CN109988151B

CN109988151B - Acetylene compound, preparation method and application thereof

Info

Publication number: CN109988151B
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 Scitech MQ Pharmaceuticals Ltd
Current assignee: Beijing Scitech MQ Pharmaceuticals Ltd
Priority date: 2017-12-29
Filing date: 2017-12-29
Publication date: 2021-04-23
Anticipated expiration: 2037-12-29
Also published as: CN109988151A

Abstract

The invention belongs to the field of chemical medicine, and particularly relates to a compound shown in a formula (I) or pharmaceutically acceptable salt, isomer, solvate, crystal or prodrug thereof, andpharmaceutical compositions containing these compounds and the use of these compounds or compositions in the manufacture of medicaments. The compound and the pharmaceutically acceptable salt, isomer, solvate, crystal or prodrug thereof can be used for treating or preventing various cancers and other diseases.

Description

Acetylene compound, preparation method and application thereof

Technical Field

The invention belongs to the field of chemical medicine, and particularly relates to a compound with BCR-ABL kinase inhibitory activity or pharmaceutically acceptable salts, isomers, solvates, crystal forms or prodrugs thereof, a pharmaceutical composition containing the compound and application of the compound or the composition in preparation of a medicament.

Background

Under the action of various carcinogenic factors, cells of a tumor are subjected to abnormal signal transduction, and certain cells of part of tissues lose regulation and control on normal growth of the cells, so that the cells are subjected to apoptosis disorder and continuous cell proliferation, and further, new organisms are generated by clonal growth. Tumor cells have the ability to grow autonomously after losing normal growth regulatory function, and tumors can continue to grow after growth of carcinogenic factors ceases. Clinically, tumors can be divided into solid tumors and non-solid tumors, the solid tumors, namely tangible tumors, are treated by surgical resection, chemotherapy and other methods, while the non-solid tumors mainly use chemical drugs to kill cancer cells, but the chemical drugs have large side effects, and cells in vivo are damaged no matter whether the cells are malignant tumor cells or not.

Leukemia is one of the malignancies, belonging to the non-solid tumors, which ranks first in the incidence of pediatric malignancies. According to the natural course of leukemia cells, they can be classified into two major categories, acute leukemia and chronic leukemia. Among them, Chronic Leukemia is classified into Chronic Myelogenous Leukemia (CML) and Chronic Lymphocytic Leukemia (CLL). Chronic myeloid leukemia accounts for about 20% of all leukemias, and occurs in all age groups.

At present, a novel anticancer drug with low toxicity and strong specificity, which selectively acts on a specific target, has become a new direction for research of antitumor drugs, and in patients with Chronic Myelogenous Leukemia (CML), the long arm of chromosome 22 is translocated to chromosome 9 to form Philadelphia chromosome, and the BCR gene and the ABL gene are fused to form a BCR-ABL fusion gene, so that BCR-ABL protein tyrosine kinase is expressed, and the kinase can cause the change of cell proliferation, adhesion and survival property, and cause the generation of various tumors. BCR-ABL is not expressed in normal cells, so that it is an ideal drug target for treating chronic granulocytic leukemia.

Currently, the most clinically used small molecule inhibitors against BCR-ABL tyrosine kinase include: the first generation drug imatinib; the second generation drugs dasatinib, nilotinib, and bosutinib; the third generation drug, ponatinib. The tyrosine kinase inhibitor plays a role in resisting chronic granulocytic leukemia mainly by inhibiting the activity of BCR-ABL fusion protein.

Imatinib (Imatinib) is a small molecule BCR-ABL tyrosine kinase inhibitor developed by novain, and was approved by the FDA for the treatment of CML in 2001. This is the first tyrosine kinase inhibitor to treat CML, which can treat cancer by targeting specific damaged genes of tumor cells. Compared with other treatment medicines, the imatinib can effectively relieve chronic granulocytic leukemia, and the 5-year survival rate of a patient after treatment can reach 90%. The imatinib has the remarkable characteristic that the imatinib can specifically inhibit the proliferation of chronic granulocytic leukemia cancer cells, hardly damages normal cells, and greatly reduces the toxic and side effects of the drug. Imatinib opens a new era for the treatment of diseases targeted at kinases.

The emergence of drug resistance greatly reduces the therapeutic efficacy of imatinib. The main reason for the occurrence of drug resistance of Imatinib is that mutation such as L248V, E255V, Y253H, E355G, E255K, T315I, F359V, M253H, G250E, F317L, H396P, M351T, Q252H and the like occurs in BCR-ABL gene, and the affinity between Imatinib and ABL kinase is reduced due to point mutation of ABL kinase, so that the treatment effect is obviously reduced.

The second generation Bcr-Abl tyrosine kinase inhibitor Nilotinib (Nilotinib) is an anilinopyrimidine derivative that was approved by the U.S. FDA for use in the treatment of CML 10 months of 2007. Its affinity for Bcr-Abl tyrosine kinase is 20-fold stronger than that for Imatinib. Nilotinib may inhibit Imatinib-resistant mutations other than the T315I mutation. However, most patients with CML treated by Nilotinib have the common adverse reactions of lipase and bilirubin increase, mild and moderate rash, bone marrow suppression, gastrointestinal reaction and the like.

Dasatinib (Dasatinib) is also a second generation Bcr-Abl tyrosine kinase inhibitor, is an oral kinase inhibitor with inhibitory effect on multiple kinases, and has good inhibitory effect on BCR-ABL kinase and SRC family kinase (SRC kinase is a target of antitumor drug action). Dasatinib was approved by the FDA for marketing at 6 months 2006 for the treatment of CML patients. Dasatinib has less structural requirements than imatinib and is able to overcome the resistance that occurs with many imatinib species (except for the T315I mutation). The dasatinib is rapidly absorbed after being orally taken, the maximum blood concentration is reached within 0.5-3h, and the average half-life period is 5-6 h. The main adverse reactions after the patient takes dasatinib are manifested by bone marrow suppression and neutrophilia.

Bosutinib (Bosutinib) is a new drug developed by hui pharmaceutical company, usa, for treating CML with 4-substituted aniline-3-quinolinecarbonitriles, which is approved by FDA to be marketed in month 2012 and 9, and is mainly a kinase inhibitor for CML patients who have failed in the treatment with imatinib, nilotinib, and dasatinib. Antiproliferative activity (IC) of bosutinib against both KU812 and K562 cells₅₀) 20nM and 5nM, respectively, and the antiproliferative activity of imatinib on KU812 and K562 cells was 210nM and 88nM, respectively. However, bosutinib also had no inhibitory effect on the T315I mutation. Adverse reactions in patients taking bosutinib mainly include nausea, vomiting, abdominal pain, diarrhea, rash, elevated liver enzyme levels, thrombocytopenia, anemia and fatigue.

The second generation of treatment CML drugs Dasatinib, Nilotinib and Bosutinib have extensive activity in patients who are resistant and intolerant to Imatinib, but all have no inhibitory activity on BCR-ABL T315I kinase mutation.

Although small molecule drugs targeting protein tyrosine kinases have met with great success in the treatment of chronic myelogenous leukemia, their use is largely limited by the emergence of drug resistance. 17 mutations in this kinase region have now been identified, including 6 known imatinib-resistant mutations (M244V, Y253H, F359C/V/I, G250E, E255K and T315I) and 11 newly added mutations (K247N, E282K, K285N, V289L, L273F, E292K, N297T, H375P, T406I, W430L and E431G). Due to the emergence of resistance to second-generation BCR-ABL tyrosine kinase inhibitors, there is a need to develop new BCR-ABL tyrosine kinase inhibitors.

The third generation BCR-ABL tyrosine kinase inhibitor Ponatinib (Ponatiniib) is an oral multi-target kinase inhibitor. It is mainly used to overcome BCR-ABL^T315IMeanwhile, the inhibitor also has good inhibition effect on the wild BCR-ABL. Ponatinib inhibits BCR-ABL kinase activity including the T315I mutation, and Praintinib inhibits wild-type BCR-ABL kinase and BCR-ABL kinase according to the description of the literature (Rabndran SK, et al. cancer Res,2004,64(11), 3958-^T315IThe binding pattern of the kinase differs only slightly, its inhibitory activity (IC) on wild-type BCR-ABL kinase₅₀) Is BCR-ABL^T315IInhibitory Activity (IC)₅₀) 5-7 times of the total weight of the product. Patients taking Ponatinib can develop serious adverse vascular events including fatal and life-threatening myocardial infarction, stroke, tissue necrosis due to interruption of limb blood flow, etc. Its serious side effects limit the clinical use of the drug.

Disclosure of Invention

The invention relates to an alkyne compound and a pharmaceutically acceptable salt, isomer, solvate, crystal form or prodrug thereof, which can be used for treating or preventing diseases caused by over-expression of BCR-ABL kinase, wherein the diseases comprise imatinib resistant mutation (M244V, Y253H, F359C/V/I, G250E, E255K and T315I) and 11 newly added mutations (K247N, E282K, K285N, V289L, L273F, E255 292K, N297T, H375P, T406I, W430L and E431G). The compounds and the pharmaceutically acceptable salts, prodrugs and other forms thereof can be used for treating or preventing a plurality of different cancers. The invention also relates to pharmaceutical compositions comprising said compounds and prodrugs thereof, as well as effective polymorphic forms thereof, intermediates to said compounds, and salts thereof, useful in methods of treatment of various forms of BCR-ABL mediated diseases.

The present invention provides an acetylenic compound and its use as a treatment or prophylaxis of a disease caused by a tyrosine kinase (e.g. ABL, VEGFR2) or a variant thereof.

The present invention provides a compound or its isomer, tautomer, solvate, hydrate or pharmaceutically acceptable salt thereof. The present invention is provided in the form of a pharmaceutical composition of its effective polymorph. The compound has the structural formula (I):

wherein the content of the first and second substances,

x is N, CH;

R¹is composed of

a) Aryl unsubstituted or substituted by 1 to 3 of the same or different Ra or a 5-6 membered heteroaromatic ring, said 5-6 membered heteroaromatic ring containing 1 to 3 heteroatoms selected from N, O and S;

b) ring A and ring B unsubstituted or substituted by 1-2 same or different Rb, wherein ring A is a 5-6 membered heteroaromatic ring or a 6 membered aromatic ring, ring B is selected from a 3-6 membered cycloalkyl, a 3-6 membered heterocycle, a 5-6 membered heteroaromatic ring or an aromatic ring, and the heterocycle and heteroaromatic ring each independently contain 1-3 heteroatoms selected from N, O and S;

ra is H, hydroxy, cyano, acyl, halogen, C unsubstituted or substituted by hydroxy or halogen₁-C₆Alkyl, -OR 'OR-NR' R ";

rb is H, halogen, hydroxy, cyano, carbonyl, acyl, C₁-C₆Alkyl, -OR';

r 'and R' are each independently H, C₃-C₆Cycloalkyl, unsubstituted or substituted by hydroxy, carboxy, halogen, C₁-C₆Alkoxy or C₁-C₆Alkylamino substituted C₁-C₆An alkyl group;

R²is H, C₁-C₆An alkyl group;

R³is composed of

D Ring is unsubstituted or optionally substituted (Rd)_mA substituted 5-6 membered cycloalkyl, a 5-6 membered heterocyclic ring containing 1-3 heteroatoms selected from N, O and S, a 5-6 membered aromatic ring or a 5-6 membered heteroaromatic ring containing 1-3 heteroatoms selected from N, O and S;

(Rd)_mrepresents m identical or different substituents Rd, m is an integer from 0 to 4, Rd is H, halogen, hydroxy, cyano, C₁-C₆Alkoxy radical, C₁-C₆Alkylamino, unsubstituted or substituted by hydroxy, halogen, C₁-C₆Alkoxy radical, C₁-C₆Alkylamino substituted C₁-C₆Alkyl, or is-L-E- (Re)_n；

L is- (CR)^l1R^l2)_q-; q is an integer of 1 to 4;

R^l1and R^l2Each independently is H or C₁-C₆An alkyl group;

e is a 3-6 membered cyclic or heterocyclic group, said 3-6 membered heterocyclic group being a saturated or unsaturated heterocyclic group containing 1-3 heteroatoms selected from N, O and S;

(Re)_nn are identical or different Re, n is an integer from 0 to 4, Re is H, halogen, hydroxy, C₁-C₆Alkoxy radical, C₁-C₆Alkylamino, unsubstituted or substituted by halogen, hydroxy, C₁-C₆Alkoxy or C₁-C₆Alkylamino substituted C₁-C₆An alkyl group.

The term "substituted" as used herein includes complex substituents (e.g., phenyl, aryl, heteroalkyl, heteroaryl), suitably 1 to 5 substituents, preferably 1 to 3 substituents, and most preferably 1 to 2 substituents, which are freely selectable from the list of substituents.

Unless otherwise specified, alkyl groups as used herein include saturated monovalent hydrocarbon groups having straight, branched or cyclic moieties. For example, alkyl groups include methyl, ethyl, propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, cyclobutyl, n-pentyl, 3- (2-methyl) butyl, 2-pentyl, 2-methylbutyl, neopentyl, cyclopentyl, n-hexyl, 2-methylpentyl and cyclohexyl. Alkoxy groups are the oxidized ethers consisting of the previously described linear, branched or cyclic alkyl groups. Similarly, alkenyl and alkynyl groups include straight, branched or cyclic alkenyl and alkynyl groups.

The term "aryl" as used herein, unless otherwise specified, refers to an unsubstituted or substituted aromatic radical, such as phenyl, naphthyl, anthracenyl. The term "aroyl" refers to-C (O) -aryl.

The term "heterocyclyl", as used herein, unless otherwise specified, represents an unsubstituted or substituted stable 3 to 8 membered monocyclic saturated ring system consisting of carbon atoms and from 1 to 3 heteroatoms selected from N, O, S, wherein the N, S heteroatoms may be optionally oxidized and the N heteroatoms may be optionally quaternized. The heterocyclic ring may be bonded to any heteroatom or carbon atom, thereby forming a stable structure. Examples of such heterocycles include, but are not limited to, aziridinyl, pyrrolidinyl, piperidinyl, piperazinyl oxide, piperidinyl oxide, azanyl, tetrahydrofuranyl, dioxolanyl, tetrahydroimidazolyl, tetrahydrothiazolyl, tetrahydrohiccup, tetrahydropyranyl, morphinyl, thiomorpholinyl sulfoxide, thiomorpholinyl sulfone, and hiccup diazolyl.

The term "heteroaryl" as used herein, unless otherwise specified, represents an unsubstituted or substituted stable 5-or 6-membered monocyclic aromatic ring system, and may also represent an unsubstituted or substituted 9-or 10-membered fused benzene heteroaromatic ring system or bicyclic heteroaromatic ring system consisting of carbon atoms and from 1 to 4 heteroatoms selected from N, O, S, wherein the N, S heteroatoms may be optionally oxidized and the N heteroatoms may also be optionally quaternized. The heteroaryl group may be attached to any heteroatom or carbon atom, thereby forming a stable structure. Examples of heteroaryl groups include, but are not limited to, thianthrenyl, furanyl, imidazolyl, isoxazolyl, hiccup, pyrazolyl, pyrrolyl, thiazolyl, thiadiazolyl, triazolyl, pyridyl, pyridazinyl, indolyl, azaindolyl, indazolyl, benzimidazolyl, benzofuranyl, benzothienyl, benzisoxazolyl, benzoxazolyl, benzpyrazolyl, benzothiazolyl, benzothiadiazolyl, benzotriazolyl, adenine, quinolinyl or isoquinolinyl.

The term "carbonyl" refers to a C (O) group.

Whenever the term "alkyl" or "aryl" or any of their prefix roots appear in the name of a substituent (e.g., aralkyl, dialkylamino), it is to be considered as encompassing those limitations given above for "alkyl" and "aryl". Specified number of carbon atoms (e.g., C)₁‐C₆) Will independently represent the number of carbon atoms in an alkyl moiety or an alkyl moiety in a larger substituent (where alkyl is taken as its prefix root).

In a preferred embodiment of the invention there is provided a compound of formula (I) or isomers, tautomers, solvates, hydrates, and pharmaceutically acceptable salts thereof,

wherein the content of the first and second substances,

x is N, CH;

R¹is composed of

a) A 5-6 membered heterocycle or 5-6 membered heteroaryl unsubstituted or substituted with 1-3 of the same or different Ra, said heterocycle and heteroaryl rings each independently containing 1-3 heteroatoms selected from N, O and S;

b) ring A and ring B being unsubstituted or substituted by 1 to 2 identical or different Rb,

ring A is a 5-6 membered heteroaromatic ring, ring B is selected from a 3-6 membered heterocyclic ring or a 3-6 membered heteroaromatic ring;

the heterocycle and heteroaryl ring each independently contain 1-3 heteroatoms selected from N, O and S;

ra is-H, hydroxy, cyano, halogen, C unsubstituted or substituted by hydroxy or halogen₁-C₃Alkyl, -OR 'OR NR' R ";

r 'and R' are each independently H, C₁-C₄Alkyl radical, C₃-C₄Cycloalkyl, unsubstituted or substituted by halogen, C₁-C₃Alkoxy or C₁-C₃Alkylamino substituted C₁-C₃An alkyl group;

rb is-H, halogen, CF₃Carbonyl, cyano, C₁-C₃An alkyl group;

R²is H, C₁-C₄A linear or branched alkyl group;

R³is composed of

D Ring is unsubstituted or optionally substituted (Rd)_mA substituted 6-membered aromatic ring or a 5-6 membered aromatic ring containing 1-3 heteroatoms selected from N, O and S;

(Rd)_mm identical or different substituents Rd, m being 0, 1,2 or 3; rd is H, halogen, hydroxy, cyano, C₁-C₄Alkoxy radical, C₁-C₄Alkylamino, unsubstituted or substituted by hydroxy, halogen, C₁-C₄Alkoxy radical, C₁-C₄Alkylamino substituted C_1-4Alkyl or-L-E- (Re)_n；

L is- (CR)^l1R^l2)_q，R^l1And R^l2Are independently H, C_1-3An alkyl group, q is an integer of 1 to 3,

e is a 3-6 membered heterocyclic group, said 3-6 membered heterocyclic group being a saturated heterocyclic group or an unsaturated heterocyclic group containing 1-3 heteroatoms selected from N, O and S;

(Re)_nn identical or different substituents Re, Re being H, halogen, hydroxy, C_1-3Alkoxy radical, C_1-3Alkylamino, unsubstituted or substituted by halogen, hydroxy, C₁-C₃Alkoxy or C₁-C₃Alkylamino substituted C₁-C₄Alkyl, n is 0, 1,2 or 3.

In another preferred embodiment, the compound or its isomer, tautomer, solvate, hydrate and pharmaceutically acceptable salt thereof, R²Is H, methyl, ethyl, propyl or isopropyl.

In one embodiment, the compound or its isomer, tautomer, solvate, hydrate and pharmaceutically acceptable salt,

R¹in order to realize the purpose,

(Ra)^pis substituted by p identical or different Ra's, p is 0, 1,2 or 3;

ra is-H, fluoro, chloro, bromo, methyl, ethyl, propyl, butyl, isopropyl, trifluoromethyl, -NHR ', R' is-H, methyl, ethyl, aminoethyl, methylaminoethyl, dimethylaminoethyl, cyclopropyl, methoxyethyl, aminopropyl, methylaminopropyl, dimethylaminopropyl, cyclobutyl or methoxypropyl;

(Rb)^rr is substituted by r, the same or different Rb, 0, 1, or 2;

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

R³is composed of

D Ring unsubstituted or substituted (Rd)_mA substituted aromatic or heteroaromatic ring, the aromatic ring being a phenyl ring, the heteroaromatic ring being selected from pyridine, pyridazine or thiazole;

(Rd)_mm identical or different substituents Rd, m is 0, 1,2 or 3, Rd is H, halogen, hydroxy, cyano, C_1‐C₄Alkoxy radical, C_1‐C₄Alkylamino, unsubstituted or substituted by hydroxy, halogen, C_1‐C₄Alkoxy radical, C_1‐C₄Alkylamino substituted C_1‐4Alkyl, or is-L-E- (Re)_n；

L is- (CH)₂)_q-q is 1,2 or 3;

e is a 3-6 membered heterocyclic group, said 3-6 membered heterocyclic group is selected from the group consisting of cycloethylethane, ethylene oxide, pyrrolidine, pyrroline, pyrrole, pyrazolidine, pyrazoline, imidazole, pyrazole, furan, tetrahydrofuran, dihydrofuran, tetrahydrothiophene, thiophene, oxazole, thiazole, thiadiazole, piperidine, pyridine, dihydropyridine, morpholine, piperazine or pyrazine;

(Re)_nn equal or different Re, Re is H, halogen, hydroxy, C_1‐3Alkoxy radical, C_1‐3Alkylamino, unsubstituted or substituted by halogen, hydroxy, C_1‐C₃Alkoxy or C_1‐C₃Alkylamino substituted C_1‐C₃Alkyl, n is 0, 1,2 or 3.

R³is composed of

D Ring unsubstituted or substituted (Rd)_mA substituted benzene ring or a pyridine,

(Rd)_mm identical or different substituents Rd, m is 0, 1 or 2, Rd is H, fluorine, chlorine,bromo, hydroxy, cyano, methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, methoxy, ethoxy, propoxy, isopropoxy, fluoromethyl, difluoromethyl, trifluoromethyl, fluoromethoxy, difluoromethoxy, trifluoromethoxy, hydroxymethyl, hydroxyethyl, hydroxypropyl, amino, methylamino, ethylamino, dimethylamino, aminomethyl, aminoethyl, aminopropyl, methylaminomethyl, methylaminoethyl, methylaminopropyl, ethylaminomethyl, ethylaminopropyl, dimethylaminomethyl, dimethylaminoethyl, dimethylaminopropyl, or is-L-E- (Re)_n，

L is- (CH)₂)_q-q is 1,2 or 3;

e is a 5-6 membered heterocyclic ring, said 5-6 membered heterocyclic ring selected from the group consisting of:

(Re)_nn identical or different substituents Re, Re being H, fluorine, chlorine, bromine, hydroxyl, methyl, ethyl, propyl, isopropyl, fluoromethyl, difluoromethyl, trifluoromethyl, methoxy, methoxymethylmethoxyethyl, amino, methylamino, dimethylamino, aminomethyl, methylaminomethyl, dimethylaminomethyl, aminoethyl, methylaminoethyl, dimethylaminoethyl, hydroxymethyl, hydroxyethyl, n being 0, 1 or 2.

It will be clear that the compounds of formula (I) and pharmaceutically acceptable salts thereof may exist in solvated as well as unsolvated forms. For example, the solvated form may be water soluble. The present invention includes all such solvated and unsolvated forms.

The compounds of the invention may have asymmetric carbon atoms and, depending on their physicochemical differences, such diastereomeric mixtures may be separated into the individual diastereomers by methods well known in the art, for example, chromatography or fractional crystallization. Enantiomers can be separated by first converting the enantiomeric mixture into a diastereomeric mixture by reaction with a suitably optically active compound (such as ethanol), separating the diastereomers, and then converting (hydrolyzing) the individual diastereomers to the corresponding pure enantiomers. All such isomers, including diastereomeric mixtures and pure enantiomers are considered as part of the invention.

The compounds of the present invention may also be present in the form of pharmaceutically acceptable salts. In pharmaceutical applications, salts of the compounds of the invention refer to non-toxic pharmaceutically acceptable salts. Pharmaceutically acceptable salt forms include pharmaceutically acceptable acidic/anionic salts or basic/cationic salts. Pharmaceutically acceptable acid/anion salts generally take the form of having the basic nitrogen protonated by an inorganic or organic acid. Representative organic or inorganic acids include hydrochloric, hydrobromic, hydroiodic, perchloric, sulfuric, nitric, phosphoric, acetic, propionic, glycolic, lactic, succinic, maleic, tartaric, citric, benzoic, mandelic, methanesulfonic, isethionic, benzenesulfonic, oxalic, palmitic, 2-naphthalenesulfonic, p-toluenesulfonic, cyclohexylsulfamic, salicylic, hexonic, trifluoroacetic acids. Pharmaceutically acceptable basic/cationic salts include, but are not limited to, aluminum, calcium, chloroprocaine, choline, diethanolamine, ethylenediamine, lithium, magnesium, potassium, sodium and zinc.

The present invention includes prodrugs of the compounds of the invention in their art. In general, such prodrugs are functional derivatives of the compounds that are readily converted in vivo to the desired drug. Thus, in the methods of treatment of the present invention, the term "administering" shall encompass the treatment of a variety of different described conditions, either with compounds that have been specifically disclosed or with compounds that may not have been specifically identified but which are capable of being converted in vivo to those specifically identified in the subject after administration of the agent. Conventional procedures for the selection and preparation of suitable prodrug derivatives have been described in books such as "design of prodrugs" (edited by h. bundgaard, Elsevier, 1985).

It will be apparent that the definition of any substituent or variable at a particular position in a molecule is independent of the definition of that substituent or variable elsewhere in that molecule. It will be readily appreciated that the substitutions and substitution patterns on the compounds of the invention may be selected by one of ordinary skill in the art to provide compounds which are chemically stable and which are readily synthesized by techniques known in the art and by the methods set forth herein.

Prodrugs of the parent compound are those derivatives and prodrugs which, when administered to a mammal, enhance the bioavailability of the compound (e.g., by allowing enhanced absorption into the blood stream upon oral administration) or permit faster delivery to the biological site of interest in the body relative to the parent compound. Preferred prodrugs of the invention include derivatives of the compounds that have enhanced aqueous solubility or active transport through the intestinal membrane relative to the parent compound.

The present invention also provides a process for the preparation of a compound of formula I as hereinbefore described, for example by the process as hereinafter described.

The invention provides a method for preparing the compound of the formula (I), which is prepared from the compound of the formula D and R¹X is reacted to give, wherein R¹，R²，R³As defined above, in the above-mentioned manner,

in a preferred embodiment, the formulae D and R¹And (3) during the reaction of X, preparing the compound shown in the formula I in an organic solvent under the conditions of a catalyst, an organic base and a fluoride ion salt.

The organic solvent includes, but is not limited to, one or a combination of two or more of toluene, xylene, tetrahydrofuran, ethyl acetate, dioxane, N-methylpyrrolidone, N-dimethylformamide, and benzene.

Wherein the catalyst includes but is not limited to cuprous iodide andpalladium bis (triphenylphosphine) dichlorideOr cuprous iodide withTetratriphenylene Palladium based phosphine；

Organic bases include, but are not limited to, one or a combination of two or more of triethylamine, diisopropylethylamine, 1, 8-diazabicycloundecen-7-ene, or N-methylmorpholine;

salts of fluoride ions include, but are not limited to, one or a combination of two or more of tetrabutylammonium fluoride, potassium fluoride, or cesium fluoride.

The invention provides a method for preparing the compound of the formula (I), which comprises the following steps that the compound of the formula D is obtained by reacting the compound of the formula C with trimethyl ((tributylstannyl) ethynyl) silane

In a preferred embodiment of the present invention,

when the compound shown in the formula C reacts with trimethyl ((tributylstannyl) ethynyl) silane, the compound shown in the formula D is prepared in an organic solvent under the condition of a catalyst.

Preferably, the catalyst comprises, but is not limited to, palladium tetrakistriphenylphosphine,Bis (dibenzylideneacetone) palladiumA mixture of palladium acetate and palladium acetate, wherein,palladium bis (triphenylphosphine) dichlorideOne or a combination of two or more of them.

Preferably, the organic solvent includes, but is not limited to, one or a combination of two or more of toluene, xylene, tetrahydrofuran, dioxane, N-methylpyrrolidone, N-dimethylformamide, and benzene.

In a preferred embodiment of the present invention,

a process for preparing a compound as hereinbefore described comprising reacting a compound of formula A with a compound of formula B to give a compound of formula C,

in a preferred embodiment, the compound of formula A is reacted with a compound of formula B, wherein formula A may be reacted with an acylating agent prior to reaction with formula B.

Preferably, the acylating agent includes but is not limited to one or a combination of two or more of phosphorus oxychloride, thionyl chloride, oxalyl chloride, phosphorus trichloride or phosphorus pentachloride.

In another embodiment, a compound of formula A is reacted with a compound of formula B in the presence of a condensing agent to provide a compound of formula C,

preferably, the condensing agent includes, but is not limited to, one or a combination of two or more of 2- (7-benzotriazole oxide) -N, N' -tetramethylurea Hexafluorophosphate (HATU), propylphosphoric anhydride (T3P), a carbodiimide type condensing agent, a carbonium type condensing agent, a urea cationic type condensing agent, and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI);

preferably, this step is carried out in an organic base including, but not limited to, one or a combination of two or more of triethylamine, diisopropylethylamine, pyridine, 4-dimethylaminopyridine, 1, 8-diazabicycloundecen-7-ene or N-methylmorpholine.

The invention provides a method for preparing a compound shown as the formula (I), which is characterized in that the compound is shown as the formula D1 and R³NH₂Obtained by reaction, wherein R¹，R²，R³As defined above, in the above-mentioned manner,

in a preferred embodiment, the compound of formula D1 is reacted with a compound of formula R³NH₂In the reaction of the compounds of the formula D1 with acylating agents and then with the compounds of the formula R³NH₂The reaction is carried out.

In another embodiment, a compound of formula D1 is reacted with a compound of formula R in the presence of a condensing agent³NH₂The compound reacts to obtain the compound shown in the formula I,

The invention provides a process for preparing a compound as defined hereinbefore, characterized in that it comprises the steps of subjecting a compound as defined in C1 to hydrolysis reaction to give a compound as defined in F1,

wherein R is¹And R²As defined above.

Hydrolyzing the compound represented by the formula C1 in a mixed solvent of an organic solvent and water in the presence or absence of an inorganic base to obtain the compound represented by the formula D1.

Preferably, the organic solvent can be both protic and aprotic organic solvents, and all common organic solvents are within the applicable range of the reaction.

The present invention provides a process for the preparation of the compounds described hereinbefore, comprising the step of reacting a compound described by B1 with R¹The compound described by C1 is obtained by X reaction,

wherein R is¹And R²As defined above.

In a preferred embodiment, the formulae B1 and R¹In the reaction of X, in an organic solvent, a catalyst, an organic base, and fluoride ionsUnder salt conditions, a compound represented by formula C1 was prepared.

Preferably, the organic solvent includes, but is not limited to, one or a combination of two or more of toluene, xylene, tetrahydrofuran, ethyl acetate, dioxane, N-methylpyrrolidone, N-dimethylformamide, and benzene.

Wherein the catalyst comprises, but is not limited to, cuprous iodide and palladium bis (triphenylphosphine) dichloride or cuprous iodide and palladium tetrakis (triphenylphosphine);

The present invention provides a process for preparing a compound as hereinbefore described comprising the steps of reacting a compound represented by a1 with trimethyl ((tributylstannyl) ethynyl) silane to give a compound represented by B1,

wherein R is²As defined above.

In a preferred embodiment, when the formula A1 is reacted with trimethyl ((tributylstannyl) ethynyl) silane, the compound shown as the formula B1 is prepared in an organic solvent under the condition of a catalyst,

preferably, the catalyst comprises but is not limited to one or a combination of more than two of tetratriphenylphosphine palladium, bis (dibenzylideneacetone) palladium, palladium acetate and palladium bistriphenylphosphine dichloride.

The present invention provides a process for the preparation of a compound of formula I as hereinbefore defined, which comprises the following steps:

reacting a compound shown in a formula A and a compound shown in a formula B in the step 1), wherein the formula A can react with an acylating reagent, and the acylating reagent comprises one or the combination of more than two of phosphorus oxychloride, thionyl chloride, oxalyl chloride, phosphorus trichloride or phosphorus pentachloride, and then reacts with the formula B; or reacting with a compound shown in a formula B under the condition of a condensing agent to prepare the compound shown in the formula C, wherein the condensing agent comprises one or more of 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethylurea Hexafluorophosphate (HATU), propyl phosphoric anhydride (T3P), a carbodiimide type condensing agent, a carbocation type condensing agent, a urea cationic type condensing agent and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI). Preferably, this step is carried out in an organic base including, but not limited to, one or a combination of two or more of triethylamine, diisopropylethylamine, pyridine, 4-dimethylaminopyridine, 1, 8-diazabicycloundecen-7-ene or N-methylmorpholine.

In the step 2), when the formula C reacts with trimethyl ((tributylstannyl) ethynyl) silane, a compound shown in the formula D is prepared in an organic solvent under the condition of a catalyst, wherein the catalyst includes but is not limited to one or a combination of two or more of tetratriphenylphosphine palladium, bis (dibenzylideneacetone) palladium, palladium acetate and triphenylphosphine palladium dichloride, and includes but is not limited to one or a combination of two or more of toluene, xylene, tetrahydrofuran, dioxane, N-methylpyrrolidone, N-dimethylformamide and benzene.

Step 3) the formulae D and R¹And (3) during the reaction of X, preparing the compound shown in the formula I in an organic solvent under the conditions of a catalyst, an organic base and a fluoride ion salt. Wherein the catalyst comprises, but is not limited to, cuprous iodide and palladium bis (triphenylphosphine) dichloride or cuprous iodide and palladium tetrakis (triphenylphosphine); the organic base includes but is not limited to one of triethylamine, diisopropylethylamine, 1, 8-diazabicycloundecen-7-ene or N-methylmorpholineOr a combination of two or more thereof; salts of fluoride ions include, but are not limited to, one or a combination of two or more of tetrabutylammonium fluoride, potassium fluoride, or cesium fluoride.

In another embodiment, the present invention provides a process for preparing a compound of formula I, comprising the following steps:

in the step 1), when the formula A1 reacts with trimethyl ((tributylstannyl) ethynyl) silane, a compound shown as a formula B1 is prepared in an organic solvent under the condition of a catalyst,

preferably, the catalyst comprises but is not limited to one or the combination of more than two of tetratriphenylphosphine palladium, bis (dibenzylideneacetone) palladium, palladium acetate and palladium bistriphenylphosphine dichloride.

Step 2) of formulae B1 and R¹When X is reacted, a compound shown as a formula C1 is prepared in an organic solvent under the condition of a catalyst, an organic base and a fluoride ion salt.

cuprous iodide and one or more than two of palladium tetratriphenylphosphine are combined;

The organic solvent includes, but is not limited to, one or a combination of two or more of toluene, xylene, tetrahydrofuran, ethyl acetate, dioxane, N-methylpyrrolidone, N-dimethylformamide, and benzene. .

Hydrolyzing the compound shown in the formula C1 in the step 3) in a mixed solvent of an organic solvent and water under the condition of inorganic base to obtain the compound shown in the formula D1.

Preferably, the inorganic base includes but is not limited to one or a combination of two or more of sodium hydroxide and lithium hydroxide;

preferably, the organic solvent is a protic or aprotic organic solvent, and all common organic solvents are within the applicable scope of the reaction, including but not limited to tetrahydrofuran and methanol.

Step 4) formula D1 and formula R³NH²The compounds shown are reacted with one another,

in one embodiment, formula D1 is different from formula R³NH²Reaction of a compound of formula D1 with an acylating agent, and reaction with R³NH²Carrying out reaction to prepare the compound shown in the formula I,

the acylating agent comprises but is not limited to one or the combination of more than two of phosphorus oxychloride, thionyl chloride, oxalyl chloride, phosphorus trichloride or phosphorus pentachloride;

in another embodiment, a compound of formula D1 is reacted with a compound of formula R under condensing agent conditions³NH²Reacting to prepare the compound shown in the formula I,

It will be clear that the compounds of formula I, isomers, crystal forms or prodrugs and pharmaceutically acceptable salts thereof may exist in solvated as well as unsolvated forms. For example, the solvated form may be water soluble. The present invention includes all such solvated and unsolvated forms.

One aspect of the invention is a pharmaceutical composition comprising any one of the compounds described herein (or a prodrug, or a pharmaceutically acceptable salt, or other pharmaceutically acceptable derivative thereof), and one or more pharmaceutically acceptable carriers or excipients. These compositions may optionally further comprise one or more additional therapeutic agents. The compounds of the invention may be co-administered to a patient in need thereof with one or more other treatment regimens (e.g., administration of sorafenib or other kinase inhibitors, interferons, bone marrow transplantation, farnesyl transferase inhibitors, bisphosphonates, thalidomide, cancer vaccines, hormonal therapy, antibodies, radiation, etc.). The pharmaceutical composition of the compound may be another anticancer agent or agents.

The compositions of the present invention comprise a compound of the present invention in combination with a pharmaceutically acceptable carrier, including any and all solvents, diluents or other vehicles, dispersing or suspending aids, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as appropriate for the particular dosage form desired. Some examples of pharmaceutically acceptable carrier materials include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; tragacanth powder; malt; gelatin; talc powder; excipients, such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; ethylene glycols, such as propylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; ringer's solution; ethanol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants, may also be present in the composition.

Consisting of a pharmaceutically acceptable carrier and any of the compounds described above. For example, the invention is a pharmaceutical composition made by mixing any of the above-described compounds with a pharmaceutically acceptable carrier. The demonstration of this invention is a process for making a pharmaceutical composition comprising any of the above-described compounds and a pharmaceutically acceptable carrier.

Such compositions can be administered to a subject in need thereof to inhibit growth, development, and/or metastasis of cancers, including solid or non-solid tumors (e.g., chronic myelogenous leukemia, gastrointestinal stromal tumors, acute myelogenous leukemia, thyroid cancer, etc.), including those resistant to treatment with imatinib, dasatinib, nilotinib, bosutinib, or other kinase inhibitors.

The term "subject" as used herein refers to an animal, particularly a mammal, most often a human, who is often the subject of treatment, observation or experiment. The term "therapeutically effective amount" refers to an amount of active compound or pharmaceutical ingredient that will elicit the biological or medical response of a tissue, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician, which includes alleviation of the symptoms of the disease or disorder being treated.

Pharmaceutical compositions containing the compounds of the present invention as active ingredients, and methods for preparing the instant compounds, are presently contemplated by the present invention. Furthermore, some crystalline forms of the compounds may exist as polymorphs and as such may be included in the present invention. In addition, some compounds may form solvates with water (i.e., hydrates) or common organic solvents, and such solvates are intended to be included within the scope of this invention.

To prepare the pharmaceutical compositions of this invention, one or more compounds or salts of formula (I) as the active ingredient may be intimately admixed with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques, which carrier may take a wide variety of forms depending on the form of preparation designed for administration by any convenient route, e.g. oral or parenteral. Suitable pharmaceutically acceptable carriers are well known in the art. A description of some of these pharmaceutically acceptable carriers can be found in the handbook of pharmaceutical excipients, which is published by the United states society of pharmacy and British pharmaceutical society.

Administration of the active ingredient may be effected by any means capable of delivering the ingredient to the site of reaction (e.g., tumor cells). Such means may include oral routes, rectal routes, intraduodenal routes, parenteral injection (including intravenous, subcutaneous, intramuscular, intravascular or infusion), topical administration, and the like. The dosage of the active ingredient to be administered will, of course, depend on the subject being treated, the severity of the pain, the mode of administration and the judgment of the prescribing physician. While effective dosages may range from about 0.001 mg to about 300 mg (usually from about 0.01 mg to about 100 mg, and more suitably from about 0.1 mg to about 30 mg), specific dosages may range from about 0.001 mg per kg of body weight per day to about 300 mg per kg of body weight per day (more usually from about 0.01 mg to about 100 mg per kg of body weight per day, and more suitably from about 0.1 mg to about 30 mg per kg of body weight per day).

The composition may be in a form suitable, for example, for oral administration, for example, as a tablet, capsule, pill, powder, sustained release form, solution or suspension; for parenteral injection such as clear solutions, suspensions, emulsions; or for topical application such as creams; or as suppositories for rectal administration. The pharmaceutical compositions may also be in unit dosage form suitable for single use administration of the precise dosage. The pharmaceutical composition will include a conventional pharmaceutical carrier or excipient and the compound as an active ingredient prepared in accordance with the present invention, and may also include other medicinal or pharmaceutical agents, carriers, adjuvants, and the like.

The primary mode of parenteral administration involves dissolving a solution or suspension of the active ingredient in a sterile aqueous solution, such as an aqueous solution of propylene glycol or dextrose. This dosage form can be suitably diluted to the desired concentration.

Suitable pharmaceutical carriers include inert diluents, fillers, water, and various organic solvents. If pre-designed, the pharmaceutical formulation may include additional ingredients such as flavorings, antidiarrheal agents, excipients, and the like. Thus, for oral administration, tablets containing various excipients, such as citric acid, may be combined with various disintegrants such as starch, alginic acid, certain silicate complexes and some antidiarrheal agents such as sucrose, gelatin and acacia. Additionally, lubricants such as magnesium stearate, sodium lauryl sulfate, talc, and the like are useful for making tablets. Solid compounds of a similar type may also be used for either soft or hard gel capsule filling. Thus, preferred materials include lactose or milk sugar and high molecular weight polyethyelene ethanol. When the aqueous suspension or the liqueur is used as a necessary product for oral administration, the active ingredients therein may be combined with various sweeteners or other flavorings and coloring agents, and if necessary, emulsifiers or suspending agents may be added, together with diluents such as water, alcohol, propylene glycol, glycerin or mixtures thereof.

Therapeutic compounds may also be administered to mammals other than humans. The dosage of the drug administered to a mammal will depend on the species of the animal and its disease state or disorder in which it is suffering. The therapeutic compound may be administered to the animal in the form of a capsule, bolus, tablet or solution. Therapeutic compounds may also be administered into the animal by injection or infusion. We prepared these pharmaceutical forms according to conventional means which meet the criteria of veterinary practice. Alternatively, the pharmaceutical composition may be mixed with animal feed for feeding to the animal, and thus, the concentrated feed supplement or premix may be prepared for mixing with conventional animal feed.

It is a further object of the present invention to provide a method for treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a composition comprising a compound of the present invention. Cancers that can be so treated are noted elsewhere herein, including, but not limited to, cancers that are resistant to imatinib, dasatinib, nilotinib, bosutinib, ponatinib. Treatment may also be combined with one or more other cancer therapies, including surgery, radiation therapy (e.g., gamma-rays, neutron beam radiation therapy, electron beam radiation therapy, proton therapy, brachytherapy, and systemic radioisotopes, etc.), endocrine therapy, biological response modifiers (e.g., interferons, interleukins, and Tumor Necrosis Factor (TNF)), hyperthermia, cryotherapy, attenuating any adverse effects (e.g., antiemetics), and other cancer chemotherapeutic drugs. Other formulations, routes of administration, and regimens which may be used may be the same as or different from the compounds of the invention.

The invention also includes the use of a compound of the invention, or a pharmaceutically acceptable derivative thereof, for the manufacture of a medicament for the treatment of cancer (including non-solid tumors, primary or metastatic cancer, as noted elsewhere herein and including one or more other treatments for which the cancer is resistant or refractory) as well as other diseases (including but not limited to ocular fundus disease, psoriasis, atheroma, pulmonary fibrosis, liver fibrosis, bone marrow fibrosis, etc.). The compounds of the present invention are useful for use in anticancer drugs. The compounds of the invention may also be used in medicine to alleviate or prevent disease by inhibiting one or more kinases (e.g., BCR-ABL, VEGFR, etc.).

The compounds of the invention are also useful as standards and reagents for the characterization of various kinases, particularly but not exclusively protein tyrosine kinases, and for the study of the role of such kinases in biological and pathological phenomena; for the study of intracellular signal transduction pathways mediated by such kinases, comparative evaluation of novel kinase inhibitors; and in cell lines and animal models for the study of different cancers.

The examples provided below are intended to better illustrate the invention, all temperatures being in degrees centigrade unless otherwise indicated.

Detailed Description

Example 1

Preparation of 1-methyl-N- (4- ((4-methylpiperazin-1-) methyl) phenyl) -7- (pyridine-3-ethynyl) -1H-indole-2-carboxamide

Step 1) preparation of 7-bromo-1-methyl-N- (4- ((4-methylpiperazin-1-) methyl) phenyl) -1H-indole-2-carboxamide

Dissolving 7-bromo-1-methyl-1H-indole-2-carboxylic acid (2.5g,10mmol), 4- ((4-methylpiperazine-1-) methyl) amine (2.0g,10mmol) and 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethylurea Hexafluorophosphate (HATU) (3.8g,10mmol) in N, N-dimethylformamide, adding diethylisopropylamine (1.65mL,10mmol), stirring until the reaction is finished, extracting with ethyl acetate and water, concentrating the organic phase, and performing column chromatography to obtain the target compound 3.1g with the yield of 70%.¹H NMR(400MHz,DMSO-d₆)δ10.48(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) preparation of 1-methyl-N- (4- ((4-methylpiperazin-1-) methyl) phenyl) -7- ((trimethylsilyl) ethynyl) -1H-indole-2-carboxamide

7-bromo-1-methyl-N- (4- ((4-methylpiperazin-1-) methyl) phenyl) -1H-indole-2-carboxamide (3.1g,7mmol), trimethyl ((tributylstannyl) ethynyl) silane (9.9g,14mmol) and palladium (0.9g,0.7mmol) bistriphenylphosphine dichloride were placed in a reaction flask, toluene was added, heating was carried out until the reaction was complete, the solvent was evaporated, and column chromatography purification gave 1.9 g of a yellow solid in 60% yield.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) preparation of 1-methyl-N- (4- ((4-methylpiperazin-1-) methyl) phenyl) -7- (pyridine-3-ethynyl) -1H-indole-2-carboxamide

Dissolving 1-methyl-N- (4- ((4-methylpiperazine-1-) methyl) phenyl) -7- ((trimethylsilyl) ethynyl) -1H-indole-2-carboxamide (92mg,0.2mmol), 3-iodopyridine (41mg,0.2mmol), palladium (7mg,0.02mmol) bistriphenylphosphine dichloride, cuprous iodide (3.8mg,0.02mmol) in a mixed solvent of triethylamine and tetrahydrofuran, heating and stirring until the reaction is finished, extracting with ethyl acetate and water, concentrating an organic phase, and carrying out column chromatography to obtain 46 mg of a product, wherein the yield is 50%.¹H NMR(400MHz,DMSO-d₆)δ10.48(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]⁺。

Example 2

Preparation of 1-methyl-N- (4- ((4-methylpiperazin-1-) methyl) -3- (trifluoromethyl) phenyl) -7- (pyridine-3-ethynyl) -1H-indole-2-carboxamide

Step 1) preparation of 7-bromo-1-methyl-N- (4- ((4-methylpiperazin-1-) methyl) -3- (trifluoromethyl) phenyl) -1H-indole-2-carboxamide

Dissolving 7-bromo-1-methyl-1H-indole-2-carboxylic acid (2.5g,10mmol), 4- ((4-methylpiperazin-1-) methyl) -3- (trifluoromethyl) aniline (2.73g,10mmol) and 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HATU) (3.8g,10mmol) in N, N-dimethylformamide, adding diethylisopropylamine (1.65mL,10mmol), stirring until the reaction is finished, extracting with ethyl acetate and water, concentrating the organic phase, and performing column chromatography to obtain a product 3.6g with a yield of 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) preparation of 1-methyl-N- (4- ((4-methylpiperazin-1-) methyl) -3- (trifluoromethyl) phenyl) -7- ((trimethylsilyl) ethynyl) -1H-indole-2-carboxamide

Placing 7-bromo-1-methyl-N- (4- ((4-methylpiperazine-1-) methyl) -3- (trifluoromethyl) phenyl) -1H-indole-2-carboxamide (3.6g,7mmol), trimethyl ((tributylstannyl) ethynyl) silane (9.9g,14mmol) and palladium (0.9g,0.7mmol) bis (triphenylphosphine) dichloride in a reaction bottle, adding toluene, heating until the reaction is finished, evaporating off the solvent, and purifying by column chromatography to obtain 2.2 g of a yellow solid product with a yield of 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) preparation of 1-methyl-N- (4- ((4-methylpiperazin-1-) methyl) -3- (trifluoromethyl) phenyl) -7- (pyridine-3-ethynyl) -1H-indole-2-carboxamide

1-methyl-N- (4- ((4-methylpiperazine-1-) methyl) -3- (trifluoromethyl) phenyl) -7- ((trimethylsilyl) ethynyl) -1H-indole-2-carboxamide (105mg,0.2mmol), 3-iodopyridine (41mg,0.2mmol), palladium (7mg,0.02mmol) of triphenylphosphine dichloride, cuprous iodide (3.8mg,0.02mmol) were dissolved in a mixed solvent of triethylamine and tetrahydrofuran, the mixture was heated and stirred until the reaction was completed, ethyl acetate and water were extracted, the organic phase was concentrated, and 53 mg of a product was obtained by column chromatography, with a yield of 50%.¹H NMR(400MHz,DMSO-d₆)δ10.78(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]⁺。

Example 3

Preparation of N- (4- ((4-methylpiperazin-1-) methyl) -3- (trifluoromethyl) phenyl) -7- (pyrimidine-5-ethynyl) -1H-indole-2-carboxamide

Step 1) preparation of 7-bromo-N- (4- ((4-methylpiperazin-1-) methyl) -3- (trifluoromethyl) phenyl) -1H-indole-2-carboxamide

7-bromo-1H-indole-2-carboxylic acid (1) (2.4g,10mmol), 4- ((4-methylpiperazin-1-) methyl) -3- (trifluoromethyl) aniline (2.73g,10mmol) and 2- (7-oxybenzotriazole) -N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HATU) (3.8g,10mmol) were dissolved in N, N-dimethylformamide and diethylisopropylamine (1.65mL,10 m) was addedmol) is added, the mixture is stirred until the reaction is finished, ethyl acetate and water are used for extraction, organic phase is concentrated, and column chromatography is carried out to obtain 3.5g of product with 70 percent of yield.¹H NMR(400MHz,DMSO-d₆)δ11.67(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) preparation of N- (4- ((4-methylpiperazin-1-) methyl) -3- (trifluoromethyl) phenyl) -7- ((trimethylsilyl) ethynyl) -1H-indole-2-carboxamide

7-bromo-N- (4- ((4-methylpiperazin-1-) methyl) -3- (trifluoromethyl) phenyl) -1H-indole-2-carboxamide (3.5g,7mmol), trimethyl ((tributylstannyl) ethynyl) silane (9.9g,14mmol) and palladium (0.9g,0.7mmol) bistriphenylphosphine dichloride were placed in a reaction flask, toluene was added, heating was carried out until the reaction was complete, the solvent was evaporated, and column chromatography purification gave 2.1 g of a yellow solid in 60% yield.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) preparation of N- (4- ((4-methylpiperazin-1-) methyl) -3- (trifluoromethyl) phenyl) -7- (pyrimidine-5-ethynyl) -1H-indole-2-carboxamide

7-bromo-N- (4- ((4-methylpiperazin-1-) methyl) -3- (trifluoromethyl) phenyl) -1H-indole-2-carboxamide (102mg,0.2mmol), 5-iodopyrimidine (41mg,0.2mmol), palladium (7mg,0.02mmol) bistriphenylphosphine dichloride, cuprous iodide (3.8mg,0.02mmol), triethylamine and tetrakisHeating and stirring in a mixed solvent of hydrogen and furan until the reaction is finished, extracting with ethyl acetate and water, concentrating an organic phase, and carrying out column chromatography to obtain a product 52 mg, wherein the yield is 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]⁺。

Example 4

Preparation of 7- ((2-aminopyrimidin-5-) ethynyl) -N- (3-chloro-4- ((4-methylpiperazin-1-) methyl) phenyl) -1-methyl-1H-indole-2-carboxamide

Step 1) preparation of Ethyl 1-methyl-7- ((trimethylsilyl) ethynyl) -1H-indole-2-carboxylate

Putting ethyl 7-bromo-1-methyl-1H-indole-2-formate (8g,28mmol), trimethyl ((tributylstannyl) ethynyl) silane (21.7g,56mmol) and palladium (2.0g,2.8mmol) triphenylphosphine dichloride into a reaction flask, adding toluene, heating and stirring until the reaction is finished, evaporating off the solvent, and purifying by column chromatography to obtain 5.4 g of a yellow solid product with the yield of 65%.¹H NMR(400MHz,DMSO-d₆)δ7.69–7.64(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]⁺。

Step 2) preparation of Ethyl 7- ((2-aminopyrimidin-5-) ethynyl) -1-methyl-1H-indole-2-carboxylate

Ethyl 1-methyl-7- ((trimethylsilyl) ethynyl) -1H-indole-2-carboxylate (3.0g,10mmol), 5-iodopyrimidin-2-amine (3.3g,15mmol), cuprous iodide (0.19g,1mmol), tetrabutylammonium fluoride(2.6g,10mmol) is dissolved in a mixed solvent of triethylamine and dimethylformamide, heated and stirred until the reaction is finished, ethyl acetate and water are extracted, an organic phase is concentrated, and column chromatography is carried out to obtain 1.6g of a yellow solid product, wherein the yield is 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]⁺。

Step 3) preparation of 7- ((2-aminopyrimidin-5-) ethynyl) -1-methyl-1H-indole-2-carboxylic acid

Dissolving ethyl 7- ((2-aminopyrimidine-5-) ethynyl) -1-methyl-1H-indole-2-formate (1.6g,5mmol) in tetrahydrofuran, adding an aqueous solution of sodium hydroxide, stirring until the reaction is finished, concentrating under reduced pressure to evaporate the tetrahydrofuran in the reaction solution, adjusting the pH value of the reaction solution to acidity by using dilute hydrochloric acid, and performing suction filtration to obtain 1.39 g of white solid with the yield of 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) preparation of 7- ((2-aminopyrimidin-5-) ethynyl) -N- (3-chloro-4- ((4-methylpiperazin-1-) methyl) phenyl) -1-methyl-1H-indole-2-carboxamide

Dissolving 7- ((2-aminopyrimidine-5-) ethynyl) -1-methyl-1H-indole-2-carboxylic acid (58mg,0.2mmol), 3-chloro-4- ((4-methylpiperazin-1-) methyl) aniline (48mg,0.2mmol), 2- (7-oxybenzotriazole) -N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HATU) (80mg,0.22mmol) in dimethylformamide, adding diethylisopropylamine (0.16mL,1mmol), stirring until the reaction is finished, extracting with ethyl acetate and water, concentrating the organic phase, and performing column chromatographyChromatography gave 51 mg of product, 50% yield.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]⁺。

Example 5

Preparation of 7- ((1H-pyrazolo [3,4-b ] pyridin-5-) ethynyl) -N- (3-chloro-4- ((4-methylpiperazin-1-) methyl) phenyl) -1-methyl-1H-indole-2-carboxamide

Step 1) As in example 4, step 1)

Step 2) preparation of ethyl 7- ((11H-pyrazolo [3,4-b ] pyridin-5-) ethynyl) -1-methyl-1H-indole-2-carboxylate

Ethyl 1-methyl-7- ((trimethylsilyl) ethynyl) -1H-indole-2-carboxylate (3.0g,10mmol), 5-bromo-1H-pyrazolo [3,4-b]Pyridine (2.9g,15mmol), cuprous iodide (0.19g,1mmol) and tetrabutylammonium fluoride (2.6g,10mmol) are dissolved in a mixed solvent of triethylamine and dimethylformamide, the mixture is heated and stirred until the reaction is finished, ethyl acetate and water are extracted, an organic phase is concentrated, and column chromatography is carried out to obtain a yellow solid product 1.4g, wherein the yield is 42%.¹H NMR(400MHz,DMSO-d₆)δ13.90(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]⁺。

Step 3) preparation of 7- ((1H-pyrazolo [3,4-b ] pyridine-5-) ethynyl) -1-methyl-1H-indole-2-carboxylic acid

Ethyl 7- ((11H-pyrazolo [3, 4-b)]Pyridine-5-) ethynyl) -1-methyl-1H-indole-2-carboxylate (1.4g,4.2mmol) in tetrahydrofuranAdding sodium hydroxide aqueous solution, stirring until the reaction is finished, concentrating under reduced pressure to evaporate tetrahydrofuran in the reaction solution, adjusting the pH value of the reaction solution to acidity by using dilute hydrochloric acid, and performing suction filtration to obtain 1.26 g of white solid with the yield of 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) preparation of 7- ((1H-pyrazolo [3,4-b ] pyridin-5-) ethynyl) -N- (3-chloro-4- ((4-methylpiperazin-1-) methyl) phenyl) -1-methyl-1H-indole-2-carboxamide

Reacting 7- ((1H-pyrazolo [3, 4-b)]Pyridine-5-) ethynyl) -1-methyl-1H-indole-2-carboxylic acid (VI-2) (63mg,0.2mmol), 3-chloro-4- ((4-methylpiperazin-1-) methyl) aniline (48mg,0.2mmol), 2- (7-benzotriazole oxide) -N, N' -tetramethylurea Hexafluorophosphate (HATU) (80mg,0.22mmol), dissolved in dimethylformamide, added diethylisopropylamine (0.16mL,1mmol), stirred until the reaction is complete, extracted with ethyl acetate and water, the organic phase concentrated, and column chromatography gave 49 mg, 46% yield. 1H NMR (300MHz, DMSO-d)₆)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]⁺。

Examples 6 to 22

Step 1) and step 2) are the same as step 1) and step 2) in the production method of example 1.

Step 3) referring to step 3) in the preparation method of example 1, the operation is exactly the same, whichIn the following table, R is present in equal molar equivalents¹X replaces 3-iodopyridine to obtain the corresponding example compound. Specific compounds are shown in the following table:

examples 23 to 45

Step 1) and step 2) are the same as step 1) and step 2) in the production method of example 2.

Step 3) reference example 2 preparation method step 3) is exactly the same operation, and the specific implementation is R in the following table in equal molar equivalents¹X replaces 3-iodopyridine to obtain the corresponding example compound. Specific compounds are shown in the following table:

examples 46 to 49

Step 1) and step 2) are the same as step 1) and step 2) in the production method of example 3.

Step 3) reference example 3 preparation method, step 3) being exactly the same, was conducted in such a way that R in the following table is in equal molar equivalents¹X replaces 5-iodopyrimidine to obtain the corresponding example compound. Specific compounds are shown in the following table:

examples 49 to 61

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

Step 4) reference example 4 the preparation process of step 4) was carried out in exactly the same way, in particular by means of R in the following table in equal molar equivalents³NH₂The 3-chloro-4- ((4-methylpiperazin-1-) methyl) aniline was replaced to give the corresponding example compound. Specific compounds are shown in the following table:

examples 62 to 63

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

example 64

Test for inhibition of ABL/ABL-T315I kinase Activity by Small molecule Compounds

The specific implementation method comprises the following steps: in the in vitro assembled enzymatic reaction, different concentrations of the test compound are added to detect the inhibition effect of the compound on the ABL/ABL-T315I enzymatic reaction, and the compound with the inhibition activity is screened by calculating the IC50 for inhibiting the ABL/ABL-T315I enzymatic reaction.

Instruments, materials and reagents

Second, Experimental scheme

The following protocol illustrates the complete protocol steps and intermediate reagent preparation methods by taking ABL as an example, and the protocol of the complete ABL-T315I kinase can be obtained by fine adjustment according to the protocol of the ABL.

1. Preparing a reagent:

preparation of EDTA (0.5M pH8.0) solution by accurately weighing 14.612g EDTA powder, adding ultrapure water, and adjusting volume to 100mL (if insoluble, heating to 37 deg.C, adjusting pH to 8.0 with NaOH solution)

1 × Kinase Assay Buffer: into a reagent bottle were added 25mL of HEPES solution (1M), 190.175mg of EGTA, and 5mL of MgCl₂The solution (1M), 1mL DTT, 50. mu.L Tween-20, and ultrapure water were added to make a volume of 500mL (pH adjusted to 7.5).

1 × Detection Buffer 1mL of 10 × Detection Buffer was added to 9mL of water and mixed.

4 × stop solution: 0.8mL of the above EDTA (0.5M, pH 8.0.0) solution, 1mL of 10 XDetection Buffer and 8.2mL of ultrapure water were mixed together.

4 × ABL Kinase solution the Kinase stock solution was diluted with 1 × Kinase Assay Buffer to a concentration of 0.62nM, mixed well and stored on ice.

4 × substrate solution: substrate ULight was diluted with 1 × Kinase Assay Buffer^TMPolyGT stock solution was brought to 200nM and mixed well.

4 × ATP solution: the ATP stock was diluted with 1 XKinase Assay Buffer to a concentration of 40. mu.M and mixed well.

4 × detection solution: the Detection Antibody Eu-W1024-labeled Anti-Phosphorylazine Antibody (PT66) was diluted with 1 XDeprotection Buffer to a concentration of 8nM, and mixed well.

2 × substrate/ATP mixture: equal amounts of 4 Xsubstrate solution and 4 XATP solution 1:1 were mixed (prepared before use). 2. Experimental procedure

1) The dilution of the compound of the present invention,

in a 96-well plate a, the compounds of examples 1-63 of the present invention were diluted with DMSO solutions at 3-fold ratio to form 11 gradients, and the 12 th gradient was pure DMSO solution (as a positive control); a new 96-well plate b was prepared, and the solution was diluted 6.25 times with ultrapure water (DMSO concentration: 16%)

2) The compounds of examples 1-63 were rotated to 384 well plates

The compound solution diluted with ultrapure water in the above 96-well plate b was transferred to the corresponding well of a 384-well plate according to a standard 2-well carousel.

3) Add 4 × kinase solution: mu.l of the 4 Xkinase solution was added to the corresponding reaction well of the 384-well plate using a line gun, and pre-reacted at room temperature for 5 minutes.

4) Add 2 Xsubstrate/ATP mix 5. mu.l of the above 2 Xsubstrate/ATP mix to the corresponding reaction well of a 384 well plate using a discharge gun.

5) Negative control: negative control wells were set in 384-well plates, and 2.5. mu.l of 4 Xsubstrate, 2.5. mu.l of 4 Xenzyme solution, 2.5. mu.l of 1 XKinase Assay Buffer, and 2.5. mu.l of ultrapure water containing 16% DMSO were added to each well.

6) And (4) centrifuging, mixing uniformly, and reacting for 2 hours at room temperature in a dark place.

7) Termination of the enzymatic reaction:

5. mu.l of the above 4 Xstop solution was pipetted into the corresponding well of 384-well plate, centrifuged and mixed, and reacted at room temperature for 5 minutes.

8) And (3) color development reaction:

then, 5. mu.l of the 4X detection solution was pipetted and added to the corresponding well of 384-well plate, and the mixture was centrifuged and mixed and reacted at room temperature for 1 hour.

9) The 384 well plate is placed in a plate reader, and a corresponding program detection signal is called.

10)IC₅₀And (3) analysis:

pore reading 10000 × EU 665/EU 615 values

Inhibition rate [1- (experimental well reading-negative control well reading)/(positive control well reading-negative control well reading) ]. 100%

Inputting the drug concentration and the corresponding inhibition rate into GraphPad Prism5 for processing to calculate the corresponding IC₅₀。

Third, experimental conditions:

1) ABL kinase activity inhibition molecule screening experimental conditions:

the final concentration of ABL kinase in the reaction system is 0.155nM, the final concentration of ATP is 10 muM, and the substrate ULight^TM-labeled polyGT final concentration of 50nM and enzymatic reaction time of 2 hours.

The highest final concentration of the compound in the reaction system is 2.5 mu M, and the compound is diluted by 3 times of gradient to total 11 concentrations, and the lowest final concentration is 0.042 nM. The final concentration of DMSO was 4%.

2) ABL-T315I kinase activity inhibition molecular screening experimental conditions:

the final concentration of ABL (T315I) kinase in the reaction system is 0.5nM, the final concentration of ATP is 10. mu.M, and the substrate ULight^TM-labeled polyGT final concentration of 50nM and enzymatic reaction time of 2 hours.

The highest final concentration of the compound in the reaction system is 2.5 mu M, and the compound is diluted by 3 times of gradient to total 11 concentrations, and the lowest final concentration is 0.042 nM. The final concentration of DMSO is 1%.

Table I shows the results of the determination of the inhibitory activity of some of the compounds of the present invention on the tyrosine kinases BCR-ABL and ABL-T315I. The IC50 values in the table below represent the concentration of the compound at which the highest inhibition of the kinase is 50%, where A represents an IC50 of less than or equal to 100nM, B represents an IC50 of greater than 100nM but less than or equal to 1000nM, C represents an IC50 of greater than 1000nM but less than or equal to 10000nM, and D represents an IC50 of greater than 10000 nM. NT means that the corresponding kinase was not tested.

TABLE I test results (IC) for ABL/ABL-T315I kinase inhibitory Activity of certain compounds of the invention₅₀)

Example 65

Assays for inhibition of VEGFR-2 kinase Activity by Small molecule Compounds

The specific test method is as follows:

1. compound dilution: a total of 12 concentrations (10000 nM for the drug used in this experiment, and 0.002384nM for the lowest concentration) were 4-fold serially diluted starting from 10000nM for the highest concentration,

2. mu.l of the compound diluted in the gradient was taken with a line gun, added to a 384-well plate,

3. adding an enzyme: adding 5 μ l 2X VEGFR-2 kinase into corresponding reaction well of 384-well plate with a discharging gun, mixing, pre-reacting at room temperature for 30min,

4. the gun was arranged to take 2.5. mu.l of 4 Xsubstrate/ATP Mix and add to the corresponding reaction well of 384-well plate,

5. negative control: add 2.5. mu.l/well 4 Xsubstrate/ATP Mix and 7.5. mu.l 1X Kinase Assay Buffer to 384 well plates

Positive control: 2.5. mu.l/well 4 Xsubstrate/ATP Mix, 2.5. mu.l/well 1 XKinase Assay Buffer containing 4% DMSO, 5. mu.l/well 2 XVEGFR-2 solution were added to 384 well plates. The final concentration of DMSO in the reaction system is 4 percent,

6. centrifuging, mixing, reacting at room temperature in dark place for 60min,

7. termination of the enzymatic reaction: adding 5 μ l 4X Stop solution into mesopore of 384-well plate with a discharge gun, centrifuging, mixing, reacting at room temperature for 5min,

8. and (3) color development reaction: adding 5 μ l 4X Detection Mix into mesopores of 384-well plate with a discharging gun for color development, centrifuging, mixing, reacting at room temperature for 60min,

9. and (3) putting the 384-well plate into an Envision plate reading instrument to read the plate, and calling a corresponding program detection signal.

10. Analysis and processing of raw data:

the drug concentration and the corresponding inhibition rate are input into GraphPad Prism5 for calculation, and the inhibition rate of the compound is calculated as follows: inhibition (%) [1- (assay well reading-negative control well reading)/(positive control well reading-negative control well reading)]x 100%. The corresponding IC was obtained by software processing of GraphPad Prism5₅₀Value (concentration of compound at which the highest inhibition of the enzyme was 50%).

Table (II) shows the results of the measurement of the inhibitory activity of a part of the compounds of the present invention on VEGFR2 tyrosine kinase, wherein A represents IC₅₀Less than or equal to 100nM, B represents IC₅₀Greater than 100nM but less than or equal to 1000nM, C represents IC₅₀Greater than 1000nM but less than or equal to 10000nM, D representing IC₅₀Greater than 10000 nM.

TABLE II results of partial compound of the invention on VEGFR2 kinase inhibitory Activity assay (IC)₅₀)

Example 66

Activity test for small molecule compounds for inhibiting cell proliferation

Adding test compounds with different concentrations into cell culture solution, and comparing the increase of target cells by the drug

Reproductive IC50 reflects the inhibitory effect of compounds on cell proliferation.

A laboratory instrument and materials:

main experimental apparatus and materials

Two experimental reagents

Experimental reagent

A compound: dissolved in DMSO to make a 10mM solution.

Three-relevant solution preparation and dilution:

1. compound dilution: dissolving all the compounds of the invention in DMSO to prepare 10mM stock solution, and completing the first gradient dilution of the compounds to be tested in the DMSO, wherein the dilution times are 3 times or 4 times; an 80-fold bulk dilution of all compounds was done in cell culture broth to give 5 × compounds, which were added to the wells of a 96-well plate containing cells to give a final cell culture broth of 1 × design final concentration.

The general compound design is 9 concentration gradient, the highest final concentration is 25000nM, 4 times diluted 9 concentrations after the lowest concentration is 0.38nM, all wells in DMSO final concentration is 0.25%.

Four experimental procedures

1) The cells were transferred to a 15mL centrifuge tube and centrifuged at 1000rpm for 4 minutes.

2) Discarding the supernatant, adding complete culture solution, blowing and beating uniformly, mixing 10 μ L of cell suspension and 10 μ L of 0.4% trypan blue uniformly, counting with a cell counter, and recording the number of cells and survival rate.

3) Each well was seeded with 80 μ L of cell suspension into 96-well plates at different cell seeding cell densities as follows:

cell density

Cell name	Culture medium	Density of inoculation
			K562	RPMI 1640+10％FBS	10000/well
BaF3-BCR-ABL-T315I	RPMI 1640+10％FBS	5000/well

4) To each well (row B to row G, column 2 to column 11) was added 20. mu.L of the above 5X compound diluted with the culture broth, and mixed and shaken. (each compound was placed in two replicates and three compounds were tested in a 96-well plate);

5) after culturing for 72 hours in an incubator at 37 ℃ containing 5% CO2, 10. mu.L of CCK-8 reagent was added to each well, and the culture was carried out for 2 hours (the reaction time was adjusted depending on the shade of the color);

6) the OD value was read at 450nm on a multifunction plate reader.

7) Data processing: cell survival (%) - (As-Ab)/(Ac-Ab) ]. 100%

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

ac: OD of control wells (medium containing cells, CCK-8);

ab: OD of blank wells (medium without cells and compounds, CCK-8).

The values were then introduced into Graphpad Prism5 software for curve fitting and IC50 was calculated.

Table (III) shows the results of the activity assay of some of the compounds of the present invention on inhibition of cell proliferation, wherein A represents IC₅₀Less than or equal to 100nM, B represents IC₅₀Greater than 100nM but less than or equal to 500nM, C represents IC₅₀Greater than 500nM but less than or equal to 1000nM, D representing IC₅₀Greater than 1000 nM.

TABLE (III) results of measurement of cell proliferation inhibitory Activity of representative Compounds of the present invention

Example 67

As a specific embodiment of an oral pharmaceutical, about 10mg of the compound of example 54 is mixed with 80-120mg of lactose, divided, and filled into capsules to obtain capsules of the compound of example 54.

The biological data provided herein demonstrate that the compounds of the invention are useful for treating or preventing diseases caused by abnormalities in ABL and/or VEGFR2 kinases. The compounds of the invention also include methods of treating diseases that are resistant to one or more other therapeutic methods, including, but not limited to, acute myelogenous leukemia, chronic myelogenous leukemia, acute myelogenous leukemia, gastrointestinal stromal tumors, acute myelogenous leukemia, non-small cell lung cancer, breast cancer, pancreatic cancer, glioma, glioblastoma, ovarian cancer, cervical cancer, colorectal cancer, melanoma, endometrial cancer, prostate cancer, bladder cancer, leukemia, gastric cancer, liver cancer, thyroid cancer, non-hodgkin's lymphoma, nasopharyngeal cancer, esophageal cancer, brain tumors, B-cell and T-cell lymphomas, lymphoma, multiple myeloma, sarcoma of biliary tract cancer, bile duct cancer, ocular fundus disease, dry eye, psoriasis, vitiligo, dermatitis, alopecia areata, rheumatoid arthritis, colitis, and combinations thereof, Multiple sclerosis, systemic lupus erythematosus, Crohn's disease, atherosclerosis, pulmonary fibrosis, hepatic fibrosis, myelofibrosis, and the like. The compounds of the present invention may be administered as monotherapy or in combination therapy, in combination with a plurality of the compounds of the present invention or in combination with other drugs other than those of the present invention.

The foregoing is a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various improvements and modifications can be made to the embodiment of the present invention without departing from the principle of the present invention, and these improvements and modifications should also be construed as the protection scope of the present invention.

Claims

1. A compound, enantiomer, diastereomer, tautomer, crystalline form or a pharmaceutically acceptable salt thereof, having a structural formula (I):

wherein the content of the first and second substances,

x is N, CH;

R¹is composed of

a) A 5-6 membered heteroaryl ring unsubstituted or substituted with 1-3 of the same or different Ra, said 5-6 membered heteroaryl ring containing 1-3 heteroatoms selected from N, O and S;

b) ring a and ring B structure unsubstituted or substituted with 1-2 same or different Rb' S, wherein ring a is a 5-6 membered heteroaromatic ring or a 6 membered aromatic ring, ring B is selected from a 3-6 membered cycloalkyl, a 3-6 membered heterocyclic ring, a 5-6 membered heteroaromatic ring, said heterocyclic and heteroaromatic rings each independently contain 1-3 heteroatoms selected from N, O and S;

rb is H, halogen, hydroxy, cyano, oxo (═ O), acyl, C₁-C₆Alkyl OR-OR';

R²is H, C₁-C₆An alkyl group;

R³is composed of

D Ring is unsubstituted or optionally substituted (Rd)_mA substituted 5-6 membered cycloalkyl, a 5-6 membered heterocyclic ring containing 1-3 heteroatoms selected from N, O and S, a 6 membered aromatic ring, or a 5-6 membered heteroaromatic ring containing 1-3 heteroatoms selected from N, O and S;

L is- (CR)^l1R^l2)_q-; q is an integer of 1 to 4;

R^l1and R^l2Each independently is H or C₁-C₆An alkyl group;

e is a 3-6 membered saturated or unsaturated heterocyclic group containing 1-3 heteroatoms selected from N, O and S;

2. The compound according to claim 1, or enantiomers, diastereomers, tautomers, crystalline forms, and pharmaceutically acceptable salts thereof,

wherein the content of the first and second substances,

x is CH;

R¹is composed of

a) A 5-6 membered heteroaryl, unsubstituted or substituted with 1-3 of the same or different Ra, containing 1-3 heteroatoms selected from N, O and S;

b) ring A and ring B structures unsubstituted or substituted by 1-2 of the same or different Rb,

ring A is a 5-6 membered heteroaromatic ring, ring B is selected from a 3-6 membered heterocyclic ring or a 5-6 membered heteroaromatic ring;

r 'and R' are each independently H, C₁-C₄Alkyl radical, C₃-C₄Cycloalkyl radicals, or by halogen, C₁-C₃Alkoxy or C₁-C₃Alkylamino substituted C₁-C₃An alkyl group;

rb is-H, halogen, oxo (═ O), cyano, C_1-C₃An alkyl group;

R²is H, C₁-C₄A linear or branched alkyl group;

R³is composed of

D Ring is unsubstituted or optionally substituted (Rd)_mA substituted 6-membered aromatic ring or a 5-6 membered heteroaromatic ring containing 1-3 heteroatoms selected from N, O and S;

(Rd)_mm identical or different substituents Rd, m being 0, 1,2 or 3; rd is H, halogen, hydroxy, cyano, C₁-C₄Alkoxy radical, C₁-C₄Alkylamino, unsubstituted or substituted by hydroxy, halogen, C₁-C₄Alkoxy radical, C₁-C₄Alkylamino substituted C₁-C₄Alkyl or-L-E- (Re)_n；

L is- (CR)^l1R^l2)_q-，R^l1And R^l2Are independently H, C_1-3Alkyl, q is an integer from 1 to 3, E is a 3-6 membered saturated or unsaturated heterocyclyl containing from 1 to 3 heteroatoms selected from N, O and S; (Re)_nN identical or different substituents Re, Re being H, halogen, hydroxy, C₁-C₃Alkoxy radical, C₁-C₃Alkyl ammoniaRadical, unsubstituted or substituted by halogen, hydroxy, C₁-C₃Alkoxy or C₁-C₃Alkylamino substituted C₁-C₄Alkyl, n is 0, 1,2 or 3.

3. The compound according to claim 1, or enantiomers, diastereomers, tautomers, crystalline forms, and pharmaceutically acceptable salts thereof,

R²is H, methyl, ethyl, propyl or isopropyl.

4. The compound according to claim 1, or enantiomers, diastereomers, tautomers, crystalline forms, and pharmaceutically acceptable salts thereof,

R¹in order to realize the purpose,

(Ra)^pis substituted by p identical or different Ra, p is 0, 1,2 or 3;

ra is-H, fluoro, chloro, bromo, methyl, ethyl, propyl, butyl, isopropyl, trifluoromethyl, -NHR ', R' is-H, methyl, ethyl, methylaminoethyl, dimethylaminoethyl, cyclopropyl, methoxyethyl, methylaminopropyl, dimethylaminopropyl, cyclobutyl or methoxypropyl;

(Rb)^rr is substituted by r, the same or different Rb, r is 0, 1, or 2;

rb is-H, fluoro, chloro, bromo, oxo (═ O), cyano, methyl, ethyl, propyl, isopropyl.

5. The compound according to claim 1, or enantiomers, diastereomers, tautomers, crystalline forms, and pharmaceutically acceptable salts thereof,

R¹is composed of

(Ra)^pIs substituted by p identical or different Ra, p is 0, 1 or 2;

ra is-H, fluoro, chloro, bromo, methyl, ethyl, propyl, butyl, isopropyl, trifluoromethyl or-NHR ', R' is-H, methyl, ethyl, methylaminoethyl, dimethylaminoethyl, cyclopropyl, methoxyethyl, methylaminopropyl, dimethylaminopropyl, cyclobutyl or methoxypropyl;

(Rb)^rr is substituted by r identical or different Rb, r is 0 or 1;

6. The compound according to claim 1, or enantiomers, diastereomers, tautomers, crystalline forms, and pharmaceutically acceptable salts thereof,

R³is composed of

Ring D is an unsubstituted or (Rd) m-substituted aromatic or heteroaromatic ring, the aromatic ring is a benzene ring, and the heteroaromatic ring is selected from pyridine, pyridazine or thiazole;

(Rd)_mm identical or different substituents Rd, m is 0, 1,2 or 3, Rd is H, halogen, hydroxy, cyano, C_1-C₄Alkoxy radical, C₁-C₄Alkylamino, unsubstituted or substituted by hydroxy, halogen, C₁-C₄Alkoxy radical, C₁-C₄Substituted by alkylaminoC_1-4Alkyl, or is-L-E- (Re)_n；

L is- (CH)₂)_q-q is 1,2 or 3;

e is selected from the group consisting of cycloethylethane, ethylene oxide, pyrrolidine, pyrroline, pyrrole, pyrazolidine, pyrazoline, imidazole, pyrazole, furan, tetrahydrofuran, dihydrofuran, tetrahydrothiophene, thiophene, oxazole, thiazole, thiadiazole, piperidine, pyridine, dihydropyridine, morpholine, piperazine or pyrazine;

(Re)_nn equal or different Re, Re is H, halogen, hydroxy, C_1-C₃Alkoxy radical, C_1-C₃Alkylamino, unsubstituted or substituted by halogen, hydroxy, C_1-C₃Alkoxy or C_1-C₃Alkylamino substituted C_1-C₃Alkyl, n is 0, 1,2 or 3.

7. The compound according to claim 1, or enantiomers, diastereomers, tautomers, crystalline forms, and pharmaceutically acceptable salts thereof,

R³is composed of

(Rd)_mis m identical or different substituents Rd, m is 0, 1 or 2, Rd is H, fluorine, chlorine, bromine, hydroxyl, cyano, methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, methoxy, ethoxy, propoxy, isopropoxy, fluoromethyl, difluoromethyl, trifluoromethyl, hydroxymethyl, hydroxyethyl, hydroxypropyl, methylamino, ethylamino, dimethylamino, methylaminomethyl, methylaminoethyl, methylaminopropyl, ethylaminomethyl, ethylaminopropyl, dimethylaminomethyl, dimethylaminoethyl, dimethylaminopropyl or is-L-E- (Re)_n，

L is- (CH)₂)_q-q is 1,2 or 3;

(Re)_nn identical or different substituents Re, Re is H, fluorine, chlorine, bromine, hydroxyl, methyl, ethyl, propyl, isopropyl, fluoromethyl, difluoromethyl, trifluoromethyl, methoxy, methoxymethyl, methoxyethyl, methylamino, dimethylamino, methylaminomethyl, dimethylaminomethyl, methylaminoethyl, dimethylaminoethyl, hydroxymethyl, hydroxyethyl, n is 0, 1 or 2.

8. The compound of claim 1, an enantiomer, diastereomer, tautomer, crystalline form, or a pharmaceutically acceptable salt thereof, selected from the group consisting of:

1-methyl-N- (4- ((4-methylpiperazin-1-) methyl) phenyl) -7- (pyridine-3-ethynyl) -1H-indole-2-carboxamide,

1-methyl-N- (4- ((4-methylpiperazin-1-) methyl) -3- (trifluoromethyl) phenyl) -7- (pyridine-3-ethynyl) -1H-indole-2-carboxamide,

n- (4- ((4-methylpiperazin-1-) methyl) -3- (trifluoromethyl) phenyl) -7- (pyrimidine-5-ethynyl) -1H-indole-2-carboxamide,

7- ((2-aminopyrimidin-5-) ethynyl) -N- (3-chloro-4- ((4-methylpiperazin-1-) methyl) phenyl) -1-methyl-1H-indole-2-carboxamide,

7- ((1H-pyrazolo [3,4-b ] pyridin-5-) ethynyl) -N- (3-chloro-4- ((4-methylpiperazin-1-) methyl) phenyl) -1-methyl-1H-indole-2-carboxamide,

7- ((6-aminopyridin-3-) ethynyl) -1-methyl-N- (4- ((4-methylpiperazin-1-) methyl) phenyl) -1H-indole-2-carboxamide,

1-methyl-N- (4- ((4-methylpiperazin-1-) methyl) phenyl) -7- ((4-methylpyridin-3-) ethynyl) -1H-indole-2-carboxamide,

7- ((6-amino-4-methylpyridin-3-) ethynyl) -1-methyl-N- (4- ((4-methylpiperazin-1-) methyl) phenyl) -1H-indole-2-carboxamide,

7- ((4-chloropyridine-3-) ethynyl) -1-methyl-N- (4- ((4-methylpiperazine-1-) methyl) phenyl) -1H-indole-2-carboxamide,

7- ((6-amino-4-chloropyridin-3-) ethynyl) -1-methyl-N- (4- ((4-methylpiperazin-1-) methyl) phenyl) -1H-indole-2-carboxamide,

1-methyl-N- (4- ((4-methylpiperazin-1-) methyl) phenyl) -7- (pyrimidine-5-ethynyl) -1H-indole-2-carboxamide,

7- ((2-aminopyrimidin-5-) ethynyl) -1-methyl-N- (4- ((4-methylpiperazin-1-) methyl) phenyl) -1H-indole-2-carboxamide,

1-methyl-N- (4- ((4-methylpiperazin-1-) methyl) phenyl) -7- ((4-methylpyrimidin-5-) ethynyl) -1H-indole-2-carboxamide,

7- ((2- (ethylamino) pyrimidine-5-ethynyl) -1-methyl-N- (4- ((4-methylpiperazin-1-) methyl) phenyl) -1H-indole-2-carboxamide,

7- ((2- ((2- (diethylamino) ethyl) amino) pyrimidin-5-) ethynyl) -1-methyl-N- (4- ((4-methylpiperazin-1-) methyl) phenyl) -1H-indole-2-carboxamide,

7- ((2- ((2-methoxyethyl) amino) pyrimidin-5-) ethynyl) -1-methyl-N- (4- ((4-methylpiperazin-1-) methyl) phenyl) -1H-indole-2-carboxamide,

1-methyl-7- ((2- (methylamino) pyrimidin-5-) ethynyl) -N- (4- ((4-methylpiperazin-1-) methyl) phenyl) -1H-indole-2-carboxamide,

7- ((2- (cyclopropylamino) pyrimidine-5-) ethynyl) -N- (4- ((4-methylpiperazin-1-) methyl) phenyl) -1H-indole-2-carboxamide,

7- ((6-amino-5-methylpyridin-3-) ethynyl) -1-methyl-N- (4- ((4-methylpiperazin-1-) methyl) phenyl) -1H-indole-2-carboxamide,

7- ((2-amino-4-methylpyrimidin-5-) ethynyl) -1-methyl-N- (4- ((4-methylpiperazin-1-) methyl) phenyl) -1H-indole-2-carboxamide,

7- ((1H-pyrrolo [3,4-b ] pyridin-5-) ethynyl) -1-methyl-N- (4- ((4-methylpiperazin-1-) methyl) phenyl) -1H-indole-2-carboxamide,

7- ((1H-pyrazolo [3,4-b ] pyrimidin-5-yl) ethynyl) -1-methyl-N- (4- ((4-methylpiperazin-1-) methyl) phenyl) -1H-indole-2-carboxamide,

7- ((6-aminopyridin-3-yl) ethynyl) -1-methyl-N- (4- ((4-methylpiperazin-1-) methyl) -3- (trifluoromethyl) phenyl) -1H-indole-2-carboxamide,

1-methyl-N- (4- ((4-methylpiperazin-1-) methyl) -3- (trifluoromethyl) phenyl) -7- ((4-methylpyridin-3-) ethynyl) -1H-indole-2-carboxamide,

7- ((6-amino-4-methylpyridin-3-) ethynyl) -1-methyl-N- (4- ((4-methylpiperazin-1-) methyl) -3- (trifluoromethyl) phenyl) -1H-indole-2-carboxamide,

7- ((4-chloropyridine-3-) ethynyl) -1-methyl-N- (4- ((4-methylpiperazin-1-) methyl) -3- (trifluoromethyl) phenyl) -1H-indole-2-carboxamide,

7- ((6-amino-4-chloropyridin-3-) ethynyl) -1-methyl-N- (4- ((4-methylpiperazin-1-) methyl) -3- (trifluoromethyl) phenyl) -1H-indole-2-carboxamide,

1-methyl-N- (4- ((4-methylpiperazin-1-) methyl) -3- (trifluoromethyl) phenyl) -7- (pyrimidine-5-ethynyl) -1H-indole-2-carboxamide,

7- ((2-aminopyrimidin-5-) ethynyl) -1-methyl-N- (4- ((4-methylpiperazin-1-) methyl) -3- (trifluoromethyl) phenyl) -1H-indole-2-carboxamide,

1-methyl-N- (4- ((4-methylpiperazin-1-) methyl) -3- (trifluoromethyl) phenyl) -7- ((4-methylpyrimidin-5-) ethynyl) -1H-indole-2-carboxamide,

7- ((2- (ethylamino) pyrimidin-5-) ethynyl) -1-methyl-N- (4- ((4-methylpiperazin-1-) methyl) -3- (trifluoromethyl) phenyl) -1H-indole-2-carboxamide,

7- ((2- ((2- (dimethylamino) ethyl) amino) pyrimidin-5-) ethynyl) -1-methyl-N- (4- ((4-methylpiperazin-1-) methyl) -3- (trifluoromethyl) phenyl) -1H-indole-2-carboxamide,

7- ((2- ((2-methoxyethyl) amino) pyrimidin-5-) ethynyl) -1-methyl-N- (4- ((4-methylpiperazin-1-) methyl) -3- (trifluoromethyl) phenyl) -1H-indole-2-carboxamide,

1-methyl-7- ((2- (methylamino) pyrimidin-5-) ethynyl) -1-methyl-N- (4- ((4-methylpiperazin-1-) methyl) -3- (trifluoromethyl) phenyl) -1H-indole-2-carboxamide,

7- ((2- (cyclopropylamino) pyrimidin-5-) ethynyl) -1-methyl-N- (4- ((4-methylpiperazin-1-) methyl) -3- (trifluoromethyl) phenyl) -1H-indole-2-carboxamide,

7- ((6-amino-5-methylpyridin-3-) ethynyl) -1-methyl-N- (4- ((4-methylpiperazin-1-) methyl) -3- (trifluoromethyl) phenyl) -1H-indole-2-carboxamide,

7- ((2-amino-4-methylpyrimidin-5-) ethynyl) -1-methyl-N- (4- ((4-methylpiperazin-1-) methyl) -3- (trifluoromethyl) phenyl) -1H-indole-2-carboxamide,

7- ((1H-pyrazole-5-) ethynyl) -1-methyl-N- (4- ((4-methylpiperazin-1-) methyl) -3- (trifluoromethyl) phenyl) -1H-indole-2-carboxamide,

7- (imidazo [1,2-b ] pyridazin-3-ethynyl) -1-methyl-N- (4- ((4-methylpiperazin-1-) methyl) -3- (trifluoromethyl) phenyl) -1H-indole-2-carboxamide,

7- ((1H-pyrazolo [3,4-b ] pyridin-5-) ethynyl) -1-methyl-N- (4- ((4-methylpiperazin-1-) methyl) -3- (trifluoromethyl) phenyl) -1H-indole-2-carboxamide,

1-methyl-N- (4- ((4-methylpiperazin-1-) methyl) -3- (trifluoromethyl) phenyl) -7- (pyrazolo [1,5-a ] pyrimidine-6-ethynyl) -1H-indole-2-carboxamide,

7- ((1H-pyrrolo [2,3-b ] pyridin-5-) ethynyl) -1-methyl-N- (4- ((4-methylpiperazin-1-) methyl) -3- (trifluoromethyl) phenyl) -1H-indole-2-carboxamide,

7- ((2, 3-dihydro-1H-pyrrolo [2,3-b ] pyridin-5-) ethynyl) -1-methyl-N- (4- ((4-methylpiperazin-1-) methyl) -3- (trifluoromethyl) phenyl) -1H-indole-2-carboxamide,

1-methyl-N- (4- ((4-methylpiperazin-1-) methyl) -3- (trifluoromethyl) phenyl) -7- ((5,6,7, 8-tetrahydro-1, 8-naphthyridin-3-) ethynyl) -1H-indole-2-carboxamide,

1-methyl-N- (4- ((4-methylpiperazin-1-) methyl) -3- (trifluoromethyl) phenyl) -7- ((2-oxo-2, 3-dihydro-1H-imidazo [4,5-b ] pyridin-6-) ethynyl) -1H-indole-2-carboxamide,

7- ((2-aminopyrimidin-5-) ethynyl) -N- (4- ((4-methylpiperazin-1-) methyl) -3- (trifluoromethyl) phenyl) -1H-indole-2-carboxamide,

7- ((6-amino-4-chloropyridin-3-) ethynyl) -N- (4- ((4-methylpiperazin-1-) methyl) -3- (trifluoromethyl) phenyl) -1H-indole-2-carboxamide,

7- ((6-amino-4-methylpyridin-3-) ethynyl) -N- (4- ((4-methylpiperazin-1-) methyl) -3- (trifluoromethyl) phenyl) -1H-indole-2-carboxamide,

7- ((2-aminopyrimidine-5-ethynyl) -N- (4- ((4- (2-hydroxyethyl) piperazin-1-) methyl) -3- (trifluoromethyl) phenyl) -1-methyl-1H-indole-2-carboxamide,

(S) -7- ((2-aminopyrimidin-5-) ethynyl) -N- (4- ((3- (dimethylamino) pyrrolidin-1-) methyl) -3- (trifluoromethyl) phenyl) -1-methyl-1H-indole-2-carboxamide,

(R) -7- ((2-Aminopyrimidin-5-) ethynyl) -N- (4- ((3- (dimethylamino) pyrrolidin-1-) methyl) -3- (trifluoromethyl) phenyl) -1-methyl-1H-indole-2-carboxamide,

n- (4- ((1H-imidazol-1-) methyl) -3- (trifluoromethyl) phenyl) -7- ((2-aminopyridine-5-) ethynyl) -1-methyl-1H-indole-2-carboxamide,

7- ((2-Aminopyrimidine-5-) ethynyl) -N- (3-fluoro-4- ((4-methylpiperazin-1-) methyl) phenyl) -1-methyl-1H-indole-2-carboxamide,

7- ((2-aminopyrimidin-5-) ethynyl) -N- (3-bromo-4- ((4-methylpiperazin-1-) methyl) phenyl) -1-methyl-1H-indole-2-carboxamide,

7- ((2-aminopyrimidin-5-) ethynyl) -1-methyl-N- (3- (trifluoromethyl) phenyl) -1-methyl-1H-indole-2-carboxamide,

7- ((2-aminopyrimidin-5-) ethynyl) -N- (4- ((dimethylamino) methyl) -3-fluorophenyl) -1-methyl-1H-indole-2-carboxamide,

7- ((2-aminopyrimidin-5-) ethynyl) -N- (4- (2- (dimethylamino) ethyl) -3- (trifluoromethyl) phenyl) -1-methyl-1H-indole-2-carboxamide,

7- ((2-amino-5-) ethynyl) -1-methyl-N- (3- ((4-methylpiperazin-1-) methyl) -5- (trifluoromethyl) phenyl) -1-methyl-1H-indole-2-carboxamide,

7- ((2-aminopyrimidin-5-) ethynyl) -N- (3- ((dimethylamino) methyl) -5- (trifluoromethyl) phenyl) -1-methyl-1H-indole-2-carboxamide,

7- ((2-aminopyrimidin-5-) ethynyl) -N- (3- (3- (dimethylamino) propyl) -5- (trifluoromethyl) phenyl) -1-methyl-1H-indole-2-carboxamide,

7- ((2-aminopyrimidin-5-) ethynyl) -N- (3- (3-methoxypropyl) -5- (trifluoromethyl) phenyl) -1-methyl-1H-indole-2-carboxamide,

7- ((1H-pyrazolo [3,4-b ] pyridin-5-) ethynyl) -N- (3-bromo-4- ((4-methylpiperazin-1-) methyl) phenyl) -1-methyl-1H-indole-2-carboxamide,

(S) -7- ((1H-pyrazolo [3,4-b ] pyridin-5-) ethynyl) -N- (4- ((3- (diethylamino) pyrrolidin-1-) methyl) -3- (trifluoromethyl) phenyl) -1-methyl-1H-indole-2-carboxamide.

9. A process for preparing a compound according to any one of claims 1 to 7, which is represented by formula D and R¹X is reacted to give, wherein R¹，R²，R³As defined in any one of claims 1 to 7,

10. the method of claim 9, further comprising the step of

11. The method of claim 10, further comprising the step of

12. A compound of formula D, an enantiomer, diastereomer, tautomer, or a pharmaceutically acceptable salt thereof,

wherein R is²And R³As defined in any one of claims 1 to 7.

13. A process for preparing a compound according to any one of claims 1 to 7, which is represented by formula D1 with R³NH₂Obtained by reaction, wherein R¹，R²，R³As defined in any one of claims 1 to 7,

14. the method of claim 13, further comprising the step of

15. The method of claim 14, further comprising the step of

16. The method of claim 15, further comprising the step of

17. A compound of formula D1, an enantiomer, a diastereomer, a tautomer, or a pharmaceutical thereofA pharmaceutically acceptable salt wherein R¹And R²As defined in any one of claims 1 to 7,

18. a compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the salt is the hydrochloride, hydrobromide, hydroiodide, perchlorate, sulphate, nitrate, phosphate, formate, acetate, propionate, glycolate, lactate, succinate, maleate, tartrate, malate, citrate, fumarate, gluconate, benzoate, mandelate, methanesulphonate, isethionate, benzenesulfonate, oxalate, palmitate, 2-naphthalenesulphonate, p-toluenesulphonate, cyclamate, salicylate, hexonate, trifluoroacetate, aluminium salt, calcium salt, chloroprocaine salt choline salt, diethanolamine salt, ethylenediamine salt, lithium salt, potassium salt, sodium salt or zinc salt of the compound.

19. A pharmaceutical composition comprising a compound of any one of claims 1-7, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

20. A pharmaceutical composition comprising a compound of formula (I) as claimed in claim 1, a pharmaceutically acceptable salt thereof as active ingredient, one or more other therapeutic agents, and one or more pharmaceutically acceptable carriers or excipients.

21. Use of a compound of formula (I) according to any one of claims 1-7 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of cancer and autoimmune diseases associated with tyrosine kinases ABL, VEGFR 2.

22. The use of claim 21, wherein the cancer and autoimmune diseases associated with tyrosine kinases ABL, VEGFR2 comprise: fundus disease, dry eye, psoriasis, vitiligo, dermatitis, alopecia areata, rheumatoid arthritis, colitis, multiple sclerosis, systemic lupus erythematosus, crohn's disease, atheroma, pulmonary fibrosis, hepatic fibrosis, myelofibrosis, non-small cell lung cancer, breast cancer, pancreatic cancer, glioma, glioblastoma, ovarian cancer, cervical cancer, colorectal cancer, melanoma, endometrial cancer, prostate cancer, bladder cancer, leukemia, gastric cancer, liver cancer, gastrointestinal stromal tumor, thyroid cancer, chronic myeloid leukemia, acute myeloid leukemia, chronic myeloid leukemia, non-hodgkin's lymphoma, nasopharyngeal cancer, esophageal cancer, brain tumor, lymphoma, multiple myeloma, biliary sarcoma, biliary tract cancer.

23. The use of claim 22, wherein the lymphoma comprises B-cell and T-cell lymphomas.