TWI523856B - BCR-ABL kinase inhibitor and its application - Google Patents

Description

BCR-ABL kinase inhibitor and its application

The present invention belongs to the field of chemical medicine, and in particular relates to a compound having BCR-ABL kinase inhibitory activity or a pharmaceutically acceptable salt, isomer, solvate, crystal or prodrug thereof, and a pharmaceutical composition containing the same and these The use of a compound or composition in the preparation of a medicament.

Protein tyrosine kinases (PTKs) are a class of kinases that catalyze the transfer of γ -phosphates on ATP to protein tyrosine residues, which initiate functional proteins by catalyzing the phosphorylation of phenolic hydroxyl groups on various protein tyrosine residues. The role of the protein enzyme system. Protein tyrosine kinases (PTKs) play an important role in the signal transduction pathways in cells, regulating a series of physiological and biochemical processes such as cell growth, differentiation and death. Abnormal expression of protein tyrosine kinase can lead to disturbances in cell proliferation regulation, which in turn leads to tumorigenesis. In addition, the abnormal expression of protein tyrosine kinase is also closely related to tumor invasion and metastasis, tumor angiogenesis, and chemotherapy resistance of tumors. Tyrosine kinase inhibitors act as competitive inhibitors of adenosine triphosphate (ATP) binding to tyrosine kinases, competitively bind to tyrosine kinases, block tyrosine kinase activity, and inhibit cell proliferation. Several tyrosines already exist. Acid protein kinase inhibitors have been successfully developed.

In patients with chronic myeloid leukemia (CML), the long arm of chromosome 22 translocates to chromosome 9, forming a Philadelphia chromosome, and leads to the fusion of BCR gene and ABL gene to form BCR-ABL fusion gene, expressing BCR-ABL protein tyrosine. Kinase, through phosphorylation in cell signal transduction and transformation, promotes the infinite proliferation of CML mature granulocytes. BCR-ABL is not expressed in normal cells and has become an ideal drug target for the treatment of CML. Currently, BCR-ABL tyrosine kinase inhibitors have become the first-line treatment for most chronic myeloid leukemias.

Imatinib is the first protein-targeted therapeutic protein tyrosine kinase inhibitor that inhibits BCR-ABL tyrosine kinase at the cellular level in vitro and in vivo, selectively inhibiting BCR-ABL-positive cell lines. And the proliferation of fresh leukemia cells in Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL) patients, induced apoptosis. The development and clinical use of imatinib has opened a new era of tumor molecular targeting, but long-term use of imatinib will produce drug resistance, leading to relapse. With the widespread clinical application of imatinib, the problem of drug resistance has become increasingly prominent. The main reason for acquired tolerance is due to point mutations in BCR-ABL that result in the inability of imatinib to bind to BCR-ABL. Moreover, hundreds of BCR-ABL point mutations have been found to be associated with imatinib resistance, with 15 to 20% of imatinib-tolerant patients having a T315I mutation.

The emergence of imatinib resistance has ignited a new generation of tyrosine The development of kinase inhibitors has led to the development of newer drugs for the treatment of leukemia, such as drug-resistant or non-resistant CML, Ph+ALL.

It is an object of the present invention to provide a class of compounds having a broad spectrum of BCR-ABL kinase inhibitory effects, or pharmaceutically acceptable salts, isomers, solvates, crystals or prodrugs thereof having the structure of Formula I:

Another object of the invention is to provide a process for the preparation of a compound of formula I of the invention, or a pharmaceutically acceptable salt, isomer, solvate, crystal or prodrug thereof.

A further object of the present invention is to provide a composition comprising a compound of Formula I of the present invention, or a pharmaceutically acceptable salt, isomer, solvate, crystal or prodrug thereof, and a pharmaceutically acceptable carrier, and a composition comprising the same A composition of a compound of formula I or a pharmaceutically acceptable salt, isomer, solvate, crystal or prodrug thereof and another protein tyrosine kinase inhibitor.

A further object of the present invention is to provide a compound of the formula I according to the invention, or a pharmaceutically acceptable salt, isomer, solvate, crystal or prodrug thereof, for treating and/or preventing a tumor, and the method of the invention Compound of formula I Or the use of a pharmaceutically acceptable salt, isomer, solvate, crystal or prodrug thereof for the manufacture of a medicament for the treatment and/or prevention of a tumor.

In view of the above, the present invention provides the following technical solution: In a first aspect, the present invention provides a compound, or a pharmaceutically acceptable salt, isomer, solvate, crystal or prodrug thereof thereof, having the structure of Formula I:

Wherein X is selected from N or C(R ₅ ), wherein R _{5 is} selected from hydrogen, halogen, C _1-6 alkyl or halogenated C _1-6 alkyl; Y is selected from N or C(R ₆ ), wherein R _{6 is} selected from the group consisting of hydrogen, halogen, C _1-6 alkyl, halo C _1-6 alkyl; R _{1 is} selected from the group consisting of hydrogen, alkyl, alkoxy, haloalkyl, haloalkoxy, -OH, -NH ₂ , halogen or CN; R _{2 is} selected from the group consisting of hydrogen, alkyl, alkoxy, haloalkyl, haloalkoxy, -OH, -NH ₂ , halogen or CN; W is selected from C _1-6 alkylene, -C ( O) -, or R ₄ form a 4-8 membered cycloalkyl or absent; R ₃ is selected from substituted or unsubstituted five-, six-, seven-membered or eight-membered nitrogen-containing heterocyclic group; and R ₄ is selected from From hydrogen, alkyl, haloalkyl, or together with W to form a 4-8 membered cycloalkyl; provided that the following compound is excluded: 3-((1H-pyrrolo[2,3-b]pyrazin-5-yl) Ethyl)-4-methyl-N-[4-((4-methylpyridazin-1-yl)methyl)-3-trifluoromethylphenyl]benzamide.

In some preferred embodiments, the compound of the invention is a compound of formula I, and pharmaceutically acceptable salts, isomers, solvates, crystals or prodrugs thereof, wherein: X is selected from N or C (R ₅ Wherein R _{5 is} selected from the group consisting of hydrogen, fluorine, chlorine, bromine, C _1-4 alkyl, halo C _1-4 alkyl; Y is selected from N or C(R ₆ ), wherein R _{6 is} selected from hydrogen, fluorine , chloro, bromo, C _1-4 alkyl, halo C _1-4 alkyl; R _{1 is} selected from hydrogen, C _1-4 alkyl, C _1-4 alkoxy, halo C _1-4 alkyl , halogenated C _1-4 alkoxy, -OH, -NH ₂ , fluorine, chlorine, bromine or CN; R _{2 is} selected from hydrogen, C _1-4 alkyl, C _1-4 alkoxy, halogen C _1-4 alkyl, halo C _1-4 alkoxy, -OH, -NH ₂ , fluorine, chlorine, bromine or CN; W is selected from C _1-4 alkylene, -C(O)-, and R ₄ forms a 4-8 membered cycloalkyl group or is absent; and R _{3 is} selected from substituted or unsubstituted azacyclohexanes having 1, 2 or 3 nitrogen atoms selected from alkyl groups, hydroxyl groups, Hydroxyalkyl, alkoxy, amino, monoalkylamino, bisalkylamino, decylamino, alkyl guanylamino, aryl decylamino, heteroaryl decylamino, halogen, halogen substituted alkyl Halogen-substituted alkoxy group The said substituents are selected from C _1-6 alkyl, hydroxyl, C _1-6 alkyl, C _1-6 alkoxy, amino, mono-C _1-6 alkylamino, di-C _1-6 alkyl Amino, decylamino, C _1-6 alkyl decylamino, aryl decylamino, heteroaryl decylamino, halogen, halogen substituted C _1-6 alkyl, halogen substituted C _1-6 alkoxy And R _{4 is} selected from the group consisting of hydrogen, C _1-6 alkyl, halogenated C _1-6 alkyl or, together with W, a cyclopentyl group, a cyclobutane group, a cyclohexane group or a cycloheptyl group.

In some preferred embodiments, the compound of the invention is a compound of formula I, and pharmaceutically acceptable salts, isomers, solvates, crystals or prodrugs thereof, wherein: X is selected from N or C (R ₅ Wherein R _{5 is} selected from the group consisting of hydrogen, fluorine, chlorine, bromine, C _1-4 alkyl, halo C _1-4 alkyl; Y is selected from N or C(R ₆ ), wherein R _{6 is} selected from hydrogen, fluorine , chloro, bromo, C _1-4 alkyl, halo C _1-4 alkyl; R _{1 is} selected from hydrogen, C _1-4 alkyl, C _1-4 alkoxy, halo C _1-4 alkyl , halogenated C _1-4 alkoxy, -OH, -NH ₂ , fluorine, chlorine, bromine or CN; R _{2 is} selected from hydrogen, C _1-4 alkyl, C _1-4 alkoxy, halogen C _1-4 alkyl, halo C _1-4 alkoxy, -OH, -NH ₂ , fluorine, chlorine, bromine or CN; W is selected from C _1-4 alkylene, -C(O)-, and R ₄ forms a 4-8 membered cycloalkyl group or is absent; R _{3 is} selected from pyridazinyl, pyridyl, azabicycloalkyl, imidazolyl, pyrazolyl, pyrrolyl, triazolyl, pyridazinyl, pyrimidine , pyrazinyl, acridinyl, triazinyl, or substituted pyridazinyl, pyridyl, azabicycloalkyl, imidazolyl, pyrazolyl, pyrrolyl, triazolyl, pyridazinyl, pyrimidinyl , pyrazinyl, acridinyl, three a group, wherein the substitution is selected from the group consisting of an alkyl group, a hydroxyl group, a hydroxyalkyl group, an alkoxy group, an amino group, a monoalkylamino group, a dialkylamino group, a decylamino group, an alkyl decylamino group, an aryl decylamino group, a hetero group Aryl decylamino, halogen, halogen-substituted alkyl, halogen-substituted alkoxy, preferably the substituent is selected from C _1-6 alkyl, hydroxy, hydroxy C _1-6 alkyl, C _1-6 Alkoxy, amino, mono C _1-6 alkylamino, bis C _1-6 alkylamino, decylamino, C _1-6 alkyl decylamino, aryl decylamino, heteroaryl decylamino , halogen, halogen-substituted C _1-6 alkyl, halogen-substituted C _1-6 alkoxy; R _{4 is} selected from hydrogen, C _1-6 alkyl, halo C _1-6 alkyl, or together with W It constitutes a cyclopentyl group, a cyclobutane group, a cyclohexane group, and a cycloheptyl group.

More preferably, the compounds of the present invention include compounds of formula I and their pharmaceutically acceptable salts, isomers, solvates, crystalline or prodrug thereof, wherein: X is selected from N or C (R _5), wherein R _{5 is} selected from the group consisting of hydrogen, fluorine, chlorine, bromine, C _1-3 alkyl, halogenated C _1-3 alkyl; Y is selected from N or C(R ₆ ), wherein R _{6 is} selected from the group consisting of hydrogen, fluorine, chlorine, and bromine , C _1-3 alkyl, halo C _1-3 alkyl; R _{1 is} selected from hydrogen, C _1-3 alkyl, C _1-3 alkoxy, halo C _1-3 alkyl, halogen C _1-3 alkoxy, -OH, -NH ₂ , fluorine, chlorine, bromine or CN; R _{2 is} selected from hydrogen, C _1-3 alkyl, C _1-3 alkoxy, halogenated C _1-3 alkane , halo C _1-3 alkoxy, -OH, -NH ₂ , fluoro, chloro, bromo or CN; W is selected from C _1-3 alkylene (eg -CH ₂ -, -CH ₂ -CH ₂ -), -C(O)-, forms a cyclopentyl group with R ₄ or is absent; R _{3 is} selected from pyridazinyl, aridin-4-yl, diazabicyclooctylalkyl, for example 3,8- Diazabicyclo[3.2.1]octyl, the pyridazinyl, acridin-4-yl, diazabicyclooctyl can be one or more C _1-6 alkyl, C _{1- 6} alkoxy substituted, for example by one or more methyl, ethyl, propyl, isopropyl, methoxy, ethoxy Substituted by a radical or a propoxy group; R _{4 is} selected from hydrogen, C _1-4 alkyl, halo C _1-4 alkyl, or together with W constitutes a cyclopentyl group.

In some embodiments of the above compounds of Formula I, wherein X is N and Y is N.

In some embodiments of the above compounds of Formula I, wherein X is N and Y is C(R ₆ ), said R _{6 is} selected from the group consisting of hydrogen, halogen, C _1-6 alkyl, halo C _1-6 alkane And preferably R _{6 is} selected from the group consisting of hydrogen, fluorine, chlorine, bromine, C _1-4 alkyl, halo C _1-4 alkyl, more preferably R _{6 is} selected from the group consisting of hydrogen, fluorine, chlorine, bromine, C _{1- 3-} alkyl, halo C _1-3 alkyl.

In some embodiments of the above compounds of Formula I, wherein X is C(R ₅ ), Y is N, and said R _{5 is} selected from the group consisting of hydrogen, halogen, C _1-6 alkyl, halo C _1-6 alkane a group, preferably R _{5 is} selected from the group consisting of hydrogen, fluorine, chlorine, bromine, C _1-4 alkyl, halogenated C _1-4 alkyl, more preferably R _{5 is} selected from the group consisting of hydrogen, fluorine, chlorine, bromine, C _{1- 3-} alkyl, halo C _1-3 alkyl.

In some embodiments of the above compounds of Formula I, wherein X is C(R ₅ ), Y is C(R ₆ ), and R ₅ and R ₆ are each independently selected from hydrogen, halogen, C _1-6 An alkyl group, a halogenated C _1-6 alkyl group, preferably R ₅ and R ₆ are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, C _1-4 alkyl, halogenated C _1-4 alkyl, more preferably R ₅ and R ₆ are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, C _1-3 alkyl, halo C _1-3 alkyl.

Preferably, the compounds provided herein, or pharmaceutically acceptable salts, isomers, solvates, crystals or prodrugs thereof, have the structure of Formula Ia:

Wherein X is selected from N or C(R ₅ ), wherein R _{5 is} selected from the group consisting of hydrogen, halogen, C _1-6 alkyl, halo C _1-6 alkyl; Y is selected from N or C(R ₆ ), wherein R _{6 is} selected from the group consisting of hydrogen, halogen, C _1-6 alkyl, halo C _1-6 alkyl; R _{1 is} selected from the group consisting of hydrogen, alkyl, alkoxy, haloalkyl, haloalkoxy, -OH, -NH ₂ , halogen or CN; R _{2 is} selected from the group consisting of hydrogen, alkyl, alkoxy, haloalkyl, haloalkoxy, -OH, -NH ₂ , halogen or CN; W is selected from C _1-6 alkylene, -C ( O) - or absent; R ₃ is selected from substituted or unsubstituted five-, six-, seven-membered or eight yuan nitrogen-containing heterocyclic group, R ₃ is preferably a substituted or unsubstituted, containing 1, 2 or 3 a nitrogen atom of azacyclohexane, further preferably R ₃ is pyridazinyl, pyridyl, azabicycloalkyl, imidazolyl, pyrazolyl, pyrrolyl, triazolyl, pyridazinyl, pyrimidinyl, pyridyl Azinyl, acridinyl, triazinyl, or substituted pyridazinyl, pyridyl, azabicycloalkyl, imidazolyl, pyrazolyl, pyrrolyl, triazolyl, pyridazinyl, pyrimidinyl, pyrazine a base, an acridine group, a triazinyl group, wherein the substituent is selected from a C _1-6 alkyl group, a hydroxyl group, a hydroxy C _1-6 alkyl group, a C _1-6 group Alkoxy, amino, mono C _1-6 alkylamino, bis C _1-6 alkylamino, decylamino, C _1-6 alkyl decylamino, aryl decylamino, heteroaryl decylamino a halogen, a halogen-substituted C _1-6 alkyl group, a halogen-substituted C _1-6 alkoxy group, more preferably R ₃ is a pyridazinyl group, an acridinium-4-yl group, a diazabicycloalkyl group such as 3, 8-diazabicyclo[3.2.1]octyl, or pyridazinyl, acridin-4-yl, diazo substituted by one or more C _1-6 alkyl, C _1-6 alkoxy Heterobicycloalkyl, for example R ₃ is 4-methylpyridazin-1-yl, acridin-4-yl, 1-methylacridin-4-yl, 3,8-diazabicyclo[3.2.1 ] octan-8-yl, 1-methyl-3,8-diazabicyclo [3.2.1] octane-8-yl; and R ₄ is selected from hydrogen, C _1-6 alkyl, halo C _1-6 alkyl; the conditions are excluded from the following compounds: 3-((1H-pyrrolo[2,3-b]pyrazin-5-yl)ethynyl)-4-methyl-N-[4-( (4-Methyloxazin-1-yl)methyl)-3-trifluoromethylphenyl]benzamide.

Preferably, the compounds provided herein, or pharmaceutically acceptable salts, isomers, solvates, crystals or prodrugs thereof, have the structure of Formula Ib:

Wherein X is selected from N or C(R ₅ ), wherein R _{5 is} selected from the group consisting of hydrogen, halogen, C _1-6 alkyl, halo C _1-6 alkyl; Y is selected from N or C(R ₆ ), wherein R _{6 is} selected from the group consisting of hydrogen, halogen, C _1-6 alkyl, halo C _1-6 alkyl; R _{1 is} selected from the group consisting of hydrogen, alkyl, alkoxy, haloalkyl, haloalkoxy, -OH, -NH ₂ , halogen or CN; R _{2 is} selected from the group consisting of hydrogen, alkyl, alkoxy, haloalkyl, haloalkoxy, -OH, -NH ₂ , halogen or CN; W is selected from C _1-6 alkylene, -C ( O)- or absent; R _{3 is} selected from substituted or unsubstituted five-, six-, seven- or eight-membered nitrogen-containing heterocyclic groups, preferably R ₃ is substituted or unsubstituted containing 1, 2 or 3 a nitrogen atom of azacyclohexane, further preferably R ₃ is pyridazinyl, pyridyl, azabicycloalkyl, imidazolyl, pyrazolyl, pyrrolyl, triazolyl, pyridazinyl, pyrimidinyl, pyridyl Azinyl, acridinyl, triazinyl, or substituted pyridazinyl, pyridyl, azabicycloalkyl, imidazolyl, pyrazolyl, pyrrolyl, triazolyl, pyridazinyl, pyrimidinyl, pyrazine yl, piperidinyl, triazinyl, wherein said substituent is selected from C _1-6 alkyl, hydroxyl, C _1-6 alkyl, C _1-6 Alkoxy, amino, mono-C _1-6 alkylamino, di-C _1-6 alkylamino group, acyl group, C _1-6 alkyl acyl group, an acyl group an aryl group, a heteroaryl acyl group, Halogen, halogen substituted C _1-6 alkyl, halogen substituted C _1-6 alkoxy, more preferably R ₃ is pyridazinyl, acridin-4-yl, diazabicycloalkyl, or one Or a plurality of C _1-6 alkyl, C _1-6 alkoxy substituted pyridazinyl, aridin-4-yl, diazabicycloalkyl, such as R ₃ is 4-methylpyridazine-1- Base, acridin-4-yl, 1-methylacridin-4-yl, 3,8-diazabicyclo[3.2.1]octane-8-yl, 1-methyl-3,8-di azabicyclo [3.2.1] octane-8-yl; and R ₄ is selected from hydrogen, C _1-6 alkyl, halo C _1-6 alkyl group.

Preferably, the compounds provided herein, or pharmaceutically acceptable salts, isomers, solvates, crystals or prodrugs thereof, have the structure of Formula Ic:

Wherein X is selected from N or C(R ₅ ), wherein R _{5 is} selected from the group consisting of hydrogen, halogen, C _1-6 alkyl, halo C _1-6 alkyl; Y is selected from N or C(R ₆ ), wherein R _{6 is} selected from the group consisting of hydrogen, halogen, C _1-6 alkyl, halo C _1-6 alkyl; R _{1 is} selected from the group consisting of hydrogen, alkyl, alkoxy, haloalkyl, haloalkoxy, -OH, -NH ₂ , halogen or CN; R _{2 is} selected from hydrogen, alkyl, alkoxy, haloalkyl, haloalkoxy, -OH, -NH ₂ , halogen or CN; and R _{3 is} selected from substituted or unsubstituted five or six a monovalent, seven- or eight-membered nitrogen-containing heterocyclic group, preferably R ₃ is a substituted or unsubstituted azacyclohexane having 1, 2 or 3 nitrogen atoms, further preferably R ₃ is pyridazinyl, pyridine , azabicycloalkyl, imidazolyl, pyrazolyl, pyrrolyl, triazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, acridinyl, triazinyl, or substituted pyridazinyl, pyridyl , azabicycloalkyl, imidazolyl, pyrazolyl, pyrrolyl, triazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, acridinyl, triazinyl, wherein said substituent is selected from C _{1- 6} alkyl, hydroxyl, C _1-6 alkyl, C _1-6 alkoxy, amino, mono-C _1-6 alkylamino , Di-C _1-6 alkylamino group, acyl group, C _1-6 alkyl acyl group, an acyl group an aryl group, a heteroaryl acyl group, halogen, halo-substituted C _1-6 alkyl, halo Substituted C _1-6 alkoxy, more preferably R ₃ is pyridazinyl, aridin-4-yl, diazabicycloalkyl, or by one or more C _1-6 alkyl, C _{1- 6} alkoxy-substituted pyridazinyl, acridin-4-yl, diazabicycloalkyl, for example R ₃ is 4-methylpyridazin-1-yl, acridin-4-yl, 1-methyl Acridine-4-yl, 3,8-diazabicyclo[3.2.1]octane-8-yl, 1-methyl-3,8-diazabicyclo[3.2.1]octane-8- base.

Preferably, the compounds provided herein, or pharmaceutically acceptable salts, isomers, solvates, crystals or prodrugs thereof, have the structure of Formula Id:

Wherein X is selected from N or C(R ₅ ), wherein R _{5 is} selected from the group consisting of hydrogen, halogen, C _1-6 alkyl, halo C _1-6 alkyl; Y is selected from N or C(R ₆ ), wherein R _{6 is} selected from the group consisting of hydrogen, halogen, C _1-6 alkyl, halo C _1-6 alkyl; R _{1 is} selected from the group consisting of hydrogen, alkyl, alkoxy, haloalkyl, haloalkoxy, -OH, -NH ₂ , halogen or CN; R _{2 is} selected from hydrogen, alkyl, alkoxy, haloalkyl, haloalkoxy, -OH, -NH ₂ , halogen or CN; and R _{3 is} selected from substituted or unsubstituted five or six a monovalent, seven- or eight-membered nitrogen-containing heterocyclic group, preferably R ₃ is a substituted or unsubstituted azacyclohexane having 1, 2 or 3 nitrogen atoms, further preferably R ₃ is pyridazinyl, pyridine , azabicycloalkyl, imidazolyl, pyrazolyl, pyrrolyl, triazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, acridinyl, triazinyl, or substituted pyridazinyl, pyridyl , azabicycloalkyl, imidazolyl, pyrazolyl, pyrrolyl, triazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, acridinyl, triazinyl, wherein said substituent is selected from C _{1- 6} alkyl, hydroxyl, C _1-6 alkyl, C _1-6 alkoxy, amino, mono-C _1-6 alkylamino , Di-C _1-6 alkylamino group, acyl group, C _1-6 alkyl acyl group, an acyl group an aryl group, a heteroaryl acyl group, halogen, halo-substituted C _1-6 alkyl, halo Substituted C _1-6 alkoxy, more preferably R ₃ is pyridazinyl, aridin-4-yl, diazabicycloalkyl, or by one or more C _1-6 alkyl, C _{1- 6} alkoxy-substituted pyridazinyl, acridin-4-yl, diazabicycloalkyl, for example R ₃ is 4-methylpyridazin-1-yl, acridin-4-yl, 1-methyl Acridine-4-yl, 3,8-diazabicyclo[3.2.1]octane-8-yl, 1-methyl-3,8-diazabicyclo[3.2.1]octane-8- base.

Preferably, in accordance with the present invention, in some embodiments, in Formula I, Ia, Ib, Ic, Id, R _{1 is} selected from hydrogen, alkyl, alkoxy, haloalkyl, halo or CN, especially R _{1 is} selected from the group consisting of hydrogen, C _1-6 alkyl, C _1-6 alkoxy, halo C _1-6 alkyl, halogen or CN.

Preferably, in accordance with the present invention, in some embodiments, in Formula I, Ia, Ib, Ic, Id, R _{2 is} selected from hydrogen, alkyl, haloalkyl, halogen or CN, particularly R _{2 is} selected from hydrogen. , C _1-6 alkyl, halo C _1-6 alkyl, halogen or CN.

Preferably, in accordance with the present invention, in some embodiments, in Formula I, Ia, Ib, Ic, Id, R _{3 is} selected from the group consisting of 4-methylpyridazin-1-yl, acridin-4-yl, 1 -methyl acridin-4-yl, 3,8-diazabicyclo[3.2.1]octane-8-yl, 1-methyl-3,8-diazabicyclo[3.2.1]octane -8-based.

Preferably, according to the present invention, in some embodiments, in Formula I, Ia, Ib, Ic, Id , R _{4 is} selected from methyl or trifluoromethyl.

Preferably, in accordance with the present invention, in some embodiments, in Formulas I, Ia, Ib, Ic, Id, W and R ₄ together form a cyclopentyl group.

Preferably, according to the present invention, in some embodiments, in Formula I, Ia, Ib, Ic, Id , R ₅ is selected from hydrogen, halogen.

Preferably, according to the present invention, in some embodiments, in Formula I, Ia, Ib, Ic, Id , R _{6 is} selected from hydrogen, halogen.

Preferably, in accordance with the present invention, in some embodiments, in Formula I, Ia, Ib, Ic, Id, R _{1 is} selected from the group consisting of hydrogen, methyl, trifluoromethyl, methoxy, ethoxy, C. Oxy or isopropoxy.

Preferably, in accordance with the present invention, in some embodiments, in Formula I, Ia, Ib, Ic, Id, R _{2 is} selected from hydrogen, methyl, ethyl, propyl or isopropyl.

Preferably, according to the present invention, in some embodiments, in Formula I, Ia, Ib, Ic, Id , R ₅ is selected from hydrogen, fluoro.

Preferably, according to the present invention, in some embodiments, in Formula I, Ia, Ib, Ic, Id , R _{6 is} selected from hydrogen, fluoro.

The present invention provides the following specific compounds:

In another aspect, the invention provides a process for the preparation of a compound of the formula of the invention. The preparation method of the compound of the formula I comprises the following steps: (1) Preparation of the intermediate of the formula 5: a) a compound of formula 1 is reacted with a compound of formula 2 to give an intermediate of formula 3; b) an intermediate of formula 3 is reacted with trimethylsulfonium acetylene to give an intermediate of formula 4; c) an intermediate of formula 4 is removed Methyl decyl group to give an intermediate of formula 5; (2) preparation of a compound of formula I: d) an intermediate of formula 5 is reacted with an intermediate of formula 6 to give a compound of formula I wherein R ₁ , R ₂ , R ₃ , R ₄ , W, X, Y have the meaning of formula I, M is selected From chlorine, bromine, iodine.

In a third aspect, the invention provides a pharmaceutical composition comprising a compound of the invention, or a pharmaceutically acceptable salt, isomer, solvate, crystal or prodrug thereof.

In some embodiments, the invention provides a compound of the invention, or a pharmaceutically acceptable salt, isomer, solvate, crystal or prodrug thereof, and a compound comprising the same, or a pharmaceutically acceptable salt, isomer thereof A pharmaceutical composition of a solvate, crystal or prodrug for use in the treatment or prevention of cancer, including solid tumors and various forms of leukemia, including leukemia resistant to other therapies.

In some embodiments, the invention provides a pharmaceutical composition comprising a compound, isomer, solvate, crystal or prodrug of the invention, further comprising one or more selected from the group consisting of a tyrosine protease inhibitor , EGFR inhibitors, VEGFR inhibitors, BCR-ABL inhibitors, c-kit inhibitors, c-Met inhibitors, Raf inhibitors, MEK inhibitors, histone deacetylase inhibitors, VEGF antibodies, EGF antibodies, HIV protein kinase inhibitor, HMG-CoA reductase inhibitor, and the like.

The compound, isomer, solvate, crystal or prodrug of the present invention may be formulated into a pharmaceutical preparation by mixing with a pharmaceutically acceptable carrier, diluent or excipient to be suitable for oral or parenteral administration. Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, and oral routes. The formulation may be administered by any route, such as by infusion or bolus injection, by transepithelial or mucous membranes of the skin (eg, oral mucosa or rectum, etc.) Route of absorption. Administration can be systemic or topical. Examples of the orally administered preparations include solid or liquid dosage forms, specifically, tablets, pills, granules, powders, capsules, syrups, emulsions, suspensions and the like. The formulations may be prepared by methods known in the art and comprise carriers, diluents or excipients conventionally employed in the field of pharmaceutical formulations.

In a fourth aspect, the present invention provides a method, a method for treating and/or preventing a tumor of a compound, an isomer, a solvate, a crystal or a prodrug of the present invention or a pharmaceutical composition of the present invention, and in the preparation of a medicament for preventing and/or treating cancer Use of a compound, isomer, solvate, crystal or prodrug of the invention or a compound, isomer, solvate, crystal or prodrug of the invention, to a tumor-prone population or tumor patient The pharmaceutical composition is effective to reduce the incidence of tumors and prolong the life of tumor patients.

In some embodiments, the invention provides methods for treating and/or preventing a tumor comprising administering to a subject in need thereof a therapeutically and/or prophylactically effective amount of a compound, isomer, solvate, crystal of the invention Or a prodrug or a pharmaceutical composition of the invention. The compounds, isomers, solvates, crystals or prodrugs of the invention or pharmaceutical compositions of the invention may be administered to a mammal in need thereof to inhibit tumor growth, progression and/or metastasis, including solid tumors , for example, breast cancer, colon cancer, gastric cancer, pancreatic cancer, central nervous system tumors, and head and neck cancers, as well as various forms of leukemia, including leukemia and other cancers that are resistant to other treatments, such as resistance to imatinib or other kinase inhibitors. The kinase is inhibited by a compound or composition of the invention.

Terminology

The "alkyl group" of the present invention means a linear, branched or cyclic saturated hydrocarbon group, preferably a C _1-6 alkyl group. For example, a suitable C _1-6 alkyl group includes, but is not limited to, a methyl group, a Base, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, tert-butyl, cyclobutyl, n-pentyl, isopentyl, cyclopentyl, cyclohexyl, n-hexyl. Herein, the alkyl group is further preferably a C _1-3 alkyl group, and a suitable C _1-3 alkyl group is a methyl group, an ethyl group, a propyl group, an isopropyl group, or a cyclopropyl group. As used herein, an alkyl group in the present invention includes a substituted or unsubstituted alkyl group, which may be optionally substituted with one or more groups selected from the group consisting of an alkyl group, an alkoxy group, an aryloxy group, Alkylamino, arylamino, halogen, hydroxy, amino, nitro, cyano, alkyl fluorenyl, amino fluorenyl, alkylamino fluorenyl, sulfonyl, sulfinyl, fluorenyl, aryl or heteroaryl .

The "alkoxy group" of the present invention means an alkyl-O- group, preferably a C _1-6 alkyl-O- group, further preferably a C _1-3 alkyl-O- group, a suitable C _1-3 alkyl group - O- is methoxy, ethoxy, propoxy or isopropoxy.

The "halogen" of the present invention means fluorine, chlorine, bromine or iodine, preferably fluorine or chlorine.

The "haloalkyl group" of the present invention means an alkyl group substituted with at least one halogen, preferably a halogenated C _1-6 alkyl group, further preferably a halogenated C _1-3 alkyl group, a suitable halogenated C _1-3 alkane. Base is chloromethyl, fluoromethyl, dichloromethyl, difluoromethyl, trichloromethyl, trifluoromethyl, chloroethyl, fluoroethyl, dichloroethyl, difluoroethyl, trichloro Ethyl, trifluoroethyl.

The "haloalkoxy group" of the present invention means an alkoxy group substituted with at least one halogen, preferably a C _1-6 alkoxy group substituted with at least one halogen, and further preferably a halogenated C _1-3 alkoxy group, suitably Halogenated C _1-3 alkoxy is chloromethoxy, fluoromethoxy, dichloromethoxy, difluoromethoxy, trichloromethoxy, trifluoromethoxy; dichloroethoxy , difluoroethoxy, trichloroethoxy, trifluoroethoxy.

The "five-membered, six-membered, seven-membered, eight-membered nitrogen-containing heterocyclic group" of the present invention means that the substituted or unsubstituted has at least one ring, and the total number of ring atoms is five, six, seven, eight and A saturated, partially saturated, and fully unsaturated heterocyclic group containing at least one nitrogen atom. Preferably, the "five-membered, six-membered, seven-membered, eight-membered nitrogen-containing heterocyclic group" is pyridazinyl, pyridyl, azabicycloalkyl, imidazolyl, pyrazolyl, pyrrolyl, triazolyl , pyridazinyl, pyrimidinyl, pyrazinyl, acridinyl, triazinyl, or substituted pyridazinyl, pyridyl, azabicycloalkyl, imidazolyl, pyrazolyl, pyrrolyl, triazolyl, Pyridazinyl, pyrimidinyl, pyrazinyl, acridinyl, triazinyl, wherein said substituent is selected from the group consisting of alkyl, hydroxy, hydroxyalkyl, alkoxy, amino, monoalkylamino, bisalkylamino a mercaptoamine group, an alkylamino group, an arylguanamine group, a heteroarylguanamine group, a halogen, a halogen-substituted alkyl group, a halogen-substituted alkoxy group, preferably the substituent is selected from C _{1- 6} alkyl, hydroxy, hydroxy C _1-6 alkyl, C _1-6 alkoxy, amino, mono C _1-6 alkylamino, bis C _1-6 alkylamino, decylamino, C _1-6 Alkyl decylamino, aryl decylamino, heteroaryl decylamino, halogen, halogen substituted C _1-6 alkyl, halogen substituted C _1-6 alkoxy.

The "4-8 membered cycloalkyl group" of the present invention means a monocyclic or bicyclic saturated hydrocarbon group having 4 to 8 carbon atoms, and a suitable cycloalkyl group includes a cyclobutyl group and a ring. A pentyl group, a cyclohexyl group, a cycloheptyl group and the like. Other suitable cycloalkyl groups include spiropentyl, bicyclo[2.1.0]pentyl and bicyclo[3.1.0]hexyl, and the like.

The "solvate" of the present invention means, in a conventional sense, a complex formed by a combination of a solute (such as an active compound, a salt of an active compound) and a solvent (such as water). Solvent refers to a solvent known or readily determinable by those skilled in the art. In the case of water, the solvate is generally referred to as a hydrate such as a monohydrate, a dihydrate, a trihydrate or the like.

"Crystalline" as used in the present invention refers to various solid forms formed by the compounds of the present invention, including crystalline forms and amorphous forms.

"Isomers" of the invention include the compounds cis conformation, conformational isomer and enantiomer. A conformational isomer refers to a cis-trans isomer of the cis or trans configuration. A conformational isomer refers to a stereoisomer resulting from the rotation of a single bond.

The "prodrug" of the present invention refers to a compound which is converted into a compound of the present invention by reaction with an enzyme, gastric acid or the like under physiological conditions of a living body, that is, a compound which is converted into a compound of the invention by oxidation, reduction, hydrolysis or the like of an enzyme. And/or a compound which converts to the compound of the invention by a hydrolysis reaction such as gastric acid or the like.

The "pharmaceutically acceptable salt" of the present invention means a pharmaceutically acceptable salt of the compound of the present invention and an acid, which includes, but is not limited to, phosphoric acid, sulfuric acid, hydrochloric acid, hydrobromic acid, citric acid, and Malay. Acid, malonic acid, mandelic acid, succinic acid, fumaric acid, acetic acid, lactic acid, nitric acid, and the like.

The "pharmaceutical composition" of the present invention means any one of the present The compounds described herein, including isomers, prodrugs, solvates, pharmaceutically acceptable salts or chemically protected forms thereof, and mixtures of one or more pharmaceutically acceptable carriers.

The "pharmaceutically acceptable carrier" of the present invention means a carrier which does not cause significant irritation to an organism and does not interfere with the biological activity and properties of the administered compound, and includes a solvent, a diluent or other excipient, a dispersing agent, and a surface active agent. Agents, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants, and the like. Unless any conventional carrier medium is incompatible with the compounds of the invention. Some examples of pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and derivatives thereof, such as sodium carboxymethylcellulose, and Cellulose and cellulose acetate; malt, gelatin, and the like.

"Excipient" as used herein refers to an inert substance that is added to a pharmaceutical composition to further facilitate administration of the compound. Excipients can include calcium carbonate, calcium phosphate, various sugars and various types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols.

The "application in the preparation of a medicament for treating and/or preventing a tumor" of the present invention means that the growth, development and/or metastasis of the tumor can be inhibited, and the therapeutically effective dose is mainly administered to a human or animal in need thereof. The compounds of the invention inhibit, slow or reverse the growth, progression or spread of a tumor in a subject.

The compound of the present invention refers to all compounds of the general formula of the present invention, including any of Formula I, Formula Ia, Formula Ib, Formula Ic or Formula Id. Compounds of the formula and specific compounds.

The following representative examples are intended to better illustrate the invention and are not intended to limit the scope of the invention.

Example 1. 3-Ethynyl-4-methyl-N-[4-((4-methylpyridazin-1-yl)methyl)-3-trifluoromethylphenyl]benzamide preparation

Step 1: Preparation of 3-iodo-4-methyl-N-[4-(4-methylpyridazin-1-yl)methyl]-3-trifluoromethylphenyl]benzamide

4-(4-Methyloxazin-1-ylmethyl)-3-trifluoromethylaniline (2.27 g, 8.3 mmol), 3-iodo-4-methyl-benzoguanidine chloride was added to the reactor. (10 mmol), 15 ml of tetrahydrofuran, 10 ml of triethylamine, and stirred at room temperature for 4 hours. Washed with saturated NaHCO ₃ solution, extracted with ethyl acetate and water were added, then washed with saturated NaCl solution, dried over anhydrous Na ₂ SO _4, solvent was distilled off under reduced pressure. The residue was purified with EtOAc EtOAc (EtOAc) _{^{1 H NMR (500MHz, CDCl 3}} ) δ: 8.39 (s, 1H, NH), 8.29 (s, 1H, Ar-H), 7.88 (d, 1H, Ar-H), 7.86 (s, 1H, Ar- H), 7.75 (d, 1H, Ar-H), 7.73 (d, 1H, Ar-H), 7.2 8 (d, 1H, Ar-H), 3.62 (s, 2H, PhCH ₂ ), 2.60 (b) , 8H, 4 × -CH ₂ ), 2.47 (s, 3H, -CH ₃ ), 2.31 (s, 3H, -CH ₃ ).

Step 2: 3-Trimethyldecyl ethynyl-4-methyl-N-[4-((4-methylpyridazin-1-yl)methyl)-3-trifluoromethylphenyl]benzene Preparation of formamide

The product obtained in Step 1 (3.1 g, 6.1 mmol), Pd(PPh ₃ ) ₂ Cl ₂ (426 mg, 0.61 mmol), CuI (231 mg, 1.21 mmol) was placed in a reactor, 30 ml of toluene was added as a solvent, triethylamine 1 ml maintains an alkaline environment. To the mixture was added trimethyldecyl acetylene (3.0 g, 30.3 mmol) under an inert atmosphere and stirred at 58 ° C for 24 hours. End of the reaction, the organic layer was extracted, and the combined reaction mixture were added ethyl acetate and water, washed with saturated NaCl solution, dried over anhydrous added Na ₂ SO _4. Concentration under reduced pressure, the title compound was crystall _{^{1 H NMR (500MHz, CDCl 3}} ) δ: 8.30 (s, 1H, NH), 7.86 (s, 1H, Ar-H), 7.83 (d, 1H, Ar-H), 7.72 (s, 1H, Ar- H), 7.55 (d, 1H, Ar-H), 7.41 (d, 1H, Ar-H), 7.24 (d, 1H, Ar-H), 3.60 (s, 2H, PhCH ₂ ), 2.48 (b, 8H, 4x-CH ₂ ), 2.45 (s, 3H, -CH ₃ ), 2.28 (s, 3H, -CH ₃ ), 0.26 (s, 9H, 3×-CH ₃ ).

Step 3: Preparation of 3-ethynyl-4-methyl-N-[4-((4-methylpyridazin-1-yl)methyl)-3-trifluoromethylphenyl]benzamide

The product of the step 2 (1.59 g, 3.3 mmol), potassium carbonate (1.82 g, 13.2 mmol), and 20 ml of methanol were mixed in the reactor, and the mixture was stirred at room temperature for 3 hours under an inert gas atmosphere. The reaction completed, the methanol was removed by rotary evaporation, ethyl acetate and the organic layer was extracted with water, combined, washed with saturated NaCl solution, dried over anhydrous added Na ₂ SO _4. The organic solution was then concentrated on a rotary evaporator. _{^{1 H NMR (500MHz, CDCl 3}} ) δ: 10.47 (s, 1H, NH), 8.19 (s, 1H, Ar-H), 8.08 (s, 1H, Ar-H), 8.04 (d, 1H, Ar- H), 7.91 (d, 1H, Ar-H), 7.70 (d, 1H, Ar-H), 7.47 (d, 1H, Ar-H), 4.50 (s, 1H, ≡CH), 3.56 (s, 2H, PhCH ₂ ), 2.50 (s, 3H, -CH ₃ ), 2.36 (b, 8H, 4 × CH ₂ ), 2.15 (s, 3H, -CH ₃ ).

Example 2. 3-((1H-pyrrolo[2,3-b]pyrazin-5-yl)ethynyl)-4-methyl-N-[4-((4-methylpyridazine-1) Of -methyl)methyl)-3-trifluoromethylphenyl]benzamide

The compound prepared in Example 1 (126 mg, 0.3 mmol), 5-bromo-1H-pyrrolo[2,3-b]pyrazine (59 mg, 0.3 mmol), Pd(PPh ₃ ) ₂ Cl was added to a 10 ml sealed tube. ₂ (63 mg, 0.006 mmol), CuI (18 mg, 0.09 mmol), 1mLEt ₃ N and 5 mL of DMF, and the mixture was stirred at 80 ° C for 8 hours under an inert gas atmosphere. End of the reaction, the mixture was extracted, the organic layer was combined with ethyl acetate and water, washed with saturated NaCl solution, dried over anhydrous Na ₂ SO _4. Concentration under reduced pressure and the residue was purified eluted eluted elut elut _{^{1 H NMR (500MHz, CDCl 3}} ) δ: 8.91 (br, 1H, -NH), 8.46 (s, 1H, Ar-H), 8.02 (d, 1H, Ar-H), 7.98 (s, 1H, Ar -H), 7.87 (s, 1H, Ar-H), 7.85 (s, -NH, 1H), 7.78-7.80 (m, 1H, Ar-H), 7.69-7.70 (d, 1H, Ar-H) , 7.60-7.62 (m, 1H, Ar-H), 7.35 (d, 1H, Ar-H), 6.72-6.73 (m, 1H, Ar-H), 3.61 (s, 2H, -CH ₂ ), 2.60 (s, 3H, -CH ₃ ), 2.54 (b, 8H, -CH ₂ ), 2.33 (s, 3H, -CH ₃ ). ESI-MS m/z: [M+H] ⁺

Example 3. 3-((1H-pyrrolo[2,3-b]pyrazin-5-yl)ethynyl)-4-methyl-N-[4-((4-methylpyridazine-1) -yl)methyl)-3-trifluoromethylphenyl]benzamide hydrochloride

Weigh 3-((1H-pyrrolo[2,3-b]pyrazin-5-yl)ethynyl)-4-methyl-N-[4-((4-methylpyridazin-1-yl) Methyl)-3-trifluoromethylphenyl]benzamide (30 mg) was dissolved in 5 mL of methanol, and ethyl acetate aqueous solution was added dropwise to pH 3, and stirred at room temperature for 3 h. The volatiles were evaporated in vacuo and dried <RTI ID=0.0> ^{1 H NMR (300MHz, DMSO-} d 6) δ: 12.34 (s, 1H), 10.61 (s, 1H), 10.25 (b, 1H), 8.56 (s, 1H), 8.26 (s, 2H), 8.14 ( d,1H), 7.96-8.01 (m, 2H), 7.73 (d, 1H), 7.56 (d, 1H), 6.67-6.69 (m, 1H), 3.70 (s, 2H), 3.37 (m, 4H) , 2.89-3.06 (m, 4H), 2.77 (s, 3H), 2.61 (s, 3H). ¹ H NMR (300 MHz, DMSO-d ₆ + D ₂ O) δ: 10.62 (s, 1H), 8.57 (s, 1H), 8.22 (s, 2H), 8.07 (d, 1H), 7.93-7.99 (m) , 2H), 7.74 (d, 1H), 7.56 (d, 1H), 6.71 (d, 1H), 3.70 (s, 2H), 3.38-3.42 (m, 2H), 2.91-3.06 (m, 4H), 2.81 (s, 3H), 2.61 (s, 3H), 2.42 (m, 2H).

Example 4. 3-((1H-pyrazolo[3,4-b]pyrazin-5-yl)ethynyl)-4-methyl-N-[4-(4-methylpyridazine-1 -yl)methyl-3-trifluoromethylphenyl]benzamide

Step 1: Preparation of methyl 3-amino-6-bromopyrazine-2-carboxylate

3-Aminopyrazine-2-carboxylic acid methyl ester (4.59 g, 30 mmol), N-bromosuccinimide (5.34 g, 30 mmol) and acetonitrile (100 mL) were added to the mixture. The filter cake was washed with EtOAc (EtOAc). ¹ H-NMR (500 MHz, DMSO-d ₆ ) δ: 8.42 (s, 1H, s-H), 7.54 (s, 2H, -NH ₂ ), 3.86 (s, 3H, -CH ₃ ).

Step 2: Preparation of 3,6-dibromopyrazine-2-carboxylic acid methyl ester

Methyl 3-amino-6-bromopyrazine-2-carboxylate (4.62 g, 20 mmol), aqueous hydrobromic acid (48%, 24 ml), acetic acid (3.2 ml) was added to the mixture and cooled to -5 °C. A solution of liquid bromine (3.2 ml, 60 mmol) in acetic acid (5 ml) and an aqueous solution of sodium nitrite (4.8 g, 70 mmol) (20 ml) were added slowly. The reaction was stirred at -5 ° C for 1 h. The reaction was completed, washed with a saturated aqueous solution of sodium sulphate, and extracted with ethyl acetate and water, and washed with a saturated NaCI solution and dried over anhydrous sodium sulfate. The residue was purified with EtOAc EtOAc (EtOAc) ¹ H-NMR (500 MHz, DMSO-d ₆ ) δ: 8.92 (s, 1H, Ar-H), 3.95 (s, 3H, -CH ₃ ).

Step 3: Preparation of 3,6-dibromo-2-hydroxymethylpyrazine

Methyl 3,6-dibromopyrazine-2-carboxylate (5.0 g, 17 mmol), tetrahydrofuran (100 ml) was added to the reactor and cooled to -78 °C. Diisobutylaluminum hydride (34 ml, 34 mmol) was slowly added dropwise, and the mixture was reacted at -78 ° C for 2 h. The reaction was completed, and quenched by the addition of glacial acetic acid at -78 ° C. The residue was dissolved in 3N hydrochloric acid, water was added and extracted with dichloromethane, and washed with saturated NaCl solution, dried over anhydrous Na ₂ SO _4, the solvent was removed by distillation under reduced pressure. The residue was purified with EtOAc EtOAc (EtOAc) ¹ H-NMR (500 MHz, DMSO-d ₆ ) δ: 8.64 (s, 1H, s-H), 5.61 (t, 1H, OH), 3.95 (d, 2H, -CH ₂ ).

Step 4: Preparation of 3,6-dibromopyrazine-2-ylformaldehyde

3,6-Dibromo-2-hydroxymethylpyrazine (1.7 g, 6.3 mmol), manganese dioxide (5.5 g, 63 mmol), dichloromethane (60 ml) was added to the mixture, and stirred at room temperature for 30 h. After completion of the reaction, the mixture was filtered, evaporated, evaporated, evaporated ¹ H-NMR (500 MHz, DMSO-d ₆ ) δ: 10.01 (s, 1H, -CHO), 5.61 (s, 1H, Ar-H).

Step 5: Preparation of 5-bromo-1H-pyrazole [3,4-b]pyrazine

3,6-dibromopyrazin-2-ylcarboxaldehyde (600 mg, 2.26 mmol) and anhydrous tetrahydrofuran (60 ml) were added to the reactor, and hydrazine hydrate (1.12 g, 22.6 mol) in tetrahydrofuran (5 ml) was slowly added with stirring at room temperature. ) solution. Heat to 65 ° C for 6 h. The solvent was evaporated under reduced pressure. ^{1 H-NMR (300MHz, DMSO} -d 6) δ: 14.35 (s, 1H, NH), 8.74 (s, 1H, Ar-H), 8.45 (s, 1H, Ar-H).

Step 6: 3-((1H-pyrazol[3,4-b]pyrazin-5-yl)ethynyl)-4-methyl-N-(4-((4-methylpyridazine-1-) Preparation of methyl)methyl)-3-trifluoromethylphenyl)benzamide

5-Bromo-1H-pyrazole [3,4-b]pyrazine (59.4 g, 0.3 mmol), 3-ethynyl-4-methyl-N-(4-((4-A) was added to the reactor. Pyridazin-1-yl)methyl)-3-trifluoromethylphenyl)benzamide (126 mg, 0.3 mmol), bistriphenylphosphinepalladium dichloride (22 mg, 0.03 mmol), tricyclic Hexylphosphine (16 mg, 0.06 mmol), cuprous iodide (6 mg, 0.03 mmol), cesium carbonate (99 mg, 0.3 mmol) and 6 drops of N,N-diisopropylethylamine were stirred at 80 ° C overnight. The reaction ended, extracted with ethyl acetate and aqueous ammonia, and washed with saturated NaCl solution, dried over anhydrous Na ₂ SO _4, the solvent was removed by distillation under reduced pressure. The residue was purified with EtOAc (EtOAc) ^{1 H-NMR (500MHz, DMSO} -d 6) δ: 14.36 (b, 1H), 10.56 (s, 1H), 8.88 (s, 1H, Ar-H), 8.52 (s, 1H, Ar-H), 8.28(d,1H,Ar-H), 8.22 (d,1H,Ar-H), 8.08 (d,1H,Ar-H), 7.99 (m,1H,Ar-H), 7.71 (d,1H, Ar-H), 7.57 (d, 1H, Ar-H), 3.60 (s, 2H, PhCH ₂ ), 2.62 (b, 8H, 4×-CH ₂ ), 2.36 (b, 6H, 2×-CH _{3 )} ). ESI-MS m/z: [M+H] ⁺ = 534.3.

Example 5. 3-((7-Fluoro-5H-pyrrolo[2,3-b]pyrazin-2-yl)ethynyl)-4-methyl-N-[4-(4-methylindole) Pyrazin-1-yl)methyl-3-trifluoromethylphenyl]benzamide

Step 1: Preparation of 2-bromo-7-fluoro-5H-pyrrolo[2,3-b]pyrazine

In a dry 100 ml eggplant-shaped flask, 2-bromo-5H-pyrrolo[2,3-b]pyrazine (500 mg, 2.52 mmol), 1-chloromethyl-4-fluoro-1,4-diazide was added. Bicyclo [2.2.2.] octane difluoroborate (SelectFluor, 893mg, 2.52mmol), acetic acid (2mL) and acetonitrile (6mL), stirred at room temperature for 2h, heated to 80 ° C, after 48h reaction, cooled to room temperature ,concentrate. Addition of water and ethyl acetate, EtOAc (EtOAc)EtOAc.

Step 2: 3-((7-Fluoro-5H-pyrrolo[2,3-b]pyrazin-2-yl)ethynyl)-4-methyl-N-[4-(4-methylpyridazine Preparation of -1-yl)methyl-3-trifluoromethylphenyl]benzamide

Using the same procedure as in Step 4 of Example 4, 3-ethynyl-4-methyl-N-[4-(4-methylpyridazin-1-ylmethyl)-3-trifluoromethylphenyl The title compound was obtained as a yellow solid, m.p.p. ^{1 H-NMR (300MHz, DMSO} -d 6) δ: 10.56 (s, 1H), 8.65 (s, 1H), 8.28 (s, 1H), 8.22 (s, 1H), 8.07 (d, 1H), 8.06 (s, 1H), 7.98 (d, 1H), 7.72 (d, 1H), 7.57 (d, 1H), 3.58 (s, 2H), 2.62 (s, 3H), 2.40 (m, 8H), 2.17 ( s, 3H). ESI-MS m/z: [M+H] ⁺ = 551.3.

Example 6. 3-((7-Fluoro-5H-pyrrolo[2,3-b]pyrazin-2-yl)ethynyl)-4-methyl-N-[4-(4-methylindole) Pyrazin-1-yl)methyl-3-trifluoromethylphenyl]benzamide hydrochloride

Weigh 3-((7-fluoro-5H-pyrrolo[2,3-b]pyrazin-2-yl)ethynyl)-4-methyl-N-[4-(4-methylpyridazine- 1-yl)methyl-3-(trifluoromethyl)phenyl]benzamide (30 mg) was dissolved in 5 mL of methanol, and a solution of hydrogen chloride in ethyl acetate was added dropwise to pH 3 and stirred at room temperature for 3 h. After that, the volatile material was evaporated under reduced pressure and dried in vacuo tolulu ^{1 H-NMR (300MHz, DMSO} -d 6) δ: 12.27 (s, 1H), 10.61 (s, 1H), 10.16 (b, 1H), 8.64 (s, 1H), 8.28 (s, 1H), 8.25 (s, 1H), 8.14 (d, 1H), 8.06 (s, 1H), 7.98 (d, 1H), 7.74 (d, 1H), 7.56 (d, 1H), 3.77 (s, 2H), 3.39 ( d, 4H), 2.92-3.03 (m, 4H), 2.77 (s, 3H), 2.61 (s, 3H). ^{1 H-NMR (300MHz, DMSO} -d 6 + D 2 O) δ: 10.60 (s, 1H), 8.65 (s, 1H), 8.26 (s, 1H), 8.23 (s, 1H), 8.11 (d, 1H), 8.02 (s, 1H), 7.97 (d, 1H), 7.74 (d, 1H), 7.56 (d, 1H), 3.72 (s, 2H), 3.39-3.42 (m, 4H), 2.93-3.07 (m, 4H), 2.80 (s, 3H), 2.62 (s, 3H).

Example 7. 3-((1H-Imidazo[4,5-b]pyrazin-5-yl)ethynyl)-4-methyl-N-[4-(4-methylpyridazine-1- -methyl-3-(trifluoromethyl)phenyl]benzamide

Step 1: Preparation of 2,3-diamino-5-bromopyrazine

2-Amino-3,5-dibromopyrazine (10 g, 0.04 mol) was added to aqueous ammonia (50 ml), and the mixture was heated to 110 ° C for 16 hours, then extracted with ethyl acetate and H ₂ O. The column was separated to give the title compound. ¹ H NMR (300 MHz, CDCl ₃ ) δ: 7.60 (s, 1H), 4.37 (b, 2H), 4.19 (b, 2H).

Step 2: Preparation of 5-bromo-1H-imidazo[4,5-b]pyrazine

2,3-Diamino-5-bromopyrazine (1 g, 0.005 mol) was added to diethoxymethyl acetate (5 ml), protected with N ₂ and heated to 120-130 ° C for 4 hours to give brown clarification after the solution was added H ₂ O and extracted with ethyl acetate, the organic layers were combined, concentrated and separated by column to give the title compound. ¹ H NMR (300 MHz, CDCl ₃ ) δ: 8.45 (s, 1H), 8.32 (s, 1H). ESI-MS m/z: [M+H] ⁺ = 199.0.

Step 3: 3-((1H-Imidazo[4,5-b]pyrazin-5-yl)ethynyl)-4-methyl-N-[4-(4-methylpyridazin-1-yl) Preparation of methyl-3-(trifluoromethyl)phenyl]benzamide

Using the same procedure as in Step 4 of Example 4, 3-ethynyl-4-methyl-N-[4-(4-methylpyridazin-1-ylmethyl)-3-trifluoromethylphenyl Benzylguanamine and 5-bromo-1H-imidazo[4,5-b]pyrazine were used as starting materials to prepare the target compound. ^{1 H NMR (300MHz, DMSO-} d 6) δ: 10.59 (s, 1H), 9.50 (b, 1H), 8.89 (s, 1H), 8.75 (s, 1H), 8.22-8.27 (m, 2H), 8.13 (d, 1H), 7.95-7.99 (m, 1H), 7.72 (d, 1H), 7.57 (d, 1H), 3.67 (s, 2H), 3.34 (b, 2H), 2.89-3.05 (m, 4H), 2.80 (s, 3H), 2.61 (s, 3H), 2.33-2.40 (m, 2H). ESI-MS m / z: [ M + H] + = 534.2.

Example 8. 3-((1H-pyrrolo[2,3-b]pyrazin-5-yl)ethynyl)-4-methyl-N-(1-(4-methylpyridazine-1- Base-2,3-dihydro-1 H -indol-5-yl)benzamide

Step 1: Preparation of 3-ethynyl-4-methyl-N-(1-oxo-2,3-dihydro-1 H -indol-5-yl)benzamide

3-ethynyl-4-methylbenzoic acid (5.0 g, 17 mmol), tetrahydrofuran (100 mL) and 2 drops of DMF were added to the reactor, and hydrazine chloride (1.9 mL, 26 mmol) was slowly added dropwise at 0 ° C, and The reaction was continued at 0 ° C for 2 h. After completion of the reaction, the mixture was concentrated under reduced pressure to give 3-ethynyl-4-methylbenzhydrin chloride. The obtained chlorobenzene was dissolved in 20 mL of THF and added dropwise to 30 mL of 2.5 g of 5-amino-1-oxo- 2,3-Dihydro-1 H -indole in tetrahydrofuran solution, 7 mL of Et ₃ N was added dropwise, and reacted at room temperature for 1 h. After the reaction was completed, the solvent was evaporated under reduced pressure, extracted with ethyl acetate and water, and saturated NaCI solution. washed, dried over anhydrous Na ₂ SO _4, filtered, rotary evaporation, purified by column chromatography to give the title compound. ESI-MS m/z: [M+H] ⁺ = 290.3.

Step 2: 3-ethynyl-4-methyl-N-(1-(4-methylpyridazin-1-yl)-2,3-dihydro-1 H -indol-5-yl)-benzene Guanamine

The step 1 was added to the reactor as 3-ethynyl-4-methyl-N-(1-oxo-2,3-dihydro-1 H -indol-5-yl)benzamide (2.9 g). , 10 mmol), methylpyrazine (1 g, 10 mmol), dichloroethane (100 mL), tetraisopropyl titanate (2.9 g, 10 mmol), and reacted for 12 h at room temperature. extracted with ethyl acetate and water, and washed with saturated NaCl solution, dried over anhydrous Na ₂ SO _4, filtered, rotary evaporation, purified by column chromatography to give the title compound. ESI-MS m/z: [M+H] ⁺ = 374.3.

Step 3: 3-((1 H -pyrrolo[2,3-b]pyrazin-5-yl)ethynyl)-4-methyl-N-(1-(4-methylpyridazine-1- Preparation of bis(2,3-dihydro-1 H -indol-5-yl)benzamide

5-Bromo-1 H -pyrrolo[2,3-b]pyrazine (59.4 g, 0.3 mmol) was added to the reactor, and the product obtained in Step 2 was 3-ethynyl-4-methyl-N-(1- (4-methylpyridazin-1-yl)-2,3-dihydro-1 H -indol-5-yl)-benzamide (112 mg, 0.3 mmol), bistriphenylphosphine palladium dichloride (22 mg, 0.03 mmol), tricyclohexylphosphine (16 mg, 0.06 mmol), cuprous iodide (6 mg, 0.03 mmol), cesium carbonate (99 mg, 0.3 mmol), 6 drops of N,N-diisopropylethylamine The reaction was carried out at 80 ° C for 12 h. After the reaction was completed, ethyl acetate and aqueous ammonia were added, and the mixture was washed with a saturated NaCI solution, dried over anhydrous Na ₂ SO ₄ , filtered, and dried. ^{1 H NMR (500MHz, DMSO-} d 6) δ: 12.30 (s, 1H, NH), 10.22 (s, 1H, NH), 8.55 (s, 1H, Ar-H), 8.21 (s, 1H, Ar- H), 7.99 (s, 1H, Ar-H), 7.92 (d, 1H, Ar-H), 7.70 (m, 1H, Ar-H), 7.54 (m, 2H, Ar-H), 7.22 (d) , 1H, Ar-H), 6.68 (d, 1H, Ar-H), 4.23 (b, 1H, -CH), 2.87 (b, 2H, -CH ₂ ), 2.77 (b, 2H, -CH ₂ ) , 2.59 (s, 3H, -CH ₃ ), 2.35 (b, 8H, 4 × -CH ₂ ), 2.15 (s, 3H, -CH ₃ ). ESI-MS m/z: [M+H] ⁺ = 491.3.

Example 9. 3-((3-Fluoro-1 H -pyrrolo[2,3-b]pyrazin-5-yl)ethynyl)-4-methyl-N-(1-(4-methyl) Pyridazin-1-yl)-2,3-dihydro-1 H -indol-5-yl)benzamide

3-Fluoro-5-bromo-1 H -pyrrolo[2,3-b]pyrazine (65 mg, 0.3 mmol) obtained in Step 1 of Example 5 was added to the reactor, and the product obtained in Step 2 of Example 8 was 3- Ethynyl-4-methyl-N-(1-(4-methylpyridazin-1-yl)-2,3-dihydro-1 H -indol-5-yl)-benzamide (112 mg, 0.3 mmol), bistriphenylphosphine palladium dichloride (22 mg, 0.03 mmol), tricyclohexylphosphine (16 mg, 0.06 mmol), cuprous iodide (6 mg, 0.03 mmol), cesium carbonate (99 mg, 0.3 mmol) 6 drops of N,N-diisopropylethylamine were reacted at 80 ° C for 12 h. After completion of the reaction, ethyl acetate and aqueous ammonia were added for extraction, and washed with a saturated NaCI solution, dried over anhydrous Na ₂ SO ₄ , filtered, concentrated, and purified by column chromatography. ^{1 H NMR (500MHz, DMSO-} d 6) δ: 10.22 (s, 1H, NH), 8.64 (s, 1H, Ar-H), 8.23 (s, 1H, Ar-H), 8.04 (s, 1H, Ar-H), 7.94 (d, 1H, Ar-H), 7.70 (s, 1H, Ar-H), 7.54 (m, 2H, Ar-H), 7.22 (d, 1H, Ar-H), 4.24 (t,1H,-CH), 2.89 (m, 2H, -CH ₂ ), 2.77 (b, 2H, -CH ₂ ), 2.60 (s, 3H, -CH ₃ ), 2.37 (b, 8H, 4×) -CH ₂ ), 2.17 (s, 3H, -CH ₃ ). ESI-MS m/z: [M+H] ⁺ = 509.2.

Example 10. 3-(( 1H -pyrrolo[2,3-b]pyrazin-5-yl)ethynyl)-4-methyl-N-(4-((3-methyl-3, 8-Diazabicyclo[3.2.1]octane-8-yl)methyl)-3-(trifluoromethyl)phenyl)benzamide

Step 1: Preparation of bromomethyl-2-trifluoromethyl-4-nitrobenzene

To the reactor were added 2-trifluoromethyl-4-nitrotoluene (10 g, 50 mmol), NBS (8.9 g, 50 mmol), carbon tetrachloride (50 mL), azobisisobutyronitrile (AIBN, 164 mg, 1 mmol), reflux reaction for 12 h. After the reaction was completed, it was filtered, and the filtrate was used directly for the next reaction.

Step 2: 1-((3-tert-Butoxycarbonyl-3,8-diazabicyclo[3.2.1]octane-8-yl)methyl)-2-trifluoromethyl-4-nitro Preparation of benzene

3-tert-Butyloxycarbonyl-3,8-diazabicyclo[3.2.1]octane (10.6 g, 50 mmol), triethylamine (14 mL, 100 mmol) was added to the reaction filtrate of Step 1, The reaction was carried out for 2 h at room temperature. After completion of the reaction, the solvent was distilled off under reduced pressure, extracted with ethyl acetate and water, and washed with saturated NaCl solution, dried over anhydrous Na ₂ SO _4, filtered, concentrated and purified by column chromatography to give the title compound. ESI-MS m/z: [M+H] ⁺ = 416.2.

Step 3: 1-((3-tert-Butoxycarbonyl-3,8-diazabicyclo[3.2.1]octane-8-yl)methyl)-2-trifluoromethyl-4-aminobenzene Preparation

Step 2 was added to the reactor to give 1-((3-tert-butoxycarbonyl-3,8-diazabicyclo[3.2.1]octane-8-yl)methyl)-2-trifluoromethyl 4-Nitrobenzene (8 g, 19.2 mmol), amine chloride (10 g, 190 mmol), iron powder (2 g), ethanol (50 mL), refluxed for 2 h. After completion of the reaction, filtration, solvent was distilled off under reduced pressure, extracted with ethyl acetate and water, and washed with saturated NaCl solution, dried over anhydrous Na ₂ SO _4, filtered, rotary evaporation, purified by column chromatography to give the title compound. ESI-MS m/z: [M+H] ⁺ = 386.2.

Step 4: 3-ethynyl-4-methyl-N-(4-((3-tert-butoxycarbonyl-3,8-diazabicyclo[3.2.1]octane-8-yl)methyl) -3-(trifluoromethyl) Preparation of phenyl)benzamide

3-ethynyl-4-methylbenzoic acid (5.0 g, 17 mmol), tetrahydrofuran (100 mL) and 2 drops of DMF were added to the reactor, and hydrazine chloride (1.9 mL, 26 mmol) was slowly added dropwise at 0 ° C, and The reaction was continued at 0 ° C for 2 h. After completion of the reaction, the mixture was concentrated under reduced pressure to give chlorobenzene, and the obtained chlorobenzene was dissolved in 20 mL of THF, and added dropwise to 30 mL of 6.5 g of the product obtained in Step 3 1-((3-tert-butoxycarbonyl-3,8- diazabicyclo [3.2.1] octan-8-yl) methyl) -2-tetrahydrofuran-trifluoromethyl-4-aminobenzene added dropwise _{Et 3 N (7mL, 51mol)} , at room IH reaction temperature, after completion of the reaction, the solvent was distilled off under reduced pressure, extracted with ethyl acetate and water, and washed with saturated NaCl solution, dried over anhydrous Na ₂ SO _4, filtered, concentrated and purified by column chromatography to give the title compound. ESI-MS m/z: [M+H] ⁺ = 528.3.

Step 5: 3-ethynyl-4-methyl-N-(4-((3-methyl-3,8-diazabicyclo[3.2.1]octane-8-yl)methyl)-3) -(trifluoromethyl)phenyl)benzamide

Step 4 was added to the reactor to give 3-ethynyl-4-methyl-N-(4-((3-tert-butoxycarbonyl-3,8-diazabicyclo[3.2.1]octane-8) -Methyl)-3-(trifluoromethyl)phenyl)benzamide (5 g, 9.4 mmol), 40% aqueous formaldehyde (10 mL), formic acid (50 mL). After completion of the reaction, ethyl acetate and extracted with water, and washed with saturated NaCl solution, dried over anhydrous Na ₂ SO _4, filtered, rotary evaporation, purified by column chromatography to give the title compound. ESI-MS m/z: [M+H] ⁺ = 442.3.

Step 6: 3-(( 1H -pyrrolo[2,3-b]pyrazin-5-yl)ethynyl)-4-methyl-N-(4-((3-methyl-3,8) -diazabicyclo[3.2.1]octane-8-yl)methyl)-3-(trifluoromethyl)phenyl)benzamide

5-Bromo-1 H -pyrrolo[2,3-b]pyrazine (59.4 mg, 0.3 mmol) was added to the reactor, and the product obtained in Step 5 was 3-ethynyl-4-methyl-N-(4- ((3-Methyl-3,8-diazabicyclo[3.2.1]octane-8-yl)methyl)-3-(trifluoromethyl)-phenyl)benzamide (112 mg, 0.3 mmol), bistriphenylphosphine palladium dichloride (22 mg, 0.03 mmol), tricyclohexylphosphine (16 mg, 0.06 mmol), cuprous iodide (6 mg, 0.03 mmol), cesium carbonate (99 mg, 0.3 mmol) 6 drops of N,N-diisopropylethylamine were reacted at 80 ° C for 12 h. After completion of the reaction, ethyl acetate and aqueous ammonia were added for extraction, and washed with a saturated NaCI solution, dried over anhydrous Na ₂ SO ₄ , filtered, ^{1 H NMR (500MHz, DMSO-} d 6) δ: 12.31 (s, 1H, NH), 10.54 (s, 1H, NH), 8.55 (s, 1H, Ar-H), 8.24 (b, 1H, Ar- H), 8.20 (b, 1H, Ar-H), 8.06 (d, 1H, Ar-H) 7.99 (d, 1H, Ar-H), 7.95 (d, 1H, Ar-H), 7.67 (d, 1H, Ar-H), 7.55 (d, 1H, Ar-H), 7.68 (d, H, Ar-H), 3.52 (s, 2H, PhCH ₂ ), 2.98 (b, 2H, 2×-CH) , 2.61(s,3H,-CH ₃ ), 2.46(b,2H,-CH2), 2.25(b,2H,-CH2), 2.15(s,3H,-CH ₃ ),1.82(b,2H,- CH ₂ ), 1.70 (b, 2H, -CH ₂ ). ESI-MS m/z: [M+H] ⁺ = 559.3.

Example 11. 3-((3-Fluoro-1 H -pyrrolo[2,3-b]pyrazin-5-yl)ethynyl)-4-methyl-N-(4-((3-) 3-,8-diazabicyclo[3.2.1]octane-8-yl)methyl)-3-(trifluoromethyl)phenyl)benzamide

5-Bromo-3-fluoro-1 H -pyrrolo[2,3-b]pyrazine (65 mg, 0.3 mmol) was added to the reactor, and the product obtained in Step 10 of Example 10 was 3-ethynyl-4-methyl. -N-(4-((3-methyl-3,8-diazabicyclo[3.2.1]octane-8-yl)methyl)-3-(trifluoromethyl)phenyl)benzene) Indoleamine (112 mg, 0.3 mmol), bistriphenylphosphine palladium dichloride (22 mg, 0.03 mmol), tricyclohexylphosphine (16 mg, 0.06 mmol), cuprous iodide (6 mg, 0.03 mmol), cesium carbonate ( 99 mg, 0.3 mmol), 6 drops of N,N-diisopropylethylamine, and reacted at 80 ° C for 12 h. After completion of the reaction, ethyl acetate and aqueous ammonia were added for extraction, and washed with a saturated NaCI solution, dried over anhydrous Na ₂ SO ₄ , filtered, ^{1 H NMR (500MHz, DMSO-} d 6) δ: 10.54 (s, 1H, NH), 8.64 (s, 1H, Ar-H), 8.27 (b, 1H, Ar-H), 8.20 (b, 1H, Ar-H), 8.07 (b, 1H, Ar-H), 8.05 (b, 1H, Ar-H), 7.96 (m, 1H, Ar-H), 7.67 (d, 1H, Ar-H), 7.55 (d, 1H, Ar-H), 3.52 (s, 2H, PhCH ₂ ), 2.98 (b, 2H, 2 × -CH), 2.61 (s, 3H, -CH ₃ ), 2.46 (b, 2H, - CH ₂ ), 2.25 (b, 2H, -CH ₂ ), 2.15 (s, 3H, -CH ₃ ), 1.82 (b, 2H, -CH ₂ ), 1.70 (b, 2H, -CH ₂ ). ESI-MS m/z: [M+H] ⁺ = 577.3.

Experimental Example 1 Evaluation of in vitro cell viability of the compound of the present invention

This experiment tested the inhibitory activity of the compounds of the invention on leukemia cells using imatinib and Ponatinib as controls. Imatinib was prepared according to the method described in Chinese Patent No. CN1043531C and identified by hydrogen spectroscopy and mass spectrometry. Ponatinib was purchased from Shanghai Xinkuo Chemical Technology Co., Ltd., and Ponatinib hydrochloride was prepared according to the method of Example 3 of the present invention and passed through hydrogen spectroscopy. Mass spectrometry identification.

1. The experimental materials used in this experimental example are as follows:

1.1 Compound

The compounds of the above representative examples 2, 4, 5, 8, 9, 10 and imatinib and Ponatinib were used. Each compound was dissolved in DMSO to 1OmM later, it was diluted to 50 μ M with complete medium, and then diluted with 0.1% DMSO in complete medium to 10 μ M, containing serially diluted 10-fold with a total of 10 concentrations.

1.2 cells

K562 leukemia cells: purchased from ATCC, USA; imatinib-tolerant K562 leukemia cells: purchased from ATCC, USA.

1.3 Reagents

Dimethyl sulfoxide (DMSO), purchased from Sigma, USA; CellTiter-Glo ^® Luminescent Cell Viability Assay Kit, purchased from Promega, USA; IMEM medium (IMEM medium), Purchased from Gibco, USA; penicillin/streptomycin (Pen/Strep), purchased from Gibco, USA; Fatal bovine serun (FBS), purchased from Gibco, USA; 25% containing EDTA trypsin (0.25% Trypsin) - EDTA), purchased from Gibco, USA; 10cm cell culture dish, purchased from Corning, USA; 50mL centrifuge tube, purchased from Corning, USA; 384-well flat-bottomed whiteboard (384 Well Flat Clear Bottom White), purchased from Corning, USA; Phosphate Buffered Saline (PBS), prepared weekly.

1.4 Instrument

Autofocus fluorescent multi-function microplate reader (PHERAstar Plus), purchased from BMG Labtech, Germany.

2. The experimental methods used in this experimental example are as follows: 1) Collect log phase cells, adjust the cell suspension concentration to 1 × 10 ⁵ /ml, add 40 μL of cell suspension per well to the 384-well plate, and the number of cells per well is 4. ×10 ³ /well, the edge wells were filled with sterile PBS; 2) 10 μL of the above concentration gradient of the compound of the invention was added. Each compound was repeated three times for each concentration. Add 10 μL of the same concentration of DMSO to the blank control; 3) Incubate the cells in a 37 ° C / 5% CO ₂ incubator; 4) Add 30 μL of the luminescence cell viability assay reagent mixture after 72 h of dosing; 5) 37 ° C / 5% CO ₂ Incubate for 10 min in the incubator; determine the chemiluminescence value on the PHERAstar microplate after centrifugation at low speed; 6) Cell Viability = (RLU _sample / RLU _negative ) × 100%, wherein the RLU _sample is the dosing hole RLU (relative light unit) value, RLU _negative is the unfilled RLU value (ie, the same concentration of DMSO-treated cell control), and the IC ₅₀ is processed using the Graphpad Prism 4.0 data processing software four-parameter logic fitting module. The IC ₅₀ value indicates the concentration of the compound corresponding to 50% cell growth inhibition of the compound compared to the compound-free treated group. The IC ₅₀ results are shown in Table 1.

As can be seen from the above experimental results, the compound of the present invention has a strong inhibitory activity against the leukemia cells with an IC ₅₀ value at the nM level. For non-resistant K562 leukemia cells, the compounds of the invention are comparable to Ponatinib and far superior to imatinib. For imatinib-tolerant K562 leukemia cells, the compounds of the invention exhibited better inhibitory activity than Ponatinib.

Experimental Example 2 Evaluation of ABL1 (T315I) Tyrosine Kinase Activity

This experiment tested the inhibition of ABL (T315I) kinase activity by the compounds prepared in the examples of the present invention, using imatinib as a control. Imatinib was prepared by the method described in Chinese Patent No. CN1043531C and identified by hydrogen spectroscopy and mass spectrometry.

ABL (T315I) tyrosine kinase activity was tested using a commercially available human ABL T315I mutant enzyme (Human ABL1 (T315I), active, Cat # 14-522, Millipore, USA). Kinase activity assays were performed according to the manufacturer's instructions. The Peptide substrate was Abltide (EAIYAAPFAKKK) and was purchased from Millipore Corporation of the United States. Ion exchange chromatography paper (P81) was purchased from Whatman Company of the United Kingdom. [γ- ³³ P] ATP was purchased from Perkin Elmer.

The compound of the present invention and imatinib were serially diluted 3 times from 1 μM, respectively, for a total of 10 concentrations (50.8 pM, 152.0 pM, 457.0 pM, 1.37 nM, 4.12 nM, 12.3 nM, 37.0 nM, 111.0 nM, 333.0 nM and 1.0). μM). 5.0 μM Abltide was added to each well and then human T315I mutant enzyme was added. [γ- ³³ P]ATP was added at room temperature to a final concentration of 1.0 μM, and the reaction was carried out for 120 minutes. A 20 μl aliquot was transferred to a P81 ion exchange chromatography paper. The chromatography paper was then washed thoroughly 3 times with a 0.75% phosphoric acid solution and once with acetone. Finally, a gamma- ^33P radioactivity assay was performed. The experimental results are shown in Table 2.

The above experimental results show that the compound of the present invention has a significantly better IC ₅₀ for the T315I mutant enzyme than imatinib, and has potent ability to inhibit the T315I mutant enzyme.

From the experimental results of the present invention, it can be concluded that the compound of the present invention not only has a very good effect on leukemia cells without mutation, but also can significantly inhibit the T315I mutant enzyme, and thus is a broad-spectrum BCR-ABL inhibitor. For patients with cancer resistant or resistant to tyrosine kinase inhibitor (TKI) treatment, such as chronic myeloid leukemia (CML) chronic, blast, accelerated, and Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia Patients with acute lymphoblastic leukemia should have good prospects.

Experimental Example 3 Drug Metabolism Experiment

1. The experimental materials used in this experimental example are as follows:

1.1 Compound

The compounds of the invention prepared using the above representative examples 3 and 6 were used. Among them, the oral drug formulation is dissolved in physiological saline to prepare a suspension of 3 mg/ml; the drug formulation in the tail vein is a mixed solution of DMSO: polyoxyethylene castor oil: physiological saline = 1:30:69, and is made into 2.5. Mg/ml solution.

1.2 Animals

Male Sprague-Dawley rats, 3 in each group, weighing 150-250 g, were provided by Shanghai Xipuer-Beikai Experimental Animal Co., Ltd. The rats in the test group were given an environmental adaptation period of 2 to 4 days before the experiment, and were fasted for 8-12 hours before administration, and given water for 2 hours after administration, and fed after 4 hours.

1.3 Reagents

Methanol (chromatographically pure): produced by Spectrum; acetonitrile (chromatographically pure): produced by Spectrum; the remaining reagents are commercially available analytical grades.

1.4 Instrument

American AB company API 4000 triple quadrupole liquid-mass instrument with electrospray ion source (ESI), LC-20AD double pump; SIL-20AC autosampler; CTO-20AC column thermostat; DGU-20A3R Degasser; Analyst QS A01.01 chromatography workstation; Milli-Q ultrapure water (Millipore Inc); Qilinbeier Vortex-5 oscillator; HITACHI CF16R X II benchtop high speed refrigerated centrifuge.

2. The experimental methods used in this experimental example are as follows: 1) SD rats were fasted but free to drink water for 12 hours, taking 0 time blank plasma; 2) taking 3 rats in step 1), intragastric (Intragastric Administration, IG) The compound of Example 3 was administered at 15 mg/kg; 3 rats in step 1) were administered, and the compound of Example 3 was administered 3 mg/kg in the intravenous vein (IV); the rats in step 1) were taken 3 Only 15 mg/kg of the compound of Example 6 was administered by gavage; 3 rats in the step 1) were administered, and the compound of Example 6 was administered 5 mg/kg in the tail vein (IV); 3) 10 min after the gavage, At 30min, 1h, 2h, 4h, 6h, 8h, 10h, 24h, the fundus venous plexus was continuously taken from the EP tube with heparin. After centrifugation at 8000rpm/min for 5min, the upper layer of plasma was taken and frozen at -20 °C. LC-MS/MS analysis; 4) According to the plasma concentration-time data obtained in step 3), the pharmacokinetic parameters were calculated using WinNonlin software, see Table 3; 5) 5 min, 15 min after tail vein injection, At 30min, 1h, 2h, 4h, 8h, and 24h, the fundus venous plexus was continuously taken from the EP tube with heparin. After centrifugation at 8000 rpm/min for 5 min, the upper layer of plasma was taken and frozen at -20 °C until LC-MS/MS analysis. 6) According to the plasma concentration-time data obtained in step 5), the pharmacokinetic parameters were calculated using WinNonlin software, as shown in Table 3.

Wei-Sheng Huang et al (Discovery of 3-[2-(Imidazo[1,2-b]pyridazin-3-yl)ethynyl]-4-methyl-N-{4-[(4-methyl-piperazin-1- Yl)-methyl]-3-(trifluoromethyl)phenyl}benzamide(AP24534),a Potent,Orally Active Pan-Inhibitor of Breakpoint Cluster Region-Abelson(BCR-ABL)Kinase Including the T315I Gatekeeper Mutant,J.Med.Chem.2010 (53) 4701-4719) reported that Ponatinib 15 mg/kg was orally administered, peak concentration ( _Cmax ) was 204.8 ng/ml, and oral bioavailability (F) was 18.2%. Thus, the compounds of the present invention have a higher _Cmax than Ponatinib and have a higher oral bioavailability.

Experimental Example 4 Subacute Toxicity Experiment

1. The experimental materials used in this experimental example are as follows:

1.1 Compound

Using the compound of the present invention prepared in the above Example 6, using Ponatinib hydrochloride as a control, the oral pharmaceutical formulation was: physiological saline dissolved.

1.2 Animals

Male mice, 24, weighing 18±2 g, were purchased from Qinglong Mountain Animal Breeding Farm in Jiangning District, Nanjing. The mice were given an environmental adaptation period of 2 to 4 days before the experiment, and were fasted for 8 to 12 hours before administration, and given water for 2 hours after administration, and fed after 4 hours.

1.3 Reagents

Ultrapure water: Milli-Q ultrapure water (Millipore Inc) instrument made.

1.4 Instrument

Milli-Q Ultra Water Purifier (Millipore Inc); LT501 Electronic Balance.

2. The experimental methods used in this experimental example are as follows: 1) 18 mice were randomly divided into 3 groups: blank control group, Ponatinib hydrochloride drug group, and Example 6 drug group, 6 rats in each group; The mice are administered orally by the method of administration: day 0-2, dose 10 Mg/kg; on the 3rd to 5th day, the dose was 20mg/kg; on the 6th to 9th day, the dose was 40mg/kg; 3) the weight of the mice was recorded, and the average body weight and relative body weight change (RCBW%) were calculated. The relative weight changes reflect the animal's body weight affected by the drug, the results are shown in Table 4 and Table 5. Calculation formula: RCBW% = (weight of one day of administration - body weight of the first day of administration) / body weight of the first day of administration × 100%.

The above subacute toxicity test results showed that the Ponatinib group had a significant effect on the body weight of the mice compared with the blank control group, and the P value was <0.05, while the compound of the present invention had no effect on the body weight of the mice and showed extremely low toxicity. Thus, the medicament of the present invention is significantly superior to Ponatinib in terms of toxic side effects.

While the invention has been described hereinabove, it will be understood by those skilled in the art that various modifications and changes of the invention may be made without departing from the spirit and scope of the invention. The scope of the invention is not limited to the detailed description set forth above, but is intended to be within the scope of the claims.

Claims (11)

A compound or a pharmaceutically acceptable salt thereof having the structure of Formula I: Wherein X is selected from N or C(R ₅ ), wherein R _{5 is} selected from the group consisting of hydrogen, halogen, C _1-6 alkyl, halo C _1-6 alkyl; Y is selected from N or C(R ₆ ), wherein R _{6 is} selected from the group consisting of hydrogen, halogen, C _1-6 alkyl, halo C _1-6 alkyl; R _{1 is} selected from hydrogen, C _1-6 alkyl, C _1-6 alkoxy, halo C _{1- 6} alkyl, halo C _1-6 alkoxy, -OH, -NH ₂ , halogen or CN; R _{2 is} selected from hydrogen, C _1-6 alkyl, C _1-6 alkoxy, halogen C _{1 1-6} alkyl, halo C _1-6 alkoxy, -OH, -NH _2, halogen, or CN; W is selected from C _1-6 alkylene, -C (O) -, or 4 and R ₄ form -8 membered cycloalkyl or absent; R _{3 is} selected from substituted or unsubstituted five-, six-, seven- or eight-membered nitrogen-containing heterocyclic groups; and R _{4 is} selected from hydrogen, C _1-6 alkyl, Halogenated C _1-6 alkyl, or together with W, form a 4-8 membered cycloalkyl; provided that the following compound is excluded: 3-((1H-pyrrolo[2,3-b]pyrazin-5-yl) Ethyl)-4-methyl-N-[4-((4-methylpyridazin-1-yl)methyl)-3-trifluoromethylphenyl]benzamide. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is a compound of the formula Ia: Wherein X is selected from N or C(R ₅ ), wherein R _{5 is} selected from the group consisting of hydrogen, halogen, C _1-6 alkyl, halo C _1-6 alkyl; Y is selected from N or C(R ₆ ), wherein R _{6 is} selected from the group consisting of hydrogen, halogen, C _1-6 alkyl, halo C _1-6 alkyl; R _{1 is} selected from hydrogen, C _1-6 alkyl, C _1-6 alkoxy, halo C _{1- 6} alkyl, halo C _1-6 alkoxy, -OH, -NH ₂ , halogen or CN; R _{2 is} selected from hydrogen, C _1-6 alkyl, C _1-6 alkoxy, halogen C _{1 -6} alkyl, halo C _1-6 alkoxy, -OH, -NH ₂ , halogen or CN; R _{3 is} selected from substituted or unsubstituted five-, six-, seven-, or eight-membered nitrogen-containing heterocycles W is selected from C _1-6 alkylene, -C(O)- or absent; and R _{4 is} selected from hydrogen, C _1-6 alkyl or halo C _1-6 alkyl; The following compound: 3-((1H-pyrrolo[2,3-b]pyrazin-5-yl)ethynyl)-4-methyl-N-[4-((4-methylpyridazine-1-) Methyl)-3-trifluoromethylphenyl]benzamide. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is a compound of the formula Ib: Wherein X is selected from N or C(R ₅ ), wherein R _{5 is} selected from the group consisting of hydrogen, halogen, C _1-6 alkyl, halo C _1-6 alkyl; Y is selected from N or C(R ₆ ), wherein R _{6 is} selected from the group consisting of hydrogen, halogen, C _1-6 alkyl, halo C _1-6 alkyl; R _{1 is} selected from hydrogen, C _1-6 alkyl, C _1-6 alkoxy, halo C _{1- 6} alkyl, halo C _1-6 alkoxy, -OH, -NH ₂ , halogen or CN; R _{2 is} selected from hydrogen, C _1-6 alkyl, C _1-6 alkoxy, halogen C _{1 -6} alkyl, halo C _1-6 alkoxy, -OH, -NH ₂ , halogen or CN; R _{3 is} selected from substituted or unsubstituted five-, six-, seven- or eight-membered nitrogen-containing heterocycles W is selected from C _1-6 alkylene, -C(O)- or absent; and R _{4 is} selected from hydrogen, C _1-6 alkyl, halo C _1-6 alkyl. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is a compound of the formula Ic: Wherein X is selected from N or C(R ₅ ), wherein R _{5 is} selected from the group consisting of hydrogen, halogen, C _1-6 alkyl, halo C _1-6 alkyl; Y is selected from N or C(R ₆ ), wherein R _{6 is} selected from the group consisting of hydrogen, halogen, C _1-6 alkyl, halo C _1-6 alkyl; R _{1 is} selected from hydrogen, C _1-6 alkyl, C _1-6 alkoxy, halo C _{1- 6} alkyl, halo C _1-6 alkoxy, -OH, -NH ₂ , halogen or CN; R _{2 is} selected from hydrogen, C _1-6 alkyl, C _1-6 alkoxy, halogen C _{1 -6} alkyl, halo C _1-6 alkoxy, -OH, -NH ₂ , halogen or CN; and R _{3 is} selected from substituted or unsubstituted five-, six-, seven- or eight-membered nitrogen-containing Ring base. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is a compound of the formula Id: Wherein X is selected from N or C(R ₅ ), wherein R _{5 is} selected from the group consisting of hydrogen, halogen, C _1-6 alkyl, halo C _1-6 alkyl; Y is selected from N or C(R ₆ ), wherein R _{6 is} selected from the group consisting of hydrogen, halogen, C _1-6 alkyl, halo C _1-6 alkyl; R _{1 is} selected from hydrogen, C _1-6 alkyl, C _1-6 alkoxy, halo C _{1- 6} alkyl, halo C _1-6 alkoxy, -OH, -NH ₂ , halogen or CN; R _{2 is} selected from hydrogen, C _1-6 alkyl, C _1-6 alkoxy, halogen C _{1 -6} alkyl, halo C _1-6 alkoxy, -OH, -NH ₂ , halogen or CN; and R _{3 is} selected from substituted or unsubstituted five-, six-, seven- or eight-membered nitrogen-containing Ring base. The compound of any one of claims 1 to 5, wherein R _{1 is} selected from the group consisting of hydrogen, methyl, trifluoromethyl, methoxy, ethoxy, propoxy or iso Propyloxy; and R _{2 is} selected from hydrogen, methyl, ethyl, propyl or isopropyl. The compound of any one of claims 1 to 5, wherein W is selected from C _1-3 alkylene, -C(O)-, or together with R ₄ constitutes cyclopentane, or a pharmaceutically acceptable salt thereof Base or non-existent; R _{3 is} selected from pyridazinyl, acridin-4-yl, 3,8-diazabicyclo[3.2.1]octyl, the pyridazinyl, the acridin-4-yl and The 3,8-diazabicyclo[3.2.1]octyl group may be substituted by one or more C _1-6 alkyl groups, C _1-6 alkoxy groups, preferably R _{3 is} selected from 4-methyl fluorene. Pyrazin-1-yl, acridin-4-yl, 1-methylacridin-4-yl, 3,8-diazabicyclo[3.2.1]octane-8-yl, 1-methyl-3 , 8-diazabicyclo[3.2.1]octane-8-yl; R _{4 is} selected from methyl, trifluoromethyl, or together with W constitutes a cyclopentyl group; R _{5 is} selected from hydrogen, halogen, preferably It is selected from the group consisting of hydrogen and fluorine; and R _{6 is} selected from the group consisting of hydrogen and halogen, preferably selected from the group consisting of hydrogen and fluorine. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is a compound selected from the group consisting of: A pharmaceutical composition comprising the compound of any one of claims 1 to 5 and 8, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. Use of a compound according to any one of claims 1 to 5 and 8, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for treating a tumor. Use of the pharmaceutical composition according to claim 9 for the preparation of a medicament for treating a tumor.