaceutically acceptable salt thereof as an active ingredient, and one or more pharmaceutically acceptable carriers.

"pharmaceutical composition" as used herein, refers to a formulation of one or more compounds of the present invention or salts thereof with a carrier generally accepted in the art for delivery of biologically active compounds to an organism (e.g., a human). The purpose of the pharmaceutical composition is to facilitate delivery of the drug to an organism.

The term "pharmaceutically acceptable carrier" refers to a substance that is co-administered with, and facilitates the administration of, an active ingredient, including, but not limited to, any glidant, sweetener, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersant, disintegrant, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier that is acceptable for use in humans or animals (e.g., livestock) as permitted by the national food and drug administration. Examples include, but are not limited to, calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

The pharmaceutical composition can be prepared into solid, semi-solid, liquid or gaseous preparations, such as tablets, pills, capsules, powder, granules, paste, emulsions, suspensions, solutions, suppositories, injections, inhalants, gels, microspheres, aerosols and the like.

The pharmaceutical compositions of the present invention may be manufactured by methods well known in the art, such as conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, lyophilizing, and the like.

The route of administration of the compounds of the present invention or pharmaceutically acceptable salts thereof or pharmaceutical compositions thereof includes, but is not limited to, oral, rectal, transmucosal, enteral, or topical, transdermal, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, intravenous administration. The preferred route of administration is oral.

For oral administration, the pharmaceutical compositions may be formulated by mixing the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, slurries, suspensions and the like, for oral administration to a patient. For example, for pharmaceutical compositions intended for oral administration, tablets may be obtained in the following manner: the active ingredient is combined with one or more solid carriers, the resulting mixture is granulated if necessary, and processed into a mixture or granules, if necessary with the addition of small amounts of excipients, to form tablets or tablet cores. The core may be combined with an optional enteric coating material and processed into a coated dosage form more readily absorbed by an organism (e.g., a human).

The invention also provides the use of a compound as described hereinbefore or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment or prevention of a disease associated with a protein kinase.

Use of a compound as described above or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment or prevention of a disease associated with anaplastic lymphoma kinase (ALK kinase).

Preferably, the ALK kinase-associated disease is selected from cell proliferative diseases, preferably tumors.

Preferably, the aforementioned cell proliferative diseases include non-small cell lung cancer, anaplastic large cell lymphoma, inflammatory myofibroblastic tumors, nasopharyngeal carcinoma, breast cancer, colorectal cancer, diffuse large B cell lymphoma, liver cancer, gastric cancer, esophageal cancer, pancreatic cancer, ovarian cancer, systemic histiocytosis and neuroblastoma.

In the invention, the inventor carries out ALK kinase inhibition activity and ALK related mutation site binding rate determination experiments on a series of boron-substituted aniline compounds obtained by synthesis, and finds that part of the compounds show higher inhibition activity on ALK, show better binding rate on ALK mutation sites (such as L1196M, F1174L and C1156Y) and have obvious inhibition activity on ROS 1; in addition, cell proliferation experiments of lung cancer cell lines and phenotype screening experiments of zebra fish are also carried out, and the anti-tumor activity of part of compounds in vivo is found to be obvious.

Compared with the prior art, the boron-substituted aniline protein kinase inhibitor provided by the invention is based on reasonable drug design of a target, and a series of compounds with novel structures are obtained through substitution modification of groups; and a series of compounds with antitumor activity are optimally screened out by combining a kinase activity experiment, a cell proliferation experiment and a zebra fish phenotype screening experiment. Therefore, the complex can be used for developing a new generation of protein kinase inhibitor, has great clinical application value for targeted treatment or prevention of ALK-mediated diseases, and has considerable market potential.

Drawings

FIG. 1 is a dorsal distribution of Albino zebrafish iris pigment cells

FIG. 2 is a dose-effect relationship diagram (mean. + -. sem) of the effect of REX-C1/REX-C3 on the pigmented cells of zebra fish iris

FIG. 3 is a graph showing the effect of REX-C1/REX-C3 on pigmented cells of iris of zebra fish

Detailed Description

The following are specific examples of the present invention and further describe the technical solutions of the present invention, but the scope of the present invention is not limited to these examples. All changes, modifications and equivalents that do not depart from the spirit of the invention are intended to be included within the scope thereof.

In the preparation method of the target compound, the liquid chromatography adopts a Waters symmetry C18 chromatographic column. Thin layer chromatography was performed using GF254(0.25 mm). Nuclear Magnetic Resonance (NMR) was measured using a Bruker-400 NMR spectrometer; liquid chromatography/Mass Spectrometry (LC/MS) Using a Waters ZQ mass spectrometer (column: Waters symmetry C18, mm, 5 μm, 35 ℃ C.), the ESI (+) ion mode was used.

In addition, all operations involving easily oxidizable or hydrolyzable raw materials were carried out under nitrogen protection. Unless otherwise indicated, the starting materials used in the present invention are all commercially available starting materials and can be used without further purification.

Example preparation of- ((5-chloro-2- ((2-isopropoxy-5-methyl-4- (4-piperidinyl) phenyl) amine) 4-pyrimidinyl) amine) benzo [ C ] [1,2] oxo 1(3H) boronic acid [ code REX-C1 ]

The synthetic route is as follows:

synthesis scheme 1: synthesis of intermediate 7- ((2, 5-dichloropyrimidinyl) amino) benzo [ c ] [1,2] oxo 1(3H) boronic acid (i.e., Compound 1-5)

Step 1: preparation of 3-nitro-2- (4,4,5, 5-tetramethyl-1, 3, 2-dioxoboryl) benzylacetic acid (i.e. Compound 1-2)

Dissolving a raw material 2-bromo-3-nitrobenzyl acetic acid (namely a compound 1-1,11.3g,40.0mmol) in 1,4 dioxane (150.0mL), slowly adding potassium acetate (13.7g,140.0mmol), 1,1' -bis (diphenylphosphino) ferrocene palladium dichloride (2.6g,3.0mmol) and bis (pinacolato) diboron (15.2g,60.0mmol), replacing with nitrogen, and reacting at 90-95 ℃ for 16 hours; after completion of the reaction, the reaction mixture was poured into 500.0mL of water, extracted with ethyl acetate, dried, concentrated, and subjected to silica gel column chromatography to obtain compound 1-2(13.0g) in yield: 98.0 percent.

MSm/z[ESI]：322.1[M+1]。

Step 2: preparation of intermediate 7-nitrobenzo [ c ] [1,2] oxo 1(3H) boronic acid (i.e. compound 1-3)

Dissolving the compound 1-2(6.4g,20.0mmol) in methanol (200.0mL), dropwise adding 5.0mol/L sodium hydroxide solution (8.0mL,40.0mmol), and reacting at 60-70 ℃ for 24 minutes; after the reaction was completed, the reaction mixture was warmed to room temperature, and 3.0mol/L hydrochloric acid water was added to destroy the reaction mixture, followed by extraction with water and ethyl acetate, and the organic phase was washed twice with sodium hydrogencarbonate, dried over anhydrous sodium sulfate, and concentrated to obtain compound 1-3(2.2g) in yield: 63.0 percent.

MSm/z[ESI]：179.2[M+1]。

And step 3: preparation of intermediate 7-aminobenzo [ c ] [1,2] oxo 1(3H) boronic acid (i.e. Compound 1-4)

Adding compounds 1-3(2.2g,12.0mmol) and 10% palladium on carbon (i.e. Pd/C,0.5g) to dry methanol (25.0mL), replacing with nitrogen, introducing hydrogen, and reacting at 25 deg.C for 12 hr; after cooling, filtration over celite, separation, drying over anhydrous sodium sulfate, and concentration gave compound 1-4(1.1g), yield: 58.0 percent.

MSm/z[ESI]：150.2[M+1]。

And 4, step 4: preparation of 7- ((2, 5-dichloropyrimidinyl) amino) benzo [ c ] [1,2] oxo 1(3H) boronic acid (i.e., Compound 1-5)

Dissolving the compounds 1-4(1.1g,7.0mmol) in DMF (30.0mL) under the condition of ice-water bath, slowly adding sodium hydride (0.18g,7.0mmol) into the reaction system, stirring at 0 ℃ for 15 minutes, and dropwise adding 2,4, 5-trichloropyrimidine (1.2g,6.0mmol) into the reaction system, and stirring at 25 ℃ overnight; after cooling, the mixture was poured into 50.0mL of water, extracted with ethyl acetate, dried, concentrated, and subjected to silica gel column chromatography to obtain compound 1-5(1.0g), yield: 53.0 percent.

MSm/z[ESI]：296.2[M+1]。¹H-NMR(400MHz,DMSO-d₆)：H NMR(400MHz,DMSO-d6):＝9.531(s,1H),8.940(s,1H),8.462(s,1H),8.021(d,1H,J＝8.4Hz),7.554-7.548(m,1H),7.218-7.199(d,1H,J＝7.6Hz),5.040(s,2H)。

Synthesis scheme 2: synthesis of intermediate 4- (4-amino-5-isopropoxy-2-methylphenyl) piperidine-1-tert-butoxy carbonate (i.e. compound 2-4)

Step 1: preparation of intermediate 1-chloro-5-isopropoxy-2-methyl-4-nitrobenzene (i.e. compound 2-2)

Dissolving the raw material 2-chloro-4-fluoro-5-nitrotoluene (compound 2-1,14.2g,74.9mmol) in isopropanol (100.0mL), adding cesium carbonate (122.0g,374.4mmol), and after addition, reacting at 60 ℃ overnight; after completion of the reaction, the reaction mixture was concentrated to remove isopropanol, poured into 500.0mL of water, extracted with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate and concentrated to obtain compound 2-2(14.3g) in yield: 82.0 percent.

MSm/z[ESI]：230.6[M+1]。¹H-NMR(400MHz,CDCl₃)：＝7.880(s,1H)，7.498(s,1H)，4.871-4.795(m,1H)，2.294(s,1H)，1.281-1.266(d,J＝6.0Hz,6H)。

Step 2: preparation of intermediate 4- (5-isopropoxy-2-methyl-4-nitro-phenyl) -pyridine (i.e. compound 2-3)

4-pyridineboronic acid (14.7g,120.0mmol) was dissolved in a mixed solution of dioxane and water (the volume ratio v/v of both was 2:1), and the mixture was stirred for 5 minutes while being replaced with nitrogen. Tris (1, 3-dibenzylideneacetone) dipalladium (10.0g,10.9mmol), 2-dicyclohexylphosphine-2 ',6' -dimethoxy-1, 1' -biphenyl (11.2g,27.2mmol), compound 2-2(25.0g,109.0mmol) and potassium phosphate (46.2g,218.0mmol) were added in this order under nitrogen protection, and the reaction was refluxed overnight. After the reaction, the reaction mixture was cooled to room temperature, washed twice with 1.0mol of saturated NaOH, and extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, concentrated and subjected to silica gel column chromatography to obtain compound 2-3(20.0g) in yield: 67.4 percent.

MSm/z[ESI]：272.6[M+1]。¹H-NMR(400MHz,DMSO-d₆)：＝8.698-8.683(dd,J₁＝1.6Hz,J₂＝4.4Hz 1H)，7.831(s,1H)，7.474-7.459(dd,J₁＝2.0Hz,J₂＝4.8Hz1H)，7.230(s,1H)，4.912-4.822(m,1H)，2.196(s,1H)，1.285-1.270(d,J＝6.0Hz,6H)。

And step 3: preparation of intermediate 4- (4-amino-5-isopropoxy-2-methylphenyl) piperidine-1-tert-butoxy carbonate (i.e. compound 2-4)

Compound 2-3(43.8g,161.0mmol) was dissolved in a mixture of acetic acid (400.0mL) and trifluoroacetic acid (25.0mL), platinum dioxide (17.6g) containing 40% of platinum was added, and the mixture was stirred for 5 minutes under nitrogen substitution and hydrogen introduction, and then pressurized to 1 atmosphere to react for 36 hours. After the reaction is finished, cooling to room temperature, filtering, removing platinum dioxide, adding 100.0mL of water for dilution, and extracting with ethyl acetate; the organic phase was washed twice with 1.0mol of saturated NaOH, dried over anhydrous sodium sulfate, concentrated, and subjected to silica gel column chromatography to obtain compound 2-4(34.0g) in yield: 60.6 percent.

MSm/z[ESI]：348.2[M+1]。¹H-NMR(400MHz,DMSO-d₆)：＝6.575(s,1H)，6.432(s,1H)，4.439-4.345(m,2H)，4.083-4.063(m,2H)，2.818-2.81(m,2H)，2.122(s,3H)，1.620-1.590(m,2H)，1.423(s,9H)，1.401-1.388(m,2H)，1.231-1.216(m,6H)。

Synthesis scheme 3: synthesis of target compound 7- ((5-chloro-2- ((2-isopropoxy-5-methyl-4- (4-piperidinyl) phenyl) amine) 4-pyrimidinyl) amine) benzo [ C ] [1,2] oxo 1(3H) boronic acid (i.e., REX-C1)

Step 1: preparation of intermediate tert-butyl 4- (4- ((1-hydroxy-1, 3 benzo [ c ] [1,2] oxo 1(3H) Borate) -2-pyrimidineamino) -5-isopropoxy-2-tolyl) piperidine-1-carboxylate (Compound 3-1)

Compound 2-4(0.9g,2.7mmol), compound 1-5(0.8g,2.7mmol), 4, 5-bis (diphenylphosphino) -9, 9-dimethylxanthene (0.3g,0.5mmol), palladium acetate (63.0mg,0.3mmol) and cesium carbonate (2.7g,8.1mmol) were dissolved in dioxane (50.0mL), and the solution was charged into a sealed tube, purged with nitrogen and reacted at 95 ℃ for 18 hours. After the reaction was completed, the solvent was spin-dried, ethyl acetate and water were added, extraction, organic phase drying, and purification by silica gel column chromatography to obtain compound 3-1(0.2g), yield: 16.0 percent.

MSm/z[ESI]：608.1[M+1]。

Step 2: preparation of target Compound (i.e., REX-C1)

Compound 3-1(0.2g, 0.4mmol) was added to dichloromethane (10.0mL) with stirring, trifluoroacetic acid (2mL) was added dropwise, and the mixture was stirred at room temperature overnight. Adjusting pH to more than 10.0 with 5.0% sodium bicarbonate water solution, extracting with ethyl acetate, drying, and spin-drying to obtain REX-C1(80.0mg) as target compound with yield: 34.0 percent.

MSm/z[ESI]：508.1[M+1]。¹H-NMR(400MHz,DMSO-d₆)：＝8.444(s,1H),8.176(m,2H),7.976(s,1H),7.600(s,1H),7.320-7.315(m,1H),7.086-7.066(m,1H),6.835(s,1H),5.007(s,2H),4.540-4.445(m,1H),3.060-3.055(m,2H),2.740-2.735(m,1H),2.630-2.628(m,2H),2.095(s,3H),1.650-1.550(m,4H),1.250-1.235(d,6H,J＝6Hz)。

Example preparation of 24- ((5-chloro-2- ((2-isopropoxy-5-methyl-4- (4-piperidinyl) phenyl) amine) 4-pyrimidinyl) amine) benzo [ C ] [1,2] oxo 1(3H) boronic acid [ code REX-C2 ]

The synthetic route is as follows:

as shown in the synthetic route provided in this example, in "synthesis of compounds 1 to 5" in synthesis scheme 1, the raw material 2-bromo-6-nitrobenzyl acetic acid in step 1 is reacted instead of 2-bromo-3-nitrobenzyl acetic acid in example 1, and the rest of the synthesis methods are the same as in synthesis scheme 1 in example 1, and after multi-step reaction, compounds 1 to 5 are obtained, with yield: 28.0 percent.

MSm/z[ESI]：296.4[M+1]。

In synthesis scheme 2 "synthesis of compound 2-4", the raw material 2-chloro-4-fluoro-5-nitrotoluene (i.e., compound 2-1), and the remaining synthesis methods are the same as in synthesis scheme 2 of example 1, to obtain compound 2-4, with yield: 62.8 percent.

MSm/z[ESI]：348.2[M+1]。

In synthesis scheme 3 "synthesis of object compound REX-C2", compounds 1 to 5 and compounds 2 to 4 prepared in this example were prepared according to synthesis scheme 3 of example 1 to obtain object compound REX-C2 with yield: 9.0 percent.

MSm/z[ESI]：508.1[M+1]。¹H-NMR(400MHz,DMSO-d₆)：＝9.478(s,1H),8.725-8.690(d,1H,J＝14Hz),8.499-8.463(m,1H),8.236(s,1H),8.188(s,1H),7.716-7.699(d,1H,J＝6.8Hz),7.475-7.741(m,2H),7.314(s,1H),6.699(s,1H),4.916(s,2H),4.514(m,1H),3.339-3.335(m,2H),3.020-3.015(m,2H),2.873-2.870(m,1H),1.850(s,3H),1.754-1.740(m,4H),1.257-1.242(d,6H,J＝6Hz)。

Example preparation of- ((5-trifluoromethoxy-2- ((2-isopropoxy-5-methyl-4- (4-piperidinyl) phenyl) amine) 4-pyrimidinyl) amine) benzo [ C ] [1,2] oxo 1(3H) boronic acid [ code REX-C3 ]

The synthetic route is as follows:

as shown in the synthetic route provided in this example, in "synthesis of compounds 1 to 5" in synthesis scheme 1,2, 4-dichloro-5-trifluoromethylpyrimidine as a raw material in step 4 is reacted instead of 2,4, 6-trichloropyrimidine in example 1, and the rest of the synthesis methods are the same as in synthesis scheme 1 of example 1, and after multi-step reactions, compounds 1 to 5 are obtained, with yield: 31.0 percent.

MSm/z[ESI]：296.4[M+1]。

In synthesis scheme 2 "synthesis of compound 2-4", the raw material 2-chloro-4-fluoro-5-nitrotoluene (i.e., compound 2-1), and the remaining synthesis methods are the same as in synthesis scheme 2 of example 1, to obtain compound 2-4, with yield: 62.8 percent.

MSm/z[ESI]：348.2[M+1]。

In synthesis scheme 3 "synthesis of object compound REX-C3", compounds 1 to 5 and compounds 2 to 4 prepared in this example were prepared according to synthesis scheme 3 of example 1 to obtain object compound REX-C3 with yield: 18.0 percent.

MSm/z[ESI]：542.1[M+1]。¹H-NMR(400MHz,DMSO-d₆)：＝9.404-9.554(m,2H),8.613(s,1H),7.751-7.902(br s,1H),7.341-7.385(m,2H),7.203-7.261(m,1H),6.851(s,1H),4.995(s,2H),4.503-4.574(m,1H),3.311-3.392(m,2H),2.962-3.093(m,2H),2.083(s,3H),1.932-1.993(m,2H),1.771-1.862(m,2H),1.241-1.262(d,J＝6Hz,6H).

Example preparation of- ((5-chloro-2- ((2-isopropoxy-5-methyl-4- (1- (morpholinylmethyl) cyclopropyl) phenyl) amine) 4-pyrimidinyl) amine) benzo [ C ] [1,2] oxo 1(3H) boronic acid [ number REX-C9 ]

The synthetic route is as follows:

as described in the synthetic route provided in this example, in synthesis scheme 1 "synthesis of compounds 1 to 5", the starting material 2-bromo-3-nitrobenzyl acetic acid (i.e., compound 2-1) and the remaining synthesis methods are the same as in synthesis scheme 1 of example 1, to obtain compounds 1 to 5, with yields: 28.0 percent.

MSm/z[ESI]：296.4[M+1]。

Synthesis scheme 2: synthesis of intermediate 2-isopropoxy-5-methyl-4- (1- (methylenemorpholinyl) cyclopropyl) aniline (compound 2-5)

Step 1: preparation of intermediate 1- (2-methyl-4-nitro-5-isopropoxy) cyclopropylphenylacetic acid (i.e., compound 2-2)

The starting material methyl 1- (2-methyl-4-nitro-5-hydroxy) cyclopropylphenylacetate (i.e., compound 2-1,1g,3.4mmol) and lithium hydroxide (571mg,13.6mmol) were dissolved in methanol (50ml) and water (10ml), and the reaction was heated to 80 ℃ and stirred for 60 minutes. After completion of the reaction, water was added and 1N hydrochloric acid was added to adjust pH 4, followed by filtration to obtain compound 2-2(750mg) in yield: 79 percent.

MSm/z[ESI]：280.2[M+1]。

Step 2: preparation of intermediate (1- (5-isopropoxy-2-methyl-4-nitro) cyclopropyl) phenylacetic acid morpholine (compound 2-3)

Compound 2-2(600mg,2.15mmol), triethylamine (653mg,6.45mmol), HATU (1.064g,2.8mmol), morpholine (281mg,3.23mmol) were dissolved in N, N-dimethylformamide (50ml) and reacted at room temperature for 3 hours. After the reaction was completed, water was added and ethyl acetate was added for extraction, and drying and spin-drying were carried out to obtain compound 2-3(673mg), yield: 90 percent.

MSm/z[ESI]：347.4[M+1]。

And step 3: preparation of intermediate 2-isopropoxy-5-methyl-4- (1- (methylenemorpholinyl) cyclopropyl) nitrobenzene (namely compound 2-4)

Compound 2-3(673mg,1.93mmol) was dissolved in tetrahydrofuran (50ml), and boron trifluoride (14.4ml,14.4mmol,1M in THF) was added dropwise at room temperature. After the reaction was completed, water was added and ethyl acetate was added for extraction, and drying and spin-drying were carried out to obtain compound 2-4(450mg) in yield: and 69 percent.

MSm/z[ESI]：333.4[M+1]。

And 4, step 4: preparation of intermediate 2-isopropoxy-5-methyl-4- (1- (methylenemorpholinyl) cyclopropyl) aniline (compound 2-5)

Compounds 2-4(450mg,1.35mmol), Pd/C (238mg, 58.8% in H₂O) was dissolved in methanol (25ml), and the solution was replaced with hydrogen overnight at room temperature. After the reaction was completed, the palladium-carbon was removed by filtration, dried and spin-dried to obtain compound 2-5(400mg) with yield: 97 percent.

MSm/z[ESI]：305.4[M+1]。

In synthesis scheme 3 "synthesis of object compound REX-C9", compounds 1 to 5 and compounds 2 to 5 prepared in this example were prepared according to synthesis scheme 3 of example 1 to obtain object compound REX-C9 with yield: 20.0 percent.

MSm/z[ESI]：564.1[M+1]。¹H-NMR(400MHz,DMSO-d₆)：＝8.382-8.360(d,J＝8.8Hz,1H),8.299(s,1H),8.061(s,1H),8.012(s,1H),7.460(s,1H),7.402-7.360(t,J＝8.4Hz,1H),6.881-6.862(d,J＝7.6Hz,1H),6.805(s,1H),4.999(s,2H),4.607-4.547(m,1H),3.721-3.698(t,J＝4.6Hz,4H),2.594(m,6H),2.478(s,3H),2.044-2.027(m,1H),1.442-1.427(d,J＝6Hz,6H),0.886(m,2H),0.811(m,2H).

Example 5 measurement of ALK kinase inhibitory Activity and binding Rate to related mutation sites

For the compounds REX-C1, REX-C2, REX-C3 and REX-C9 prepared in examples 1 to 4, use was made of

(FRET) method for determining ALK kinase inhibitory activity of the aforementioned compound, the inhibitory activity being IC₅₀Expressed by this index, IC₅₀I.e., the concentration of the compound at which the activity of ALK kinase is inhibited by 50%.

At the same time adopt

Eu Kinase binding Assay (TR-FRET) Assay for determining the binding rate of the compounds of the present invention to ALK-associated mutation sites, such as ALK L1196M, and also using IC₅₀This index is used to express. Lantha screen Eu kinase binding assay Alexa Fluor conjugate or kinase "tracer" binding was detected by addition of Eu-labeled antibody or anti-tag antibody. Binding of the tracer and antibody to the kinase results in a high degree of FRET, whereas the use of a kinase inhibitor instead of the tracer results in loss of FRET.

The invention utilizes the kinase assay platform of Life technology company to carry out the determination, and the determination result is shown in the table I. The result shows that the compound provided by the invention has better ALK inhibitory activity and better binding rate to ALK mutation sites (such as ALK L1196M).

TABLE ALK inhibitory Activity and ALK L1196M binding Rate determination of the Compounds of the examples

	ALK IC50(nM)	ALK F1196M IC50(nM)
			REX-C1	80.9	92.3
REX-C2	31.5	51.3
			REX-C3	32.6	17.6
REX-C9	1070	1040

Further, the activity of the selected compound REX-C1 at 100nM was measured using the kinase assay platform of Life technology, Inc., and the results are shown in Table II. The results show that the compounds REX-B2, REX-B3 and REX-B4 provided by the invention show good binding rate to ALK F1174L and ALK C1156Y, and show high inhibitory activity to LTK and ROS 1.

TABLE II example determination of the Activity of Compounds on the relevant kinases at a concentration of 100nM

Example 6 Cell proliferation assay (Cell Titer GLO assay)

Test compounds: the compounds REX-C1, REX-C2, REX-C3 and REX-C9 prepared in the embodiments 1-4 of the invention.

Cell lines: the human metaplasia large cell lymphoma cell strain Karpas-299 and the non-small cell lung cancer cell strain NCI-H3122 were purchased from Bai Biotech, Inc. of Nanjing Ke.

The method comprises the following steps: collecting cells in logarithmic growth phase, counting, resuspending the cells in complete medium, adjusting cell concentration to the appropriate concentration (determined by cell density optimization assay), inoculating 96-well plates, adding 100. mu.l/wellA suspension of cells. Cells were incubated at 37 ℃ and 100% relative humidity, 5% CO₂Incubate in incubator for 24 hours. The test compound was diluted with the medium to the set corresponding effect concentration and the cells were added at 25. mu.l/well. The final concentration of compound was started at 10. mu.M, and diluted in 3-fold gradient for 10 concentration points. Cells were incubated at 37 ℃ and 100% relative humidity, 5% CO₂Incubate in incubator for 72 hours. After the incubation is finished, the prepared reagent is added into the Cell plate according to the specification of the Cell Titer Glo reagent, and the Cell plate is incubated for 10 minutes at room temperature in a dark place after being fully mixed. The cell plate was placed in a plate reader for analysis, and the chemiluminescence read was set and data recorded. And calculating the inhibition rate. The IC of each compound on each cell was calculated using graphad 6.0 based on the inhibition of each cell growth by different concentrations of each compound₅₀The value is obtained.

The calculation formula is as follows:

as a result: see table three.

Detection of Cell proliferation IC of example compound to Karpas-299/NCI-H3122 Cell line by Episcell Titer GLO method₅₀Value of

IC50(μM)	Karpas-299	NCI-H3122
			REX-C1	<1	<0.2
REX-C2	<1	<1
			REX-C3	<0.3	<0.05
REX-C9	<0.5	<0.5

Example 8 Zebra fish phenotype screening experiment

The zebra fish is a vertebrate, has 85 percent of gene homology with human, has a signal transduction path basically similar to that of the human, and has a biological structure and a physiological function which are highly similar to those of mammals; the zebra fish is small in size, rapid in development, transparent in embryo and high in egg laying amount, and the unique advantages enable the zebra fish to become one of the best model organisms for human disease research and living high-throughput drug screening. Among them, anaplastic lymphoma kinase ALK (anaplastic lymphoma kinase) has 76% of gene homology with zebrafish in human. The leucocyte tyrosine kinase ltk (leucocyte tyrosine kinase) of the zebra fish regulates and controls the generation of the pigmented cells of the iris of the zebra fish (Lopes, S.S., Yang, X., et al (2008). Leucocyte tyrosine kinase functions in pigment cell definition. PLoS Gene, 4.), the pigmented cells of the iris are expressed as a kind of silver corpuscle in the zebra fish and are distributed at the outer side of the head, eyes and spine (observed by reflected light), and the silver corpuscle is observed to be black in the transmitted light by utilizing the albono zebra fish with the loss of the melanin, which is shown in figure 1. ALK and LTK are sitter kinase, researchers found that injection of exogenous ALK plasmid also regulated iridescent pigment cell production, and experimental results also showed that most ALK inhibitors had LTK activity that inhibited iridescent pigment cell production (Rodrigues, F.S., Yang, X., Nikaido, M., Liu, Q., & Kelsh, R.N. (2012). A simple, highly visual in vivo screen for and adaptive fluorescence kinase inhibition. ACS Chem Biol,7, 1968-.

Therefore, by using this principle, we examined the effect of the compound on normal zebra fish iris pigmented cells to investigate the strength of the anti-ALK activity of the compound in vivo.

Effect of the test Compound (I) on Normal Zebra Fish iridescent pigmented cells

The scheme is as follows: selecting 6hpf (waters post fertilization) roes, randomly grouping, adding tested drugs with various concentrations, carrying out image acquisition when the volume reaches 3dpf (days post fertilization), analyzing IOD (integrated optimization) value of iris pigmented cells on the dorsal side of a zebra fish spine from a cloaca to a tail fin part by using ImageJ software, carrying out Dunnett's T-test by using Graphpad prism6.0 for statistical analysis, wherein p <0.05 shows that the statistical difference exists, and the iris pigment inhibition rate calculation formula is as follows:

the calculation results are shown in the fourth table and the fifth table, a dose-effect relationship graph (mean +/-sem) of the compound REX-C1/REX-C3 on the zebra fish iris pigmented cells is shown in the figure 2, and an effect graph of the compound REX-C1/REX-C3 on the zebra fish iris pigmented cells is shown in the figure 3.

TABLE four Effect of the Compound REX-C1 on pigmented cells of Iris membrane of Zebra Fish (mean. + -. sem)

compared with control,*,p<0.05；**,p<0.01

TABLE pentacompound REX-C3 Effect on pigmented cells of Iris membrane of Zebra fish (mean. + -. sem)

compared with control,*,p<0.05；**,p<0.01 。

Claims (7)

1. A boron-substituted aniline protein kinase inhibitor is selected from the following characteristic compounds:

7- ((5-chloro-2- ((2-isopropoxy-5-methyl-4- (4-piperidinyl) phenyl) amine) 4-pyrimidinyl) amine) benzo [ c ] [1,2] oxo 1(3H) boronic acid;

4- ((5-chloro-2- ((2-isopropoxy-5-methyl-4- (4-piperidinyl) phenyl) amine) 4-pyrimidinyl) amine) benzo [ c ] [1,2] oxo 1(3H) boronic acid;

7- ((5-trifluoromethoxy-2- ((2-isopropoxy-5-methyl-4- (4-piperidinyl) phenyl) amine) 4-pyrimidinyl) amine) benzo [ c ] [1,2] oxo 1(3H) boronic acid;

7- ((5-chloro-2- ((2-isopropoxy-5-methyl-4- (1- (morpholinylmethyl) cyclopropyl) phenyl) amine) 4-pyrimidinyl) amine) benzo [ c ] [1,2] oxo 1(3H) boronic acid.

2. A pharmaceutical composition comprising a compound as defined in claim 1, or a pharmaceutically acceptable salt thereof, as an active ingredient, together with one or more pharmaceutically acceptable carriers.

3. Use of a compound as defined in claim 1, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment or prevention of a disease associated with a protein kinase.

4. Use of a compound as defined in claim 1, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment or prevention of a disease associated with anaplastic lymphoma kinase.

5. Use according to claim 3 or 4, characterized in that: the disease is selected from cell proliferative diseases.

6. Use according to claim 5, characterized in that: the disease is selected from tumors.

7. Use according to claim 5, characterized in that: the cell proliferative disease is selected from non-small cell lung cancer, anaplastic large cell lymphoma, inflammatory myofibroblastic tumor, nasopharyngeal carcinoma, breast cancer, colorectal cancer, diffuse large B cell lymphoma, liver cancer, gastric cancer, esophageal cancer, pancreatic cancer, ovarian cancer, systemic histiocytosis or neuroblastoma.

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