CN108117551A

CN108117551A - Substitute (1H- pyrazoles [3,4-b] pyridine) carbamide compounds and its anticancer usage

Info

Publication number: CN108117551A
Application number: CN201611073936.2A
Authority: CN
Inventors: 李剑; 鲁彦丽; 毛斐; 朱进
Original assignee: East China University of Science and Technology
Current assignee: East China University of Science and Technology
Priority date: 2016-11-29
Filing date: 2016-11-29
Publication date: 2018-06-05
Anticipated expiration: 2036-11-29
Also published as: CN108117551B

Abstract

The present invention provides a kind of substitution (1H pyrazoles [3,4 b] pyridine) carbamide compounds and its anticancer usages.Specifically, the present invention provides compound of formula I and its pharmaceutically acceptable salt, wherein, the definition of each group is as noted in the discussion.The present invention compound of formula I can inhibit with the relevant receptor tyrosine kinase of tumorigenesis (including c Kit, PDGFR α and VEGFR2), for the treatment of tumour.

Description

Substituted (1H-pyrazolo [3,4-b ] pyridine) urea compound and anti-tumor application thereof

Technical Field

The invention relates to the field of medicinal chemistry and pharmacotherapeutics, in particular to a substituted (1H-pyrazolo [3,4-b ] pyridine) urea compound and an anti-tumor application thereof.

Background

Cancer (malignant tumor) is one of chronic non-infectious diseases that seriously threaten human health, and has become an increasingly serious global public health problem. Statistical data show that the number of cancer morbidity in China is increasing day by day, the current cancer morbidity is 285.91/10 ten thousand, and the mortality is 180.54/10 ten thousand. Therefore, the development of strong and effective therapies for malignant tumors is not always feasible. With the continuous development of molecular pharmacology and molecular oncology, the elucidation of the nature of tumors is gradually clarified; meanwhile, the wide application of advanced technologies such as medicines, genetic engineering, combinatorial chemistry, large-scale rapid screening and the like accelerates and powerfully develops novel antitumor medicines. At present, the focus of research and development of antitumor drugs is shifting from cytotoxic drugs to novel antitumor drugs targeting multiple link effects, and such as antitumor drugs targeting multiple targets such as key genes, regulatory molecules, cell receptors and the like have begun to enter the clinic from the laboratory. The molecular targeted therapy method has the advantages of orientation and positioning, so that the medicine selectively reacts with target tissues at a cellular or subcellular level, the medicine can be controllably distributed and continuously and slowly released at a target area, the medicine dosage can be reduced, the therapeutic effect is improved, the toxic and side effects are reduced, and the molecular targeted therapy method is an effective way for treating cancers all over the world.

In recent ten years, more than ten targeted antitumor drugs have been successfully applied to clinic, and kinase is the most important molecular targeted therapeutic drug, and about one third of the drugs in research or development projects worldwide are related to kinase. The Protein Tyrosine Kinase (PTK), the most common growth factor receptor for most tumors, participates in the regulation, signal transmission and development of normal cells, is closely related to the proliferation, differentiation, migration and apoptosis of tumor cells, can destroy the signal transmission of tumor cells by inhibiting the activity of the PTK, selectively inhibits the tumor cells, and has no influence on the normal cells. c-KIT is a typical type III receptor tyrosine kinase, is one of ATP competitive kinases, plays an important role in the processes of occurrence, development, invasion, migration and recurrence of tumors, is one of hot targets of tumor molecule targeted therapy at present, and an inhibitor of the C-KIT also becomes a hotspot for research and development of antitumor drugs.

c-KIT (also called CD117) is discovered in 1987, is a transmembrane receptor protein with tyrosine kinase activity encoded by a retrovirus proto-oncogene c-KIT, and forms a type III receptor tyrosine kinase superfamily together with a Platelet Derived Growth Factor Receptor (PDGFR), a macrophage colony stimulating factor-1 receptor (CSF-1R) and an Fms-like tyrosine kinase receptor 3(FLT3), and plays an important role in the process of tumorigenesis and development. There are more than 30 gain-of-function (GOF) mutant forms of c-KIT kinase, which is a receptor for Stem Cell Factor (SCF). After being activated by stem cell growth factors, the protein forms a dimer, leads to transmembrane phosphorylation of Tyr568 and Tyr570 in JMD, thereby changing the three-dimensional structure of JMD, weakening the interaction with the active site of kinase, namely weakening the self-inhibition effect of the kinase, leading the phosphorylation of the kinase area to further recruit downstream signal molecules, finally activating downstream signal pathways, and regulating the growth and proliferation of cells. It is known that c-KIT receptor activation is abnormal in various tumor tissues such as gastrointestinal stromal tumor (GIST), Acute Myeloid Leukemia (AML), and lung cancer. This is mainly due to c-KIT mutation leading to its overexpression and persistent activation. Functional mutations of the C-KIT gene mainly occur in an extracellular domain (exons 8 and 9), an intracellular JMD (exon 11) and an A-loop region (exon 17) in a kinase structure domain, the mutation occurrence rate of the exon 9 is 3-21%, the mutation mainly occurs in codons 501-503, and the main type is repeated insertion. The mutation rate of the exon 11 is 20-92%, and is concentrated between codons 550-599, and the mutation types comprise: point mutation, deletion mutation, insertion mutation, substitution mutation, frame shift mutation, etc.; the mutation site of the exon 13 is mainly codon 642, and the mutation rate is 0.8-4.1%. It is worth noting that: the common c-KIT mutation sites of different tumors are different. For example: in GIST, mutations occur mainly in exon 11 (e.g., V560D), Acute Myeloid Leukemia (AML) cells, and c-KIT activating mutations occur mainly in exon 17 (e.g., D816V/H/Y, etc.). And studies have shown that about 80% of GISTs have c-KIT activating mutations.

at present, more than 30 factors related to tumor angiogenesis are found, such as platelet-derived growth factor (PDGF), Vascular Endothelial Growth Factor (VEGF), fibroblast factor (FGF), angiostatin and the like, wherein the PDGF can recruit peripheral cells to stimulate tumor angiogenesis, can induce the proliferation and migration of vascular endothelial cells, smooth muscle cells and tumor cells, promote the generation of connective tissue interstitium, provide good support for new vessels, and play a direct role in tumor angiogenesis.

Therefore, the development of multi-target (c-KIT, PDGFR, VEGFR and FGFR) therapeutic drugs targeting tumorigenesis, developing relevant signaling pathways, is expected to produce better antitumor effects, especially against gastrointestinal stromal tumors.

Disclosure of Invention

The invention aims to provide a substituted (1H-pyrazolo [3,4-b ] pyridine) urea compound which has a structural general formula shown in the specification, or pharmaceutically acceptable salt thereof;

still another object of the present invention is to provide the use of substituted (1H-pyrazolo [3,4-b ] pyridine) ureas as dual c-KIT and PDGFR-a inhibitors in the prevention or treatment of tyrosine kinase receptor inhibitor-related tumor diseases (e.g., gastrointestinal stromal tumors).

In a first aspect of the invention, there is provided a compound of formula I, or a pharmaceutically acceptable salt thereof,

wherein,

x is O or S;

R¹is C₁～C₄Straight or branched alkyl or C₁～C₄A linear or branched haloalkyl;

R²is substituted or unsubstituted C₁～C₆Straight or branched alkyl, substituted or unsubstituted C₃～C₈Cycloalkyl, substituted or unsubstituted C₆～C₁₀Aryl, substituted or unsubstituted 4-10 membered heteroaryl;

wherein said substitution refers to having 1-3 substituents selected from the group consisting of: halogen, halogen,Hydroxy, nitro, cyano, C₁-C₄Alkyl radical, C₁-C₄Haloalkyl, C₁-C₄Alkoxy, -NRaRb, 4-10 membered heteroaryl, 4-8 membered heterocycloalkyl, C₆～C₁₀An aryl group;

and C in the above substituents₆～C₁₀The aryl group may optionally be further substituted by 1 to 3 substituents selected from halogen, hydroxy, nitro, cyano, C₁-C₆Alkyl radical, C₁-C₄Haloalkyl, C₁-C₄Substituent in alkoxy;

and C in the above substituents₁-C₄Alkyl may optionally be further substituted with 1-3 substituents selected from halogen, hydroxy, nitro, cyano, -NRaRb;

ra and Rb are each independently H and C₁-C₄An alkyl group;

said 4-10 membered heteroaryl and 4-8 membered heterocycloalkyl each independently contain 1-3 heteroatoms selected from N, O and S;

and the halogen is fluorine, chlorine, bromine or iodine.

In another preferred embodiment, R is¹Is C₁～C₄Straight or branched alkyl or C₁～C₄Linear or branched perfluoroalkyl, preferably, said R¹Is methyl or perfluoro-substituted methyl.

In another preferred embodiment, the compound of formula I has one or more characteristics selected from the group consisting of:

(a) said C₁～C₆The linear or branched alkyl group is selected from: ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isobutyl, and neopentyl;

(b) said C is₃～C₈Cycloalkyl is selected from: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptylA cyclooctyl group;

(c) said C₆～C₁₀Aryl is selected from: phenyl, naphthyl, indanyl, preferably, said C₆～C₁₀Aryl is selected from: phenyl, 2, 3-dihydro-1H-inden-2-yl, naphthalen-1-yl, naphthalen-2-yl;

(d) said 4-10 membered heteroaryl is selected from: pyrazolyl, pyridyl, quinolinyl, furyl, thienyl, preferably said 4-10 membered heteroaryl is selected from: 1H-pyrazol-5-yl, pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, quinolin-3-yl, furan-2-yl, thiophen-2-yl;

(e) the 4-8 membered heterocycloalkyl group is selected from: piperidinyl, morpholinyl, piperazinyl (4-methylpiperazinyl), pyrrolidinyl, tetrahydropyranyl, preferably said 4-8 membered heterocycloalkyl is selected from: 2-piperidin-1-yl, 2-morpholin-4-yl, 4-methylpiperazin-1-yl.

In another preferred embodiment, the heterocycloalkyl group is a saturated heterocycloalkyl group.

In another preferred embodiment, Ra and Rb are the same.

In another preferred embodiment, Ra and Rb are both methyl or ethyl.

In another preferred embodiment, said substituted C₁～C₆The straight chain or branched alkyl group has the structure of formula II,

wherein R is³Is 5-6 membered heteroaryl, 5-6 membered heterocycloalkyl, C₆～C₁₀An aryl group;

and said C₆～C₁₀The aryl group may optionally be further substituted by 1-3 groups selected from halogen, C₁-C₆Alkyl radical, C₁-C₄Substituent in alkoxy;

said 5-6 membered heteroaryl and 5-6 membered heterocycloalkyl each independently contain 1-3 heteroatoms selected from N, O and S;

and n is an integer between 1 and 4.

In another preferred embodiment, the 5-6 membered heteroaryl is selected from: pyrazolyl, pyridyl, furanyl, thienyl, preferably said 4-to 10-membered heteroaryl is selected from: 1H-pyrazol-5-yl, pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, furan-2-yl, thiophen-2-yl.

In another preferred embodiment, the 5-6 membered heterocycloalkyl group is selected from: piperidinyl, morpholinyl, piperazinyl (4-methylpiperazinyl), pyrrolidinyl, tetrahydropyranyl, preferably said 5-6 membered heterocycloalkyl is selected from: 2-piperidin-1-yl, 2-morpholin-4-yl, 4-methylpiperazin-1-yl.

In another preferred embodiment, R is³Is furyl, thienyl, pyridyl, piperidyl, morpholinyl, or phenyl, and the phenyl may optionally be further substituted by 1-3 substituents selected from halogen, C₁-C₆Alkyl radical, C₁-C₄Substituents in alkoxy groups.

In another preferred embodiment, R is³Is furan-2-yl, thiophen-2-yl, pyridin-4-yl, 2-piperidin-1-yl, 2-morpholin-4-yl, phenyl (unsubstituted phenyl), 2-methylphenyl, 4-methylphenyl, 2-fluorophenyl, 2-chlorophenyl, 2, 6-difluorophenyl, 4-fluorophenyl, 4-chlorophenyl, or 4, 5-dimethoxyphenyl.

In another preferred embodiment, said substituted C₁～C₆The linear or branched alkyl group has the structure of formula III,

wherein R is⁴Is C₁-C₂Alkyl, and n is a whole number of 1 to 3And (4) counting.

In another preferred embodiment, R is⁴Is methyl or ethyl.

In another preferred embodiment, said substituted C₆～C₁₀Aryl is substituted phenyl and has the structure of formula IV,

wherein R is⁵Is hydrogen or C₁～C₄An alkyl group; r⁶Is 5-6 membered heterocycloalkyl, orWherein said 5-6 membered heterocycloalkyl may optionally be further substituted with 1-3 substituents selected from halogen, C₁-C₄Substituent in alkyl; and R is⁴Is C₁-C₂An alkyl group, and n is an integer of 0 to 3.

In another preferred embodiment, R is⁶Is 4-methylpiperazinyl, morpholinyl, orAnd R is⁴Is C₁-C₂An alkyl group, and n is an integer of 0 to 3.

In another preferred embodiment, R is⁶Is 4-methylpiperazin-1-yl, morpholin-4-yl, orAnd R is⁴Is C₁-C₂An alkyl group, and n is an integer of 0 to 3.

In another preferred embodiment, the structure of formula II is selected from: furan-2-ylmethyl, thien-2-ylmethyl, pyridin-4-ylmethyl, 2-piperidin-1-ylethyl, 2-morpholin-4-ylethyl, benzyl, phenethyl, phenylpropyl, phenylbutyl, 2-methylbenzyl, 4-methylbenzyl, 2-fluorobenzyl, 2-chlorobenzyl, 2, 6-difluorobenzyl, 4-fluorophenethyl, 4-chlorophenethyl, 4, 5-dimethoxyphenethyl.

In another preferred embodiment, the structure of formula III is selected from: 2-dimethylaminoethyl, 2-diethylaminoethyl, 2-dimethylaminopropyl, 2-diethylaminopropyl.

In another preferred embodiment, the structure of formula IV is selected from: 2- (4-methylpiperazin-1-yl) phenyl group, 3- (4-methylpiperazin-1-yl) phenyl group, 4- (4-methylpiperazin-1-yl) phenyl group, 2- (morpholin-4-yl) phenyl group, 2-methyl-4- (4-methylpiperazin-1-yl) phenyl group, 3-methyl-4- (4-methylpiperazin-1-yl) phenyl group, 5-methyl-2- (4-methylpiperazin-1-yl) phenyl group, 3-methyl-4- (morpholin-4-yl) phenyl group, 5-methyl-2- (morpholin-4-yl) phenyl group, (4-dimethylamino) phenyl group, (3-dimethylamino) phenyl, (4-diethylamino) phenyl, (3-diethylamino) phenyl, (4-dimethylamino) benzyl, (3-dimethylamino) benzyl, (4-diethylamino) benzyl, and (3-diethylamino) benzyl.

In another preferred embodiment, R is²Selected from the group consisting of:

ethyl, n-propyl, n-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclobutyl, 1H-pyrazol-5-yl, pyridin-2-yl, pyridin-3-yl, quinolin-3-yl, 2, 3-dihydro-1H-inden-2-yl, naphthalen-1-yl, naphthalen-2-yl, furan-2-ylmethyl, thiophen-2-ylmethyl, pyridin-4-ylmethyl, 2-piperidin-1-ylethyl, 2-morpholin-4-ylethyl, benzyl, phenethyl, phenylpropyl, phenylbutyl, 2-methylbenzyl, 4-methylbenzyl, 2-fluorobenzyl, 2-chlorobenzyl, 2, 6-difluorobenzyl group, 4-fluorophenethyl group, 4-chlorophenethyl group, 4, 5-dimethoxyphenethyl group, 2-dimethylaminoethyl group, 2-diethylaminoethyl group, 2-dimethylaminopropyl group, 2-diethylaminopropyl group, 2- (4-methylpiperazin-1-yl) phenyl group, 3- (4-methylpiperazin-1-yl) phenyl group, 4- (4-methylpiperazin-1-yl) phenyl group, 2- (morpholin-4-yl) phenyl group, 2-methyl-4- (4-methylpiperazin-1-yl) phenyl group, 3-methyl-4- (4-methylpiperazin-1-yl) phenyl group, 5-methyl-2- (4-methylpiperazin-1-yl) phenyl group, 4-chlorophenylethyl group, 2-dimethylaminoethyl group, 2-dimethylaminopropyl group, 2-ethylpiperazin-1-yl group, 3-methyl-4- (morpholin-4-yl) phenyl, 5-methyl-2- (morpholin-4-yl) phenyl, (4-dimethylamino) phenyl, (3-dimethylamino) phenyl, (4-diethylamino) phenyl, (3-diethylamino) phenyl, (4-dimethylamino) benzyl, (3-dimethylamino) benzyl, (4-diethylamino) benzyl, and (3-diethylamino) benzyl.

In another preferred embodiment, the compound of formula I is selected from the group consisting of:

N-ethyl-N ' - (4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N-propyl-N ' - (4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N-butyl-N ' - (4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N-isobutyl-N ' - (4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N-tert-butyl-N ' - (4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea 4-b ] pyridin-4-yl) phenyl) urea, N-N-pentyl-N '- (4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N-N-hexyl-N' - (4- (3-trifluoromethyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N-cyclohexyl-N '- (4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N-N' - (4- (3-methyl-1H-pyrazolo [3,4-b ], N-cyclopropyl-N '- (4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N-cyclobutyl-N' - (4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N-cyclopentyl-N '- (4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (pyridin-3-yl) -N' - (4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N-cyclopropyl-N '- (4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N-cyclobutyl-N' - (4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-, N- (pyridin-2-yl) -N '- (4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (1H-pyrazol-5-yl) -N' - (4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (naphthalen-2-yl) -N '- (4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (2, 3-dihydro-1H-inden-2-yl) -N' - (4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (quinolin-3-yl) -N '- (4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (thien-2-ylmethyl) -N' - (4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (furan-2-ylmethyl) -N '- (4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (pyridin-4-ylmethyl) -N' - (4- (3-methyl-1H-pyrido) phenyl) urea Oxazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (2-piperidin-1-ylethyl) -N '- (4- (3-trifluoromethyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (2-morpholin-4-ylethyl) -N' - (4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, and mixtures thereof, N-benzyl-N '- (4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (3-methylbenzyl) -N' - (4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (4-fluorophenethyl) -N '- (4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (3, 4-dimethoxyphenethyl) -N' - (4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (3-methyl-1H-pyrazolo [3,4-, N- (2-chlorobenzyl) -N '- (4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (4-methylbenzyl) -N' - (4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (4-chlorophenethyl) -N '- (4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (2-fluorobenzyl) -N' - (4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (4-chlorobenzyl) -N '- (4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (4-methylbenzyl) -N' - (4- (3-methyl-1H, N- (4-phenylbutyl) -N '- (4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (3-phenylpropyl) -N' - (4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (2, 6-difluorobenzyl) -N '- (4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (2-phenylethyl) -N' - (4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (2-phenylethyl) -N '- (4- (3-trifluoromethyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (3-phenylpropyl) -N' - (4- (3-trifluoromethyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (4-chlorophenethyl) -N '- (4- (3-trifluoromethyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (2-dimethylaminoethyl) -N' - (4- (3-trifluoromethyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (2-diethylaminoethyl) -N '- (4- (3-trifluoromethyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (3-dimethylaminopropyl) -N' - (4- (3-trifluoromethyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (3-diethylaminopropyl) -N '- (4- (3-trifluoromethyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (2-dimethylaminoethyl) -N' - (4- (3-methyl-1H-pyrido [3,4-b ] pyridin-4-yl) phenyl) urea Oxazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (4- (4-methylpiperazin-1-yl) phenyl) -N ' - (4- (3-trifluoromethyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) thiourea, N- (4- (4-methylpiperazin-1-yl) phenyl) -N ' - (4- (3-trifluoromethyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (2-methyl-4- (4-methylpiperazin-1-yl) phenyl) -N ' - (4- (3-trifluoromethyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (3-methyl-4- (4-methylpiperazin-1-yl) phenyl) -N ' - (4- (3-trifluoromethyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (3- (4-methylpiperazin-1-yl) phenyl) -N ' - (4- (3-trifluoromethyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (2- (4-methylpiperazin-1-yl) phenyl) -N ' - (4- (3-trifluoromethyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (5-methyl-2- (4-methylpiperazin-1-yl) phenyl) -N ' - (4- (3-trifluoromethyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (3-methyl-4- (morpholin-4-yl) phenyl) -N ' - (4- (3-trifluoromethyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (2- (morpholin-4-yl) phenyl) -N ' - (4- (3-trifluoromethyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (5-methyl-2- (morpholin-4-yl) phenyl) -N ' - (4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (2-methyl-4- (4-methylpiperazin-1-yl) phenyl) -N ' - (4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (3-methyl-4- (4-methylpiperazin-1-yl) phenyl) -N ' - (4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (3- (4-methylpiperazin-1-yl) phenyl) -N ' - (4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (2- (4-methylpiperazin-1-yl) phenyl) -N ' - (4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (5-methyl-2- (4-methylpiperazin-1-yl) phenyl) -N ' - (4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (3-methyl-4- (morpholin-4-yl) phenyl) -N ' - (4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (2- (morpholin-4-yl) phenyl) -N ' - (4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (5-methyl-2- (morpholin-4-yl) phenyl) -N ' - (4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- ((4-diethylamino) phenyl) -N ' - (4- (3-trifluoromethyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- ((3-dimethylamino) phenyl) -N ' - (4- (3-trifluoromethyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (4- ((dimethylamino) methyl) phenyl) -N ' - (4- (3- trifluoromethyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (4- ((diethylamino) methyl) phenyl) -N ' - (4- (3-trifluoromethyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (3- ((dimethylamino) methyl) phenyl) -N ' - (4- (3-trifluoromethyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (3- ((diethylamino) methyl) phenyl) -N ' - (4- (3-trifluoromethyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- ((4-dimethylamino) phenyl) -N ' - (4- (3-trifluoromethyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- ((4-diethylamino) phenyl) -N ' - (4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- ((3-dimethylamino) phenyl) -N ' - (4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea -pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (4- ((dimethylamino) methyl) phenyl) -N ' - (4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (4- ((diethylamino) methyl) phenyl) -N ' - (4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (3- ((dimethylamino) methyl) phenyl) -N ' - (4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea, N- (4- ((dimethylamino) methyl) phenyl) urea, N- (4-methyl-, N- ((4-dimethylamino) phenyl) -N' - (4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) phenyl) urea.

In another preferred embodiment, X is O and R¹Is methyl.

In another preferred embodiment, R is²Is 4-10 membered heteroaryl or phenyl, wherein said phenyl may be further substituted by halogen, C₁～C₄Alkyl, -NRaRb, 4-8 membered heterocycloalkyl, and said Ra and Rb are both methyl or ethyl.

In another preferred embodiment, R is²Is 4-10 membered heteroaryl or phenyl substituted C₁～C₆Alkyl, wherein said phenyl may optionally be further substituted with halogen.

in a second aspect of the present invention, there is provided a pharmaceutical composition comprising:

(a) a therapeutically effective amount of a compound of formula I according to the first aspect of the invention, or a pharmaceutically acceptable salt thereof, and optionally (b) a pharmaceutically acceptable carrier.

In another preferred embodiment, the pharmaceutical composition comprises 0.001-99 wt%, preferably 0.1-90 wt%, more preferably 1-80 wt% of the compound of formula I, or its pharmaceutically acceptable salt, based on the total weight of the composition.

In another preferred embodiment, the dosage form of the pharmaceutical composition is an oral dosage form or an injection.

In another preferred embodiment, the pharmaceutical composition is used for inhibiting tumor cells or treating tumors.

In another preferred embodiment, the tumor is selected from the group consisting of: gastrointestinal stromal tumor, acute myeloid leukemia, lung cancer, melanoma, acute myeloid leukemia, or a combination thereof.

in another preferred embodiment, the tumor cells comprise tumor cells in which c-KIT and PDGFR α are wild-type and mutant.

In another preferred embodiment, the tumor cell is selected from the group consisting of: BaF3 cells, GIST-T1 cells, GIST-882 cells, MOLM14, MV4-11, or a combination thereof.

in another preferred embodiment, the BaF cells are selected from the group consisting of BaF cells, C-KIT-A829-BaF cells, C-KIT-L576-BaF cells, C-KIT-C674-BaF cells, C-KIT-D816-BaF cells, C-KIT-T670-BaF cells, C-KIT-V559-T670-BaF cells, C-KIT-V654-BaF cells, C-KIT-N822-BaF cells, C-KIT-V559-V654-BaF cells, PDGFR α -T-674-BaF cells, FLT-BaF cells, and FLT-ITD-BaF cells.

In another preferred embodiment, the pharmaceutical composition is a pharmaceutical composition of a multi-target receptor tyrosine kinase inhibitor.

In another preferred embodiment, the pharmaceutical composition may be used to inhibit receptor tyrosine kinases.

In another preferred embodiment, the receptor tyrosine kinase is a type III receptor tyrosine kinase.

in another preferred embodiment, the receptor tyrosine kinase is selected from the group consisting of c-KIT, PDGFR α, VEGFR2 or a combination thereof.

In another preferred embodiment, the pharmaceutical composition may be administered in vivo or in vitro.

In a third aspect of the present invention, there is provided a use of a compound of formula I according to the first aspect of the present invention, or a pharmaceutically acceptable salt thereof, for preparing (I) a pharmaceutical composition for inhibiting tumor cells or treating tumors, and (ii) a receptor tyrosine kinase inhibitor.

In a fourth aspect of the invention, there is provided a method of inhibiting a receptor tyrosine kinase in vitro comprising the steps of:

(a) contacting a receptor tyrosine kinase with a compound of formula I according to the first aspect of the invention, or a pharmaceutically acceptable salt thereof, such that the activity of the receptor tyrosine kinase is inhibited.

In another preferred embodiment, in step (a), the compound of formula I according to the first aspect of the present invention, or a pharmaceutically acceptable salt thereof, is added to a cell culture system, such that it is contacted with a receptor tyrosine kinase.

In another preferred embodiment, the cell is a normal cell or a tumor cell.

In another preferred embodiment, the cell is a mammalian cell.

In another preferred embodiment, the cell is a human cell.

In another preferred embodiment, the method is non-therapeutic and non-diagnostic.

In a fifth aspect of the present invention, there is provided a method of treating a tumor, comprising the steps of: administering a compound of formula I according to the first aspect of the invention, or a pharmaceutically acceptable salt thereof, to a subject in need thereof.

In another preferred embodiment, the subject includes human and non-human mammals.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

Figure 1 shows the individual and mean plasma concentration-time curves (IV-2mg/kg, n-3) following a single intravenous administration of IA-41 in rats.

FIG. 2 is a graph showing the average body weight change of the mice in each group in example 94.

FIG. 3 is a graph showing the average relative body weight change of the mice in each group in example 94.

FIG. 4 is a graph showing the change in tumor volume in each group of mice in example 94.

FIG. 5 is a graph showing the relative tumor volume change in the groups of mice in example 94.

FIG. 6 shows groups of mice GIST-T1 mouse I in example 94_A-41 photograph of tumor for efficacy test.

FIG. 7 shows a tumor weight plot of tumors from each group of mice in example 94.

FIG. 8 shows Compound I_A-41 pharmacodynamic tests HE, Ki67, Tunel staining

Detailed Description

the invention provides a compound with optimal kinase activity, wherein the compound can obviously inhibit the activity of receptor tyrosine kinases (including c-KIT, PDGFR- α and VEGFR-2) related to tumorigenesis and tumor development, and experiments show that the compound has strong antiproliferative activity on three kinases, particularly on c-KIT and PDGFR- α receptors, and simultaneously shows high-selectivity and effective antiproliferative activity on 8 real tumor cell lines, particularly on gastrointestinal stromal tumor (GIST) cell lines in cell level research.

Term(s) for

The term "C₁-C₆Alkyl "means a straight or branched chain alkyl group having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, or the like.

The term "C₁-C₄Alkoxy "means a straight or branched chain alkoxy group having 1 to 4 carbon atoms, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, or the like.

The term "cycloalkyl" refers to a 3 to 8 membered all carbon monocyclic, all carbon 5/6 or 6/6 membered fused ring or polycyclic fused ring group, wherein one or more rings may contain one or more double bonds, but none of the rings has a completely conjugated pi-electron system. Examples of cycloalkyl groups are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexadienyl, adamantyl, cycloheptyl, cycloheptatrienyl, and the like.

The term "heterocycloalkyl" refers to a saturated or unsaturated ring in which at least one heteroatom selected from the group consisting of: n, S, O or P, wherein one or more of the rings may contain one or more double bonds. Such as pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, or the like.

The term "aromatic ring" refers to aromatic rings having a conjugated pi-electron system and includes carbocyclic aryl, heteroaryl groups.

The term "aryl" denotes a hydrocarbyl moiety comprising one or more aromatic rings. Examples of aryl moieties include phenyl (Ph), phenylene, naphthyl, naphthylene, pyrenyl, anthracenyl, and phenanthrenyl.

The term "heteroaryl" refers to aryl groups having at least 1 heteroatom as a ring atom, the remaining ring atoms being carbon, the heteroatoms including oxygen, sulfur, nitrogen. The ring may be a 5-or 6-or 7-membered ring. Examples of heteroaryl groups include, but are not limited to, furyl, furanylene, fluorenyl, pyrrolyl, N-alkylpyrrolyl, thienyl, oxazolyl, imidazolyl, thiazolyl, benzofuranyl, benzothienyl, pyridyl, pyrimidinyl, quinazolinyl, quinolinyl, isoquinolinyl, and indolyl.

The term "amino" denotes-NH₂、-NH-(C₁-C₆Alkyl) or-N (C)₁-C₆Alkyl radical)₂。

The term "halogen" refers to fluorine, chlorine, bromine, iodine. The term "halogenated" refers to fluorinated, chlorinated, brominated, and iodinated.

Unless otherwise specified herein, alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl groups described herein include both substituted and unsubstituted moieties. In addition, cycloalkyl, heterocycloalkyl, aryl and heteroaryl groups may also be fused to one another.

Unless otherwise indicated, the term "substituted" means that one or more hydrogen atoms on a group are replaced with a substituent selected from the group consisting of: c₁-C₁₀Alkyl radical, C₃-C₁₀Cycloalkyl radical, C₁-C₁₀Alkoxy, halogen, hydroxy, carboxyl (-COOH), C₁-C₁₀Aldehyde group, C₂-C₁₀Acyl radical, C₂-C₁₀Ester group, amino group, phenyl group; the phenyl group includes an unsubstituted phenyl group or a substituted phenyl group having 1 to 3 substituents selected from: halogen, C₁-C₁₀Alkyl, cyano, OH, nitro, C₃-C₁₀Cycloalkyl radical, C₁-C₁₀Alkoxy, amino.

Unless otherwise specified, each chiral carbon atom in all compounds of the invention may optionally be in the R configuration or the S configuration, or a mixture of the R configuration and the S configuration.

A compound of formula I

As used herein, the terms "compound of the present invention", "compound of formula I" and "substituted (1H-pyrazolo [3,4-b ] pyridine) urea compound" are used interchangeably and refer to a compound according to the first aspect of the invention having the structure shown in formula I.

The compounds of the invention may contain asymmetric or chiral centers and may therefore exist in different stereoisomeric forms. All stereoisomeric forms of the compounds of the present invention, including but not limited to diastereomers, enantiomers, and atropisomers, and mixtures thereof (e.g., racemic mixtures), are included within the scope of the present invention.

The compounds of the invention may also exist in different tautomeric forms, all of which are included within the scope of the invention. The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that are interconverted via a low energy barrier.

The compounds of the present invention may exist in unsolvated forms as well as solvated forms containing pharmaceutically acceptable solvents such as water, ethanol, and the like, including solvated as well as unsolvated forms.

The compounds of the present invention have a basic group and thus can form "pharmaceutically acceptable salts" with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid and sulfuric acid or organic acids such as ascorbic acid, nicotinic acid, citric acid, tartaric acid, lactic acid, maleic acid, malonic acid, fumaric acid, oxalic acid, malic acid, glycolic acid, succinic acid, propionic acid, acetic acid, methanesulfonic acid, trifluoromethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and the like, including pharmaceutically acceptable acid addition salts, by treating the free base of the compounds of the present invention with the inorganic or organic acids.

The invention also encompasses isotopically-labeled compounds of the invention, which are identical to those recited herein, except for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine and chlorine, such as:²hydrogen, hydrogen,³Hydrogen, hydrogen,¹¹Carbon, carbon,¹³Carbon, carbon,¹⁴Carbon, carbon,¹³Nitrogen, nitrogen,¹⁵Nitrogen, nitrogen,¹⁵Oxygen, oxygen,¹⁷Oxygen, oxygen,¹⁸Oxygen, oxygen,³¹Phosphorus, phosphorus,³²Phosphorus, phosphorus,³⁵Sulfur, sulfur,¹⁸Fluorine,¹²³Iodine,¹²⁵Iodine and³⁶chlorine.

Certain isotopically-labelled compounds of the invention (e.g. with³H and¹⁴c-labeled those) for compound and/or substrate tissue distribution assays. Tritiation (i.e., tritiation) is particularly preferred³H) And carbon-14 (i.e.¹⁴C) Isotopes because they are easy to prepare and detect. Also, heavier isotopes such as deuterium (i.e., deuterium)²H) The substitution may provide a substitution ofCertain therapeutic advantages resulting from large metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements) may therefore be preferred in certain circumstances. Positron emitting isotopes, e.g.¹⁵O、¹³N、¹¹C and¹⁸f was used in Positron Emission Tomography (PET) studies to examine substrate receptor occupancy. Isotopically labeled compounds of the present invention can generally be prepared by following procedures analogous to those disclosed in the schemes and/or in the examples below, by substituting an isotopically labeled reagent for a non-isotopically labeled reagent.

the compound has a brand-new (1H-pyrazolo [3,4-b ] pyridine) urea structure, and is subjected to inhibition activity tests of three receptor tyrosine kinases (including c-KIT, PDGFR- α and VEGFR-2) closely related to tumor tissue generation, antiproliferative activity tests of wild-type and multiple mutant cell strains of c-KIT and PDGFR-a, inhibition activity tests of 8 real tumor cell strains, pharmacokinetic characteristics, hERG and animal pharmacodynamic model experiment tests and the like.

In another aspect of the present invention, there is provided a substituted (1H-pyrazolo [3,4-b ] pyridine) urea compound having the following general formula (I):

in formula I: x is O or S; r¹Is C₁～C₄Straight or branched chainAlkyl or C₁～C₄A linear or branched perfluoroalkyl group; r²Is C₁～C₆Straight or branched alkyl, C₃～C₆Cycloalkyl radical, C₅～C₆Aromatic heterocyclic groups, fused rings or groups selected from the following structures II, III, IV:

wherein, in the formula II, R³Is C₅～C₆Aromatic heterocyclic group, substituted or unsubstituted piperidine and morpholine ring group, substituted or unsubstituted six-membered aromatic ring group, n is an integer between 1 and 4;

the substituent of the substituted phenyl is selected from: c₁～C₆Straight-chain or branched alkyl, halogen, C₁～C₃Alkoxy, wherein the number of the substituent groups is an integer of 0-3;

wherein, in the formula III, R⁴Is C₁～C₂An alkyl group, n is an integer of 1 to 3;

wherein, in the formula IV, R⁵Is hydrogen or C₁～C₃An alkyl group; r⁶Is a substituted or unsubstituted 4-methylpiperazino or morpholinyl radical, orn is an integer of 0 to 3.

A first preferred embodiment of the compounds of the formula (I) according to the invention is the compound (I)_A)：

R²Is C₁～C₆Straight or branched alkyl, C₃～C₆Cycloalkyl radical, C₅～C₆Aromatic heterocyclic groups, fused rings or groups selected from the following structures II, III, IV:

A second preferred embodiment of the compounds of the formula (I) according to the invention are the compounds (I)_B)：

X is O or S; r²Is C₁～C₆Straight or branched alkyl, C₃～C₆Cycloalkyl radical, C₅～C₆Aromatic heterocyclic groups, fused rings or groups selected from the following structures II, III, IV:

wherein,in the formula II, R³Is C₅～C₆Aromatic heterocyclic group, substituted or unsubstituted piperidine and morpholine ring group, substituted or unsubstituted six-membered aromatic ring group, n is an integer between 1 and 4;

Specifically, the compounds of the present invention include, but are not limited to, compounds selected from the group consisting of:

preparation method

The invention also provides substituted (1H-pyrazole [3,4-b ] with the structure of the general formula (I)]Pyridine) urea compound I_A～I_BAnd the preparation method of the intermediates V to XIV thereof, which comprises the following specific synthesis method.

I_AThe synthesis of (2):

in the formula, R²The meaning of (a) is the same as previously described.

The method specifically comprises the following steps:

1) adding 3-methyl-5-amino-1H-pyrazole, p-nitrobenzaldehyde and 2, 2-dimethyl-1, 3-dioxane-4, 6-dione into a round-bottom flask, dissolving with N, N-dimethylformamide, and heating at its boiling temperature until no CO is present₂Until discharge. Solid precipitates in the solution, and the solution is cooled to room temperature and diluted by adding a proper amount of isopropanol. Suction filtration, ultrasonic treatment for 15 minutes after dissolving the solid in N, N-dimethylformamide, and suction filtration to obtain an intermediate 3-methyl-4- (4-nitrophenyl) -4, 5-dihydro-1H-pyrazolo [3,4-b]Pyridin-6 (7H) -one (intermediate V).

2) Intermediate V was dissolved in dioxane, 2, 3-dichloro-5, 6-dicyano-1, 4-benzoquinone and N, O-bis (trimethylsilyl) trifluoroacetamide were added and refluxed overnight. After the reaction is finished, cooling, pouring the reaction liquid into a proper amount of water, and adding saturated NaHCO₃The solution was made alkaline and a large amount of solid was precipitated from the solution. Performing suction filtration, dissolving the filter cake with N, N-dimethylformamide, performing ultrasonic treatment for 15 min, continuing suction filtration, washing the filter cake with ethyl acetate, and drying the filter cake to obtain an intermediate 3-methyl-4- (4-nitrophenyl) -1H-pyrazolo [3,4-b]Pyridin-6 (7H) -one (intermediate VI).

3) Intermediate VI was dissolved in the appropriate amount of phenylphosphoryl dichloride and reacted overnight at 110 ℃. And cooling after the reaction is finished, pouring the system into a proper amount of ice water, stirring for half an hour, performing suction filtration, adding water into a filter cake, uniformly stirring, and adjusting to be alkalescent by using a saturated sodium bicarbonate solution. Filtering, drying the filter cake to obtain an intermediate 6-chloro-3-methyl-4- (4-nitrophenyl) -6, 7-dihydro-1H-pyrazolo [3,4-b ] pyridine (intermediate VII)

4) Mixing zinc powder, hydrazine hydrate and tetrahydrofuran in a three-neck flask, dropwise adding the tetrahydrofuran solution dissolved with the intermediate VII into a reaction system at a constant speed under the protection of nitrogen, and carrying out reflux reaction for 4 hours after the addition is finished. After the reaction is finished, cooling, carrying out suction filtration, extracting the filtrate for three times by using tetrahydrofuran, carrying out reduced pressure distillation on the extract, and carrying out column chromatography separation on the residue to obtain an intermediate 4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) aniline (an intermediate VIII).

5) Dissolving the intermediate VIII in proper amount of dimethyl sulfoxide, and adding commercial or self-made isocyanate compound (R)²-NCO), triethylamine was added, and the reaction was carried out overnight at room temperature. After the reaction is finished, cooling, concentrating, and separating the residue by column chromatography to obtain the compound N-R²-N' - [4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl]Urea (I)_A)。

I_BThe synthesis of (2):

in the formula, R²The meaning of (a) is the same as previously described.

The method specifically comprises the following steps:

1) dissolving sodium hydride in a proper amount of anhydrous tetrahydrofuran, heating and refluxing, then dropwise adding a mixed tetrahydrofuran solution of ethyl trifluoroacetate and acetonitrile, and reacting for 15h at 70 ℃. After the reaction, the reaction mixture was cooled to room temperature, poured into water, extracted with an appropriate amount of anhydrous ether, and the aqueous phase was adjusted to pH 2 with 2M hydrochloric acid solution and extracted again with anhydrous ether. Drying the total organic phase with anhydrous sodium sulfate, filtering, and distilling the filtrate under reduced pressure to obtain crude product 4,4, 4-trifluoro-3-carbonyl butyronitrile (intermediate IX);

2) intermediate IX was mixed with hydrazine hydrate, methanesulfonic acid was slowly added dropwise and after the addition was complete the reaction was carried out overnight at 80 ℃. After the reaction is finished, reduced pressure distillation is carried out, the residue is dissolved by ethyl acetate and n-hexane which are 1:1, stirring is carried out for 5 minutes, suction filtration is carried out, the filtrate is reduced pressure distillation, and column chromatography separation is carried out to obtain 3-trifluoromethyl-5-amino-1H-pyrazole (an intermediate X);

3) dissolving p-nitrobenzaldehyde in ethanol, adding 2, 2-dimethyl-1, 3-dioxane-4, 6-diketone, reacting for 3 hours at 80 ℃, adding an ethanol solution of an intermediate X, and reacting for half an hour after a solid is separated out. After the reaction is finished, cooling to room temperature, and adding a proper amount of isopropanol for dilution. And (3) carrying out suction filtration, dissolving the solid in methanol, carrying out ultrasonic treatment for 15 minutes, and carrying out suction filtration again to obtain an intermediate 3-trifluoromethyl-4- (4-nitrophenyl) -4, 5-dihydro-1H-pyrazolo [3,4-b ] pyridin-6 (7H) -one (intermediate XI).

4) Intermediate XI was dissolved in chlorobenzene, 2, 3-dichloro-5, 6-dicyano-1, 4-benzoquinone and N, O-bis (trimethylsilyl) trifluoroacetamide were added and refluxed overnight. After the reaction is finished, cooling to room temperature, pouring the reaction liquid into a proper amount of water, and adding saturated NaHCO₃The solution was made alkaline and a large amount of solid was precipitated from the solution. Filtering, washing the filter cake with water, drying the filter cake to obtain the intermediate 3-trifluoromethyl-4- (4-nitrophenyl) -1H-pyrazole [3,4-b]Pyridin-6 (7H) -one (intermediate XII).

5) Intermediate XII was dissolved in the appropriate amount of phenylphosphoryl dichloride and reacted overnight at 110 ℃. And cooling after the reaction is finished, pouring the system into a proper amount of ice water, stirring for half an hour, performing suction filtration, adding water into a filter cake, uniformly stirring, and adjusting to be alkalescent by using a saturated sodium bicarbonate solution. Filtering, drying the filter cake, adding phenylphosphonyl dichloride continuously if the raw materials are left, reacting according to the initial conditions until the intermediate XII is completely reacted, and performing post-treatment again to obtain the intermediate 6-chloro-3-trifluoromethyl-4- (4-nitrophenyl) -6, 7-dihydro-1H-pyrazolo [3,4-b ] pyridine (intermediate XIII)

6) Mixing zinc powder, hydrazine hydrate and tetrahydrofuran in a three-neck flask, dropwise adding the tetrahydrofuran solution dissolved with the intermediate XIII into a reaction system at a constant speed under the protection of nitrogen, and carrying out reflux reaction overnight after the addition is finished. After the reaction is finished, cooling, carrying out suction filtration, extracting the filtrate for three times by using tetrahydrofuran, carrying out reduced pressure distillation on the extract, and carrying out column chromatography separation on the residue to obtain an intermediate 4- (3-trifluoromethyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) aniline (intermediate XIV).

7) Dissolving intermediate XIV in appropriate amount of dimethyl sulfoxide or anhydrous THF, and adding commercial or self-made isocyanate compound (R)²-NCO), triethylamine was added, and the reaction was carried out overnight at room temperature. After the reaction is finished, cooling, concentrating, and separating the residue by column chromatography to obtain the compound N-R²-N' - [4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl]Urea (I)_B)。

All compounds encompassed by formula I can be obtained by one of ordinary skill in the art without inventive effort based on the teachings of the above preparation methods.

Pharmaceutical compositions and methods of administration

In the present invention, the pharmaceutical composition can be directly used for disease treatment, for example, for antitumor treatment. When the medicinal preparation is used, other therapeutic agents such as antitumor medicaments and the like can be used simultaneously.

The present invention provides a pharmaceutical composition comprising a safe and effective amount of a compound of the present invention and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, dextrose, water, glycerol, ethanol, powders, and combinations thereof. The pharmaceutical preparation should be compatible with the mode of administration.

In the case of pharmaceutical compositions, the compositions of the present invention may be prepared in the form of injections, for example, by conventional methods using physiological saline or aqueous solutions containing glucose and other adjuvants. Pharmaceutical compositions, such as tablets and capsules, can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions. The pharmaceutical combination of the present invention may also be formulated as a powder for inhalation by nebulization. The amount of active ingredient administered is a therapeutically effective amount, for example from about 1 microgram per kilogram of body weight to about 5 milligrams per kilogram of body weight per day. In addition, the compounds of the present invention may also be used with other therapeutic agents.

For the pharmaceutical compositions of the present invention, administration to a subject in need thereof (e.g., human and non-human mammals) can be by conventional means. Representative modes of administration include (but are not limited to): oral administration, injection, aerosol inhalation, etc.

In the case of pharmaceutical compositions, a safe and effective amount of the drug is administered to the mammal, wherein the safe and effective amount is generally at least about 10 micrograms/kg body weight, and in most cases no more than about 8 mg/kg body weight, preferably the dose is from about 10 micrograms/kg body weight to about 1 mg/kg body weight. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

The main advantages of the invention include:

(a) the compounds of the invention inhibit a variety of receptor tyrosine kinases (including c-Kit, PDGFR α and VEGFR 2).

(b) The compounds of the invention exhibit high selectivity and potent antiproliferative activity against a variety of tumor cell lines.

(c) The compound of the invention has good kinetic characteristics and medication safety.

(d) The compound of the invention can effectively inhibit GIST-T1 tumor cells in vivo, and the tumor growth inhibition can reach 82.2%.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight.

Example 1

Preparation of 3-methyl-4- (4-nitrophenyl) -4, 5-dihydro-1H-pyrazolo [3,4-b ] pyridin-6 (7H) -one (intermediate V)

4.85 g of 3-methyl-5-amino-1H-pyrazole, 7.55 g of p-nitrobenzaldehyde and 7.2 g of 2, 2-dimethyl-1, 3-dioxane-4, 6-dione were put into a 100 ml round-bottomed flask, dissolved in 20 ml of N, N-dimethylformamide and reacted at 110 ℃ with heating until no CO was present₂Until discharge. After 30 minutes of reaction, a solid precipitates out from the solution, and after cooling to room temperature, 50 ml of isopropanol is added for dilution. Suction filtration, sonication of the solid with N, N-dimethylformamide for 15 minutes, continued filtration after completion, and drying of the solid gave the title compound in 72% yield as a white solid (intermediate V).

¹H-NMR(400MHz,DMSO-d₆)δ:11.92(s,1H),10.41(s,1H),8.20(d,J＝8.7Hz,2H),7.45(d,J＝8.7Hz,2H),4.37(t,J＝6.3Hz,1H),2.88(dd,J＝15.9,7.3Hz,1H),2.58(dd,J＝16.0,5.6Hz,1H),1.86(s,3H).

Example 2

Preparation of 3-methyl-4- (4-nitrophenyl) -1H-pyrazolo [3,4-b ] pyridin-6 (7H) -one (intermediate VI)

14.9 g of intermediate V was put into a 250 ml flask, 100 ml of dioxane was added and dissolved, 66 ml of N, O-bis (trimethylsilyl) trifluoroacetamide was added, and 15 g of 2, 3-dichloro-5, 6-dicyano-1, 4-benzoquinone was added and refluxed overnight. After the reaction is finished, pouring the reaction liquidAdding into proper amount of water and adding saturated NaHCO₃The solution was made alkaline and a solid precipitated from the solution. Suction filtration, washing the filter cake with N, N-dimethylformamide and ethyl acetate, drying the filter cake at 40 ℃ to obtain 11.3 g of light yellow solid (intermediate VI), with the yield of 76%.

¹H-NMR(400MHz,DMSO-d₆)δ:13.00(s,1H),11.78(s,1H),8.33(d,J＝8.6Hz,2H),7.80(d,J＝8.6Hz,2H),6.00(s,1H),2.01(s,3H).

Example 3

Preparation of 6-chloro-3-methyl-4- (4-nitrophenyl) -6, 7-dihydro-1H-pyrazolo [3,4-b ] pyridine (intermediate VII)

5 g of intermediate VI was placed in a 100 ml flask, dissolved by adding 15 ml of phenylphosphoryl dichloride and reacted at 110 ℃ overnight. After the reaction is finished, a proper amount of ice water is poured into the system after the system is cooled, and the mixture is stirred for 1 hour, so that a large amount of solid is separated out. And (3) carrying out suction filtration, adjusting the filter cake to be alkalescent by using a saturated sodium bicarbonate solution, carrying out secondary suction filtration, rinsing the filter cake by using a large amount of water, and drying at 40 ℃ to obtain 3.1 g of a gray yellow solid (an intermediate VII), wherein the yield is 58%.

¹H-NMR(400MHz,DMSO-d₆)δ:13.71(s,1H),8.39(d,J＝8.7Hz,2H),7.90(d,J＝8.7Hz,2H),7.25(s,1H),2.16(s,3H).

Example 4

Preparation of 4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) aniline (intermediate VIII)

25 g of zinc powder, 70 ml of hydrazine monohydrate and 10ml of tetrahydrofuran are mixed in a 250 ml three-neck flask, 70 ml of tetrahydrofuran solution dissolved with 7 g of intermediate VII is dropwise added into a reaction system at a constant speed under the protection of nitrogen, and reflux reaction is carried out for 4 hours after the addition is finished. After the reaction, the reaction mixture was filtered, the filtrate was extracted three times with tetrahydrofuran, the extract was distilled under reduced pressure, and the residue was separated by column chromatography using an eluent (tetrahydrofuran: dichloromethane ═ 1:3, v/v) to give 3.1 g of a white solid (intermediate VIII) in a yield of 57%.

¹H-NMR(400MHz,DMSO-d₆)δ:13.20(s,1H),8.38(d,J＝4.7Hz,1H),7.24(d,J＝7.7Hz,2H),6.93(d,J＝4.7Hz,1H),6.69(d,J＝7.7Hz,2H),5.45(s,2H),2.27(s,3H).

Example 5

Preparation of 4,4, 4-trifluoro-3-carbonylbutyronitrile (intermediate IX)

Dissolving 0.5 g of sodium hydride in 5ml of anhydrous tetrahydrofuran, heating and refluxing to obtain a reaction system 1, then dissolving 0.4 g of acetonitrile in 2ml of anhydrous tetrahydrofuran, adding 1.2g of ethyl trifluoroacetate, dropwise adding the mixed solution into the reaction system 1, and reacting for 15 hours at 70 ℃ after the addition. After the reaction, the reaction mixture was cooled to room temperature, poured into water, extracted with an appropriate amount of anhydrous ether, and the aqueous phase was adjusted to pH 2 with 2M hydrochloric acid solution and extracted again with anhydrous ether. The total organic phase was dried over anhydrous sodium sulfate, filtered and the filtrate was distilled under reduced pressure to give a crude product of 4,4, 4-trifluoro-3-carbonylbutyronitrile (intermediate IX). The product has a low boiling point, no obvious fluorescence in ultraviolet, is difficult to monitor, and is not further purified, so the yield is calculated with the next step, and in the next step, the yield is calculated according to the complete reaction of ethyl trifluoroacetate, namely 100%.

Example 6

Preparation of 3-trifluoromethyl-5-amino-1H-pyrazole (intermediate X)

1.16 g of intermediate IX and 4.5ml of hydrazine hydrate are mixed, 5.7ml of methanesulfonic acid are added dropwise and, after the addition, the reaction is carried out overnight at 80 ℃. After completion of the reaction, the reaction mixture was distilled under reduced pressure, and the residue was dissolved in ethyl acetate/n-hexane 1:1, stirred for 5 minutes, filtered under suction, and the filtrate was distilled under reduced pressure, concentrated and subjected to column chromatography using an eluent (methanol/dichloromethane 1:60, v/v) to obtain 102 mg of a pale yellow oily substance (intermediate X). The overall yield in the two steps is 8% for examples 5 and 6.

¹H NMR(400MHz,DMSO-d₆)δ:12.15(s,1H),5.53(s,1H),5.35(s,2H).

Example 7

Preparation of intermediate 3-trifluoromethyl-4- (4-nitrophenyl) -4, 5-dihydro-1H-pyrazolo [3,4-b ] pyridin-6 (7H) -one (intermediate XI)

0.99 g of p-nitrobenzaldehyde and 0.95 g of 2, 2-dimethyl-1, 3-dioxane-4, 6-dione were put into a 50 ml round-bottomed flask, dissolved in 20 ml of ethanol, and then reacted at 80 ℃ for 3 hours with heating, followed by addition of 0.9 g of an ethanol solution (5ml) of intermediate X. After a solid had precipitated, the reaction was carried out for half an hour. After the reaction was complete, the reaction mixture was cooled to room temperature and diluted with 10ml of isopropanol. Suction filtration, sonication for 15 minutes after dissolution of the solid in methanol, followed by suction filtration and drying of the solid gave the title compound in 38% yield as a pale yellow solid (intermediate XI).

1H NMR(400MHz,DMSO-d₆)δ：13.66(s,1H),11.02(s,1H),8.19(d,J＝8.8Hz,2H),7.36(d,J＝8.8Hz,2H),4.55(t,J＝5.6Hz,1H),3.19(dd,J＝5.6Hz,2.8Hz,1H),2.56(d,J＝2.8Hz,1H).

Example 8

Preparation of 3-trifluoromethyl-4- (4-nitrophenyl) -1H-pyrazolo [3,4-b ] pyridin-6 (7H) -one (intermediate XII)

0.736 g of intermediate XII was placed in a 50 ml flask, dissolved in 15 ml of chlorobenzene, added with 2.7 ml of N, O-bis (trimethylsilyl) trifluoroacetamide and then 0.975 g of 2, 3-dichloro-5, 6-dicyano-1, 4-benzoquinone and refluxed overnight. After the reaction is finished, the reaction liquid is poured into a proper amount of water and saturated NaHCO is used₃The solution was made alkaline and a solid precipitated from the solution. Suction filtration, washing of the filter cake with water and drying at 60 ℃ gave 0.587 g of a pale yellow solid (intermediate XII) in 80% yield.

¹H-NMR(400MHz,DMSO-d₆)δ:14.15(s,1H),12.09(s,1H),8.28(d,J＝8.6Hz,2H),7.68(d,J＝8.6Hz,2H),6.46(s,1H).

Example 9

Preparation of 6-chloro-3-trifluoromethyl-4- (4-nitrophenyl) -6, 7-dihydro-1H-pyrazolo [3,4-b ] pyridine (intermediate XIII)

0.587 g of intermediate XII was placed in a 100 ml flask, dissolved by adding 10ml of phenylphosphoryl dichloride and reacted at 110 ℃ overnight. After the reaction is finished, a proper amount of ice water is poured into the system after the system is cooled, and the mixture is stirred for 1 hour, so that a large amount of solid is separated out. And (3) performing suction filtration, adjusting the filter cake to be alkalescent by using a saturated sodium bicarbonate solution, performing secondary suction filtration, rinsing the filter cake by using a large amount of water, drying at 60 ℃, dotting the plate, repeating the reaction operation if the intermediate XII does not completely react, and finally obtaining 300 mg of a gray yellow solid (the intermediate XIII), wherein the yield is 48%.

¹H-NMR(400MHz,DMSO-d₆)δ:14.96(s,1H),8.32(d,J＝8.4Hz,2H),7.74(d,J＝8.4Hz,2H),7.44(s,1H).

Example 10

Preparation of 4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) aniline (intermediate XIV)

342 mg of zinc powder, 2.6 ml of hydrazine monohydrate and 2ml of tetrahydrofuran are mixed in a 25 ml three-neck flask, 2ml of tetrahydrofuran solution dissolved with 200 mg of intermediate XIII is dropwise added into the reaction system at a constant speed under the protection of nitrogen, and after the addition is finished, reflux reaction is carried out overnight. After the reaction, the reaction mixture was filtered with suction, the filtrate was extracted three times with tetrahydrofuran, the extract was distilled under reduced pressure, and the residue was separated by column chromatography using an eluent (ethyl acetate: petroleum ether ═ 1:3, v/v) to give 90 mg of a white solid (intermediate XIV) with a yield of 55%.

¹H-NMR(400MHz,DMSO-d₆)δ:14.61(s,1H),8.60(d,J＝4.6Hz,1H),7.18(dd,J＝4.6Hz,8.2Hz,3H),6.66(d,J＝8.2Hz,2H),5.47(s,2H).

Example 11

N-ethyl-N' - (4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (Compound I_APreparation of (1)

50 mg of 4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) aniline (intermediate VIII) was dissolved in 1 ml of dimethyl sulfoxide, and 22. mu.l of ethyl isocyanate and 10. mu.l of triethylamine were added thereto and reacted at room temperature overnight. After the reaction was completed, an appropriate amount of water was added to the system, extracted three times with ethyl acetate, dried over anhydrous magnesium sulfate, filtered, distilled under reduced pressure, and the residue was separated by column chromatography (ethyl acetate: petroleum ether ═ 1:3, v/v), and the resulting solid was washed with ether to give the title compound in the form of 45 mg of yellow powder with a yield of 69%. And Mp: 211-213 ℃; purity: 93 percent;

¹H NMR(400MHz,CDCl₃)δ:9.17(s,1H),8.49(d,J＝4.8Hz,2H),7.30(d,J＝8.0Hz,2H),7.12(d,J＝4.7Hz,1H),6.83(d,J＝8.0Hz,2H),3.62(q,2H),2.36(s,3H),1.35(t,J＝7.2Hz,3H)；HRMS(ESI)m/z calcd forC₁₆H₁₈N₅O[M+H]⁺296.1511,found 296.1506.

example 12

N-propyl-N' - (4- (3-methyl-1H-pyrazolo [3, 4-b))]Pyridin-4-yl) phenyl) urea (Compound I_APreparation of (2)

Except that the ethyl isocyanate is replaced by the propyl isocyanate, other required raw materials, reagents and preparation methods are the same as those of the raw material I_A-1 to give the title compound as yellow powder 44 mg, yield 64%. And Mp: 208-210 ℃; purity: 92 percent;

¹H NMR(400MHz,CDCl₃)δ:9.15(t,J＝5.5Hz,1H),8.56(d,J＝4.9Hz,1H),7.28(d,J＝8.2Hz,2H),7.22(d,J＝5.0Hz,1H),6.72(d,J＝8.3Hz,2H),3.42-3.35(t,2H),2.30(s,3H),1.68-1.55(m,2H),0.95(t,J＝7.4Hz,3H)；HRMS(ESI)m/z calcd for C₁₇H₂₀N₅O[M+H]⁺310.1668,found 310.1662.

example 13

N-butyl-N' - (4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (Compound I_APreparation of (3)

Except that the ethyl isocyanate is replaced by the butyl isocyanate, other required raw materials, reagents and preparation methods are the same as those of the raw material I_A-1 to give the title compound as a yellow powder in 62 mg, 72% yield. And Mp: 177 to 180 ℃; purity: 95 percent;

¹H NMR(400MHz,CDCl₃)δ:9.20(s,1H),8.49(d,J＝4.8Hz),7.27(d,J＝7.8Hz),7.12(d,J＝4.8Hz),6.81(d,J＝7.8Hz,2H),3.58(dd,J＝13.0,6.4Hz,2H),2.38(s,3H),1.81-1.64(m,2H),1.48(m,2H),0.99(t,J＝7.3Hz)；HRMS(ESI)m/z calcd for C₁₈H₂₂N₅O[M+H]⁺324.1824,found 324.1819.

example 14

N-isobutyl-N' - (4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (Compound I_APreparation of (4)

After dissolving 302. mu.l of isobutylamine in 2ml of dimethyl sulfoxide, 583 mg of carbonyldiimidazole was added and reacted at room temperature for two hours, 150 mg of 4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) aniline (intermediate VIII) and 40. mu.l of triethylamine were added thereto and reacted at room temperature overnight without further treatment. After the reaction was completed, an appropriate amount of water was added to the system, extracted three times with ethyl acetate, dried over anhydrous magnesium sulfate, filtered, distilled under reduced pressure, and the residue was separated by column chromatography (methanol: dichloromethane ═ 1:30, v/v) to obtain 150 mg of the title compound in a yield of 69%. And Mp: 262 to 265 ℃; purity: 96 percent;

¹H NMR(400MHz,DMSO-d₆)δ:13.30(s,1H),8.64(s,1H),8.44(d,J＝4.7Hz,1H),7.56(d,J＝8.5Hz,2H),7.43(d,J＝8.5Hz,2H),7.00(d,J＝4.7Hz,1H),6.27(t,J＝5.8Hz,1H),2.95(t,J＝6.2Hz,2H),2.23(s,3H),1.71(dt,J＝13.5,6.6Hz,1H),0.89(d,J＝6.7Hz,6H)；HRMS(ESI)m/z calcd for C₁₈H₂₂N₅O[M+H]⁺324.1825,found 324.1819.

example 15

N-tert-butyl-N' - (4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (Compound I_APreparation of (5)

Except for replacing isobutylamine with tert-butylamine, the other needed raw materials, reagents and preparation method are the same as I_A-4 to give the title compound 120 mg in 56% yield. And Mp: 244 to 251 ℃; purity: 100 percent;

¹H NMR(400MHz,DMSO-d₆)δ:13.30(s,1H),8.47(s,1H),8.44(d,J＝4.7Hz,1H),7.52(d,J＝8.5Hz,2H),7.42(d,J＝8.5Hz,2H),6.99(d,J＝4.7Hz,1H),6.08(s,1H),2.23(s,3H),1.31(s,9H)；HRMS(ESI)m/z calcd for C₁₈H₂₁N₅NaO[M+Na]⁺346.1644,found346.1638.

example 16

N-N-pentyl-N' - (4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (Compound I_APreparation of (6)

Except that the iso-butylamine is replaced by the n-pentylamine, other required raw materials, reagents and preparation methods are the same as those of the formula I_A-4 to give the title compound 150 mg in 68% yield. And Mp: 233 to 236 ℃; purity: 98 percent;

¹H NMR(400MHz,DMSO-d₆)δ:13.31(s,1H),8.64(s,1H),8.45(d,1H,J＝4.5Hz),7.56(d,J＝8.3Hz,2H),7.43(d,J＝8.2Hz,2H),7.00(d,J＝4.6Hz,1H),6.22(t,1H),3.10(dd,2H),2.24(s,3H),1.45(m,2H),1.30(d,4H),0.89(t,3H)；HRMS(ESI)m/z calcd forC₁₉H₂₄N₅O[M+H]⁺338.1981,found 338.1975.

example 17

N-N-hexyl-N' - (4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (Compound I_APreparation of (7)

Except that n-hexylamine is replaced by isobutylamine, other required raw materials, reagents and preparation methods are the same as those of I_A-4 to give the title compound 120 mg in 52% yield. And Mp: 213-215 ℃; purity: 98 percent;

¹H NMR(400MHz,DMSO-d6)δ:13.30(s,1H,NH),8.63(s,1H,NH),8.44(d,J＝4.7Hz,1H,ArH),7.56(d,J＝8.5Hz,2H,ArH),7.42(d,J＝8.4Hz,2H,ArH),7.00(d,J＝4.7Hz,1H,ArH),6.21(t,J＝5.5Hz,1H,NH),3.10(dd,J＝12.8,6.5Hz,2H,CH2),2.23(s,3H,CH3),1.43(d,J＝6.5Hz,2H,CH2),1.29(m,6H,CH2),0.88(t,J＝6.4Hz,3H,CH3)；HRMS(ESI)m/z calcdfor C20H25N5NaO[M+Na]⁺374.1957,found 374.1951.

example 18

N-cyclopropyl-N' - (4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (Compound I_APreparation of (1) to (8)

Except for replacing isobutylamine with cyclopropylamine, the other required raw materials, reagents and preparation method are the same as those in I_A-4 to give the title compound 130 mg, yield 63%. And Mp: 247-250 ℃; purity: 97 percent;

¹H NMR(400MHz,DMSO-d₆)δ:13.30(s,1H),8.53(s,1H),8.45(d,J＝4.7Hz,1H),7.58(d,J＝8.5Hz,2H),7.43(d,J＝8.5Hz,2H),7.00(d,J＝4.7Hz,1H),6.49(d,1H),2.60-2.55(m,1H),2.23(s,3H),0.65(q,J＝6.7Hz,2H),0.46-0.39(m,2H)；HRMS(ESI)m/z calcdfor C₁₇H₁₈N₅O[M+H]+308.1511,found 308.1506.

example 19

N-cyclobutyl-N' - (4- (3-methyl-1H-pyrazolo [3, 4-b))]Pyridin-4-yl) phenyl) urea (Compound I_APreparation of (2) to (9)

Except for replacing isobutylamine with cyclobutylamine, other required raw materials, reagents and preparation method are the same as those of I_A-4 to give the title compound 160 mg, yield 67%. And Mp: 240-243 ℃; purity: 99 percent;

¹H NMR(400MHz,DMSO-d₆)δ:13.30(s,1H),8.56(s,1H),8.44(d,J＝4.7Hz,1H),7.55(d,J＝8.5Hz,2H),7.42(d,J＝8.4Hz,2H,ArH),7.00(d,J＝4.6Hz,1H,ArH),6.50(d,J＝8.1Hz,1H,NH),4.15(dd,J＝16.2,8.3Hz,1H,CH),2.23(s,3H,CH3),2.19(dd,2H,CH2),1.86(dd,J＝19.6,10.3Hz,2H,CH2),1.70-1.55(m,2H,CH2)；HRMS(ESI)m/z calcd forC18H20N5O[M+H]+322.1668,found 322.1662.

example 20

N-cyclopentyl-N' - (4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (Compound I_APreparation of (1) to (10)

Except that the isobutylamine is replaced by the cyclopentylamine, other required raw materials, reagents and preparation methods are the same as those of the formula I_A-4 to give the title compound 200 mg, 89% yield. And Mp: 258 to 262 ℃; purity: 99 percent;

¹H NMR(400MHz,DMSO-d₆)δ:13.30(s,1H),8.49(s,1H),8.44(d,J＝4.7Hz,1H),7.55(d,J＝8.5Hz,2H),7.42(d,J＝8.5Hz,2H),7.00(d,J＝4.7Hz,1H),6.25(d,J＝7.2Hz,1H),3.96(dq,J＝13.5,6.7Hz,1H),2.23(s,3H),1.85(dt,J＝12.2,6.0Hz,2H),1.62(dd,J＝16.4,5.9Hz,2H),1.56(dd,J＝14.2,7.2Hz,2H),1.39(dt,J＝12.2,6.3Hz,2H)；HRMS(ESI)m/z calcd for C₁₉H₂₂N₅O[M+H]⁺336.1824,found 336.1819.

example 21

N-cyclohexyl-N' - (4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (Compound I_APreparation of (11)

Except that the ethyl isocyanate is replaced by the cyclohexyl isocyanate, other required raw materials, reagents and preparation methods are the same as those of the raw material I_A-1，The title compound was obtained in the form of a yellow powder (40 mg) with a yield of 52%. And Mp: 233 to 237 ℃; purity: 95 percent;

¹H NMR(400MHz,DMSO-d₆)δ:13.30(s,1H),8.53(s,1H),8.44(d,J＝4.6Hz,1H),7.54(d,J＝8.4Hz,2H),7.42(d,J＝8.4Hz,2H),7.00(d,J＝4.7Hz,1H),6.17(d,J＝7.8Hz,1H),3.54-3.41(m,1H),2.23(s,3H),1.82(d,J＝8.9Hz,2H),1.67(d,J＝12.8Hz,2H),1.55(d,J＝12.0Hz,1H),1.32(dd,J＝23.6,11.7Hz,2H),1.18(dd,J＝21.8,12.1Hz,3H)；HRMS(ESI)m/z calcd for C₂₀H₂₄N₅O[M+H]⁺390.1981,found 350.1975.

example 22

N- (furan-2-ylmethyl) -N' - (4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (Compound I_APreparation of (12)

Except that the isobutylamine is replaced by 2-furanmethylamine, the other needed raw materials, reagents and preparation method are the same as I_A-4 to give the title compound 160 mg in 70% yield. And Mp: 232-234 ℃; purity: 100 percent;

¹H NMR(400MHz,DMSO-d₆)δ:13.31(s,1H),8.77(s,1H),8.45(d,J＝4.7Hz,1H),7.60(s,1H),7.57(d,J＝8.5Hz,2H),7.44(d,J＝8.5Hz,2H),7.00(d,J＝4.7Hz,1H),6.65(t,J＝5.5Hz,1H),6.41(s,1H),6.28(s,1H),4.32(d,J＝5.6Hz,2H),2.23(s,3H)；HRMS(ESI)m/z calcd for C₁₉H₁₈N₅O₂[M+H]⁺348.1460,found 348.1455.

example 23

N- (thien-2-ylmethyl) -N' - (4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (Compound I_APreparation of (E) -13)

Except that isobutylamine is replaced by 2-thiophenemethylamine, other needed raw materials, reagents and preparation method are the same as those in the step I_A-4 to give the title compound 160 mg, yield 67%. And Mp: 233 to 235 ℃; purity: 100 percent;

¹H NMR(400MHz,DMSO-d₆)δ:13.31(s,1H),8.81(s,1H),8.45(d,J＝4.7Hz,1H),7.58(d,J＝8.5Hz,2H),7.44(d,J＝8.4Hz,2H),7.40(d,J＝4.8Hz,1H),7.04-6.94(m,3H),6.77(t,J＝5.8Hz,1H),4.49(d,J＝5.8Hz,2H),2.23(s,3H)；HRMS(ESI)m/z calcd forC₁₉H₁₇N₅NaOS[M+Na]⁺386.1052,found 386.1046.

example 24

N- (pyridin-4-ylmethyl) -N' - (4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (Compound I_APreparation of (1) to (14)

Except that isobutylamine is replaced by 4-methylaminopyridine, other required raw materials, reagents and preparation methods are the same as those in the step I_A-4 to give the title compound 160 mg, yield 68%. And Mp: 218-222 ℃; purity: 97 percent;

¹H NMR(400MHz,DMSO-d₆)δ:13.31(s,1H),8.97(s,1H),8.52(d,J＝4.7Hz,2H),8.45(d,J＝4.7Hz,1H),7.59(d,J＝8.4Hz,2H),7.44(d,J＝8.4Hz,2H),7.32(d,J＝5.0Hz,2H),7.00(d,J＝4.6Hz,1H),6.85(t,J＝5.9Hz,1H),4.36(d,J＝5.9Hz,2H),2.23(s,3H)；HRMS(ESI)m/z calcd for C₂₀H₁₉N₆O[M+H]⁺359.1620,found 359.1615.

example 25

N-benzyl-N' - (4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (Compound I_APreparation of (E) -15)

Except that the ethyl isocyanate is replaced by the benzyl isocyanate, other required raw materials, reagents and preparation methods are the same as those of the formula I_A-1, to give the title compound as yellow powder 40 mg, yield 50%. And Mp: 91-97 ℃; purity: 93 percent;

¹H NMR(400MHz,DMSO-d₆)δ:9.56(t,J＝5.9Hz,1H),8.55(d,J＝5.0Hz,1H),7.37(m,4H),7.27(t,J＝7.8Hz,3H),7.23(d,J＝5.0Hz,1H),6.71(d,J＝8.4Hz,2H),4.63(d,J＝6.0Hz,2H),2.32(s,3H)；HRMS(ESI)m/z calcd for C₂₁H₂₀N₅O[M+H]⁺358.1668,found358.1662.

example 26

N- (3-methylbenzyl) -N' - (4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (Compound I_APreparation of-16)

Except that the ethyl isocyanate is replaced by the 3-methylbenzyl isocyanate, other required raw materials, reagents and preparation methods are the same as those of the formula I_A-1, to give the title compound as yellow powder 50 mg, yield 60%. And Mp: 140-145 ℃; purity: 97 percent;

¹H NMR(400MHz,DMSO-d₆)δ:9.54(t,J＝6.0Hz,1H),8.55(d,J＝5.0Hz,1H),7.28(d,J＝8.4Hz,2H),7.25-7.16(m,4H),7.08(d,J＝7.2Hz,1H),6.71(d,J＝8.4Hz,2H),4.59(d,J＝5.9Hz,2H),2.31(d,J＝6.1Hz,3H),2.29(s,3H)；HRMS(ESI)m/z calcd for C₂₂H₂₂N₅O[M+H]⁺372.1824,found 372.1819.

example 27

N- (4-methylbenzyl) -N' - (4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (Compound I_APreparation of (E) -17)

Except for replacing the isobutylamine by the p-methylbenzylamine, other needed raw materials, reagents and preparation methods are the same as I_A-4 to give the title compound 170 mg in 69% yield. And Mp: 275-276 ℃; purity: 99 percent;

¹H NMR(400MHz,DMSO-d₆)δ:13.31(s,1H),8.78(s,1H,NH),8.45(d,J＝4.7Hz,1H),7.58(d,J＝8.5Hz,2H),7.44(d,J＝8.5Hz,2H),7.21(d,J＝7.9Hz,2H),7.15(d,J＝7.9Hz,2H),7.00(d,J＝4.7Hz,1H),6.66(t,J＝5.7Hz,1H),4.28(d,J＝5.8Hz,2H),2.29(s,3H),2.23(s,3H)；HRMS(ESI)m/z calcd forC₂₂H₂₂N₅O[M+H]⁺372.1824,found 372.1819.

example 28

N- (2-fluorobenzyl) -N' - (4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (Compound I_APreparation of (18)

Except for replacing isobutylamine with o-fluorobenzylamine, other needed raw materials, reagents and preparation methodsFangtong I_A-4 to give the title compound 210 mg, 84% yield. And Mp: 248-251 ℃; purity: 97 percent;

¹H NMR(400MHz,DMSO-d₆)δ:13.31(s,1H),8.85(s,1H),8.45(d,J＝4.7Hz,1H),7.57(d,J＝8.5Hz,2H),7.44(d,J＝8.5Hz,2H),7.40(d,J＝7.5Hz,1H),7.33(dd,J＝13.5,6.8Hz,1H),7.20(t,J＝8.0Hz,2H),7.00(d,J＝4.7Hz,1H),6.73(t,J＝5.8Hz,1H),4.38(d,J＝5.8Hz,2H),2.23(s,3H)；HRMS(ESI)m/z calcd for C₂₁H₁₈FN₅NaO[M+Na]⁺398.1393,found 398.1388.

example 29

N- (2-chlorobenzyl) -N' - (4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (Compound I_APreparation of (E) -19)

Except for replacing isobutylamine by o-chlorobenzylamine, other needed raw materials, reagents and preparation method are the same as I_A-4 to give the title compound 210 mg in 79% yield. And Mp: 254 to 256 ℃; purity: 96 percent;

¹H NMR(400MHz,DMSO-d₆)δ:13.31(s,1H),8.94(s,1H),8.45(d,J＝4.7Hz,1H),7.58(d,J＝8.3Hz,2H),7.45(m,4H),7.34(dt,J＝14.5,6.9Hz,2H),7.00(d,J＝4.7Hz,1H),6.77(t,J＝5.7Hz,1H),4.40(d,J＝5.7Hz,2H),2.23(s,3H)；HRMS(ESI)m/z calcd forC₂₁H₁₉ClN₅O[M+H]⁺392.1278,found 392.1273.

example 30

N- (2, 6-difluorobenzyl) -N' - (4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (Compound I_APreparation of (E) -20)

Except that isobutylamine is replaced by 2, 6-difluorobenzylamine, the other required raw materials, reagents and preparation method are the same as those in the step I_A-4 to give the title compound 200 mg, yield 77%. And Mp: 254 to 256 ℃; purity: 97 percent;

¹H NMR(400MHz,DMSO-d₆)δ:8.68(s,1H),8.44(d,J＝4.7Hz,1H),7.54(d,J＝8.5Hz,2H),7.40(dd,3H),7.10(dt,J＝19.6,7.8Hz,3H),6.99(d,J＝4.7Hz,1H),6.70(t,J＝5.6Hz,1H),4.41(d,J＝5.6Hz,2H),2.22(s,3H)；HRMS(ESI)m/z calcd for C₂₁H₁₈F₂N₅O[M+H]⁺394.1479,found 394.1474.

example 31

N- (2-phenylethyl) -N' - (4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (Compound I_APreparation of (1) to (21)

Except that the isobutylamine is replaced by phenethylamine, other required raw materials, reagents and preparation methods are the same as those of the formula I_A-4 to give the title compound 200 mg, yield 82%. And Mp: 221-224 ℃; purity: 99 percent;

¹H NMR(400MHz,DMSO-d₆)δ:13.31(s,1H),8.73(s,1H),8.45(d,J＝4.7Hz,1H),7.56(d,J＝8.5Hz,2H),7.43(d,J＝8.4Hz,2H),7.32(d,J＝7.4Hz,2H),7.24(dd,J＝18.6,7.2Hz,3H),7.00(d,J＝4.7Hz,1H),6.22(t,J＝5.6Hz,1H),3.37(t,2H),2.78(t,J＝7.1Hz,2H),2.23(s,3H)；HRMS(ESI)m/z calcd for C₂₂H₂₂N₅O[M+H]⁺372.1824,found 372.1819.

example 32

N- (4-fluorophenethyl) -N' - (4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (Compound I_APreparation of-22)

Except that ethyl isocyanate is replaced by 4-fluorophenethyl isocyanate, other needed raw materials, reagents and preparation methods are the same as those in the step I_A-1, to give 60 mg of the title compound in 23% yield. And Mp: 185-189 ℃ and purity: 97 percent;

¹H NMR(400MHz,CDCl₃)δ:9.26(s,1H),8.38(d,J＝4.9Hz,1H),7.26(dd,J＝8.3,3.5Hz,4H),7.11(d,J＝4.9Hz,1H),6.99(dd,J＝16.8,8.3Hz,2H),6.81(d,J＝8.2Hz,2H),3.86-3.77(t,2H),3.00(t,J＝6.9Hz,2H),2.36(s,3H)；HRMS(ESI)m/z calcd forC₂₂H₂₁FN₅O[M+H]⁺390.1730,found 390.1725.

example 33

N- (4-Chlorobenzoethyl) -N' - (4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (Compound I_APreparation of (E) -23)

Except that isobutylamine is replaced by 4-chlorophenylethylamine, other required raw materials, reagents and preparation method are the same as those in the step I_A-4 to give the title compound 220 mg in 82% yield. And Mp: 244 to 247 ℃; purity: 99 percent;

¹H NMR(400MHz,DMSO-d₆)δ:13.30(s,1H),8.71(s,1H),8.45(d,J＝4.7Hz,1H),7.55(d,J＝8.5Hz,2H),7.43(d,J＝8.5Hz,2H),7.38(d,J＝8.3Hz,2H),7.29(d,J＝8.3Hz,2H),7.00(d,J＝4.7Hz,1H),6.21(t,J＝5.6Hz,1H),3.36(t,2H),2.77(t,J＝7.0Hz,2H),2.23(s,3H)；HRMS(ESI)m/z calcd for C₂₂H₂₁ClN₅O[M+H]⁺406.1435,found 406.1429.

example 34

N- (3, 4-Dimethoxyphenethyl) -N' - (4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (Compound I_APreparation of (E) -24)

Except that ethyl isocyanate is replaced by 3, 4-dimethoxy phenethyl isocyanate, other required raw materials, reagents and preparation methods are the same as those of the raw material I_A-1, to give the title compound 50 mg, yield 18%. And Mp: 178-181 ℃; purity: 92 percent;

¹H NMR(400MHz,CDCl₃)δ:9.18(t,J＝5.4Hz,1H),8.48(d,J＝5.0Hz,1H),7.27(d,J＝8.3Hz,2H),7.22(d,J＝5.0Hz,1H),6.93-6.86(m,2H),6.81(d,J＝7.9Hz,1H),6.71(d,J＝8.3Hz,2H),3.72(s,3H),3.69(s,3H),3.64(dd,J＝12.6,6.6Hz,2H),2.86(t,J＝6.9Hz,2H),2.31(s,3H)；HRMS(ESI)m/z calcd for C₂₄H₂₆N₅O₃[M+H]⁺432.2036,found 432.2030.

example 35

N- (3-phenylpropyl) -N' - (4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (Compound I_APreparation of (E) -25)

In addition to the replacement of isobutylamine by amphetamineThe starting materials, reagents and methods of preparation were as in example I_A-4, to give the title compound 180 mg, yield 70%. And Mp: 223-225 ℃; purity: 98 percent;

¹H NMR(400MHz,DMSO-d₆)δ:13.31(s,1H),8.68(s,1H),8.45(d,J＝4.7Hz,1H),7.57(d,J＝8.5Hz,2H),7.43(d,J＝8.5Hz,2H),7.30(t,J＝7.4Hz,2H),7.23(d,J＝7.2Hz,2H),7.19(t,J＝7.1Hz,1H),7.00(d,J＝4.7Hz,1H),6.31(t,J＝5.5Hz,1H),3.13(dd,J＝12.8,6.5Hz,2H),2.63(t,J＝7.6Hz,2H),2.24(s,3H),1.83-1.67(m,2H)；HRMS(ESI)m/zcalcd for C₂₃H₂₃N₅NaO[M+Na]⁺408.1800,found 408.1795.

example 36

N- (4-phenylbutyl) -N' - (4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (Compound I_APreparation of (2) to (26)

Except for replacing isobutylamine with phentermine, the other required raw materials, reagents and preparation method are the same as those in I_A-4 to give the title compound 200 mg, yield 75%. And Mp: 224 to 227 ℃; purity: 92 percent;

¹H NMR(400MHz,DMSO-d₆)δ:13.30(s,1H),8.63(s,1H),8.44(d,J＝4.6Hz,1H),7.55(d,J＝8.4Hz,2H),7.42(d,J＝8.3Hz,2H),7.28(t,J＝7.4Hz,2H),7.24-7.13(m,3H),6.99(d,J＝4.6Hz,1H),6.24(t,J＝5.5Hz,1H),3.13(dd,J＝12.6,6.4Hz,2H),2.61(t,J＝7.4Hz,2H),2.23(s,3H),1.60(dd,J＝14.8,7.3Hz,2H),1.47(dd,J＝14.5,7.0Hz,2H)；HRMS(ESI)m/z calcd for C₂₄H₂₅N₅NaO[M+Na]⁺422.1957,found 422.1951.

example 37

N- (1H-pyrazol-5-yl) -N' - (4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (Compound I_APreparation of (E) -27)

Except that isobutylamine is replaced by 3-aminopyrazole, other required raw materials, reagents and preparation methods are the same as I_A-4, to give the title compound 60 mg in 30% yield. And Mp: 260-263 ℃; purity: 99 percent;

¹H NMR(400MHz,DMSO-d₆)δ:13.33(s,1H),12.28(s,1H),9.26(s,1H),9.06(s,1H),8.46(d,J＝4.7Hz,1H),7.63(d,J＝8.4Hz,3H),7.49(d,J＝8.5Hz,2H),7.03(d,J＝4.7Hz,1H),6.29(s,1H),2.24(s,3H)；HRMS(ESI)m/z calcd for C₁₇H₁₅N₇NaO[M+Na]⁺356.1236,found 356.1230.

example 38

N- (pyridin-3-yl) -N' - (4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (Compound I_APreparation of (E) -28)

Except that 3-pyridylamine is replaced by isobutylamine, the other required raw materials, reagents and preparation method are the same as those in the step I_A-4 to give the title compound 120 mg in 52% yield. And Mp: 245-247 ℃; purity: 98 percent;

¹H NMR(400MHz,DMSO-d₆)δ:13.33(s,1H),9.06(s,1H),8.64(s,1H),8.47(d,J＝4.7Hz,1H),8.21(d,J＝4.1Hz,1H),7.97(d,J＝7.6Hz,1H),7.65(d,J＝8.5Hz,2H),7.51(d,J＝8.5Hz,2H),7.34(dd,J＝8.3,4.7Hz,1H),7.03(d,J＝4.7Hz,1H),6.66(s,1H),2.25(s,3H)；HRMS(ESI)m/z calcd for C₁₉H₁₆N₆NaO[M+Na]⁺367.1283,found 367.1278.

example 39

N- (pyridin-2-yl) -N' - (4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (Compound I_APreparation of (E) -29)

Except that the isobutylamine is replaced by 2-pyridylamine, the other required raw materials, reagents and preparation method are the same as those in the step I_A-4 to give the title compound 46 mg in 30% yield. And Mp: 244 to 248 ℃; purity: 96 percent;

¹H NMR(400MHz,DMSO-d₆)δ:13.32(s,1H),10.74(s,1H),9.53(s,1H),8.46(d,J＝4.7Hz,1H),8.30(d,J＝4.9Hz,1H),7.76(t,J＝7.8Hz,1H),7.70(d,J＝8.4Hz,2H),7.51(t,J＝8.0Hz,3H),7.02(dd,J＝8.1,4.0Hz,2H),2.23(s,3H)；HRMS(ESI)m/z calcd forC₁₉H₁₆N₆NaO[M+Na]⁺367.1283,found 367.1278.

example 40

N- (2, 3-dihydro-1H-inden-2-yl) -N' - (4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (Compound I_APreparation of (E) -30)

Except that the isobutylamine is replaced by 2-aminoindene, the other required raw materials, reagents and preparation method are the same as those in the step I_A-4 to give 50 mg of the title compound in 20% yield. And Mp: 249-251 ℃; purity: 96 percent;

¹H NMR(400MHz,DMSO-d₆)δ:13.30(s,1H),8.55(s,1H),8.44(d,J＝4.7Hz,1H),7.55(d,J＝8.5Hz,2H),7.43(d,J＝8.5Hz,2H),7.30-7.22(m,2H),7.21-7.14(m,2H),7.00(d,J＝4.7Hz,1H),6.55(d,J＝7.3Hz,1H),4.45(m,1H),3.21(dd,J＝15.9,7.0Hz,2H),2.80(dd,J＝15.9,5.0Hz,2H),2.22(d,J＝5.9Hz,3H)；HRMS(ESI)m/z calcd for C₂₃H₂₂N₅O[M+H]⁺384.1824,found 384.1819.

EXAMPLE 41

N- (naphthalen-2-yl) -N' - (4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (Compound I_APreparation of (E) -31)

Except that the isobutylamine is replaced by 2-naphthylamine, the other required raw materials, reagents and preparation method are the same as those in the step I_A-4 to give 90 mg of the title compound in 34% yield. And Mp: 232-235 ℃; purity: 100 percent;

¹H NMR(400MHz,DMSO-d₆)δ:13.32(s,1H),8.99(d,J＝5.5Hz,2H),8.47(d,J＝4.7Hz,1H),8.14(s,1H),7.89-7.78(m,3H),7.68(d,J＝8.6Hz,2H),7.55-7.49(m,3H),7.49-7.43(m,1H),7.37(t,J＝7.5Hz,1H),7.04(d,J＝4.7Hz,1H),2.25(d,J＝8.6Hz,3H)；HRMS(ESI)m/z calcd for C₂₄H₂₀N₅O[M+1]⁺394.1668,found 394.1662.

example 42

N- (quinolin-3-yl) -N' - (4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (Compound I_APreparation of (2) to (32)

Except that isobutylamine is replaced by 3-aminoquinoline, other required raw materials, reagents and preparation method are the same as those in the step I_A-4 to give the title compound 80 mg in 30% yield. And Mp: 226-229 ℃; purity: 99 percent;

¹H NMR(400MHz,DMSO-d₆)δ:13.33(s,1H),9.23(s,1H),9.15(s,1H),8.87(d,J＝2.5Hz,1H),8.56(d,J＝2.3Hz,1H),8.47(d,J＝4.7Hz,1H),7.93(dd,J＝13.8,8.3Hz,2H),7.69(d,J＝8.5Hz,2H),7.64-7.55(m,2H),7.53(d,J＝8.5Hz,2H),7.04(d,J＝4.7Hz,1H),2.26(s,3H)；HRMS(ESI)m/z calcd for C₂₃H₁₉N₆O[M+H]⁺395.1620,found 395.1615.

example 43

N- (naphthalen-1-yl) -N' - (4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (Compound I_APreparation of (E) -33)

Except that isobutylamine is replaced by 1-naphthylamine, the other required raw materials, reagents and preparation method are the same as those in the step I_A-4 to give the title compound 170 mg in 69% yield. And Mp: 225-228 ℃; purity: 99 percent;

¹H NMR(400MHz,DMSO-d6)δ:13.34(s,1H),8.87(s,1H),8.48(d,J＝4.6Hz,1H),8.15(d,J＝8.4Hz,1H),8.03(d,J＝7.5Hz,1H),7.96(d,J＝8.0Hz,1H),7.75-7.47(m,9H),7.04(d,J＝4.6Hz,1H),2.26(s,3H)；HRMS(ESI)m/z calcd forC₂₄H₂₀N₅O[M+H]⁺394.1668,found 394.1662.

example 44

N- (2-morpholin-4-ylethyl) -N' - (4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (I)_APreparation of (E) -34)

395 microliter of N- (2-aminoethyl) morpholine was dissolved in 4.6 ml of N, N-dimethylformamide, and 2.25 ml of pyridine was added to obtain reaction system 1. 452 microliters of phenyl chloroformate was dissolved in 2 milliliters of tetrahydrofuran solution and added dropwise to the reaction system 1 while being cooled on ice. Reacting at normal temperature for 3 hours, after the reaction is finished, adding a proper amount of water into the system, extracting with ethyl acetate for three times, washing with saturated salt water, drying with anhydrous magnesium sulfate, filtering, and concentrating to obtain a residue. The residue was dissolved in 5ml dioxane, and 60 mg 4- (3-methyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) aniline (intermediate VIII) and 20. mu.l 1-methylpyrrolidine were added to the solution and the mixture was refluxed overnight. After completion of the reaction, it was cooled to room temperature, distilled under reduced pressure, and the residue was separated by column chromatography (methanol: dichloromethane ═ 1:15, v/v) to give the title compound in 44% yield as a yellow solid. And Mp: 98-101 parts of; purity: 95 percent.

¹H-NMR(400MHz,DMSO-d₆)δ:13.30(s,1H),8.88(s,1H),8.45(d,J＝4.8Hz,1H),7.57(d,J＝8.4Hz,2H),7.43(d,J＝8.4Hz,2H),7.00(d,J＝4.8Hz,1H),6.18(t,J＝5.2Hz,1H),3.61(brs,4H),3.55(t,J＝5.6Hz,2H),3.24(q,J＝11.6Hz,5.6Hz,2H),2.41(brs,4H),2.23(s,3H)；HRMS(ESI)m/z calcd for C₂₀H₂₅N₆O₂[M+H]⁺381.2034,found 381.2029.

Example 45

N- (2-dimethylaminoethyl) -N' - (4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (I)_APreparation of (E) -35)

Except that N- (2-aminoethyl)Except for replacing morpholine by N, N-dimethylethylenediamine, the other needed raw materials, reagents and preparation method are the same as those of I_A-34 to give the title compound 39 mg as yellow oil in 43% yield. And Mp: 54-56 ℃; purity: 99 percent;

¹H-NMR(400MHz,DMSO-d₆)δ::13.32(s,1H),9.01(s,1H),8.46(d,J＝4.8Hz,1H),7.56(d,J＝8.4Hz,2H),7.43(d,J＝8.4Hz,2H),7.00(d,J＝4.8Hz,1H),6.29(t,J＝5.6Hz,1H),3.24(q,J＝11.6Hz,6.0Hz,2H),2.45(t,J＝6.0Hz,2H),2.27(s,6H),2.24(s,3H)；HRMS(ESI)m/z calcd forC₁₈H₂₃N₆O[M+H]⁺339.1928,found339.1927.

example 46

N- (2-methyl-4- (4-methylpiperazin-1-yl) phenyl) -N' - (4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (I)_APreparation of (E) -36)

1 g of 5-fluoro-2-nitrotoluene and 1.783 g of potassium carbonate were dissolved in 7ml of dimethyl sulfoxide, 1.072 ml of N-methylpiperazine was added, and the mixture was stirred at 122 ℃ overnight. After the reaction is finished, cooling to room temperature, adding a proper amount of water into the system, extracting with ethyl acetate for three times, washing with saturated salt water, drying with anhydrous magnesium sulfate, filtering, and concentrating to obtain an intermediate XV: 1.2 g.

1.2g of intermediate XV are dissolved in methanol, 120 mg of 10% Pd/C are added, hydrogen is introduced and the reaction is carried out overnight at room temperature. After the reaction is complete, it is filtered, concentrated and the residue is isolated by column chromatography (ethyl acetate: petroleum ether: 1:2, v/v) to give intermediate XVI: 0.935 g.

Except for exchanging N- (2-aminoethyl) morpholine for intermediate XVI, the other required raw materials, reagents and preparation method are the same as I_A-34 to give the title compound in a 24% yield as a 29 mg pink solid. And Mp: 263 to 264; purity: 95 percent.

¹H-NMR(400MHz,DMSO-d₆)δ::13.33(s,1H),9.09(s,1H),8.46(d,J＝4.8Hz,1H),7.86(s,1H),7.62(d,J＝8.4Hz,1H),7.48(d,J＝8.4Hz,3H),7.02(d,J＝4.8Hz,1H),6.81(s,1H),6.75(d,J＝8.8Hz,1H),3.07(t,J＝4.4Hz,4H),2.45(t,J＝4.4Hz,4H),2.25(s,3H),2.21(s,6H)；HRMS(ESI)m/z calcd for C₂₆H₃₀N₇O[M+H]⁺456.2506,found 456.2504.

Example 47

N- (3-methyl-4- (4-methylpiperazin-1-yl) phenyl) -N' - (4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (I)_APreparation of (E) -37)

Except for changing 5-fluoro-2-nitrotoluene into 2-fluoro-5-nitrotoluene, other required raw materials, reagents and preparation method are the same as I_A-36 to give the title compound 24 mg as a milky white solid in 20% yield. And Mp: 236-238; purity: 95 percent;

¹H-NMR(400MHz,DMSO-d₆)δ::13.34(s,1H),8.87(s,1H),8.58(s,1H),8.46(d,J＝4.8Hz,1H),7.62(d,J＝8.6Hz,2H),7.48(d,J＝8.6Hz,2H),7.27(s,1H),7.24(d,J＝8.4Hz,1H),7.02(d,J＝4.8Hz,1H),6.97(d,J＝8.8Hz,1H),2.79(t,J＝4.4Hz,4H),2.45(brs,4H),2.24(t,9H)；HRMS(ESI)m/z calcd for C₂₆H₃₀N₇O[M+H]⁺456.2506,found 456.2506.

example 48

N- (3- (4-methylpiperazin-1-yl) phenyl) -N' - (4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (I)_APreparation of (E) -38)

Except for changing 5-fluoro-2-nitrotoluene into 3-fluoronitrobenzene, other required raw materials, reagents and preparation method are the same as I_A-36 to give the title compound 32 mg as a yellow solid in 27% yield. And Mp: 108-109; purity: 95 percent;

¹H-NMR(400MHz,DMSO-d₆)δ:13.33(s,1H),8.91(s,1H),8.68(s,1H),8.46(d,J＝4.8Hz,1H),7.63(d,J＝8.4Hz,3H),7.49(d,J＝8.4Hz,2H),7.20(s,1H),7.12(t,J＝8.0Hz,1H),7.02(d,J＝4.8Hz,1H),6.81(d,J＝7.6Hz,1H),6.59(d,J＝8.0Hz,1H),3.13(brs,4H),2.50(brs,4H),2.25(d,6H)；HRMS(ESI)m/z calcd for C₂₅H₂₈N₇O[M+H]⁺442.2350,found442.2348.

example 49

N- (2- (4-methylpiperazin-1-yl) phenyl) -N' - (4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (I)_APreparation of (1) to (39)

Except for changing 5-fluoro-2-nitrotoluene into 2-fluoronitrobenzene, other required raw materials, reagents and preparation method are the same as I_A-36 to give the title compound 105 mg as a white solid in 89% yield. And Mp: 253-253; purity: 99 percent;

¹H-NMR(400MHz,DMSO-d₆)δ:13.33(s,1H),8.91(s,1H),8.68(s,1H),8.46(d,J＝4.8Hz,1H),7.63(d,J＝8.4Hz,3H),7.49(d,J＝8.4Hz,2H),7.20(s,1H),7.12(t,J＝8.0Hz,1H),7.02(d,J＝4.8Hz,1H),6.81(d,J＝7.6Hz,1H),6.59(d,J＝8.0Hz,1H),3.13(brs,4H),2.50(brs,4H),2.25(d,6H)；HRMS(ESI)m/z calcd for C₂₅H₂₈N₇O[M+H]⁺442.2350,found442.2346.

example 50

N- (5-methyl-2- (4-methylpiperazin-1-yl) phenyl) -N' - (4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (I)_APreparation of (4) to (40)

Except for changing 5-fluoro-2-nitrotoluene into 4-fluoro-5-nitrotoluene, other required raw materials, reagents and preparation method are the same as I_A-36 to give the title compound 23 mg as a white solid in 19% yield. And Mp: 247 to 250; purity: 95 percent;

¹H-NMR(400MHz,DMSO-d₆)δ:13.33(s,1H),9.81(s,1H),8.47(d,J＝4.8Hz,1H),8.08(s,1H),7.89(s,1H),7.68(d,J＝8.4Hz,2H),7.50(d,J＝8.4Hz,2H),7.07(d,J＝8.0Hz,1H),7.03(d,J＝4.8Hz,1H),6.79(d,J＝8.0Hz,1H),2.79(t,J＝4.0Hz,4H),2.64(brs,4H),2.31(s,3H),2.25(s,6H)；HRMS(ESI)m/z calcd for C₂₆H₃₀N₇O[M+H]⁺456.2506,found 456.2504.

example 51

N- (3-methyl-4- (morpholin-4-yl) phenyl) -N' - (4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (I)_APreparation of (E) -41)

Except that 5-fluoro-2-nitrotoluene is changed into 2-fluoro-5-nitrotoluene, and N-methylpiperazine is changed into morpholine, the other required raw materials, reagents and preparation method are the same as I_A-36 to give the title compound in 11% yield as a white solid. And Mp:253 to 255; purity: 90 percent.

¹H-NMR(400MHz,DMSO-d₆)δ:13.32(s,1H),8.85(s,1H),8.57(s,1H),8.46(d,J＝4.8Hz,1H),7.62(d,J＝8.4Hz,2H),7.48(d,J＝8.4Hz,2H),7.28(s,1H),7.26(d,J＝8.4Hz,1H),7.03(d,J＝4.8Hz,1H),6.98(d,J＝8.4Hz,1H),3.73(t,J＝4.4Hz,4H),2.79(t,J＝4.4Hz,4H),2.25(s,6H).；HRMS(ESI)m/z calcd for C₂₅H₂₇N₆O₂[M+H]⁺443.2190,found443.2188.

Example 52

N- (2- (morpholin-4-yl) phenyl) -N' - (4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (I)_APreparation of (E) -42)

Except that 5-fluoro-2-nitrotoluene is changed into 2-fluoronitrobenzene and N-methylpiperazine is changed into morpholine, the other required raw materials, reagents and preparation method are the same as those of I_A-36 to give the title compound 45 mg as a milky white solid in 39% yield. And Mp: 225-228 ℃; purity: 99 percent;

¹H-NMR(400MHz,DMSO-d₆)δ:13.33(s,1H),9.76(s,1H),8.47(d,J＝4.4Hz,1H),8.20(s,1H),8.08(d,J＝8.0Hz,1H),7.68(d,J＝8.4Hz,2H),7.51(d,J＝8.4Hz,2H),7.20(d,J＝7.2Hz,1H),7.10(t,J＝7.2Hz,1H),7.03(d,J＝4.4Hz,1H),6.99(d,J＝8.0Hz,1H),3.87(brs,4H),2.83(brs,4H),2.25(s,3H).HRMS(ESI)m/z calcd for C₂₄H₂₅N₆O₂[M+H]⁺429.2034,found 429.2033.

example 53

N- (5-methyl-2- (morpholin-4-yl) phenyl) -N' - (4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (I)_APreparation of (E) -43)

Except that 5-fluoro-2-nitrotoluene is replaced by 4-fluoro-5-nitrotoluene, and N-methylpiperazine is replaced by morpholine, the other required raw materials, reagents and preparation method are the same as those in I_A-36 to give the title compound 57 mg as a yellow solid in 48% yield. And Mp: 268-270; purity: 98 percent;

¹H-NMR(400MHz,DMSO-d₆)δ:13.34(s,1H),9.76(s,1H),8.47(d,J＝4.4Hz,1H),8.19(s,1H),7.94(s,1H),7.68(d,J＝8.4Hz,2H),7.51(d,J＝8.4Hz,2H),7.09(d,J＝8.0Hz,1H),7.03(d,J＝4.4Hz,1H),6.81(d,J＝8.0Hz,1H),3.86(t,J＝3.6Hz,4H),2.79(t,J＝3.6Hz,4H),2.26(d,6H)；HRMS(ESI)m/z calcd for C₂₅H₂₇N₆O₂[M+H]⁺443.2190,found443.2188.

example 54

N- ((4-diethylamino) phenyl) -N' - (4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (I)_APreparation of-44)

Except that 5-fluoro-2-nitrotoluene is changed into parafluoronitrobenzene and N-methylpiperazine is changed into diethylamine, the other required raw materials, reagents and preparation method are the same as I_A-36 to give the title compound 81 mg as a tan solid in 73% yield. And Mp: 242 to 246 ℃; purity: 95 percent;

¹H-NMR(400MHz,DMSO-d₆)δ:13.32(s,1H),8.76(s,1H),8.46(d,J＝4.8Hz,1H),8.33(s,1H),7.63(d,J＝8.4Hz,2H),7.47(d,J＝8.4Hz,2H),7.25(d,J＝8.8Hz,2H),7.00(d,J＝4.8Hz,1H),6.64(d,J＝8.8Hz,2H),3.27(q,J＝6.8Hz,4H),2.26(s,3H),1.07(t,J＝6.8Hz,6H)；HRMS(ESI)m/z calcd for C₂₄H₂₇N₆O[M+H]⁺415.2241,found 415.2238.

example 55

N- ((3-dimethylamino) phenyl) -N' - (4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (I)_APreparation of-45)

Stirring 7.2 ml of 40% formaldehyde solution and 2.56 ml of concentrated sulfuric acid in an ice bath to obtain a system 1, slowly adding 2.4 g of m-nitroaniline and 4.24 g of sodium borohydride tetrahydrofuran (80 ml) solution into the system 1, reacting for 2 hours in the ice bath, adding 1.92 g of sodium hydroxide, and stirring for half an hour. After the reaction, an appropriate amount of water was added to the system, extracted three times with ethyl acetate, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure, and the residue was separated by column chromatography (ethyl acetate: petroleum ether ═ 1:20, v/v) to give 1.8 g of an intermediate.

1.8 g of intermediate XVII are dissolved in methanol, 900 mg of 10% Pd/C are added, hydrogen is introduced and the reaction is carried out overnight at room temperature. After the reaction is complete, it is filtered, concentrated and the residue is isolated by column chromatography (ethyl acetate: petroleum ether: 1:10, v/v) to yield 1.3 g of intermediate XVIII.

Except for exchanging N- (2-aminoethyl) morpholine for intermediate XVIII, the other required raw materials, reagents and preparation method are the same as I_A-34 to give the title compound 20 mg as a pale yellow solid in 19% yield. And Mp: 226-228 deg.C; purity: 98 percent;

¹H-NMR(400MHz,DMSO-d₆)δ:13.33(s,1H),8.84(s,1H),8.61(s,1H),8.46(d,J＝4.8Hz,1H),7.63(d,J＝8.4Hz,2H),7.49(d,J＝8.4Hz,2H),7.08(t,J＝8.0Hz,1H),7.03(d,J＝4.8Hz,1H),6.95(s,1H),6.74(d,J＝8.0Hz,1H),6.38(d,J＝8.0Hz,1H),2.89(s,7H),2.25(s,3H)；HRMS(ESI)m/z calcd for C₂₂H₂₃N₆O[M+H]⁺387.1928,found 387.1925.

example 56

N- ((3-diethylamino) phenyl) -N' - (4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (I)_APreparation of (E) -46)

Except that 40% formaldehyde solution is replaced by 40% acetaldehyde solution, the other required raw materials, reagents and preparation method are the same as those in the step I_A-45 to give the title compound 29 mg as a brownish solid in 26% yield. And Mp: 125-128 ℃; purity: 97 percent;

¹H-NMR(400MHz,DMSO-d₆)δ:13.34(s,1H),8.84(s,1H),8.59(s,1H),8.47(d,J＝4.8Hz,1H),7.63(d,J＝8.4Hz,2H),7.49(d,J＝8.4Hz,2H),7.03(t,J＝4.8Hz,8.4Hz,2H),6.92(s,1H),6.63(d,J＝8.4Hz,1H),6.31(d,J＝8.4Hz,1H),3.31(q,J＝7.2Hz,4H),2.25(s,3H),1.10(t,J＝7.2Hz,6H)；HRMS(ESI)m/z calcd for C₂₄H₂₇N₆O[M+H]⁺415.2241,found415.2241.

example 57

N- (4- ((dimethylamino) methyl) phenyl) -N' - (4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (I)_APreparation of (E) -47)

Except that 5-fluoro-2-nitrotoluene is changed into p-nitrobenzyl chloride, and N-methylpiperazine is changed into dimethylamine, the other needed raw materials, reagents and preparation method are the same as those in I_A-36 to give the title compound as yellow solid 100mg, yield 93%. And Mp: 59-62 ℃; purity: 95 percent;

¹H-NMR(400MHz,DMSO-d₆)δ:13.34(s,1H),8.96(s,1H),8.80(s,1H),8.47(d,J＝4.8Hz,1H),7.64(d,J＝8.4Hz,H2),7.49(d,J＝8.4Hz,2H),7.43(d,J＝8.4Hz,2H),7.20(d,J＝8.4Hz,2H),7.00(t,J＝4.8Hz,1H),3.34–3.33(s,2H),2.25(s,3H),2.13(s,6H)；HRMS(ESI)m/z calcd for C₂₃H₂₅N₆O[M+H]⁺401.2084,found 401.2083.

example 58

N- (4- ((diethylamino) methyl) phenyl) -N' - (4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (I)_APreparation of (E) -48)

Except that 5-fluoro-2-nitrotoluene is changed into nitrobenzyl chloride, and N-methylpiperazine is changed into diethylamine, the other required raw materials, reagents and preparation method are the same as I_A-36 to give the title compound as yellow solid 50 mg, yield 44%. And Mp: 123-125 ℃; purity: 97 percent;

¹H-NMR(400MHz,DMSO-d₆)δ:13.34(s,1H),8.92(s,1H),8.74(s,1H),8.46(d,J＝4.8Hz,1H),7.63(d,J＝8.4Hz,2H),7.49(d,J＝8.4Hz,2H),7.41(d,J＝7.6Hz,2H),7.23(d,J＝7.6Hz,2H),7.03(d,J＝4.8Hz,1H),3.46(s,2H),2.44(s,4H),2.25(s,3H),0.98(s,6H)；HRMS(ESI)m/z calcd for C₂₅H₂₉N₆O[M+H]⁺429.2397,found 429.2396.

example 59

N- (3- ((dimethylamino) methyl) phenyl) -N' - (4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (I)_A-49) ofPreparation of

Except that 5-fluoro-2-nitrotoluene is changed into m-nitrobenzyl bromide, and N-methylpiperazine is changed into dimethylamine, the other required raw materials, reagents and preparation method are the same as those of I_A-36 to give the title compound as a yellow solid 25mg, yield 23%. And Mp: 185 to 186 ℃; purity: 90 percent;

¹H-NMR(400MHz,DMSO-d₆)δ:13.39(s,1H),9.64(s,1H),9.25(s,1H),8.52(d,J＝2.8Hz,1H),7.95(d,J＝7.6Hz,1H),7.73(d,J＝7.2Hz,2H),7.55(d,J＝7.2Hz,2H),7.31(t,J＝6.4Hz,1H),7.23(d,J＝6.4Hz,1H),7.08(d,J＝2.8Hz,1H),7.04(t,J＝6.4Hz,1H),3.50(s,2H),2.30(s,3H),2.24(s,6H)；HRMS(ESI)m/z calcd for C₂₃H₂₅N₆O[M+H]⁺401.2084,found 401.2082.

example 60

N- ((4-dimethylamino) phenyl) -N' - (4- (3-methyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (I)_APreparation of (E) -50)

Except that 5-fluoro-2-nitrotoluene is changed into p-fluoronitrobenzene and N-methylpiperazine is changed into dimethylamine, the other needed raw materials, reagents and preparation method are the same as those of I_A-36 to give the title compound as a tan solid in 10 mg, yield 10%. And Mp: 218-221 ℃; purity: 90 percent;

¹H-NMR(400MHz,DMSO-d₆)δ:13.33(s,1H),8.78(s,1H),8.46(d,J＝4.4Hz,1H),8.41(s,1H),7.61(d,J＝8.4Hz,2H),7.48(d,J＝8.4Hz,2H),7.29(d,J＝8.8Hz,2H),7.02(d,J＝4.4Hz,1H),6.72(d,J＝8.8Hz,2H),2.82(d,6H),2.23(s,3H)；HRMS(ESI)m/z calcdfor C₂₂H₂₃N₆O[M+H]⁺387.1928,found 387.1928.

example 61

N-N-pentyl-N' - (4- (3-trifluoromethyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (I)_BPreparation of (1)

Except that n-hexylamine is replaced by isobutylamine and XIV is replaced by intermediate VIII, the other required raw materials, reagents and preparation method are the same as those in I_A-4 to give the title compound as a white powder in 20 mg yield 20. And Mp: 215-216 ℃; purity: 99 percent;

¹H NMR(400MHz,DMSO-d6)δ:14.73(s,1H),8.66(d,J＝3.6Hz,2H),7.53(d,J＝8.4Hz,2H),7.35(d,J＝8.4Hz,2H),7.24(d,J＝3.6Hz,1H),6.24(t,J＝5.4Hz,1H),3.10(q,J＝12.8,6.4Hz,2H),1.43(m,2H),1.29(m,6H),0.87(t,J＝6.8Hz,3H).HRMS(ESI)m/zcalcd for C₂₀H₂₂N₅OF₃[M+H]⁺405.1776,found 405.1777.

example 62

N- (3-phenylpropyl) -N' - (4- (3-trifluoromethyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (I)_BPreparation of (2)

Except that isobutylamine is replaced by phenethylamine and the intermediate VIII is replaced by XIV, the other required raw materials, reagents and preparation method are the same as I_A-4 to give the title compound 27 mg, white solid, yield 25%. And Mp: 225-227 ℃; purity: 95 percent；

1H NMR(400MHz,DMSO-d6)δ:14.69(s,1H),8.76(s,1H),8.67(d,J＝4.8Hz,1H),7.54(d,J＝8.4Hz,2H),7.34(dd,J＝12.8,8.0Hz,4H),7.25(m,4H),6.25(t,J＝5.6Hz,1H),2.78(t,J＝7.0Hz,2H),1.26(m,1H),0.87(brs,1H).HRMS(ESI)m/z calcd for C₂₂H₁₈N₅OF₃[M+H]⁺425.1463,found 425.1465.

Example 63

N- (3-phenylpropyl) -N' - (4- (3-trifluoromethyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (I)_BPreparation of (3)

Except that the isobutylamine is replaced by the amphetamine and the intermediate VIII is replaced by the intermediate XIV, the other required raw materials, reagents and preparation method are the same as those in the step I_A-4 to give the title compound as a white powder in 18 mg, 16% yield. And Mp: 212-214 ℃; purity: 100 percent;

1H NMR(400MHz,DMSO-d6)δ:14.71(s,1H),8.70(s,1H),8.67(d,J＝4.8Hz,1H),7.55(d,J＝8.4Hz,2H),7.36(d,J＝8.4Hz,2H),7.30(t,J＝7.4Hz,2H),7.24(d,J＝4.4Hz,3H),7.19(t,J＝7.2Hz,1H),6.33(t,J＝5.6Hz,1H),3.13(dd,J＝12.8,6.4Hz,2H),2.63(t,J＝7.6Hz,2H),1.76(m,2H).HRMS(ESI)m/z calcd for C₂₃H₂₀N₅OF₃[M+H]⁺439.1620,found439.1613.

example 64

N- (4-Chlorobenzoethyl) -N' - (4- (3-trifluoromethyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (I)_BPreparation of (4)

Except that isobutylamine is replaced by 4-chlorophenylethylamine and the intermediate VIII is replaced by XIV, the other required raw materials, reagents and preparation method are the same as I_A-4 to give the title compound as a pale yellow powder, 26 mg, yield 23%. And Mp: 228 to 232 ℃; purity: 97 percent;

1H NMR(400MHz,DMSO-d6)δ:14.71(s,1H),8.72(s,1H),8.67(d,J＝4.8Hz,1H),7.53(d,J＝8.4Hz,2H),7.37(dd,J＝12.4,8.4Hz,4H),7.29(d,J＝8.0Hz,2H),7.24(d,J＝4.8Hz,1H),6.23(t,J＝5.6Hz,1H),2.77(t,J＝6.8Hz,2H),1.24(s,1H),0.84(t,1H).HRMS(ESI)m/z calcd for C₂₂H₁₇N₅OF₃Na[M+Na]⁺482.0971,found 482.0973.

example 65

N- (2-dimethylaminoethyl) -N' - (4- (3-trifluoromethyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (I)_BPreparation of (5)

Except that isobutylamine is replaced by N, N-dimethylethylenediamine and the intermediate VIII is replaced by the intermediate XIV, the other required raw materials, reagents and preparation method are the same as those in I_A-4 to give the title compound 41 mg as a milky white solid in 42% yield. And Mp: 165-168 ℃; purity: 97 percent;

¹H-NMR(400MHz,DMSO-d₆)δ:8.93(s,1H),8.67(d,J＝4.4Hz,1H),7.53(d,J＝8.4Hz,2H),7.35(d,J＝8.4Hz,2H),7.24(d,J＝4.4Hz,1H),6.22(t,J＝5.2Hz,1H),3.22(dd,J＝11.6,6.0Hz,2H),2.39(t,J＝6.0Hz,2H),2.22(s,6H).HRMS(ESI)m/z calcd forC₁₈H₂₀N₆OF₃[M+H]⁺393.1651,found 393.1651.

example 66

N- (2-diethylaminoethyl) -N' - (4- (3-trifluoromethyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (I)_BPreparation of (6)

Except that isobutylamine is replaced by N, N-diethylethylenediamine and the intermediate VIII is replaced by the intermediate XIV, the other required raw materials, reagents and preparation method are the same as those in I_A-4 to give the title compound as a cream white powder in 35 mg, yield 33%. And Mp: 128-132 ℃; purity: 97 percent;

¹H-NMR(400MHz,DMSO-d₆)δ:9.04(s,1H),8.67(d,J＝4.4Hz,1H),7.55(d,J＝8.4Hz,2H),7.35(d,J＝8.4Hz,2H),7.24(d,J＝4.4Hz,1H),6.29(s,1H),3.24(q,J＝5.6Hz,2H),2.65(s,6H),1.03(t,J＝7.2,8.9Hz,6H).HRMS(ESI)m/z calcd for C₂₀H₂₄N₆OF₃[M+H]⁺421.1964,found 421.1967.

example 67

N- (3-dimethylaminopropyl) -N' - (4- (3-trifluoromethyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (I)_BPreparation of (7)

Except that isobutylamine is replaced by 3-dimethylaminopropylamine and intermediate VIII is replaced by intermediate XIV, the other required raw materials, reagents and preparation method are the same as I_A-4 to give the title compound as a pale yellow powder 28 mg, yield 27%. And Mp: 186-189 ℃; purity: 96 percent;

¹H-NMR(400MHz,DMSO-d₆)δ:8.79(s,1H),8.66(d,J＝4.8Hz,1H),7.54(d,J＝8.4Hz,2H),7.35(d,J＝8.4Hz,2H),7.24(d,J＝4.8Hz,1H),6.33(t,J＝5.6Hz,1H),3.14(dd,J＝12.4,6.4Hz,2H),2.35(t,J＝6.0Hz,2H),2.21(s,6H),1.61(m,2H).HRMS(ESI)m/zcalcd for C₁₉H₂₁N₆OF₃[M+H]⁺406.1729,found 406.1731.

example 68

N- (3-diethylaminopropyl) -N' - (4- (3-trifluoromethyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (I)_BPreparation of (1) to (8)

Except that isobutylamine is replaced by 3-diethylaminopropylamine and intermediate VIII is replaced by intermediate XIV, the other required raw materials, reagents and preparation method are the same as those in I_A-4 to give the title compound as a cream white powder 12 mg, yield 11%. And Mp: 217-220 ℃; purity: 99 percent;

¹H-NMR(400MHz,DMSO-d₆)δ:14.71(s,1H),9.15(s,1H),8.67(d,J＝4.4Hz,1H),7.55(d,J＝8.4Hz,2H),7.35(d,J＝8.4Hz,2H),7.24(d,J＝4.4Hz,1H),6.69(s,1H),3.20(q,J＝11.6,6.0Hz,2H),3.06(brs,6H),1.82(brs,2H),1.21(m,6H).HRMS(ESI)m/z calcdfor C₂₁H₂₆N₆OF₃[M+H]⁺435.2120,found 435.2118.

example 69

N- (2-piperidin-1-ylethyl) -N' - (4- (3-trifluoromethyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (I)_BPreparation of (2) to (9)

Except that isobutylamineThe 1- (2-aminoethyl) piperidine is replaced, the intermediate VIII is replaced by the intermediate XIV, and other required raw materials, reagents and preparation methods are the same as those of the intermediate I_A-4 to give the title compound as a pale yellow powder 34 mg, yield 31%. And Mp: 120-122 ℃; purity: 93 percent;

¹H-NMR(400MHz,DMSO-d₆)δ:8.91(s,1H),8.67(d,J＝4.4Hz,1H),7.54(d,J＝8.4Hz,2H),7.35(d,J＝8.4Hz,2H),7.24(d,J＝4.4Hz,1H),6.17(t,J＝4.8Hz,1H),3.23(dd,J＝11.6,6.0Hz,2H),2.36(t,6H),1.53(m,4H),1.40(m,2H).HRMS(ESI)m/z calcd forC₂₁H₂₄N₆OF₃[M+H]⁺433.1964,found 433.1963.

example 70

N- (2-morpholin-4-ylethyl) -N' - (4- (3-trifluoromethyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (I)_BPreparation of (1) to (10)

Except that isobutylamine is replaced by N- (2-aminoethyl) morpholine and intermediate VIII is replaced by intermediate XIV, the other required raw materials, reagents and preparation method are the same as those in I_A-4 to give the title compound as a pale yellow powder 35 mg, yield 32%. And Mp: 217-219 ℃; purity: 96 percent;

¹H-NMR(400MHz,DMSO-d₆)δ:14.70(s,1H),8.92(s,1H),8.66(d,J＝4.8Hz,1H),7.54(d,J＝8.4Hz,2H),7.35(d,J＝8.4Hz,2H),7.24(d,J＝4.8Hz,1H),6.21(t,J＝5.2Hz,1H),3.61(t,J＝4.4Hz,4H),3.24(dd,J＝11.6,6.0Hz,2H),2.41(t,6H).HRMS(ESI)m/zcalcd for C₂₀H₂₂N₆O₂F₃[M+H]⁺435.1756,found 435.1754.

example 71

N- (4- (4-methylpiperazin-1-yl) phenyl) -N' - (4- (3-trifluoromethyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (I)_BPreparation of (11)

Except that 5-fluoro-2-nitrotoluene is replaced by 4-fluoronitrobenzene and the intermediate VIII is replaced by the intermediate XIV, the other required raw materials, reagents and preparation method are the same as those in I_A-36 to give the title compound 37 mg as a silver gray solid in 30% yield. And Mp: 267-268 ℃; purity: 96 percent;

¹H-NMR(400MHz,DMSO-d₆)δ:14.73(s,1H),8.83(s,1H),8.68(d,J＝4.8Hz,1H),8.52(s,1H),7.59(d,J＝8.4Hz,2H),7.40(d,J＝8.4Hz,2H),7.32(d,J＝8.8Hz,2H),7.26(d,J＝4.8Hz,1H),6.90(d,J＝8.8Hz,2H),3.06(s,4H),2.44(s,4H),2.24(s,3H).HRMS(ESI)m/z calcd for C₂₅H₂₅N₇OF₃[M+H]⁺496.2073,found 496.2079.

example 72

N- (2-methyl-4- (4-methylpiperazin-1-yl) phenyl) -N' - (4- (3-trifluoromethyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (I)_BPreparation of (12)

Except that the intermediate VIII is replaced by the intermediate XIV, other required raw materials, reagents and preparation methods are the same as those of the intermediate VIII_A-36 to give the title compound as a tan powder 22 mg, yield 17%. And Mp: 250-253 deg.C; purity: 91%;

¹H-NMR(400MHz,DMSO-d₆)δ:14.74(s,1H),9.07(s,1H),8.68(d,J＝4.4Hz,1H),7.86(s,1H),7.59(d,J＝8.4Hz,2H),7.48(d,J＝8.8Hz,1H),7.40(d,J＝8.4Hz,2H),7.26(d,J＝4.4Hz,1H),6.81(s,1H),6.76(d,J＝8.8Hz,1H),3.09(s,4H),2.46(s,4H),2.23(d,6H).HRMS(ESI)m/z calcd for C₂₆H₂₇N₇OF₃[M+H]⁺510.2229,found 510.2232.

example 73

N- (3-methyl-4- (4-methylpiperazin-1-yl) phenyl) -N' - (4- (3-trifluoromethyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (I)_BPreparation of (E) -13)

Except that 5-fluoro-2-nitrotoluene is replaced by 2-fluoro-5-nitrotoluene, and the intermediate VIII is replaced by the intermediate XIV, the other required raw materials, reagents and preparation method are the same as I_A-36 to give the title compound as a pale yellow powder 69 mg, 54% yield. And Mp: 209-211 ℃; purity: 97 percent;

¹H-NMR(400MHz,DMSO-d₆)δ:14.73(s,1H),8.87(s,1H),8.68(d,J＝4.8Hz,1H),8.59(s,1H),7.59(d,J＝8.4Hz,2H),7.40(d,J＝8.4Hz,2H),7.25(m,3H),6.97(d,J＝8.4Hz,1H),2.80(s,4H),2.44(s,4H),2.24(d,6H).HRMS(ESI)m/z calcd for C₂₆H₂₇N₇OF₃[M+H]⁺510.2229,found 510.2230.

example 74

N- (3- (4-methylpiperazin-1-yl) phenyl) -N' - (4- (3-trifluoromethyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (I)_BPreparation of (1) to (14)

Except for the addition of 5-fluoro-2-nitrotoluene is replaced by 3-fluoronitrobenzene, the intermediate VIII is replaced by the intermediate XIV, and other required raw materials, reagents and preparation methods are the same as those of the intermediate I_B-11 to give the title compound as yellow powder 57 mg, yield 46%. And Mp: 229-231 ℃; purity: 95 percent;

¹H-NMR(400MHz,DMSO-d₆)δ:14.73(s,1H),8.90(s,1H),8.69(d,J＝4.8Hz,2H),7.60(d,J＝8.4Hz,2H),7.41(d,J＝8.4Hz,2H),7.27(d,J＝4.8Hz,1H),7.20(s,1H),7.12(t,J＝8.0Hz,1H),6.82(d,J＝8.0Hz,1H),6.59(d,J＝8.0Hz,1H),3.12(s,4H),2.48(s,4H),2.24(s,3H).HRMS(ESI)m/z calcd for C₂₅H₂₅N₇OF₃[M+H]⁺496.2073,found496.2072..

example 75

N- (2- (4-methylpiperazin-1-yl) phenyl) -N' - (4- (3-trifluoromethyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (I)_BPreparation of (E) -15)

Except that 5-fluoro-2-nitrotoluene is replaced by 2-fluoronitrobenzene and the intermediate VIII is replaced by the intermediate XIV, the other required raw materials, reagents and preparation method are the same as those in I_A-36 to give the title compound as a white powder 40 mg, yield 32%. And Mp: 283 to 286 ℃; purity: 98 percent;

¹H-NMR(400MHz,DMSO-d₆)δ:14.73(s,1H),9.80(s,1H),8.69(d,J＝4.4Hz,1H),8.09(s,1H),8.03(d,J＝8.0Hz,1H),7.64(d,J＝8.0Hz,2H),7.43(d,J＝8.0Hz,2H),7.27(d,J＝4.4Hz,1H),7.19(d,J＝7.6Hz,1H),7.08(t,J＝8.0Hz,1H),6.99(t,J＝7.6Hz,1H),2.84(s,4H),2.63(s,4H),2.30(s,3H).HRMS(ESI)m/z calcd for C₂₅H₂₅N₇OF₃[M+H]⁺496.2073,found 496.2071.

example 76

N- (5-methyl-2- (4-methylpiperazin-1-yl) phenyl) -N' - (4- (3-trifluoromethyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (I)_BPreparation of-16)

Except that 5-fluoro-2-nitrotoluene is replaced by 4-fluoro-3-nitrotoluene, and the intermediate VIII is replaced by the intermediate XIV, the other required raw materials, reagents and preparation method are the same as I_A-36 to give the title compound as yellow powder 31 mg, yield 24%. And Mp: 274-276 ℃; purity: 96 percent;

¹H-NMR(400MHz,DMSO-d₆)δ:14.73(s,1H),9.82(s,1H),8.69(d,J＝4.8Hz,1H),8.09(s,1H),7.90(s,1H),7.65(d,J＝8.4Hz,2H),7.43(d,J＝8.4Hz,2H),7.27(d,J＝4.8Hz,1H),7.07(d,J＝8.0Hz,1H),6.79(d,J＝8.0Hz,1H),2.80(s,4H),2.65(s,4H),2.31(s,3H),2.26(s,3H).HRMS(ESI)m/z calcd for C₂₆H₂₇N₇OF₃[M+H]⁺510.2229,found510.2228.

example 77

N- (3-methyl-4- (morpholin-4-yl) phenyl) -N' - (4- (3-trifluoromethyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (I)_BPreparation of (E) -17)

Except that 5-fluoro-2-nitrotoluene is replaced by 2-fluoro-5-nitrotoluene, N-methylpiperazine is replaced by morpholine, intermediate VIII is replaced by intermediate XIV, and the other required raw materials, reagents and preparation method are the same as those in I_A-36 to give the title compound as a cream white powder, 105 mg, 84% yield. And Mp: 85-87 ℃; purity:99％；

¹H-NMR(400MHz,DMSO-d₆)δ:14.69(s,1H),8.87(s,1H),8.69(d,J＝4.4Hz,1H),8.60(s,1H),7.61(d,J＝8.4Hz,2H),7.41(d,J＝8.4Hz,2H),7.30(s,1H),7.27(d,J＝4.4Hz,1H),7.04(s,1H),6.99(d,J＝8.4Hz,1H),3.73(t,4H),2.79(t,4H),2.26(s,3H).HRMS(ESI)m/z calcd for C₂₅H₂₄N₆O₂F₃[M+H]⁺497.1913,found 497.1914.

example 78

N- (2- (morpholin-4-yl) phenyl) -N' - (4- (3-trifluoromethyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (I)_BPreparation of (18)

Except that 5-fluoro-2-nitrotoluene is replaced by 2-fluoronitrobenzene, N-methylpiperazine is replaced by morpholine, and intermediate VIII is replaced by intermediate XIV, the other required raw materials, reagents and preparation method are the same as those in I_A-36 to give the title compound as a white powder in 36 mg, yield 30%. And Mp: 224 to 226 ℃; purity: 100 percent;

¹H-NMR(400MHz,DMSO-d₆)δ:14.74(s,1H),9.77(s,1H),8.69(d,J＝4.4Hz,1H),8.21(s,1H),8.08(d,J＝8.0Hz,1H),7.65(d,J＝8.4Hz,2H),7.44(d,J＝8.4Hz,2H),7.27(d,J＝4.4Hz,1H),7.21(d,J＝8.0Hz,1H),7.10(t,J＝7.6Hz,1H),7.00(t,J＝7.6Hz,1H),3.87(s,4H),2.83(s,4H).HRMS(ESI)m/z calcd for C₂₄H₂₂N₆O₂F₃[M+H]⁺483.1756,found483.1756.

example 79

N- (5-methyl-2- (morpholin-4-yl) phenyl) -N' - (4- (3-trifluoromethyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (I)_BPreparation of (E) -19)

Except that 5-fluoro-2-nitrotoluene is replaced by 2-fluoro-5-nitrotoluene, N-methylpiperazine is replaced by morpholine, intermediate VIII is replaced by intermediate XIV, and the other required raw materials, reagents and preparation method are the same as those in I_A-36 to give the title compound as yellow powder 43 mg, yield 34%. And Mp: 153-155 ℃; purity: 91%;

¹H-NMR(400MHz,DMSO-d₆)δ:14.74(s,1H),9.77(s,1H),8.69(d,J＝4.4Hz,1H),8.22(s,1H),7.94(s,1H),7.65(d,J＝8.4Hz,2H),7.43(d,J＝8.4Hz,2H),7.27(d,J＝4.4Hz,1H),7.09(d,J＝8.0Hz,1H),6.81(d,J＝8.0Hz,1H),3.86(s,4H),2.79(s,4H),2.26(s,3H).HRMS(ESI)m/z calcd for C₂₅H₂₄N₆O₂F₃[M+H]⁺497.1913,found 497.1914.

example 80

N- (4- (4-methylpiperazin-1-yl) phenyl) -N' - (4- (3-trifluoromethyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) thiourea (I)_BPreparation of (E) -20)

Except for replacing 5-fluoro-2-nitrotoluene with 4-fluoronitrobenzene, other required raw materials, reagents and preparation methods are the same as the intermediate XVI, so that an intermediate XIX: 4- (4-methylpiperazin-1-yl) aniline.

200 mg of intermediate XIX was dissolved in 4 ml of dichloromethane, and then added dropwise to a dichloromethane (6 ml) solution of sulfur dichloride (240. mu.l) under nitrogen atmosphere at 0 ℃ and 124 mg of sodium hydroxide were added to react at room temperature for 3 hours. After the reaction, the reaction mixture was filtered, washed with dichloromethane, and distilled under reduced pressure to obtain a residue. The residue was dissolved in 5ml of ethanol, and 70 mg of 4- (3-trifluoromethyl-1H-pyrazolo [3,4-b ] pyridin-4-yl) aniline (intermediate XIV) was added thereto and reacted at ordinary temperature for 1 hour. After completion of the reaction, distillation was performed under reduced pressure, and the residue was separated by column chromatography (methanol: dichloromethane ═ 1:30, v/v) to give 76 mg of the title compound in 59% yield. And Mp: 153-155 ℃; purity: 99 percent;

¹H-NMR(400MHz,DMSO-d₆)δ:14.76(s,1H),9.76(d,J＝15.2Hz,2H),8.71(d,J＝4.4Hz,1H),7.70(d,J＝7.6Hz,2H),7.44(d,J＝7.6Hz,2H),7.28(d,J＝5.6Hz,3H),6.94(d,J＝8.0Hz,2H),3.13(s,4H),2.45(s,4H),2.24(s,3H).HRMS(ESI)m/z calcd forC₂₅H₂₄N₇OF₃S[M+H]⁺512.1844,found 512.1974.

example 81

N- ((4-diethylamino) phenyl) -N' - (4- (3-trifluoromethyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (I)_BPreparation of (1) to (21)

Except that 5-fluoro-2-nitrotoluene is replaced by parafluoronitrobenzene, N-methylpiperazine is replaced by diethylamine, and the intermediate VIII is replaced by the intermediate XIV, the other required raw materials, reagents and preparation method are the same as I_A-36 to give the title compound 23 mg as a tan solid in 20% yield. And Mp: 253-254 ℃; purity: 99 percent;

¹H-NMR(400MHz,DMSO-d₆)δ:14.70(s,1H),8.76(s,1H),8.68(d,J＝4.0Hz,1H),8.33(s,1H),7.58(d,J＝8.0Hz,2H),7.39(d,J＝8.0Hz,2H),7.25(d,J＝8.0Hz,3H),6.64(d,J＝8.0Hz,2H),3.29(dd,J＝13.2,6.4Hz,4H),1.07(t,J＝6.4Hz,6H).HRMS(ESI)m/zcalcd for C₂₄H₂₄N₆OF₃[M+H]⁺469.1964,found 469.1962.

example 82

N- ((3-dimethylamino) phenyl) -N' - (4- (3-trifluoromethyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (I)_BPreparation of-22)

Except for replacing the intermediate VIII by the intermediate XIV, other required raw materials, reagents and preparation methods are the same as those of the intermediate I_A-45 to give the title compound 80 mg as a yellow solid in 72% yield. And Mp: 82-84 ℃; purity: 98 percent;

¹H-NMR(400MHz,DMSO-d₆)δ:14.73(s,1H),8.84(s,1H),8.69(d,J＝4.8Hz,1H),8.62(s,1H),7.60(d,J＝8.4Hz,2H),7.41(d,J＝8.4Hz,2H),7.27(d,J＝4.8Hz,1H),7.08(t,J＝8.0Hz,1H),6.95(s,1H),6.74(d,J＝8.0Hz,1H),6.39(d,J＝8.0Hz,1H),2.89(s,6H).HRMS(ESI)m/z calcd for C₂₂H₂₀N₆OF₃[M+H]⁺441.1650,found 441.1651.

example 83

N- ((3-diethylamino) phenyl) -N' - (4- (3-trifluoromethyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (I)_BPreparation of (E) -23)

Except that the intermediate VIII is replaced by the intermediate XIV, and the formaldehyde solution is replaced by the acetaldehyde solution, the other required raw materials, reagents and preparation method are the same as those in the step I_A-45 to give the title compound as a tan powder 54 mg, yield 46%. And Mp: 151-153 ℃; purity: 98 percent;

¹H-NMR(400MHz,DMSO-d₆)δ:14.72(s,1H),8.81(s,1H),8.68(d,J＝4.4Hz,1H),8.56(s,1H),7.59(d,J＝8.4Hz,2H),7.41(d,J＝8.4Hz,2H),7.26(d,J＝4.4Hz,1H),7.04(t,J＝8.0Hz,1H),6.91(t,J＝2.0Hz,1H),6.63(dd,J＝8.0,1.2Hz,1H),6.31(dd,J＝8.0,2.0Hz,1H),3.31(t,J＝7.2,4H),1.10(t,J＝7.2,6H).HRMS(ESI)m/z calcd forC₂₄H₂₄N₆OF₃[M+H]⁺469.1964,found 469.1962.

example 84

N- (4- ((dimethylamino) methyl) phenyl) -N' - (4- (3-trifluoromethyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (I)_BPreparation of (E) -24)

Except that 5-fluoro-2-nitrotoluene is replaced by p-nitrobenzyl chloride, N-methylpiperazine is replaced by dimethylamine, and the intermediate VIII is replaced by the intermediate XIV, the other required raw materials, reagents and preparation method are the same as those in the step I_A-36 to give the title compound as a yellow solid 86 mg, yield 75%. And Mp: 200-202 ℃; purity: 95 percent;

¹H-NMR(400MHz,DMSO-d₆)δ:14.52(s,1H),8.93(s,1H),8.78(s,1H),8.69(d,J＝4.4Hz,1H),7.61(d,J＝8.0Hz,2H),7.43(t,J＝8.0Hz,4H),7.26(d,J＝4.4Hz,1H),7.21(d,J＝8.0Hz,2H),3.34(s,3H),2.15(s,6H).HRMS(ESI)m/z calcd for C₂₃H₂₂N₆OF₃[M+H]⁺455.1807,found 455.1809.

example 85

N- (4- ((diethylamino) methyl) phenyl) -N' - (4- (3-trifluoromethyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (I)_BPreparation of (E) -25)

Except that 5-fluoro-2-nitrotoluene is replaced by p-nitrobenzyl chloride, N-methylpiperazine is replaced by diethylamine, and the intermediate VIII is replaced by the intermediate XIV, the other required raw materials, reagents and preparation method are the same as I_A-36 to give the title compound as a yellow solid 94 mg in 74% yield. And Mp: 90-93 ℃; purity: 95 percent;

¹H-NMR(400MHz,DMSO-d₆)δ:14.52(s,1H),8.93(s,1H),8.77(s,1H),8.68(d,J＝4.4Hz,1H),7.61(d,J＝8.0Hz,2H),7.42(d,J＝4.8Hz,4H),7.26(d,J＝4.4Hz,1H),7.23(d,J＝8.0Hz,1H),3.50(s,2H),2.47(t,J＝6.8Hz,4H),0.99(t,J＝6.8Hz,6H).HRMS(ESI)m/zcalcd for C₂₅H₂₆N₆OF₃[M+H]⁺483.21210,found 483.2121.

example 86

N- (3- ((dimethylamino) methyl) phenyl) -N' - (4- (3-trifluoromethyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (I)_BPreparation of (2) to (26)

Except that 5-fluoro-2-nitrotoluene is replaced by m-nitrobenzyl bromide, N-methylpiperazine is replaced by dimethylamine, and the intermediate VIII is replaced by the intermediate XIV, the other required raw materials, reagents and preparation method are the same as those in I_A-36 to give the title compound as a yellow solid, 26 mg, yield 23%. And Mp: 96-98 ℃; purity: 99 percent;

¹H-NMR(400MHz,DMSO-d₆)δ:14.39(s,1H),8.92(s,1H),8.82(s,1H),8.69(d,J＝4.4Hz,1H),7.62(d,J＝8.4Hz,2H),7.49(s,1H),7.42(d,J＝8.4Hz,2H),7.34(d,J＝8.0Hz,1H),7.27(d,J＝4.4Hz,1H),7.23(d,J＝7.6Hz,1H),6.91(d,J＝7.6Hz,1H),3.40(s,2H),2.17(s,6H).HRMS(ESI)m/z calcd for C₂₃H₂₂N₆OF₃[M+H]⁺455.1807,found 455.1806.

example 87

N- ((4-dimethylamino) phenyl) -N' - (4- (3-trifluoromethyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (I)_BPreparation of (E) -27)

Except that 5-fluoro-2-nitrotoluene is replaced by parafluoronitrobenzene, N-methylpiperazine is replaced by dimethylamine, and the intermediate VIII is replaced by the intermediate XIV, the other required raw materials, reagents and preparation method are the same as those in I_A-36 to give the title compound as a brownish solid 10 mg, yield 9%. And Mp: 219-221 ℃; purity: 80 percent;

¹H-NMR(400MHz,DMSO-d₆)δ::14.71(s,1H),8.80(s,1H),8.68(d,J＝4.8Hz,1H),8.43(s,1H),7.58(d,J＝8.8Hz,2H),7.39(d,J＝8.8Hz,2H),7.27(d,J＝9.2Hz,2H),6.87(s,1H),6.71(d,J＝9.2Hz,2H),2.84(s,6H).HRMS(ESI)m/z calcd for C₂₃H₂₂N₆OF₃[M+H]⁺441.1651,found 441.1649.

example 88

N- (3- ((diethylamino) methyl) phenyl) -N' - (4- (3-trifluoromethyl-1H-pyrazolo [3, 4-b)]Pyridin-4-yl) phenyl) urea (I)_BPreparation of (E) -28)

Except that 5-fluoro-2-nitrotoluene is replaced by m-nitrobenzyl bromide, N-methylpiperazine is replaced by diethylamine, andexcept for replacing the intermediate VIII by the intermediate XIV, other required raw materials, reagents and preparation methods are the same as those of the intermediate I_A-36 to give the title compound as a yellow solid 32 mg in 26% yield. And Mp: 70-72 ℃; purity: 98 percent;

¹H-NMR(400MHz,DMSO-d₆)δ:14.73(s,1H),8.94(d,2H),8.69(d,J＝4.4Hz,1H),7.61(d,J＝8.4Hz,2H),7.49(s,1H),7.42(d,J＝8.4Hz,2H),7.37(d,J＝8.4Hz,2H),7.27(d,J＝7.6,1H),7.24(d,J＝4.4,1H),6.95(d,J＝7.6Hz,1H),3.60(s,2H),2.54(q,J＝7.2Hz,3H),1.02(t,J＝7.2Hz,6H).HRMS(ESI)m/z calcd for C₂₅H₂₆N₆OF₃[M+H]⁺483.2120,found 483.2120.

example 89

Experimental method and result of inhibiting activity of three receptor tyrosine kinases by using compound

the method for testing the kinase activity of the compound adopts a Mobility Shift Assay (Mobility Shift Assay) method under the condition of KmATP concentration, selects three in-vitro kinases of VEGFR-2, PGDFR- α and c-KIT for activity test, uses a compound Imatinib as a standard control, prepares a 50 mu M solution for each compound, and dilutes the solution into 10 concentration points (the concentration difference between each concentration point is 4 times) for single-hole detection.

The data of the activity of the compounds of the invention on the 3 tyrosine receptor kinases are shown in Table 1, and a total of 29 compounds of the invention are found to have better c-KIT inhibitory activity and IC₅₀<100nM, 17 of them have strong inhibitory activity to c-KIT, half of the effective inhibitory concentration is 1nM<IC₅₀<10nM, 33 compounds of the invention have good inhibitory activity to PDGFR α, and half effective inhibitory concentration IC₅₀<1000nM, 8 of the compounds of the invention have better inhibitory activity to PDGFR α, half the effective inhibitory concentration is 10nM<IC₅₀<100nM, 1 compound of the invention has strong inhibitory activity to PDGFR α, and half effective inhibitory concentration is 1nM<IC₅₀<10 nM; no good inhibitory activity (IC) on VEGFR-2 was found₅₀<1000nM), more than half (41) of which have certain inhibitory activity on VEGFR-2, half the effective inhibitory concentration being 1000nM<IC50<10000 nM; the activity data are detailed in table 1 and attached table 1: the compound of the invention is hopeful to be developed into antineoplastic drugs of multi-target receptor tyrosine kinase inhibitors.

in Table 1, c-KIT represents the type III tyrosine kinase receptor, PDGFRa represents the platelet-derived growth factor receptor α, VEGFR2 represents the vascular endothelial growth factor receptor 2, Linifanib, which is an antitumor drug candidate proposed by Yapei, USA, and imatinib mesylate, which is a GISTs approved by FDA in 2002 for unresectable and/or metastatic tumors, is the first cell signaling inhibitor for clinical treatment of malignancies.

TABLE 1 inhibitory Activity data (IC) of the Compounds of the invention on three tyrosine kinases₅₀，nM)

Attached table 1

Example 90

GI of antiproliferative experiments with compounds of the invention against multiple cognate BaF3 cell lines₅₀Measurement test method and results

Compounds of the invention are provided as c-KIT and PDGFRA kinasesAll the best I_A41 for example, compounds were tested for antiproliferative activity on various cognate BaF3 cell lines. In the experiment, 10mM of a compound mother liquor (DMSO) to be detected is diluted according to 7 concentrations by 3 times to prepare a drug plate, the drug plate is stored under appropriate conditions, and meanwhile, the DMSO solvent with the same volume is used as a blank control, and imatinib is used as a positive control. Preparing the corresponding logarithmic phase cells into a cell forming plate, culturing under appropriate conditions, adding medicine, and culturing at 37 deg.C with 5% CO₂And (3) incubating in an incubator for 72 hours, adding 10 mu L of CellTiter-Glo cell proliferation fluorescence detection reagent, standing for 10 minutes, and detecting the action condition of the drug and the cells through Envision Plate-Reader reading. The activity data are shown in table 2 below,

TABLE 2 Compounds I of the invention_A41 pairs of homologous BaFs₃Antiproliferative experimental activity data (GI) of cell lines₅₀，μM)

GI₅₀(μM)	I_A41	Imatinib
			BaF3	4.031	4.686
c-KIT-BaF3	0.0181	0.3819
			c-KIT-A829P-BaF3	0.01159	0.1482
c-KIT-L576P-BaF3	0.01056	0.04508
			c-KIT-C674S-BaF3	0.01045	0.02493
c-KIT-D816H-BaF3	0.1341	1.245
			c-KIT-D816V-BaF3	＞10	＞10
c-KIT-T670I-BaF3	0.03924	＞10
			c-KIT-V559D-T670I-BaF3	0.03637	＞10
c-KIT-V654A-BaF3	0.1365	0.8031
			c-KIT-N822K-BaF3	0.01109	1.466
c-KIT-V559D-BaF3	0.09645	0.03598
			c-KIT-V559D-V654A-BaF3	0.3303	0.803
PDGFRα-BaF3	0.01044	0.01066
			PDGFRα-T674I-BaF3	0.1348	＞10
FLT3-BaF3	0.0107	＞10
			FLT3-ITD-BaF3	0.01043	2.014

As can be seen from table 2: compound I_A-41 shows a strong inhibitory effect on most of c-KIT, PDGFR α and mutant cell lines, but no significant inhibitory effect on c-KIT-D816V-BaF3, wherein Compound I has an antiproliferative activity compared to that of the marketed drug imatinib_Athe gene-41 has a breakthrough improvement on the antiproliferative effect of C-KIT-T670I-BaF3, C-KIT-V559D-T670I-BaF3, C-KIT-N822K-BaF3, PDGFR α -T674I-BaF3, FLT3-BaF3 and FLT3-ITD-BaF3 cells, a great improvement on the antiproliferative effect of C-KIT-BaF3, C-KIT-A829P-BaF3, C-KIT-D816H-BaF3, C-KIT-V559D-V654D-BaF D and other targets, and a good antiproliferative effect on C-KIT-L D-BaF D, C-KIT-C674-BaF D, C-KIT-V654-BaF D-D cells.

Example 91

Anti-proliferation experiment GI (GI) of compound of the invention aiming at multiple real tumor cell lines₅₀Measurement method and results of

In view of example 90, Compound I_A41 shows stronger antiproliferative effect on kinases such as c-KIT, PDGFRa and the like and mutation thereof, and the compound is shown in the antiproliferative experimental method aiming at 8 real tumor cell lines in the embodiment. The cell lines included hamster ovary cell lines (CHL, CHO), GIST cell lines (GIST-T1, GIST-882, GIST-48B), BCR-ABL kinase class cell lines (MEG-01, K562, KU812), and the cells were specifically subjected to the same procedures as in example 90 except that 10. mu.L CCK8 cell proliferation assay reagent was added to GIST-882 under the respective suitable culture conditions, and the activity data are shown in Table 3.

TABLE 3 Compounds I of the invention_AAnti-proliferative Experimental Activity data (GI) against various real tumor cell lines (41)₅₀，μM)

Cell lines	I_A41(GI₅₀μM)	Imatinib (GI)₅₀μM)
			CHO	7.036	>10
CHL	>10	>10
			GIST-48B	>10	6.986
K562	>10	0.147
			MEG-01	9.092	0.0453
GIST-T1	<0.003	0.003
			GIST-882	<0.003	0.0202
KU812	>10	0.11
			MOLM14	0.0107	6.452
MV4-11	0.0105	3.589

As can be seen from Table 3, Compound I_A-41 pairs of GIST-T1(<0.003μM)、GIST-882(<0.003 mu M), MOLM14(0.0107 mu M) and MV4-11(0.0105 mu M) cell lines have obvious effect, and have no effect or insignificant effect on KU812, MEG-01, K562, GIST-48B, CHL and CHO cell lines. This result shows that the compounds I of the invention_A-41 No proliferation inhibition on Normal cellsThe preparation has high-efficiency selectivity on the treatment of gastrointestinal stromal tumor, and provides a direction for further developing anti-gastrointestinal stromal tumor targeted drugs.

Example 92

The invention relates to a method for testing the pharmacokinetic characteristics of a compound and the results thereof

In view of the compounds I of the invention_A41 good kinase inhibition activity and tumor cell inhibition activity, and then the pharmacokinetic characteristics of the kinase inhibition activity and the tumor cell inhibition activity are investigated, 30% HP- β -CD, PEG400 and trace DMSO (11:8:1) are selected as a solvent, and a preparation I is prepared_AA final concentration of-41 of 2mg/mL and administered intravenously at a dose of 2mg/kg to 6 male SD rats; sample collection (about 0.2mL blood for jugular vein) and data analysis were performed at appropriate times; single intravenous administration of rats I_AAfter 41, individuals and average plasma concentration-time curves (n ═ 3) are shown in fig. 1. pharmacokinetic parameters of the test substances were calculated according to the plasma concentration data of the drugs using the pharmacokinetic calculation software winnonlin6.2.1 non-compartmental model, respectively, see table 4. The experimental data show that: in intravenous injection, Compound I_A-41 shows acceptable half-life and clearance.

Table 4 single intravenous administration of rats I_A-41 after I_A-41 major pharmacokinetic parameters

NA: not applicable; -: no calculated value

Example 93

Test method and result of effect of compound on hERG potassium channel by using full-automatic patch clamp QPatch detection method

Is selected from_A-41 As a representative Compound, CHO-hERCulturing G cells in proper conditions and performing proper post-treatment to ensure the cell density to be 2-5 × 10⁶and/mL. Adding 2 mu L of 20mM compound mother liquor into 998 mu L of extracellular fluid, sequentially carrying out 3-time continuous dilution in the extracellular fluid containing 0.2% DMSO to obtain the final concentration to be tested, simultaneously selecting Cisapride as a positive control, and analyzing the effect of the medicament on CHO-hERG cells by utilizing the electrophysiological process recorded by a full-automatic patch clamp QPatch detection method, wherein the experimental data are shown in Table 5.

TABLE 5 Compounds I_AResults of inhibition of hERG Potassium Current by-41

Compound (I)	Maximum concentration inhibition (%)	IC₅₀(μm)
			I_A-41	39.78	>40
Cisapride	98.91	0.13

Note: > 40. mu.M means that the inhibitory effect of the compound is less than 50% at 40. mu.M

As can be seen from the above experimental results, the compounds I of the present invention_AThe inhibition effect of the-41 on the hERG potassium channel is very weak, which indicates that the cardiac toxicity is very low, and the application safety window is better, thus being beneficial to developing into clinical drugs.

Example 94

Drug effect experimental method and result of compound in GIST-T1 tumor cell mouse transplantation model

Selection of I in this experiment_A-41 as representative compound, 0.5% MC + 0.4% Tween80 as vehicle, vehicle group as blank control, and Imatinib as positive control. Mice were inoculated subcutaneously with GIST-T1 cells 1 x 10 at the right anterior shoulder blade⁶When the average tumor volume of the tumor-bearing mice reaches 100-200mm³At the time, the mice were randomly divided into 5 groups. The pharmacodynamic experimental design was performed as shown in Table 6, and the compound concentration was prepared as shown in Table 7. The drug was injected intraperitoneally once daily and tumor volume and body weight were measured. Experimental data the tumor volume and tumor weight of the treated group were compared with those of the control group for significant differences by one way ANOVA test, and the experimental results are shown in fig. 2-7. Finally, TUNEL staining, Ki67 staining and HE staining analysis were performed on each group of mouse GIST-T1 cells to observe the compound I of the present invention_A-41 effects on tumor cell proliferation and apoptosis (see FIG. 8).

TABLE 6 pharmacodynamic experimental design

TABLE 7 preparation method of test drug and reference drug

From the above results, it can be seen that:

1) weight: during the experiment, the body weight of each group of mice increased to some extent from the beginning to the end of the experiment, and the others were not abnormal.

2) Swelling and swelling treating medicineTumor volume: blank control group and low dose group (I) during the test period_A4125 mg/kg) mice showed a large increase in tumor appearance. The tumor volume of the high-dose group mice showed strong tumor inhibition.

3) Tumor volume relative percentage: the high-dose group drugs have an inhibitory effect on mouse tumors. The drug effect of the low-dose group is not obvious. It shows that the medicine can not penetrate the tumor well, and only when the concentration in the body fluid reaches a certain degree, the medicine can penetrate the tumor to play the inhibiting effect of the medicine.

4) Tumor weight of tumor: the experimental data result shows that the compound I of the invention_A41 the inhibition of gastrointestinal stromal tumor is in the same order of magnitude as that of the marketed drug imatinib.

5) Immunohistochemical experimental results: according to the staining phenomenon, HE staining results show that: the cell morphology and the tissue are relatively complete without necrosis, thereby providing a basis for explaining the drug effect later. As can be seen from the results of Ki67 staining, the tumor cells proliferated faster in the low dose group, while the tumor cells were significantly decreased in the high dose group and the Imatinib group due to the inhibition of the tumor cells. As can be seen from the TUNEL staining, the high dose group stained significantly more brown cells, and the majority of cells in the tumor were in an apoptotic state.

The general indication is that: compounds I of the invention_A41 in case of high dose 100mg/kg i.p., it has significant antiproliferative and pro-apoptotic effects on GIST-T1 tumor and almost corresponds to the marketed drug imatinib, as seen by I_A-41 is expected to be developed into a drug targeting gastrointestinal stromal tumor.

The substituted (1H-pyrazolo [3,4-b ] pyridine) urea antitumor compound is a micromolecule compound with a new structure, has multi-target tyrosine kinase inhibitory activity, has antiproliferative capability on various homologous BaF3 cell lines and real tumor cells, has good pharmacokinetic characteristics and no cardiotoxicity, shows better antitumor proliferation phenomenon in animal drug effect experiments, and has simple preparation process and low production cost, so the compound is expected to be developed into a multi-target tyrosine kinase antitumor drug with a new structure, and is particularly expected to be developed into a clinical candidate drug for resisting gastrointestinal stromal tumors.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. A compound of formula I, or a pharmaceutically acceptable salt thereof,

wherein,

x is O or S;

wherein said substitution refers to having 1-3 substituents selected from the group consisting of: halogen, hydroxy, nitro, cyano, C₁-C₄Alkyl radical, C₁-C₄Haloalkyl, C₁-C₄Alkoxy, -NRaRb, 4-10 membered heteroaryl, 4-8 membered heterocycloalkyl, C₆～C₁₀An aryl group;

ra and Rb are each independently H and C₁-C₄An alkyl group;

and the halogen is fluorine, chlorine, bromine or iodine.

2. A compound of formula I, or a pharmaceutically acceptable salt thereof, as claimed in claim 1 wherein R is¹Is C₁～C₄Straight or branched alkyl or C₁～C₄Linear or branched perfluoroalkyl, preferably, said R¹Is methyl or perfluoro-substituted methyl.

3. The compound of formula I, or a pharmaceutically acceptable salt thereof, according to claim 1, wherein the compound of formula I has one or more characteristics selected from the group consisting of:

(b) said C is₃～C₈Cycloalkyl is selected from: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl;

4. The compound of formula I, or a pharmaceutically acceptable salt thereof, according to claim 1, wherein the substituted C is₁～C₆The straight chain or branched alkyl group has the structure of formula II,

and said C₆～C₁₀The aryl group may optionally be further substituted by 1-3 groups selected from halogen, C₁-C₆Alkyl radical, C₁-C₄Substituent in alkoxyGeneration;

and n is an integer between 1 and 4.

5. The compound of formula I, or a pharmaceutically acceptable salt thereof, according to claim 1, wherein the substituted C is₁～C₆The linear or branched alkyl group has the structure of formula III,

wherein R is⁴Is C₁-C₂An alkyl group, and n is an integer of 1 to 3.

6. The compound of formula I, or a pharmaceutically acceptable salt thereof, according to claim 1, wherein the substituted C is₆～C₁₀Aryl is substituted phenyl and has the structure of formula IV,

7. The compound of formula I, or a pharmaceutically acceptable salt thereof, according to claim 1, wherein the compound of formula I is selected from the group consisting of:

8. A pharmaceutical composition, comprising:

(a) a therapeutically effective amount of a compound of formula I according to claim 1, or a pharmaceutically acceptable salt thereof,

and optionally (b) a pharmaceutically acceptable carrier.

9. Use of a compound of formula I according to claim 1, or a pharmaceutically acceptable salt thereof, for the preparation of (I) a pharmaceutical composition for inhibiting tumor cells or treating tumors, and (ii) a receptor tyrosine kinase inhibitor.

10. A method of inhibiting a receptor tyrosine kinase in vitro comprising the steps of:

(a) contacting a receptor tyrosine kinase with a compound of formula I as defined in claim 1, or a pharmaceutically acceptable salt thereof, thereby inhibiting the activity of the receptor tyrosine kinase.