CN109988151A

CN109988151A - A kind of acetylene compound, preparation method and applications

Info

Publication number: CN109988151A
Application number: CN201711498588.8A
Authority: CN
Inventors: 张强; 张宏波; 周利凯; 冯守业; 杨海龙; 王中祥
Original assignee: Beijing Saite Mingqiang Medicine Technology Co Ltd
Current assignee: Beijing Saite Mingqiang Medicine Technology Co Ltd
Priority date: 2017-12-29
Filing date: 2017-12-29
Publication date: 2019-07-09
Anticipated expiration: 2037-12-29
Also published as: CN109988151B

Abstract

The invention belongs to chemical medicines, more particularly to formula (I) compound or its pharmaceutically acceptable salt, isomers, solvate, crystallization or prodrug, and the pharmaceutical composition containing these compounds and application of these compound or compositions in medicine preparation.The forms such as this kind of compound and its pharmaceutically acceptable salt, isomers, solvate, crystallization or prodrug can be used for the treatment or prevention of many different cancers and Other diseases.

Description

A kind of acetylene compound, preparation method and applications

Technical field

The invention belongs to chemical medicines, and in particular to one kind have BCR-ABL kinase inhibiting activity compound or Its pharmaceutically acceptable salt, isomers, solvate, crystal form or prodrug, and the pharmaceutical composition containing these compounds and Application of these compound or compositions in medicine preparation.

Background technique

Tumour is body under the action of all kinds of carcinogens, and cell is abnormal signal transduction, portion of tissue some Cell loses the regulation to its normal growth, leads to the imbalance of Apoptosis and the continuous proliferation of cell, and then lead to neoformation It is Clonal growth and formed.Tumour cell possesses autonomous growth ability, and cause after losing normal growth regulating function After the cancer factor stops growing, tumour still is able to continued propagation.Tumour can be clinically divided into solid tumor and non-physical knurl, entity Tumor, that is, tangible tumor is treated by the methods of operation excision, chemotherapy, and non-physical knurl mainly kills cancer with chemicals Cell, but these chemicals side effects are larger, intracorporal cell, all can be by broken regardless of whether be malignant cell It is bad.

Leukaemia is one of malignant tumour, belongs to non-physical knurl, is arranged in the disease incidence of pediatric malignancies the first.Root According to the natural history of leukaemia cell, acute leukemia and chronic leukemia two major classes can be classified as.Wherein chronic leukemia Chronic granulocytic leukemia (Chronic Myelogenous Leukemia, CML) and chronic lymphocytic can be divided into again Leukaemia (Chronic Lymphocytic Leukemia, CLL).Chronic myelocytic leukemia accounts for 20% left side of all leukaemia The right side is fallen ill in whole age groups.

Currently, selectively acting in specific target spot low toxicity and high specificity new anticancer drug have become it is antitumor The new direction of drug research, in chronic myelocytic leukemia (CML) patient, No. 22 chromosome long arm transpositions to No. 9 chromosomes, shape At Philadelphia chromosome, and BCR gene and abl gene is caused to merge to form BCR-ABL fusion, expresses BCR-ABL protein junket Histidine kinase, the kinases can cause cell Proliferation, stick the change with nature of life, lead to the generation of kinds of tumors.BCR-ABL It is not expressed in normal cell, so it is the treatment ideal drug targets of chronic myelocytic leukemia.

At present clinically it is most common for BCR-ABL tyrosine kinase micromolecular inhibitor include: first generation drug she Imatinib；Second generation drug Dasatinib, nilotinib and Bosutinib；Third generation drug Ponatinib.Tyrosine kinase suppression Preparation mainly passes through the activity for inhibiting BCR-ABL fusion protein, to play the effect of anti-chronic myelocytic leukemia.

Imatinib (Imatinib) is the small molecule BCR-ABL tyrosine kinase developed by Novartis Co., Ltd, in It is used to treat in CML by FDA approval within 2001.This is the tyrosine kinase inhibitor of first treatment CML, it can pass through targeting The specific defective gene of tumour cell carrys out treating cancer.Compared with other therapeutic agents, Imatinib can effectively be alleviated slowly Property granulocytic leukemia, 5 annual survival rate of patient is up to 90% after treatment.One distinguishing feature of its Imatinib is that it can be special The proliferation of anisotropic inhibition chronic myelocytic leukemia cancer cell, to normal cell then almost without injury, this is greatly reduced The toxic side effect of drug.Imatinib has been started using kinases as the new era of target treatment disease.

The appearance of drug resistance largely reduces the therapeutic effect of Imatinib.There is drug resistance in Imatinib Main cause be BCR-ABL gene have occurred including L248V, E255V, Y253H, E355G, E255K, T315I, F359V, The mutation such as M253H, G250E, F317L, H396P, M351T, Q252H, due to the point mutation of ABL kinases, reduces Imatinib Affinity between ABL kinases causes its therapeutic effect to be decreased obviously.

Second generation Bcr-Abl tyrosine kinase inhibitor nilotinib (Nilotinib) is that a kind of aniline pyrimidine class is derivative Object is approved listing by U.S. FDA in October, 2007 for treating CML.Its affinity ratio to Bcr-Abl tyrosine kinase Imatinib is 20 times strong.Nilotinib can inhibit the drug resistant mutation of Imatinib in addition to T315I is mutated.But it uses There are lipase and bilirubin raising, light Mild skin rashes, bone marrow suppression, stomach and intestine by most of patient of CML of Nilotinib treatment The common adverse reactions such as road reaction.

Dasatinib (Dasatinib) is also second generation Bcr-Abl tyrosine kinase inhibitor, it is a kind of to a variety of sharp Enzyme has the oral kinase inhibitor of inhibiting effect, and to BCR-ABL kinases and SRC family kinase, (SRC kinases is antineoplastic One target spot of object effect) there is good inhibitory effect.Dasatinib obtains FDA approval listing in June, 2006 and is used to control Treat CML patient.Dasatinib requires the drug resistance few, it can overcome a variety of Imatinibs to occur compared with Imatinib specificity structure Property (except T315I be mutated).Dasatinib by oral administration after be absorbed rapidly, maximum plasma concentration is reached in 0.5-3h, be averaged Half-life period is 5-6h.Patient takes the main adverse reaction after Dasatinib and shows bone marrow suppression and hyperhypercytosis.

Bosutinib (Bosutinib) is the 4- substituted aniline -3- quinolinecarbonitriles class researched and developed by Wyeth Pharmaceuticals, the U.S. The new drug of CML is treated, in September, 2012 is ratified to list, be controlled primarily directed to Imatinib, nilotinib and Dasatinib by FDA Treat the kinase inhibitor of the CML patient of failure.Antiproliferative activity (IC of the Bosutinib to two kinds of cells of KU812 and K562₅₀) point Not Wei 20nM and 5nM, and Imatinib is respectively 210nM and 88nM to the antiproliferative activity of KU812 and K562 cell.But uncle is relaxed No inhibitory effect equally is mutated to T315I for Buddhist nun.The adverse reaction that the patient for taking Bosutinib occurs specifically includes that evil The heart, vomiting, abdominal pain, diarrhea, fash, liver enzyme level raising, decrease of platelet and anaemia and fatigue.

Second generation treatment CML drug Dasatinib, Nilotinib and Bosutinib to Imatinib drug resistance and is not resistant to There is extensive activity in the patient received, but all do not have inhibitory activity to BCR-ABL T315I kinase mutant.

Although being obtained using protein tyrosine kinase as the small-molecule drug of target for treating chronic granulocytic leukemia Very big success is used since the appearance of drug resistance greatly limits it.At present oneself identify 17 kinds this swash Mutation in enzyme area, including 6 kinds of known imatinib-resistants mutation (M244V, Y253H, F359C/V/I, G250E, E255K and T315I) and 11 kinds of newly-increased mutation (K247N, E282K, K285N, V289L, L273F, E292K, N297T, H375P, T406I, W430L and E431G).Due to the appearance of second generation BCR-ABL tyrosine kinase drug resistance, exploitation Novel B CR-ABL tyrosine kinase inhibitor is necessary out.

Third generation BCR-ABL tyrosine kinase Ponatinib (Ponatinib) is that a kind of oral type multiple target point is sharp Enzyme inhibitor.It is primarily used to overcome BCR-ABL^T315IIt requires also to have the BCR-ABL of wild type simultaneously to inhibit effect well Fruit.Ponatinib can inhibit containing the BCR-ABL kinase activity including T315I mutation, according to document (Rabindran SK, et Al.Cancer Res, 2004,64 (11), 3958-3965) record, Ponatinib is to wild type BCR-ABL kinases and BCR- ABL^T315IThe binding pattern of kinases only has small difference, to the inhibitory activity (IC of wild type BCR-ABL kinases₅₀) be BCR-ABL^T315IInhibitory activity (IC₅₀) 5-7 times.The patient for taking Ponatinib will appear serious bad vascular events, packet The myocardial infarction, stroke, limb blood flow for including mortality and threat to life, which are interrupted, causes tissue necrosis etc..Its serious side effect limitation The clinical application of the drug.

Summary of the invention

The present invention relates to a kind of acetylene compound and its pharmaceutically acceptable salt, isomers, solvate, crystal forms or preceding Medicine can be used as treating or preventing the disease as caused by BCR-ABL kinases overexpression, including imatinib-resistant mutation (M244V, Y253H, F359C/V/I, G250E, E255K and T315I) and 11 kinds of newly-increased mutation (K247N, E282K, K285N, V289L, L273F, E292K, N297T, H375P, T406I, W430L and E431G).This kind of compound and its pharmaceutically acceptable The forms such as salt, prodrug can be used for the treatment or prevention of many different cancers.The invention further relates to remove to include the compound And its prodrug, it further include the pharmaceutical composition of its effective polymorphic forms, the intermediate of the compound, in various not similar shapes The compound and its salt used in the treatment method for the disease that the BCR-ABL of formula is mediated.

The present invention provides a kind of acetylene compound and its be used as treat or prevent by tyrosine kinase (such as ABL, ) or the purposes of disease caused by its mutation VEGFR2.

The present invention provides a kind of compound or its isomers, tautomer, solvate, hydrate or its pharmaceutically Acceptable salt.The present invention is provided in the form of its effective polymorphous pharmaceutical composition.The compound has structure Formula (I):

Wherein,

X is N, CH；

R¹For

A) aryl or 5-6 member hetero-aromatic ring unsubstituted or replaced by 1-3 identical or different Ra, the 5-6 member are miscellaneous Aromatic ring is the hetero atom that N, O and S are selected from containing 1-3；

B) unsubstituted or replaces ring A and ring B by 1-2 identical or different Rb, wherein ring A for 5-6 member hetero-aromatic ring or 6 yuan of aromatic rings, ring B are selected from 3-6 member naphthenic base, 3-6 circle heterocyclic ring, 5-6 member hetero-aromatic ring or aromatic ring, and the heterocycle and hetero-aromatic ring difference are only The vertical hetero atom that N, O and S are selected from containing 1-3；

Ra is H, hydroxyl, cyano, acyl group, halogen, the unsubstituted or C that is replaced by hydroxyl or halogen₁-C₆Alkyl ,-OR' Or-NR'R "；

Rb is H, halogen, hydroxyl, cyano, carbonyl, acyl group, C₁-C₆Alkyl ,-OR'；

R' and R " is independently H, C₃-C₆Naphthenic base, it is unsubstituted or by hydroxyl, carboxyl, halogen, C₁-C₆Alkoxy Or C₁-C₆The C that alkylamino replaces₁-C₆Alkyl；

R²For H, C₁-C₆Alkyl；

R³For

D ring is unsubstituted or optionally by (Rd)_mSubstituted 5-6 member naphthenic base, 5-6 member contain 1-3 selected from N, O and S Heteroatomic heterocycle, 5-6 member aromatic ring or 5-6 member contain 1-3 selected from the heteroatomic hetero-aromatic ring of N, O and S；

(Rd)_mRepresenting m identical or different substituent R d, m is the integer of 0-4, Rd H, halogen, hydroxyl, cyano, C₁- C₆Alkoxy, C₁-C₆Alkylamino, it is unsubstituted or by hydroxyl, halogen, C₁-C₆Alkoxy, C₁-C₆The C that alkylamino replaces₁-C₆Alkane Base, or be-L-E- (Re)_n；

L is-(CR^l1R^l2)_q-；Q is the integer of 1-4；

R^l1And R^l2It is independently H or C₁-C₆Alkyl；

E is 3-6 member ring group or heterocycle, and the 3-6 circle heterocyclic ring base is to be selected from the heteroatomic saturation of N, O and S containing 1-3 Heterocycle or unsaturated heterocycle base；

(Re)_nIt is the integer of 0-4, Re H, halogen, hydroxyl, C for n identical or different Re, n₁-C₆Alkoxy, C₁-C₆ Alkylamino, it is unsubstituted or by halogen, hydroxyl, C₁-C₆Alkoxy or C₁-C₆The C that alkylamino replaces₁-C₆Alkyl.

Term " substitution " referred to here, including complicated substituent group (for example, phenyl, aryl, miscellaneous alkyl, heteroaryl), Proper is 1 to 5 substituent group, preferably 1 to 3, preferably 1 to 2, can freely be selected from substituent group list It selects.

Unless there are specified otherwise, alkyl as used herein includes the alkyl of saturation unit price, these alkyl have a straight chain, branch or Annulus.For example, alkyl includes methyl, ethyl, propyl, isopropyl, cyclopropyl, n- butyl, isobutyl group, sec-butyl, tert- Butyl, cyclobutyl, n- amyl, 3- (2- methyl) butyl, 2- amyl, 2- methyl butyl, neopentyl, cyclopenta, n- hexyl, 2- oneself Base, 2- methyl amyl and cyclohexyl.Alkoxy is by previously described straight chain, the oxide ether of branched chain or cyclic alkyl composition.Class As, alkenyl and alkynyl include straight chain, branched chain or cyclic alkenyl radical and alkynyl.

Term " aryl " used herein refers to unsubstituted or substituted virtue unless otherwise specified Perfume base, such as phenyl, naphthalene, anthryl.Term " aroyl " refers to-C (O)-aryl.

Term " heterocycle " used herein represents unsubstituted or substituted steady unless there are specified otherwise 3 to 8 fixed unit monocycle saturated ring systems, they are made of carbon atom and 1 to 3 hetero atom selected from N, O, S, wherein N, S hetero atom can be aoxidized arbitrarily, and N hetero atom can also be by arbitrarily quaternized.Heterocycle can be with any hetero atom or carbon atom In conjunction with thus one stable structure of composition.The example of this kind of heterocycle includes but is not limited to azacyclo- ethyl group, pyrroles Alkyl, piperidyl, piperazinyl aoxidize piperazinyl, and oxyl base aoxidizes azepine base, azepine base, tetrahydrofuran base, dioxy penta Ring group, imidazolidine base, tetrahydro-thiazoles base, tetrahydro oh oxazolyl, THP trtrahydropyranyl, morpholine base, thio-morpholine group, thiophene morphine Quinoline sulfoxide, thiophene morpholine sulfone and oh di azoly.

Term " heteroaryl " used herein represents unsubstituted or substituted stabilization unless otherwise specified 5 or 6 unit monocycle aromatic ring systems, unsubstituted or substituted thick benzene heteroaromatic ring systems of 9 or 10 yuan of benzene can also be represented Or bicyclic heteroaromatic ring system, they are formed by carbon atom and by 1 to 4 hetero atom selected from N, O, S, wherein N, the miscellaneous original of S Son can be aoxidized arbitrarily, and N hetero atom can also be by arbitrarily quaternized.Heteroaryl can stick with any hetero atom or carbon atom Get up, thus one stable structure of composition.The example of heteroaryl includes but is not limited to thianthrene group, furyl, imidazoles Base, isoxazolyl, oh oxazolyl, pyrazolyl, pyrrole radicals, thiazolyl, thiadiazolyl group, triazolyl, pyridyl group, pyridazinyl, indyl, Azaindolyl, indazolyl, benzimidazolyl, benzofuranyl, benzothienyl, benzo isoxazolyl, benzoxazolyl, benzene And pyrazolyl, benzothiazolyl, diazosulfide base, benzotriazole base, adenyl, quinolyl or isoquinolyl.

Term " carbonyl " refers to C (O) base.

No matter when term " alkyl " or " aryl " or they any prefix root appear in the title of a substituent In (for example, aralkyl, dialkyl amino), those of it will be considered containing the above are " alkyl " and " aryl " and provide limit System.The specified quantity of carbon atom is (for example, C₁‐C₆) indicate independent in a moieties or in a bigger substituent group In moieties (wherein alkyl is as its prefix root) in carbon atom quantity.

Formula (I) compound or its isomers, tautomer, solvation are provided in a preferred embodiment of the invention Object, hydrate and its pharmaceutically acceptable salt,

Wherein,

X is N, CH；

R¹For

A) unsubstituted or replace 5-6 circle heterocyclic ring or 5-6 unit's heteroaryl, the heterocycle by 1-3 identical or different Ra With the hetero atom for being selected from N, O and S containing 1-3 of hetero-aromatic ring independently；

It is b) unsubstituted or replaces ring A simultaneously ring B by 1-2 identical or different Rb,

Ring A is 5-6 member hetero-aromatic ring, and ring B is selected from 3-6 circle heterocyclic ring or 3-6 member hetero-aromatic ring；

The hetero atom that N, O and S are selected from containing 1-3 of the heterocycle and hetero-aromatic ring independently；

Ra is-H, hydroxyl, cyano, halogen, the unsubstituted or C that is replaced by hydroxyl or halogen₁-C₃Alkyl ,-OR' or NR' R"；

R' and R " is independently H, C₁-C₄Alkyl, C₃-C₄Naphthenic base, it is unsubstituted or by halogen, C₁-C₃Alkoxy Or C₁-C₃The C that alkylamino replaces₁-C₃Alkyl；

Rb is-H, halogen, CF₃, carbonyl, cyano, C₁-C₃Alkyl；

R²For H, C₁-C₄The alkyl of linear chain or branched chain；

R³For

D ring is unsubstituted or optionally by (Rd)_mThe 6 yuan of aromatic rings or 5-6 member replaced contain 1-3 selected from the miscellaneous original of N, O and S The aromatic ring of son；

(Rd)_mFor m identical or different substituent R d, m 0,1,2 or 3；Rd is H, halogen, hydroxyl, cyano, C₁-C₄ Alkoxy, C₁-C₄Alkylamino, it is unsubstituted or by hydroxyl, halogen, C₁-C₄Alkoxy, C₁-C₄The C that alkylamino replaces_1-4Alkyl, It or is-L-E- (Re)_n；

L is-(CR^l1R^l2)_q, R^l1And R^l2It is independently H, C_1-3Alkyl, q are the integer of 1-3,

E is 3-6 circle heterocyclic ring base, and the 3-6 circle heterocyclic ring base is to be selected from the heteroatomic saturated heterocyclic of N, O and S containing 1-3 Base or unsaturated heterocycle base；

(Re)_nFor n identical or different substituent R e, Re H, halogen, hydroxyl, C_1-3Alkoxy, C_1-3Alkylamino, not It is substituted or by halogen, hydroxyl, C₁-C₃Alkoxy or C₁-C₃The C that alkylamino replaces₁-C₄Alkyl, n 0,1,2 or 3.

In another preferred embodiment, the compound or its isomers, tautomer, solvate, Hydrate and its pharmaceutically acceptable salt, R²For H, methyl, ethyl, propyl or isopropyl.

In one of scheme, the compound or its isomers, tautomer, solvate, hydrate and its Pharmaceutically acceptable salt,

R¹For,

(Ra)^pTo be replaced by p identical or different Ra, p 0,1,2 or 3；

Ra is-H, and fluorine, chlorine, bromine, methyl, ethyl, propyl, butyl, isopropyl, trifluoromethyl ,-NHR', R' are-H, first Base, ethyl, amino-ethyl, methylaminoethyl, dimethylaminoethyl, cyclopropyl, methoxy ethyl, aminopropyl, methylamino third Base, dimethylamino-propyl, cyclobutyl or methoxy-propyl；

(Rb)^rTo be replaced by r identical or different Rb, r 0,1 or 2；

Rb is-H, fluorine, chlorine, bromine, trifluoromethyl, carbonyl, cyano, methyl, ethyl, propyl, isopropyl.

R³For

D ring is unsubstituted or by (Rd)_mSubstituted aromatic ring or hetero-aromatic ring, the aromatic ring are phenyl ring, the hetero-aromatic ring choosing From pyridine, pyridazine or thiazole；

(Rd)_mFor m identical or different substituent R d, m 0,1,2 or 3, Rd H, halogen, hydroxyl, cyano, C_1‐C₄ Alkoxy, C_1‐C₄Alkylamino, it is unsubstituted or by hydroxyl, halogen, C_1‐C₄Alkoxy, C_1‐C₄The C that alkylamino replaces_1‐4Alkyl, or Person is-L-E- (Re)_n；

L is-(CH₂)_q, q 1,2 or 3；

E is 3-6 circle heterocyclic ring base, and the 3-6 circle heterocyclic ring is selected from azirane, ethylene oxide, pyrrolidines, pyrrolin, pyrrole It coughs up, pyrazolidine, pyrazoline, imidazoles, pyrazoles, furans, tetrahydrofuran, dihydrofuran, thiophane, thiophene, oxazole, thiazole, thiophene Diazole, piperidines, pyridine, dihydropyridine, morpholine, piperazine or pyrazine；

(Re)_nFor n identical or different Re, Re H, halogen, hydroxyl, C_1‐3Alkoxy, C_1‐3Alkylamino, it is unsubstituted Or by halogen, hydroxyl, C_1‐C₃Alkoxy or C_1‐C₃The C that alkylamino replaces_1‐C₃Alkyl, n 0,1,2 or 3.

R³For

D ring is unsubstituted or by (Rd)_mSubstituted phenyl ring or pyridine,

(Rd)_mFor m identical or different substituent R d, m 0,1 or 2, Rd H, fluorine, chlorine, bromine, hydroxyl, cyano, first Base, ethyl, propyl, isopropyl, butyl, tert-butyl, methoxyl group, ethyoxyl, propoxyl group, isopropoxy, methyl fluoride, difluoro first Base, trifluoromethyl, fluorine methoxyl group, difluoro-methoxy, trifluoromethoxy, methylol, ethoxy, hydroxypropyl, amino, methylamino, Ethylamino, dimethylamino, amino methyl, amino-ethyl, aminopropyl, Methyaminomethyl, methylaminoethyl, methylaminopropyl, Ethylaminomethyl, ethylaminoethyl, ethylaminopropyl, dimethylamino methyl, dimethylaminoethyl, dimethylamino-propyl, or For-L-E- (Re)_n,

L is-(CH₂)_q, q 1,2 or 3；

E is 5-6 circle heterocyclic ring, and the 5-6 circle heterocyclic ring is selected from:

(Re)_nFor identical or different substituent R e, the Re H of n, fluorine, chlorine, bromine, hydroxyl, methyl, ethyl, propyl is different Propyl, a methyl fluoride, difluoromethyl, trifluoromethyl, methoxyl group, methoxy methoxy ethyl, amino, methylamino, diformazan Amino, amino methyl, Methyaminomethyl, dimethylamino methyl, amino-ethyl, methylaminoethyl, dimethylaminoethyl, hydroxyl first Base, ethoxy, n 0,1 or 2.

It is clear that the compound and its officinal salt of molecule formula (I) may exist solvation form and nonsolvated forms. Such as solvation form can be water-soluble form.The present invention includes all these solvations and non-solvated form.

The compound of the present invention may have asymmetric carbon atom, according to their physical and chemical difference, by known technology Mature method, for example, this diastereoisomeric mixture can be separated into single by chromatography or Steppecd crystallization Diastereoisomer.The separation of enantiomter can be by first being carried out instead with the compound (as ethyl alcohol) for suitably having optically active It answers, the mixture of enantiomerism is converted to diastereoisomeric mixture, separates diastereoisomer, then single diastereomeric Isomers converts (hydrolysis) into corresponding pure enantiomter.All such isomers, including diastereoisomer mixing Object and pure enantiomer are considered as a part of the invention.

The compound of the present invention can also exist in the form of pharmaceutically acceptable salt class.In pharmaceutical applications, this hair The salt of bright compound refers to avirulent pharmaceutically acceptable salt class.Pharmaceutically acceptable salt class form includes pharmaceutically may be used Acidity/the anion salt or basic/cationic salts of receiving.The form that pharmaceutically acceptable acidity/anion salt is usually taken It is that basic nitrogen therein is allowed to be protonated by inorganic or organic acid.Representative organic or inorganic acid includes hydrochloric acid, hydrobromic acid, hydrogen iodine Acid, perchloric acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, propionic acid, hydroxyacetic acid, lactic acid, succinic acid, maleic acid, tartaric acid, citric acid, Benzoic acid, mandelic acid, methanesulfonic acid, isethionic acid, benzene sulfonic acid, oxalic acid, palmitinic acid, 2- naphthalene sulfonic acids, p-methyl benzenesulfonic acid, hexamethylene Amidosulfonic acid, salicylic acid, saccharinic acid, trifluoroacetic acid.Pharmaceutically acceptable basic/cationic salts class includes (not only limiting certainly In this) aluminium, calcium, chloroprocanine, choline, diethanol amine, ethylenediamine, lithium, magnesium, potassium, sodium and zinc.

The present invention includes the pro-drug that the compound of the invention in its field is constituted.In general, this kind of pro-drug It is the functional derivatives that those are easy the compound of drug required for being converted into vivo.So in treatment of the invention In method, term " administration " will can use compound disclosed in having defined can also comprising treating disease described by various differences With with may be without it is expressly intended that but can be converted into vivo what those had been made clear at it after main body is given these drugs The compound of drug.The traditional program of selection and preparation about suitable prodrug derivatives, as " pro-drug Design " it described in (H.Bundgaard chief editor, Elsevier, 1985.) this reference book.

It will be apparent that the definition of any substituent group or variable in a molecule on specific position and the substituent group or variable The definition in other places in that molecule is irrelevant.We are readily appreciated that, the substitution in the compound of the present invention and Substitute mode can be selected with very common technology on a kind of subject, chemically stable and easy on subject to provide Existing technology and methods presented herein are come the compound that synthesizes.

The prodrug of parent compound refers to that those derivatives and prodrug can be improved the compound when giving mammal Bioavilability (for example, being absorbed into blood by allowing to enhance after oral) or can be faster relative to parent compound It is transferred to vivo biodistribution position of interest.Relative to parent compound, currently preferred prodrug includes that there is enhancing to pass through The derivative of the compound of goldbeater's skin, water solubility or active transport.

The present invention also provides the methods for preparing compound of formula I described previously, are such as prepared by following methods.

The present invention provides a kind of method for preparing aforementioned formula (I) compound, the compound as shown in formula D and R¹X reacts It arrives, wherein R¹, R², R³As hereinbefore defined,

In a preferred scheme, formula D and R¹When X reacts, in organic solvent, catalyst, organic base and fluorine ion Salt under the conditions of, compound shown in preparation formula I.

Organic solvent is including but not limited to toluene, dimethylbenzene, tetrahydrofuran, ethyl acetate, dioxane, N- methylpyrrole A combination of one or more in alkanone, n,N-Dimethylformamide and benzene.

Wherein, catalyst including but not limited to cuprous iodide withPalladium diphenyl phosphine dichlorideOr cuprous iodide withFour triphens Base phosphine palladium；

Organic base is including but not limited to triethylamine, diisopropyl ethyl amine, 1,8- diazabicylo, 11 carbon -7- alkene or N- Methyl morpholine a combination of one or more；

The salt of fluorine ion is including but not limited to one or more of tetrabutyl ammonium fluoride, potassium fluoride or cesium fluoride Combination.

The present invention provides a kind of method for preparing aforementioned formula (I) compound, and it includes following steps, i.e., changes as shown in formula C It closes object and trimethyl ((tributylstamlyl) acetenyl) silane reaction obtains formula D compound

In a preferred scheme,

When formula C compound represented and trimethyl ((tributylstamlyl) acetenyl) silane reaction, in organic solvent In, under catalysts conditions, compound shown in formula D is prepared.

Preferably, the catalyst including but not limited to tetra-triphenylphosphine palladium,Double (bis- Ya Benzyl benzylacetones) palladium, palladium acetate,Palladium diphenyl phosphine dichlorideMiddle a combination of one or more.

Preferably, organic solvent is including but not limited to toluene, dimethylbenzene, tetrahydrofuran, dioxane, N- crassitude A combination of one or more in ketone, n,N-Dimethylformamide and benzene.

In a preferred scheme,

A method of compound described previously being prepared, it includes the compounds as shown in formula A to react with compound shown in formula B Formula C compound is obtained,

In a preferred scheme, when compound shown in formula A is reacted with compound shown in formula B, wherein formula A can be with acylation Reagent reaction, then reacted with formula B.

Preferably, the acylating reagent include but is not limited to phosphorus oxychloride, thionyl chloride, oxalyl chloride, phosphorus trichloride or A combination of one or more in phosphorus pentachloride.

In another embodiment, compound shown in formula A reacts in the presence of condensing agent with compound shown in formula B, Compound shown in formula C is obtained,

Preferably, the condensing agent includes but is not limited to 2- (7- aoxidizes benzotriazole)-N, N, N', N'- tetramethylurea Hexafluorophosphoric acid ester (HATU), propylphosphonic anhydride (T3P), carbodiimide type condensing agent, carbonium ion type condensing agent, urea ionic Condensing agent, a combination of one or more in 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (EDCI)；

Preferably, this step can carry out in organic base, and the organic base is including but not limited to triethylamine, diisopropyl Ethylamine, pyridine, 4-dimethylaminopyridine, 1,8- diazabicylo, 11 carbon -7- alkene or N-methylmorpholine one or two with On combination.

The present invention provides a kind of method for preparing formula (I) described compound above, which is characterized in that it changes as shown in formula D1 Close object and R³NH₂Reaction obtains, wherein R¹, R², R³As hereinbefore defined,

In a preferred scheme, compound shown in formula D1 and formula R³NH₂When shown compound reacts, wherein formula D1 can Reacted with acylating reagent, then with formula R³NH₂It is reacted.

In another embodiment, compound shown in formula D1, in the presence of condensing agent, with formula R³NH₂Shown compound is anti- It answers, obtains compound shown in Formulas I,

The present invention provides a kind of method for preparing compound described previously, which is characterized in that it includes following steps, i.e., by Compound described in C1 obtains compound described in F1 through hydrolysis,

Wherein, wherein R¹And R²As hereinbefore defined.

Compound shown in formula C1, existing for the inorganic base under the conditions of or under conditions of be not present, organic solvent and water it is mixed In bonding solvent, hydrolysis obtains compound shown in formula D1.

Preferably, above-mentioned reaction can be achieved using protic and non-proton organic solvent in organic solvent, all common Organic solvent this reaction can application range.

The present invention provides a kind of method for preparing compound described previously, and it includes following steps, i.e. the change as described in B1 Close object and R¹X reacts to obtain compound described in C1,

Wherein R¹And R²As hereinbefore defined.

In a preferred embodiment, formula B1 and R¹When X reacts, in organic solvent, catalyst, organic base and fluorine ion Salt under the conditions of, compound shown in preparation formula C1.

Preferably, organic solvent is including but not limited to toluene, dimethylbenzene, tetrahydrofuran, ethyl acetate, dioxane, N- A combination of one or more in methyl pyrrolidone, n,N-Dimethylformamide and benzene.

Wherein, catalyst is including but not limited to cuprous iodide and palladium diphenyl phosphine dichloride or cuprous iodide and four triphens Base phosphine palladium；

The present invention provides a kind of method for preparing compound described previously, and it includes following steps, i.e. compound shown in A1 Compound shown in B1 is obtained with trimethyl ((tributylstamlyl) acetenyl) silane reaction,

Wherein R²As hereinbefore defined.

In a preferred embodiment, when formula A1 is with trimethyl ((tributylstamlyl) acetenyl) silane reaction, having In solvent, under catalysts conditions, compound shown in formula B1 is prepared,

Preferably, the catalyst is including but not limited to tetra-triphenylphosphine palladium, double (bis- Ya Benzyl benzylacetones) palladium, palladium acetate, A combination of one or more in palladium diphenyl phosphine dichloride.

The present invention provides the preparation method of compound shown in Formulas I above, reaction process is as follows:

When step 1) Chinese style A is reacted with compound shown in formula B, wherein formula A can be reacted with acylating reagent, the acylated examination Agent includes but is not limited to group one or more kinds of in phosphorus oxychloride, thionyl chloride, oxalyl chloride, phosphorus trichloride or phosphorus pentachloride It closes, then is reacted with formula B；Or under the conditions of condensing agent, reacted with formula B, compound shown in formula C is prepared, wherein institute Stating condensing agent includes but is not limited to 2- (7- aoxidize benzotriazole)-N, N, N', N'- tetramethylurea hexafluorophosphoric acid ester (HATU), Propylphosphonic anhydride (T3P), carbodiimide type condensing agent, carbonium ion type condensing agent, urea ionic condensing agent, 1- (3- diformazan Aminopropyl) a combination of one or more in -3- ethyl-carbodiimide hydrochloride (EDCI).Preferably, this step can be It is carried out in organic base, the organic base is including but not limited to triethylamine, diisopropyl ethyl amine, pyridine, 4- dimethylamino pyrrole Pyridine, 1,8- diazabicylo, 11 carbon -7- alkene or N-methylmorpholine a combination of one or more.

When step 2) Chinese style C is with trimethyl ((tributylstamlyl) acetenyl) silane reaction, in organic solvent, urge Under the conditions of agent, compound shown in formula D is prepared, wherein catalyst is including but not limited to tetra-triphenylphosphine palladium, double (bis- Ya Benzyl Benzylacetone) palladium, palladium acetate, a combination of one or more in palladium diphenyl phosphine dichloride, including but not limited to toluene, two It is one or more kinds of in toluene, tetrahydrofuran, dioxane, N-Methyl pyrrolidone, n,N-Dimethylformamide and benzene Combination.

Step 3) Chinese style D and R¹When X reacts, in organic solvent, under the conditions of the salt of catalyst, organic base and fluorine ion, Compound shown in preparation formula I.Wherein, catalyst is sub- including but not limited to cuprous iodide and palladium diphenyl phosphine dichloride or iodate Copper and tetra-triphenylphosphine palladium；Organic base is including but not limited to triethylamine, diisopropyl ethyl amine, 1,8- diazabicylo 11 Carbon -7- alkene or N-methylmorpholine a combination of one or more；The salt of fluorine ion is including but not limited to tetrabutyl ammonium fluoride, fluorine Change the combination of one or more of potassium or cesium fluoride.

In another embodiment, the present invention provides the preparation methods of compound shown in Formulas I, and reaction process is such as Under:

When step 1) Chinese style A1 is with trimethyl ((tributylstamlyl) acetenyl) silane reaction, in organic solvent, Under catalysts conditions, compound shown in formula B1 is prepared,

It is preferred that wherein catalyst is including but not limited to tetra-triphenylphosphine palladium, double (bis- Ya Benzyl benzylacetones) palladium, palladium acetate, two A combination of one or more in triphenylphosphine palladium.

Step 2) Chinese style B1 and R¹When X reacts, in organic solvent, the salt condition of catalyst, organic base and fluorine ion Under, compound shown in preparation formula C1.

A combination of one or more in cuprous iodide and tetra-triphenylphosphine palladium；

Compound shown in step 3) Chinese style C1, under conditions of inorganic base, the in the mixed solvent of organic solvent and water, hydrolysis Obtain compound shown in formula D1.

It is preferred that wherein inorganic base is including but not limited to a combination of one or more in sodium hydroxide, lithium hydroxide；

It is preferred that above-mentioned reaction, institute can be achieved using protic and non-proton organic solvent in organic solvent organic solvent Have common organic solvent this reaction can application range, including but not limited to one or more kinds of in tetrahydrofuran, methanol Combination.

Step 4) Chinese style D1 and formula R³NH²Shown compound reaction,

In an arrangement, formula D1 and formula R³NH²Shown compound reaction, wherein formula D1 can be reacted with acylating reagent, then With R³NH²It is reacted, compound shown in Formulas I is prepared,

The acylating reagent includes but is not limited to phosphorus oxychloride, thionyl chloride, oxalyl chloride, phosphorus trichloride or phosphorus pentachloride Middle a combination of one or more；

In another embodiment, compound shown in formula D1, under the conditions of condensing agent, with formula R³NH²Reaction, is prepared into To compound shown in Formulas I,

It is clear that the compound of molecular formula I, isomers, crystal form or prodrug and its officinal salt may exist solvation shape Formula and nonsolvated forms.Such as solvation form can be water-soluble form.The present invention include all these solvations and not The form of solvation.

Any one of one aspect of the present invention is a kind of pharmaceutical composition, including compound described herein (or prodrug, Or its pharmaceutically acceptable salt or other pharmaceutically acceptable derivates) and it is one or more pharmaceutically acceptable Carrier or excipient.These compositions can optionally further include one or more other therapeutic agents.The compound of the present invention It can be with one or more other therapeutic schemes (for example, Sorafenib or other kinase inhibitors, interferon, bone-marrow transplantation, method Farnesyl transferase enzyme inhibitor, diphosphonate, the application combination of Thalidomide, cancer vaccine, hormonotherapy, antibody, radiation etc.) altogether It is same to be applied to required patient.The pharmaceutical composition of compound can be one or more anticancer agents.

Composition of the invention includes the compound of the present invention and pharmaceutically acceptable carrier, including any and all molten Agent, diluent or other carriers, dispersion or suspension aids, surfactant, isotonic agent, thickener or emulsifier, are consolidated preservative Body adhesive, lubricant etc., to be suitable for required particular dosage form.The example packet of some pharmaceutically acceptable carrier materials It includes, but is not limited to, sugar, such as lactose, dextrose and saccharose；Starch such as cornstarch and potato starch；Cellulose and its derivative Object such as sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate；Powdered tragacanth；Malt；Gelatin；Talcum powder；Excipient, Such as cocoa butter and suppository wax；Oil such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil；Second two Alcohol, such as propylene glycol；Esters such as ethyl oleate and ethyl laurate, agar；Buffer such as magnesium hydroxide and aluminium hydroxide；Algae Acid；Apirogen water；Isotonic saline solution；Ringer's solution；Ethyl alcohol and phosphate buffer solution and other nontoxic compatibility profits Lubrication prescription such as NaLS and magnesium stearate and colorant, releasing agent, coating agent, sweetener, flavoring agent and aromatic, Preservative and antioxidant can also may be present in composition.

It is made of pharmaceutically acceptable carrier and any compound described above.For example the invention is logical It crosses and mixes a kind of Pharmaceutical Compositions made by any compound described above and pharmaceutically acceptable carrier.To the invention Demonstration be one the Pharmaceutical Compositions as composed by any compound and pharmaceutically acceptable carrier described above be made Process.

This composition can be applied to subject in need to inhibit its growth, the cancer and/or transfer of development, including Solid tumor or non-physical knurl (such as chronic granulocytic leukemia, gastrointestinal stromal tumor, acute myeloblastic leukemia, thyroid gland Cancer etc.), including those by Imatinib, Dasatinib, nilotinib, Bosutinib or other kinase inhibitor for treating have it is anti- The cancer of pharmacological property.

Term " main body " referred to here, refers to a kind of animal, especially mammal, most often refers to the mankind, The mankind are often taken as the object for the treatment of, observation or test.Term " dose therapeutically effective ", refers to reactive compound or pharmacy The dosage of ingredient, these dosage can organized, and lead to biology or reaction medically in animal or human body, and such reaction What exactly studied member, animal doctor, physician or other clinicians were looked for, including just treated disease or mix Symptom mitigation.

As the Pharmaceutical Compositions including the compound of the present invention of active constituent, and prepare the side of the instant compound Method is all the current contents of the present invention.Moreover, the crystalline forms of some compounds can be used as polycrystal presence, this form It may also be included in that in current invention.In addition, some compounds can be with water (i.e. hydrate) or commonly organic molten Agent is formed together solvate, and this solvate also is attempted to include in the scope of the present invention.

In order to prepare the Pharmaceutical Compositions of this invention, one or more chemical combination of the molecule formula (I) as its active constituent Object or salt can be mixed closely with pharmaceutical carriers, this be carried out according to traditional pharmacy ingredients technical, wherein Carrier can be used a variety of more according to by different administration mode (for example, oral or parenteral administration) designed preparation form The form of sample.Pharmaceutically acceptable carrier appropriate is technically well-known.To some of such pharmaceutically acceptable The description of carrier can be found " pharmaceutical excipient handbook " is inner, and the book is by American Pharmaceutical Association and pharmacy society, Britain combined publication.

The administration of active constituent can be by any mode institute that the ingredient can be transferred to reacting environment's (e.g., tumour cell) It influences.These modes may include oral cavity route, anal route, 12 intrarectal routes, parental injection (including intravenous, skin Under, muscle, intravascular or perfusion), local administration, etc..Certainly, the dosage for the active constituent being given will be controlled dependent on receiving The judgement of the main body for the treatment of, the severity of pain, administration mode and the doctor to prescribe.However effective dose about exists (usually between 0.01 milligram to 100 milligrams, more suitable situation is about in 0.1 milli between 0.001 milligram to 300 milligrams Gram between 30 milligrams), specific dosage can be from 0.001 milligram of daily per kilogram of body weight to daily per kilogram of body weight 300 milligrams (more commonly used is 0.01 milligram to 100 milligrams of daily per kilogram of body weight, is further suitable that daily per kilogram of body weight 0.1 milligram to 30 milligrams).

The ingredient can have following form, such as, it is suitble to be administered orally, such as tablet, capsule, pill, medicinal powder continues The form of release, solution or suspension；For parental injection such as transparent liquid, suspension, emulsion；Or it is used for local application Such as cream, frost；Or rectally is used for as suppository.Pharmaceutical Compositions can also be suitable for exact dose in the form of unit dose Once daily.The Pharmaceutical Compositions will include a kind of traditional pharmaceutical carriers or excipient and are made according to current invention The compound as active constituent, alternatively, it is also possible to include other medicine or pharmaceutical formulations, carrier, adjuvant, etc..

Main parenteral dosage regimen include the solution of active constituent or suspension are dissolved in aseptic aqueous solution, such as Propyleneglycoles aqueous solution or glucose solution.This dosage form can be by the concentration appropriate for being diluted to needs.

Pharmaceutical carriers appropriate include inert diluent, filler, water and a variety of different organic solvents.If pressed Preparatory design, pharmaceutical formulation may include some supplementary elements such as flavouring, antidiarrheic, the east of excipient and the like West.Therefore, for oral medicine, include the tablet of various excipient, such as citric acid, can such as form sediment with various disintegrants Powder, alginic acid, certain silicate complex and some antidiarrheics such as sucrose, gel and gum arabic combine.In addition, As magnesium stearate, sodium lauryl sulfate, the lubricants such as talcum are also quite useful for tablet is made.The solid-state of similar type Compound can also be used for soft or hard gel capsule filling.Therefore, preferred material includes lactose or toffee and high molecular weight Polyvinyl alcohol.When necessity as oral medicine of aqueous suspension or elixir, the active constituent in there face may be tied Different sweetener or other flavouring and toner and coloring agent are closed, can also add emulsifier or suspending agent when necessary, one And the also dilution being added, as water, alcohol, propyleneglycoles, glycerol or their mixture.

Therapeutic compound can also award mammal and non-human.It will be taken to drug dose used in a mammal Certainly in the type of the animal and its disease condition or the de-synchronization state locating for it.Therapeutic compound can be with capsule, greatly The form of pill, tablet liquid medicine is fed for animal.Therapeutic compound can also be allowed to enter animal by way of injecting or inculcating In vivo.We prepare these medicament forms according to the traditional mode for meeting veterinary practice standard.As a kind of selectable Mode, pharmacy synthetic drug can mix with animal feed and be fed for animal, therefore, the feed addictive of concentration or mix and stir in advance Material can be in case of to mix common animal feed.

A further object of the present invention is to be to provide a kind of method for treating cancer in subject in need, packet Include a kind of method that the therapeutically effective amount of the composition containing the compound of the present invention is applied to subject.The cancer that can be treated in this way Disease can indicate in the other places this paper, including be not limited to have and replace to Imatinib, Dasatinib, nilotinib, Bosutinib, Pu Na Buddhist nun has the cancer of drug resistance.One or more other cancer therapies can also be applied in combination in treatment, including operation, radiotherapy are (such as Gamma-radiation, neutron beam radiotherapy, electron beam evaporation treatment, proton therapeutic, plesioradiotherapy and the same position of body radioactivity Element etc.), endocrinotherapy, biological response modifier (for example, interferon, interleukin and tumor necrosis factor (TNF)), thermotherapy, Cold therapy weakens any adverse effect (for example, antemetic) and other cancer chemotherapeutic drugs.Other workable systems Agent, administration route and dosage regimen can be identical or different with the compound of the present invention as the compound of the present invention.

The invention also includes the uses of the compound of the present invention or its pharmaceutically acceptable derivates, manufacture for treating Cancer (including non-physical knurl, solid tumor, primary or metastatic cancer, have as pointed by the other places this paper and including cancer anti- Property or refractory one or more other treatments) and Other diseases (including but not limited to fundus oculi disease, psoriasis, artery congee Sample, pulmonary fibrosis, liver fibrosis, myelofibrosis etc.) medicament.Purposes of the compound of the present invention in anticancer drug be Useful.The compound of the present invention can also be used in drug by inhibiting one or more kinases (such as BCR-ABL, VEGFR Deng) to mitigate or prevent disease.

The compound of the present invention also can be used as standard items and reagent for characterizing various kinases, be especially but not limited to albumen Tyrosine kinase, and it is highly useful to study effect of this kinases in biology and pathological phenomena；For study by This kind of kinase mediated intracellular signalling pathway, for the comparative evaluation of new kinase inhibitor；And for studying difference In the cell line and animal model of cancer.

Examples provided below can better illustrate the present invention, and unless stated otherwise, all temperature are degree Celsius.

Specific embodiment

Embodiment 1

1- methyl-N- (4- ((4- methyl piperazine -1-) methyl) phenyl) -7- (pyridine -3- acetenyl) -1H- indoles -2- first The preparation of amide

The bromo- 1- methyl-N- of step 1) 7- (4- ((4- methyl piperazine -1-) methyl) phenyl) -1H- indole 2-carboxamides Preparation

By the bromo- 1- Methyl-1H-indole -2- formic acid (2.5g, 10mmol) of 7-, 4- ((4- methyl piperazine -1-) methyl) amine (2.0g, 10mmol) and 2- (7- aoxidizes benzotriazole)-N, N, N', N'- tetramethylurea hexafluorophosphoric acid ester (HATU) (3.8g, It 10mmol) is dissolved in n,N-Dimethylformamide, is added diethyl isopropyl amine (1.65mL, 10mmol), stirring is to having reacted Finish, ethyl acetate and water extraction, organic phase concentration, column chromatograph to obtain 3.1 grams of target compound, yield 70%.¹H NMR (400MHz,DMSO-d₆) δ 10.48 (s, 1H), 7.76-7.68 (m, 3H), 7.53-7.49 (m, 1H), 7.27 (d, J=8.4Hz, 2H), 7.23 (s, 1H), 7.04 (t, J=7.7,7.7Hz, 1H), 4.28 (s, 3H), 3.43 (s, 2H), 2.48-2.27 (m, 8H), 2.19(s,3H)；MS:441[M+H]⁺。

Step 2) 1- methyl-N- (4- ((4- methyl piperazine -1-) methyl) phenyl) -7- ((trimethyl silicon substrate) acetenyl) - The preparation of 1H- indole 2-carboxamides

By the bromo- 1- methyl-N- of 7- (4- ((4- methyl piperazine -1-) methyl) phenyl) -1H- indole 2-carboxamides (3.1g, 7mmol) with trimethyl ((tributylstamlyl) acetenyl) silane (9.9g, 14mmol) and palladium diphenyl phosphine dichloride (0.9g, 0.7mmol) is placed in a reaction flask, and toluene is added, is heated to end of reaction, boils off solvent, column chromatographic purifying obtains yellow 1.9 grams of solid, yield 60%.¹H NMR(400MHz,DMSO-d₆)δ10.30(s,1H),7.67–7.63(m,1H),7.63– 7.58(m,2H),7.32–7.27(m,1H),7.19–7.12(m,3H),7.02–6.95(m,1H),4.24(s,3H),3.30(s, 2H),2.38–2.07(m,8H),2.04(s,3H),0.16(s,9H)；MS:459[M+H]⁺。

Step 3) 1- methyl-N- (4- ((4- methyl piperazine -1-) methyl) phenyl) -7- (pyridine -3- acetenyl) -1H- Yin The preparation of diindyl -2- formamide

By 1- methyl-N- (4- ((4- methyl piperazine -1-) methyl) phenyl) -7- ((trimethyl silicon substrate) acetenyl) -1H- Yin Diindyl -2- formamide (92mg, 0.2mmol), 3- iodine pyridine (41mg, 0.2mmol), palladium diphenyl phosphine dichloride (7mg, 0.02mmol), cuprous iodide (3.8mg, 0.02mmol), the in the mixed solvent of molten triethylamine and tetrahydrofuran, heating stirring is extremely End of reaction, ethyl acetate and water extraction, organic phase concentration, column chromatograph to obtain 46 milligrams of product, yield 50%.¹H NMR (400MHz,DMSO-d₆) δ 10.48 (s, 1H), 8.86-8.82 (m, 1H), 8.62 (dd, J=4.9,1.7Hz, 1H), 8.08- 8.03(m,1H),7.85–7.79(m,1H),7.78–7.71(m,2H),7.59–7.54(m,1H),7.53–7.47(m,1H), 7.34–7.26(m,3H),7.23–7.15(m,1H),4.44(s,3H),3.49(s,2H),2.83–2.60(m,4H),2.51(s, 3H),2.46–2.37(m,4H)；¹³C NMR(101MHz,DMSO-d6)δ160.48,151.71,149.46,138.64, 134.53,134.13,130.58,129.62,127.34,124.25,124.16,120.80,120.41,120.08,106.71, 105.93,91.00,62.15,55.21,52.97,46.23,33.95；MS:464[M+H]⁺。

Embodiment 2

1- methyl-N- (4- ((4- methyl piperazine -1-) methyl) -3- (trifluoromethyl) phenyl) -7- (pyridine -3- acetylene Base) -1H- indole 2-carboxamides preparation

The bromo- 1- methyl-N- of step 1) 7- (4- ((4- methyl piperazine -1-) methyl) -3- (trifluoromethyl) phenyl) -1H- Yin The preparation of diindyl -2- formamide

By the bromo- 1- Methyl-1H-indole -2- formic acid (2.5g, 10mmol) of 7-, 4- ((4- methyl piperazine -1-) methyl) -3- (trifluoromethyl) aniline (2.73g, 10mmol) and 2- (7- aoxidizes benzotriazole)-N, N, N', N'- tetramethylurea hexafluorophosphoric acid Ester (HATU) (3.8g, 10mmol) is dissolved in n,N-Dimethylformamide, addition diethyl isopropyl amine (1.65mL, 10mmol), stirring to end of reaction, ethyl acetate and water extraction, organic phase concentration, column chromatographs to obtain 3.6 grams of product, yield 70%.¹H NMR(400MHz,DMSO-d₆)δ10.74(s,1H),8.22(s,1H),8.05–7.99(m,1H),7.77–7.69 (m, 2H), 7.53 (d, J=7.5Hz, 1H), 7.30 (s, 1H), 7.05 (t, J=7.7,7.7Hz, 1H), 4.30 (s, 3H), 3.57 (s,2H),2.47–2.23(m,8H),2.17(s,3H)；MS:509[M+H]⁺。

Step 2) 1- methyl-N- (4- ((4- methyl piperazine -1-) methyl) -3- (trifluoromethyl) phenyl) -7- ((trimethyl Silicon substrate) acetenyl) -1H- indole 2-carboxamides preparation

By the bromo- 1- methyl-N- of 7- (4- ((4- methyl piperazine -1-) methyl) -3- (trifluoromethyl) phenyl) -1H- indoles -2- Formamide (3.6g, 7mmol) and trimethyl ((tributylstamlyl) acetenyl) silane (9.9g, 14mmol) and two triphens Base phosphine dichloride palladium (0.9g, 0.7mmol) is placed in a reaction flask, and toluene is added, is heated to end of reaction, boils off solvent, column layer Analysis purifies to obtain 2.2 grams of yellow solid product, yield 60%.¹H NMR(400MHz,DMSO-d₆)δ10.67(s,1H),8.22(d,J =2.2Hz, 1H), 8.05-7.99 (m, 1H), 7.80-7.75 (m, 1H), 7.72 (d, J=8.5Hz, 1H), 7.45-7.40 (m, 1H), 7.34 (s, 1H), 7.11 (t, J=7.7,7.7Hz, 1H), 4.37 (s, 3H), 3.58 (s, 2H), 2.49-2.31 (m, 8H), 2.22(s,3H),0.27(s,9H)；MS:527[M+H]⁺。

Step 3) 1- methyl-N- (4- ((4- methyl piperazine -1-) methyl) -3- (trifluoromethyl) phenyl) -7- (pyridine -3- Acetenyl) -1H- indole 2-carboxamides preparation

By 1- methyl-N- (4- ((4- methyl piperazine -1-) methyl) -3- (trifluoromethyl) phenyl) -7- ((trimethyl silicon substrate) Acetenyl) -1H- indole 2-carboxamides (105mg, 0.2mmol), 3- iodine pyridine (41mg, 0.2mmol), two triphenylphosphine dichloros Change palladium (7mg, 0.02mmol), cuprous iodide (3.8mg, 0.02mmol), the in the mixed solvent of molten triethylamine and tetrahydrofuran adds Thermal agitation to end of reaction, ethyl acetate and water extraction, organic phase concentration, column chromatographs to obtain 53 milligrams of product, yield 50%.¹H NMR(400MHz,DMSO-d₆) δ 10.78 (s, 1H), 8.84 (d, J=2.1Hz, 1H), 8.62 (dd, J=4.9,1.7Hz, 1H), 8.25 (d, J=2.2Hz, 1H), 8.14-8.01 (m, 2H), 7.83 (dd, J=8.0,1.2Hz, 1H), 7.72 (d, J=8.6Hz, 1H), 7.58 (dd, J=7.3,1.2Hz, 1H), 7.51 (dd, J=7.9,4.9Hz, 1H), 7.41 (d, J=1.2Hz, 1H), 7.20 (t, J=7.6,7.6Hz, 1H), 4.45 (s, 3H), 3.65 (s, 2H), 3.10-2.76 (m, 4H), 2.71-2.52 (m, 7H)；¹³CNMR(101MHz,DMSO-d₆)δ160.85,151.69,149.47,138.75,138.63,137.21,131.85, 130.87,127.26,124.38,124.16,123.86,120.88,120.06,107.48,105.99,90.94,90.72, 57.36,53.74,50.85,43.73,34.00.MS:532[M+H]⁺。

Embodiment 3

N- (4- ((4- methyl piperazine -1-) methyl) -3- (trifluoromethyl) phenyl) -7- (pyrimidine -5- acetenyl) -1H- Yin The preparation of diindyl -2- formamide

The bromo- N- of step 1) 7- (4- ((4- methyl piperazine -1-) methyl) -3- (trifluoromethyl) phenyl) -1H- indoles -2- first The preparation of amide

By the bromo- 1H- indole-2-carboxylic acid (1) (2.4g, 10mmol) of 7-, 4- ((4- methyl piperazine -1-) methyl) -3- (trifluoro Methyl) aniline (2.73g, 10mmol) and 2- (7- aoxidizes benzotriazole)-N, N, N', N'- tetramethylurea hexafluorophosphoric acid ester (HATU) (3.8g, 10mmol) is dissolved in n,N-Dimethylformamide, is added diethyl isopropyl amine (1.65mL, 10mmol), Stirring to end of reaction, ethyl acetate and water extraction, organic phase concentration, column chromatographs to obtain 3.5 grams of product, yield 70%.¹H NMR (400MHz,DMSO-d₆) δ 11.67 (s, 1H), 10.65 (s, 1H), 8.23 (d, J=2.1Hz, 1H), 8.10-8.02 (m, 1H), 7.80-7.70 (m, 2H), 7.55-7.45 (m, 2H), 7.06 (t, J=7.8Hz, 1H), 3.58 (s, 2H), 2.49-2.24 (m, 8H),2.18(s,3H)；MS:495[M+H]⁺。

Step 2) N- (4- ((4- methyl piperazine -1-) methyl) -3- (trifluoromethyl) phenyl) -7- ((trimethyl silicon substrate) second Alkynyl) -1H- indole 2-carboxamides preparation

By the bromo- N- of 7- (4- ((4- methyl piperazine -1-) methyl) -3- (trifluoromethyl) phenyl) -1H- indole 2-carboxamides (3.5g, 7mmol) and trimethyl ((tributylstamlyl) acetenyl) silane (9.9g, 14mmol) and two triphenylphosphines two Palladium chloride (0.9g, 0.7mmol) is placed in a reaction flask, and toluene is added, is heated to end of reaction, boils off solvent, column chromatographic purifying 2.1 grams of yellow solid are obtained, yield 60%.¹H NMR(400MHz,DMSO-d₆)δ11.51(s,1H),10.67(s,1H),8.28 (d, J=2.2Hz, 1H), 8.10-8.00 (m, 1H), 7.83-7.71 (m, 2H), 7.47 (s, 1H), 7.42-7.36 (m, 1H), 7.11 (t, J=7.7Hz, 1H), 3.58 (s, 2H), 2.46-2.26 (m, 8H), 2.18 (s, 3H), 0.31 (s, 9H)；MS:513[M +H]⁺。

Step 3) N- (4- ((4- methyl piperazine -1-) methyl) -3- (trifluoromethyl) phenyl) -7- (pyrimidine -5- acetenyl) - The preparation of 1H- indole 2-carboxamides

By the bromo- N- of 7- (4- ((4- methyl piperazine -1-) methyl) -3- (trifluoromethyl) phenyl) -1H- indole 2-carboxamides (102mg, 0.2mmol), 5- iodine pyrimidine (41mg, 0.2mmol), palladium diphenyl phosphine dichloride (7mg, 0.02mmol), iodate Cuprous (3.8mg, 0.02mmol), the in the mixed solvent of molten triethylamine and tetrahydrofuran, heating stirring to end of reaction, acetic acid second Ester and water extraction, organic phase concentration, column chromatograph to obtain 52 milligrams of product, yield 50%.¹H NMR(400MHz,DMSO-d₆)δ12.12 (s,1H),10.63(s,1H),9.24–9.15(m,3H),8.30–8.26(m,1H),8.12–8.06(m,1H),7.88–7.84 (m,1H),7.77–7.71(m,1H),7.57–7.51(m,2H),7.23–7.17(m,1H),3.59(s,2H),2.49–2.40 (m,8H),2.25(s,3H)；MS:519[M+H]⁺。

Embodiment 4

7- ((2- aminopyrimidine -5-) acetenyl)-N- (the chloro- 4- of 3- ((4- methyl piperazine -1-) methyl) phenyl) -1- first The preparation of base -1H- indole 2-carboxamides

The preparation of step 1) ethyl 1- methyl -7- ((trimethyl silicon substrate) acetenyl) -1H- indole-2-carboxylic acid ester

By the bromo- 1- Methyl-1H-indole -2- formic acid esters (8g, 28mmol) of ethyl 7- and trimethyl ((tributylstannyl Base) acetenyl) silane (21.7g, 56mmol) and palladium diphenyl phosphine dichloride (2.0g, 2.8mmol) be placed in a reaction flask, Toluene is added, heating stirring to end of reaction boils off solvent, and column chromatographic purifying obtains 5.4 grams of yellow solid product, yield 65%.¹H NMR(400MHz,DMSO-d₆) δ 7.69-7.64 (m, 1H), 7.38-7.35 (m, 1H), 7.24 (s, 1H), 7.01 (t, J= 7.6,7.6Hz,1H),4.33(s,3H),4.27–4.21(m,2H),1.52–1.48(m,3H),0.19(s,9H)；MS:300[M+ H]⁺。

The preparation of step 2) ethyl 7- ((2- aminopyrimidine -5-) acetenyl) -1- Methyl-1H-indole -2- formic acid esters

By ethyl 1- methyl -7- ((trimethyl silicon substrate) acetenyl) -1H- indole-2-carboxylic acid ester (3.0g, 10mmol), 5- Iodine pyrimidine -2- amine (3.3g, 15mmol), cuprous iodide (0.19g, 1mmol), tetrabutyl ammonium fluoride (2.6g, 10mmol) are dissolved in The in the mixed solvent of triethylamine and dimethylformamide, heating stirring to end of reaction, ethyl acetate and water extract, and organic phase is dense Contracting, column chromatograph to obtain 1.6 grams of yellow solid product, yield 50%.¹H NMR(400MHz,DMSO-d₆)δ8.51(s,2H),7.96 (s, 1H), 7.76-7.71 (m, 1H), 7.65-7.61 (m, 1H), 7.51-7.48 (m, 1H), 7.34 (s, 1H), 7.14 (d, J= 7.6Hz, 1H), 4.46 (s, 3H), 4.32 (t, J=7.1,7.1Hz, 2H), 1.29-1.26 (m, 3H)；MS:321[M+H]⁺。

The preparation of step 3) 7- ((2- aminopyrimidine -5-) acetenyl) -1- Methyl-1H-indole -2- formic acid

Ethyl 7- ((2- aminopyrimidine -5-) acetenyl) -1- Methyl-1H-indole -2- formic acid esters (1.6g, 5mmol) is molten In tetrahydrofuran, sodium hydrate aqueous solution is added, the tetrahydro furan boiled off in reaction solution is concentrated under reduced pressure in stirring to end of reaction It mutters, adjusts reaction solution pH value to acidity with dilute hydrochloric acid, filter, obtain 1.39 grams of white solid, yield 95%.¹H NMR(400MHz, DMSO-d₆)δ13.13(s,1H),8.54(s,1H),7.75–7.50(m,3H),7.50–7.34(m,2H),7.17–7.04(m, 2H),4.48(s,3H)；MS:293[M+H]⁺。

Step 4) 7- ((2- aminopyrimidine -5-) acetenyl)-N- (the chloro- 4- of 3- ((4- methyl piperazine -1-) methyl) phenyl) - The preparation of 1- Methyl-1H-indole -2- formamide

By 7- ((2- aminopyrimidine -5-) acetenyl) -1- Methyl-1H-indole -2- formic acid (58mg, 0.2mmol), 3- is chloro- 4- ((4- methyl piperazine -1-) methyl) aniline (48mg, 0.2mmol), 2- (7- aoxidizes benzotriazole)-N, N, N', N'- tetramethyl Base urea hexafluorophosphoric acid ester (HATU) (80mg, 0.22mmol), is dissolved in dimethylformamide, and diethyl isopropyl amine is added (0.16mL, 1mmol), stirring to end of reaction, ethyl acetate and water extraction, organic phase concentration, column chromatograph to obtain 51 milligrams of product, Yield 50%.¹H NMR(300MHz,DMSO-d₆)δ10.58(s,1H),8.51(s,2H),7.97(s,1H),7.85–7.64(m, 2H), 7.54-7.38 (m, 2H), 7.31 (d, J=3.5Hz, 1H), 7.25-7.07 (m, 3H), 4.39 (s, 3H), 3.52 (s, 2H),2.44–2.23(m,8H),2.19–2.10(m,3H)；MS:514[M+H]⁺。

Embodiment 5

7- ((1H- pyrazolo [3,4-b] pyridine -5-) acetenyl)-N- (the chloro- 4- of 3- ((4- methyl piperazine -1-) methyl) benzene Base) -1- Methyl-1H-indole -2- formamide preparation

Step 1) is the same as 4 step 1) of embodiment

Step 2) ethyl 7- ((11H- pyrazolo [3,4-b] pyridine -5-) acetenyl) -1- Methyl-1H-indole -2- formic acid The preparation of ester

By ethyl 1- methyl -7- ((trimethyl silicon substrate) acetenyl) -1H- indole-2-carboxylic acid ester (3.0g, 10mmol), 5- Bromo- 1H- pyrazolo [3,4-b] pyridine (2.9g, 15mmol), cuprous iodide (0.19g, 1mmol), tetrabutyl ammonium fluoride (2.6g, 10mmol), it is dissolved in the in the mixed solvent of triethylamine and dimethylformamide, heating stirring to end of reaction, ethyl acetate and water Extraction, organic phase concentration, column chromatograph to obtain 1.4 grams of yellow solid product, yield 42%.¹H NMR(400MHz,DMSO-d₆)δ 13.90 (s, 1H), 8.76 (s, 1H), 8.60 (s, 1H), 8.15 (s, 1H), 7.79 (d, J=8.1,1.9Hz, 1H), 7.59 (d, 1H), 7.36 (s, 1H), 7.19 (t, J=7.9,2.1Hz, 1H), 4.54 (s, 3H), 4.38-4.24 (m, 2H), 1.36 (t, 3H)； MS:345[M+H]⁺。

The preparation of step 3) 7- ((1H- pyrazolo [3,4-b] pyridine -5-) acetenyl) -1- Methyl-1H-indole -2- formic acid

By ethyl 7- ((11H- pyrazolo [3,4-b] pyridine -5-) acetenyl) -1- Methyl-1H-indole -2- formic acid esters (1.4g, 4.2mmol) is dissolved in tetrahydrofuran, and sodium hydrate aqueous solution, stirring to end of reaction is added, and reduced pressure boils off anti- The tetrahydrofuran in liquid is answered, reaction solution pH value is adjusted to acidity with dilute hydrochloric acid, filters, obtain 1.26 grams of white solid, yield 95% 。¹H NMR(400MHz,DMSO-d₆)δ13.94(s,1H),8.75(s,1H),8.60(s,1H),8.15(s,1H),7.71(d,J =8.0Hz, 1H), 7.51 (d, J=7.3Hz, 1H), 7.19-7.07 (m, 2H), 4.57 (s, 3H)；MS:317[M+H]⁺。

Step 4) 7- ((1H- pyrazolo [3,4-b] pyridine -5-) acetenyl)-N- (chloro- 4- of 3- ((4- methyl piperazine -1-) Methyl) phenyl) -1- Methyl-1H-indole -2- formamide preparation

By 7- ((1H- pyrazolo [3,4-b] pyridine -5-) acetenyl) -1- Methyl-1H-indole -2- formic acid (VI-2) (63mg, 0.2mmol), the chloro- 4- of 3- ((4- methyl piperazine -1-) methyl) aniline (48mg, 0.2mmol), (7- aoxidizes benzo three to 2- Nitrogen azoles)-N, N, N', N'- tetramethylurea hexafluorophosphoric acid ester (HATU) (80mg, 0.22mmol) is dissolved in dimethylformamide, adds Enter diethyl isopropyl amine (0.16mL, 1mmol), stirring to end of reaction, ethyl acetate and water extraction, organic phase concentration, column 49 milligrams of product are chromatographed to obtain, yield 46%.1H NMR(300MHz,DMSO-d₆)δ13.94(s,1H),10.59(s,1H),8.76 (d, J=2.1Hz, 1H), 8.55 (d, J=2.1Hz, 1H), 8.22 (s, 1H), 7.97 (d, J=2.1Hz, 1H), 7.82-7.77 (m,1H),7.72–7.66(m,1H),7.59–7.53(m,1H),7.46–7.40(m,1H),7.34(s,1H),7.22–7.14 (m,1H),4.46(s,3H),3.52(s,2H),2.44–2.30(m,8H),2.17(s,3H)；MS:538[M+H]⁺。

Embodiment 6-22

Step 1) and step 2) are the same as the step 1) and step 2) in the preparation method of embodiment 1.

Step 3) in the preparation method of step 3) reference implementation example 1, operate it is identical, specific embodiment be with R in the following table of equal mole equivalent¹X substitutes 3- iodine pyridine, obtains corresponding embodiment compound.Chemical combination is embodied Object is as shown in the table:

Embodiment 23-45

Step 1) and step 2) are the same as the step 1) and step 2) in the preparation method of embodiment 2.

Step 3) in the preparation method of step 3) reference implementation example 2, operate it is identical, specific embodiment be with R in the following table of equal mole equivalent¹X substitutes 3- iodine pyridine, obtains corresponding embodiment compound.Chemical combination is embodied Object is as shown in the table:

Embodiment 46-49

Step 1) and step 2) are the same as the step 1) and step 2) in the preparation method of embodiment 3.

Step 3) in the preparation method of step 3) reference implementation example 3, operate it is identical, specific embodiment be with R in the following table of equal mole equivalent¹X substitutes 5- iodine pyrimidine, obtains corresponding embodiment compound.Chemical combination is embodied Object is as shown in the table:

Embodiment 49-61

Step 1) and step 3) are the same as the step 1) in the preparation method of embodiment 4 to step 3).

Step 4) in the preparation method of step 4) reference implementation example 4, operate it is identical, specific embodiment be with R in the following table of equal mole equivalent³NH₂The chloro- 4- of 3- ((4- methyl piperazine -1-) methyl) aniline is substituted, is obtained corresponding Embodiment compound.It is as shown in the table that compound is embodied:

Embodiment 62-63

Step 1) and step 3) are the same as the step 1) in the preparation method of embodiment 5 to step 3).

Embodiment 64

The test of small molecule compound inhibition ABL/ABL-T315I kinase activity

Specific implementation method is as follows: in the enzymatic reaction assembled in vitro, the untested compound of various concentration is added, with inspection Compound is surveyed to the inhibiting effect of ABL/ABL-T315I enzymatic reaction, passes through what is inhibited to ABL/ABL-T315I enzymatic reaction The calculating of IC50 is to screen the compound for having inhibitory activity.

One, instrument, material and reagent

Two, experimental programs

Following experimental programs illustrate the preparation of reagents method of complete experimental procedure and midway, according to ABL by taking ABL as an example Experimental program fine tuning the experimental program of complete ABL-T315I kinases can be obtained.

1. preparation of reagents:

EDTA (0.5M pH8.0) solution is prepared: precise 14.612g EDTA powder, constant volume arrives after ultrapure water is added 100mL (if having it is insoluble be heated to 37 DEG C, with NaOH solution tune pH to 8.0)

1 × Kinase Assay Buffer: 25mL HEPES solution (1M), 190.175mg are separately added into reagent bottle EGTA、5mL MgCl₂Solution (1M), 1mL DTT, 50 μ L Tween-20 add ultrapure water constant volume to 500mL (adjusting pH to 7.5).

1 × Detection Buffer: it takes 10 × Detection of 1mL Buffer that 9mL water is added and mixes.

4 × terminate liquid: by 0.8mL above-mentioned EDTA (0.5M, pH 8.0) solution, 10 × Detection of 1mL Buffer and 8.2mL ultrapure water mixes.

4 × ABL kinase solution: diluting kinases stoste to concentration with 1 × Kinase Assay Buffer is 0.62nM, is mixed It is even, it saves on ice.

4 × substrate solution: substrate ULight is diluted with 1 × Kinase Assay Buffer^TMPolyGT stoste arrives 200nM is mixed.

4 × ATP solution: diluting ATP stoste to concentration with 1 × Kinase Assay Buffer is 40 μM, is mixed.

4 × detection liquid: detection antibody Eu-W1024-labeled Anti- is diluted with 1 × Detection Buffer Phosphotyrosine Antibody (PT66) is 8nM to concentration, is mixed.

2 × substrate/ATP mixed liquor: 4 × substrate solution and 4 × ATP solution 1:1 equivalent are mixed and (uses preceding preparation).2, Experimental procedure

1) dilution of the compounds of this invention,

In 96 orifice plate a, the compound of 1-63 of embodiment of the present invention DMSO solution is pressed into 3 times of dilution proportions, forms 11 A gradient, the 12nd gradient are pure DMSO solution (as positive control)；One piece of 96 new orifice plate b is taken, above-mentioned solution is used super Pure water dilutes 6.25 times (DMSO concentration is 16%)

2) by the compound turntable of embodiment 1-63 to 384 orifice plates

By the compound solution diluted in above-mentioned 96 orifice plate b with ultrapure water according to the standard turntable of 2 multiple holes to 384 orifice plates In corresponding hole.

3) add 4 × kinase solution: taking the 2.5 above-mentioned 4 × kinase solutions of μ l to be added to the corresponding reacting hole of 384 orifice plates with the volley of rifle fire In, it mixes room temperature pre-reaction 5 minutes.

4) add 2 × substrate/ATP mixed liquor: taking the 5 above-mentioned 2 × substrates of μ l/ATP mixed liquor corresponding to 384 orifice plates with the volley of rifle fire In reacting hole.

5) negative control: being arranged negative control hole in 384 orifice plates, every hole be added 2.5 4 × substrates of μ l, 2.5 μ l 4 × Enzyme solutions, 2.5 μ l 1 × Kinase Assay Buffer and 2.5 ultrapure waters of the μ l containing 16%DMSO.

6) centrifugation mixes, and is protected from light room temperature reaction 2 hours.

7) enzymatic reaction is terminated:

The 5 above-mentioned 4 × terminate liquids of μ l to 384 orifice plate corresponding apertures are drawn, centrifugation mixes, and reacts at room temperature 5 minutes.

8) chromogenic reaction:

It draws 5 μ l above-mentioned 4 × detection liquid and is added to 384 orifice plate corresponding apertures, centrifugation mixes, and reacts at room temperature 1 hour.

9) 384 orifice plates are put into plate reader, transfer corresponding Programmable detection signal.

10)IC₅₀Analysis:

Hole readings=10000*EU665 value/EU615 value

Inhibiting rate=[1- (experimental port readings-negative control hole readings)/(Positive control wells readings-negative control hole is read Value)] * 100%

Drug concentration and the input processing of GraphPad Prism 5 of corresponding inhibiting rate are calculated into corresponding IC₅₀。

Three, experiment conditions:

1) ABL kinase activity inhibits molecule to screen experiment condition:

10 μM of the final concentration of the final concentration of 0.155nM of ABL kinases in reaction system, ATP, substrate ULight^TM-labeled The final concentration of 50nM of PolyGT, time of enzymatic reacting are 2 hours.

Final concentration of 2.5 μM of compound highest in reaction system, totally 11 concentration, minimum end are dense after 3 times of gradient dilutions Degree is 0.042nM.DMSO final concentration of 4%.

2) ABL-T315I kinase activity inhibits molecule to screen experiment condition:

10 μM of final concentration of ABL (T315I) kinases final concentration of 0.5nM, ATP, substrate ULight in reaction system^TM- The final concentration of 50nM of labeled PolyGT, time of enzymatic reacting are 2 hours.

Final concentration of 2.5 μM of compound highest in reaction system, totally 11 concentration, minimum end are dense after 3 times of gradient dilutions Degree is 0.042nM.DMSO final concentration of 1%.

Table (one) lists in the present invention part of compounds to tyrosine kinase BCR-ABL and ABL-T315I inhibitory activity Measurement result.IC50 value indicates compound concentration when kinases highest inhibiting rate is 50% in following table, and wherein A indicates that IC50 is small In or equal to 100nM, B indicates IC50 greater than 100nM but is less than or equal to 1000nM, and C indicates IC50 greater than 1000nM but is less than Or it is equal to 10000nM, D indicates that IC50 is greater than 10000nM.NT is indicated without testing corresponding kinases.

Table (one), part of compounds of the present invention are to ABL/ABL-T315I kinase inhibiting activity test result (IC₅₀)

Embodiment 65

The test of small molecule compound inhibition VEGFR-2 kinase activity

The specific test method is as follows:

1. diluted chemical compound: carrying out totally 12 concentration (this experiments after 4 times of gradient dilutions since maximum concentration 10000nM The maximum final concentration of 10000nM, minimum final concentration of 0.002384nM of the drug used),

2. taking 2.5 compounds of the μ l through gradient dilution with the volley of rifle fire, it is added in 384 orifice plates,

3. enzyme: taking 5 μ l 2X VEGFR-2 kinases to be added in the corresponding reacting hole of 384 orifice plates with the volley of rifle fire, mix rear chamber Warm pre-reaction 30min,

4. the volley of rifle fire takes 2.5 μ l 4X substrates/ATP Mix to be added in the corresponding reacting hole of 384 orifice plates,

5. negative control: 2.5 μ l/ hole 4X substrates/ATP Mix and 7.5 μ l 1X Kinase is added in 384 orifice bores Assay Buffer

Positive control: 2.5 μ l/ hole 4X substrates/ATP Mix, 2.5 1Xs of the hole μ l/ containing 4%DMSO is added in 384 orifice plates Kinase Assay Buffer, 5 hole μ l/ 2X VEGFR-2solution.Final concentration of the 4% of DMSO in reaction system,

6. centrifugation mixes, it is protected from light room temperature reaction 60min,

7. terminating enzymatic reaction: taking 5 μ l 4X Stop solution to be added in 384 orifice plate mesoporous with the volley of rifle fire, centrifugation is mixed It is even, 5min is reacted at room temperature,

8. chromogenic reaction: it takes 5 μ l 4X Detection Mix to be added in 384 orifice plate mesoporous with the volley of rifle fire and develops the color, from The heart mixes, and reacts at room temperature 60min,

9. 384 orifice plates are put into Envision plate reader read plate, corresponding Programmable detection signal is transferred.

10. the analysis and processing of initial data:

Drug concentration and corresponding inhibiting rate are inputted into GraphPad Prism5 calculation processing, the meter of the inhibiting rate of compound Calculation method is as follows: inhibiting rate (%)=[1- (experimental port readings-negative control hole readings)/(Positive control wells readings-feminine gender is right According to hole readings)] x100%.It is handled with GraphPad Prism5 software and obtains corresponding IC₅₀Value is (when enzyme highest inhibiting rate 50% Compound concentration).

Table (two) lists part of compounds in the present invention to the measurement result of VEGFR2 tyrosine-kinase enzyme inhibition activity, Middle A indicates IC₅₀IC is indicated less than or equal to 100nM, B₅₀Greater than 100nM but it is less than or equal to 1000nM, C indicates IC₅₀It is greater than 1000nM but be less than or equal to 10000nM, D indicate IC₅₀Greater than 10000nM.

Table (two), part of compounds of the present invention are to VEGFR2 kinase inhibiting activity test result (IC₅₀)

Embodiment 66

The active testing of small molecule compound inhibition cell Proliferation

The untested compound of various concentration, the increasing by comparing drug to target cell are added in cell culture fluid

The IC50 grown come reflect compound on intracellular proliferation inhibitory effect.

One laboratory apparatus and material:

Major experimental instrument and material

Two experiment reagents

Experiment reagent

Compound: it is dissolved in DMSO and is made into 10mM solution.

Three related solution allocations and dilution:

1. diluted chemical compound: all the compounds of this invention are dissolved in DMSO and are made into 10mM stock solution, complete in DMSO to be measured The first time gradient dilution of compound, extension rate are 3 times or 4 times；80 times that all compounds are completed in cell culture fluid Whole dilution, obtained compound are 5 × compound, which will be added in the hole of celliferous 96 orifice plate, so that most It is 1 × design final concentration in whole cell culture fluid.

General compound is designed as 9 concentration gradients, wherein the final concentration of 25000nM of highest, through 4 times of dilutions, 9 concentration Minimum concentration is 0.38nM, DMSO final concentration of 0.25% in all holes afterwards.

Four experimental procedures

1) cell is transferred in 15mL centrifuge tube, with 1000rpm centrifugation 4 minutes.

2) liquid is discarded supernatant, complete culture solution is added, piping and druming uniformly, takes 10 μ L cell suspending liquids and 10 μ L, 0.4% tire to expect Indigo plant mixes, and is counted with cell counter, and cell number and survival rate are recorded.

3) for the cell suspension of 80 μ L of every hole inoculation into 96 orifice plates, different cell inoculation cell densities are as follows:

Cell density

Cell Name	Culture medium	Inoculum density
			K562	RPMI 1640+10%FBS	10000/well
BaF3-BCR-ABL-T315I	RPMI 1640+10%FBS	5000/well

4) the above-mentioned 5 × chemical combination diluted with culture solution of 20 μ L is added in (B row to G row, the 2nd column to the 11st column) in every hole Object, mixing shake up.(each compound setting two is parallel, and 96 orifice plates can test three compounds)；

5) 10 μ L CCK-8 reagents are added in every hole after cultivating 72 hours in 37 DEG C of incubators containing 5%CO2, and culture 2 is small When (can be according to shade come adjusting reaction time)；

6) its OD value is read at 450nm in multi-functional plate reading machine.

7) data processing: cell survival rate (%)=[(As-Ab)/(Ac-Ab)] * 100%

As: the OD value of experimental port (containing cell culture medium, CCK-8, compound)；

Ac: the OD value of control wells (containing cell culture medium, CCK-8)；

Ab: the OD value of blank well (culture medium, CCK-8 without cell and compound).

Then numerical value importing Graphpad Prism5 software is carried out curve fitting, calculates IC50.

Table (three) lists the determination of activity that part of compounds cell proliferation inhibits in the present invention as a result, wherein A is indicated IC₅₀IC is indicated less than or equal to 100nM, B₅₀Greater than 100nM but it is less than or equal to 500nM, C indicates IC₅₀Greater than 500nM but small In or equal to 1000nM, D expression IC₅₀Greater than 1000nM.

The determination of activity result of table (three), the representative compound on intracellular Proliferation Ability of the present invention

Embodiment 67

As the specific materialization of an oral drugs, 80-120mg cream is added in the compound of about 10mg embodiment 54 Sugar mixing, packing fill capsule, obtain the capsule of 54 compound of embodiment.

Biological data provided by the present invention show the compound of the present invention be conducive to treat or prevent due to ABL and/ Or disease caused by VEGFR2 kinases exception.The compound of the present invention also includes the resistance to one or more other treatment methods for the treatment of Disease, including but not limited to acute myelocytic leukemia, chronic granulocytic leukemia, chronic granulocytic leukemia, Acute myeloblastic leukemia, gastrointestinal stromal tumor, acute myeloblastic leukemia, non-small cell lung cancer, Small Cell Lung Cancer, mammary gland Cancer, cancer of pancreas, glioma, glioblastoma, oophoroma, cervix cancer, colorectal cancer, melanoma, intrauterine Film cancer, prostate cancer, bladder cancer, leukaemia, gastric cancer, liver cancer, thyroid cancer, non-Hodgkin lymphoma, nasopharyngeal carcinoma, cancer of the esophagus, Brain tumor, B cell and t cell lymphoma, lymthoma, Huppert's disease, biliary tract carcinosarcoma, cholangiocarcinoma, fundus oculi disease, dry eyes Disease, psoriasis, leucoderma, dermatitis, alopecia areata, rheumatoid arthritis, colitis, multiple sclerosis, systemic loupus erythematosus, Crow Grace disease, atheroma, pulmonary fibrosis, liver fibrosis, myelofibrosis, in it is any etc..The compound of the present invention can be with As monotherapy or conjoint therapy, can with multiple the compound of the present invention drug combinations or with its other medicine other than the present invention Object drug combination.

The above is a preferred embodiment of the present invention, it is noted that for those skilled in the art For, under the premise of not departing from principle of the present invention, embodiments of the present invention can also make several improvements and modify, These improvement and modification also should be regarded as protection scope of the present invention.

Claims

1. a kind of compound, isomers, tautomer, solvate, hydrate, crystal form or its is pharmaceutically acceptable Salt, the compound have structure formula (I):

Wherein,

X is N, CH；

R¹For

A) aryl or 5-6 member hetero-aromatic ring unsubstituted or replaced by 1-3 identical or different Ra, the 5-6 member hetero-aromatic ring For the hetero atom for being selected from N, O and S containing 1-3；

B) unsubstituted or replaces ring A and ring B by 1-2 identical or different Rb, wherein ring A is 5-6 member hetero-aromatic ring or 6 yuan Aromatic ring, ring B are selected from 3-6 member naphthenic base, 3-6 circle heterocyclic ring, 5-6 member hetero-aromatic ring or aromatic ring, and the heterocycle and hetero-aromatic ring are independently The hetero atoms that N, O and S are selected from containing 1-3；

Ra is H, hydroxyl, cyano, acyl group, halogen, the unsubstituted or C that is replaced by hydroxyl or halogen₁-C₆Alkyl ,-OR' or-NR' R"；

Rb is H, halogen, hydroxyl, cyano, carbonyl, acyl group, C₁-C₆Alkyl, or-OR'；

R' and R " is independently H, C₃-C₆Naphthenic base, it is unsubstituted or by hydroxyl, carboxyl, halogen, C₁-C₆Alkoxy or C₁- C₆The C that alkylamino replaces₁-C₆Alkyl；

R²For H, C₁-C₆Alkyl；

R³For

D ring is unsubstituted or optionally by (Rd)_mSubstituted 5-6 member naphthenic base, 5-6 member contain 1-3 selected from N, O and S hetero atom Heterocycle, 5-6 member aromatic ring or 5-6 member contain 1-3 selected from the heteroatomic hetero-aromatic ring of N, O and S；

(Rd)_mRepresenting m identical or different substituent R d, m is the integer of 0-4, Rd H, halogen, hydroxyl, cyano, C₁-C₆Alkane Oxygroup, C₁-C₆Alkylamino, it is unsubstituted or by hydroxyl, halogen, C₁-C₆Alkoxy, C₁-C₆The C that alkylamino replaces₁-C₆Alkyl, or Person is-L-E- (Re)_n；

L is-(CR^l1R^l2)_q-；Q is the integer of 1-4；

R^l1And R^l2It is independently H or C₁-C₆Alkyl；

E is 3-6 member ring group or heterocycle, and the 3-6 circle heterocyclic ring base is to be selected from the heteroatomic saturated heterocyclic of N, O and S containing 1-3 Base or unsaturated heterocycle base；

(Re)_nIt is the integer of 0-4, Re H, halogen, hydroxyl, C for n identical or different Re, n₁-C₆Alkoxy, C₁-C₆Alkane ammonia Base, it is unsubstituted or by halogen, hydroxyl, C₁-C₆Alkoxy or C₁-C₆The C that alkylamino replaces₁-C₆Alkyl.

2. compound according to claim 1 or its isomers, tautomer, solvate, hydrate, crystal form and its Pharmaceutically acceptable salt,

Wherein,

X is N, CH；

R1 is

A) unsubstituted or replace 5-6 circle heterocyclic ring or 5-6 unit's heteroaryl by 1-3 identical or different Ra, the heterocycle and miscellaneous The hetero atom that N, O and S are selected from containing 1-3 of aromatic ring independently；

Ra is-H, hydroxyl, cyano, halogen, the unsubstituted or C that is replaced by hydroxyl or halogen₁-C₃Alkyl ,-OR' or NR'R "；

R' and R " is independently H, C₁-C₄Alkyl, C₃-C₄Naphthenic base, it is unsubstituted or by halogen, C₁-C₃Alkoxy or C₁- C₃The C that alkylamino replaces₁-C₃Alkyl；

Rb is-H, halogen, CF₃, carbonyl, cyano, C₁-C₃Alkyl；

R₂For H, C₁-C₄The alkyl of linear chain or branched chain；

R3 is

D ring is unsubstituted or optionally by (Rd)_mIt is a heteroatomic selected from N, O and S that the 6 yuan of aromatic rings or 5-6 member replaced contain 1-3 Aromatic ring；

(Rd)_mFor m identical or different substituent R d, m 0,1,2 or 3；Rd is H, halogen, hydroxyl, cyano, C₁-C₄Alcoxyl Base, C₁-C₄Alkylamino, it is unsubstituted or by hydroxyl, halogen, C₁-C₄Alkoxy, C₁-C₄The C that alkylamino replaces_1-4Alkyl or For-L-E- (Re)_n；

E is 3-6 circle heterocyclic ring base, the 3-6 circle heterocyclic ring base be containing 1-3 selected from the heteroatomic saturated heterocyclyl of N, O and S or Unsaturated heterocycle base；

(Re)_nFor n identical or different substituent R e, Re H, halogen, hydroxyl, C_1-3Alkoxy, C_1-3Alkylamino is not taken Generation or by halogen, hydroxyl, C₁-C₃Alkoxy or C₁-C₃The C that alkylamino replaces₁-C₄Alkyl, n 0,1,2 or 3.

3. compound according to claim 1 or its isomers, tautomer, solvate, hydrate, crystal form and its Pharmaceutically acceptable salt,

R²For H, methyl, ethyl, propyl or isopropyl.

4. compound according to claim 1 or its isomers, tautomer, solvate, hydrate, crystal form and its Pharmaceutically acceptable salt,

R¹For,

(Ra)^pTo be replaced by p identical or different Ra, p 0,1,2 or 3；

Ra is-H, and fluorine, chlorine, bromine, methyl, ethyl, propyl, butyl, isopropyl, trifluoromethyl ,-NHR', R' are-H, methyl, second Base, amino-ethyl, methylaminoethyl, dimethylaminoethyl, cyclopropyl, methoxy ethyl, aminopropyl, methylaminopropyl, two Methylaminopropyl, cyclobutyl or methoxy-propyl；

(Rb)^rTo be replaced by r identical or different Rb, r 0,1 or 2；

5. compound according to claim 1 or its isomers, tautomer, solvate, hydrate, crystal form and its Pharmaceutically acceptable salt,

R¹For

(Ra)^pTo be replaced by p identical or different Ra, p 0,1 or 2；

Ra is-H, fluorine, and chlorine, bromine, methyl, ethyl, propyl, butyl, isopropyl, trifluoromethyl or-NHR', R' are-H, methyl, second Base, amino-ethyl, methylaminoethyl, dimethylaminoethyl, cyclopropyl, methoxy ethyl, aminopropyl, methylaminopropyl, two Methylaminopropyl, cyclobutyl or methoxy-propyl；

(Rb)^rTo be replaced by r identical or different Rb, r is 0 or 1；

6. compound according to claim 1 or its isomers, tautomer, solvate, hydrate, crystal form and its Pharmaceutically acceptable salt,

R3 is

D ring is aromatic ring or hetero-aromatic ring that be unsubstituted or being replaced by (Rd) m, and the aromatic ring is phenyl ring, and the hetero-aromatic ring is selected from pyrrole Pyridine, pyridazine or thiazole；

(Rd)_mFor m identical or different substituent R d, m 0,1,2 or 3, Rd H, halogen, hydroxyl, cyano, C₁-C₄Alcoxyl Base, C₁-C₄Alkylamino, it is unsubstituted or by hydroxyl, halogen, C₁-C₄Alkoxy, C₁-C₄The C that alkylamino replaces_1-4Alkyl, or For-L-E- (Re)_n；

L is-(CH₂)_q, q 1,2 or 3；

E is 3-6 circle heterocyclic ring base, and the 3-6 circle heterocyclic ring is selected from azirane, ethylene oxide, pyrrolidines, pyrrolin, pyrroles, pyrrole Oxazolidine, pyrazoline, imidazoles, pyrazoles, furans, tetrahydrofuran, dihydrofuran, thiophane, thiophene, oxazole, thiazole, thiadiazoles, Piperidines, pyridine, dihydropyridine, morpholine, piperazine or pyrazine；(Re)_nFor the identical or different Re of n, Re H, halogen, hydroxyl, C_1-3Alkoxy, C_1-3Alkylamino, it is unsubstituted or by halogen, hydroxyl, C₁-C₃Alkoxy or C₁-C₃The C that alkylamino replaces₁-C₃Alkane Base, n 0,1,2 or 3.

7. compound according to claim 1 or its isomers, tautomer, solvate, hydrate, crystal form and its Pharmaceutically acceptable salt,

R³For

D ring is unsubstituted or by (Rd)_mSubstituted phenyl ring or pyridine,

(Rd)_mFor m identical or different substituent R d, m 0,1 or 2, Rd H, fluorine, chlorine, bromine, hydroxyl, cyano, methyl, second Base, propyl, isopropyl, butyl, tert-butyl, methoxyl group, ethyoxyl, propoxyl group, isopropoxy, methyl fluoride, difluoromethyl, trifluoro Methyl, fluorine methoxyl group, difluoro-methoxy, trifluoromethoxy, methylol, ethoxy, hydroxypropyl, amino, methylamino, ethylamino, Dimethylamino, amino methyl, amino-ethyl, aminopropyl, Methyaminomethyl, methylaminoethyl, methylaminopropyl, ethylamino first Base, ethylaminoethyl, ethylaminopropyl, dimethylamino methyl, dimethylaminoethyl, dimethylamino-propyl, or be-L-E- (Re)_n,

L is-(CH₂)_q, q 1,2 or 3；

(Re)_nFor identical or different substituent R e, the Re H of n, fluorine, chlorine, bromine, hydroxyl, methyl, ethyl, propyl, isopropyl, One methyl fluoride, difluoromethyl, trifluoromethyl, methoxyl group, methoxy methoxy ethyl, amino, methylamino, dimethylamino, Amino methyl, Methyaminomethyl, dimethylamino methyl, amino-ethyl, methylaminoethyl, dimethylaminoethyl, methylol, hydroxyl second Base, n 0,1 or 2.

8. a kind of method for preparing compound described in any one of claim 1-7, which is characterized in that its chemical combination as shown in formula D Object and R¹X reacts to obtain, wherein R¹, R², R³If claim 1-7 is defined,

9. a kind of method for preparing compound described in any one of claim 1-7, which is characterized in that it includes following steps, That is the compound as shown in formula C and trimethyl ((tributylstamlyl) acetenyl) silane reaction obtains formula D compound, wherein R²And R³If claim 1-7 is defined,

10. a kind of method for preparing compound described in any one of claim 1-7, which is characterized in that it includes following steps, I.e. the compound as shown in formula A reacts to obtain formula C compound with compound shown in formula B, wherein R²And R³As claim 1-7 determines Justice,

11. a kind of formula D compound represented, isomers, tautomer, solvate, hydrate or its can pharmaceutically connect The salt received,

Wherein R²And R³As claim 1-7 is defined.

12. a kind of method for preparing compound described in any one of claim 1-7, which is characterized in that it changes as shown in formula D1 Close object and R³NH₂Reaction obtains, wherein R¹, R², R³If claim 1-7 is defined,

13. a kind of method for preparing compound described in any one of claim 1-7, which is characterized in that it includes following steps, I.e. the compound as described in C1 obtains compound described in D1 through hydrolysis,

Wherein, wherein R¹And R²As claim 1-7 is defined.

14. a kind of method for preparing compound described in any one of claim 1-7, which is characterized in that it includes following steps, That is the compound as described in B1 and R¹X reacts to obtain compound described in C1,

Wherein R¹And R²As claim 1-7 is defined.

15. a kind of method for preparing compound described in any one of claim 1-7, which is characterized in that it includes following steps, That is compound shown in A1 and trimethyl ((tributylstamlyl) acetenyl) silane reaction obtains compound shown in B1,

Wherein R²As claim 1-7 is defined.

16. a kind of formula D1 compound represented, isomers, tautomer, solvate, hydrate or its can pharmaceutically connect The salt received, wherein R¹And R²If claim 1-7 is defined,

17. the compound or its pharmaceutical salt of formula (I) according to claim 1, wherein the salt be acid/yin from Alite or basic/cationic salts；The form that pharmaceutically acceptable acidity/anion salt is usually taken is to allow basic nitrogen therein It being protonated by inorganic or organic acid, representative organic or inorganic acid includes hydrochloric acid, hydrobromic acid, hydroiodic acid, perchloric acid, sulfuric acid, Nitric acid, phosphoric acid, formic acid, acetic acid, propionic acid, hydroxyacetic acid, lactic acid, succinic acid, maleic acid, tartaric acid, malic acid, citric acid are rich Horse acid, gluconic acid benzoic acid, mandelic acid, methanesulfonic acid, isethionic acid, benzene sulfonic acid, oxalic acid, palmitinic acid, 2- naphthalene sulfonic acids are right Toluenesulfonic acid, cyclohexylamino sulfonic acid, salicylic acid, saccharinic acid, trifluoroacetic acid.Pharmaceutically acceptable basic/cationic salts class packet (this is of course not solely limited to include) aluminium, calcium, chloroprocanine, choline, diethanol amine, ethylenediamine, lithium, magnesium, potassium, sodium and zinc.

18. a kind of Pharmaceutical composition, it includes compound described in claim 1-7 or its pharmaceutically acceptable salts or its water Close object or its solvate or its prodrug and pharmaceutically acceptable carrier or excipient.

19. a kind of Pharmaceutical composition, wherein the compound comprising the formula (I) as described in claim 1-, pharmaceutically acceptable Salt, hydrate, solvate or prodrug are as active constituent, one or more of the other therapeutic agent and one or more Pharmaceutically acceptable carrier or excipient.

20. the compound or its pharmaceutically acceptable salt or its prodrug of formula described in any one of -7 (I) according to claim 1 Application in the drug that cancer relevant to tyrosine kinase and autoimmune disease are treated in preparation.It is preferred that the junket ammonia Acid kinase is including but not limited to ABL, VEGFR2；It is preferred that the cancer relevant to tyrosine kinase and autoimmune disease packet It includes but is not limited to: is fundus oculi disease, xerophthalmia, psoriasis, leucoderma, dermatitis, alopecia areata, rheumatoid arthritis, colitis, multiple Hardening, systemic loupus erythematosus, Crohn disease, atheroma, pulmonary fibrosis, liver fibrosis, myelofibrosis, non-small cell Lung cancer, Small Cell Lung Cancer, breast cancer, cancer of pancreas, glioma, glioblastoma, oophoroma, cervix cancer, colon are straight Intestinal cancer, melanoma, carcinoma of endometrium, prostate cancer, bladder cancer, leukaemia, gastric cancer, liver cancer, gastrointestinal stromal tumor, thyroid gland It is cancer, chronic myelocytic leukemia, acute myeloblastic leukemia, acute myelocytic leukemia, chronic granulocytic leukemia, non- Hodgkin lymphoma, nasopharyngeal carcinoma, cancer of the esophagus, brain tumor, B cell and t cell lymphoma, lymthoma, Huppert's disease, cancer of bile ducts It is any etc. in sarcoma, cholangiocarcinoma.