Dipyrazolopyrimidine compound as well as synthesis method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a dipyrazolopyrimidine compound as well as a synthesis method and application thereof. The structure of the dipyrazolopyrimidine compound is as shown in the following formula (1). According to the invention, different fat heterocycles are introduced to the C5 site of a pyrimidine ring to inspect the size of the ring and the influence of substituent groups on the ring on the activity of the compound, and dipyrazolopyrimidine compounds 1-20 are designed and synthesized. Wherein the inhibition rate of the compound 2 is the highest and reaches 69.67%, and the compound 2 shows a remarkable inhibition effect. In addition, the compounds 4, 5, 13, 14, 15 and 17 also have high activity (the inhibition rate exceeds 30%). Therefore, the dipyrazolopyrimidine compound prepared by the invention is a KRASG12D inhibitor with relatively high activity and can be used for preparing medicines for treating pancreatic cancer, intestinal cancer and lung cancer. Formula (1).

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and specifically relates to a dipyrazolopyrimidine compound, its synthesis method, and its application. Background Technology

[0002] RAS proteins are small membrane-bound guanine nucleotide-binding proteins that act as molecular switches by cycling between active GTP-binding and inactive GDP-binding conformations. RAS signaling is regulated by a balance between activation of guanine nucleotide exchange factor (GEF) and inactivation of GTPase-activated proteins. RAS proteins play a crucial role in the regulation of cell proliferation, differentiation, and survival by mediating numerous signaling pathways, including the RAF-MEK-ERK (RAF-MAPK / ERK kinase-extracellular signal-regulated kinase), PI3K-AKT-mTOR (phosphoinositol 3-kinase-AKT-rapamycin mechanism target), and RALGDS-RAL (RAL guanine nucleotide dissociation stimulant-RAL) pathway.

[0003] The KRAS gene is located on the short arm of chromosome 4 and encodes six exons. Alternating splicing on its fourth exon produces KRAS-4A and KRAS-4B, with molecular weights of approximately 21 kDa and containing 188 and 189 amino acid residues, respectively. KRAS-4B is the major transcript in human cells and has received more attention than KRAS-4A; therefore, current research mainly focuses on the KRAS-4B isoform. KRAS consists of two domains: the N-terminal G domain and the C-terminal hypervariable region, the latter responsible for anchoring KRAS to the cell membrane. The catalytic G-domain, composed of the P-loop and switch regions I and II, acts as a GTP-GDP switch by promoting GTP-GDP exchange. Mutations at codon 12 in KRAS, particularly the glycine mutation, lead to increased steric hindrance, affecting the efficient binding of KRAS to GAP and reducing GTP hydrolysis. This keeps KRAS in a persistently activated state, stimulating downstream signaling to remain highly active, ultimately disrupting signal transduction and leading to cell carcinogenesis. Recent studies have shown that there are no significant differences among oncogenic KRAS mutants, but the degree of GTPase activity reduction varies considerably. G12C Almost unaffected, while KRAS G12D The rate decreased by 50 times.

[0004] Of all RAS mutations, KRAS mutations account for 85%, primarily found in lung cancer (30%), pancreatic cancer (70%), and colon cancer (40%). KRAS 12-position mutations include G12C, G12D, and G12V, among others. G12D Mutations are most common in pancreatic ductal carcinoma and colorectal cancer. KRASG12D Patients with KRAS mutations have a very low survival rate, and to date, there is no targeted therapy for KRAS. G12D With the market availability of drugs targeting KRAS, it's important to understand that KRAS participates in numerous physiological functions within the body and shares high homology with HRAS and NRAS. Drugs targeting KRAS can simultaneously affect the normal physiological functions of both HRAS and NRAS, resulting in strong toxic side effects. Therefore, developing drugs that selectively target KRAS is crucial. G12D Drugs targeting mutants are urgently needed.

[0005] Due to KRAS G12D Since there are few known binding sites for proteins, it is necessary to design and study novel KRAS. G12D Small molecule inhibitors remain a significant challenge. Therefore, there is still a need in this field to develop KRAS inhibitors with better selectivity and lower toxicity. G12D Inhibitors. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a dipyrazolopyrimidine compound, its synthesis method, and its applications.

[0007] The technical solution of the present invention is as follows:

[0008] A dipyrazolopyrimidine compound as shown in formula (1), or a pharmaceutically acceptable salt thereof,

[0009]

[0010] Equation (1)

[0011] Wherein, R1 is selected from one of the following groups:

[0012] ;

[0013] R2 is selected from one of the following groups:

[0014] ;

[0015] R3 is selected from one of the following groups:

[0016] .

[0017] According to a preferred embodiment of the present invention, the dipyrazolopyrimidine compound is specifically selected from one of the following 20 compounds, or a pharmaceutically acceptable salt, prodrug, hydrate or solvent compound, polymorph, stereoisomer, tautomer, resonance, enantiomer or diastereomer:

[0018] .

[0019] The present invention also provides a method for preparing the above-mentioned dipyrazolopyrimidine compounds.

[0020] The synthesis procedures for compounds 1-4 are as follows:

[0021] .

[0022] The synthesis process for compounds 5-20 is as follows:

[0023] .

[0024] According to a preferred embodiment of the present invention, the specific preparation method of compounds 1-4 includes the following steps:

[0025] (1) 5,7-dichloropyrazolo[1,5-a]pyrimidine and N-bromosuccinimide were dissolved in chloroform and reacted at room temperature for 12 h. The reaction solution was washed with saturated brine, dried with anhydrous sodium sulfate and filtered to remove impurities, and crude product was obtained. The crude product was then purified by silica gel column chromatography with a mixture of petroleum ether and ethyl acetate and eluted to obtain 3-bromo-5,7-dichloropyrazolo[1,5-a]pyrimidine.

[0026] (2) Dissolve the 3-bromo-5,7-dichloropyrazole[1,5-a]pyrimidine obtained in step (1) in dichloromethane, add N,N-diisopropylethylamine and amine compounds, and react at -40℃ under nitrogen protection for 0.5 h. Remove impurities by washing the reaction solution with saturated brine, drying with anhydrous sodium sulfate and filtering to obtain crude product; then purify the crude product by silica gel column chromatography with a mixture of petroleum ether and ethyl acetate and elute to obtain intermediate 1.

[0027] The amine compound is selected from (1R,5S)-3,8-diazabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester or N-methylpiperazine;

[0028] (3) The intermediate 1 and alcohol compound obtained in step (2) were dissolved in a solvent of tetrahydrofuran and NN dimethylformamide. Sodium hydride was added at 0°C, and the reaction was stirred at room temperature. The reaction solution was washed with saturated brine, dried with anhydrous sodium sulfate, and filtered to remove impurities, and crude product was obtained. The crude product was then purified by silica gel column chromatography using a mixture of dichloromethane and methanol and eluted to obtain intermediate 2.

[0029] The alcohol compounds are selected from N-methyl-4-hydroxypiperidine, 3-hydroxy-1-methyltetrahydropyrrole, 1-methyl-2-pyrrolidinemethanol or N-ethyl-4-hydroxypiperidine;

[0030] (4) The intermediate 2 and 3-hydroxy-1-naphthoic acid pinacol ester obtained in step (3) were dissolved in 1,4-dioxane solvent, and Pd(dppf)Cl2 and cesium carbonate were added as catalysts. The reaction was carried out at 85°C for 3 h. The reaction solution was dried with anhydrous sodium sulfate, filtered and evaporated to dryness. Then, the product was hydrolyzed with hydrochloric acid-1,4-dioxane and acetonitrile. The hydrolysate was washed with saturated brine, dried with anhydrous sodium sulfate and filtered to remove impurities, and crude product was obtained. The crude product was then purified by silica gel column chromatography with a mixture of dichloromethane and methanol and eluted to obtain compounds 1 to 4.

[0031] According to a preferred embodiment of the present invention, the specific preparation method of compounds 5-20 includes the following steps:

[0032] 1) Dissolve 5,7-dichloropyrazolo[1,5-a]pyrimidine in chloroform, add N-bromosuccinimide, heat to 40°C, react for 12 hours, and after the reaction is complete as detected by TLC, add water to quench the reaction, extract three times with ethyl acetate, combine the organic phases, wash with saturated brine, dry with anhydrous sodium sulfate, filter, add silica gel and concentrate, and purify by silica gel column chromatography to obtain 3-bromo-5,7-dichloropyrazolo[1,5-a]pyrimidine;

[0033] 2) Dissolve the 3-bromo-5,7-dichloropyrazole[1,5-a]pyrimidine obtained in step 1) in dichloromethane, cool to -40°C, add amine compounds, add diisopropylethylamine dropwise, and after the addition is complete, allow the temperature to rise naturally to room temperature. After the reaction is complete as detected by TLC, concentrate under reduced pressure and dry to obtain intermediate 3.

[0034] The amine compounds are selected from 2-tert-butoxycarbonyl-2,5-diazabicyclo[2.2.1]heptane, 1-tert-butoxycarbonyl-2-methylpiperazine, 1-(tert-butoxycarbonyl)-1,4-diazabicycloheptane, 3,4-dimethylpiperazine, 6-(tert-butyl)-3,6-diazabicyclo[3.1.1]heptane, 1-tert-butoxycarbonyl-2-piperazinone, and 8-tert-butoxycarbonyl-3,8-diazabicyclo[3.2.1]octane;

[0035] 3) Add intermediate 3 obtained in step 2) and an alcohol compound to tetrahydrofuran. After cooling to 0°C in an ice-water bath, add sodium hydride, allow to warm naturally to room temperature, stir for 0.5 hours, heat to 100°C in an oil bath, react for 2 hours, cool to 0°C in an ice-water bath, quench with water, extract with dichloromethane, and dry with anhydrous sodium sulfate. Purify by silica gel column chromatography to obtain intermediate 4;

[0036] The alcohol compounds are selected from (1-methylpyrrolidone-2-yl)methanol, 8-methyl-8-azabicyclo[3.2.1]octane-3-ol, 1-ethylpyrrolidone-3-ol, 1-methylpiperidin-4-ol, 1-isopropylpiperidin-4-ol, 1-cyclopropylpiperidin-4-ol, (2S,7aR)-7a-((hydroxy)methyl)-2-fluorohexahydro-1H-pyrrolazine, benzyl alcohol, and N-ethyl-4-hydroxypiperidine;

[0037] 4) Add intermediate 4 obtained in step 3), 7-fluoro-3-(methoxymethoxy)-8-[(triisopropylsilyl)ethynyl]naphthalene-1-boronic acid pinacol ester, cesium carbonate and Ad2nBuP-Pd-G3 sequentially to 1,4-dioxane solvent. Purge with nitrogen for 15 min to replace air, microwave heat to 82℃ for 1 hour, and after the reaction is complete by TLC monitoring, dry with anhydrous sodium sulfate, filter, wash three times with methanol, concentrate under reduced pressure to dryness with silica gel, and purify by silica gel column chromatography to obtain intermediate 5;

[0038] 5) Add intermediate 5 obtained in step 4) and CsF to DMF, stir overnight at room temperature, monitor the reaction by TLC until complete, add silica gel, concentrate directly to dryness, and purify by silica gel column chromatography to obtain intermediate 6;

[0039] 6) Add intermediate 6 obtained in step 5) to methanol, cool to 0℃, add 4 M 1,4-dioxane hydrochloride dropwise, and after the addition is complete, allow to rise naturally to room temperature and react for 2 h. After the reaction is complete as monitored by TLC, concentrate directly to dryness, dissolve in methanol, concentrate again, repeat three times, and purify by high performance chromatography to obtain compounds 5-20.

[0040] This invention provides the above-mentioned dipyrazolopyrimidine compounds and their isomers or pharmaceutically acceptable salts thereof for the preparation of KRAS G12D Application in drugs for treating diseases related to protein dysfunction.

[0041] According to a preferred embodiment of the invention, the one with KRAS G12D Diseases associated with protein dysfunction include pancreatic cancer, colorectal cancer, and lung cancer.

[0042] Of these, approximately 90% of pancreatic cancers have KRAS mutations, with nearly half being the G12D type. About 40% of colorectal cancers have KRAS mutations, with G12D being one of the major subtypes. The presence of KRAS mutations can lead to resistance to commonly used targeted therapies (such as cetuximab). G12D also accounts for a significant proportion of KRAS mutations in lung cancer.

[0043] This invention provides a KRAS G12DProtein inhibitors, comprising an effective amount of the above-mentioned dipyrazolopyrimidine compounds or their stereoisomers, tautomers, resonances, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates, or crystal forms.

[0044] The present invention provides a pharmaceutical composition comprising the above-mentioned dipyrazolopyrimidine compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient.

[0045] Technical features and beneficial effects of the present invention:

[0046] This invention introduces different aliphatic heterocycles at the C5 position of the pyrimidine ring to investigate the effect of ring size and substituents on compound activity. Using N-methylpiperazine as a polar head and a 3-hydroxynaphthyl ring as a hydrophobic binding group, different cyclic amines are introduced at the C5 position of the pyrimidine ring using O atoms as linkers. It is hypothesized that the conformational constraint of the bridged ring allows the compounds to better bind to protein pockets, leading to the design and synthesis of dipyrazolopyrimidine compounds 1-20. Compound 2 exhibits the highest inhibition rate, reaching 69.67%, demonstrating a significant inhibitory effect. Furthermore, compounds 4, 5, 13, 14, 15, and 17 also show high activity (inhibition rates all exceeding 30%). This indicates that the dipyrazolopyrimidine compounds prepared in this invention represent a class of highly active KRAS inhibitors. G12D Inhibitors can be used to develop drugs for treating pancreatic cancer, colorectal cancer, and lung cancer. Attached Figure Description

[0047] Figure 1 Molecular mechanism analysis of compound 5 and its effect on cycle distribution in human gastric adenocarcinoma cells AGS;

[0048] In the figure, A shows the levels of AKT, p-AKT, ERK, and p-ERK in human gastric adenocarcinoma cells AGS treated with different concentrations of compound 5 for 24 hours by Western blot analysis; B shows the quantitative analysis of the Western blot results; and C shows the effect of compound 5 at different concentrations on inducing apoptosis in human gastric adenocarcinoma cells AGS.

[0049] Figure 2 Compound 5 was used to induce apoptosis in human gastric adenocarcinoma cells AGS and inhibit their long-term proliferation.

[0050] In the figure, A shows the cell cycle distribution obtained by PI staining after two-dimensional culture of human gastric adenocarcinoma cells AGS with compound 5 for 48 hours; B shows the hematoxylin-eosin staining results of human gastric adenocarcinoma cells AGS after treatment with compound 5 at a specified concentration for 14 days; and C shows the statistical analysis of the number of clones. Detailed Implementation

[0051] To make the objectives, technical solutions, and beneficial effects of the embodiments in this disclosure clearer, the technical solutions in the embodiments of this disclosure will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this disclosure, not all embodiments. Based on the embodiments in this disclosure, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this disclosure.

[0052] All reagents and equipment used in this embodiment are existing reagents and equipment that can be purchased commercially.

[0053] In this invention, compounds 1-4 were prepared according to the method described in Example 1, and compounds 5-20 were prepared according to the method described in Example 5.

[0054] Human pancreatic cancer cells Aspc-1 (G12D), human pancreatic cancer cells PANC-1 (G12D), human gastric adenocarcinoma cells AGS (G12D), lung adenocarcinoma cells A549 (G12S), human non-small cell lung cancer cells H23 (G12C), human liver cancer cells Hep-G2 (NRASQ61L), human breast cancer cells MDA-MB-231 (G13D), human normal colonic epithelial cells NCM460, rat adrenal pheochromocytoma cells PC-12, and African green monkey kidney cells VERO are all available at Jinan Dengke Biotechnology Co., Ltd.

[0055] Example 1

[0056] A method for preparing compound 1, the specific steps of which are as follows:

[0057] (1) Synthesis of 3-bromo-5,7-dichloropyrazole[1,5-a]pyrimidine

[0058]

[0059] 5,7-Dichloropyrazolo[1,5-a]pyrimidine (A, 200 mg, 1.06 mmol, 1 eq) and N-bromosuccinimide (379 mg, 2.13 mmol, 2 eq) were dissolved in chloroform (2 ml) and reacted at room temperature (25 °C) for 12 h. The reaction solution was washed with saturated brine, dried over anhydrous sodium sulfate, and filtered to remove impurities, yielding a crude product. The crude product was then purified by silica gel column chromatography using a mixture of petroleum ether and ethyl acetate (20:1 v / v) and eluted to obtain a light green solid (75% yield), which was 3-bromo-5,7-dichloropyrazolo[1,5-a]pyrimidine.

[0060] (2) Synthesis of (1R,5S)-3-(3-bromo-5-chloropyrazolo[1,5-a]pyrimidin-7-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester

[0061]

[0062] 3-Bromo-5,7-dichloropyrazole[1,5-a]pyrimidine (200 mg, 0.75 mmol, 1 eq) was dissolved in dichloromethane (4 mL), and N,N-diisopropylethylamine (DIPEA, 193 mg, 1.50 mmol, 2 eq) and (1R,5S)-3,8-diazabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester (174 mg, 0.82 mmol, 1.1 eq) were added. The reaction mixture was reacted at -40℃ under nitrogen protection for 0.5 h. The reaction solution was washed with saturated brine, dried with anhydrous sodium sulfate, and filtered to remove impurities, yielding a crude product. The crude product was then purified by silica gel column chromatography using a mixture of petroleum ether and ethyl acetate (volume ratio 10:1) and eluted to obtain a pale yellow oil (yield 80%), which is (1R,5S)-3-(3-bromo-5-chloropyrazolo[1,5-a]pyrimidin-7-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester.

[0063] (3) Synthesis of 8-tert-butoxycarbonyl-3-(3-bromo-5-((1-methylpiperidin-4-yl)oxy)pyrazolo[1,5-a]pyrimidin-7-yl)-3,8-diazabicyclo[3.2.1]octane

[0064]

[0065] (1R,5S)-3-(3-bromo-5-chloropyrazolo[1,5-a]pyrimidin-7-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester (150 mg) and N-methyl-4-hydroxypiperidine (59 mg) were dissolved in a solvent (2 mL) consisting of tetrahydrofuran:NN dimethylformamide = 3:1. Sodium hydride (3.0 mmol) was added at 0 °C, and the mixture was stirred at room temperature (25 °C) for 2 h. The reaction solution was washed with saturated brine, dried over anhydrous sodium sulfate, and filtered to remove impurities, yielding a crude product. The crude product was then purified by silica gel column chromatography using a mixture of dichloromethane and methanol (50:1 v / v), and eluted to obtain a white solid (133 mg). mg, yield 70%), which is 8-tert-butoxycarbonyl-3-(3-bromo-5-((1-methylpiperidin-4-yl)oxy)pyrazolo[1,5-a]pyrimidin-7-yl)-3,8-diazabicyclo[3.2.1]octane;

[0066] In the mixed solvent of tetrahydrofuran and N,N dimethylformamide, the volume ratio of tetrahydrofuran to N,N dimethylformamide is 3:1.

[0067] (4) Synthesis of compound 1

[0068]

[0069] 8-tert-butoxycarbonyl-3-(3-bromo-5-((1-methylpiperidin-4-yl)oxy)pyrazolo[1,5-a]pyrimidin-7-yl)-3,8-diazabicyclo[3.2.1]octane (123 mg, 0.25 mmol, 1 eq) and pinacol ester of 3-hydroxy-1-naphthoborate (63.75 mg, 0.25 mmol, 1 eq) were dissolved in a solvent (2 ml) of 1,4-dioxane:water = 3:1. Pd(dppf)Cl2 (22.2 mg, 0.025 mmol, 0.1 eq) and cesium carbonate (188.5 mg, 0.5 mmol, 0.5 eq) were added. Under the catalysis of mg, 2 eq), the reaction was carried out at 85 °C for 3 h. The reaction solution was dried with anhydrous sodium sulfate, filtered and evaporated to dryness. Then, the product was hydrolyzed with hydrochloric acid-1,4-dioxane and acetonitrile and evaporated to dryness. The hydrolysate was washed with saturated brine, dried with anhydrous sodium sulfate and filtered to remove impurities, and the crude product was obtained. The crude product was then purified by silica gel column chromatography with a mixture of dichloromethane and methanol (volume ratio 10:1) and eluted to obtain a grayish-white solid (yield 77%), which was designated as compound 1.

[0070] The characterization data of compound 1 are as follows:

[0071] 1H NMR (800 MHz, DMSO-d6) δ 8.24 (s, 1H), 7.94 (s, 1H), 7.90 - 7.89(d, J = 8.0 Hz,1H), 7.71 - 7.70 (d, J = 8.0 Hz, 1H), 7.38 (t, J = 16.0 Hz,1H), 7.21 - 7.18 (m, 2H), 7.10 (s, 1H), 6.22 - 6.21 (d, J = 8.0 Hz, 1H), 4.22- 4.21 (d, J = 8.0 Hz, 2H), 4.17 - 4.15 (d, J = 16.0 Hz, 2H), 3.56 - 3.53 (d,J = 24.0 Hz, 5H), 3.09 - 3.06 (m, 6H), 3.00 - 2.98 (d, J = 16.0 Hz, 2H), 2.22- 2.20 (d, J = 16.0 Hz, 4H), 2.09 - 2.08 (d, J = 8.0 Hz, 2H) ppm;

[0072] 13C NMR (200 MHz, DMSO-d6) δ 162.2, 162.1, 155.3, 152.5, 152.2,149.1, 146.0, 143.7, 135.7, 126.9, 126.3, 122.7, 120.2, 108.5, 106.5, 93.9,83.9, 83.3, 54.1, 52.0, 49.0, 46.1 (3C), 30.7, 26.6, 18.1, 9.6 ppm;

[0073] HRMS (ESI): m / z [M+H]+calcd for C 28 H 33 N6O2 + 485.2660; found 485.2663.

[0074] Its chemical structural formula is:

[0075] .

[0076] The chemical name is: 4-(7-((1R,5S)-3,8-diazabicyclo[3.2.1]octane-3-yl)-5-((1-methylpiperidin-4-yl)oxy)pyrazolo[1,5-a]pyrimidin-3-yl)naphth-2-ol.

[0077] Example 2

[0078] Compound 2 was synthesized according to the flowchart and method described in Example 1, except that N-methyl-4-hydroxypiperidine in step (3) was replaced with 3-hydroxy-1-methyltetrahydropyrrole. The resulting product was a gray solid (yield 77%), denoted as Compound 2.

[0079] The characterization data of compound 2 are as follows:

[0080] 1H NMR (800 MHz, DMSO-d6) δ 9.76 (s, 1H), 8.26 (s, 1H), 7.91 - 7.90 (d, J = 8.0 Hz, 1H), 7.73 - 7.72 (d, J = 8.0 Hz, 1H), 7.39 (t, J = 16.0 Hz,1H), 7.25 (s, 1H), 7.21 (t, J = 16.0 Hz, 1H), 7.11 (s, 1H), 5.73 (s, 1H),4.22 - 4.20 (d, J = 16.0 Hz, 2H), 3.58 (s, 2H), 3.40 (s, 4H), 3.07 - 3.05 (d,J = 16.0 Hz, 2H), 2.63 - 2.57 (m, 3H), 2.21 (s, 3H), 1.98 - 1.97 (d, J = 8.0Hz, 2H), 1.81 - 1.77 (m, 1H), 1.76 - 1.75 (m, 2H) ppm;

[0081] 13C NMR (200 MHz, DMSO-d6) δ 162.6, 155.2, 153.0, 146.2, 143.9,135.7, 132.0, 126.9, 126.6, 126.5, 126.3, 122.8, 120.2, 108.4, 106.3, 83.0,76.2, 62.4, 54.9, 54.5, 54.0 (2C), 42.1 (2C), 32.7, 28.5 ppm;

[0082] HRMS (ESI): m / z [M+H]+ calcd for C 37 H 31 N6O2 + 471.2503; found 471.2509.

[0083] Its chemical structural formula is:

[0084] .

[0085] The chemical name is: 4-(7-((1R,5S)-3,8-diazabicyclo[3.2.1]octane-3-yl)-5-((1-methylpyrrolidine-3-yl)oxy)pyrazolo[1,5-a]pyrimidin-3-yl)naphth-2-ol.

[0086] Example 3

[0087] Compound 3 was synthesized according to the flowchart and method described in Example 1, except that (1R,5S)-3,8-diazabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester in step (2) was replaced with N-methylpiperazine, and N-methyl-4-hydroxypiperidine in step (3) was replaced with 1-methyl-2-pyrrolidinemethanol. The resulting product was a brown oily liquid (yield 64%), and was designated as compound 3.

[0088] The characterization data of compound 3 are as follows:

[0089] 1H NMR (400 MHz, DMSO-d6) δ 9.80 (s, 1H), 8.26 (s, 1H), 7.84 - 7.82 (d, J = 16. Hz, 1H), 7.73 - 7.71 (d, J = 16.0 Hz, 1H), 7.41 - 7.37 (m, 1H),7.23 - 7.18 (m, 1H), 7.12 - 7.12 (d, J = 4.9 Hz, 1H), 5.94 (s, 1H), 4.01 (s,2H), 3.72 (s, 4H), 2.57 - 2.54 (t, J = 24.0 Hz, 4H), 2.27 (s, 3H), 1.92 -1.84 (m, 4H), 1.77 - 1.75 (m, 2H), 1.65 (s, 2H), 1.23 (s, 4H) ppm;

[0090] 13C NMR (200 MHz, DMSO-d6) δ 163.2, 155.1, 152.4, 146.1, 144.0,135.7, 131.9, 126.9, 126.5, 126.4, 126.3, 122.8, 120.3, 108.4, 106.4, 83.2,68.8, 63.5, 57.3, 54.4 (2C), 48.2 (2C), 46.2 (2C), 41.5 (2C), 28.8, 22.9 (2C)ppm;

[0091] HRMS (ESI): m / z [M+H] + calcd for C 29 H 35 N6O2 + 499.2816; found 499.2826.

[0092] Its chemical structural formula is:

[0093] .

[0094] The chemical name is: 4-(7-(2-methylpiperazin-1-yl)-5-((tetrahydro-1H-pyrrolazin-7a(5H)-yl)methoxy)pyrazolo[1,5-a]pyrimidin-3-yl)naphth-2-ol-2-ol.

[0095] Example 4

[0096] Compound 4 was synthesized according to the flowchart and method described in Example 1, except that (1R,5S)-3,8-diazabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester in step (3) was replaced with N-ethyl-4-hydroxypiperidine. The resulting product was a brown oily liquid (yield 70%), and was designated as compound 4.

[0097] The characterization data of compound 4 are as follows:

[0098] 1H NMR (800 MHz, MeOD) δ 10.55 (s, 1H), 9.07 (s, 1H), 8.73 - 8.72 (d,J = 8.0Hz, 1H), 8.53 - 8.52 (d, J = 8.0 Hz, 1H), 8.20 (t, J = 16.0 Hz, 1H), 8.04 - 8.04 (d, J = 2.4 Hz, 1H), 7.99 (t, J = 16.0 Hz, 1H), 7.91 - 7.91 (d, J= 2.6 Hz, 1H), 6.53 (s, 1H), 5.62 (s, 1H), 5.04 - 5.02 (d, J = 16.0 Hz, 1H),4.37 (s, 2H), 4.17 (s, 4H), 3.99 (s, 1H), 3.87 - 3.86 (d, J = 8.0 Hz, 1H),3.48 (s, 2H), 3.09 - 3.07 (m, 2H), 2.79 - 2.78 (d, J = 8.0 Hz, 1H), 2.56 -2.55 (m, 2H), 2.42 - 2.41 (m, 2H), 1.77 (t, J = 16.0 Hz, 3H) ppm;

[0099] 13C NMR (200 MHz, MeOD) δ 163.2, 156.0, 153.9, 147.0, 144.7, 136.5,132.9, 127.7, 127.5, 127.2, 127.1, 123.5, 121.0, 109.2, 107.0, 83.9, 55.5(2C), 54.8 (2C), 52.7 (2C), 49.8, 31.8, 29.4 (2C), 13.5 (3C) ppm;

[0100] HRMS (ESI): m / z [M+H] + calcd for C29H35N6O2 + 499.2816; found 499.2819.

[0101] Its chemical structural formula is:

[0102] .

[0103] The chemical name is: 4-(7-((1R,5S)-3,8-diazabicyclo[3.2.1]octane-3-yl)-5-((1-methylpyrrolidine-3-yl)oxy)pyrazolo[1,5-a]pyrimidin-3-yl)naphth-2-ol.

[0104] Example 5

[0105] A method for preparing compound 5, the specific steps of which are as follows:

[0106] 1) Synthesis of 3-bromo-5,7-dichloropyrazole[1,5-a]pyrimidine

[0107]

[0108] 5,7-Dichloropyrazolo[1,5-a]pyrimidine (200 mg, 1.06 mmol, 1 eq) was dissolved in chloroform (2 mL), and N-bromosuccinimide (NBS, 379 mg, 2.13 mmol, 2 eq) was added. The mixture was heated to 40 °C in an oil bath and reacted for 12 hours. The reaction was detected by TLC (electrolyte: petroleum ether / ethyl acetate = 8:1). After the reaction was complete, water was added to quench the reaction. The mixture was extracted three times with ethyl acetate, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated with silica gel, and purified by silica gel column chromatography (mobile phase: petroleum ether / ethyl acetate = 20:1) to give a yellow solid (227 mg, yield 80%), which was 3-bromo-5,7-dichloropyrazolo[1,5-a]pyrimidine.

[0109] 2) Synthesis of 5-(3-bromo-5-chloropyrazolo[1,5-a]pyrimidin-7-yl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid tert-butyl ester

[0110]

[0111] 3-Bromo-5,7-dichloropyrazole[1,5-a]pyrimidine (200 mg, 0.75 mmol, 1 eq) was dissolved in dichloromethane (4 mL), cooled to -40 °C using a cold trap, and then 2-tert-butoxycarbonyl-2,5-diazabicyclo[2.2.1]heptane (163 mg, 0.82 mmol, 1.1 eq) was added. Diisopropylethylamine (DIPEA, 193 mg, 1.50 mmol, 2 eq) was then slowly added dropwise. After the addition was complete, the mixture was allowed to warm to room temperature naturally, and the mixture was detected by TLC (developing solvent: petroleum ether / ethyl acetate = 4:1). After the reaction was complete, the product was concentrated to dryness under reduced pressure and dried in a vacuum drying oven to obtain crude tert-butyl 5-(3-bromo-5-chloropyrazolo[1,5-a]pyrimidin-7-yl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid, which (356 mg) did not require purification and was used directly in the next step.

[0112] 3) Synthesis of 5-(3-bromo-5-((1-methylpyrrolidone-2-yl)methoxy)pyrazolo[1,5-a]pyrimidin-7-yl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid tert-butyl ester

[0113]

[0114] Add 2 mL of tetrahydrofuran to a pressure-resistant tube, then add 100 mg of crude tert-butyl 5-(3-bromo-5-chloropyrazolo[1,5-a]pyrimidin-7-yl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid and 48 mg of (1-methylpyrrolidone-2-yl)methanol. After cooling to 0°C in an ice-water bath, slowly add sodium hydride, allow to warm naturally to room temperature, stir for 0.5 hours, seal the tube, heat to 100°C in an oil bath, and react for 2 hours. Monitor by TLC (developing solvent: dichloromethane / methanol = 10:1). After cooling to 0°C in an ice-water bath, open the tube, quench with water dropwise, extract with dichloromethane, and dry with anhydrous sodium sulfate. The compound was purified by silica gel column chromatography (mobile phase: dichloromethane / methanol = 50 / 1) to give a white solid (yield 79.76%), which is 5-(3-bromo-5-((1-methylpyrrolidone-2-yl)methoxy)pyrazolo[1,5-a]pyrimidin-7-yl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid tert-butyl ester;

[0115] 4) Synthesis of 5-(3-(7-fluoro-3-(methoxymethoxy)-8-((triisopropylsilyl)ethynyl)naphth-1-yl)-5-((1-methylpyrrolidone-2-yl)methoxypyrazolo[1,5-a]pyrimidin-7-yl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid tert-butyl ester

[0116]

[0117] Add 1,4-dioxane:water = 5:1 to a microwave tube, then add 5-(3-bromo-5-((1-methylpyrrolidone-2-yl)methoxy)pyrazolo[1,5-a]pyrimidin-7-yl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid tert-butyl ester, 7-fluoro-3-(methoxymethoxy)-8-[(triisopropylsilyl)ethynyl]naphthalene-1-boronic acid pinacol ester, and cesium carbonate (120 mg, 124 mg, 152 mg 2 eq). Finally, add Ad2nBuP-Pd-G3 (17 mg 0.1 eq). Purge with nitrogen for 15 min to replace air, and heat to 82°C (50°F) using a microwave reactor. W) The reaction was carried out for 1 hour, and the reaction was monitored by TLC (developing solvent: DCM / MeOH=10:1). After the reaction was complete, the product was dried with anhydrous sodium sulfate, filtered, washed three times with methanol, concentrated to dryness under reduced pressure with silica gel, and purified by silica gel column chromatography (mobile phase: dichloromethane / methanol=50:1 increased to 20:1). The resulting orange-yellow solid was 5-(3-(7-fluoro-3-(methoxymethoxy)-8-((triisopropylsilyl)ethynyl)naphth-1-yl)-5-((1-methylpyrrolidone-2-yl)methoxypyrazolo[1,5-a]pyrimidin-7-yl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid tert-butyl ester.

[0118] 5) Synthesis of 5-(3-(8-ethynyl-7-fluoro-3-(methoxymethoxy)naphth-1-yl)-5-((1-methylpyrrolidine-2-yl)methoxypyrazolo[1,5-a]pyrimidin-7-yl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid tert-butyl ester

[0119]

[0120] DMF was added to the reaction flask, followed by 5-(3-(7-fluoro-3-(methoxymethoxy)-8-((triisopropylsilyl)ethynyl)naphth-1-yl)-5-((1-methylpyrrolidone-2-yl)methoxypyrazolo[1,5-a]pyrimidin-7-yl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid tert-butyl ester (101 mg) and CsF (192 mg 10 eq). The mixture was stirred overnight at room temperature and monitored by TLC. After the reaction was complete, silica gel was added, and the mixture was concentrated directly to dryness. The solution was then purified by silica gel column chromatography (mobile phase: dichloromethane / methanol = 50 / 1 increased to 20 / 1) to obtain a yellow solid (74 mg 10 eq). mg, yield 88%), namely 5-(3-(8-ethynyl-7-fluoro-3-(methoxymethoxy)naphth-1-yl)-5-((1-methylpyrrolidine-2-yl)methoxypyrazolo[1,5-a]pyrimidin-7-yl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid tert-butyl ester;

[0121] 6) Synthesize compound 5

[0122]

[0123] Methanol (1 mL) was added to the reaction flask, followed by 70 mg of 5-(3-(8-ethynyl-7-fluoro-3-(methoxymethoxy)naphth-1-yl)-5-((1-methylpyrrolidine-2-yl)methoxypyrazolo[1,5-a]pyrimidin-7-yl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid tert-butyl ester. The mixture was cooled to 0°C using an ice-water bath, and 700 μl of 4 M 1,4-dioxane hydrochloride (HCl in dioxane) was slowly added dropwise. After the addition was complete, the mixture was allowed to warm to room temperature naturally. The reaction was allowed to proceed for 2 h. After the reaction was confirmed to be complete by TLC, the mixture was concentrated to dryness. Methanol (2 mL) was added to dissolve the residue, and the mixture was concentrated again. This process was repeated three times. The mixture was then purified using a Shimadzu LC-20AR reversed-phase high-performance chromatography (HPLC) under the following conditions: column elution with methanol-water (0.1% HCOOH) gradient from 35% to 100% for 50 minutes, followed by 100% ACN for 10 minutes at a flow rate of 500 mcg / mL. The concentration was measured at mL / min, and UV detection was performed (λ=254 nm). A white compound (35 mg) was obtained and stored under nitrogen at low temperature. This compound was designated as compound 5.

[0124] The characterization data of compound 5 are as follows:

[0125] 1H NMR (800 MHz, DMSO-d6) δ 10.0 (s, 1H), 7.9 (s, 1H), 7.8 (dd, J =9.2, 5.9 Hz, 1H), 7.4 (t, J = 8.9 Hz, 1H), 7.2 (d, J = 2.8 Hz, 1H), 7.1 (d, J= 2.7 Hz, 1H), 5.4 (s, 1H), 4.1 – 3.8 (m, 4H), 3.7 (s, 1H), 3.4 (s, 5H), 3.0 (d, J = 10.3 Hz, 1H), 2.9 (d, J = 10.1 Hz, 1H), 2.8 – 2.8 (m, 1H), 2.4 (p, J= 6.2 Hz, 1H), 2.1 – 2.0 (m, 4H), 1.9 (d, J = 9.2 Hz, 1H), 1.7 (d, J = 9.5Hz, 1H), 1.7 – 1.6 (m, 1H), 1.6 (dt, J = 8.6, 4.7 Hz, 2H), 1.5 – 1.4 (m, 1H);

[0126] 13C NMR (200 MHz, DMSO) δ 162.6 (d, J = 248.9 Hz), 154.6, 148.1,143.6, 132.2 (d, J = 5.9 Hz,2C), 132.2, 130.4 (d, J = 8.9 Hz), 127.5, 124.2,115.8 (d, J = 25.9 Hz), 110.2, 107.6, 105.5 (d, J = 15.6 Hz), 91.2, 75.9,68.3, 63.5, 57.3, 55.8, 28.8, 22.9;

[0127] HRMS (ESI): m / z [M+H] + calcd for C 29 H 29 FN6O2 + 513.2414; found 513.2419.

[0128] Its chemical structure is:

[0129] .

[0130] The chemical name is: 4-(7-(2,5-diazabicyclo[2.2.1]heptane-2-yl)-5-((1-methylpyrrolidone-2-yl)methoxy)pyrazolo[1,5-a]pyrimidin-3-yl)-5-ethynyl-6-fluoronaphthyl-2-ol.

[0131] Example 6

[0132] Compound 6 was synthesized according to the flowchart and method described in Example 6, except that 2-tert-butoxycarbonyl-2,5-diazabicyclo[2.2.1]heptane in step 2) was replaced with 1-tert-butoxycarbonyl-2-methylpiperazine. The resulting product was a brown oily liquid (yield 71%), and was designated as compound 6.

[0133] The characterization data of compound 6 are as follows:

[0134] 1H NMR (800 MHz, DMSO-d6) δ 9.9 (s, 1H), 8.0 (s, 1H), 7.9 (dd, J =9.2, 5.8 Hz, 1H), 7.4 (t, J = 9.0 Hz, 1H), 7.2 (d, J = 2.8 Hz, 1H), 7.1 (d, J= 2.8 Hz, 1H), 5.8 (s, 1H), 4.3 (s, 3H), 4.1 – 3.9 (m, 2H), 3.8 (s, 1H), 3.1– 3.0 (m, 1H), 3.0 – 2.9 (m, 3H), 2.9 – 2.8 (m, 1H), 2.6 (t, J = 11.2 Hz,1H), 2.4 (p, J = 5.7 Hz, 1H), 2.1 (q, J = 8.8 Hz, 4H), 1.7 (s, 1H), 1.6 (q, J= 8.5 Hz, 2H), 1.5 – 1.4 (m, 1H), 1.1 (d, J = 6.4 Hz, 3H);

[0135] 13C NMR (200 MHz, DMSO) δ 162.8, 162.6 (d, J = 245.2 Hz), 154.6,152.5, 147.6, 143.5, 133.1, 131.9 (d, J = 5.9 Hz, 2C), 130.4 (d, J = 9.6 Hz), 127.5, 124.3, 115.9 (d, J = 25.9 Hz), 110.4, 108.5, 105.4 (d, J = 15.6 Hz), 91.1 (d, J = 7.4 Hz), 82.6, 75.9, 68.5, 63.4, 57.3, 55.0, 50.2, 48.5, 45.1, 28.7, 22.9, 19.4;

[0136] HRMS (ESI): m / z [M+H] + calcd for C 29 H 31 FN6O2 + 515.2571; found 515.2575.

[0137] Its chemical structural formula is:

[0138]

[0139] The chemical name is: 5-ethynyl-6-fluoro-4-[7-(3-methylpiperazin-1-yl)-5-[(1-methylpyrrolidone-2-yl)methoxy]pyrazolo[1,5-a]pyrimidin-3-yl]-2-naphthol.

[0140] Example 7

[0141] Compound 7 was synthesized according to the flowchart and method described in Example 5, except that 2-tert-butoxycarbonyl-2,5-diazabicyclo[2.2.1]heptane in step 2) was replaced with 1-(tert-butoxycarbonyl)-1,4-diazabicycloheptane. The resulting product was a white powder (yield 84%), and was designated as compound 7.

[0142] The characterization data of compound 7 are as follows:

[0143] 1H NMR (800 MHz, DMSO-d6) δ 10.1 (s, 1H), 8.0 (s, 1H), 7.9 (dd, J =9.2, 5.8 Hz, 1H), 7.4 (t, J = 8.9 Hz, 1H), 7.2 (d, J = 2.7 Hz, 1H), 7.1 (d, J= 2.8 Hz, 1H), 5.6 (s, 1H), 4.0 – 3.8 (m, 2H), 3.7 (s, 1H), 3.0 (s, 3H), 2.8(tt, J = 5.4, 3.1 Hz, 1H), 2.4 – 2.4 (m, 1H), 2.1 – 1.9 (m, 6H), 1.8 – 1.7(m, 3H), 1.5 (p, J = 8.2 Hz, 2H), 1.5 – 1.4 (m, 1H);

[0144] 13C NMR (200 MHz, DMSO) δ 162.8, 162.6 (d, J = 245.2 Hz), 154.7,153.2, 147.7, 143.4, 133.2, 131.9 (d, J = 5.9 Hz, 2C), 130.4 (d, J = 8.9 Hz), 130.1, 127.5, 124.3, 115.8 (d, J = 26.7 Hz), 110.4, 108.3, 105.4 (d, J = 15.6Hz), 91.0 (d, J = 7.4 Hz), 82.0, 75.8, 68.5, 63.3(2C), 57.3, 54.9, 54.0(2C),28.9, 28.7, 22.9(2C);

[0145] HRMS (ESI): m / z [M+H] + calcd for C 30 H 31 FN6O2 + 527.2571; found 527.2574.

[0146] Its chemical structural formula is:

[0147]

[0148] The chemical name is: 4-[7-(3,8-diazabicyclo[3.2.1]octane-3-yl)-5-[(1-methylpyrrolidone-2-yl)methoxy]pyrazolo[1,5-a]pyrimidin-3-yl]-5-ethynyl-6-fluoro-2-naphthol.

[0149] Example 8

[0150] Compound 8 was synthesized according to the flowchart and method described in Example 5, except that 2-tert-butoxycarbonyl-2,5-diazabicyclo[2.2.1]heptane in step 2) was replaced with 3,4-dimethylpiperazine. The resulting product was a white powder (yield 81%), and was designated as compound 8.

[0151] The characterization data of compound 8 are as follows:

[0152] 1H NMR (800 MHz, DMSO-d6) δ 10.0 (s, 1H), 8.0 (s, 1H), 7.9 (dd, J =9.2, 5.9 Hz, 1H), 7.4 (t, J = 8.9 Hz, 1H), 7.2 (d, J = 2.6 Hz, 1H), 7.1 (d, J= 2.7 Hz, 1H), 5.8 (s, 1H), 4.4 – 3.9 (m, 4H), 3.8 (s, 1H), 3.1 (t, J = 11.4Hz, 1H), 2.9 (dt, J = 11.7, 3.0 Hz, 1H), 2.9 – 2.8 (m, 1H), 2.8 (t, J = 11.1Hz, 1H), 2.4 – 2.4 (m, 1H), 2.4 (td, J = 11.6, 3.3 Hz, 1H), 2.3 (tt, J = 6.5,3.3 Hz, 1H), 2.3 (s, 3H), 2.1 – 2.0 (m, 4H), 1.7 – 1.6 (m, 1H), 1.6 (q, J =8.1 Hz, 2H), 1.5 – 1.4 (m, 1H), 1.1 (d, J = 6.2 Hz, 3H);

[0153] 13C NMR (200 MHz, DMSO) δ 162.0, 162.6 (d, J = 245.2 Hz), 152.2,147.5, 143.5, 133.2, 131.8 (d, J = 5.9 Hz, 2C), 130.4 (d, J = 9.6 Hz), 130.1,127.4, 124.3, 115.9 (d, J = 26.7 Hz), 110.4, 108.5, 105.4 (d, J = 15.6 Hz),91.1 (d, J = 6.7 Hz), 82.6, 75.9, 68.6, 63.3, 57.3, 57.1, 54.7, 54.4, 48.4,42.6, 28.7, 22.9, 17.0;

[0154] HRMS (ESI):m / z [M+H] + calcd for C 30 H 33 FN6O2 + 529.2727; found 529.2730。

[0155] Its chemical structural formula is:

[0156]

[0157] The chemical name is: 4-[7-(3,4-dimethylpiperazin-1-yl)-5-[(1-methylpyrrolidone-2-yl)methoxy]pyrazolo[1,5-a]pyrimidin-3-yl]-5-ethynyl-6-fluoro-2-naphthol.

[0158] Example 9

[0159] Compound 9 was synthesized according to the flowchart and method described in Example 5, except that 2-tert-butoxycarbonyl-2,5-diazabicyclo[2.2.1]heptane in step 2) was replaced with 6-(tert-butyl)-3,6-diazabicyclo[3.1.1]heptane. The resulting product was a white powder (yield 86%), denoted as compound 9.

[0160] The characterization data of compound 9 are as follows:

[0161] 1H NMR (800 MHz, DMSO-d6) δ 9.9 (s, 1H), 7.9 (s, 1H), 7.9 (dd, J =9.1, 5.9 Hz, 1H), 7.4 (t, J = 8.9 Hz, 1H), 7.2 (d, J = 2.7 Hz, 1H), 7.1 (d, J = 2.8 Hz, 1H), 5.5 (s, 1H), 4.4 (s, 2H), 4.0 (d, J = 10.0 Hz, 2H), 3.9 (s,1H), 3.7 (d, J = 6.3 Hz, 2H), 3.4 (s, 6H), 2.9 – 2.8 (m, 1H), 2.6 – 2.5 (m, 2H), 2.4 (p, J = 6.0 Hz, 1H), 2.1 – 2.0 (m, 4H), 1.7 (d, J = 8.6 Hz, 2H), 1.6(q, J = 8.4 Hz, 2H), 1.5 – 1.4 (m, 1H);

[0162] 13C NMR (200 MHz, DMSO) δ 163.3, 162.6 (d, J = 245.2 Hz), 154.6,151.8, 148.6, 143.3, 133.2, 132.2 (d, J = 5.9 Hz, 2C), 130.4 (d, J = 8.9 Hz),127.6, 124.2, 115.8 (d, J = 25.9 Hz), 110.2, 107.5, 105.5 (d, J = 15.6 Hz),91.2 (d, J = 7.4 Hz), 77.8, 75.9, 68.3, 63.4(2C), 57.3, 55.1(2C), 53.9, 30.2, 28.7, 22.9;

[0163] HRMS (ESI): m / z [M+H] + calcd for C 29 H 29 FN6O2 + 513.2414; found 513.2419.

[0164] Its chemical structural formula is:

[0165]

[0166] The chemical name is: 4-[7-(3,6-diazabicyclo[3.1.1]heptane-3-yl)-5-[(1-methylpyrrolidone-2-yl)methoxy]pyrazolo[1,5-a]pyrimidin-3-yl]-5-ethynyl-6-fluoro-2-naphthol.

[0167] Example 10

[0168] Compound 10 was synthesized according to the flowchart and method described in Example 5, except that 2-tert-butoxycarbonyl-2,5-diazabicyclo[2.2.1]heptane in step 2) was replaced with 1-tert-butoxycarbonyl-2-piperazinone. The resulting product was a white powder (yield 81%), and was designated as compound 10.

[0169] The characterization data of compound 10 are as follows:

[0170] 1H NMR (800 MHz, DMSO-d6) δ 10.0 (s, 1H), 8.2 (t, J = 2.6 Hz, 1H), 8.0 (s, 1H), 7.9 (dd, J = 9.2, 6.0 Hz, 1H), 7.4 (t, J = 8.9 Hz, 1H), 7.2 (d,J = 2.7 Hz, 1H), 7.1 (d, J = 2.7 Hz, 1H), 5.8 (s, 1H), 4.3 – 4.2 (m, 2H), 4.0(s, 3H), 3.8 (s, 1H), 3.4 (td, J = 5.4, 2.5 Hz, 2H), 2.9 – 2.8 (m, 1H), 2.4 –2.4 (m, 1H), 2.1 – 1.9 (m, 4H), 1.8 – 1.6 (m, 1H), 1.6 (q, J = 8.1 Hz, 2H), 1.5 – 1.4 (m, 1H);

[0171] 13C NMR (200 MHz, DMSO) δ 166.4, 162.8, 162.6 (d, J = 245.2 Hz), 154.7, 150.9, 147.6, 143.7, 133.2, 131.7 (d, J = 5.9 Hz, 2C), 130.4 (d, J =9.6 Hz), 130.1, 127.5, 124.3, 115.9 (d, J = 25.9 Hz), 110.5, 108.7, 105.4 (d, J = 15.6 Hz), 91.2 (d, J = 6.7 Hz), 82.5, 75.8, 68.7, 63.3, 57.3, 51.7, 45.0, 28.7, 22.9;

[0172] HRMS (ESI): m / z [M+H] + calcd for C 28 H 27 FN6O3 + 515.2207; found 515.2205.

[0173] Its chemical structural formula is:

[0174]

[0175] The chemical name is: 4-[3-(8-ethynyl-7-fluoro-3-hydroxy-1-naphthyl)-5-[(1-methylpyrrolidone-2-yl)methoxy]pyrazolo[1,5-a]pyrimidin-7-yl]piperazin-2-one.

[0176] Example 11

[0177] Compound 11 was synthesized according to the flowchart and method described in Example 5, except that (1-methylpyrrolidone-2-yl)methanol in step 3) was replaced with 8-methyl-8-azabicyclo[3.2.1]octane-3-ol. The resulting product was a white powder (yield 79%), denoted as compound 11.

[0178] The characterization data of compound 11 are as follows:

[0179] 1H NMR (800 MHz, DMSO-d6) δ 10.0 (s, 1H), 7.9 – 7.9 (m, 1H), 7.8 (dd,J = 9.1, 5.9 Hz, 1H), 7.4 (t, J = 8.9 Hz, 1H), 7.2 (d, J = 2.6 Hz, 1H), 7.1(d, J = 2.7 Hz, 1H), 5.3 (s, 1H), 4.8 (t, J = 6.4 Hz, 1H), 3.9 (s, 1H), 3.8(s, 1H), 3.7 (s, 1H), 3.5 (s, 2H), 3.2 (s, 1H), 3.1 – 3.0 (m, 1H), 3.0 (d, J= 10.0 Hz, 3H), 2.1 (s, 3H), 2.0 – 1.9 (m, 5H), 1.8 – 1.6 (m, 4H);

[0180] 13C NMR (201 MHz, DMSO-d6) δ 163.1, 162.4, 161.9, 154.7, 148.7,148.1, 143.0, 133.1, 132.3, 132.3, 130.4, 130.3, 127.3, 123.8, 115.8, 115.7,110.0, 107.7, 105.7, 105.6, 90.8, 77.5, 76.2, 70.9, 55.8, 53.2, 49.1, 46.2,30.9;

[0181] HRMS (ESI): m / z [M+H] + calcd for C 31 H32 FN6O2 + 539.2571; found 539.2575.

[0182] Its chemical structural formula is:

[0183]

[0184] The chemical name is: 4-[7-(2,5-diazabicyclo[2.2.1]heptane-2-yl)-5-[(8-methyl-8-azabicyclo[3.2.1]octane-3-yl)oxy]pyrazolo[1,5-a]pyrimidin-3-yl]-5-ethynyl-6-fluoro-2-naphthol.

[0185] Example 12

[0186] Compound 12 was synthesized according to the flowchart and method described in Example 5, except that (1-methylpyrrolidine-2-yl)methanol in step 3) was replaced with 1-ethylpyrrolidine-3-ol. The resulting product was a white powder (yield 82%), denoted as compound 12.

[0187] The characterization data of compound 12 are as follows:

[0188] 1H NMR (800 MHz, DMSO-d6) δ 10.0 (s, 1H), 7.9 (s, 1H), 7.9 (dd, J =9.1, 5.9 Hz, 1H), 7.4 (t, J = 8.9 Hz, 1H), 7.2 (d, J = 2.6 Hz, 1H), 7.1 (dd,J = 4.6, 2.3 Hz, 1H), 5.4 (s, 1H), 4.9 (s, 1H), 3.8 (d, J = 21.4 Hz, 1H), 3.7(s, 1H), 3.2 (s, 1H), 3.0 (d, J = 11.2 Hz, 1H), 2.9 (d, J = 10.8 Hz, 1H), 2.6(d, J = 8.0 Hz, 1H), 2.3 – 2.3 (m, 2H), 2.2 (q, J = 7.2 Hz, 1H), 1.9 (s, 1H),1.9 (d, J = 8.7 Hz, 1H), 1.7 (d, J = 9.4 Hz, 1H), 1.7 – 1.7 (m, 1H), 1.0 (t,J = 7.2 Hz, 3H);

[0189] 13C NMR (201 MHz, DMSO-d6) δ 163.2, 162.7, 161.9, 154.7, 148.7,148.2, 143.2, 133.1, 132.3, 132.2, 130.4, 130.4, 127.4, 124.0, 115.8, 115.7,110.1, 107.8, 105.7, 105.6, 76.1, 75.0, 60.1, 55.8, 52.4, 49.7, 49.6, 49.1,14.0, 13.9;

[0190] HRMS (ESI): m / z [M+H] + calcd for C 29 H 30 FN6O2 + 513.2414; found 513.2414.

[0191] Its chemical structural formula is:

[0192]

[0193] The chemical name is: 4-(7-(2,5-diazabicyclo[2.2.1]heptane-2-yl)-5-((1-ethylpyrrolidine-3-yl)oxy)pyrazolo[1,5-a]pyrimidin-3-yl)-5-ethynyl-6-fluoro-2-naphthol.

[0194] Example 13

[0195] Compound 13 was synthesized according to the flowchart and method described in Example 5, except that (1-methylpyrrolidone-2-yl)methanol in step 3) was replaced with 1-methylpiperidin-4-ol. The resulting product was a white powder (yield 82%), and was designated as compound 13.

[0196] The characterization data of compound 13 are as follows:

[0197] 1H NMR (800 MHz, DMSO-d6) δ 10.0 (s, 1H), 7.9 (s, 1H), 7.9 (dd, J =9.2, 6.0 Hz, 1H), 7.4 (t, J = 9.0 Hz, 1H), 7.2 (d, J = 2.6 Hz, 1H), 7.1 (d, J= 2.7 Hz, 1H), 5.4 (s, 1H), 4.5 (p, J = 5.0 Hz, 1H), 3.8 (s, 1H), 3.7 (s,1H), 3.5 – 3.4 (m, 2H), 3.2 (s, 1H), 3.0 (d, J = 9.8 Hz, 1H), 2.9 (d, J = 9.4Hz, 1H), 2.5 – 2.5 (m, 1H), 2.1 (s, 3H), 1.9 – 1.7 (m, 6H), 1.6 – 1.5 (m, 2H);

[0198] 13C NMR (201 MHz, DMSO-d6) δ 163.1, 162.4, 161.9, 154.7, 148.7,148.1, 143.0, 133.1, 132.3, 132.3, 130.4, 130.3, 127.3, 123.8, 115.8, 115.7,110.0, 107.7, 105.7, 105.6, 90.8, 77.5, 76.2, 70.9, 55.8, 53.2, 49.1, 46.2,30.9;

[0199] HRMS (ESI): m / z [M+H] + calcd for C 29 H 29 FN6O2 + 513.2414; found 513.2419.

[0200] Its chemical structural formula is:

[0201]

[0202] The chemical name is: 4-(7-(2,5-diazabicyclo[2.2.1]heptane-2-yl)-5-((1-methylpiperidin-4-yl)oxy)pyrazolo[1,5-a]pyrimidin-3-yl)-5-ethynyl-6-fluoro-2-naphthol.

[0203] Example 14

[0204] Compound 14 was synthesized according to the flowchart and method described in Example 5, except that (1-methylpyrrolidone-2-yl)methanol in step 3) was replaced with 1-isopropylpiperidin-4-ol. The resulting product was a white powder (yield 84%), and was designated as compound 14.

[0205] The characterization data of compound 14 are as follows:

[0206] 1H NMR (800 MHz, DMSO-d6) δ 10.0 (s, 1H), 7.9 (s, 1H), 7.9 (dd, J =9.2, 5.8 Hz, 1H), 7.4 (t, J = 8.9 Hz, 1H), 7.2 (d, J = 2.7 Hz, 1H), 7.1 (d, J= 2.7 Hz, 1H), 5.4 (s, 1H), 4.5 (p, J = 4.9 Hz, 1H), 3.8 (s, 1H), 3.7 (s,1H), 3.2 (s, 1H), 3.0 (d, J = 11.3 Hz, 1H), 2.9 (d, J = 10.1 Hz, 1H), 2.6(tt, J = 10.2, 5.2 Hz, 3H), 2.0;

[0207] 13C NMR (201 MHz, DMSO-d6) δ 163.1, 162.5, 161.9, 154.7, 148.7,148.1, 143.0, 133.2, 130.3, 130.3, 127.3, 123.8, 115.7, 115.6, 110.0, 105.7,105.6, 90.9, 77.5, 76.2, 71.8, 55.8, 53.9, 49.1, 46.1, 31.5, 18.5, 18.5.

[0208] HRMS (ESI): m / z [M+H] + calcd for C31H34FN6O2 + 541.2727; found 541.2730.

[0209] Its chemical structural formula is:

[0210]

[0211] The chemical name is: 4-(7-(2,5-diazabicyclo[2.2.1]heptane-2-yl)-5-((1-isopropylpiperidin-4-yl)oxy)pyrazolo[1,5-a]pyrimidin-3-yl)-5-ethynyl-6-fluoro-2-naphthol.

[0212] Example 15

[0213] Compound 15 was synthesized according to the flowchart and method described in Example 5, except that (1-methylpyrrolidone-2-yl)methanol in step 3) was replaced with 1-cyclopropylpiperidin-4-ol. The resulting product was a white powder (yield 87%), which was designated as compound 15.

[0214] The characterization data of compound 15 are as follows:

[0215] 1H NMR (800 MHz, DMSO-d6) δ 10.0 (s, 1H), 7.9 (d, J = 7.4 Hz, 1H), 7.9 (dd, J = 9.2, 6.0 Hz, 1H), 7.4 (t, J = 8.9 Hz, 1H), 7.2 (d, J = 2.7 Hz,1H), 7.1 (d, J = 2.7 Hz, 1H), 5.4 (s, 1H), 4.6 (hept, J = 4.3 Hz, 1H), 3.8(s, 1H), 3.7 (s, 1H), 3.2 (s, 1H), 3.0 (d, J = 10.1 Hz, 1H), 2.9 (d, J = 10.1Hz, 1H), 2.7 (t, J = 7.6 Hz, 2H), 2.0 (s, 2H), 1.9 – 1.7 (m, 5H), 1.5 – 1.5(m, J = 3.7 Hz, 1H), 1.5 – 1.4 (m, 2H), 0.4 – 0.3 (m, 2H), 0.3 – 0.2 (m, 2H);

[0216] 13C NMR (201 MHz, DMSO-d6) δ 163.1, 162.4, 161.9, 154.7, 148.7,148.1, 143.1, 133.2, 132.3, 132.3, 130.4, 130.3, 127.3, 123.8, 115.8, 115.7,110.0, 107.7, 105.7, 105.6, 90.9, 77.5, 76.2, 71.4, 55.8, 51.1, 49.1, 38.3,30.9, 6.4;

[0217] HRMS (ESI): m / z [M+H] + calcd for C 31 H 32 FN6O2 + 539.2571; found 539.2575.

[0218] Its chemical structural formula is:

[0219]

[0220] The chemical name is: 4-(7-(2,5-diazabicyclo[2.2.1]heptane-2-yl)-5-(((2S,7aR)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl)methoxy)pyrazolo[1,5-a]pyrimidin-3-yl)-5-ethynyl-6-fluoro-2-naphthol.

[0221] Example 16

[0222] Compound 16 was synthesized according to the flowchart and method described in Example 5, except that (1-methylpyrrolidine-2-yl)methanol in step 3) was replaced with (2S,7aR)-7a-((hydroxy)methyl)-2-fluorohexahydro-1H-pyrrolazine. The resulting product was a white powder (yield 84%), and was designated as compound 16.

[0223] The characterization data of compound 16 are as follows:

[0224] 1H NMR (800 MHz, DMSO-d6) δ 10.0 (s, 1H), 7.9 – 7.8 (m, 2H), 7.4 (t,J = 8.9 Hz, 1H), 7.2 (d, J = 2.6 Hz, 1H), 7.1 (d, J = 2.7 Hz, 1H), 5.4 (s,1H), 5.2 – 5.1 (m, 1H), 3.9 (s, 2H), 3.7 (s, 4H), 3.2 (s, 1H), 3.1 (d, J =10.3 Hz, 1H), 3.0 – 2.8 (m, 4H), 2.7 (td, J = 9.3, 6.2 Hz, 1H), 2.0 – 1.8 (m,4H), 1.7 (t, J = 10.3 Hz, 2H), 1.6 (dq, J = 12.1, 6.6 Hz, 1H), 1.6 (d, J =13.2 Hz, 1H);

[0225] 13C NMR (201 MHz, DMSO-d6) δ 163.6, 163.2, 162.0, 154.7, 148.0,143.8, 133.2, 130.5, 127.5, 124.3, 115.9, 115.8, 110.3, 105.4, 98.7, 97.8,75.8, 72.4, 71.7, 60.2, 60.1, 60.1, 56.7, 49.1, 42.8, 42.7, 36.0, 25.4, 25.4;

[0226] HRMS (ESI): m / z [M+H] + calcd for C 31 H 31 F2N6O2 + 557.2477; found 557.2482.

[0227] Its chemical structural formula is:

[0228]

[0229] The chemical name is: 4-(7-(2,5-diazabicyclo[2.2.1]heptane-2-yl)-5-(((2S,7aR)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl)methoxy)pyrazolo[1,5-a]pyrimidin-3-yl)-5-ethynyl-6-fluoro-2-naphthol.

[0230] Example 17

[0231] Compound 17 was synthesized according to the flowchart and method described in Example 5, except that: in step 2), 2-tert-butoxycarbonyl-2,5-diazabicyclo[2.2.1]heptane was replaced with 8-tert-butoxycarbonyl-3,8-diazabicyclo[3.2.1]octane, and in step 3), (1-methylpyrrolidine-2-yl)methanol was replaced with 1-cyclopropylpiperidin-4-ol. The resulting product was a white powder (yield 85%), and was designated as compound 17.

[0232] The characterization data of compound 17 are as follows:

[0233] 1H NMR (800 MHz, DMSO-d6) δ 10.0 (s, 1H), 8.2 (s, 1H), 8.1 (s, 1H), 7.9 (dd, J = 9.2, 6.0 Hz, 1H), 7.4 (t, J = 8.9 Hz, 1H), 7.3 (d, J = 2.6 Hz,1H), 7.2 (d, J = 2.7 Hz, 1H), 5.9 (s, 1H), 4.7 (s, 1H), 4.2 – 4.2 (m, 2H), 3.8 (s, 1H), 3.5 – 3.4 (m, 3H), 3.2 (s, 2H), 2.9 (d, J = 6.0 Hz, 2H), 2.2 (s,2H), 2.1 – 2.0 (m, 2H), 1.9 – 1.9 (m, 2H), 1.6 (s, 2H), 0.6 – 0.4 (m, 4H);

[0234] 13C NMR (201 MHz, DMSO-d6) δ 163.5, 163.2, 161.9, 161.8, 154.7,152.1, 147.4, 143.5, 133.2, 131.6, 131.6, 130.6, 130.5, 127.3, 124.0, 115.9,115.8, 110.4, 108.8, 105.4, 105.3, 91.0, 91.0, 83.9, 76.0, 54.2, 50.6, 49.1,38.4, 25.5, 25.4;

[0235] HRMS (ESI): m / z [M+H] + calcd for C 32 H 34 N6O2F + 553.2727; found 553.2727.

[0236] Its chemical structural formula is:

[0237]

[0238] The chemical name is: 4-(7-(3,8-diazabicyclo[3.2.1]octane-3-yl)-5-((1-cyclopropylpiperidin-4-yl)oxy)pyrazolo[1,5-a]pyrimidin-3-yl)-5-ethynyl-6-fluoro-2-naphthol.

[0239] Example 18

[0240] Compound 18 was synthesized according to the flowchart and method described in Example 5, except that: in step 2), 2-tert-butoxycarbonyl-2,5-diazabicyclo[2.2.1]heptane was replaced with 8-tert-butoxycarbonyl-3,8-diazabicyclo[3.2.1]octane, and in step 3), (1-methylpyrrolidine-2-yl)methanol was replaced with benzyl alcohol. The resulting product was a white powder (yield 88%), and was designated as compound 18.

[0241] The characterization data of compound 18 are as follows:

[0242] 1H NMR (800 MHz, DMSO-d6) δ 8.32 (s, 1H), 8.00 (s, 1H), 7.89 (dd, J =9.1, 5.9 Hz, 1H), 7.41 (t, J = 8.9 Hz, 1H), 7.27 (ddt, J = 10.1, 4.0, 2.7 Hz,6H), 7.15 (d, J = 2.6 Hz, 1H), 5.78 (s, 1H), 5.09 (s, 2H), 3.81 (s, 3H), 3.18(s, 2H), 2.08 (s, 3H), 1.86 (d, J = 7.0 Hz, 2H);

[0243] 13C NMR (201 MHz, DMSO) δ 165.2, 163.3, 162.6, 162.1, 154.7, 152.7,147.5, 143.9, 136.9, 133.2, 131.7, 131.7, 130.5, 130.5, 129.2, 128.6, 128.4,127.7, 124.5, 116.0, 115.9, 110.6, 108.5, 105.4, 105.3, 91.4, 91.4, 82.6,75.7, 67.4, 53.9, 53.0, 49.1, 27.4;

[0244] HRMS (ESI): m / z [M+H] + calcd for C 31 H 27 N5O2F + 520.2149; found 520.2154.

[0245] Its chemical structural formula is:

[0246]

[0247] The chemical name is: 4-(5-(benzyloxy)-7-(3,8-diazabicyclo[3.2.1]octane-3-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-5-ethynyl-6-fluoro-2-naphthol.

[0248] Example 19

[0249] Compound 19 was synthesized according to the flowchart and method described in Example 5, except that: in step 2), 2-tert-butoxycarbonyl-2,5-diazabicyclo[2.2.1]heptane was replaced with 8-tert-butoxycarbonyl-3,8-diazabicyclo[3.2.1]octane, and in step 3), (1-methylpyrrolidine-2-yl)methanol was replaced with N-ethyl-4-hydroxypiperidine. The resulting product was a white powder (yield 82%), and was designated as compound 19.

[0250] The characterization data of compound 19 are as follows:

[0251] 1H NMR (800 MHz, DMSO-d6) δ 8.23 ​​(s, 1H), 8.06 (d, J = 5.2 Hz, 1H), 7.88 (dd, J = 9.1, 5.9 Hz, 1H), 7.39 (t, J = 8.9 Hz, 1H), 7.25 (d, J = 2.6Hz, 1H), 7.15 (d, J = 2.6 Hz, 1H), 5.82 (s, 1H), 4.71 (dt, J = 8.7, 4.4 Hz, 1H), 4.38 (dd, J = 11.0, 5.3 Hz, 1H), 4.29 (dd, J = 11.1, 5.5 Hz, 1H), 4.14 –4.08 (m, 2H), 3.44 – 3.37 (m, 2H), 2.88 (d, J = 14.7 Hz, 2H), 2.61 – 2.56 (m,2H), 2.49 (s, 1H), 2.39 (q, J = 9.7 Hz, 1H), 2.18 (s, 1H), 2.00 (dp, J = 8.1,3.9 Hz, 2H), 1.98 – 1.89 (m, 3H), 1.77 – 1.64 (m, 3H), 1.39 (s, 1H), 1.36 (s,1H), 1.06 (t, J = 7.2 Hz, 3H);

[0252] 13C NMR (201 MHz, DMSO) δ 164.4, 164.1, 163.1, 161.9, 161.8, 154.8,152.3, 147.4, 143.5, 143.4, 133.2, 131.6, 131.6, 130.6, 130.5, 127.3, 124.1,115.9, 115.7, 110.4, 108.8, 105.4, 105.3, 91.1, 91.0, 83.7, 79.8, 76.0, 67.7,64.4, 57.3, 54.1, 51.3, 51.2, 49.4, 28.6, 28.5, 28.5, 26.0, 22.9, 11.2;

[0253] HRMS (ESI): m / z [M+H] + calcd for C 31 H 34 N6O2F + 541.2727; found 541.2725.

[0254] Its chemical structural formula is:

[0255]

[0256] Chemical name: 4-(7-(3,8-diazabicyclo[3.2.1]octane-3-yl)-5-((1-ethylpiperidin-4-yl)oxy)pyrazolo[1,5-a]pyrimidin-3-yl)-5-ethynyl-6-fluoro-2-naphthol.

[0257] Example 20

[0258] Compound 20 was synthesized according to the flowchart and method described in Example 5, except that (1-methylpyrrolidone-2-yl)methanol in step 3) was replaced with N-ethyl-4-hydroxypiperidine. The resulting product was a white powder (yield 86%), denoted as compound 20.

[0259] The characterization data of compound 20 are as follows:

[0260] 1H NMR (800 MHz, DMSO-d6) δ 9.84 (s, 1H), 8.25 (s, 1H), 7.89 (d, J =8.5 Hz, 1H), 7.72 (d, J = 8.2 Hz, 1H), 7.40 (t, J = 7.5 Hz, 1H), 7.26 (d, J =2.4 Hz, 1H), 7.21 (td, J = 6.8, 3.4 Hz, 1H), 7.12 (d, J = 2.5 Hz, 1H), 5.73(s, 1H), 5.00 (s, 1H), 4.52 (s, 1H), 4.14 (d, J = 61.2 Hz, 2H), 3.96 (s, 1H),3.74 – 3.65 (m, 1H), 3.53 – 3.46 (m, 2H), 3.34 (d, J = 11.2 Hz, 1H), 2.99 –2.89 (m, 3H), 2.26 – 2.14 (m, 3H), 2.02 (dd, J = 47.4, 11.9 Hz, 3H), 1.20 (t,J = 7.3 Hz, 3H), 1.07 (s, 6H);

[0261] 13C NMR (201 MHz, DMSO) δ 163.8, 162.4, 155.3, 148.3, 146.3, 144.5,135.8, 132.0, 127.0, 126.6, 126.5, 126.4, 122.8, 120.2, 108.5, 106.1, 78.6,74.0, 72.6, 71.0, 60.6, 58.7, 57.3, 50.9, 44.1, 35.7, 28.1, 25.4, 9.9;

[0262] HRMS (ESI): m / z [M+H] + calcd for C 30 H 31 FN6O2 + 527.2571; found 527.2574.

[0263] Its chemical structural formula is:

[0264]

[0265] The chemical name is: 4-(7-(2,5-diazabicyclo[2.2.1]heptane-2-yl)-5-((1-ethylpiperidin-4-yl)oxy)pyrazolo[1,5-a]pyrimidin-3-yl)-5-ethynyl-6-fluoro-2-naphthol.

[0266] Experimental Example 1

[0267] The protein-level activity of compounds 1-20 synthesized in Examples 1-20 was evaluated to investigate their effect on KRAS at a concentration of 10 μM. G12D The inhibitory activity was measured, and the results are shown in Table 1.

[0268] For KRAS G12D The methods for evaluating inhibitory activity are as follows:

[0269] (1) Dilute the positive control drug according to the reference concentration, and use the diluent as the first concentration point to perform a 3-fold serial dilution, setting a total of 10 concentration points;

[0270] (2) Transfer 100 μL of the compound to a 384-well plate, with two replicates per column; centrifuge the 384-well plate at 1000 rpm; add 2.5 μL of KRAS- to each well. G12D >P solution, centrifuge the 384-well detection plate at 1000 rpm;

[0271] (3) Add 2.5 μL of cRAF solution to each well, centrifuge the 384-well plate at 1000 rpm, and incubate at 25°C for 15 minutes; add 5 μL of SA-Tb&GST-XL665 solution to the 384-well plate, incubate at 25°C for 1 hour, and read the HTRF signal (665 / 620 nm ratio) using a BMG instrument.

[0272] Table 1. Effects of compounds 1-20 on KRAS G12D inhibition rate

[0273]

[0274] Table 1 above shows the effects of 20 compounds on KRAS at a concentration of 10 μM. G12D Inhibitory activity of the target.

[0275] As shown in Table 1, compound 2 exhibited the highest inhibition rate (69.67%), demonstrating a significant inhibitory effect. Furthermore, compounds 4, 5, 13, 14, 15, and 17 also showed high activity (inhibition rates all exceeding 30%). Meanwhile, one compound showed a negative inhibition rate, with compound 1 exhibiting the lowest (-6.3%), suggesting that this compound did not show inhibitory activity under these conditions, or that it may have been affected by experimental error. The inhibition rates of the remaining compounds were generally low, mostly falling within the weak activity range of 0%–30%.

[0276] Experimental Example 2

[0277] Bioactivity tests and mechanism of action studies were conducted on compounds 5, 13, and 14, which showed good inhibitory effects.

[0278] 1. First, the CCK-8 assay was used, with KRAS as the reference. G12D The inhibitor MRTX1133 was used as a positive control to evaluate the effects of compounds 5, 13, and 14 on KRAS. G12D Mutant cell lines, non-KRAS G12D The antiproliferative activities of mutant cell lines, wild-type cells, and normal cells were evaluated, and the results are shown in Table 2.

[0279] The KRAS G12D Mutant cell lines include: human pancreatic cancer cells Aspc-1 (G12D), human pancreatic cancer cells PANC-1 (G12D), and human gastric adenocarcinoma cells AGS (G12D).

[0280] The non-KRAS G12D Mutant cell lines include: human lung adenocarcinoma cell line A549 (G12S), human non-small cell lung cancer cell line H23 (G12C), human liver cancer cell line Hep-G2 (NRAS Q61L), and human breast cancer cell line MDA-MB-231 (G13D).

[0281] The wild-type cells were human normal colonic epithelial cells NCM 460 and rat adrenal pheochromocytoma cells PC-12; the normal cells were African green monkey kidney cells VERO.

[0282] The CCK-8 assay is a conventional method and can be performed according to existing technology or using a CCK-8 assay kit (TargetMol, C0005).

[0283] Table 2

[0284]

[0285] As shown in Table 2, compound 5 affects KRAS G12D Mutant cell lines (Aspc-1, PANC-1, and AGS) exhibited moderate antiproliferative activity, IC50...50 The concentrations were 8.7 μM, 4.7 μM, and 7.8 μM, respectively. In contrast, compounds 13 and 14 exhibited excellent antiproliferative activity, particularly against PANC-1 cells, with IC50 values ​​of 8.7 μM, 4.7 μM, and 7.8 μM. 50 The value is approximately 1 μM.

[0286] Then compound 5 was used against non-KRAS G12D The mutant cell lines A549 (G12S), H23 (G12C), Hep-G2 (NRASQ61L) and MDA-MB-231 (G13D) showed no inhibitory activity, and compounds 13 and 14 also showed good selectivity.

[0287] Finally, compound 5 continued to target NCM 460 (wild type, IC50). 50 > 20 μM), PC-12 (wild type, IC 50 > 20 μM), or VERO (normal cells, IC50). 50 Compounds > 20 μM exhibited low toxicity. In contrast, compounds 13 and 14 showed relatively poor selectivity, IC50... 50 The value ranges from 10 to 20 μM. Based on KRAS... G12D To investigate the activity and selectivity of mutant cells, compound 5 was selected for further biological research.

[0288] Experimental Example 3

[0289] 1. Compound 5 blocks pro-survival and proliferative signals by inhibiting downstream KRAS signaling pathways.

[0290] To investigate the molecular mechanism of action of compound 5, Western blotting was used to detect the levels and phosphorylation status of key signaling proteins ERK and AKT in human gastric adenocarcinoma cells AGS. Furthermore, the effects of compound 5 on the MAPK / ERK and PI3K / AKT pathways (two key downstream oncogenic signaling pathways of KRAS) in human gastric adenocarcinoma cells AGS were analyzed. The results are as follows: Figure 1 As shown in A and B.

[0291] The specific method was as follows: Human gastric adenocarcinoma cells (AGS) were co-incubated with different concentrations of compound 5 (0, 1, 5, 10 μM) for 24 h. Cells were then collected and proteins were extracted. The expression levels of ERK, AKT, and their phosphorylated forms p-ERK and p-AKT were detected by Western blotting, with Tubulin and β-actin used as internal controls. Further, grayscale analysis was used to quantify each protein band, and the relative ratios of p-AKT / AKT and p-ERK / ERK were calculated to evaluate the effects of compound 5 on the MAPK / ERK and PI3K / AKT signaling pathways.

[0292] Depend on Figure 1 As shown in A and B, treatment with compound 5 resulted in a concentration-dependent decrease in p-ERK protein levels. Simultaneously, total ERK protein levels remained stable across all groups, indicating that compound 5 specifically inhibits ERK phosphorylation and activation. Similar inhibition was observed in the PI3K / AKT pathway, where drug concentration-dependent inhibition of AKT phosphorylation was observed. This simultaneous inactivation of both pathways can synergistically lead to cell proliferation inhibition and weakened survival signaling, providing a theoretical basis for its potential as a KRAS-driven cancer treatment strategy.

[0293] 2. Inhibition of the signaling pathway triggered by compound 5 leads to cell cycle arrest in G1 phase.

[0294] Given the crucial roles of MAPK and PI3K-AKT signaling in cell cycle progression, the effect of compound 5 on cell cycle distribution was investigated.

[0295] The specific method was as follows: Based on the grouping and drug treatment in point 1, flow cytometry was used to detect the DNA content of propidium iodide-stained cells to assess the effect of compound 5 on cell cycle distribution. The results are as follows: Figure 1 As shown in C.

[0296] Depend on Figure 1 As shown in Figure C, treatment with 5 μM or 10 μM of compound 5 significantly altered the cell cycle distribution of human gastric adenocarcinoma AGS cells. Treatment induced significant G1 phase arrest, with a significant increase in the number of G1 phase cells (0 μM: 45.3%, 5 μM: 51.4%, 10 μM: 53.1%). Conversely, the proportion of S phase cells decreased accordingly (0 μM: 33.4%, 5 μM: 24.4%, 10 μM: 23.6%). The proportion of G2 phase cells remained relatively stable (0 μM: 19.9%, 5 μM: 22.0%, 10 μM: 20.4%). These changes indicate that compound 5 treatment significantly disrupted the cell cycle progression of human gastric adenocarcinoma AGS cells, and the inhibition of the aforementioned signaling pathways successfully arrested AGS cells in the G1 phase. By maintaining G1 phase arrest and preventing AGS cells from entering the S phase for DNA replication, this treatment inhibited DNA replication and proliferation of AGS cells.

[0297] 3. Compound 5 synergistically induces apoptosis by promoting cell cycle arrest and signaling pathway inactivation.

[0298] Cell cycle arrest is usually accompanied by the initiation of the apoptotic program. Annexin V / PI double staining further confirmed the pro-apoptotic effect of compound 5.

[0299] The specific method was as follows: Human gastric adenocarcinoma cells AGS at different stages (G1, S, and G2 phases) were co-incubated with different concentrations of compound 5 (0, 5 μM, and 10 μM) for 48 hours, and then analyzed by flow cytometry. The results are as follows: Figure 2 As shown in Figure A.

[0300] Depend on Figure 2 As shown in Figure A, compound 5 effectively induced apoptosis in cancer cells in a concentration-dependent manner. With increasing compound 5 concentration, the proportion of normal / surviving cells in quadrant Q3 significantly decreased (5 μM: 47.215%, 10 μM: 32.207%, 20 μM: 9.423%); the proportion of early apoptotic cells (Q4) slightly increased (5 μM: 21.415%, 10 μM: 31.332%, 20 μM: 32.031%); while the increase in the proportion of late apoptotic / necrotic cells (Q2) was the most significant (5 μM: 29.385%, 10 μM: 35.359%, 20 μM: 56.620%). This clearly demonstrates that compound 5 not only effectively induced apoptosis in cancer cells, but also, at higher concentrations, caused most cells to enter irreversible late apoptosis or directly lead to necrosis. Ultimately, the synergistic effect of signaling pathway inhibition and cell cycle arrest induced by compound 5 resulted in large-scale, irreversible cell death.

[0301] 2. Compound 5 significantly inhibited the long-term proliferation and self-regeneration capacity of cancer cells in a concentration-dependent manner.

[0302] To assess the fundamental effect of compound 5 on the long-term survival of cancer cell populations, the following method was used: human gastric adenocarcinoma cells (AGS) were co-incubated with different concentrations of compound 5 (0, 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, and 10 μM) for 14 days to form visible colonies. Hematoxylin-eosin staining, counting, and statistical analysis were then performed. The results are as follows: Figure 2 As shown in B and C.

[0303] Depend on Figure 2 As shown in B and C, compound 5 can eliminate the long-term proliferative potential of cancer cells. Untreated negative control human gastric adenocarcinoma cells (AGS) exhibited strong colony-forming ability, while compound 5 significantly reduced the number of colonies formed in a concentration-dependent manner. The colony formation assay results clearly demonstrate that compound 5 not only kills cells in the short term but also effectively eliminates the long-term proliferative capacity and self-renewal potential of cancer cells.

[0304] Overall, about 30% of the dipyrazolopyrimidine compounds prepared in this invention exhibit strong inhibitory potential, representing a class of highly active KRAS compounds. G12D Inhibitors can be used to prepare drugs for treating pancreatic cancer, colorectal cancer, and lung cancer.

[0305] The embodiments described above are merely illustrative descriptions of this disclosure and are not intended to limit the scope of this disclosure. Any modifications and improvements made by those skilled in the art to the technical solutions of this disclosure without departing from the spirit of this disclosure should fall within the protection scope defined by this disclosure.

Claims

1. A dipyrazolopyrimidine compound as shown in formula (1), or a pharmaceutically acceptable salt thereof, Equation (1) in, R1 is selected from one of the following groups: ； R2 is selected from one of the following groups: ； R3 is selected from one of the following groups: 。 2. The dipyrazolopyrimidine compound as described in claim 1, characterized in that, The dipyrazolopyrimidine compounds are specifically selected from one of the following 20 compounds, or their pharmaceutically acceptable salts, prodrugs, hydrates or solvents, polymorphs, stereoisomers, tautomers, resonances, enantiomers or diastereomers: 。 3. The dipyrazolopyrimidine compound as described in claim 2, characterized in that, The specific preparation method of compounds 1-4 includes the following steps: (1) 5,7-dichloropyrazolo[1,5-a]pyrimidine and N-bromosuccinimide were dissolved in chloroform and reacted at room temperature for 12 h. The reaction solution was washed with saturated brine, dried with anhydrous sodium sulfate and filtered to remove impurities, and crude product was obtained. The crude product was then purified by silica gel column chromatography with a mixture of petroleum ether and ethyl acetate and eluted to obtain 3-bromo-5,7-dichloropyrazolo[1,5-a]pyrimidine. (2) Dissolve the 3-bromo-5,7-dichloropyrazole[1,5-a]pyrimidine obtained in step (1) in dichloromethane, add N,N-diisopropylethylamine and amine compounds, and react at -40℃ under nitrogen protection for 0.5 h. Remove impurities by washing the reaction solution with saturated brine, drying with anhydrous sodium sulfate and filtering to obtain crude product; then purify the crude product by silica gel column chromatography with a mixture of petroleum ether and ethyl acetate and elute to obtain intermediate 1. (3) The intermediate 1 and alcohol compound obtained in step (2) were dissolved in a solvent of tetrahydrofuran and NN dimethylformamide. Sodium hydride was added at 0°C, and the reaction was stirred at room temperature. The reaction solution was washed with saturated brine, dried with anhydrous sodium sulfate, and filtered to remove impurities, and crude product was obtained. The crude product was then purified by silica gel column chromatography using a mixture of dichloromethane and methanol and eluted to obtain intermediate 2. (4) The intermediate 2 and 3-hydroxy-1-naphthoic acid pinacol ester obtained in step (3) were dissolved in 1,4-dioxane solvent, and Pd(dppf)Cl2 and cesium carbonate were added as catalysts. The reaction was carried out at 85°C for 3 h. The reaction solution was dried with anhydrous sodium sulfate, filtered and evaporated to dryness. Then, the product was hydrolyzed with hydrochloric acid-1,4-dioxane and acetonitrile. The hydrolysate was washed with saturated brine, dried with anhydrous sodium sulfate and filtered to remove impurities, and crude product was obtained. The crude product was then purified by silica gel column chromatography with a mixture of dichloromethane and methanol and eluted to obtain compounds 1 to 4.

4. The dipyrazolopyrimidine compound as described in claim 3, characterized in that, In step (2), the amine compound is selected from (1R,5S)-3,8-diazabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester, N-methylpiperazine, or N-ethyl-4-hydroxypiperidine; In step (3), the alcohol compound is selected from N-methyl-4-hydroxypiperidine, 3-hydroxy-1-methyltetrahydropyrrole, 1-methyl-2-pyrrolidinemethanol or N-ethyl-4-hydroxypiperidine.

5. The dipyrazolopyrimidine compound as described in claim 2, characterized in that, The specific preparation method of compounds 5-20 includes the following steps: 1) Dissolve 5,7-dichloropyrazolo[1,5-a]pyrimidine in chloroform, add N-bromosuccinimide, heat to 40°C, react for 12 hours, and after the reaction is complete as detected by TLC, add water to quench the reaction, extract three times with ethyl acetate, combine the organic phases, wash with saturated brine, dry with anhydrous sodium sulfate, filter, add silica gel and concentrate, and purify by silica gel column chromatography to obtain 3-bromo-5,7-dichloropyrazolo[1,5-a]pyrimidine; 2) Dissolve the 3-bromo-5,7-dichloropyrazole[1,5-a]pyrimidine obtained in step 1) in dichloromethane, cool to -40°C, add amine compounds, add diisopropylethylamine dropwise, and after the addition is complete, allow the temperature to rise naturally to room temperature. After the reaction is complete as detected by TLC, concentrate under reduced pressure and dry to obtain intermediate 3. 3) Add intermediate 3 obtained in step 2) and an alcohol compound to tetrahydrofuran. After cooling to 0°C in an ice-water bath, add sodium hydride, allow to warm naturally to room temperature, stir for 0.5 hours, heat to 100°C in an oil bath, react for 2 hours, cool to 0°C in an ice-water bath, quench with water, extract with dichloromethane, and dry with anhydrous sodium sulfate. Purify by silica gel column chromatography to obtain intermediate 4; 4) Add intermediate 4 obtained in step 3), 7-fluoro-3-(methoxymethoxy)-8-[(triisopropylsilyl)ethynyl]naphthalene-1-boronic acid pinacol ester, cesium carbonate and Ad2nBuP-Pd-G3 sequentially to 1,4-dioxane solvent. Purge with nitrogen for 15 min to replace air, microwave heat to 82°C and react for 1 hour. After the reaction is complete, monitor the reaction by TLC and dry with anhydrous sodium sulfate. Filter, wash three times with methanol, add silica gel and concentrate under reduced pressure to dryness, and purify by silica gel column chromatography to obtain intermediate 5. 5) Add intermediate 5 obtained in step 4) and CsF to DMF, stir overnight at room temperature, monitor the reaction by TLC until complete, add silica gel, concentrate directly to dryness, and purify by silica gel column chromatography to obtain intermediate 6; 6) Add intermediate 6 obtained in step 5) to methanol, cool to 0℃, add 4 M hydrochloric acid 1,4-dioxane dropwise, and after the addition is complete, naturally warm to room temperature and react for 2 h. After the reaction is complete by TLC monitoring, concentrate directly to dryness, add methanol to dissolve and concentrate again, repeat three times, and purify by high performance chromatography to obtain compounds 5-20.

6. The dipyrazolopyrimidine compound as described in claim 5, characterized in that, In step 2), the amine compound is selected from 2-tert-butoxycarbonyl-2,5-diazabicyclo[2.2.1]heptane, 1-tert-butoxycarbonyl-2-methylpiperazine, 1-(tert-butoxycarbonyl)-1,4-diazabicycloheptane, 3,4-dimethylpiperazine, 6-(tert-butoxycarbonyl)-3,6-diazabicyclo[3.1.1]heptane, 1-tert-butoxycarbonyl-2-piperazinone, and 8-tert-butoxycarbonyl-3,8-diazabicyclo[3.2.1]octane; In step 3), the alcohol compound is selected from (1-methylpyrrolidone-2-yl)methanol, 8-methyl-8-azabicyclo[3.2.1]octane-3-ol, 1-ethylpyrrolidone-3-ol, 1-methylpiperidin-4-ol, 1-isopropylpiperidin-4-ol, 1-cyclopropylpiperidin-4-ol, (2S,7aR)-7a-((hydroxy)methyl)-2-fluorohexahydro-1H-pyrrolazine, benzyl alcohol, and N-ethyl-4-hydroxypiperidine.

7. The dipyrazolopyrimidine compound of claim 1, its isomers, or pharmaceutically acceptable salts thereof, in the preparation of KRAS G12D Application in drugs for treating diseases related to protein dysfunction.

8. The application as described in claim 7, characterized in that, The above is related to KRAS G12D Diseases associated with protein dysfunction include pancreatic cancer, colorectal cancer, and lung cancer.

9. A KRAS G12D Protein inhibitors, characterized in that, The KRAS G12D Protein inhibitors comprise an effective amount of the dipyrazolopyrimidine compound of claim 1 or its stereoisomers, tautomers, resonators, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates, or crystal forms thereof.

10. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the dipyrazolopyrimidine compound of claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient.

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