Pyrazolopyrimidine-5-amine compound as well as preparation method and application thereof

Abstract

The invention relates to pyrazolopyrimidine-5-amine compounds as well as a preparation method and application thereof, and belongs to the technical field of compounds and medicines. The preparation method comprises the following steps: protecting 5 # amino of pyrazolo [1, 5-a] pyrimidine-5-amine by adopting butyric acid methyl ester amide with large steric hindrance, enabling an electrophilic reagent (such as a halogenating agent) to highly selectively attack a 7 # site with small steric hindrance by utilizing a space shielding effect so as to obtain a key 7 # halogenated intermediate, and modifying the 5 # amino by virtue of a hydrolysis reaction, so that the pyrazolo [1, 5-a] pyrimidine-5-amine is obtained. According to the pyrazolo [1, 5-a] pyrimidine compound, the 5 # site and the 7 # site of the pyrazolo [1, 5-a] pyrimidine are accurately substituted, the 5 # site amino substituted pyrazolo [1, 5-a] pyrimidine compound is obtained, and the compound has better anti-tumor activity, provides a new drug choice for tumor treatment and enriches an anti-tumor drug library.

Description

TECHNICAL FIELD

[0001] The present application relates to the fields of compounds and medicine, and in particular to a pyrazolo pyrimidine-5-amine compound and a preparation method and application thereof. BACKGROUND

[0002] c-Met (mesenchymal epithelial transition factor) is a receptor tyrosine kinase encoded by the proto-oncogene MET, and is also a specific receptor of hepatocyte growth factor (HGF), which is involved in the regulation of physiological processes such as cell proliferation, migration, survival and angiogenesis. Abnormal activation of the c-Met signaling pathway (such as gene amplification, overexpression or mutation) is closely related to the invasiveness and drug resistance of various solid tumors, especially in non-small cell lung cancer (NSCLC), gastric cancer, colorectal cancer and other cancers. Therefore, the development of highly efficient and highly selective c-Met inhibitors has become an important direction for the research and development of anti-tumor drugs.

[0003] The c-Met inhibitors currently in clinical research or approved for marketing include multi-target inhibitors such as Crizotinib, Cabozantinib and Tepotinib. Pyrazolo[1,5-a]pyrimidine compounds are also of interest due to their excellent kinase inhibition activity and drugability, but no compounds with this structure have entered clinical trials. Among pyrazolo[1,5-a]pyrimidine compounds, the c-Met inhibitors reported in the prior art are mainly of type II, with the exception of oxygen atoms linking the aromatic ring structure at position 7, and other modifications are mainly at position 3 of pyrazolo[1,5-a]pyrimidine. Based on the model structure of type II, in addition to a small number of methyl and chlorine atoms, there are few other types of structural modifications at position 5 of pyrazolo[1,5-a]pyrimidine. Since the amine group (-NH) at position 5 is a strong electron-donating group, excessive activation of position 5 and adjacent position 7 leads to poor regioselectivity (difficulty in controlling the occurrence at position 5 or position 7) and easy over-substitution (such as 5,7-disubstitution) when directly modified. In addition, in terms of technology, it is difficult to accurately control single-site substitution (position 5 vs position 7), the reaction is difficult to control, and multiple substitution side reactions are prone to occur. Therefore, in addition to a small number of methyl and chlorine atoms, there are few other types of structural modifications, which makes the structure of this type of compound relatively simple, limiting the space for improving drug performance through structural optimization. And at position 7, the aromatic ring structure is basically linked by an oxygen atom, and this relatively single connection method also limits the structural changes of the compounds, making it difficult to produce more new structures with different properties and activities.

[0004] Due to the relative concentration and singleness of the structural type, it is difficult for pyrazolo[1,5-a]pyrimidine compounds to achieve higher levels in terms of kinase inhibition activity. Compared with some multi-target c-Met inhibitors that have entered the clinical or market, the advantages of the c-Met kinase activity inhibition of the pyrazolo[1,5-a]pyrimidine compounds may not be obvious enough, thereby affecting the drugability and clinical application prospect. The singleness of the structure may lead to insufficient selectivity of the compound to c-Met kinase. While inhibiting c-Met, the compound may have a certain inhibitory effect on other kinases, thereby increasing the potential toxic side effects and reducing the safety and tolerability of the drug. Therefore, it is necessary to develop pyrazolo[1,5-a]pyrimidine compounds with higher activity and new structures. SUMMARY

[0005] The present application aims to overcome the deficiencies of the prior art and provide a pyrazolopyrimidine-5-amine compound and a preparation method and application thereof.

[0006] To achieve the above-mentioned object, the technical scheme adopted by the present application is as follows: In a first aspect, the present application provides a pyrazolopyrimidine-5-amine compound, the structural formula of which is shown as formula (I), wherein R is selected from (substituted phenyl)pyridyl, (substituted phenyl)oxodihydropyridyl, ((substituted phenyl)dioxotetrahydropyrimidyl, and the substituted phenyl is selected from methyl, methoxy, halogen mono-substituted or poly-substituted phenyl.

[0007] In the present application, an amine group is introduced at the 5th position of pyrazolo[1,5-a]pyrimidine, a large steric hindrance methyl butyrate amide is used to protect the 5th amine group, the steric shielding effect is utilized to make the electrophilic reagent (such as a halogenating agent) attack the 7th position with high selectivity, a key 7-halogenated intermediate is obtained, and then the 5th amine group is modified through a hydrolysis reaction.

[0008] There are multiple nitrogen atoms or carbon sites with similar properties on the pyrazolo[1,5-a]pyrimidine nucleus. How to realize high selectivity of introducing amine group only at the 5th position without reaction at other positions (such as the 3rd position or the 7th position) is the primary problem of synthesis. Meanwhile, the amination method itself has limitations: direct amination of C-H bond (the most atom-economic method) usually requires harsh conditions (such as strong oxidants, high temperature) and expensive catalysts (such as rhodium, palladium, etc.), which may be poor in chemical compatibility with the complex pyrazolo-pyrimidine nucleus, leading to decomposition of the nucleus or other side reactions. The present application uses chloroformyl butyric acid methyl ester as a key reagent to react with specific nitrogen atoms on the pyrazolo[1,5-a]pyrimidine nucleus to construct a butyric acid methyl ester bridging intermediate with activation and guiding functions. The intermediate precisely guides the nucleophilic attack of the amination reagent on the 5th carbon atom through the coordination of its ester carbonyl group or the space constraint effect, thereby realizing almost absolute regioselectivity and introducing amine group only at the 5th position without reaction at other positions (such as the 3rd position or the 7th position). By introducing a "temporary directing protecting group" - chloroformyl butyric acid methyl ester, it is converted into a two-step efficient and highly controllable classical reaction (amidation + nucleophilic substitution / amination + hydrolysis), overcoming the limitations of the amination method itself.

[0009] The present application introduces amine group at the 5th position of pyrazolo[1,5-a]pyrimidine, providing more possibilities for structural modification. The introduction of amine group at the 5th position opens up a new modification site, which can break through the limitations of traditional structures and explore new structure-activity relationships, providing more space for the structural optimization of c-Met inhibitors. Amine group can be introduced into various functional groups and substituents through various chemical reactions such as acylation, alkylation, sulfonylation, etc., thereby synthesizing a variety of derivatives with diverse structures, which are expected to find compounds with higher activity and better selectivity. Drugs with amine structure often interact with receptors, enzymes and other targets in the body. The amine group in the pyrazolo[1,5-a]pyrimidine-5-amine compounds of the present application can form stronger hydrogen bonds, electrostatic interactions, etc. with the active site of c-Met tyrosine kinase, thereby enhancing the inhibition activity of c-Met and achieving higher inhibition effect.

[0010] By rationally designing the substituents on the amine group at the 5th position, the specificity of the compound in binding to c-Met tyrosine kinase is improved, and the interaction with other kinases or non-target proteins is reduced, thereby improving the selectivity of c-Met and reducing the toxic side effects of drugs.

[0011] The presence of amine group can improve the water solubility and solubility of the compound by forming hydrogen bond with solvent molecules and other interactions. It is of great significance for the absorption, distribution and subsequent formulation development of drugs, and is expected to improve the bioavailability of drugs and make it easier to develop drugs into formulations suitable for various administration routes such as oral or injection. Appropriate substitution of amine group can change the electronic properties and spatial structure of the compound, making it more difficult to be recognized and metabolized by metabolic enzymes, and enhancing the metabolic stability. This helps to prolong the half-life of the drug in the body, reduce the frequency of administration, improve the compliance of patients, and thus has more advantages in clinical application.

[0012] As a preferred embodiment of the pyrazolopyrimidine-5-amine compound of the present application, the substituted phenyl group is selected from the group consisting of methyl, methoxy, halogen mono- or poly-substituted phenyl at 2, 3, 4 or 5 positions.

[0013] As a preferred embodiment of the pyrazolopyrimidine-5-amine compound of the present application, the halogen is fluorine.

[0014] As a preferred embodiment of the pyrazolopyrimidine-5-amine compound of the present application, the substituted phenyl group is selected from at least one of 2-fluorophenyl, 3-fluorophenyl, 4-fluorophenyl, 4-methylphenyl, 2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl, 3,5-dimethoxyphenyl, 3,4-difluorophenyl, 2,4-difluorophenyl, 3-fluoro-4-methoxyphenyl or 4-fluoro-3-methoxyphenyl.

[0015] As a preferred embodiment of the pyrazolo pyrimidine-5-amine compound according to the present application, the pyrazolo pyrimidine-5-amine compound includes at least one of N-(4-((5-aminopyrazolo[1,5-a]pyrimidin-7-yl)oxy)-3-fluorophenyl)-6-(4-fluorophenyl)picolinamide, N-(4-((5-aminopyrazolo[1,5-a]pyrimidin-7-yl)oxy)-3-fluorophenyl)-6-(4-methoxyphenyl)picolinamide, N-(4-((5-aminopyrazolo[1,5-a]pyrimidin-7-yl)oxy)-3-fluorophenyl)-6-(3-fluoro-4-methoxyphenyl)picolinamide, N-(4-((5-aminopyrazolo[1,5-a]pyrimidin-7-yl)oxy)-3-fluorophenyl)-6-(3-fluorophenyl)picolinamide, N-(4-((5-aminopyrazolo[1,5-a]pyrimidin-7-yl)oxy)-3-fluorophenyl)-6-(2-fluorophenyl)picolinamide, N-(4-((5-aminopyrazolo[1,5-a]pyrimidin-7-yl)oxy)-3-fluorophenyl)-6-(3-methoxyphenyl)picolinamide, N-(4-((5-aminopyrazolo[1,5-a]pyrimidin-7-yl)oxy)-3-fluorophenyl)-6-(2-methoxyphenyl)picolinamide, N-(4-((5-aminopyrazolo[1,5-a]pyrimidin-7-yl)oxy)-3-fluorophenyl)-6-(3,5-dimethoxyphenyl)picolinamide, N-(4-((5-aminopyrazolo[1,5-a]pyrimidin-7-yl)oxy)-3-fluorophenyl)-6-(3,4-difluorophenyl)picolinamide, N-(4-((5-aminopyrazolo[1,5-a]pyrimidin-7-yl)oxy)-3-fluorophenyl)-6-(4-fluoro-3-methoxyphenyl)picolinamide, N-(4-((5-aminopyrazolo[1,5-a]pyrimidin-7-yl)oxy)-3-fluorophenyl)-6-(2,4-difluorophenyl)picolinamide, N-(4-((5-aminopyrazolo[1,5-a]pyrimidin-7-yl)oxy)-3-fluorophenyl)-6-(p-tolyl)picolinamide, N-(4-((5-aminopyrazolo[1,5-a]pyrimidin-7-yl)oxy)-3-fluorophenyl)-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide, or N-(4-((5-aminopyrazolo[1,5-a]pyrimidin-7-yl)oxy)-3-fluorophenyl)-3-(4-fluorophenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide.

[0016] As a preferred embodiment of the pyrazolo pyrimidine-5-amine compound of the present application, the pyrazolo pyrimidine-5-amine compound is N-(4-((5-amino pyrazolo [1, 5-a] pyrimidin-7-yl) oxy)-3-fluorophenyl)-6-(3-fluorophenyl) picolinamide, which has better anti-tumor effect on lung cancer and breast cancer.

[0017] As a preferred embodiment of the pyrazolo pyrimidine-5-amine compound of the present application, the pyrazolo pyrimidine-5-amine compound is N-(4-((5-amino pyrazolo [1, 5-a] pyrimidin-7-yl) oxy)-3-fluorophenyl)-6-(3-fluorophenyl) picolinamide, which has better anti-tumor effect on lung cancer and breast cancer.

[0018] As a preferred embodiment of the pyrazolo pyrimidine-5-amine compound of the present application, the pyrazolo pyrimidine-5-amine compound is N-(4-((5-amino pyrazolo [1, 5-a] pyrimidin-7-yl) oxy)-3-fluorophenyl)-6-(3-fluorophenyl) picolinamide, which has better anti-tumor effect on lung cancer and breast cancer.

[0019] In a second aspect, the present application provides a preparation method of the pyrazolo pyrimidine-5-amine compound, comprising the following steps: S1, 3-ethoxy-3-iminopropionic acid ethyl ester hydrochloride isopropanol suspension is added with triethylamine and 3-amino pyrazole successively, mixed thoroughly, heated to reflux and reacted sufficiently, the obtained reaction product is cooled to room temperature, filtered, the solid is collected and washed with ethyl acetate to remove part of impurities, to obtain intermediate A; S2, intermediate A obtained in step S1 is mixed with phosphorus oxychloride, heated to reflux at 100-120℃ for 1.5-2.5h to quench the reaction, to obtain the reaction product, the obtained reaction product is adjusted to pH 8.5-9.5, then extracted with n-butanol, the collected extract is concentrated and washed, to obtain intermediate B; S3, intermediate B obtained in step S2 is dissolved in dichloromethane, mixed with pyridine and N, N-dimethylformamide, then mixed with chloroformic methyl butyrate, reacted at room temperature for 3.5-4.5h to quench the reaction, to obtain the reaction product which is extracted with dichloromethane, the collected extract is concentrated and purified, to obtain intermediate C; S4, the intermediate C obtained in step S3 is dissolved in chlorobenzene, then 2-fluoro-4-nitrophenol, pyridine are added, and the reaction is carried out at 128-132°C, the reaction mixture is obtained after removing chlorobenzene, water is added and mixed, then extracted with ethyl acetate, the extract is collected, dried, filtered, concentrated, purified to obtain intermediate D; S5, intermediate D obtained in step S4, ammonium chloride, iron powder are dissolved in an ethanol-water mixture (in step S5, the volume ratio of ethanol to water in the ethanol-water mixture is 5:1), and the reaction is carried out at 80-100°C, the reaction mixture is filtered, the filtrate is concentrated, extracted with ethyl acetate, the extract is collected, dried, filtered, concentrated, purified to obtain intermediate E; S6, the acyl donor is dissolved in solvent dichloromethane, pyridine 1 and N,N-dimethylformamide are added, then oxalyl chloride is added, mixed and reacted, and the solid is obtained after removing the solvent; intermediate E obtained in step S5 is dissolved in solvent dichloromethane, the obtained solid, pyridine 2 and N,N-dimethylformamide are added, and the reaction is carried out under a protective gas, the reaction mixture is added with water, extracted with dichloromethane, the extract is collected, dried, filtered, concentrated, purified to obtain intermediate F; S7, lithium hydroxide is dissolved in a tetrahydrofuran-water mixture (the volume ratio of tetrahydrofuran to water is 3:1) to obtain a mixture, the mixture is added to intermediate F, the reaction is carried out at room temperature, and the pH value is adjusted to 4.5-5.5 to obtain the reaction mixture, the reaction mixture is extracted with ethyl acetate, the extract is washed to neutral pH, then dried, concentrated, and purified to obtain a pyrazolo pyrimidine-5-amine compound.

[0020] The present application uses 3-ethoxy-3-iminopropionic acid ethyl ester hydrochloride and 3-amino pyrazole as raw materials, connects with various pyridine carboxylic acid derivatives (acyl donor) through chlorination, etherification, reduction and other reactions, and then obtains the target compound through hydrolysis reaction.

[0021] As a preferred embodiment of the preparation method of the present application, in step S1, the molar ratio of 3-ethoxy-3-iminopropionic acid ethyl ester hydrochloride, 3-amino pyrazole and triethylamine is 1-1.5:1-1.5:1. The heating reflux reaction is carried out at 85°C for 12h.

[0022] As a preferred embodiment of the preparation method of the present application, in step S1, the molar ratio of 3-ethoxy-3-iminopropionic acid ethyl ester hydrochloride, 3-amino pyrazole and triethylamine is 1:1.17:1.

[0023] As a preferred embodiment of the preparation method of the present application, in step S2, the molar ratio of intermediate A and phosphorus oxychloride is 1:6.5-7.5.

[0024] In a preferred embodiment of the preparation method of the present invention, in step S2, the molar ratio of intermediate A to phosphorus oxychloride is 1:7.

[0025] In a preferred embodiment of the preparation method described in this invention, in step S2, intermediate A obtained in step S1 is mixed with phosphorus oxychloride, and the reaction is quenched by heating and refluxing at 110°C for 2 hours to obtain the reactant.

[0026] In a preferred embodiment of the preparation method of the present invention, in step S3, the molar ratio of intermediate B to pyridine and methyl chloroformylbutyrate is 1:1 to 2:1 to 2.

[0027] In a preferred embodiment of the preparation method described in this invention, in step S3, the molar ratio of intermediate B to pyridine and methyl chloroformylbutyrate is 1:1.5:1.5.

[0028] In a preferred embodiment of the preparation method described in this invention, in step S4, the molar ratio of intermediate C, 2-fluoro-4-nitrophenol, and pyridine is 1:2.5-3.5:2.5-3.5.

[0029] In a preferred embodiment of the preparation method of the present invention, in step S4, the molar ratio of intermediate C, 2-fluoro-4-nitrophenol, and pyridine is 1:3:3.

[0030] In a preferred embodiment of the preparation method of the present invention, in step S5, the molar ratio of intermediate D, iron powder and ammonium chloride is 1:3.5-4.5:7.5-8.5.

[0031] In a preferred embodiment of the preparation method of the present invention, in step S5, the molar ratio of intermediate D, iron powder and ammonium chloride is 1:4:8.

[0032] In a preferred embodiment of the preparation method described in this invention, in step S6, the molar ratio of the acyl donor, pyridine 1, oxalyl chloride, intermediate E, and pyridine 2 is 1:1 to 1.5:1 to 1.5:1.2:1 to 1.5.

[0033] In a preferred embodiment of the preparation method described in this invention, in step S6, the molar ratio of the acyl donor, pyridine 1, oxalyl chloride, intermediate E, and pyridine 2 is 1:1.3:1.5:1.2:1.5.

[0034] In a preferred embodiment of the preparation method of the present invention, in step S6, the acyl donor is a pyridine carboxylic acid derivative.

[0035] In a preferred embodiment of the preparation method described in this invention, in step S6, the acyl donor includes 6-(4-fluorophenyl)pyridinecarboxylic acid, 6-(4-methoxyphenyl)pyridinecarboxylic acid, 6-(3-fluoro-4-methoxyphenyl)pyridinecarboxylic acid, 6-(3-fluorophenyl)pyridinecarboxylic acid, 6-(2-fluorophenyl)pyridinecarboxylic acid, 6-(3-methoxyphenyl)pyridinecarboxylic acid, 6-(2-methoxyphenyl)pyridinecarboxylic acid, 6-(3-fluoro ... ,5-Dimethoxyphenyl)pyridinecarboxylic acid, 6-(3,4-difluorophenyl)pyridinecarboxylic acid, 6-(3-methoxy-4-fluorophenyl)pyridinecarboxylic acid, 6-(2,4-difluorophenyl)pyridinecarboxylic acid, 6-(4-methylphenyl)pyridinecarboxylic acid, 1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxylic acid or 3-(4-fluorophenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid.

[0036] In a preferred embodiment of the preparation method described in this invention, in step S7, the molar ratio of lithium hydroxide to intermediate F is 2.5 to 3.5:1.

[0037] In a preferred embodiment of the preparation method described in this invention, in step S7, the molar ratio of lithium hydroxide to intermediate F is 3:1.

[0038] Thirdly, the present invention provides the use of the above-mentioned pyrazolopyrimidine-5-amine compounds in the preparation of medicaments for treating tumors.

[0039] As a preferred embodiment of the application described in this invention, the tumor includes lung cancer, breast cancer, gastric cancer, colorectal cancer, prostate cancer, ovarian cancer, etc.

[0040] In a preferred embodiment of the application described in this invention, the tumor includes lung cancer or breast cancer.

[0041] In a preferred embodiment of the application described in this invention, the tumor is lung cancer.

[0042] Fourthly, the present invention provides a medicament for treating tumors, comprising the above-mentioned pyrazolopyrimidine-5-amine compounds.

[0043] In a preferred embodiment of the medicament described in this invention, the medicament further includes a pharmaceutically acceptable carrier.

[0044] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention uses ethyl 3-ethoxy-3-iminopropionate hydrochloride and 3-aminopyrazole as raw materials. After chlorination, etherification, and reduction reactions, the compounds are linked with various pyridine carboxylic acid derivatives as acyl donors, followed by hydrolysis to obtain pyrazolo[1,5-a]pyrimidine compounds. This invention employs a sterically hindered methyl butyrate amide to protect the 5-position amino group of pyrazolo[1,5-a]pyrimidine-5-amine. Utilizing the steric shielding effect, electrophilic reagents (such as halogenating agents) selectively attack the less sterically hindered 7-position, obtaining a crucial 7-position halogenated intermediate. Further hydrolysis then modifies the 5-position amino group, achieving precise substitution at both the 5 and 7 positions of pyrazolo[1,5-a]pyrimidine, resulting in pyrazolo[1,5-a]pyrimidine compounds with 5-position amino substitution. These compounds exhibit enhanced antitumor activity, providing new drug options for tumor treatment and enriching the antitumor drug library. Attached Figure Description

[0045] Figure 1 The above is the 1H NMR spectrum of intermediate A in Example 1 of this invention; Figure 2 The carbon NMR spectrum of intermediate A in Example 1 of this invention; Figure 3 This is the mass spectrum of intermediate A in Example 1 of the present invention; Figure 4 The 1H NMR spectrum of intermediate B in Example 1 of this invention; Figure 5 The carbon NMR spectrum of intermediate B in Example 1 of this invention; Figure 6 This is the mass spectrum of intermediate B in Example 1 of the present invention; Figure 7 The above is the 1H NMR spectrum of intermediate C in Example 1 of this invention; Figure 8 The image shows the carbon NMR spectrum of intermediate C in Example 1 of this invention. Figure 9 This is the mass spectrum of intermediate C in Example 1 of the present invention; Figure 10 The above is the 1H NMR spectrum of intermediate D in Example 1 of this invention; Figure 11 The carbon NMR spectrum of intermediate D in Example 1 of this invention; Figure 12 The NMR fluorine spectrum of intermediate D in Example 1 of this invention; Figure 13 This is the mass spectrum of intermediate D in Example 1 of the present invention; Figure 14 The 1H NMR spectrum of intermediate E in Example 1 of this invention; Figure 15The carbon NMR spectrum of intermediate E in Example 1 of this invention; Figure 16 The NMR fluorine spectrum of intermediate E in Example 1 of this invention; Figure 17 This is the mass spectrum of intermediate E in Example 1 of the present invention; Figure 18 The above is the 1H NMR spectrum of intermediate F in Example 1 of this invention; Figure 19 The image shows the carbon NMR spectrum of intermediate F in Example 1 of this invention. Figure 20 The NMR fluorine spectrum of intermediate F in Example 1 of this invention; Figure 21 This is the mass spectrum of intermediate F in Example 1 of the present invention; Figure 22 The 1H NMR spectrum of the target product in Example 1 of this invention; Figure 23 The carbon NMR spectrum of the target product in Example 1 of this invention; Figure 24 The NMR fluorine spectrum of the target product in Example 1 of this invention; Figure 25 This is the mass spectrometry spectrum of the target product in Example 1 of the present invention; Figure 26 This is a process flow diagram for preparing the pyrazolopyrimidine compounds of this invention. Detailed Implementation

[0046] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0047] Unless otherwise specified, all other materials and reagents used in the examples are commercially available.

[0048] Example 1 A method for preparing a pyrazolopyrimidine compound, the molecular formula of which is: C 24 H 16 F2N6O2, systematically named N-(4-((5-aminopyrazolo[1,5-a]pyrimidin-7-yl)oxy)-3-fluorophenyl)-6-(4-fluorophenyl)pyridineamide, has the structural formula shown in formula (I). Where R is

[0049] The preparation method of this compound includes the following steps: (1) Preparation of intermediate A: At 0°C, triethylamine (2.20 mL) and 3-aminopyrazole (0.0175 mol) were added sequentially to a suspension of ethyl 3-ethoxy-3-iminopropionic acid hydrochloride (0.015 mol) in isopropanol (50 mL). The mixture was stirred for 30 minutes to obtain a mixture. The mixture was heated to reflux at 85°C overnight (12 h). The reactants were cooled to room temperature, filtered, and the solid was collected and washed with ethyl acetate to remove the ethyl acetate and obtain a yellow solid product, i.e., intermediate A, with a yield of 65.5%. The obtained intermediate A is 5-aminopyrazolo[1,5-a]pyrimidin-7(4H)-one. The 1H NMR spectrum of intermediate A is shown below. Figure 1 As shown, the carbon NMR spectrum is as follows: Figure 2 As shown, the mass spectrum is as follows: Figure 3 As shown, its NMR and mass spectrometry information is as follows: 1 H NMR (400MHz, DMSO-d6) δ7.61 (d, J = 1.9 Hz, 1H), 6.55 (s, 2H), 5.90 (d, J = 1.9 Hz, 1H), 4.72 (s, 1H). 13 C NMR (101MHz, DMSO-d6) δ158.1,154.2,141.6,139.9,85.8,74.7. m / z(HR-ESI-MS)Calcd.for C6H7N4O[M+Na] + :173.0434.found:173.0432. (2) Preparation of intermediate B: At 0°C, intermediate A (0.023 mol) obtained in step (1) was mixed with phosphorus oxychloride (30 mL), heated to reflux at 110°C for 2 hours and cooled to room temperature. Then, an ice-water mixture was added to quench the reaction and the reactant was obtained. The pH of the reactant was adjusted to 9 and then extracted with n-butanol. The n-butanol extract was collected and concentrated to remove n-butanol. The concentrate was obtained and washed with ethyl acetate. The ethyl acetate was removed to obtain a yellow solid product, namely intermediate B, with a yield of 67%. The obtained intermediate B is 7-chloropyrazolo[1,5-a]pyrimidine-5-amine, and the 1H NMR spectrum of intermediate B is shown below. Figure 4 As shown, the carbon NMR spectrum is as follows: Figure 5 As shown, the mass spectrum is as follows: Figure 6 As shown, its NMR and mass spectrometry information is as follows: 1 H NMR (400MHz, Chloroform-d) δ8.62(dd,J=7.2,2.1Hz,1H),7.69(dd,J=6.7,2.1Hz,1H),7.40(dd,J=8.9,4.6Hz,2H),6.69–6.66(m,1H).13 C NMR (101MHz, Chloroform-d) δ158.8,148.4,144.9,139.1,100.3,94.3. m / z(HR-ESI-MS)Calcd.for C6H5ClN4[M+H] + :169.0276.found:169.0276. (3) Preparation of intermediate C The intermediate B (2.0 mmol) obtained in step (2) was dissolved in 15 mL of dichloromethane, and pyridine (4.0 mmol) and N,N-dimethylformamide (2-3 drops) were added and mixed. Then, methyl chloroformylbutyrate (2.1 mmol) was added dropwise at 0 °C and mixed. The mixture was stirred at room temperature for 4 hours, and then 100 mL of saturated sodium bicarbonate solution was added to quench the reaction. The reactants were obtained and extracted with dichloromethane. The dichloromethane extract was concentrated to obtain the crude product. The crude product was purified by column chromatography to obtain intermediate C with a yield of 70%. The obtained intermediate C is methyl 5-((7-chloropyrazolo[1,5-a]pyrimidin-5-yl)amino)-5-oxovalerate, and the 1H NMR spectrum of intermediate C is shown below. Figure 7 As shown, the carbon NMR spectrum is as follows: Figure 8 As shown, the mass spectrum is as follows: Figure 9 As shown, its NMR and mass spectrometry information is as follows: 1 H NMR (400MHz, Chloroform-d) δ8.45 (s, 1H), 8.18–8.09 (m, 2H), 6.46 (dd, J = 2.3, 0.7Hz, 1H), 3.69 (s, 3H), 2.49 (dt, J = 30.0, 7.1Hz, 4H), 2.09–2.01 (m, 2H). 13 C NMR (101MHz, Chloroform-d) δ173.6,171.5,149.6,147.8,146.0,140.3,100.8,96.3,51.9,36.5,32.9,20.3. m / z(HR-ESI-MS)Calcd.for C 12 H 13 ClN4O3[M+H] + :297.0749.found:297.0744. (4) Preparation of intermediate D The intermediate C (0.5 mmol) obtained in step (3) was dissolved in chlorobenzene (3 mL), and then 2-fluoro-4-nitrophenol (1.5 mmol) and pyridine (1.5 mmol) were added. The mixture was stirred at 130 °C for 3 h. The reaction was monitored by thin-layer chromatography. No reaction spots were found, indicating that the reaction was complete. After the reaction was completed, chlorobenzene was removed by rotary evaporation. 50 mL of H2O was added, and the mixture was extracted three times with ethyl acetate (50 mL × 3). The organic phases were combined, dried over anhydrous Na2SO4, filtered, concentrated to remove ethyl acetate, and then purified by column chromatography to obtain a white solid product, namely intermediate D, with a yield of 43.8%. The obtained intermediate D is methyl 5-((7-(2-fluoro-4-nitrophenoxy)pyrazolo[1,5-a]pyrimidin-5-yl)amino)-5-oxovalerate. The 1H NMR spectrum of intermediate D is shown below. Figure 10 As shown, the carbon NMR spectrum is as follows: Figure 11 As shown, the NMR fluorine spectrum is as follows: Figure 12 As shown, the mass spectrum is as follows: Figure 13 As shown, its NMR and mass spectrometry information is as follows: 1 H NMR(400MHz,Chloroform-d)δ8.45(s,1H),8.22–8.17(m,2H),8.12(d,J=2.3Hz,1H),7.54(dd,J=9.2,7.5Hz,1H),7 .29(d,J=0.9Hz,1H),6.44(dd,J=2.3,0.7Hz,1H),3.66(s,3H),2.42(dt,J=27.6,7.2Hz,4H),1.97(t,J=7.2Hz,2H). 13 C NMR (101MHz, Chloroform-d) δ173.5,171.6,151.4,148.8,146.5,146.4,144.5,114.2,96.1,51.9,36.5,32.8,20.1. 19 F NMR(376MHz,Chloroform-d)δ-122.4.m / z(HR-ESI-MS)Calcd.for C 12 H 13 ClN4O3[M+H] + :418.1157.found:418.1154. (5) Preparation of intermediate E The intermediate D (1 mmol), ammonium chloride (8 mmol), and iron powder (5 mmol) obtained in step (4) were dissolved in an ethanol-water mixture (ethanol to water volume ratio of 5:1) and reacted at 90°C for 1 hour. The reaction was monitored by thin-layer chromatography. If no reaction spot was found, the reaction was complete. After the reaction was completed, the mixture was filtered, and the filtrate was concentrated and extracted three times with ethyl acetate (50 mL × 3). The organic phases were combined, dried over anhydrous Na2SO4, filtered, concentrated to remove ethyl acetate, and then purified by column chromatography to obtain a pale yellow solid product, namely intermediate E, with a yield of 55%. The obtained intermediate E is methyl 5-((7-(4-amino-2-fluorophenoxy)pyrazolo[1,5-a]pyrimidin-5-yl)amino)-5-oxovalerate. The 1H NMR spectrum of intermediate E is shown below. Figure 14 As shown, the carbon NMR spectrum is as follows: Figure 15 As shown, the NMR fluorine spectrum is as follows: Figure 16 As shown, the mass spectrum is as follows: Figure 17 As shown, its NMR and mass spectrometry information is as follows: 1 H NMR(400MHz, Acetone-d6)δ8.06(dd,J=2.2,0.8Hz,1H),7.26(s,1H),7.25–7.18(m,1H),6.70–6.60(m,2H),6.32(d d,J=2.2,0.8Hz,1H),3.59(d,J=0.8Hz,3H),2.56(dd,J=7.2,0.8Hz,2H),2.35(d,J=7.4Hz,2H),1.93–1.86(m,2H). 13 C NMR (101MHz, Acetone-d6) δ172.9,172.0,156.4,152.4,149.5,149.4,149.1,145.3,123.8,110.6,102.2,102.0,93.8,80.8,50.8,35.6,32.7,20.1. 19 F NMR(376MHz,Acetone-d6)δ-127.8. m / z(HR-ESI-MS)Calcd.for C 18 H 19 FN5O4[M+H] + :388.1416.found:388.1415. (6) Preparation of intermediate F 6-(4-fluorophenyl)pyridinecarboxylic acid (1 mmol) was dissolved in dichloromethane (6 mL). Pyridine (1.3 mmol) and 2 drops of N,N-dimethylformamide were added dropwise at 0 °C, followed by oxaloyl chloride (1.5 mmol). The mixture was stirred for 30 minutes to mix thoroughly. The mixture was stirred at room temperature until the reaction was complete. After the reaction was completed, the dichloromethane solvent was removed to obtain a pale yellow solid. The intermediate E (0.465 g, 1.2 mmol) obtained in step (5) was dissolved in dichloromethane (6 mL). The obtained pale yellow solid (1 mmol) was added at 0 °C. 1), pyridine (0.12 mL, 1.5 mmol), and 2 drops of N,N-dimethylformamide were reacted under nitrogen protection for 4 hours. The reaction status was monitored in real time by thin-layer chromatography. After the reaction was confirmed to be complete, 20 mL of water was added to the reaction system and extracted with dichloromethane. The organic phases were combined, dried and filtered with anhydrous sodium sulfate, and concentrated to remove dichloromethane to obtain crude product. The crude product was purified and separated by preparative thin-layer chromatography (dichloromethane:methanol = 60:1, v / v) to obtain a white solid, i.e., intermediate F, with a yield of 43%. The intermediate F obtained is methyl 5-((7-(2-fluoro-4-(6-(3-fluorophenyl)pyridineamino)phenoxy)pyrazolo[1,5-a]pyrimidin-5-yl)amino)-5-oxovalerate; The 1H NMR spectrum of intermediate F is shown below. Figure 18 As shown, the carbon NMR spectrum is as follows: Figure 19 As shown, the NMR fluorine spectrum is as follows: Figure 20 As shown, the mass spectrum is as follows: Figure 21 As shown, its NMR and mass spectrometry information is as follows: 1 H NMR(400MHz,DMSO-d6)δ10.95(d,J=3.1Hz,1H),10.87(d,J=6.8Hz,1H),8.47–8.4 3(m,1H),8.26–8.19(m,2H),8.22–8.12(m,3H),8.10(dd,J=8.1,2.1Hz,1H),8.02 –7.89(m,2H),7.73–7.65(m,1H),7.39–7.33(m,1H),7.15(d,J=10.2Hz,1H),6.48 –6.45(m,1H),2.40(t,J=7.2Hz,2H),2.27(t,J=6.8Hz,2H),1.72(q,J=7.3Hz,2H). 13C NMR(101MHz,DMSO-d6)δ173.4,173.0,163.6,162.5,155.3,154.8,152.6,151.5,149.9,149.0,146.2,141.6,140.7 ,139.7,138.9,134.4,132.1,130.2,124.2,122.9,121.6,118.4,116.2,116.0,110.1,94.6,51.7,35.6,33.0,20.2. 19 FNMR(376MHz,DMSO-d6)δ-112.0,-128.9. m / z(HR-ESI-MS)Calcd.for C 30 H 24 F2N6O5[M+H] + :587.1849.found:587.1864. (7) Preparation of pyrazolopyrimidine compounds: At 0°C, lithium hydroxide (0.072 g, 3 mmol) was dissolved in a tetrahydrofuran-water mixture (tetrahydrofuran to water volume ratio of 3:1) to obtain a mixture. The mixture was added dropwise to intermediate F and stirred overnight at room temperature. The pH was adjusted to 5 with dilute hydrochloric acid solution to obtain the reactant. The reactant was extracted three times with ethyl acetate (50 mL × 3). The organic phases were combined and washed with saturated brine until the pH was neutral. The mixture was then dried with anhydrous sodium sulfate, concentrated to remove ethyl acetate, and purified by preparative thin-layer chromatography (dichloromethane:methanol = 60:1, volume ratio) to obtain a white solid, which was the target product. The yield of the target product was 34%.

[0050] The 1H NMR spectrum of the target product is shown below. Figure 22 As shown, the carbon NMR spectrum is as follows: Figure 23 As shown, the NMR fluorine spectrum is as follows: Figure 24 As shown, the mass spectrum is as follows: Figure 25 As shown, its NMR and mass spectrometry information is as follows: 1 H NMR(400M Hz,DMSO-d6)δ10.83(s,1H),8.41(q,J=3.4Hz,1H),8.25–8.08(m,4H),8.00–7.77(m,4H) ,7.65–7.58(m,1H),7.35(s,1H),6.67(s,2H),5.94–5.92(m,1H),5.38(d,J=9.1Hz,1H). 13C NMR(101MHz,DMSO-d6)δ163.6,162.5,159.2,154.9,154.6,151.5,151.1,150.0,144.9,1 41.7,139.8,138.7,132.2,130.2,124.4,123.9,122.3,118.3,116.2,110.1,91.4,79.0. 19 F NMR(376MHz,DMSO-d6)δ-111.9,-128.9. m / z(HR-ESI-MS)Calcd.for C 24 H 16 F2N6O2[M+H] + :459.1376.found:459.1287. The target product was named N-(4-((5-aminopyrazolo[1,5-a]pyrimidin-7-yl)oxy)-3-fluorophenyl)-6-(4-fluorophenyl)pyridineamide.

[0051] Example 2 A method for preparing a pyrazolopyrimidine compound, the only difference between this embodiment and Example 1 is the reaction raw material in step (6). Compared with Example 1, in this embodiment, 6-(4-fluorophenyl)pyridinecarboxylic acid in step (6) of Example 1 is changed to 6-(4-methoxyphenyl)pyridinecarboxylic acid, and the target product is obtained with a yield of 53%; its NMR and mass spectrometry information are as follows: 1 H NMR (400MHz, DMSO-d6) δ10.84(s,1H),8.37–8.18(m,4H),8.16–7.90(m,4H),7.66(d,J= 10.7Hz,1H),7.10(d,J=8.7Hz,2H),6.70(s,2H),5.96(s,1H),5.44(s,1H),3.85(s,3H). 13 C NMR(101MHz,DMSO-d6)δ163.8,161.1,159.2,155.7,154.5,151.0,149.7,144.9,141.7,140.8,139 .5,138.7,134.6,130.3,129.3,124.3,123.1,120.9,118.2,114.6,110.0,91.4,79.0,55.8,45.7. 19 F NMR(376MHz,DMSO-d6)δ-129.0. m / z(HR-ESI-MS)Calcd.for C 24 H18 F2N6O2[M+H] + :471.1575.found:471.1539. The target product was named N-(4-((5-aminopyrazolo[1,5-a]pyrimidin-7-yl)oxy)-3-fluorophenyl)-6-(4-methoxyphenyl)pyridineamide.

[0052] Example 3 A method for preparing a pyrazolopyrimidine compound, the only difference between this embodiment and Example 1 is the reaction raw material in step (6). Compared with Example 1, in this embodiment, 6-(4-fluorophenyl)pyridinecarboxylic acid in step (6) of Example 1 is changed to 6-(3-fluoro-4-methoxyphenyl)pyridinecarboxylic acid, and the target product is obtained with a yield of 34%; its NMR and mass spectrometry information are as follows: 1 H NMR (400MHz, DMSO-d6) δ10.87(s,1H),8.17–8.14(m,1H),8.06–8.00(m,1H),7.99–7.91(m,2H),7.87(dd,J=3. 1,2.1Hz,3H),7.68–7.61(m,2H),7.34–7.28(m,1H),5.96(t,J=2.3Hz,2H),5.42(d,J=9.0Hz,2H),3.93(s,3H). 13 C NMR(101MHz,DMSO-d6)δ163.8,163.1,162.1,159.2,155.4,154.0,151.8,150.7,144.9,142.3,141 .1,139.1,135.6,133.7,131.8,130.6,129.2,124.4,123.0,118.4,114.8,114.0,91.4,79.0,56.7. 19 FNMR(376MHz,DMSO-d6)δ-128.8,-134.9. m / z(HR-ESI-MS)Calcd.for C 25 H 18 F2N6O3[M+H] + :489.1481.found:489.1442. The target product was named N-(4-((5-aminopyrazolo[1,5-a]pyrimidin-7-yl)oxy)-3-fluorophenyl)-6-(3-fluoro-4-methoxyphenyl)pyridineamide.

[0053] Example 4 A method for preparing a pyrazolopyrimidine compound, the only difference between this embodiment and Example 1 is the reaction raw material in step (6). Compared with Example 1, in this embodiment, 6-(4-fluorophenyl)pyridinecarboxylic acid in step (6) of Example 1 is changed to 6-(3-fluorophenyl)pyridinecarboxylic acid, and the target product is obtained with a yield of 33%; its NMR and mass spectrometry information are as follows: 1 H NMR(400MHz,DMSO-d6)δ10.89(s,1H),8.37–8.30(m,1H),8.24–8.13(m,4H), 8.04–7.87(m,4H),7.69–7.61(m,2H),6.72(s,2H),5.96(s,1H),5.40(s,1H). 13 C NMR(101MHz,DMSO-d6)δ167.6,163.6,162.5,159.2,154.5,154.5,151.5,151.1,150.1,145.0,1 41.8,140.9,138.7,134.7,132.2,132.2,129.2,124.4,123.8,123.0,122.5,118.4,110.2,91.4. 19 F NMR(376MHz,DMSO-d6)δ-112.5,-128.8. m / z(HR-ESI-MS)Calcd.for C 24 H 16 F2N6O2[M+H] + :459.1376.found:459.1366. The target product was named N-(4-((5-aminopyrazolo[1,5-a]pyrimidin-7-yl)oxy)-3-fluorophenyl)-6-(3-fluorophenyl)pyridineamide.

[0054] Example 5 A method for preparing a pyrazolopyrimidine compound, the only difference between this embodiment and Example 1 is the reaction raw material in step (6). Compared with Example 1, in this embodiment, 6-(4-fluorophenyl)pyridinecarboxylic acid in step (6) of Example 1 is changed to 6-(2-fluorophenyl)pyridinecarboxylic acid, and the target product is prepared with a yield of 29%; its NMR and mass spectrometry information are as follows: 1H NMR(400MHz,DMSO-d6)δ10.86(s,1H),8.22–8.14(m,3H),8.04–7.99(m,1H),7.97 –7.82(m,4H),7.68–7.41(m,2H),6.69(s,2H),5.96(d,J=2.1Hz,1H),5.42(s,1H). 13 C NMR(101MHz,DMSO-d6)δ163.6,162.5,159.2,154.6,154.5,152.1,151.5,151.1,150.4,144.9,1 41.7,140.8,139.5,138.7,134.8,132.2,125.5,124.4,123.0,122.1,118.3,110.1,91.4,79.0. 19 F NMR(376MHz,DMSO-d6)δ-117.0,-128.8. m / z(HR-ESI-MS)Calcd.for C 24 H 16 F2N6O2[M+H] + :459.1376.found:459.1286. The target product was named N-(4-((5-aminopyrazolo[1,5-a]pyrimidin-7-yl)oxy)-3-fluorophenyl)-6-(2-fluorophenyl)pyridineamide.

[0055] Example 6 A method for preparing a pyrazolopyrimidine compound, the only difference between this embodiment and Example 1 is the reaction raw material in step (6). Compared with Example 1, in this embodiment, 6-(4-fluorophenyl)pyridinecarboxylic acid in step (6) of Example 1 is changed to 6-(3-methoxyphenyl)pyridinecarboxylic acid, and the target product is obtained with a yield of 29%; its NMR and mass spectrometry information are as follows: 1 H NMR (400MHz, DMSO-d6) δ10.90(s,1H),8.31–8.26(m,1H),8.25–8.19(m,1H),8.17–8.13(m,2H),7.94–7.90(m,3H),7.89–7.88( m,1H),7.69–7.64(m,1H),7.50–7.45(m,1H),7.11–7.07(m,1H),6.70(s,2H),5.97(d,J=2.1Hz,1H),5.41(s,1H),3.90(s,3H). 13CNMR(101MHz,DMSO-d6)δ163.8,160.3,159.2,155.8,154.6,151.1,150.0,145.0,141.7,140.9,139 .6,138.7,134.7,130.5,124.5,123.0,122.0,120.1,118.3,115.9,113.3,110.1,91.4,79.0,55.8. 19 F NMR(376MHz,DMSO-d6)δ-128.8. m / z(HR-ESI-MS)Calcd.for C 25 H 19 FN6O3[M+H]+:471.1575.found:471.1570. The target product was named N-(4-((5-aminopyrazolo[1,5-a]pyrimidin-7-yl)oxy)-3-fluorophenyl)-6-(3-methoxyphenyl)pyridineamide.

[0056] Example 7 A method for preparing a pyrazolopyrimidine compound, the only difference between this embodiment and Example 1 is the reaction raw material in step (6). Compared with Example 1, in this embodiment, 6-(4-fluorophenyl)pyridinecarboxylic acid in step (6) of Example 1 is changed to 6-(2-methoxyphenyl)pyridinecarboxylic acid, and the target product is obtained with a yield of 34%; its NMR and mass spectrometry information are as follows: 1 H NMR (400MHz, DMSO-d6) δ10.81(s,1H),8.21–8.18(m,1H),8.14–8.08(m,3H),8.06–8.03(m,1H),7.93–7.86(m,3H),7.65(d,J=8. 9Hz,1H),7.50–7.46(m,1H),7.17–7.13(m,1H),6.69(d,J=4.1Hz,2H),5.97(d,J=2.0Hz,1H),5.41(d,J=3.1Hz,1H),3.87(s,3H). 13 C NMR(101MHz,DMSO-d6)δ170.9,163.9,159.2,157.5,154.8,151.1,150.0,145.0,141.7,138.9,138 .3,132.0,131.3,128.7,127.7,124.4,123.0,121.4,121.2,118.1,112.4,109.9,91.4,78.9,56.2. 19F NMR(376MHz,DMSO-d6)δ-128.9. m / z(HR-ESI-MS)Calcd.for C 25 H 19 FN6O3[M+H] + :471.1575.found:471.1536. The target product was named N-(4-((5-aminopyrazolo[1,5-a]pyrimidin-7-yl)oxy)-3-fluorophenyl)-6-(2-methoxyphenyl)pyridineamide.

[0057] Example 8 A method for preparing a pyrazolopyrimidine compound, the only difference between this embodiment and Example 1 is the reaction raw material in step (6). Compared with Example 1, in this embodiment, 6-(4-fluorophenyl)pyridinecarboxylic acid in step (6) of Example 1 is changed to 6-(3,5-dimethoxyphenyl)pyridinecarboxylic acid, and the target product is obtained with a yield of 37%; its NMR and mass spectrometry information are as follows: 1 H NMR(400MHz,DMSO-d6)δ10.93(s,1H),10.87(s,1H),8.19–8.13(m,4H),8.05–8.00(m,1H),7.69–7.61(m, 2H),7.52–7.48(m,2H),6.65(d,J=2.4Hz,1H),5.97(d,J=2.1Hz,2H),5.41(d,J=5.7Hz,2H),3.88(s,6H). 13 C NMR(101MHz,DMSO-d6)δ163.8,162.5,161.4,159.2,155.7,154.6,151.1,150.0,145.0,141 .7,140.9,140.0,139.7,132.2,124.4,123.0,122.1,118.3,106.0,102.1,91.4,79.0,55.9. 19 F NMR(376MHz,DMSO-d6)δ-128.8.m / z(HR-ESI-MS)Calcd.for C 26 H 21 FN6O4[M+H] + :501.1681.found:501.1645. The target product was named N-(4-((5-aminopyrazolo[1,5-a]pyrimidin-7-yl)oxy)-3-fluorophenyl)-6-(3,5-dimethoxyphenyl)pyridinecarboxamide.

[0058] Example 9 A method for preparing a pyrazolopyrimidine compound, the only difference between this embodiment and Example 1 is the reaction raw material in step (6). Compared with Example 1, in this embodiment, 6-(4-fluorophenyl)pyridinecarboxylic acid in step (6) of Example 1 is changed to 6-(3,4-difluorophenyl)pyridinecarboxylic acid, and the target product is obtained with a yield of 25%; its NMR and mass spectrometry information are as follows: 1 H NMR(400MHz,DMSO-d6)δ10.83(s,1H),8.16–8.11(m,3H),7.98(d,J=7.6Hz,1H),7.9 2–7.83(m,4H),7.65–7.54(m,2H),6.67(s,3H),5.93(d,J=2.1Hz,1H),5.35(s,1H). 13 C NMR(101MHz,DMSO-d6)δ163.5,162.5,159.2,154.5,153.7,152.1,151.5,151.1,150.0,145.0,1 41.7,140.9,140.0,138.7,138.6,134.8,134.7,132.2,124.4,123.0,118.3,110.1,91.4,79.0. 19 F NMR(376MHz,DMSO-d6)δ-128.8,-137.3,-137.9. m / z(HR-ESI-MS)Calcd.for C 24 H 15 F3N6O2[M+H] + :477.1281.found:477.1242. The target product was named N-(4-((5-aminopyrazolo[1,5-a]pyrimidin-7-yl)oxy)-3-fluorophenyl)-6-(3,4-difluorophenyl)pyridineamide.

[0059] Example 10 A method for preparing a pyrazolopyrimidine compound, the only difference between this embodiment and Example 1 is the reaction raw material in step (6). Compared with Example 1, in this embodiment, 6-(4-fluorophenyl)pyridinecarboxylic acid in step (6) of Example 1 is changed to 6-(4-fluoro-3-methoxyphenyl)pyridinecarboxylic acid, and the target product is obtained with a yield of 37%; its NMR and mass spectrometry information are as follows: 1H NMR(400MHz,DMSO-d6)δ10.94(s,1H),8.34–8.32(m,1H),8.19–8.12(m,4H),7.96–7.87(m, 4H),7.70–7.63(m,2H),7.42–7.38(m,1H),6.74(s,1H),5.98(d,J=2.2Hz,1H),4.04(s,3H). 13 C NMR(101MHz,DMSO-d6)δ163.7,159.2,155.0,154.6,151.0,149.9,148.0,145.0,139.8,132 .1,132.0,129.4,129.3,124.5,124.2,121.8,118.2,116.6,113.5,109.8,91.4,79.0,56.7. 19 F NMR(376MHz,DMSO-d6)δ-128.8,-133.8. The target product was named N-(4-((5-aminopyrazolo[1,5-a]pyrimidin-7-yl)oxy)-3-fluorophenyl)-6-(4-fluoro-3-methoxyphenyl)pyridineamide.

[0060] Example 11 A method for preparing a pyrazolopyrimidine compound, the only difference between this embodiment and Example 1 is the reaction raw material in step (6). Compared with Example 1, in this embodiment, 6-(4-fluorophenyl)pyridinecarboxylic acid in step (6) of Example 1 is changed to 6-(2,4-difluorophenyl)pyridinecarboxylic acid, and the target product is obtained with a yield of 57%; its NMR and mass spectrometry information are as follows: 1 H NMR(400MHz,DMSO-d6)δ10.86(s,1H),8.21–8.15(m,3H),8.08–7.87(m,4H),7.65 (t,J=8.7Hz,1H),7.48–7.32(m,1H),6.70(s,2H),5.98–5.96(m,1H),5.42(s,1H). 13 C NMR(101MHz,DMSO-d6)δ178.0,168.3,167.2,160.1,159.2,157.4,156.7,156.3,156.0,155.1,1 53.8,151.0,145.5,144.3,143.6,139.2,138.7,129.0,127.7,126.9,123.0,117.5,99.3,86.2. 19F NMR(376MHz,DMSO-d6)δ-108.1,-112.2,-128.8. m / z(HR-ESI-MS)Calcd.for C 24 H 15 F3N6O2[M+H] + :477.1281.found:477.1242. The target product was named N-(4-((5-aminopyrazolo[1,5-a]pyrimidin-7-yl)oxy)-3-fluorophenyl)-6-(2,4-difluorophenyl)pyridineamide.

[0061] Example 12 A method for preparing a pyrazolopyrimidine compound, the only difference between this embodiment and Example 1 is the reaction raw material in step (6). Compared with Example 1, in this embodiment, 6-(4-fluorophenyl)pyridinecarboxylic acid in step (6) of Example 1 is changed to 6-(4-methylphenyl)pyridinecarboxylic acid, and the target product is obtained with a yield of 44%; its NMR and mass spectrometry information are as follows: 1 H NMR (400MHz, DMSO-d6) δ10.88(s,2H),8.26–7.89(m,10H),7.35(d,J=7.8Hz,2H),6.73(s,3H),5.96(s,2H),5.44(d,J=8.2Hz,2H),2.38(s,3H). 13 C NMR (101MHz, DMSO-d6)δ 163.8,162.5,159.2,155.9,154.5,151.5,151.1,149.9,144.9,141.7,140.9,139.8,135.1,132.2,130.0,127.8,124.4,123.0,118.3,110.1,91.4,79.0,21.5. 19 F NMR(376MHz,DMSO-d6)δ-128.9. m / z(HR-ESI-MS)Calcd.for C 25 H 19 FN6O2[M+H] + :455.1626; found:455.1536. The target product was named N-(4-((5-aminopyrazolo[1,5-a]pyrimidin-7-yl)oxy)-3-fluorophenyl)-6-(4-methylphenyl)pyridineamide.

[0062] Example 13 A method for preparing a pyrazolopyrimidine compound, the only difference between this embodiment and Example 1 is the reaction raw material in step (6). Compared with Example 1, in this embodiment, 6-(4-fluorophenyl)pyridinecarboxylic acid in step (6) of Example 1 is replaced with 1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxylic acid, and the target product is obtained with a yield of 34%; its NMR and mass spectrometry information are as follows: 1 H NMR(400MHz,DMSO-d6)δ12.15(s,1H),8.60(d,J=7.4Hz,1H),8.17–8.06(m,2H),7.86 (s,1H),7.63–7.40(m,6H),6.76–6.70(m,1H),6.64(s,2H),5.95(s,1H),5.39(s,1H). 13 C NMR(101MHz,DMSO-d6)δ162.4,162.3,159.2,154.5,153.7,151.1,145.7,144.9,144.9,1 38.7,136.7,134.5,129.9,124.7,120.5,117.3,117.3,116.7,109.3,107.6,91.4,79.0. 19 F NMR(376MHz,DMSO-d6)δ-112.5,-128.3. m / z(HR-ESI-MS)Calcd.for C 24 H 16 F2N6O3[M+H] + :475.1325.found:475.1286. The target product was named N-(4-((5-aminopyrazolo[1,5-a]pyrimidin-7-yl)oxy)-3-fluorophenyl)-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide.

[0063] Example 14 A method for preparing a pyrazolopyrimidine compound, the only difference between this embodiment and Example 1 is the reaction raw material in step (6). Compared with Example 1, in this embodiment, 6-(4-fluorophenyl)pyridinecarboxylic acid in step (6) of Example 1 is adjusted to 3-(4-fluorophenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid, and the target product is obtained with a yield of 56%; its NMR and mass spectrometry information are as follows: 1H NMR (400MHz, DMSO-d6) δ11.15(s,1H),9.63(s,1H),7.02(d,J=5.5Hz,1H),6.68 –6.54(m,3H),6.18–6.15(m,2H),6.03–5.84(m,5H),4.81(s,1H),4.13(s,1H). 13 C NMR (101MHz, DMSO-d6) δ172.5,163.5,162.4,159.1,154.5,152.1,151.3,150.3,144.9,141. 6,140.8,139.5,133.8,132.1,124.3,123.0,122.1,118.2,112.7,110.0,105.1,91.3,79.0. 19 F NMR(376MHz,DMSO-d6)δ-113.3,-128.2. m / z(HR-ESI-MS)Calcd.for C 23 H 15 F2N7O4[M+H] + :492.1226.found:492.1213. The target product was named N-(4-((5-aminopyrazolo[1,5-a]pyrimidin-7-yl)oxy)-3-fluorophenyl)-3-(4-fluorophenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-carboxamide.

[0064] The process flow diagram for preparing the pyrazolopyrimidine compounds of this invention is shown below. Figure 26 As shown, the reaction reagents and conditions are as follows: (a) TEA, IPA, 85℃, 12h; (b) POCl3, reflux, 110℃, 2h; (c) Py, DMF, DCM, 0℃→RT, 4h; (d) Py, Chlorobenzene, 130℃, 3h; (e) Fe, NH4Cl, EtOH, H2O, 0℃→RT, 12h; (f) HATU, DIPEA, DCM, 0℃→RT, 24h; (g) LiOH, THF, H2O; In the figure, compound AF represents intermediate AF, and compound 11 is the target product.

[0065] The information on the reactants and target products in step (6) of Examples 1-14 of this invention is shown in Table 1. Test case The antitumor activity of the drugs was tested using the pyrazolopyrimidine compounds of Examples 1-14 as test drugs and 5-fluorouracil as a positive control drug.

[0066] 1. Preparation of the test drug: Dissolve the drug in DMSO to prepare a 100mM stock solution. Dilute to 0-500μM with serum-free culture medium before testing. Prepare and use immediately.

[0067] 2. Tumor cells include: human lung cancer cells (PC-9 cells), human non-small cell lung cancer cells (A549 cells), and human breast cancer cells (MCF7 cells).

[0068] 3. Antitumor activity test (1) PC-9 and A549 cells were cultured in RPMI 1640 medium, 10% fetal bovine serum (FBS), 100 U / mL penicillin and 100 mg / mL streptomycin, at 37°C in a 5% CO2 incubator. When the cells were in good condition, they were seeded into 96-well plates.

[0069] (2) MCF-7 cells were cultured in high-glucose DMEM medium, 10% fetal bovine serum (FBS), 100 U / mL penicillin and 100 mg / mL streptomycin, at 37°C in a 5% CO2 incubator. When the cells were in good condition, they were seeded into 96-well plates.

[0070] PC-9, A549, and MCF-7 cells, cultured to optimal condition, were seeded into 96-well plates at densities of 6500 cells / well for PC-9, 6500 cells / well for A549, and 4000 cells / well for MCF-7. After seeding, the cells were incubated at 37°C in a 5% CO2 incubator until complete adhesion. Then, 100 μL of serum-free medium containing different compound concentrations was added. After 72 h of incubation, the culture medium was removed, and 110 μL of a mixture containing 10 μL of CCK8 and 100 μL of medium was added. Incubation was continued for 1–4 h. A blank control group (medium medium + solvent) and a zeroing group (cells + medium + equal volume of solvent) were established. Absorbance was measured at 450 nm and 630 nm using a microplate reader. The data were processed to calculate the inhibition rate of each compound. The inhibition rate was plotted against the corresponding concentrations of different compounds. The data were processed using statistical software to calculate the IC50. 50 The inhibition rate was calculated using the formula: [1 - (experimental group - background group) / (blank group - zeroing group)] * 100%. Each experimental group had 6 replicates, and the results were expressed as mean ± SD of 3 independent experiments.

[0071] 4. Results The results of the antitumor activity of Examples 1-14 against different tumor cells are shown in Table 2. Table 2 Table 2 shows that several new compounds exhibited significant and selective antitumor activity, with potency comparable to or even superior to the positive control 5-fluorouracil in some cases. In particular, Examples 4, 11, and 12 are considered the most promising candidate compounds in this series.

[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A pyrazolopyrimidine-5-amine compound, characterized in that, Its structural formula is shown in equation (I). R is selected from (substituted phenyl)pyridyl, (substituted phenyl)oxodihydropyridyl, and (substituted phenyl)dioxotetrahydropyrimidinyl, and the substituted phenyl is selected from methyl, methoxy, halogen monosubstituted or polysubstituted phenyl.

2. The pyrazolopyrimidine-5-amine compound as described in claim 1, characterized in that, The substituted phenyl group is selected from methyl, methoxy, halogen monosubstituted or polysubstituted phenyl groups at positions 2, 3, 4, and 5.

3. The pyrazolopyrimidine-5-amine compound as described in claim 1 or 2, characterized in that, The halogen is fluorine.

4. The pyrazolopyrimidine-5-amine compound as described in claim 3, characterized in that, The substituted phenyl group is selected from at least one of 2-fluorophenyl, 3-fluorophenyl, 4-fluorophenyl, 4-methylphenyl, 2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl, 3,5-dimethoxyphenyl, 3,4-difluorophenyl, 2,4-difluorophenyl, 3-fluoro-4-methoxyphenyl or 4-fluoro-3-methoxyphenyl.

5. The pyrazolopyrimidine-5-amine compound as described in claim 4, characterized in that, The pyrazolopyrimidine-5-amine compounds include N-(4-((5-aminopyrazolo[1,5-a]pyrimidine-7-yl)oxy)-3-fluorophenyl)-6-(4-fluorophenyl)pyridineamide, N-(4-((5-aminopyrazolo[1,5-a]pyrimidine-7-yl)oxy)-3-fluorophenyl)-6-(4-methoxyphenyl)pyridineamide, N-(4-((5-aminopyrazolo[1,5-a]pyrimidine-7-yl)oxy)-3-fluorophenyl)-6-(3-fluoro-4-methoxyphenyl)pyridineamide, N-(4-((5-aminopyrazolo[1,5-a]pyrimidine-7-yl)oxy)-3-fluorophenyl)-6-(3-fluoro-4-methoxyphenyl)pyridineamide, N-(4-((5-aminopyrazolo[1,5-a]pyrimidine- 7-yl)oxy)-3-fluorophenyl)-6-(3-fluorophenyl)pyridine amide, N-(4-((5-aminopyrazolo[1,5-a]pyrimidin-7-yl)oxy)-3-fluorophenyl)-6-(2-fluorophenyl)pyridine amide, N-(4-((5-aminopyrazolo[1,5-a]pyrimidin-7-yl)oxy)-3-fluorophenyl)-6-(3-methoxyphenyl)pyridine amide, N-(4-((5-aminopyrazolo[1,5-a]pyrimidin-7-yl)oxy)-3-fluorophenyl)-6-(2 ... [5-a]pyrimidin-7-yl)oxy)-3-fluorophenyl)-6-(3,5-dimethoxyphenyl)pyridine amide, N-(4-((5-aminopyrazolo[1,5-a]pyrimidin-7-yl)oxy)-3-fluorophenyl)-6-(3,4-difluorophenyl)pyridine amide, N-(4-((5-aminopyrazolo[1,5-a]pyrimidin-7-yl)oxy)-3-fluorophenyl)-6-(4-fluoro-3-methoxyphenyl)pyridine amide, N-(4-((5-aminopyrazolo[1,5-a]pyrimidin-7-yl)oxy)-3-fluorophenyl)-6-(2,4-difluorophenyl) At least one of pyridine amide, N-(4-((5-aminopyrazolo[1,5-a]pyrimidin-7-yl)oxy)-3-fluorophenyl)-6-(p-tolyl)pyridine amide, N-(4-((5-aminopyrazolo[1,5-a]pyrimidin-7-yl)oxy)-3-fluorophenyl)-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide, or N-(4-((5-aminopyrazolo[1,5-a]pyrimidin-7-yl)oxy)-3-fluorophenyl)-3-(4-fluorophenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-carboxamide).

6. The method for preparing the pyrazolopyrimidine-5-amine compound according to any one of claims 1 to 5, characterized in that, Includes the following steps: Triethylamine and 3-aminopyrazole were added sequentially to the isopropanol suspension of S1, 3-ethoxy-3-iminopropionic acid ethyl ester hydrochloride, mixed thoroughly, and heated under reflux to react completely. The resulting reactant was cooled to room temperature, filtered, and the solid was collected and washed with ethyl acetate to remove some impurities, yielding intermediate A. S2. Mix intermediate A obtained in step S1 with phosphorus oxychloride, heat and reflux at 100-120°C for 1.5-2.5 h to quench the reaction, and obtain the reactant. Adjust the pH of the reactant to 8.5-9.5, and then extract with n-butanol. Collect the extract, concentrate and wash to obtain intermediate B. S3. The intermediate B obtained in step S2 is dissolved in dichloromethane, pyridine and N,N-dimethylformamide are added and mixed, then methyl chloroformylbutyrate is added and mixed. The reaction is quenched after reacting at room temperature for 3.5-4.5 h, and the reactant is obtained and extracted with dichloromethane. The extract is collected, concentrated and purified to obtain intermediate C. S4. The intermediate C obtained in step S3 is dissolved in chlorobenzene, and then 2-fluoro-4-nitrophenol and pyridine are added. The mixture is reacted at 128-132℃ to obtain the reactant and remove the chlorobenzene. Water is added and mixed well, and then extracted with ethyl acetate. The extract is collected, dried, filtered, concentrated and purified to obtain intermediate D. S5. Dissolve intermediate D obtained in step S4, ammonium chloride, and iron powder in an ethanol-water mixture and react fully at 80-100℃. Filter the reactants, concentrate the filtrate, extract with ethyl acetate, collect the extract, dry, filter, concentrate, and purify to obtain intermediate E. S6. The acyl donor is dissolved in dichloromethane, pyridine 1 and N,N-dimethylformamide are added, then oxalyl chloride is added, and the mixture is stirred until fully reacted. The solvent is removed to obtain a solid. The intermediate E obtained in step S5 is dissolved in dichloromethane, the obtained solid, pyridine 2 and N,N-dimethylformamide are added, and the mixture is fully reacted under a protective gas. Water is added to the reactants, and the mixture is extracted with dichloromethane. The extract is collected, dried, filtered, concentrated, and purified to obtain intermediate F. S7. Lithium hydroxide was dissolved in a tetrahydrofuran-water mixture to obtain a mixture. The mixture was added to intermediate F and allowed to react fully at room temperature. The pH was adjusted to 4.5–5.5 to obtain the reactant. The reactant was extracted with ethyl acetate. The extract was washed until the pH was neutral. Then it was dried, concentrated, and purified to obtain pyrazolopyrimidine-5-amine compounds.

7. The preparation method according to claim 6, characterized in that, In step S1, the molar ratio of ethyl 3-ethoxy-3-iminopropionic acid hydrochloride to 3-aminopyrazole and triethylamine is 1-1.5:1-1.5:1; And / or, in step S2, the molar ratio of intermediate A to phosphorus oxychloride is 1:6.5 to 7.5; And / or, in step S3, the molar ratio of intermediate B to pyridine and methyl chloroformylbutyrate is 1:1 to 2:1 to 2; And / or, in step S4, the molar ratio of intermediate C, 2-fluoro-4-nitrophenol, and pyridine is 1:2.5-3.5:2.5-3.5; And / or, in step S5, the molar ratio of intermediate D, iron powder, and ammonium chloride is 1:3.5-4.5:7.5-8.5; And / or, in step S6, the molar ratio of the acyl donor, pyridine 1, oxalyl chloride, intermediate E, and pyridine 2 is 1:1 to 1.5:1 to 1.5:1.2:1 to 1.5; And / or, in step S7, the molar ratio of lithium hydroxide to intermediate F is 2.5 to 3.5:

1.

8. The use of the pyrazolopyrimidine-5-amine compound according to any one of claims 1 to 5 in the preparation of a medicament for treating tumors.

9. The application as described in claim 8, characterized in that, The tumors include lung cancer, breast cancer, stomach cancer, colorectal cancer, prostate cancer, or ovarian cancer.

10. A drug for treating tumors, characterized in that, Includes the pyrazolopyrimidine-5-amine compounds according to any one of claims 1 to 5.

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