CN108117551B - Substituted (1H-pyrazolo[3,4-b]pyridine) urea compounds and their antitumor use - Google Patents

Abstract

The invention provides a substituted (1H-pyrazole [3,4-b ]]The compound of the formula I can inhibit receptor tyrosine kinases (including c-Kit, PDGFR α and VEGFR2) related to tumorigenesis and tumor development, and is used for treating tumors.

Description

Substituted (1H-pyrazolo[3,4-b]pyridine) urea compounds and their antitumor use

technical field

The present invention relates to the fields of medicinal chemistry and pharmacotherapy, and more particularly to substituted (1H-pyrazo[3,4-b]pyridine) urea compounds and their antitumor uses.

Background technique

Cancer (malignant tumor) is one of the chronic non-communicable diseases that seriously threaten human health and has become an increasingly serious global public health problem. According to statistics, the number of cancer patients in my country is increasing. The current cancer incidence rate is 285.91/100,000 and the mortality rate is 1.8054/100,000. Moreover, it is expected that in the next few decades, the cancer incidence and mortality in my country will continue to rise. trend. Therefore, it is urgent to develop strong and effective treatment methods for malignant tumors. With the continuous development of molecular pharmacology and molecular oncology, the elucidation of the nature of tumors has gradually become clearer. Development of oncology drugs. At present, the focus of anti-tumor drug research and development is shifting from cytotoxic drugs to new anti-tumor drugs targeting multiple links. For example, anti-tumor drugs targeting key genes, regulatory molecules and cell receptors have begun to develop room into the clinic. Molecular targeted therapy has the advantages of orientation and localization, which enables the drug to selectively react with the target tissue at the cellular or subcellular level, so that the drug can be distributed in a controllable manner, and the drug can be continuously and slowly released in the target area. Reducing the dose of medication, improving the therapeutic effect, and reducing the toxic and side effects is an effective way of cancer treatment all over the world.

In the past ten years, more than ten targeted anti-tumor drugs have been successfully applied in the clinic, and kinases are the most important molecular targeted therapy drugs. related to kinases. The first tyrosine kinase inhibitor to enter the clinic, protein tyrosine kinase (PTK), is the most common growth factor receptor for most tumors. It is involved in the regulation, signal transmission and development of normal cells, and is also associated with tumor cells. It is closely related to the proliferation, differentiation, migration and apoptosis of tumor cells. By inhibiting its activity, it can destroy the signal transmission of tumor cells and selectively inhibit tumor cells without affecting normal cells. c-KIT is a typical type III receptor tyrosine kinase, which is a kind of ATP-competitive kinase. It plays a very important role in the occurrence and development of tumors, as well as in the process of invasion, migration and recurrence. It is the current molecular targeted therapy for tumors. One of the most popular targets of cancer, its inhibitors have also become a hot spot in the research and development of anti-tumor drugs.

c-KIT (also known as CD117) was discovered in 1987 and is a transmembrane receptor protein with tyrosine kinase activity encoded by the retrovirus proto-oncogene c-KIT. PDGFR), macrophage colony-stimulating factor-1 receptor (CSF-1R), and Fms-like tyrosine kinase receptor 3 (FLT3) together constitute the type III receptor tyrosine kinase superfamily, which play a key role in tumorigenesis and development. plays a very important role. There are more than 30 gain-of-function (GOF) mutant forms of c-KIT kinase, and it is the receptor of stem cell factor (SCF). After it is activated by stem cell growth factor, it forms a dimer, which leads to the transmembrane phosphorylation of Tyr568 and Tyr570 in JMD, thereby changing the three-dimensional structure of JMD and weakening its interaction with the active site of the kinase, that is, weakening the auto-inhibitory effect of the kinase , resulting in phosphorylation of the kinase domain, which in turn recruits downstream signaling molecules, and finally activates downstream signaling pathways to regulate cell growth and proliferation. Currently, it is known that c-KIT receptors are abnormally activated in various tumor tissues such as gastrointestinal stromal tumor (GIST), acute myeloid leukemia (AML), and lung cancer. This is mainly due to the overexpression and persistent activation of c-KIT mutation. Functional mutations of the C-KIT gene mainly occur in the extracellular domain (exons 8 and 9), the intracellular JMD (exon 11) and the A-loop region (exon 17) in the kinase domain. The incidence of 9 mutations was 3-21%, mainly in codons 501-503, and the main type was repeated insertion. The mutation rate of exon 11 is between 20% and 92%, which is concentrated between codons 550 and 599. The mutation types include: point mutation, deletion mutation, insertion mutation, substitution mutation, frame shift mutation, etc.; exon 13 The mutation site is mainly codon 642, and its mutation rate is 0.8-4.1%. It is worth noting that there are differences in the common c-KIT mutation sites in different tumors. For example, in GIST, mutations mainly occur in exon 11 (such as V560D), and in acute myeloid leukemia (AML) cells, c-KIT-activating mutations mainly occur in exon 17, such as D816V/H/Y, etc. And studies have shown that about 80% of GISTs have c-KIT-activating mutations.

Furthermore, angiogenesis is thought to play an important role in tumor initiation, progression, invasion and metastasis. On the one hand, angiogenesis lays a material foundation for nutrients and oxygen to enter tumor tissue and transport metabolites out of tissue cells; on the other hand, it provides a transport pathway for tumor cells to migrate to target organs and promote their continued growth. At present, studies have found that there are more than 30 factors related to tumor angiogenesis, such as platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), fibroblast factor (FGF), angiostatin, etc. PDGF can recruit Pericytes stimulate tumor angiogenesis, induce the proliferation and migration of vascular endothelial cells, smooth muscle cells and tumor cells, promote the production of connective tissue stroma, provide good support for new blood vessels, and play a direct role in tumor angiogenesis . Studies have found that PDGFR signal transduction is involved in a variety of tumors, of which 7-12% have mutations in the PDGFR-α gene, and the mutation sites are concentrated in exons 18 (Kinase II) and 12 (near-membrane regulation). region) and 14 (kinase region), and PDGFRa mutants are mostly low-risk, most common in gastric GIST. Further studies have shown that the signaling cascade mediated by VEGF/VEGFR-2 can regulate the proliferation, migration, survival and permeability of vascular endothelial cells, and promote angiogenesis. Fibroblast growth factor receptor (FGFR) binds to FGFs, which can trigger various responses of target cells and play a key role in tumorigenesis and angiogenesis.

Therefore, the development of multi-target (c-KIT, PDGFR, VEGFR and FGFR) therapeutic drugs targeting tumorigenesis and development of related signaling pathways is expected to produce better anti-tumor effects, especially for gastrointestinal stromal tumors. Effect.

SUMMARY OF THE INVENTION

One object of the present invention is to provide a substituted (1H-pyrazo[3,4-b]pyridine) urea compound, the general structure of which is shown in (I), or a pharmaceutically acceptable salt thereof;

Another object of the present invention is to provide substituted (1H-pyrazo[3,4-b]pyridine) ureas as dual inhibitors of c-KIT and PDGFR-a in the prevention or treatment of tyrosine kinase receptor inhibitors Related tumor diseases (such as gastrointestinal stromal tumors) applications.

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

in,

X is O or S;

R ¹ is C ₁ -C ₄ straight or branched chain alkyl or C ₁ -C ₄ straight or branched chain haloalkyl;

R ² is a substituted or unsubstituted C ₁ -C ₆ linear or branched chain alkyl group, a substituted or unsubstituted C ₃ -C ₈ cycloalkyl group, a substituted or unsubstituted C ₆ -C 10 aryl group, a substituted or unsubstituted C 6 -C ₁₀ aryl group, a substituted or unsubstituted C 3 -C 8 cycloalkyl group unsubstituted 4-10 membered heteroaryl;

Wherein, the substitution refers to having 1-3 substituents selected from the group consisting of halogen, hydroxyl, nitro, cyano, C ₁ -C ₄ alkyl, C ₁ -C ₄ haloalkyl, C ₁ -C ₄ Alkoxy, -NRaRb, 4-10-membered heteroaryl, 4-8-membered heterocycloalkyl, C ₆ -C ₁₀ aryl;

And the C ₆ -C ₁₀ aryl group in the above-mentioned substituents can optionally be further selected from halogen, hydroxyl, nitro, cyano, C ₁ -C ₆ alkyl, C ₁ -C ₄ haloalkyl by 1-3 groups. , Substituent substitution in C ₁ -C ₄ alkoxy;

And the C ₁ -C ₄ alkyl group in the above-mentioned substituents can optionally be further substituted by 1-3 substituents selected from halogen, hydroxyl, nitro, cyano, -NRaRb;

Ra and Rb are each independently H and _C1 - _C4 alkyl;

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

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

In another preferred example, the R ¹ is a C ₁ -C ₄ linear or branched chain alkyl or a C ₁ -C ₄ linear or branched perfluoroalkyl group, preferably, the R ¹ is methyl or perfluoro-substituted methyl.

In another preference, the compound shown in the described formula I has one or more features selected from the following group:

(a) the C ₁ -C ₆ straight or branched chain alkyl group is selected from: ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl base, neopentyl, n-hexyl, isobutyl, neobutyl;

(b) the C ₃ -C ₈ cycloalkyl group is selected from: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl;

(c) The C ₆ -C ₁₀ aryl group is selected from: phenyl, naphthyl, and indenyl, preferably, the C ₆ -C ₁₀ aryl group is selected from: phenyl, 2,3 -Dihydro-1H-inden-2-yl, naphth-1-yl, naphth-2-yl;

(d) the 4-10-membered heteroaryl group is selected from: pyrazolyl, pyridyl, quinolyl, furanyl, thienyl, preferably, the 4-10-membered heteroaryl group 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 Preferably, the 4-8 membered heterocycloalkyl is selected from: 2-piperidin-1-yl, 2-morpholin-4-yl, morpholin-4-yl, 4-methylpiperazine-1-yl base.

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

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

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

In another preferred example, the substituted C ₁ -C ₆ linear or branched alkyl group has the structure of formula II,

Wherein, R ³ is a 5-6 membered heteroaryl group, a 5-6 membered heterocycloalkyl group, or a C ₆ -C ₁₀ aryl group;

And the C ₆ -C ₁₀ aryl group may optionally be further substituted by 1-3 substituents selected from halogen, C ₁ -C ₆ alkyl group, and C ₁ -C ₄ alkoxy group;

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

In addition, n is an integer between 1 and 4.

In another preferred example, the 5-6-membered heteroaryl group is selected from: pyrazolyl, pyridyl, furyl, and thienyl, preferably, the 4-10-membered heteroaryl group 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 example, the 5-6 membered heterocycloalkyl is selected from: piperidinyl, morpholinyl, piperazinyl (4-methylpiperazinyl), pyrrolidinyl, tetrahydropyran Preferably, the 5-6 membered heterocycloalkyl is selected from: 2-piperidin-1-yl, 2-morpholin-4-yl, morpholin-4-yl, 4-methylpiperyl oxazin-1-yl.

In another preferred embodiment, the R ³ is furanyl, thienyl, pyridyl, piperidinyl, morpholinyl, or phenyl, and the phenyl may optionally be further replaced by 1-3 Substituents selected from halogen, C ₁ -C ₆ alkyl, and C ₁ -C ₄ alkoxy are substituted.

In another preferred example, the R ³ is furan-2-yl, thiophen-2-yl, pyridin-4-yl, 2-piperidin-1-yl, 2-morpholin-4-yl, benzene (unsubstituted phenyl), 2-methylphenyl, 4-methylphenyl, 2-fluorophenyl, 2-chlorophenyl, 2,6-difluorophenyl, 4-fluorophenyl, 4-chlorophenyl, or 4,5-dimethoxyphenyl.

In another preferred example, the substituted C ₁ -C ₆ linear or branched alkyl group has the structure of formula III,

Wherein, R ⁴ is a C ₁ -C ₂ alkyl group, and n is an integer between 1 and 3.

In another preferred embodiment, the R ⁴ is methyl or ethyl.

In another preferred example, the substituted C ₆ -C ₁₀ aryl group is a substituted phenyl group and has the structure of formula IV,

其中，所述的5-6元杂环烷基可非必须地进一步被1-3个选自卤素、C₁-C₄烷基中的取代基取代；并且R⁴为C₁-C₂烷基，且n为0～3之间的整数。Wherein, R ⁵ is hydrogen or C ₁ -C ₄ alkyl; R ⁶ is 5-6 membered heterocycloalkyl, or

Wherein, the 5-6 membered heterocycloalkyl may optionally be further substituted by 1-3 substituents selected from halogen and C ₁ -C ₄ alkyl; and R ⁴ is C ₁ -C ₂ alkane base, and n is an integer between 0 and 3.

并且R⁴为C₁-C₂烷基，且n为0～3之间的整数。In another preferred embodiment, the R ⁶ is 4-methylpiperazinyl, morpholinyl, or

And R ⁴ is a C ₁ -C ₂ alkyl group, and n is an integer between 0 and 3.

并且R⁴为C₁-C₂烷基，且n为0～3之间的整数。In another preferred embodiment, the R ⁶ is 4-methylpiperazin-1-yl, morpholin-4-yl, or

And R ⁴ is a C ₁ -C ₂ alkyl group, and n is an integer between 0 and 3.

In another preferred example, the structure of formula II is selected from: furan-2-ylmethyl, thiophen-2-ylmethyl, pyridin-4-ylmethyl, 2-piperidin-1-ylethyl , 2-morpholin-4-ylethyl, benzyl, phenethyl, phenylpropyl, phenbutyl, 2-methylbenzyl, 4-methylbenzyl, 2-fluorobenzyl, 2-chloro Benzyl, 2,6-difluorobenzyl, 4-fluorophenethyl, 4-chlorophenethyl, 4,5-dimethoxyphenethyl.

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

In another preferred example, the structure of formula IV is selected from: 2-(4-methylpiperazin-1-yl)phenyl, 3-(4-methylpiperazin-1-yl)phenyl, 4-(4-Methylpiperazin-1-yl)phenyl, 2-(morpholin-4-yl)phenyl, 2-methyl-4-(4-methylpiperazin-1-yl)benzene base, 3-methyl-4-(4-methylpiperazin-1-yl)phenyl, 5-methyl-2-(4-methylpiperazin-1-yl)phenyl, 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, (3-diethylamino)benzyl.

In another preferred embodiment, the R ² is selected from the following group:

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, naphth-1-yl, naphth-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, 4-fluorophenethyl, 4-Chlorophenethyl, 4,5-dimethoxyphenethyl, 2-dimethylaminoethyl, 2-diethylaminoethyl, 2-dimethylaminopropyl, 2-diethylaminopropyl , 2-(4-methylpiperazin-1-yl)phenyl, 3-(4-methylpiperazin-1-yl)phenyl, 4-(4-methylpiperazin-1-yl)benzene base, 2-(morpholin-4-yl)phenyl, 2-methyl-4-(4-methylpiperazin-1-yl)phenyl, 3-methyl-4-(4-methylpiperyl) oxazin-1-yl)phenyl, 5-methyl-2-(4-methylpiperazin-1-yl)phenyl, 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, (3-diethylamine) group) benzyl.

In another preference, the compound of formula I is selected from the following group:

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-pyridyl) Azolo[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, N-n-pentyl-N'-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea, N-n-hexyl-N' -(4-(3-Methyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea, N-n-hexyl-N'-(4-(3-trifluoromethyl) yl-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-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-ring Amyl-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-(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) yl-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-(thiophen-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-pyrazolo[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-trifluoromethyl-1H-pyrazole) [3,4-b]pyridin-4-yl)phenyl)urea, N-(2-morpholin-4-ylethyl)-N'-(4-(3-methyl-1H-pyrazole) [3,4-b]pyridin-4-yl)phenyl)urea, N-benzyl-N'-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridine- 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-(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-chlorobenzene ethyl)-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-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-pyridyl) azolo[3,4-b]pyridin-4-yl)phenyl)urea, N-(2-phenethyl)-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]pyridine) -4-yl)phenyl)urea, N-(3-dimethylaminopropyl)-N'-(4-(3-trifluoromethyl-1H-pyrazolo[3,4-b]pyridine- 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-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea, N -(4-(4-Methylpiperazin-1-yl)phenyl)-N'-(4-(3-trifluoromethyl-1H-pyrazolo[3,4-b]pyridine-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]pyridine-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-(morpholine-4- base)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-pyrazole) [3,4-b]pyridin-4-yl)phenyl)urea, N-(2-(4-methylpiperazin-1-yl)phenyl)-N'-(4-(3-methyl) yl-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-diethylamino) Phenyl)-N'-(4-(3-trifluoromethyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea, N-((3-dimethylamine yl)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-pyrazole) [3,4-b]pyridin-4-yl)phenyl)urea, N-((4-diethylamino)phenyl)-N'-(4-(3-methyl-1H-pyrazole) [3,4-b]pyridin-4-yl)phenyl)urea, N-((3-diethylamino)phenyl)-N'-(4-(3-methyl-1H-pyrazole) [3,4-b]pyridin-4-yl)phenyl)urea, N-((3-dimethylamino)phenyl)-N'-(4-(3-methyl-1H-pyrazole) [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)phenyl)-N'-( 4-(3-Methyl-1H-pyrazolo[ 3,4-b]pyridin-4-yl)phenyl)urea.

In another preferred embodiment, the X is O and R ¹ is methyl.

In another preferred example, the R ² is a 4-10-membered heteroaryl group or a phenyl group, wherein the phenyl group can be further substituted by halogen, C ₁ -C ₄ alkyl, -NRaRb, 4-8-membered Heterocycloalkyl, and said Ra and Rb are both methyl or ethyl.

In another preferred embodiment, the R ² is a 4-10-membered heteroaryl group or a phenyl-substituted C ₁ -C ₆ alkyl group, wherein the phenyl group may optionally be further substituted by halogen.

In another preference, the compound of formula I is selected from the following group:

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

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

In another preferred example, the pharmaceutical composition contains 0.001-99wt%, preferably 0.1-90wt%, more preferably 1-80wt% of the compound represented by formula I, or a pharmaceutically acceptable salt thereof, 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 include wild-type and mutant-type tumor cells of c-KIT and PDGFRα.

In another preferred embodiment, the tumor cells are 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 BaF3 cells are selected from the following group: BaF3 cells, c-KIT-BaF3 cells, c-KIT-A829P-BaF3 cells, c-KIT-L576P-BaF3 cells, c-KIT-C674S -BaF3 cells, c-KIT-D816H-BaF3 cells, c-KIT-D816V-BaF3 cells, c-KIT-T670I-BaF3 cells, c-KIT-V559D-T670I-BaF3 cells, c-KIT-V654A-BaF3 cells , c-KIT-N822K-BaF3 cells, c-KIT-V559D-BaF3 cells, c-KIT-V559D-V654A-BaF3 cells, PDGFRα-BaF3 cells, PDGFRα-T674I-BaF3 cells, FLT3-BaF3 cells, and FLT3- ITD-BaF3 cells.

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

In another preferred embodiment, the pharmaceutical composition can be used to inhibit receptor tyrosine kinase.

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, PDGFRα, VEGFR2 or a combination thereof.

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

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

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

(a) contacting the receptor tyrosine kinase with the compound represented by formula I according to the first aspect of the present invention, or a pharmaceutically acceptable salt thereof, thereby inhibiting the activity of the receptor tyrosine kinase.

In another preferred example, in step (a), the compound represented by the formula I described in the first aspect of the present invention, or a pharmaceutically acceptable salt thereof, is added to the cell culture system, so that it interacts with the receptor. tyrosine kinase contacts.

In another preferred embodiment, the cells are normal cells or tumor cells.

In another preferred embodiment, the cells are mammalian cells.

In another preferred embodiment, the cells are human cells.

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

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

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

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (eg, the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, it is not repeated here.

Description of drawings

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

FIG. 2 shows a graph of the average body weight change of each group of mice in Example 94. FIG.

FIG. 3 shows a graph of the average relative body weight change of each group of mice in Example 94. FIG.

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

FIG. 5 shows a graph showing the change in relative tumor volume of each group of mice in Example 94. FIG.

FIG. 6 shows the tumor photos of the GIST-T1 mouse IA _- 41 drug efficacy test of each group of mice in Example 94. FIG.

FIG. 7 shows the tumor weight map of each group of mouse tumors in Example 94. FIG.

Figure 8 shows HE, Ki67, Tunel staining of compound IA _- 41 efficacy test

Detailed ways

After extensive and in-depth research, the inventors unexpectedly found for the first time that the compound of formula I can significantly inhibit the activity of receptor tyrosine kinases (including c-KIT, PDGFR-α and VEGFR-2) related to the occurrence and development of tumors . Experiments show that the compound of formula I has inhibitory activity on all three kinases, especially on c-KIT and PDGFR-α receptors. At the same time, in the cell level study, an optimal kinase activity compound of the present invention showed strong anti-proliferative ability to the wild-type and various mutant cell lines of c-KIT and PDGFR-a, as well as to 8 real tumors. The cell line exhibits high selectivity and potent anti-proliferative activity, especially for gastrointestinal stromal tumor (GIST) cell lines. In pharmacokinetic and hERG studies, the compound showed acceptable kinetic characteristics and a good drug safety profile. In the follow-up GIST-T1 animal pharmacodynamic model test, the compound also showed good anti-tumor proliferation effect. Therefore, the compounds of the present invention can be used as multi-targeted tyrosine kinase inhibitors, and can be selectively developed into multi-targeted antitumor drugs for gastrointestinal stromal tumors.

the term

The term " _C1 - _C6 alkyl" refers to straight or branched chain alkyl groups having 1-6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl , tert-butyl, or similar groups.

The term "C ₁ -C ₄ alkoxy" refers to 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 similar groups.

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 a or multiple double bonds, but none of the rings have a fully 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 is present in the ring backbone, wherein one or more of the rings may contain one or more double bonds. For example, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, or the like.

The term "aromatic ring" refers to an aromatic ring having a conjugated pi electron system, including carbocyclic aryl, heteroaryl.

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

The term "heteroaryl" refers to an aryl group having at least one heteroatom as a ring atom and the remaining ring atoms being carbon, 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, furanyl, furanylene, fluorenyl, pyrrolyl, N-alkylpyrrolyl, thienyl, oxazolyl, imidazolyl, thiazolyl, benzofuranyl, benzene thienyl, pyridyl, pyrimidinyl, quinazolinyl, quinolinyl, isoquinolinyl and indolyl.

The term "amino" means _-NH2 , -NH-( _C1 - _C6 alkyl) or -N( _C1 - _C6 alkyl) ₂ .

The term "halogen" refers to fluorine, chlorine, bromine, iodine. The term "halo" refers to fluoro, chloro, bromo, iodo.

As used herein, unless otherwise specified, the alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl groups described herein include both substituted and unsubstituted moieties. In addition, cycloalkyl, heterocycloalkyl, aryl and heteroaryl groups can also be fused to each other.

Unless otherwise specified, the term "substituted" refers to the replacement of one or more hydrogen atoms on a group with a substituent selected from the group consisting of _C1 - _C10 alkyl, C3 _{- C10} cycloalkyl, _C1 -C ₁₀ alkoxy, halogen, hydroxyl, carboxyl (-COOH), C ₁ -C ₁₀ aldehyde, C ₂ -C ₁₀ acyl, C ₂ -C ₁₀ ester, amino, phenyl; said phenyl Including unsubstituted phenyl or substituted phenyl with 1-3 substituents selected from: halogen, C ₁ -C ₁₀ alkyl, cyano, OH, nitro, C ₃ -C ₁₀ ring Alkyl, C ₁ -C ₁₀ alkoxy, amino.

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

Compounds 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 all refer to the first compound of the present invention. The compound having the structure shown in formula I described in the aspect.

The compounds of the present invention may contain asymmetric or chiral centers and thus may 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 (eg, racemic mixtures), are included within the scope of the present invention. within the range.

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

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

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

The present invention also encompasses isotopically-labeled compounds of the present invention, which, apart from the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from that normally found in nature, are the same as the narrator. Examples of isotopes that may be incorporated into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as: ² hydrogen, ³ hydrogen, ¹¹ carbon, ¹³ carbon, ¹⁴ carbon, respectively , ¹³ nitrogen, ¹⁵ nitrogen, ¹⁵ oxygen, ¹⁷ oxygen, ¹⁸ oxygen, ³¹ phosphorus, ³² phosphorus, ³⁵ sulfur, ¹⁸ fluorine, ¹²³ iodine, ¹²⁵ iodine and ³⁶ chlorine.

Certain isotopically-labeled compounds of the invention (eg, those labeled ^{with3H and14C} ) are useful in compound and/or substrate tissue distribution assays. Tritiated (ie ³ H) and carbon-14 (ie ¹⁴ C) isotopes are particularly preferred because of their ease of preparation and detection. Furthermore, substitution with heavier isotopes such as deuterium (ie, ² H) may provide certain therapeutic advantages (eg, increased in vivo half-life or reduced dosage requirements) resulting from greater metabolic stability, and thus may in some cases be preferred. Positron emitting isotopes such ^as15O , ^13N , ^{11C and18F} are used in positron emission tomography (PET) studies to examine substrate acceptor occupancy. Isotopically labeled compounds of the invention can generally be prepared following methods 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 compounds involved in the present invention have a novel substituted (1H-pyrazo[3,4-b]pyridine) urea structure, and are subjected to three receptor tyrosine kinases (including c - Inhibitory activity test of KIT, PDGFR-α and VEGFR-2), anti-proliferative activity test of wild-type and various mutant cell lines of c-KIT and PDGFR-a, inhibitory activity test of 8 real tumor cell lines , as well as pharmacokinetic characteristics, hERG and animal pharmacodynamic model experimental tests, etc., various activity results show that some compounds of the present invention have inhibitory activities on three kinases, especially on c-KIT and PDGFR-α receptors. The inhibitory activity is particularly obvious. The compound with the best kinase inhibitory activity has inhibitory activity on most wild and mutant c-KIT and PDGFR-a cell lines, and has a good drug safety window, which can selectively act on tumor cells. It also showed excellent anti-tumor effect in the later animal pharmacodynamic model, laying a foundation for the further design and development of new multi-target receptor tyrosine kinase inhibitor anti-tumor drugs in the future.

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

In formula I: X is O or S; R ¹ is C ₁ -C ₄ straight or branched chain alkyl or C ₁ -C ₄ straight or branched perfluoroalkyl; R ² is C ₁ -C ₆ straight chain Chain or branched alkyl, C ₃ -C ₆ cycloalkyl, C ₅ -C ₆ aromatic heterocyclyl, fused ring or selected from the following structures II, III, IV:

Wherein, in formula II, R ³ is a C ₅ -C ₆ aromatic heterocyclic group, a substituted or unsubstituted piperidine and morpholine ring group, a substituted or unsubstituted six-membered aromatic ring group, and n is between 1 and 4. integer;

The substituent of the substituted phenyl group is selected from: C ₁ -C ₆ straight or branched chain alkyl, halogen, C ₁ -C ₃ alkoxy, and the number of the substituent is an integer of 0-3;

Wherein, in formula III, R ⁴ is a C ₁ -C ₂ alkyl group, and n is an integer between 1 and 3;

n为0～3之间的整数。Wherein, in formula IV, R ⁵ is hydrogen or C ₁ -C ₃ alkyl; R ⁶ is substituted or unsubstituted 4-methylpiperazinyl or morpholine ring group, or

n is an integer between 0 and 3.

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

R ² is C ₁ -C ₆ straight or branched chain alkyl, C ₃ -C ₆ cycloalkyl, C ₅ -C ₆ aromatic heterocyclic group, fused ring or selected from the following structures II, III, IV:

Wherein, in formula II, R ³ is a C ₅ -C ₆ aromatic heterocyclic group, a substituted or unsubstituted piperidine and morpholine ring group, a substituted or unsubstituted six-membered aromatic ring group, and n is between 1 and 4. integer;

The substituent of the substituted phenyl group is selected from: C ₁ -C ₆ straight or branched chain alkyl, halogen, C ₁ -C ₃ alkoxy, and the number of the substituent is an integer of 0-3;

Wherein, in formula III, R ⁴ is a C ₁ -C ₂ alkyl group, and n is an integer between 1 and 3;

n为0～3之间的整数。Wherein, in formula IV, R ⁵ is hydrogen or C ₁ -C ₃ alkyl; R ⁶ is substituted or unsubstituted 4-methylpiperazinyl or morpholine ring group, or

n is an integer between 0 and 3.

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

X is O or S; R ² is C ₁ -C ₆ straight or branched chain alkyl, C ₃ -C ₆ cycloalkyl, C ₅ -C ₆ aromatic heterocyclic group, fused ring or selected from the following structure II, III, IV:

Wherein, in formula II, R ³ is a C ₅ -C ₆ aromatic heterocyclic group, a substituted or unsubstituted piperidine and morpholine ring group, a substituted or unsubstituted six-membered aromatic ring group, and n is between 1 and 4. integer;

The substituent of the substituted phenyl group is selected from: C ₁ -C ₆ straight or branched chain alkyl, halogen, C ₁ -C ₃ alkoxy, and the number of the substituent is an integer of 0-3;

Wherein, in formula III, R ⁴ is a C ₁ -C ₂ alkyl group, and n is an integer between 1 and 3;

n为0～3之间的整数。Wherein, in formula IV, R ⁵ is hydrogen or C ₁ -C ₃ alkyl; R ⁶ is substituted or unsubstituted 4-methylpiperazinyl or morpholine ring group, or

n is an integer between 0 and 3.

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

Preparation

The present invention also provides preparation methods for the substituted (1H-pyrazo[3,4-b]pyridine) urea compounds IA _{- IB} and intermediates V-XIV of the general formula (I), the specific synthesis methods are as follows.

Synthesis of _IA :

In the formula, R ² has the same meaning as described above.

Specifically include the following steps:

1) Add 3-methyl-5-amino-1H-pyrazole, p-nitrobenzaldehyde and 2,2-dimethyl-1,3-dioxane-4,6-dione to a round bottom flask , dissolved in N,N-dimethylformamide and heated at its boiling temperature until no _CO2 was released. A solid precipitated out in the solution, and after cooling to room temperature, an appropriate amount of isopropanol was added to dilute. Suction filtration, the solid was dissolved in N,N-dimethylformamide and then sonicated for 15 minutes, and after suction filtration, the intermediate 3-methyl-4-(4-nitrophenyl)-4,5-dihydro was obtained -1H-Pyrazolo[3,4-b]pyridin-6(7H)-one (Intermediate V).

2) Dissolve intermediate V in dioxane, add 2,3-dichloro-5,6-dicyano-1,4-benzoquinone and N,O-bis(trimethylsilyl)tris Fluoroacetamide, reflux overnight. After the reaction was completed, it was cooled, the reaction solution was poured into an appropriate amount of water, and made alkaline with saturated NaHCO ₃ solution, and a large amount of solid was precipitated from the solution. Suction filtration, the filter cake was dissolved with N,N-dimethylformamide, sonicated for 15 minutes and then suction filtration was continued, the filter cake was washed with ethyl acetate, and the filter cake was dried to obtain the intermediate 3-methyl-4-(4- Nitrophenyl)-1H-pyrazolo[3,4-b]pyridin-6(7H)-one (Intermediate VI).

3) The intermediate VI was dissolved in an appropriate amount of phenylphosphoryl dichloride, and the reaction was carried out at 110° C. overnight. After the reaction was completed, it was cooled, the system was poured into an appropriate amount of ice water, stirred for half an hour, filtered with suction, the filter cake was added with water, stirred evenly, and then adjusted to weakly alkaline with saturated sodium bicarbonate solution. Suction filtration, the filter cake is dried to obtain the intermediate 6-chloro-3-methyl-4-(4-nitrophenyl)-6,7-dihydro-1H-pyrazolo[3,4-b]pyridine ( Intermediate VII)

4) Mix zinc powder, hydrazine hydrate and tetrahydrofuran in a three-necked flask, under nitrogen protection, add the tetrahydrofuran solution dissolved with intermediate VII into the reaction system dropwise at a uniform speed, and reflux for 4 hours after adding. After the reaction was completed, it was cooled, 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 to obtain the intermediate 4-(3-methyl-1H-pyrazole[3,4-b]] Pyridin-4-yl)aniline (Intermediate VIII).

5) Dissolve the intermediate VIII in an appropriate amount of dimethyl sulfoxide, add a commercial or self-made isocyanate compound (R ² -NCO), and then add triethylamine, and react at room temperature overnight. After the reaction was completed, it was cooled and concentrated, and the residue was separated by column chromatography to obtain compound NR ² -N'-[4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl) Phenyl]urea ( _IA ).

Synthesis of _IB :

In the formula, R ² has the same meaning as described above.

Specifically include the following steps:

1) Dissolve sodium hydride in an appropriate amount of anhydrous tetrahydrofuran, heat to reflux, then add dropwise a mixed solution of ethyl trifluoroacetate and acetonitrile in tetrahydrofuran, and react at 70°C for 15h after the addition. After the reaction is completed, cool to room temperature, pour the reaction solution into water, extract with an appropriate amount of anhydrous ether, adjust the pH of the aqueous phase to 2 with 2M hydrochloric acid solution, and extract again with anhydrous ether. The total organic phase was dried with anhydrous sodium sulfate, filtered, and the filtrate was distilled under reduced pressure to obtain the crude product 4,4,4-trifluoro-3-carbonylbutyronitrile (Intermediate IX);

2) Mix intermediate IX and hydrazine hydrate, slowly add methanesulfonic acid dropwise, and react overnight at 80°C after the addition. After the reaction was completed, distillation under reduced pressure, the residue was dissolved in ethyl acetate:n-hexane=1:1, stirred for 5 minutes, suction filtered, the filtrate was distilled under reduced pressure, and separated by column chromatography to obtain 3-trifluoromethyl-5 - amino-1H-pyrazole (Intermediate X);

3) Dissolve p-nitrobenzaldehyde in ethanol, add 2,2-dimethyl-1,3-dioxane-4,6-dione, react at 80°C for 3h, then add intermediate X ethanol solution, after a solid precipitated out, the reaction was carried out for half an hour. After the reaction was completed, it was cooled to room temperature and diluted with an appropriate amount of isopropanol. Suction filtration, the solid was dissolved in methanol, ultrasonicated for 15 minutes, and then suction filtered to obtain the intermediate 3-trifluoromethyl-4-(4-nitrophenyl)-4,5-dihydro-1H-pyrazole[3 ,4-b]pyridin-6(7H)-one (Intermediate XI).

4) Dissolve intermediate XI in chlorobenzene, add 2,3-dichloro-5,6-dicyano-1,4-benzoquinone and N,O-bis(trimethylsilyl)trifluoroethyl amide, reflux overnight. After the reaction was completed, it was cooled to room temperature, the reaction solution was poured into an appropriate amount of water, and made alkaline with saturated NaHCO ₃ solution, and a large amount of solid was precipitated from the solution. Suction filtration, washing the filter cake with water, and drying the filter cake to obtain the intermediate 3-trifluoromethyl-4-(4-nitrophenyl)-1H-pyrazo[3,4-b]pyridine-6(7H) - Ketone (Intermediate XII).

5) The intermediate XII was dissolved in an appropriate amount of phenylphosphoryl dichloride and reacted at 110°C overnight. After the reaction was completed, it was cooled, the system was poured into an appropriate amount of ice water, stirred for half an hour, filtered with suction, the filter cake was added with water, stirred evenly, and then adjusted to weakly alkaline with saturated sodium bicarbonate solution. Suction filtration, drying the filter cake, if there is any remaining raw material, continue to add phenylphosphonyl dichloride, and react according to the initial conditions until the reaction of the intermediate XII is complete, and after post-processing again, the intermediate 6-chloro-3- Trifluoromethyl-4-(4-nitrophenyl)-6,7-dihydro-1H-pyrazolo[3,4-b]pyridine (Intermediate XIII)

6) Mix zinc powder, hydrazine hydrate and tetrahydrofuran in a three-necked flask, under nitrogen protection, add the tetrahydrofuran solution dissolved with intermediate XIII into the reaction system dropwise at a uniform speed, and after adding, reflux reaction overnight. After the reaction was completed, it was cooled, 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 to obtain the intermediate 4-(3-trifluoromethyl-1H-pyrazo[3,4- b] Pyridin-4-yl)aniline (Intermediate XIV).

7) Dissolve intermediate XIV in an appropriate amount of dimethyl sulfoxide or anhydrous THF, add a commercial or self-made isocyanate compound (R ² -NCO), add triethylamine, and react at room temperature overnight. After the reaction was completed, it was cooled and concentrated, and the residue was separated by column chromatography to obtain compound NR ² -N'-[4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl) Phenyl]urea ( _IB ).

According to the teaching of the above preparation methods, those of ordinary skill in the art can obtain all the compounds contained in formula I without creative efforts.

Pharmaceutical compositions and methods of administration

In the present invention, the pharmaceutical composition can be directly used for disease treatment, for example, for anti-tumor treatment. When using the pharmaceutical preparation of the present invention, other therapeutic agents, such as antitumor drugs, can also be used at the same time.

The present invention provides a pharmaceutical composition comprising a safe and effective amount of the compound of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, powders, and combinations thereof. The drug formulation should match the mode of administration.

Taking a pharmaceutical composition as an example, the composition of the present invention can be prepared in the form of injection, for example, prepared by a conventional method with a physiological saline or an aqueous solution 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 nebulization. The active ingredient is administered in a therapeutically effective amount, eg, about 1 microgram/kg body weight to about 5 mg/kg body weight per day. In addition, the compounds of the present invention may also be used with other therapeutic agents.

The pharmaceutical compositions of the present invention can be administered to a desired subject (eg, humans and non-human mammals) by conventional means. Representative modes of administration include, but are not limited to: oral, injection, aerosol inhalation, and the like.

When a pharmaceutical composition is used, 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 about 10 micrograms/kg body weight to about 1 mg/kg body weight. Of course, the specific dosage should also take into account the route of administration, the patient's health and other factors, which are all within the skill of the skilled physician.

The main advantages of the present invention include:

(a) The compounds of the present invention can inhibit various receptor tyrosine kinases (including c-Kit, PDGFRα and VEGFR2).

(b) The compounds of the present invention exhibit highly selective and potent anti-proliferative activity against various tumor cell lines.

(c) The compounds of the present invention have good kinetic characteristics and drug safety.

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

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In the following examples, the experimental methods without specific conditions are usually in accordance with conventional conditions, or in accordance with the conditions suggested by the manufacturer. Percentages and parts are by weight unless otherwise indicated.

Example 1

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

Combine 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 Put it into a 100 ml round-bottomed flask, dissolve it with 20 ml of N,N-dimethylformamide, and then heat the reaction at 110 °C until no CO ₂ is released. After 30 minutes of reaction, a solid precipitated out of the solution, which was cooled to room temperature and diluted by adding 50 ml of isopropanol. Suction filtration, the solid was sonicated with N,N-dimethylformamide for 15 minutes, continued suction filtration after completion, and the solid was dried to obtain the title compound, 9.8 g of white solid (Intermediate V), yield 72%.

¹ H-NMR (400MHz, DMSO-d ₆ )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)

Put 14.9g of intermediate V into a 250ml flask, add 100ml of dioxane to dissolve, then add 66ml of N,O-bis(trimethylsilyl)trifluoroacetamide, and then add 15g of 2,3 -Dichloro-5,6-dicyano-1,4-benzoquinone, reflux overnight. After the reaction, the reaction solution was poured into an appropriate amount of water and made alkaline with saturated NaHCO ₃ solution, and the solid was precipitated from the solution. Suction filtration, the filter cake was washed with N,N-dimethylformamide and ethyl acetate successively, and the filter cake was dried at 40° C. to obtain 11.3 g of light yellow solid (intermediate VI) with a yield of 76%.

¹ H-NMR (400MHz, DMSO-d ₆ )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)

Put 5 g of intermediate VI into a 100-ml flask, add 15 ml of phenylphosphoryl dichloride to dissolve, and react at 110° C. overnight. After the reaction was completed, pour the system into an appropriate amount of ice water after cooling, and stir for 1 hour, and a large amount of solid was precipitated. Suction filtration, the filter cake was adjusted to weakly alkaline with saturated sodium bicarbonate solution, and the second suction filtration was performed, and the filter cake was rinsed with a large amount of water, and dried at 40 ° C to obtain 3.1 grams of gray-yellow solid (intermediate VII), the yield is 58%.

¹ H-NMR (400MHz, DMSO-d ₆ )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 grams of zinc powder, 70 milliliters of hydrazine monohydrate, and 10 milliliters of tetrahydrofuran were mixed in a 250 milliliter three-necked flask, and under nitrogen protection, 70 milliliters of tetrahydrofuran solution dissolved with 7 grams of intermediate VII was added dropwise to the reaction system at a uniform rate, and added. After completion, the reaction was refluxed for 4 hours. After the reaction was completed, suction filtration, the filtrate was extracted three times with tetrahydrofuran, the extract was distilled under reduced pressure, and the residue was separated by column chromatography with an eluent (tetrahydrofuran:dichloromethane=1:3, v/v) to obtain a white solid (middle). Body VIII) 3.1 g, 57% yield.

¹ H-NMR (400MHz, DMSO-d ₆ )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)

Dissolve 0.5 g of sodium hydride in 5 ml of anhydrous tetrahydrofuran, heat under reflux to obtain reaction system 1, then dissolve 0.4 g of acetonitrile in 2 ml of anhydrous tetrahydrofuran, add 1.2 g of ethyl trifluoroacetate, and add the mixed solution dropwise to the reaction system In system 1, the reaction was carried out at 70 °C for 15 h after the addition. After the reaction is completed, cool to room temperature, pour the reaction solution into water, extract with an appropriate amount of anhydrous ether, adjust the pH of the aqueous phase to 2 with 2M hydrochloric acid solution, and extract again with anhydrous ether. The total organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was distilled under reduced pressure to obtain the crude product 4,4,4-trifluoro-3-carbonylbutyronitrile (Intermediate IX). Because this product has a low boiling point, no obvious fluorescence under ultraviolet light, it is difficult to monitor, and it is not further purified, so the yield is calculated with the next step. 100% calculated.

Example 6

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

Mix 1.16 g of intermediate IX and 4.5 ml of hydrazine hydrate, add 5.7 ml of methanesulfonic acid dropwise, and react at 80° C. overnight after the addition is complete. After the reaction was completed, it was distilled under reduced pressure, the residue was dissolved in ethyl acetate: n-hexane = 1: 1, stirred for 5 minutes, suction filtered, the filtrate was distilled under reduced pressure, and after concentration, the eluent (methanol: dichloromethane = 1: 1) was used. 60, v/v) and subjected to column chromatography to obtain 102 mg of pale yellow oil (Intermediate X). Example 5 and Example 6, the two-step total yield is 8%.

¹ H NMR (400MHz, DMSO-d ₆ )δ: 12.15(s, 1H), 5.53(s, 1H), 5.35(s, 2H).

Example 7

Intermediate 3-Trifluoromethyl-4-(4-nitrophenyl)-4,5-dihydro-1H-pyrazolo[3,4-b]pyridin-6(7H)-one (Intermediate XI ) preparation

Put 0.99 g of p-nitrobenzaldehyde and 0.95 g of 2,2-dimethyl-1,3-dioxane-4,6-dione into a 50-ml round-bottomed flask, dissolve with 20 ml of ethanol, The reaction was heated at 80 °C for 3 h, and then 0.9 g of intermediate X in ethanol solution (5 ml) was added. After a solid was precipitated, the reaction was carried out for half an hour. After the reaction was completed, it was cooled to room temperature and diluted with 10 ml of isopropanol. After suction filtration, the solid was dissolved in methanol and sonicated for 15 minutes, and then suction filtered. The solid was dried to obtain the title compound, 0.376 g of a pale yellow solid (intermediate XI), with a yield of 38%.

1H NMR (400MHz, DMSO-d ₆ )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)

Put 0.736 g of intermediate XII into a 50 ml flask, add 15 ml of chlorobenzene to dissolve, then add 2.7 ml of N,O-bis(trimethylsilyl) trifluoroacetamide, and then add 0.975 g of 2,3-difluoroacetamide Chloro-5,6-dicyano-1,4-benzoquinone, reflux overnight. After the reaction, the reaction solution was poured into an appropriate amount of water and made alkaline with saturated NaHCO ₃ solution, and the solid was precipitated from the solution. Suction filtration, the filter cake was washed with water, and dried at 60° C. to obtain 0.587 g of a light yellow solid (intermediate XII) with a yield of 80%.

¹ H-NMR (400MHz, DMSO-d ₆ )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)

Put 0.587 g of intermediate XII into a 100-ml flask, add 10 ml of phenylphosphoryl dichloride to dissolve, and react at 110° C. overnight. After the reaction was completed, pour the system into an appropriate amount of ice water after cooling, and stir for 1 hour, and a large amount of solid was precipitated. Suction filtration, the filter cake is adjusted to weakly alkaline with saturated sodium bicarbonate solution, and then suction filtered for a second time, rinse the filter cake with a large amount of water, dry at 60 ° C, and point the plate, if there is still intermediate XII, if the reaction is not complete, The reaction operation was repeated, and the final 300 mg of gray-yellow solid (Intermediate XIII), the yield was 48%.

¹ H-NMR (400MHz, DMSO-d ₆ )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 2 ml of tetrahydrofuran were mixed in a 25-ml three-necked flask, and under nitrogen protection, 2 ml of tetrahydrofuran solution containing 200 mg of Intermediate XIII was added dropwise to the reaction system at a uniform rate, and added After completion, the reaction was refluxed overnight. After the reaction was completed, suction filtration, the filtrate was extracted three times with tetrahydrofuran, the extract was distilled under reduced pressure, and the residue was separated by column chromatography with an eluent (ethyl acetate:petroleum ether=1:3, v/v) to obtain a white solid ( Intermediate XIV) 90 mg in 55% yield.

¹ H-NMR (400MHz, DMSO-d ₆ )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

Preparation of N-ethyl-N'-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (Compound IA _- 1)

50 mg of 4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl)aniline (Intermediate VIII) was dissolved in 1 mL of dimethylsulfoxide and 22 μL of ethyl acetate was added. Base isocyanate and 10 microliters of triethylamine were reacted overnight at room temperature. After the reaction, 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), the resulting solid was washed with diethyl ether to give the title compound as a yellow powder, 45 mg, 69% yield. Mp: 211-213°C; Purity: 93%;

¹ H NMR (400 MHz, CDCl ₃ ) δ: 9.17 (s, 1H), 8.49 (d, J=4.8 Hz, 2H), 7.30 (d, J=8.0 Hz, 2H), 7.12 (d, J=4.7 Hz ,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 forC ₁₆ H ₁₈ N ₅ O[M+H] ⁺ 296.1511, found 296.1506.

Example 12

Preparation of N-propyl-N'-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (Compound IA _- 2)

Except replacing ethyl isocyanate with propyl isocyanate, the other required raw materials, reagents and preparation methods are the same as in IA _- 1, to obtain the title compound, 44 mg of yellow powder, yield 64%. Mp: 208-210°C; Purity: 92%;

¹ H NMR (400 MHz, CDCl ₃ ) δ: 9.15 (t, J=5.5 Hz, 1H), 8.56 (d, J=4.9 Hz, 1H), 7.28 (d, J=8.2 Hz, 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 C ₁₇ H ₂₀ N ₅ O[M+H] ⁺ 310.1668, found 310.1662.

Example 13

Preparation of N-butyl-N'-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (Compound IA _- 3)

Except replacing ethyl isocyanate with butyl isocyanate, the other required raw materials, reagents and preparation methods are the same as in IA _- 1, to obtain the title compound, 62 mg of yellow powder, yield 72%. Mp: 177-180°C; Purity: 95%;

¹ H NMR (400 MHz, CDCl ₃ ) δ: 9.20 (s, 1H), 8.49 (d, J=4.8 Hz), 7.27 (d, J=7.8 Hz), 7.12 (d, J=4.8 Hz), 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 C ₁₈ H ₂₂ N ₅ O[M+H] ⁺ 324.1824, found 324.1819.

Example 14

Preparation of N-isobutyl-N'-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (Compound IA _- 4)

Dissolve 302 μl of isobutylamine in 2 ml of dimethyl sulfoxide, add 583 mg of carbonyldiimidazole, and react for two hours at room temperature without treatment, add 150 mg of 4-(3-methyl-1H-pyridine oxazol[3,4-b]pyridin-4-yl)aniline (intermediate VIII), 40 microliters of triethylamine, reacted overnight at room temperature. After the reaction, 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 with a yield of 69%. Mp: 262-265°C; Purity: 96%;

¹ H NMR (400 MHz, DMSO-d ₆ ) δ: 13.30 (s, 1H), 8.64 (s, 1H), 8.44 (d, J=4.7 Hz, 1H), 7.56 (d, J=8.5 Hz, 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 C ₁₈ H ₂₂ N ₅ O[M +H] ⁺ 324.1825, found 324.1819.

Example 15

Preparation of N-tert-butyl-N'-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (Compound IA _- 5)

Except that isobutylamine was replaced with tert-butylamine, the other required raw materials, reagents and preparation methods were the same as in IA _- 4, to obtain 120 mg of the title compound with a yield of 56%. Mp: 244-251°C; Purity: 100%;

¹ H NMR (400 MHz, DMSO-d ₆ ) δ: 13.30 (s, 1H), 8.47 (s, 1H), 8.44 (d, J=4.7 Hz, 1H), 7.52 (d, J=8.5 Hz, 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 C ₁₈ H ₂₁ N ₅ NaO[M+Na] ⁺ 346.1644, found346.1638.

Example 16

Preparation of N-n-pentyl-N'-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (Compound IA _- 6)

Except that isobutylamine was replaced by n-pentylamine, the other required raw materials, reagents and preparation methods were the same as in IA _- 4, to obtain 150 mg of the title compound with a yield of 68%. Mp: 233-236°C; Purity: 98%;

¹ H NMR (400MHz, DMSO-d ₆ ) 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 for C ₁₉ H ₂₄ N ₅ O[M+H] ⁺ 338.1981, found 338.1975.

Example 17

Preparation of N-n-hexyl-N'-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (Compound IA _- 7)

Except that isobutylamine was replaced with n-hexylamine, the other required raw materials, reagents and preparation methods were the same as in IA _- 4, to obtain 120 mg of the title compound, with a yield of 52%. Mp: 213-215°C; Purity: 98%;

¹ H NMR (400MHz, DMSO-d6) δ: 13.30 (s, 1H, NH), 8.63 (s, 1H, NH), 8.44 (d, J=4.7 Hz, 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 calcd for C20H25N5NaO[M+Na] ⁺ 374.1957, found 374.1951.

Example 18

Preparation of N-cyclopropyl-N'-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (Compound IA _- 8)

Except that isobutylamine was replaced with cyclopropylamine, other required raw materials, reagents and preparation methods were the same as in IA _- 4 to obtain 130 mg of the title compound with a yield of 63%. Mp: 247-250°C; Purity: 97%;

¹ H NMR (400MHz, DMSO-d ₆ )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 calcd for C ₁₇ H ₁₈ N ₅ O[M+H]+308.1511, found 308.1506.

Example 19

Preparation of N-cyclobutyl-N'-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (Compound IA _- 9)

Except that isobutylamine was replaced with cyclobutylamine, the other required raw materials, reagents and preparation methods were the same as in IA _- 4, to obtain 160 mg of the title compound with a yield of 67%. Mp: 240-243°C; Purity: 99%;

¹ H NMR (400 MHz, DMSO-d ₆ ) δ: 13.30 (s, 1H), 8.56 (s, 1H), 8.44 (d, J=4.7 Hz, 1H), 7.55 (d, J=8.5 Hz, 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 for C18H20N5O[M+H]+322.1668, found 322.1662.

Example 20

Preparation of N-cyclopentyl-N'-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (Compound IA _- 10)

Except that isobutylamine was replaced with cyclopentylamine, the other required raw materials, reagents and preparation methods were the same as in IA _- 4, to obtain 200 mg of the title compound with a yield of 89%. Mp: 258-262°C; Purity: 99%;

¹ H NMR (400 MHz, DMSO-d ₆ ) δ: 13.30 (s, 1H), 8.49 (s, 1H), 8.44 (d, J=4.7 Hz, 1H), 7.55 (d, J=8.5 Hz, 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, 6.3 Hz, 2H); HRMS(ESI) m/z calcd for C ₁₉ H ₂₂ N ₅ O[M+H] ⁺ 336.1824, found 336.1819.

Example 21

Preparation of N-cyclohexyl-N'-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (Compound IA _- 11)

Except replacing ethyl isocyanate with cyclohexyl isocyanate, the other required raw materials, reagents and preparation method are the same as in IA _- 1, to obtain the title compound, 40 mg of yellow powder, yield 52%. Mp: 233-237°C; Purity: 95%;

¹ H NMR (400 MHz, DMSO-d ₆ ) δ: 13.30 (s, 1H), 8.53 (s, 1H), 8.44 (d, J=4.6 Hz, 1H), 7.54 (d, J=8.4 Hz, 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, 12.1 Hz, 3H); HRMS(ESI) m/z calcd for C ₂₀ H ₂₄ N ₅ O[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 _IA -12) Preparation of

Except that isobutylamine was replaced with 2-furanmethylamine, the other required raw materials, reagents and preparation methods were the same as in IA _- 4, to obtain 160 mg of the title compound with a yield of 70%. Mp: 232-234°C; Purity: 100%;

¹ H NMR (400MHz, DMSO-d ₆ )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 C ₁₉ H ₁₈ N ₅ O ₂ [M+H] ⁺ 348.1460, found 348.1455.

Example 23

N-(thiophen-2-ylmethyl)-N'-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (Compound _IA -13) Preparation of

Except that isobutylamine was replaced by 2-thienylmethylamine, the other required raw materials, reagents and preparation methods were the same as in IA _- 4, to obtain 160 mg of the title compound with a yield of 67%. Mp: 233-235°C; Purity: 100%;

¹ H NMR (400 MHz, DMSO-d ₆ ) δ: 13.31 (s, 1H), 8.81 (s, 1H), 8.45 (d, J=4.7 Hz, 1H), 7.58 (d, J=8.5 Hz, 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 for C ₁₉ H ₁₇ N ₅ NaOS[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 _IA -14) Preparation of

Except that isobutylamine was replaced with 4-methylaminopyridine, the other required raw materials, reagents and preparation methods were the same as in IA _- 4, to obtain 160 mg of the title compound, with a yield of 68%. Mp: 218-222°C; Purity: 97%;

¹ H NMR (400 MHz, DMSO-d ₆ ) δ: 13.31 (s, 1H), 8.97 (s, 1H), 8.52 (d, J=4.7 Hz, 2H), 8.45 (d, J=4.7 Hz, 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 C ₂₀ H ₁₉ N ₆ O[M+H ] ⁺ 359.1620, found 359.1615.

Example 25

Preparation of N-benzyl-N'-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (Compound IA _- 15)

Except for replacing ethyl isocyanate with benzyl isocyanate, the other required raw materials, reagents and preparation methods are the same as in IA _- 1, to obtain the title compound, 40 mg of yellow powder, yield 50%. Mp: 91-97°C; Purity: 93%;

¹ H NMR (400 MHz, DMSO-d ₆ ) δ: 9.56 (t, J=5.9 Hz, 1H), 8.55 (d, J=5.0 Hz, 1H), 7.37 (m, 4H), 7.27 (t, J= HRMS (ESI)m/z calcd for C ₂₁ H ₂₀ N ₅ O[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 _IA - 16) Preparation

Except for replacing ethyl isocyanate with 3-methylbenzyl isocyanate, the other required raw materials, reagents and preparation methods are the same as in IA _- 1, to obtain the title compound as a yellow powder, 50 mg, yield 60%. Mp: 140-145°C; Purity: 97%;

¹ H NMR (400 MHz, DMSO-d ₆ ) δ: 9.54 (t, J=6.0 Hz, 1H), 8.55 (d, J=5.0 Hz, 1H), 7.28 (d, J=8.4 Hz, 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 C ₂₂ H ₂₂ N ₅ O[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 _IA - 17) Preparation

Except that isobutylamine was replaced by p-methylbenzylamine, the other required raw materials, reagents and preparation methods were the same as in IA _- 4, to obtain 170 mg of the title compound with a yield of 69%. Mp: 275-276°C; Purity: 99%;

¹ H NMR (400MHz, DMSO-d ₆ )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 for C ₂₂ H ₂₂ N ₅ O[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 IA _- 18 ) preparation

Except that isobutylamine was replaced by o-fluorobenzylamine, the other required raw materials, reagents and preparation methods were the same as in IA _- 4, to obtain 210 mg of the title compound with a yield of 84%. Mp: 248-251°C; Purity: 97%;

¹ H NMR (400 MHz, DMSO-d ₆ ) δ: 13.31 (s, 1H), 8.85 (s, 1H), 8.45 (d, J=4.7 Hz, 1H), 7.57 (d, J=8.5 Hz, 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 C ₂₁ H ₁₈ FN ₅ NaO[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 IA _- 19 ) preparation

Except that isobutylamine was replaced with o-chlorobenzylamine, the other required raw materials, reagents and preparation methods were the same as in IA _- 4, to obtain 210 mg of the title compound with a yield of 79%. Mp: 254-256°C; Purity: 96%;

¹ H NMR (400 MHz, DMSO-d ₆ ) δ: 13.31 (s, 1H), 8.94 (s, 1H), 8.45 (d, J=4.7 Hz, 1H), 7.58 (d, J=8.3 Hz, 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 for C ₂₁ H ₁₉ ClN ₅ O[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 Preparation of _A -20)

Except that isobutylamine was replaced with 2,6-difluorobenzylamine, the other required raw materials, reagents and preparation methods were the same as in IA _- 4, to obtain 200 mg of the title compound with a yield of 77%. Mp: 254-256°C; Purity: 97%;

¹ H NMR (400 MHz, DMSO-d ₆ ) δ: 8.68 (s, 1H), 8.44 (d, J=4.7 Hz, 1H), 7.54 (d, J=8.5 Hz, 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 C ₂₁ H ₁₈ F ₂ N ₅ O[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 _IA - 21) Preparation

Except replacing isobutylamine with phenethylamine, the other required raw materials, reagents and preparation methods are the same as in IA _- 4 to obtain 200 mg of the title compound with a yield of 82%. Mp: 221-224°C; Purity: 99%;

¹ H NMR (400MHz, DMSO-d ₆ ) 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 C ₂₂ H ₂₂ N ₅ O[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 _IA - 22) Preparation

Except for replacing ethyl isocyanate with 4-fluorophenethyl isocyanate, the other required raw materials, reagents and preparation methods were the same as in IA _- 1 to obtain 60 mg of the title compound with a yield of 23%. Mp: 185-189°C, purity: 97%;

¹ H NMR (400 MHz, CDCl ₃ ) δ: 9.26 (s, 1H), 8.38 (d, J=4.9 Hz, 1H), 7.26 (dd, J=8.3, 3.5 Hz, 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 for C ₂₂ H ₂₁ FN ₅ O[M+H] ⁺ 390.1730, found 390.1725.

Example 33

N-(4-Chlorophenethyl)-N'-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (Compound _IA - 23) Preparation

Except that isobutylamine was replaced with 4-chlorophenethylamine, the other required raw materials, reagents and preparation methods were the same as in IA _- 4, to obtain 220 mg of the title compound with a yield of 82%. Mp: 244-247°C; Purity: 99%;

¹ H NMR (400MHz, DMSO-d ₆ )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 C ₂₂ H ₂₁ ClN ₅ O[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 Preparation of (Compound IA _- 24)

Except for replacing ethyl isocyanate with 3,4-dimethoxyphenethyl isocyanate, the other required raw materials, reagents and preparation methods are the same as in IA _- 1, to obtain 50 mg of the title compound with a yield of 18%. Mp: 178-181°C; Purity: 92%;

¹ H NMR (400 MHz, CDCl ₃ ) δ: 9.18 (t, J=5.4 Hz, 1H), 8.48 (d, J=5.0 Hz, 1H), 7.27 (d, J=8.3 Hz, 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 C ₂₄ H ₂₆ N ₅ O ₃ [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 _IA - 25) Preparation

Except that isobutylamine was replaced into amphetamine, all the other required raw materials, reagents and preparation method were the same as in Example 1 _A -4, to obtain 180 mg of title compound, yield 70%. Mp: 223-225°C; Purity: 98%;

¹ H NMR (400 MHz, DMSO-d ₆ ) δ: 13.31 (s, 1H), 8.68 (s, 1H), 8.45 (d, J=4.7 Hz, 1H), 7.57 (d, J=8.5 Hz, 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 C ₂₃ H ₂₃ N ₅ NaO[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 _IA - 26) Preparation

Except replacing isobutylamine with phentermine, the other required raw materials, reagents and preparation methods are the same as in IA _- 4, to obtain 200 mg of the title compound with a yield of 75%. Mp: 224-227°C; Purity: 92%;

¹ H NMR (400 MHz, DMSO-d ₆ ) δ: 13.30 (s, 1H), 8.63 (s, 1H), 8.44 (d, J=4.6 Hz, 1H), 7.55 (d, J=8.4 Hz, 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, 7.3 Hz, 2H), 1.47 (dd, J=14.5, 7.0 Hz, 2H); HRMS(ESI) m/z calcd for C ₂₄ H ₂₅ N ₅ NaO[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 Preparation of _A -27)

Except that isobutylamine was replaced with 3-aminopyrazole, the other required raw materials, reagents and preparation methods were the same as in IA _- 4, to obtain 60 mg of the title compound with a yield of 30%. Mp: 260-263°C; Purity: 99%;

¹ H NMR (400MHz, DMSO-d ₆ )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 C ₁₇ H ₁₅ N ₇ NaO[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 IA _- 28 ) preparation

Except that isobutylamine was replaced with 3-pyridylamine, the other required raw materials, reagents and preparation methods were the same as in IA _- 4, to obtain 120 mg of the title compound with a yield of 52%. Mp: 245-247°C; Purity: 98%;

¹ H NMR (400MHz, DMSO-d ₆ )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 C ₁₉ H ₁₆ N ₆ NaO [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 IA _- 29 ) preparation

Except that isobutylamine was replaced with 2-pyridylamine, the other required raw materials, reagents and preparation methods were the same as in IA _- 4, to obtain 46 mg of the title compound with a yield of 30%. Mp: 244-248°C; Purity: 96%;

¹ H NMR (400MHz, DMSO-d ₆ )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 for C ₁₉ H ₁₆ N ₆ NaO[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)benzene Preparation of urea (compound IA _- 30)

Except that isobutylamine was replaced by 2-aminoindene, the other required raw materials, reagents and preparation methods were the same as in IA _- 4, to obtain 50 mg of the title compound with a yield of 20%. Mp: 249-251°C; Purity: 96%;

¹ H NMR (400MHz, DMSO-d ₆ )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＝7.3 Hz, 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 C ₂₃ H ₂₂ N ₅ O[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 IA _- 31 ) preparation

Except that isobutylamine was replaced with 2-naphthylamine, the other required raw materials, reagents and preparation methods were the same as in IA _- 4 to obtain 90 mg of the title compound with a yield of 34%. Mp: 232-235°C; Purity: 100%;

¹ H NMR (400 MHz, DMSO-d ₆ ) δ: 13.32 (s, 1H), 8.99 (d, J=5.5 Hz, 2H), 8.47 (d, J=4.7 Hz, 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 C ₂₄ H ₂₀ N ₅ O[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 _IA - 32) Preparation

Except that isobutylamine was replaced with 3-aminoquinoline, the other required raw materials, reagents and preparation methods were the same as in IA _- 4, to obtain 80 mg of the title compound with a yield of 30%. Mp: 226-229°C; Purity: 99%;

¹ H NMR (400MHz, DMSO-d ₆ )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 C ₂₃ H ₁₉ N ₆ O[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 IA _- 33 ) preparation

Except that isobutylamine was replaced with 1-naphthylamine, the other required raw materials, reagents and preparation methods were the same as in IA _- 4, to obtain 170 mg of the title compound with a yield of 69%. Mp: 225-228°C; Purity: 99%;

¹ H 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 for C ₂₄ H ₂₀ N ₅ O[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 ( IA _- 34) preparation

395 μl 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 μl of phenyl chloroformate was dissolved in 2 ml of tetrahydrofuran solution, and added dropwise to Reaction System 1 under an ice bath. The reaction was carried out at room temperature for 3 hours. After the reaction was completed, an appropriate amount of water was added to the system, extracted three times with ethyl acetate, washed with saturated brine, dried over anhydrous magnesium sulfate, filtered and concentrated to obtain a residue. The residue was dissolved in 5 mL of dioxane, 60 mg of 4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl)aniline (Intermediate VIII) and 20 μg were added. 1-Methylpyrrolidine was added, and the reaction was refluxed overnight. After the reaction was completed, 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 obtain the title compound, 45 mg of a yellow solid with a yield of 44%. Mp: 98-101; Purity: 95%.

¹ H-NMR (400MHz, DMSO-d ₆ )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 C ₂₀ H ₂₅ N ₆ O ₂ [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 ( _IA- 35) Preparation

Except that N-(2-aminoethyl)morpholine was replaced by N,N-dimethylethylenediamine, the other required raw materials, reagents and preparation methods were the same as in IA _- 34, to obtain 39 mg of the title compound, yellow Oil, 43% yield. Mp: 54-56°C; Purity: 99%;

¹ H-NMR (400MHz, DMSO-d ₆ )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 for C ₁₈ H ₂₃ N ₆ O[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] Preparation of pyridin-4-yl)phenyl)urea (IA _- 36)

1 g of 5-fluoro-2-nitrotoluene and 1.783 g of potassium carbonate were dissolved in 7 ml of dimethyl sulfoxide, 1.072 ml of N-methylpiperazine was added, and the mixture was stirred at 122°C overnight. After the reaction was completed, it was cooled to room temperature, an appropriate amount of water was added to the system, extracted three times with ethyl acetate, washed with saturated brine, dried over anhydrous magnesium sulfate, filtered and concentrated to obtain Intermediate XV: 1.2 g.

Dissolve 1.2 g of intermediate XV in methanol, add 120 mg of 10% Pd/C, pass hydrogen, and react overnight at room temperature. After the reaction was completed, it was filtered, concentrated, and the residue was separated by column chromatography (ethyl acetate:petroleum ether=1:2, v/v) to obtain Intermediate XVI: 0.935 g.

Except that N-(2-aminoethyl)morpholine was replaced with intermediate XVI, the other required raw materials, reagents and preparation methods were the same as in IA _- 34, to obtain the title compound, 29 mg of pink solid, the yield was 24 %. Mp: 263-264; Purity: 95%.

¹ H-NMR (400MHz, DMSO-d ₆ )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 C ₂₆ H ₃₀ N ₇ O[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] Preparation of pyridin-4-yl)phenyl)urea (IA _- 37)

Except that the 5-fluoro-2-nitrotoluene was replaced by 2-fluoro-5-nitrotoluene, the other required raw materials, reagents and preparation methods were the same as in IA _- 36 to obtain 24 mg of the title compound as a milky white solid, which was obtained as rate 20%. Mp: 236-238; Purity: 95%;

¹ H-NMR (400MHz, DMSO-d ₆ )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 C ₂₆ H ₃₀ N ₇ O[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 (IA _- 38) preparation

Except that 5-fluoro-2-nitrotoluene was replaced by 3-fluoronitrobenzene, the other required raw materials, reagents and preparation methods were the same as in IA _- 36, to obtain 32 mg of the title compound, yellow solid, yield 27% . Mp: 108-109; Purity: 95%;

¹ H-NMR (400MHz, DMSO-d ₆ )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 C ₂₅ H ₂₈ N ₇ O[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 (IA _- 39) preparation

Except that 5-fluoro-2-nitrotoluene was replaced with 2-fluoronitrobenzene, the other required raw materials, reagents and preparation methods were the same as in IA _- 36, to obtain 105 mg of the title compound, white solid, yield 89% . Mp: 253-253; Purity: 99%;

¹ H-NMR (400MHz, DMSO-d ₆ )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 C ₂₅ H ₂₈ N ₇ O[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] Preparation of pyridin-4-yl)phenyl)urea (IA _- 40)

Except that the 5-fluoro-2-nitrotoluene was replaced by 4-fluoro-5-nitrotoluene, the other required raw materials, reagents and preparation methods were the same as in IA _- 36, to obtain 23 mg of the title compound as a white solid, which was obtained as a rate 19%. Mp: 247-250; Purity: 95%;

¹ H-NMR (400MHz, DMSO-d ₆ )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 C ₂₆ H ₃₀ N ₇ O[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]pyridine-4- Preparation of phenyl) phenyl) urea (IA _- 41)

Except that 5-fluoro-2-nitrotoluene was replaced by 2-fluoro-5-nitrotoluene, and N-methylpiperazine was replaced by morpholine, other required raw materials, reagents and preparation methods were the same as in IA _- 36 , the title compound was obtained as 13 mg of white solid in 11% yield. Mp: 253-255; Purity: 90%.

¹ H-NMR (400MHz, DMSO-d ₆ )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 C ₂₅ H ₂₇ N ₆ O ₂ [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) Preparation of Urea (IA _- 42)

Except that 5-fluoro-2-nitrotoluene was replaced by 2-fluoronitrobenzene, and N-methylpiperazine was replaced by morpholine, other required raw materials, reagents and preparation methods were the same as in IA _- 36, to obtain the title Compound 45 mg, milky white solid, yield 39%. Mp: 225-228°C; Purity: 99%;

¹ H-NMR (400MHz, DMSO-d ₆ )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 C ₂₄ H ₂₅ N ₆ O ₂ [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]pyridine-4- Preparation of phenyl) phenyl) urea (IA _- 43)

Except that 5-fluoro-2-nitrotoluene was replaced by 4-fluoro-5-nitrotoluene, and N-methylpiperazine was replaced by morpholine, other required raw materials, reagents and preparation methods were the same as in IA _- 36 , 57 mg of the title compound was obtained as a yellow solid with a yield of 48%. Mp: 268-270; Purity: 98%;

¹ H-NMR (400MHz, DMSO-d ₆ )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 C ₂₅ H ₂₇ N ₆ O ₂ [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 ( IA _- 44) preparation

Except replacing 5-fluoro-2-nitrotoluene with p-fluoronitrobenzene, and replacing N-methylpiperazine with diethylamine, other required raw materials, reagents and preparation methods are the same as in IA _- 36, and obtain the title Compound 81 mg, tan solid, yield 73%. Mp: 242-246°C; Purity: 95%;

¹ H-NMR (400MHz, DMSO-d ₆ )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 C ₂₄ H ₂₇ N ₆ O[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 ( IA _- 45) preparation

7.2 ml of 40% formaldehyde solution and 2.56 ml of concentrated sulfuric acid were stirred under an ice bath to obtain system 1. A solution of 2.4 g m-nitroaniline and 4.24 g sodium borohydride in tetrahydrofuran (80 ml) was slowly added to system 1, and the ice The reaction was carried out under the bath for 2 hours, 1.92 g of sodium hydroxide was added, and the mixture was stirred 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 get 1.8 g of intermediate.

1.8 g of intermediate XVII was dissolved in methanol, 900 mg of 10% Pd/C was added, hydrogen was passed through, and the reaction was carried out at room temperature overnight. After the reaction was completed, it was filtered, concentrated, and the residue was separated by column chromatography (ethyl acetate:petroleum ether=1:10, v/v) to obtain 1.3 g of intermediate XVIII.

Except that N-(2-aminoethyl)morpholine was replaced with intermediate XVIII, the other required raw materials, reagents and preparation methods were the same as in IA _- 34, to obtain 20 mg of the title compound, pale yellow solid, yield 19% . Mp: 226-228°C; Purity: 98%;

¹ H-NMR (400MHz, DMSO-d ₆ )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 C ₂₂ H ₂₃ N ₆ O[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 ( IA _- 46) preparation

Except for replacing 40% formaldehyde solution with 40% acetaldehyde solution, the other required raw materials, reagents and preparation methods are the same as IA _- 45, to obtain 29 mg of the title compound as a cyan solid, yield 26%. Mp: 125-128°C; Purity: 97%;

¹ H-NMR (400MHz, DMSO-d ₆ )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=8.4 Hz, 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 C ₂₄ H ₂₇ N ₆ O[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)benzene Preparation of urea (IA _- 47)

Except replacing 5-fluoro-2-nitrotoluene with p-nitrobenzyl chloride, and replacing N-methylpiperazine with dimethylamine, other required raw materials, reagents and preparation methods are the same as in IA _- 36, to obtain the title Compound, yellow solid 100 mg, yield 93%. Mp: 59-62°C; Purity: 95%;

¹ H-NMR (400MHz, DMSO-d ₆ )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 C ₂₃ H ₂₅ N ₆ O[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)benzene Preparation of urea (IA _- 48)

Except that 5-fluoro-2-nitrotoluene was replaced by nitrobenzyl chloride, and N-methylpiperazine was replaced by diethylamine, other required raw materials, reagents and preparation methods were the same as in IA _- 36, to obtain the title compound , 50 mg of yellow solid, yield 44%. Mp: 123-125°C; Purity: 97%;

¹ H-NMR (400MHz, DMSO-d ₆ )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 C ₂₅ H ₂₉ N ₆ O[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)benzene Preparation of urea (IA _- 49)

Except that 5-fluoro-2-nitrotoluene was replaced by m-nitrobenzyl bromide, and N-methylpiperazine was replaced by dimethylamine, other required raw materials, reagents and preparation methods were the same as in IA _- 36, to obtain the title Compound, yellow solid 25 mg, yield 23%. Mp: 185-186°C; Purity: 90%;

¹ H-NMR (400MHz, DMSO-d ₆ )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 C ₂₃ H ₂₅ N ₆ O[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 ( IA _- 50) preparation

Except that 5-fluoro-2-nitrotoluene was replaced by p-fluoronitrobenzene, and N-methylpiperazine was replaced by dimethylamine, other required raw materials, reagents and preparation methods were the same as in IA _- 36, to obtain the title Compound, 10 mg of tan solid, yield 10%. Mp: 218-221°C; Purity: 90%;

¹ H-NMR (400MHz, DMSO-d ₆ )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 calcd for C ₂₂ H ₂₃ N ₆ O[M+H] ⁺ 387.1928, found 387.1928.

Example 61

Preparation of N-n-pentyl-N'-(4-(3-trifluoromethyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea ( _IB -1)

Except that isobutylamine was replaced by n-hexylamine, and the intermediate VIII was replaced by XIV, the other required raw materials, reagents and preparation methods were the same as in IA _- 4, to obtain the title compound, 20 mg of white powder, and the yield was 20. Mp: 215-216°C; Purity: 99%;

¹ H 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=8.4 Hz, 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 C ₂₀ H ₂₂ N ₅ OF ₃ [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 ( _IB- 2) Preparation

Except that isobutylamine was replaced by phenethylamine and intermediate VIII was replaced by XIV, the other required raw materials, reagents and preparation methods were the same as in IA _- 4, to obtain 27 mg of the title compound as a white solid with a yield of 25%. Mp: 225-227°C; Purity: 95%;

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 C ₂₂ H ₁₈ N ₅ OF ₃ [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 ( _IB- 3) Preparation

Except that isobutylamine was replaced by amphetamine, and intermediate VIII was replaced by XIV, all other required raw materials, reagents and preparation methods were the same as in IA _- 4, to obtain the title compound, 18 mg of white powder, yield 16%. Mp: 212-214°C; Purity: 100%;

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 C ₂₃ H ₂₀ N ₅ OF ₃ [M+H] ⁺ 439.1620, found439.1613.

Example 64

N-(4-Chlorophenethyl)-N'-(4-(3-trifluoromethyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea ( _IB -4) Preparation

Except that isobutylamine was replaced by 4-chlorophenethylamine, and intermediate VIII was replaced by XIV, other required raw materials, reagents and preparation methods were the same as in IA _- 4, to obtain the title compound, 26 mg of pale yellow powder, yield twenty three%. Mp: 228-232°C; Purity: 97%;

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 C ₂₂ H ₁₇ N ₅ OF ₃ Na[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 Preparation of _B- 5)

Except that isobutylamine was replaced by N,N-dimethylethylenediamine, and intermediate VIII was replaced by intermediate XIV, other required raw materials, reagents and preparation methods were the same as in IA _- 4, to obtain 41 mg of the title compound, Milky white solid, yield 42%. Mp: 165-168°C; Purity: 97%;

¹ H-NMR (400MHz, DMSO-d ₆ )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 for C ₁₈ H ₂₀ N ₆ OF ₃ [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 Preparation of _B- 6)

Except that isobutylamine was replaced by N,N-diethylethylenediamine, and intermediate VIII was replaced by intermediate XIV, the other required raw materials, reagents and preparation methods were the same as in IA _- 4, to obtain the title compound as a milky white powder 35 mg, 33% yield. Mp: 128-132°C; Purity: 97%;

¹ H-NMR (400MHz, DMSO-d ₆ )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 C ₂₀ H ₂₄ N ₆ OF ₃ [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 Preparation of _B -7)

Except that isobutylamine was replaced by 3-dimethylaminopropylamine, and intermediate VIII was replaced by intermediate XIV, other required raw materials, reagents and preparation methods were the same as in IA _- 4, to obtain the title compound, 28 mg of pale yellow powder, Yield 27%. Mp: 186-189°C; Purity: 96%;

¹ H-NMR (400MHz, DMSO-d ₆ )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 C ₁₉ H ₂₁ N ₆ OF ₃ [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 Preparation of _B- 8)

Except that isobutylamine was replaced by 3-diethylaminopropylamine, and intermediate VIII was replaced by intermediate XIV, the other required raw materials, reagents and preparation methods were the same as in IA _- 4, to obtain the title compound, 12 mg of milky white powder, collected rate 11%. Mp: 217-220°C; Purity: 99%;

¹ H-NMR (400MHz, DMSO-d ₆ )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 calcd for C ₂₁ H ₂₆ N ₆ OF ₃ [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) Preparation of Urea ( _IB -9)

Except replacing isobutylamine with 1-(2-aminoethyl)piperidine, and replacing intermediate VIII with intermediate XIV, other required raw materials, reagents and preparation methods are the same as in IA _- 4, to obtain the title compound, light 34 mg of yellow powder, yield 31%. Mp: 120-122°C; Purity: 93%;

¹ H-NMR (400MHz, DMSO-d ₆ )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 for C ₂₁ H ₂₄ N ₆ OF ₃ [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) Preparation of Urea ( _IB- 10)

Except that isobutylamine was replaced by N-(2-aminoethyl)morpholine, and intermediate VIII was replaced by intermediate XIV, other required raw materials, reagents and preparation methods were the same as in IA _- 4, to obtain the title compound, pale 35 mg of yellow powder, yield 32%. Mp: 217-219°C; Purity: 96%;

¹ H-NMR (400MHz, DMSO-d ₆ )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 C ₂₀ H ₂₂ N ₆ O ₂ F ₃ [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]pyridine-4 Preparation of -yl)phenyl)urea ( _IB -11)

Except that 5-fluoro-2-nitrotoluene was replaced by 4-fluoronitrobenzene, and intermediate VIII was replaced by intermediate XIV, the other required raw materials, reagents and preparation methods were the same as in IA _- 36 to obtain the title compound 37 mg, silver-gray solid, yield 30%. Mp: 267-268°C; Purity: 96%;

¹ H-NMR (400MHz, DMSO-d ₆ )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 C ₂₅ H ₂₅ N ₇ OF ₃ [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- Preparation of b]pyridin-4-yl)phenyl)urea ( _IB- 12)

Except that the intermediate VIII was replaced by the intermediate XIV, the other required raw materials, reagents and preparation methods were the same as in IA _- 36, to obtain the title compound as a brown powder, 22 mg, the yield was 17%. Mp: 250-253°C; Purity: 91%;

¹ H-NMR (400MHz, DMSO-d ₆ )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 C ₂₆ H ₂₇ N ₇ OF ₃ [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- Preparation of b]pyridin-4-yl)phenyl)urea ( _IB- 13)

Except that 5-fluoro-2-nitrotoluene was replaced by 2-fluoro-5-nitrotoluene, and intermediate VIII was replaced by intermediate XIV, other required raw materials, reagents and preparation methods were the same as in IA _- 36, obtaining The title compound, pale yellow powder, 69 mg, yield 54%. Mp: 209-211°C; Purity: 97%;

¹ H-NMR (400MHz, DMSO-d ₆ )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 C ₂₆ H ₂₇ N ₇ OF ₃ [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]pyridine-4 Preparation of -yl)phenyl)urea ( _IB- 14)

Except that 5-fluoro-2-nitrotoluene was replaced by 3-fluoronitrobenzene, and intermediate VIII was replaced by intermediate XIV, other required raw materials, reagents and preparation methods were the same as in _IB -11 to obtain the title compound, 57 mg of yellow powder, yield 46%. Mp: 229-231°C; Purity: 95%;

¹ H-NMR (400MHz, DMSO-d ₆ )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 C ₂₅ H ₂₅ N ₇ OF ₃ [M+H] ⁺ 496.2073,found496.2072..

Example 75

N-(2-(4-Methylpiperazin-1-yl)phenyl)-N'-(4-(3-trifluoromethyl-1H-pyrazolo[3,4-b]pyridine-4 Preparation of -yl)phenyl)urea ( _IB -15)

Except that 5-fluoro-2-nitrotoluene was replaced by 2-fluoronitrobenzene, and intermediate VIII was replaced by intermediate XIV, other required raw materials, reagents and preparation methods were the same as in IA _- 36 to obtain the title compound, 40 mg of white powder, yield 32%. Mp: 283-286°C; Purity: 98%;

¹ H-NMR (400MHz, DMSO-d ₆ )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 C ₂₅ H ₂₅ N ₇ OF ₃ [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- Preparation of b]pyridin-4-yl)phenyl)urea ( _IB- 16)

Except replacing 5-fluoro-2-nitrotoluene with 4-fluoro-3-nitrotoluene, and replacing intermediate VIII with intermediate XIV, other required raw materials, reagents and preparation methods are the same as in IA _- 36, to obtain The title compound, yellow powder 31 mg, yield 24%. Mp: 274-276°C; Purity: 96%;

¹ H-NMR (400MHz, DMSO-d ₆ )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 C ₂₆ H ₂₇ N ₇ OF ₃ [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]pyridine- Preparation of 4-yl)phenyl)urea ( _IB -17)

Except that 5-fluoro-2-nitrotoluene was replaced by 2-fluoro-5-nitrotoluene, N-methylpiperazine was replaced by morpholine, and intermediate VIII was replaced by intermediate XIV, the other required raw materials, The reagents and preparation method were the same as in IA _- 36, and the title compound was obtained as a milky white powder, 105 mg, with a yield of 84%. Mp: 85-87°C; Purity: 99%;

¹ H-NMR (400MHz, DMSO-d ₆ )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 C ₂₅ H ₂₄ N ₆ O ₂ F ₃ [ 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)benzene Preparation of urea ( _IB- 18)

Except that 5-fluoro-2-nitrotoluene was replaced by 2-fluoronitrobenzene, N-methylpiperazine was replaced by morpholine, and intermediate VIII was replaced by intermediate XIV, the other required raw materials, reagents and preparations The method is the same as IA _- 36, to obtain the title compound, 36 mg of white powder, the yield is 30%. Mp: 224-226°C; Purity: 100%;

¹ H-NMR (400MHz, DMSO-d ₆ )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 C ₂₄ H ₂₂ N ₆ O ₂ F ₃ [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]pyridine- Preparation of 4-yl)phenyl)urea ( _IB- 19)

Except that 5-fluoro-2-nitrotoluene was replaced by 2-fluoro-5-nitrotoluene, N-methylpiperazine was replaced by morpholine, and intermediate VIII was replaced by intermediate XIV, the other required raw materials, The reagents and preparation method were the same as in IA _- 36, and the title compound was obtained as a yellow powder, 43 mg, with a yield of 34%. Mp: 153-155°C; Purity: 91%;

¹ H-NMR (400MHz, DMSO-d ₆ )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 C ₂₅ H ₂₄ N ₆ O ₂ F ₃ [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]pyridine-4 Preparation of -yl)phenyl)thiourea ( _IB- 20)

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

Take intermediate XIX, dissolve 200 mg in 4 ml of dichloromethane, add sulfur dichloride (240 μl) in dichloromethane (6 ml) dropwise under nitrogen protection at 0°C, and add 124 mg of hydrogen Sodium oxide, react at room temperature for 3 hours. After completion of the reaction, suction filtration, washing with dichloromethane, and distillation under reduced pressure to obtain a residue. The residue was dissolved in 5 mL of ethanol, to which 70 mg of 4-(3-trifluoromethyl-1H-pyrazolo[3,4-b]pyridin-4-yl)aniline (intermediate XIV) was added, at room temperature React for 1 hour. After the reaction was completed, the mixture was distilled under reduced pressure, and the residue was separated by column chromatography (methanol:dichloromethane=1:30, v/v) to obtain 76 mg of the title compound with a yield of 59%. Mp: 153-155°C; Purity: 99%;

¹ H-NMR (400MHz, DMSO-d ₆ )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 for C ₂₅ H ₂₄ N ₇ OF ₃ S[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) Preparation of Urea ( _IB -21)

Except that 5-fluoro-2-nitrotoluene was replaced by p-fluoronitrobenzene, N-methylpiperazine was replaced by diethylamine, and intermediate VIII was replaced by intermediate XIV, the other required raw materials, reagents and preparations The method is the same as that of IA _- 36, to obtain 23 mg of the title compound as a purple-brown solid with a yield of 20%. Mp: 253-254°C; Purity: 99%;

¹ H-NMR (400MHz, DMSO-d ₆ )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 C ₂₄ H ₂₄ N ₆ OF ₃ [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) Preparation of Urea ( _IB -22)

Except that the intermediate VIII was replaced by the intermediate XIV, the other required raw materials, reagents and preparation methods were the same as in IA _- 45, to obtain 80 mg of the title compound as a yellow solid with a yield of 72%. Mp: 82-84°C; Purity: 98%;

¹ H-NMR (400MHz, DMSO-d ₆ )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 C ₂₂ H ₂₀ N ₆ OF ₃ [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) Preparation of Urea ( _IB -23)

Except that intermediate VIII was replaced by intermediate XIV, and the formaldehyde solution was replaced by acetaldehyde solution, other required raw materials, reagents and preparation methods were the same as in IA _- 45, to obtain the title compound, 54 mg of brown powder, yield 46% . Mp: 151-153°C; Purity: 98%;

¹ H-NMR (400MHz, DMSO-d ₆ )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 for C ₂₄ H ₂₄ N ₆ OF ₃ [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 ( _IB -24) preparation

Except that 5-fluoro-2-nitrotoluene was replaced by p-nitrobenzyl chloride, N-methylpiperazine was replaced by dimethylamine, and intermediate VIII was replaced by intermediate XIV, the other required raw materials, reagents and preparations The method is the same as IA _- 36, the title compound is obtained as a yellow solid, 86 mg, the yield is 75%. Mp: 200-202°C; Purity: 95%;

¹ H-NMR (400MHz, DMSO-d ₆ )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 C ₂₃ H ₂₂ N ₆ OF ₃ [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 ( _IB- 25) preparation

Except that 5-fluoro-2-nitrotoluene was replaced by p-nitrobenzyl chloride, N-methylpiperazine was replaced by diethylamine, and intermediate VIII was replaced by intermediate XIV, the other required raw materials, reagents and preparations The method is the same as IA _- 36, the title compound is obtained as a yellow solid, 94 mg, the yield is 74%. Mp: 90-93°C; Purity: 95%;

¹ H-NMR (400MHz, DMSO-d ₆ )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 C ₂₅ H ₂₆ N ₆ OF ₃ [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 ( _IB -26) preparation

Except that 5-fluoro-2-nitrotoluene was replaced by m-nitrobenzyl bromide, N-methylpiperazine was replaced by dimethylamine, and intermediate VIII was replaced by intermediate XIV, the other required raw materials, reagents and preparations The method is the same as IA _- 36, the title compound is obtained as a yellow solid, 26 mg, the yield is 23%. Mp: 96-98°C; Purity: 99%;

¹ H-NMR (400MHz, DMSO-d ₆ )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 C ₂₃ H ₂₂ N ₆ OF ₃ [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) Preparation of Urea ( _IB -27)

Except for replacing 5-fluoro-2-nitrotoluene with p-fluoronitrobenzene, replacing N-methylpiperazine with dimethylamine, and replacing intermediate VIII with intermediate XIV, other required raw materials, reagents and preparations The method is the same as IA _- 36, to obtain the title compound, 10 mg of green-brown solid, yield 9%. Mp: 219-221°C; Purity: 80%;

¹ H-NMR (400MHz, DMSO-d ₆ )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 C ₂₃ H ₂₂ N ₆ OF ₃ [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 ( _IB- 28) preparation

Except that 5-fluoro-2-nitrotoluene was replaced by m-nitrobenzyl bromide, N-methylpiperazine was replaced by diethylamine, and intermediate VIII was replaced by intermediate XIV, the other required raw materials, reagents and preparations The method is the same as IA _- 36, the title compound is obtained as a yellow solid, 32 mg, the yield is 26%. Mp: 70-72°C; Purity: 98%;

¹ H-NMR (400MHz, DMSO-d ₆ )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 C ₂₅ H ₂₆ N ₆ OF ₃ [M+H] ⁺ 483.2120, found 483.2120.

Example 89

Experimental methods and results of the compounds of the present invention inhibiting the activity of three receptor tyrosine kinases

The method for testing the kinase activity of the compound of the present invention is to use the mobility shift assay (Mobility Shift Assay) method under the condition of the concentration of KmATP, select three in vitro kinases of VEGFR-2, PGDFR-α and c-KIT for activity testing, and use the compound Imatinib as a standard control. Each compound was first prepared into a 50 μM solution, diluted to 10 concentration points (the concentration difference between each concentration point was 4 times) for single-well detection.

The activity data of the compounds of the present invention on the above three tyrosine receptor kinases are shown in Table 1. A total of 29 compounds of the present invention have been found to have good inhibitory activity on c-KIT with IC ₅₀ <100nM, among which 17 derivatives It has extremely strong inhibitory activity against c-KIT, and the half effective inhibitory concentration is 1nM<IC ₅₀ <10nM; a total of 33 compounds of the present invention have been found to have good inhibitory activity against PDGFRα, and the half effective inhibitory concentration IC ₅₀ <1000nM, of which 8 The compounds of the present invention have good inhibitory activity on PDGFRα, and the half effective inhibitory concentration is 10nM<IC ₅₀ <100nM; one compound of the present invention has extremely strong inhibitory activity on PDGFRα, and the half effective inhibitory concentration is 1nM<IC ₅₀ <10nM; no discovery was found Derivatives with good inhibitory activity against VEGFR-2 (IC ₅₀ <1000nM), more than half (41) of the derivatives have certain inhibitory activity against VEGFR-2, and half of the effective inhibitory concentrations are 1000nM<IC50<10000nM; activity The data are detailed in Table 1 and Supplementary Table 1: it shows that the compounds of the present invention are expected to be developed as multi-target receptor tyrosine kinase inhibitors anti-tumor drugs.

In Table 1, c-KIT stands for type III tyrosine kinase receptor, PDGFRa stands for platelet-derived growth factor receptor alpha, VEGFR2 stands for vascular endothelial growth factor receptor 2, and linifanib and imatinib mesylate are positive controls Among them, Linifanib is an anti-tumor candidate drug launched by Abbott in the United States, and imatinib mesylate was approved by the FDA in 2002 for unresectable and/or metastatic GISTs, and is the first clinical treatment for malignant tumors. Tumor cell signaling inhibitor.

Table 1 Inhibitory activity data of the compounds of the present invention on three tyrosine kinases (IC ₅₀ , nM)

Schedule 1

Example 90

Experimental methods and results of the GI ₅₀ determination of the anti-proliferation experiments of the compounds of the present invention against a variety of homologous BaF3 cell lines

Taking IA _- 41 with the best c-KIT and PDGFRA kinase activities as an example for the compounds of the present invention, the anti-proliferative activities of the compounds on various homologous BaF3 cell lines were tested. In this experiment, 10 mM stock solution of the test compound (DMSO) was prepared into a drug plate according to 7 concentrations of 3-fold dilution, and stored under suitable conditions. At the same time, the same volume of DMSO solvent was used as a blank control, and imatinib was used as a positive control. Take the corresponding logarithmic growth phase cells to prepare a cell plate, culture under appropriate conditions, after adding drugs, incubate in a 37 °C, 5% CO ₂ incubator for 72 hours, add 10 μL of CellTiter-Glo Cell Proliferation Fluorescence Detection Reagent, and let stand. After 10 minutes, the effect of drug and cells was detected by Envision Plate-Reader reading. The activity data are shown in Table 2,

Table 2 Anti-proliferation experimental activity data (GI ₅₀ , μM) of compound IA _- 41 of the present invention on various homologous BaF ₃ cell lines

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

It can be seen from Table 2: Compound IA _- 41 has strong inhibitory effect on most c-KIT, PDGFRα and mutant cell lines, but has no obvious inhibitory effect on c-KIT-D816V-BaF3. Among them, compared with the anti-proliferative activity of the marketed drug imatinib, Compound IA _- 41 has a strong effect on c-KIT-T670I-BaF3, c-KIT-V559D-T670I-BaF3, c-KIT-N822K-BaF3, PDGFRα - The anti-proliferative effect of T674I-BaF3, FLT3-BaF3, FLT3-ITD-BaF3 cells has a breakthrough improvement; c-KIT-BaF3, c-KIT-A829P-BaF3, c-KIT-D816H-BaF3, c-KIT - The anti-proliferation effect of V559D-V654A-BaF3 and other targets has been greatly improved; it has a good anti-proliferation effect on c-KIT-L576P-BaF3, c-KIT-C674S-BaF3, c-KIT-V654A-BaF3 cells improvement.

Example 91

Determination method and results of the anti-proliferation test GI ₅₀ of the compounds of the present invention against various genuine tumor cell lines

In view of the fact that in Example 90, compound IA _- 41 showed strong antiproliferative effect on kinases such as c-KIT, PDGFRa and their mutations, this example will show the antiproliferative effect of this compound against 8 real tumor cell lines. Proliferation assay methods. Cell lines include hamster ovary cell lines (CHL, CHO), GIST cell lines (GIST-T1, GIST-882, GIST-48B), BCR-ABL kinase cell lines (MEG-01, K562, KU812), where appropriate. Under the same culture conditions, the specific implementation steps of other cells are the same as those in Example 90, except that 10 μL CCK8 cell proliferation detection reagent is added to GIST-882 for detection, and the activity data are shown in Table 3.

Table 3 Anti-proliferation experimental activity data (GI ₅₀ , μM) of the compound IA _- 41 of the present invention on various genuine tumor cell lines

cell line I_A41(GI₅₀μM) Imatinib (GI₅₀μM) 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

It can be seen from Table 3 that Compound IA _- 41 has a significant effect on GIST-T1 (<0.003 μM), GIST-882 (<0.003 μM), MOLM14 (0.0107 μM), and MV4-11 (0.0105 μM) cell lines. It has no effect or obvious effect on KU812, MEG-01, K562, GIST-48B, CHL and CHO cell lines. The results show that the compound IA _- 41 of the present invention has no inhibitory effect on the proliferation of normal cells, and has high efficiency and selectivity for the treatment of gastrointestinal stromal tumors, which provides a direction for the further development of anti-gastrointestinal stromal tumor targeted drugs.

Example 92

The method and results of the experimental determination of the pharmacokinetic characteristics of the compounds of the present invention

In view of the good kinase inhibitory activity and tumor cell inhibitory activity of the compound IA _- 41 of the present invention, its pharmacokinetic characteristics were investigated. Select 30% HP-β-CD, PEG400 and trace amount of DMSO (11:8:1) as the solvent, configure the final concentration of IA _- 41 as 2mg/mL, and intravenously administer it to 6 males at a dose of 2mg/kg SD rats; sample collection (about 0.2 mL of blood from the jugular vein) and data analysis at appropriate time; individual and average blood concentration-time curves (n=3) of rats after a single intravenous administration of IA _- 41 are shown in the figure 1. According to the blood concentration data of the drug, the pharmacokinetic parameters of the test substance were calculated respectively using the non-compartmental model of the pharmacokinetic calculation software WinNonlin 6.2.1, as shown in Table 4. Experimental data show that Compound IA _- 41 exhibits acceptable half-life and clearance in intravenous injection.

Table 4 The main pharmacokinetic parameters of IA _- 41 after a single intravenous administration of IA _- 41 to rats

NA: not applicable; -: no calculated value

Example 93

Test method and results of the compound of the present invention using automatic patch clamp QPatch detection method on hERG potassium channel

IA _- 41 was selected as a representative compound, CHO-hERG cells were cultured in appropriate conditions and subjected to appropriate subsequent treatment to ensure that the cell density was 2-5×10 ⁶ /mL. Take 2 μL of 20 mM compound stock solution and add 998 μL of extracellular fluid, and then perform 3-fold serial dilution in extracellular fluid containing 0.2% DMSO to obtain the final concentration to be tested. The electrophysiological process recorded by the detection method was used to analyze the effect of drugs on CHO-hERG cells. The experimental data are shown in Table 5.

Table 5 Inhibition results of compound IA _- 41 on hERG potassium current

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

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

It can be seen from the above experimental results that the inhibitory effect of the compound IA _- 41 of the present invention on hERG potassium channel is very weak, indicating that the cardiotoxicity is very low, and it has a good drug safety window, which is beneficial to be developed into a clinical drug.

Example 94

Experimental methods and results of pharmacodynamics of the compounds of the present invention in GIST-T1 tumor cell transplantation model in mice

In this experiment, IA _- 41 was selected as the representative compound, 0.5% MC+0.4% Tween80 was used as vehicle, vehicle group was used as blank control, and Imatinib was used as positive control. GIST-T1 cells were subcutaneously inoculated at the right anterior scapula of the tested mice (1*10 ⁶ / mouse). When the average tumor volume of the tumor-bearing mice reached 100-200 mm ³ , the mice were randomly divided into 5 groups. Pharmacodynamic experimental design was carried out according to Table 6, and compound concentration preparation was carried out in Table 7. The drug was injected intraperitoneally once a day, and tumor volume and body weight were measured. The experimental data were compared by one way ANOVA test method to compare whether the tumor volume and tumor weight of the treatment group were significantly different from those of the control group. The experimental results are shown in Figures 2-7. Finally, TUNEL staining, Ki67 staining and HE staining were performed on the GIST-T1 cells of each group of mice to observe the effects of the compound IA _- 41 of the present invention on tumor cell proliferation and apoptosis (see Figure 8).

Table 6 Pharmacodynamic experimental design

Table 7 test drug, control drug preparation method

From the above results it can be seen that:

1) Body weight: During the experiment, the body weight of the mice in each group increased to a certain extent from the beginning to the end of the experiment, and no other abnormalities were found.

2) Tumor volume: During the test period, the tumors of the mice in the blank control group and the low-dose group (IA _- 41 25 mg/kg) increased significantly. The tumor volume of the mice in the high-dose group showed a strong tumor suppressive effect.

3) The relative percentage of tumor volume: the drug in the high-dose group has an inhibitory effect on the tumor in mice. The effect of the drug in the low-dose group was not obvious. It is shown that the drug cannot penetrate the tumor well, and only when the concentration in the body fluid reaches a certain level can it penetrate the tumor and exert the inhibitory effect of the drug.

4) The tumor weight of the tumor: the experimental data shows that the compound IA _- 41 of the present invention has an inhibitory effect on gastrointestinal stromal tumor in the same order of magnitude as the marketed drug imatinib.

5) Immunohistochemical test results: According to the staining phenomenon, the HE staining results showed that the cell morphology and organization were relatively complete, and there was no necrosis, which provided a basis for the subsequent description of the efficacy of the drug. It can be seen from the staining results of Ki 67 that tumor cells proliferate faster in the low-dose group, while in the high-dose group and Imatinib group, the number of cells in the proliferation state is significantly reduced due to the inhibition of tumor cells. From the results of TUNEL staining, it can be seen that in the high-dose group, there were significantly more cells stained brown, and most of the cells in the tumor were in a state of apoptosis.

Overall, the compound IA _- 41 of the present invention has obvious anti-proliferation and pro-apoptotic effects on GIST-T1 tumors under the condition of high-dose 100 mg/kg intraperitoneal injection, and is almost equivalent to the marketed drug imatinib It can be seen that IA _- 41 is expected to be developed as a drug targeting gastrointestinal stromal tumors.

The substituted (1H-pyrazo[3,4-b]pyridine) urea antitumor compounds of the present invention are small molecular compounds with a new structure, which have multi-target tyrosine kinase inhibitory activity, and can inhibit a variety of homologous tyrosine kinases. The BaF3 cell line and real tumor cells have anti-proliferation ability. They have good pharmacokinetic characteristics and no cardiotoxicity. They also show good anti-tumor proliferation in animal efficacy experiments, and their preparation process is simple. , and the production cost is low, so it is expected to develop into a new class of multi-targeted tyrosine kinase anti-tumor drugs, especially clinical candidate drugs against gastrointestinal stromal tumors.

All documents mentioned herein are incorporated by reference in this application as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (5)

1. a compound shown in formula I or its pharmaceutically acceptable salt,

Wherein, the compound of formula I is selected from the group consisting of:

2. compound shown in formula I as claimed in claim 1 or its pharmaceutically acceptable salt is characterized in that, described compound of formula I is selected from following group:

3. a pharmaceutical composition, is characterized in that, described pharmaceutical composition comprises: (a) the compound shown in formula I of claim 1 of therapeutically effective dose, or its pharmaceutically acceptable salt, and optionally (b) a pharmaceutically acceptable carrier. 4. a compound shown in the formula I according to claim 1, or the purposes of its pharmaceutically acceptable salt, is characterized in that, for preparing (i) suppressing the pharmaceutical composition of tumor cell or treating tumor, (ii) ) receptor tyrosine kinase inhibitors. 5. a method for inhibiting receptor tyrosine kinase in vitro, characterized in that, comprising the steps: (a) contacting the receptor tyrosine kinase with the compound of formula I according to claim 1, or a pharmaceutically acceptable salt thereof, thereby inhibiting the activity of the receptor tyrosine kinase.

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