Heterocyclic pyrimidine-containing compound and use thereof

Abstract

The application provides a heterocyclic pyrimidine compound and application thereof, and belongs to the technical field of medicines.The compound has strong inhibition on CDK4 / 6 kinase, and therefore can be used as an active ingredient to prepare a therapeutic drug for diseases caused by abnormal activation of CDK4 / 6 kinase, and also shows potential application value in preparation of a drug for treating and / or preventing proliferative diseases and cancers.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and in particular relates to a heterocyclic pyrimidine compound and its applications. Background Technology

[0002] Cancer seriously endangers human life and health, affecting hundreds of millions of people worldwide. According to statistics from the World Health Organization (WHO), cancer causes millions of deaths globally each year, and its incidence rate continues to rise, making it the second leading cause of death worldwide. Breast cancer is one of the most common malignant tumors among women worldwide, and its incidence rate has been rising in recent years, becoming one of the major diseases threatening women's health. Due to changes in lifestyle, environmental factors, and genetic susceptibility, the age of onset of breast cancer is trending younger, and more and more patients are being diagnosed at a younger age. Furthermore, although breast cancer is relatively rare in men, accounting for only 0.5%-1% of all breast cancer cases, its incidence rate in women is far higher than other malignant tumors. The distribution of different subtypes of breast cancer varies significantly; the most common subtype among female patients is HR (high-risk breast cancer). + / HER2 - (i.e., Luminal type A) accounts for approximately 50%-60%, and this type is more sensitive to endocrine therapy and has a relatively better prognosis. HR + / HER2 + This subtype (Luminal B) accounts for approximately 15%-20%. HER2-positive breast cancer (HR...) - / HER2 + HER2-negative breast cancer accounts for approximately 15%-20%. This type is highly aggressive and requires HER2-targeted treatment strategies to improve prognosis. Triple-negative breast cancer (TNBC) is also highly malignant, accounting for 10%-15%. In contrast, the subtypes of male breast cancer are more concentrated, with HR... + / HER2 - The subtype accounts for 80%-90%, meaning that most male breast cancer patients belong to Luminal A, indicating that they are highly sensitive to hormone therapy. In contrast, the proportion of HER2-positive and TNBC subtypes in male breast cancer is extremely low, accounting for only 5%-10%.

[0003] In the development of tumor treatment methods, molecular targeted therapy has received widespread attention and is gradually becoming a cutting-edge field in tumor treatment. Targeted therapy has the advantages of being targeted, having low toxicity, requiring small doses, and having good efficacy. This method can not only inhibit the proliferation of tumor cells by inhibiting signaling pathways, but also reduce toxicity and damage to normal cells. In the research of small molecule inhibitor targeted therapy for tumors, the cyclin-dependent protein kinase (CDK) family, especially CDK4 / 6, is gradually becoming an important target for cancer treatment.

[0004] Cyclin-dependent protein kinases (CDKs) belong to the GMGC family of serine / threonine kinases. CDKs include cycle regulators (CDKs 1-6, 11, 14-18) that regulate different stages of the cell cycle and transcription regulators (CDKs 7-13, 19, 20) that participate in transcriptional elongation and regulation. CDK family members not only play a central role in cell cycle control but also participate in various biological processes such as transcriptional regulation, metabolic regulation, and neuronal differentiation. CDK4 / 6 is overexpressed in various cancers, and inhibiting CDK4 / 6 expression can induce tumor cell apoptosis; therefore, CDK4 / 6 is considered a promising target for cancer therapy. CDK4 / 6 inhibitors (CDKIs) can not only arrest cancer cells in the G1 phase, thus quiescent, but also induce apoptosis in related cancer cells. Extensive research on CDKIs began in the 1990s.

[0005] Past studies have found that first- and second-generation CDK inhibitors are multi-target inhibitors with low selectivity and significant toxicity in clinical trials, causing substantial damage to normal cells and tissues. Therefore, the development of CDK inhibitors with high selectivity and low toxicity is urgently needed. Third-generation CDK inhibitors offer higher selectivity than first- and second-generation inhibitors, specifically targeting CDK4 / 6 with high selectivity. These inhibitors not only target ATP binding sites but may also bind to CDKs through other non-competitive mechanisms, providing higher selectivity and fewer side effects. Among them, the marketed drug Abemaciclib for treating breast cancer has been extensively studied; its structural formula is as follows:

[0006]

[0007] Abemaciclib selectively inhibits CDK4 / 6 at nanomolar concentrations. At the same concentration, the IC50 of Abemaciclib against CDK4 is [not specified]. 50 Abemaciclib's IC for CDK6 is larger than that of CDK6. 50 Approximately 15 times. Clinical studies have shown that abemaciclib monotherapy is ineffective in treating HR+ / HER2 metastatic breast cancer. Abemaciclib needs to be used in combination with fulvestrant to demonstrate better therapeutic efficacy in treating HR+ metastatic breast cancer. Abemaciclib has significant side effects in clinical practice, including gastrointestinal reactions, joint pain, bone marrow suppression, and fatigue. The most common side effects are diarrhea, bone marrow suppression, and hepatotoxicity, which require attention.

[0008] Therefore, developing safer and more effective novel CDK4 / 6 inhibitors for cancer treatment has significant social and economic value and is currently a research hotspot for major pharmaceutical companies. Modifying compound structures to design new compounds and improve their drug-likeness, thereby enhancing their biological activity and bioavailability, is crucial for identifying a new class of CDK4 / 6 inhibitors and is of great significance for the clinical treatment of diseases caused by CDK4 / 6 mutations. Therefore, current CDK4 / 6 inhibitors still require further improvement. Summary of the Invention

[0009] In order to develop novel and highly efficient CDK4 / 6 single-target antitumor inhibitors, this invention has conducted extensive research on heterocyclic pyrimidine compounds. By continuously modifying the structure of the compounds through molecular docking results and cell and kinase inhibitory activities, a series of novel heterocyclic pyrimidine compounds have been designed and synthesized.

[0010] To achieve the above objectives, the present invention provides the following technical solution:

[0011] One of the technical solutions of the present invention:

[0012] A heterocyclic pyrimidine compound, with the structural formula shown in Formula I or Formula II:

[0013] The structural formula is shown in Formula I or Formula II:

[0014]

[0015] In Equations I and II, R1 is independently selected from...

[0016] One of them;

[0017] In Equation II, the fused ring containing the X ring is selected from... One of them;

[0018] When the condensed ring containing the X ring is selected When R2 is -N, R3 is -CH;

[0019] When the condensed ring containing the X ring is selected When R2 is -CH and R3 is -N.

[0020] Furthermore, the heterocyclic pyrimidine-containing compound includes:

[0021] N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-2-(4-(methanesulfonyl)phenyl)pyrimidin-4-amine, N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-2-(4-(isopropylsulfonyl)phenyl)pyrimidin-4-amine, 4-(4-((5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidin-2-yl)-N,N-dimethylbenzenesulfonamide, N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidin-2-yl)-N,N-dimethylbenzenesulfonamide, N-(5-((4-ethylpiperazin-1-yl)) N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-2-(4-(trifluoromethyl)phenyl)pyrimidin-4-amine, N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-2-(4-(trifluoromethoxy)phenyl)pyrimidin-4-amine, N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-2-(4-(trifluoromethoxy)phenyl)pyrimidin-4-amine, N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-2-(4-(methoxymethyl)phenyl) 4-Pyrimidine-4-amine, N-(5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-2-(4-(methoxymethoxy)phenyl)pyrimidine-4-amine, 4-(4-((5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidine-2-yl)benzaldehyde, 1-(4-(4-((5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidine-2-yl)phenyl)acetone, N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidine-2-yl)phenyl)acetone 2-(4-ethoxyphenyl)-N-(5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoropyrimidin-4-amine, N-(5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoropyrimidin-4-amine, 4'-(4-((5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-2-(4-methoxyphenyl)pyrimidin-4-amine, 4'-(4-((5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidin-2-yl)-[1,Methyl 1'-biphenyl]-4-carboxylate, N-(5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-2-(p-tolyl)pyrimidin-4-amine, 2-(4-ethylphenyl)-N-(5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoropyrimidin-4-amine, N-(5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-2-(4-isopropylphenyl)pyrimidin-4-amine, N-(5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-2-(4-propylphenyl)pyrimidin-4-amine, 4-(4-((5-(((4-ethylpiperazin-1-yl)methyl)pyridin)pyridine -2-yl)amino)-5-fluoropyrimidin-2-yl)benzonitrile, 4-(4-((5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidin-2-yl)-N,N-dimethylbenzamide, 2-([1,1'-biphenyl]-4-yl)-N-(5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoropyrimidin-4-amine, 2-(4'-chloro-[1,1'-biphenyl]-4-yl)-N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoropyrimidin-4-amine, N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-2-(4'-methyl) -[1,1'-biphenyl]-4-yl)pyrimidin-4-amine, (4-(4-(((5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidin-2-yl)phenyl)(phenyl)methyl ketone, N-(5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-2-(4-morpholinophenyl)pyrimidin-4-amine, N-(5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-2-(4-fluorophenyl)thieno[3,2-d]pyrimidin-4-amine, N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-2-(4-methoxyphenyl)thieno[3,2-d]pyrimidin-4-amine , N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-2-(p-tolyl)thieno[3,2-d]pyrimidin-4-amine, N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-2-(4-isopropylphenyl)thieno[3,2-d]pyrimidin-4-amine, 4-(4-((5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)thieno[3,2-d]pyrimidin-2-yl)benzonitrile, 2-([1,1'-biphenyl]-4-yl)-N-(5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)thieno[3,2-d]pyrimidin-4-amine, 2-(4'-chloro-[1,1'-Biphenyl]-4-yl)-N-(5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)thieno[3,2-d]pyrimidin-4-amine, N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-2-(4'-methyl-[1,1'-biphenyl]-4-yl)thieno[3,2-d]pyrimidin-4-amine, N-(5 -((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-2-(4-morpholinophenyl)thieno[3,2-d]pyrimidin-4-amine, N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-(4-fluorophenyl)pyrazolo[1,5-a]pyrimidin-7-amine, N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-(4-(trifluoromethyl)phenyl)pyrazolo[1,5-a]pyrimidin-7-amine, N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-(p-tolyl)pyrazolo[1,5-a]pyrimidin-7-amine, N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-(4-isopropoxyphenyl) 4-(7-((5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)pyrazolo[1,5-a]pyrimidin-5-yl)benzonitrile and 4-(7-((5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)pyrazolo[1,5-a]pyrimidin-5-yl)benzoate.

[0022] The second technical solution of the present invention:

[0023] A method for preparing the heterocyclic pyrimidine compound described above involves using 2-chloro-5-chloromethylpyridine as a starting material, obtaining 2-amino-5-chloromethylpyridine via a Buchwald-Hartwig coupling reaction; then preparing the intermediate 5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-amine via a nucleophilic substitution reaction; obtaining 2-chloro-N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)pyrimidin-4-amine, 5-chloro-N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)pyrazolopyrimidin-7-amine and 2-chloro-N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)thieno[3,2-d]pyrimidin-4-amine via a Suzuki-Miyaura coupling reaction; and finally obtaining the target product via a Suzuki-Miyaura coupling reaction.

[0024] The third technical solution of the present invention:

[0025] A pharmaceutical composition comprising the heterocyclic pyrimidine compound as the active ingredient.

[0026] The fourth technical solution of the present invention:

[0027] The use of the heterocyclic pyrimidine compound or the pharmaceutical composition thereof in the preparation of CDK4 / 6 antitumor inhibitor drugs.

[0028] Furthermore, the CDK4 / 6 antitumor inhibitor also includes a pharmaceutically acceptable carrier or excipient.

[0029] Furthermore, the dosage forms of the CDK4 / 6 antitumor inhibitors include injections, tablets, capsules, aerosols, suppositories, films, pellets, topical liniments, or ointments.

[0030] This invention relates to the preparation of pharmaceutical compositions by mixing heterocyclic pyrimidine compounds with pharmaceutically acceptable carriers or excipients, and to the formulation of clinically acceptable dosage forms. The aforementioned pharmaceutically acceptable excipients refer to any diluent, adjuvant, and / or carrier that can be used in the pharmaceutical field. The derivatives of this invention can be used in combination with other active ingredients, provided they do not produce other adverse effects, such as allergic reactions.

[0031] Specifically, the pharmaceutical compositions of the present invention can be formulated into several dosage forms, containing some commonly used excipients in the pharmaceutical field. The dosage forms described above can be injections, tablets, capsules, aerosols, suppositories, films, pellets, topical liniments, ointments, etc. The carriers used in the pharmaceutical compositions of the present invention are common types available in the pharmaceutical field, including: binders, lubricants, disintegrants, solubilizers, diluents, stabilizers, suspending agents, flavoring agents, preservatives, solvents, and matrices. The pharmaceutical formulations can be administered orally or via non-gastrointestinal routes (e.g., intravenously, subcutaneously, intraperitoneally, or locally). If certain drugs are unstable under gastric conditions, they can be formulated into enteric-coated tablets.

[0032] The fifth technical solution of the present invention:

[0033] The use of the heterocyclic pyrimidine compound or the pharmaceutical composition thereof in the preparation of a drug for treating and / or preventing cancer.

[0034] Furthermore, the cancers mentioned include breast cancer, lung cancer, melanoma, pancreatic cancer, and prostate cancer.

[0035] The sixth technical solution of the present invention:

[0036] The use of the heterocyclic pyrimidine compound or the pharmaceutical composition thereof in the preparation of a medicament for the treatment and / or prevention of proliferative diseases.

[0037] The clinical dosage of the heterocyclic pyrimidine compounds described above for use in patients can be appropriately adjusted based on: the therapeutic efficacy and bioavailability of the active ingredients in vivo, their metabolic and excretion rates, and the patient's age, sex, and disease stage. However, the daily dose for adults should generally be 10-500 mg, preferably 50-300 mg. These preparations can be administered in divided doses at certain intervals, preferably one to six times, as directed by a doctor or pharmacist.

[0038] Compared with the prior art, the present invention has the following advantages and technical effects:

[0039] (1) The present invention provides a heterocyclic pyrimidine compound that has a strong ability to inhibit CDK4 / 6 kinase. Therefore, it can be used as an active ingredient to prepare a therapeutic drug for diseases caused by abnormal activation of CDK4 / 6 kinase, and also shows potential application value in the preparation of drugs for the treatment and / or prevention of proliferative diseases and cancer.

[0040] (2) This invention provides important technical support for the development of more efficient and selective anticancer drugs. Attached Figure Description

[0041] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0042] Figure 1 The hemolytic safety evaluation results of compound 37 in Example 37 are shown in the illustration (the illustration shows a photograph of the hemolysis test results).

[0043] Figure 2 The results of the in vivo toxicity test of each group on KM mice are shown. A represents the biochemical indicators of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and serum creatinine (CREA); B represents the biochemical indicators of total protein (TP), urea (UREA) and blood glucose (GLU); and C represents the biochemical indicators of lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and creatine kinase (CK).

[0044] Figure 3 HE staining results of various organs and tissues in each group of Kunming mice to show the effects of biotoxicity.

[0045] Figure 4 The results of the time-dependent test of the compound in Example 37 on MCF-7;

[0046] Figure 5 The results of wound healing experiments for each group treated with MCF-7;

[0047] Figure 6The results of cloning experiments on MCF-7 in each group;

[0048] Figure 7 The results of the AO staining experiment on MCF-7 in each group;

[0049] Figure 8 The results of JC-1 staining experiments on MCF-7 in each group;

[0050] Figure 9 The results of apoptosis experiments on MCF-7 cells in each group;

[0051] Figure 10 The results of cell cycle experiments on MCF-7 in each group;

[0052] Figure 11 The results of ROS staining experiments on MCF-7 in each group are shown. Detailed Implementation

[0053] All heterocyclic pyrimidine compounds in this invention were prepared by the methods described in the synthetic routes of the examples, or by similar methods, which are well known to those skilled in the art of organic chemistry. All variable factors used in the synthetic routes are defined below.

[0054] In the embodiments of the present invention, the proton NMR spectra of the compounds were determined using a Bruker ARX-400, and the mass spectra were determined using an Agilent 1100 LC / MSD; all reagents used were analytical grade or chemically pure.

[0055] The technical solution of the present invention will be further illustrated by the following embodiments.

[0056] Example 1

[0057] Preparation of N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-2-(4-(methanesulfonyl)phenyl)pyrimidin-4-amine:

[0058] Step 1: Preparation of 2-amino-5-chloromethylpyridine (2)

[0059] 1 g of 2-chloro-5-chloromethylpyridine (1, representing structural formula 1 below, the same below), 0.15 g of palladium catalyst Pd2(dba)3, 0.2 g of ligand Xantphos, 1.48 g of sodium tert-butoxide and 20 mL of toluene were added to a reaction flask, followed by 2.08 mL of ammonia. The reaction flask was sealed, and the air was replaced with nitrogen three times to ensure that the reaction system was in an oxygen-free environment. The reaction was heated to 90 °C and reacted for 8 h. The reaction progress was monitored by TLC every 1-2 h. After the reaction was completed, the reaction solution was cooled to room temperature and extracted. The extract was concentrated by rotary evaporator to remove the solvent and then recrystallized to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain 2-amino-5-chloromethylpyridine (2).

[0060]

[0061] Step 2: Preparation of 5-((4-ethylpiperazin-1-yl)methyl)pyridine-2-amine (3)

[0062] 1 g of 2-amino-5-chloromethylpyridine prepared in step one, 1.21 g of 1-ethylpiperazine, 1.95 g of K2CO3 and 20 mL of acetonitrile were added to the reaction flask. The mixture was heated at 80 °C for 12 h, and the reaction progress was monitored by TLC during the reaction. After the reaction was completed, the mixture was cooled to room temperature and filtered. The filtrate was concentrated by rotary evaporation to remove the solvent. The crude product was purified by silica gel column chromatography using methanol / dichloromethane as the eluent, and the key intermediate 5-((4-ethylpiperazin-1-yl)methyl)pyridine-2-amine (3) was finally obtained.

[0063]

[0064] Step 3: Preparation of 2-chloro-N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)pyrimidin-4-amine (4)

[0065] Starting with 0.79 g of 5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-amine (3) prepared in step two and 0.5 g of 2,4-dichloro-5-fluoropyrimidine, 1.24 g of anhydrous potassium carbonate was added as a basic reagent to neutralize the acidic byproducts generated during the reaction under the condition of 20 mL of dichloromethane as solvent. The mixture was heated under reflux at 80 °C for 6 hours to promote the substitution of chlorine with amino groups. The reaction was monitored by thin-layer chromatography (TLC). After the reaction was completed, the mixture was cooled to room temperature, extracted with water and ethyl acetate, and the organic phase was retained. Anhydrous sodium sulfate was added to the liquid for drying, filtration, and rotary evaporation concentration. The crude product was purified by silica gel column chromatography to finally obtain 2-chloro-N-(5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)pyrimidine-4-amine (4), i.e., key intermediate 4;

[0066]

[0067] Step 4: Preparation of N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-2-(4-(methanesulfonyl)phenyl)pyrimidin-4-amine

[0068]

[0069] 0.3 g of 2-chloro-N-(5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)pyrimidin-4-amine (4) prepared in step 3 and 0.27 g of 4-methanesulfonylphenylboronic acid were dissolved in 20 mL of anhydrous dioxane, and 0.37 g of cesium carbonate was added. Subsequently, 0.5 g of Pd(PPh3)Cl2 was added to promote the coupling reaction. The reaction was heated at 95 °C for 8 h. The reaction solution was cooled to room temperature, and the supernatant was extracted with water and ethyl acetate. The supernatant was dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation. Finally, the target product was purified by silica gel column chromatography to obtain the final target compound.

[0070] TOF MS ES+(m / z):(M+H)+,calcd for C 23 H 27 FN6O2S:471.1978,found,471.1991.HPLC(CHCN / KHPO:=1:1):tR:9.142min,puity95.9879%, 1 H NMR(400MHz, Acetonitrile-d3)δ9.35(s,1H),8.79(s,1H),8.76(d,J=8.5Hz,2H),8.04(d,J=8.0Hz,2H),7.67 (d,J=9.3Hz,2H),7.63(d,J=9.9Hz,1H),3.56(s,2H),3.10(s,3H),2.53-2.33(m,10H),1.00(t,J=7.2Hz,3H). 13 C NMR(151MHz, Acetonitrile-d3)δ154.47,150.72,149.44,143.47,142.31,136.46,135.21,129.02(2 C),128.27(2C),124.41,124.21,117.90,59.35,53.37,53.19(2C),52.43(2C),44.13,40.96,11.99.

[0071] Example 2

[0072] The preparation of N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-2-(4-(isopropylsulfonyl)phenyl)pyrimidin-4-amine was performed in the same manner as in Example 1, except that 4-methanesulfonylphenylboronic acid in step four was replaced with an equimolar amount of 4-isopropylsulfonylphenylboronic acid.

[0073] TOF MS ES+(m / z):(M+H)+,calcd for C 25 H 31 FN6O2S: 499.2286, found, 499.2291. HPLC (CHCN / KHPO: = 1:1): tR: 10.896min, purity: 96.6224%, 1 H NMR(400MHz, Acetonitrile-d3)δ9.41(s,1H),8.84(d,J=8.7Hz,3H),8.80(s,1H),8.03(d,J=8.6Hz,2H),7.70(d ,J=6.8Hz,2H),3.61(s,2H),3.39–3.32(m,1H),2.58–2.34(m,10H),1.28(d,J=6.8Hz,6H),1.04(t,J=7.2Hz,3H). 13 C NMR(151MHz,DMSO-d6)δ153.69,149.92,149.44,147.04,142.76,138.15,136.66,134.52,129.65(2C) ,128.72(2C),124.28,123.89,118.36,58.66,54.70,52.92,52.79(2C),52.04(2C),15.67(2C),12.42.

[0074] Example 3

[0075] The preparation of 4-(4-((5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidin-2-yl)-N,N-dimethylbenzenesulfonamide was carried out in the same manner as in Example 1, except that 4-methanesulfonylbenzeneboronic acid in step four was replaced with an equimolar amount of 4-(N,N-dimethylsulfonyl)benzeneboronic acid.

[0076] TOF MS ES+(m / z):(M+H)+,calcd for C 24 H 30FN7O2S: 500.2244, found, 500.2270, HPLC (CHCN / KHPO: = 1:1): tR: 11.366min, purity: 98.0621%, 1 H NMR(400MHz, Acetonitrile-d3)δ9.41(s,1H),8.84(s,1H),8.81(d,J=4.6Hz,3H),7.95(d,J=8.6H z,2H),7.71(d,J=6.7Hz,2H),3.62(s,2H),2.73(s,6H),2.57–2.37(m,10H),1.05(t,J=7.2Hz,3H). 13 C NMR(151MHz,DMSO-d6)δ153.84,149.87,149.48,147.06,142.03,136.62,136.01,134.47,128.78( 2C),128.55(2C),124.28,123.82,118.38,58.61,52.77(2C),52.71,52.00(2C),38.10(2C),12.29.

[0077] Example 4

[0078] The preparation of N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-2-(4-fluorophenyl)pyrimidin-4-amine was performed in the same manner as in Example 1, except that 4-methanesulfonylphenylboronic acid in step four was replaced with an equimolar amount of 4-fluorophenylboronic acid.

[0079] TOF MS ES+(m / z):(M+H)+,calcd for C 22 H 24 F2N6:411.2109,found,411.2100.HPLC(CHCN / KHPO:=1:1):tR:11.386min,puity:95.5416%, 1 H NMR(400MHz, Acetonitrile-d3)δ9.35(s,1H),8.77(s,1H),8.68(d,J=8.7Hz,2H),7.68(d,J =4.5Hz,3H),7.32(d,J=9.0Hz,2H),3.61(s,2H),2.55–2.37(m,10H),1.05(t,J=7.1Hz,3H).

[0080] Example 5

[0081] The preparation of N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-2-(4-(trifluoromethyl)phenyl)pyrimidin-4-amine was performed in the same manner as in Example 1, except that 4-methanesulfonylphenylboronic acid in step four was replaced with an equimolar amount of 4-trifluoromethylphenylboronic acid.

[0082] TOF MS ES+(m / z):(M+H)+,calcd for C 23 H 24 F4N6:461.2077,found,461.2072,HPLC(CHCN / KHPO:=1:1):tR:13.746min,puity:96.2696%, 1 H NMR(400MHz, Acetonitrile-d3)δ9.38(s,1H),8.79(d,J=7.2Hz,3H),7.88(d,J=8.2Hz, 2H),7.69(q,J=9.4Hz,3H),3.61(s,2H),2.57–2.35(m,10H),1.04(t,J=7.2Hz,3H).13C NMR(151MHz,DMSO)δ160.81,155.87,149.44,149.05,147.02,135.86,133.42,130.41,129.77(2 C),124.08,123.36,118.32,114.95(2C),63.72(2C),58.70,52.79,52.03(2C),15.12(2C),0.58.

[0083] Example 6

[0084] The preparation of N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-2-(4-(trifluoromethoxy)phenyl)pyrimidin-4-amine was performed in the same manner as in Example 1, except that 4-methanesulfonylphenylboronic acid in step four was replaced with an equimolar amount of 4-trifluoromethoxyphenylboronic acid.

[0085] TOF MS ES+(m / z):(M+H)+,calcd for C 23 H 24 F4N6O: 477.2026, found, 477.2029, HPLC (CHCN / KHPO: = 1:1): tR: 13.949min, puity: 96.7041%, 1H NMR(400MHz, Acetonitrile-d3)δ9.35(s,1H),8.76(s,1H),8.70(d,J=8.9Hz,2H),7.67(d,J=5 .1Hz,2H),7.47(d,J=8.2Hz,3H),3.60(s,2H),2.56–2.35(m,10H),1.04(t,J=7.2Hz,3H).13CNM R(151MHz,DMSO)δ153.20,149.11,149.09,148.52,148.39,145.95,136.08,135.27,133.03,1 29.01(2C),123.10,122.61,120.50(2C),117.28,57.61,51.84(2C),51.72(2C),50.97,11.34.

[0086] Example 7

[0087] The preparation of NN-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-2-(4-(methoxymethyl)phenyl)pyrimidine-4-amine was carried out in the same manner as in Example 1, except that 4-methanesulfonylphenylboronic acid in step four was replaced with an equimolar amount of 4-(methoxymethyl)phenylboronic acid.

[0088] TOF MS ES+(m / z):(M+H)+,calcd for C 24 H 29 FN6O: 437.2414, found, 437.2444, HPLC (CHCN / KHPO: = 1:1): tR: 10.446min, puity: 97.9700%, 1 H NMR (400MHz, Acetonitrile-d3) δ9.36 (s, 1H), 8.78 (s, 1H), 8.62 (d, J = 8.4Hz, 3H), 7.67 (d, J = 3.3Hz, 2H),7.53(d,J=8.5Hz,2H),3.60(s,3H),3.42(s,3H),2.54–2.34(m,10H),1.04(t,J=7.1Hz,3H).13C NMR (151MHz, DMSO) δ155.50,149.40,149.37,147.03,140.91,137.15,136.16,133.92,128.15,128 .08(2C),124.20(2C),123.54,118.32,73.75,58.69,58.17(2C),52.95(2C),52.81,52.05,12.45.

[0089] Example 8

[0090] The preparation of 4-(4-((5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidin-2-yl)benzaldehyde was carried out in the same manner as in Example 1, except that 4-methanesulfonylphenylboronic acid in step four was replaced with an equimolar amount of 4-carboxyphenylboronic acid.

[0091] TOF MS ES+(m / z):(M+H)+,calcd for C 23 H 25 FN6O: 421.2152, found, 421.2186, HPLC (CHCN / KHPO: = 1:1): tR: 10.076min, puity: 96.2183%, 1 H NMR (400MHz, Acetonitrile-d3) δ10.12(s,1H),9.40(s,1H),8.81(d,J=8.3Hz,4H),8.07(d,J=7 .9Hz,3H),7.71(t,J=8.4Hz,2H),3.62(s,2H),2.43(t,J=7.2Hz,10H),1.06(d,J=7.2Hz,3H).13C NMR(151MHz, Acetonitrile-d3)δ156.68,149.99,149.35,147.68,140.88,135.97,135.77 ,134.40,129.93(2C),128.33(2C),124.06,123.84,59.31,53.05(2C),52.39(2C),21.01.

[0092] Example 9

[0093] The preparation of 1-(4-(4-((5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidin-2-yl)phenyl)ethyl ketone was carried out in the same manner as in Example 1, except that 4-methanesulfonylphenylboronic acid in step four was replaced with an equimolar amount of 4-ethyl ketone phenylboronic acid.

[0094] TOF MS ES+(m / z):(M+H)+,calcd for C 24 H 27 FN6O: 435.2309, found, 435.2282, HPLC (CHCN / KHPO: = 1:1): tR: 10.249min, puity: 97.4835%, 1H NMR(400MHz, Acetonitrile-d3)δ9.40(s,1H),8.82(s,1H),8.73(d,J=8.5Hz,2H),8.14(d,J=8.5Hz,3H),7.7 0(d,J=6.1Hz,2H),3.66(s,2H),2.74–2.67(m,5H),2.66(s,3H),2.66–2.59(m,5H),1.16(t,J=7.2Hz,3H).13C NMR(151MHz,DMSO)δ155.82,149.40,149.26,147.02,140.34,136.07,135.33,133.78,129.83( 2C),128.12(2C),124.17,123.49,118.32,58.73,53.02(2C),52.86(2C),52.07,21.47,12.51.

[0095] Example 10

[0096] The preparation of N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-2-(4-isopropoxyphenyl)pyrimidine-4-amine was performed in the same manner as in Example 1, except that 4-methanesulfonylphenylboronic acid in step four was replaced with an equimolar amount of 4-isopropoxyphenylboronic acid.

[0097] TOF MS ES+(m / z):(M+H)+,calcd for C 25 H 31 FN6O: 451.2622, found, 465.2642, HPLC (CHCN / KHPO: = 1:1): tR: 9.499min, puity: 95.5692%, 1 H NMR(400MHz, Acetonitrile-d3)δ9.30(s,1H),8.74(s,1H),8.54(d,J=8.9Hz,3H),7.64(d,J=3.0Hz,2H),7.07(d,J =8.9Hz,2H),4.74(p,J=6.1Hz,1H),3.59(s,2H),2.55–2.35(m,10H),1.38(d,J=6.0Hz,6H),1.04(t,J=7.2Hz,3H). 13C NMR(151MHz, Acetonitrile-d3)δ154.97,150.60,149.40,139.56,137.87,136.33,135.40,130.40(2C),129 .09(2C),128.82,124.22,117.90,114.09,59.53,59.23,53.11(2C),52.91,52.45,40.94(2C),18.41,11.72.

[0098] Example 11

[0099] The preparation of 2-(4-ethoxyphenyl)-N-(5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoropyrimidine-4-amine was carried out in the same manner as in Example 1, except that 4-methanesulfonylphenylboronic acid in step four was replaced with an equimolar amount of 4-ethoxyphenylboronic acid.

[0100] TOF MS ES+(m / z):(M+H)+,calcd for C 24 H 29 FN6O: 437.2465, found, 437.2440, HPLC (CHCN / KHPO: = 1:1): tR: 12.005min, puity: 95.2710%, 1 H NMR (400MHz, Acetonitrile-d3) δ9.31(d,J=1.8Hz,1H),8.74(s,1H),8.55(d,J=8.9Hz,2H),7.66–7.64(d,2H),7.08(d ,J=8.9Hz,2H),4.16(q,J=7.0Hz,2H),3.60(s,2H),2.57–2.37(m,10H),1.46–1.42(t,3H),1.05(t,J=7.1,1.7Hz,3H). 13 C NMR(151MHz, Acetonitrile-d3)δ161.36,156.66,149.80,149.39(2C),147.70,139.45,135.62,131.12 ,129.95(2C),124.03,117.89(2C),115.01,64.10,59.62,59.35,53.11(2C),52.40(2C),14.66,11.83.

[0101] Example 12

[0102] The preparation of N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-2-(4-methoxyphenyl)pyrimidin-4-amine was performed in the same manner as in Example 1, except that 4-methanesulfonylphenylboronic acid in step four was replaced with an equimolar amount of 4-methoxyphenylboronic acid.

[0103] TOF MS ES+(m / z):(M+H)+,calcd for C 23 H 27 FN6O: 423.2309, found, 423.2248, HPLC (CHCN / KHPO: = 1:1): tR: 10.612min, puity: 98.2115%, 1 H NMR(400MHz, Acetonitrile-d3)δ9.31(s,1H),8.75(s,1H),8.57(d,J=8.8Hz,3H),7.65(d,J=3.8H z,2H),7.11(d,J=8.9Hz,2H),3.90(s,3H),3.59(s,2H),2.55–2.35(m,10H),1.04(t,J=7.2Hz,4H). 13 C NMR(151MHz, Acetonitrile-d3)δ162.03,156.64,149.83,149.40,147.71,135.64,134.11,131.29,1 29.95(2C),124.04,117.90,114.56(2C),59.36,55.68(2C),53.12(2C),52.41,40.96,14.08,11.84.

[0104] Example 13

[0105] The preparation of methyl 4'-(4-((5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidin-2-yl)-[1,1'-biphenyl]-4-carboxylate was carried out in the same manner as in Example 1, except that 4-methanesulfonylphenylboronic acid in step four was replaced with an equimolar amount of (methyl 4-carboxylate)-[1,1'-biphenyl]-4-boronic acid.

[0106] TOF MS ES+(m / z):(M+H)+,calcd for C 30 H 31 FN6O2:527.2571,found,527.2567,HPLC(CHCN / KHPO:=1:1):tR:14.406min,puity:96.1706%, 1HNMR (400MHz, DMSO-d6) δ9.50(s,1H),8.95(s,1H),8.69(d,J=8.2Hz,2H),8.08(d,J=8.3Hz,2H),7.96(dd, J=8.5,2.8Hz,5H),7.80–7.69(m,3H),3.90(s,3H),3.62(s,2H),2.49–2.19(m,10H),0.97(d,J=7.1Hz,3H). 13 C NMR(151MHz, Acetonitrile-d3)δ167.07,155.93,150.26,149.42,147.75,145.16,141.46,138.60,136.04,134.72,130.52(2C ),130.01,128.98(2C),128.07(2C),127.62(2C),124.13,124.11,117.90,59.39,53.38(2C),53.19,52.42(2C),52.34,11.99.

[0107] Example 14

[0108] The preparation of N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-2-(p-tolyl)pyrimidin-4-amine was performed in the same manner as in Example 1, except that 4-methanesulfonylphenylboronic acid in step four was replaced with an equimolar amount of 4-methylphenylboronic acid.

[0109] TOF MS ES+(m / z):(M+H)+,calcd for C 23 H 27 FN6:407.2359,found,407.2301,HPLC(CHCN / KHPO:=1:1):tR:11.739min,puity:96.2503%, 1 H NMR(400MHz, Acetonitrile-d3)δ9.32(s,1H),8.74(s,1H),8.51(d,J=8.2Hz,3H),7.65(d,J=3.6Hz,2H),7.3 8(d,J=8.0Hz,2H),3.58(s,2H),2.63–2.45(m,6H),2.44(s,3H),2.36(q,J=7.2Hz,4H),1.03(t,J=7.2Hz,3H). 13C NMR(151MHz,DMSO-d6)δ155.82,149.40,149.26,147.02,140.34,136.07,135.33,133.78,129.8 3(2C),128.12(2C),124.17,123.49,118.32,58.73,53.02(2C),52.86(2C),52.07,21.47,12.51.

[0110] Example 15

[0111] The preparation of 2-(4-ethylphenyl)-N-(5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoropyrimidine-4-amine was carried out in the same manner as in Example 1, except that 4-methanesulfonylphenylboronic acid in step four was replaced with an equimolar amount of 4-ethylphenylboronic acid.

[0112] TOF MS ES+(m / z):(M+H)+,calcd for C 24 H 29 FN6:437.2465,found,421.2518,HPLC(CHCN / KHPO:=1:1):tR:14.894min,puity:95.0913%, 1 H NMR(400MHz, Acetonitrile-d3)δ9.33(s,1H),8.75(s,1H),8.54(d,J=8.3Hz,3H),7.67–7.64(m,2H),7.42(d,J= 7.9Hz,2H),3.60(s,2H),2.76(q,J=7.6Hz,2H),2.57–2.38(m,10H),1.31(t,J=7.6Hz,3H),1.05(t,J=7.2Hz,3H). 13 C NMR (151MHz, Acetonitrile-d3) δ156.65,149.30,147.62,147.18,136.20,135.77,135.75,134.36,1 28.74(2C),128.41(2C),123.95,117.89,59.43,53.50(2C),53.24(2C),52.43,28.91,15.48,12.10.

[0113] Example 16

[0114] The preparation of N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-2-(4-isopropylphenyl)pyrimidin-4-amine was performed in the same manner as in Example 1, except that 4-methanesulfonylphenylboronic acid in step four was replaced with an equimolar amount of 4-isopropylphenylboronic acid.

[0115] TOF MS ES+(m / z):(M+H)+,calcd for C 25 H 31 FN6: 435.2672, found, 435.2649, HPLC (CHCN / KHPO: = 1:1): tR: 13.917min, puity: 95.7677%, 1 H NMR (400MHz, Acetonitrile-d3) δ9.34(s,1H),8.76(s,1H),8.55(d,J=8.3Hz,3H),7.66(d,J=3.5Hz,2H),7.46(d ,J=8.2Hz,2H),3.60(s,2H),3.08–3.01(m,1H),2.56–2.35(m,10H),1.33(d,J=6.9Hz,6H),1.04(t,J=7.2Hz,3H). 13 C NMR(151MHz,DMSO-d6)δ155.85,151.11,149.39,149.23,146.99,135.99,135.72,133.75,128.24(2C) ,127.13(2C),124.06,123.47,118.31,58.66,52.88(2C),52.77(2C),52.02,33.81,24.21(2C),12.37.

[0116] Example 17

[0117] The preparation of N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-2-(4-propylphenyl)pyrimidin-4-amine was performed in the same manner as in Example 1, except that 4-methanesulfonylphenylboronic acid in step four was replaced with an equimolar amount of 4-propylphenylboronic acid.

[0118] TOF MS ES+(m / z):(M+H)+,calcd for C 25 H 31 FN6:447.2672,found,447.2665,HPLC(CHCN / KHPO:=1:1):tR:14.189min,puity:94.4645%, 1H NMR (400MHz, Acetonitrile-d3) δ9.34(s,1H),8.76(s,1H),8.54(d,J=8.3Hz,3H),7.66(d,J=3.5Hz,2H),7.40( d,J=8.2Hz,3H),3.60(s,2H),2.70(d,J=7.8Hz,2H),2.55–2.36(m,10H),1.75–1.69(m,2H),1.06–0.98(m,6H). 13 C NMR(151MHz, Acetonitrile-d3)δ156.71,150.01,149.36,147.71,145.63,136.26,135.80,134.42,129.39( 2C),128.34(2C),124.02,123.95,117.90,59.41,53.42,53.21(2C),52.43(2C),37.97,24.88,13.67,12.03.

[0119] Example 18

[0120] The preparation of 4-(4-((5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidin-2-yl)benzonitrile was carried out in the same manner as in Example 1, except that 4-methanesulfonylphenylboronic acid in step four was replaced with an equimolar amount of 4-formonitrilephenylboronic acid.

[0121] TOF MS ES+(m / z):(M+H)+,calcd for C 23 H 24 FN7:418.2155found,418.2164,HPLC(CHCN / KHPO:=1:1):tR:10.998min,puity:95.4774%, 1 HNMR (400MHz, DMSO-d6) δ9.52(s,1H),8.96(s,1H),8.72(d,J=8.4Hz,2H),8.04(d,J=8.4Hz,2H),7.93(d,J=7. 8Hz,1H),7.77(d,J=5.2Hz,2H),3.61(s,2H),2.46–2.36(m,5H),2.31(q,J=6.9Hz,5H),0.98(t,J=7.1Hz,3H). 13C NMR(151MHz, Acetonitrile-d3)δ154.38150.73,149.41,147.71,142.81,136.44,135.17,133.25(2C),131.7 1,128.78(2C),124.37,124.19,119.35,117.89,113.37,60.53,59.31,53.16(2C),52.43(2C),29.91,11.89.

[0122] Example 19

[0123] The preparation of 4-(4-((5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidin-2-yl)-N,N-dimethylbenzamide was carried out in the same manner as in Example 1, except that 4-methanesulfonylphenylboronic acid in step four was replaced with an equimolar amount of 4-(N,N-dimethylformamide)phenylboronic acid.

[0124] TOF MS ES+(m / z):(M+H)+,calcd for C 25 H 30 FN7O: 464.2574, found, 463.2560, HPLC (CHCN / KHPO: = 1:1): tR: 9.977min, puity: 95.3366%, 1 H NMR((400MHz, Acetonitrile-d3)δ9.37(d,J=2.0Hz,1H),8.78(s,1H),8.67(d,J=8.4,2.0Hz,2H),7.68(d,J=2.3Hz,3H),7.59 (d,J=1.5Hz,2H),7.57(d,J=1.7Hz,1H),3.61(d,J=2.2Hz,2H),3.02(d,J=11.2Hz,6H),2.56–2.38(m,10H),1.07–1.04(m,3H). 13 C NMR (151MHz, DMSO-d6) δ170.27,154.87,149.56,149.45,147.05,138.72,138.30,136.34,134.37,127. 93(2C),126.27(2C),124.26,123.62,118.34,58.65,52.83,52.75(2C),52.02(2C),35.24(2C),12.34.

[0125] Example 20

[0126] The preparation of 2-([1,1'-biphenyl]-4-yl)-N-(5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoropyrimidine-4-amine was carried out in the same manner as in Example 1, except that 4-methanesulfonylphenylboronic acid in step four was replaced with an equimolar amount of [1,1'-biphenyl]-4-boronic acid.

[0127] TOF MS ES+(m / z):(M+H)+,calcd for C 28 H 29 FN6:469.2516,found,469.2519,HPLC(CHCN / KHPO:=1:1):tR:9.142min,puity:95.9879%, 1 H NMR (400MHz, DMSO-d6) δ9.50(s,1H),8.95(s,1H),8.67(d,J=8.3Hz,2H),7.89(d,J=8.2Hz,2H),7.81–7.75(m,4H ),7.54(s,2H),7.43(d,J=7.8Hz,1H),3.62(s,2H),2.47–2.38(m,5H),2.38–2.26(m,5H),0.99(t,J=7.0Hz,3H). 13 C NMR(151MHz,DMSO-d6)δ155.38,149.46,149.43,147.08,142.08,139.88,137.04,136.21,133.96,129.52(2C),12 8.75(2C),128.37,127.45(2C),127.21(2C),124.23,123.57,118.35,58.67,52.88(2C),52.78(2C),52.04,12.39.

[0128] Example 21

[0129] The preparation of 2-(4'-chloro-[1,1'-biphenyl]-4-yl)-N-(5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoropyrimidine-4-amine was carried out in the same manner as in Example 1, except that 4-methanesulfonylphenylboronic acid in step four was replaced with an equimolar amount of 4'-chloro-[1,1'-biphenyl]-4-boronic acid.

[0130] TOF MS ES+(m / z):(M+H)+,calcd for C 28 H 28ClFN6:503.2026,found,503.2094,HPLC(CHCN / KHPO:=1:1):tR:15.448min,puity:98.1275%, 1 HNMR (400MHz, DMSO-d6) δ9.48(s,1H),8.92(s,1H),8.65(d,J=8.4Hz,2H),7.88(d,J=8.4Hz,2H),7.82(d,J=8.4Hz,2H),7.74( q,J=9.4Hz,3H),7.56(d,J=8.4Hz,3H),3.61(s,2H),2.42(q,J=13.3,4.2Hz,5H),2.32(q,J=6.5Hz,5H),0.98(t,J=7.1Hz,3H). 13 C NMR(151MHz,DMSO-d6)δ155.21,149.46(2C),147.07,140.66,138.67,137.36,136.22,134.01,133.21,129.46(2 C),128.97(2C),128.78(2C),127.41(2C),124.22,123.62,118.35,58.69,52.98,52.83(2C),52.06(2C),12.47.

[0131] Example 22

[0132] The preparation of N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-2-(4'-methyl-[1,1'-biphenyl]-4-yl)pyrimidin-4-amine was performed in the same manner as in Example 1, except that in step four, 4-methanesulfonylphenylboronic acid was replaced with an equimolar amount of 4'-methyl-[1,1'-biphenyl]-4-boronic acid.

[0133] TOF MS ES+(m / z):(M+H)+,calcd for C 29 H 31 FN6:483.2662found,487.2672,HPLC(CHCN / KHPO:=1:1):tR:15.190min,puity:95.7946%, 1H NMR(400MHz, Acetonitrile-d3)δ9.38(s,1H),8.81(s,1H),8.70(d,J=8.3Hz,2H),7.84(d,J=8.3Hz,2H ),7.68(d,J=5.5Hz,5H),7.35(d,J=7.8Hz,3H),3.63(s,2H),2.60–2.41(m,13H),1.08(d,J=7.3Hz,3H). 13 CNMR(151MHz, Acetonitrile-d3)δ156.25,150.14,149.40,147.74,142.77,138.39,137.82,137.46,135.93, 134.58,130.21,128.85,127.50,127.32,124.09,124.05,117.90,59.42,53.45,53.23,52.44,20.72,12.05.

[0134] Example 23

[0135] The preparation of (4-(4-((5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidin-2-yl)phenyl)(phenyl)methyl ketone was performed in the same manner as in Example 1, except that 4-methanesulfonylphenylboronic acid in step four was replaced with an equimolar amount of 4-benzoylphenylboronic acid.

[0136] TOF MS ES+(m / z):(M+H)+,calcd for C 29 H 29 FN6O: 497.2465, found, 497.2431, HPLC (CHCN / KHPO: = 1:1): tR: 13.697min, puity: 95.4871%, 1 HNMR (400MHz, DMSO-d6) δ9.53(s,1H),8.93(s,1H),8.73(d,J=8.3Hz,2H),7.93(d,J=8.3Hz,2H),7.82(d,J=7.2Hz,3H),7.77(d,J=7. 4Hz,2H),7.71(d,J=7.6Hz,2H),7.62(d,J=7.6Hz,2H),3.62(s,2H),2.48–2.39(m,5H),2.33(q,J=7.1Hz,5H),0.99(t,J=7.1Hz,3H). 13C NMR(151MHz,DMSO-d6)δ195.95,154.39,149.78,149.46,147.08,141.64,138.35,137.50,136.52,134.35,133.31,130 .66(2C),130.13(2C),129.14(2C),128.12(2C),124.26,123.79,118.37,58.68,52.93,52.80(2C),52.04(2C),12.43.

[0137] Example 24

[0138] The preparation of N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-2-(4-morpholinophenyl)pyrimidin-4-amine was performed in the same manner as in Example 1, except that in step four, 4-methanesulfonylphenylboronic acid was replaced with an equimolar amount of 4-morpholinophenylboronic acid.

[0139] TOF MS ES+(m / z):(M+H)+,calcd for C 26 H 32 FN7O: 478.2731, found, 478.2752, HPLC (CHCN / KHPO: = 1:1): tR: 9.809min, puity: 96.4436%, 1 H NMR (400MHz, DMSO-d6) δ9.37(s,1H),8.83(s,1H),8.43(d,J=8.3Hz,2H),7.68(q,J=9.3Hz,2H),7.08(d,J=8.4H z,2H),3.78(s,4H),3.58(s,2H),3.26(s,4H),2.49–2.38(m,5H),2.31(q,J=6.6Hz,5H),0.98(t,J=6.9Hz,3H). 13 C NMR(151MHz,DMSO-d6)δ156.30,152.77,149.43,148.85,147.02,135.67,133.19,129.28(2C),128.25,12 4.04,123.27,118.32,114.64(2C),66.47(2C),66.43,58.69,52.79,52.04(2C),47.98(2C),47.94,12.41.

[0140] Example 25

[0141] The preparation of N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-2-(4-fluorophenyl)thieno[3,2-d]pyrimidine-4-amine was performed in the same manner as in Example 1, except that in step three, 2,4-dichloropyrimidine was replaced with an equimolar amount of 2,4-dichlorothieno[3,2-d]pyrimidine, and in step four, 4-methanesulfonylphenylboronic acid was replaced with an equimolar amount of 4-fluorophenylboronic acid.

[0142] TOF MS ES+(m / z):(M+H)+,calcd for C 24 H 25 FN6S: 449.1924, found, 449.1927, HPLC (CHCN / KHPO: = 1:1): tR: 10.880min, puity: 95.9452%, 1H NMR(400MHz,Chloroform-d)δ10.54(s,1H),8.46(d,J=8.7Hz,2H),8.28(d,J=5.4Hz,3H),8.17(d,J=8.5Hz,1H) ,7.81(d,J=6.5Hz,1H),7.53(d,J=5.4Hz,1H),7.34(d,J=8.9Hz,3H),2.47–2.27(m,10H),0.98(t,J=7.1Hz,3H). 13 C NMR(151MHz,DMSO-d6)δ163.20,162.40,158.86,155.06,151.92,148.19,139.12,136.50,134.93,130.5 0,130.44,128.94,124.69,115.97,115.82,115.30,115.09,59.26,52.96(2C),52.79(2C),52.05,12.43.

[0143] Example 26

[0144] The preparation of N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-2-(4-methoxyphenyl)thieno[3,2-d]pyrimidine-4-amine was performed in the same manner as in Example 1, except that in step three, 2,4-dichloropyrimidine was replaced with an equimolar amount of 2,4-dichlorothieno[3,2-d]pyrimidine, and in step four, 4-methanesulfonylphenylboronic acid was replaced with an equimolar amount of 4-methoxyphenylboronic acid.

[0145] TOF MS ES+(m / z):(M+H)+,calcd for C 25 H 28N6OS:461.2124,found,461.2119,HPLC(CHCN / KHPO:=1:1):tR:9.563min,puity:97.0245%, 1 H NMR (400MHz, Acetonitrile-d3) δ8.46(d,J=8.9Hz,2H),8.38(d,J=8.0Hz,1H),8.26(d,J=1.8Hz,1H),8.03(d,J=5.4Hz,1H),7.82(d,J=8.5Hz ,1H),7.46(d,J=5.4Hz,1H),7.08(d,J=8.9Hz,2H),3.89(s,3H),3.50(s,2H),2.54–2.39(m,6H),2.35(q,J=7.2Hz,4H),1.02(t,J=7.2Hz,3H).

[0146] Example 27

[0147] The preparation of N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-2-(p-tolyl)thieno[3,2-d]pyrimidine-4-amine was performed in the same manner as in Example 1, except that in step three, 2,4-dichloropyrimidine was replaced with an equimolar amount of 2,4-dichlorothieno[3,2-d]pyrimidine, and in step four, 4-methanesulfonylphenylboronic acid was replaced with an equimolar amount of 4-methylphenylboronic acid.

[0148] TOF MS ES+(m / z):(M+H)+,calcd for C 25 H 28 N6S: 445.2174, found, 445.2127, HPLC (CHCN / KHPO: = 1:1): tR: 10.547min, puity: 97.6186%, 1 H NMR (400MHz, DMSO-d6) δ10.48(s,1H),8.32(d,J=7.9Hz,2H),8.26(d,J=6.8Hz,2H),8.20(d,J=8.5Hz,1H),7.80(d,J=8.6,2.4Hz ,1H),7.52(d,J=5.6Hz,1H),7.33(d,J=7.9Hz,2H),3.48(s,2H),2.48–2.36(m,8H),2.31(q,J=7.2Hz,5H),0.97(t,J=7.1Hz,3H). 13C NMR(151MHz, Acetonitrile-d3)δ162.81,155.02,148.78,148.77,141.07,139.39,136.18,134.77,129.80( 2C),129.75(2C),128.55(2C),125.36,117.89,115.46,114.83,59.77,53.49,53.24(2C),52.44(2C),12.07.

[0149] Example 28

[0150] The preparation of N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-2-(4-isopropylphenyl)thieno[3,2-d]pyrimidine-4-amine was performed in the same manner as in Example 1, except that in step three, 2,4-dichloropyrimidine was replaced with an equimolar amount of 2,4-dichlorothieno[3,2-d]pyrimidine, and in step four, 4-methanesulfonylphenylboronic acid was replaced with an equimolar amount of 4-isopropylphenylboronic acid.

[0151] TOF MS ES+(m / z):(M+H)+,calcd for C 27 H 32 N6S:473.2487,found,473.2456,HPLC(CHCN / KHPO:=1:1):tR:10.597min,puity:99.5710%, 1 H NMR (400MHz, DMSO-d6) δ10.46(s,1H),8.36(d,J=7.7Hz,2H),8.28(d,J=6.1Hz,2H),8.23(s,1H),7.82(d,J=9.0Hz,1H),7.53(d,J=5.3Hz,2 H),7.41(d,J=8.0Hz,1H),3.50(s,2H),3.01–2.94(m,1H),2.47–2.38(m,6H),2.37–2.29(m,4H),1.28–1.25(m,6H),0.99(t,J=6.5Hz,3H).

[0152] Example 29

[0153] The preparation of 4-(4-((5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)thieno[3,2-d]pyrimidin-2-yl)benzonitrile was carried out in the same manner as in Example 1, except that 2,4-dichloropyrimidine in step 3 was replaced with an equimolar amount of 2,4-dichlorothieno[3,2-d]pyrimidine, and 4-methanesulfonylphenylboronic acid in step 4 was replaced with an equimolar amount of 4-formonitrilephenylboronic acid.

[0154] TOF MS ES+(m / z):(M+H)+,calcd for C 25 H 25 N7S: 456.1970, found, 456.1929, HPLC (CHCN / KHPO: = 1:1): tR: 9.279min, puity: 96.0106%, 1 H NMR (400MHz, DMSO-d6) δ10.97(s,1H),8.43(d,J=1.8Hz,1H),8.32(d,J=5.6,2.5Hz,2H),8.28(d,J=2.3Hz,2H),8.00(d,J=8.4Hz,1H),7.92(d ,J=1.6Hz,1H),7.78(d,J=2.0Hz,1H),7.43(dd,J=5.6,1.7Hz,1H),3.48(s,2H),2.45–2.37(m,5H),2.35–2.27(m,5H),0.97(t,J=7.0Hz,3H). 13 C NMR(151MHz, Acetonitrile-d3)δ163.36,156.61,156.24,151.08,148.50,139.58(2C),136.68(2C),134.99,1 33.08,131.71,130.55,129.08,125.41,124.33,117.89,115.36,59.50,53.11(2C),53.04,52.45(2C),11.73.

[0155] Example 30

[0156] The preparation of 2-([1,1'-biphenyl]-4-yl)-N-(5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)thieno[3,2-d]pyrimidin-4-amine was carried out in the same manner as in Example 1, except that in step three, 2,4-dichloropyrimidinium was replaced with an equimolar amount of 2,4-dichlorothieno[3,2-d]pyrimidinium, and in step four, 4-methanesulfonylphenylboronic acid was replaced with an equimolar amount of [1,1'-biphenyl]-4-boronic acid.

[0157] TOF MS ES+(m / z):(M+H)+,calcd for C 30 H 30 N6S:507.2331,found,507.2330,HPLC(CHCN / KHPO:=1:1):tR:10.020min,puity:97.4488%,1 H NMR (400MHz, Acetonitrile-d3) δ10.52(s,1H),8.53(d,J=8.0Hz,2H),8.29(s,2H),8.24(d,J=8.5Hz,1H),7.85(d,J=8.1Hz,3H),7.77(d, J=7.7Hz,2H),7.56(d,J=5.3Hz,1H),7.50(d,J=7.4Hz,2H),7.42(d,J=7.0Hz,2H),3.50(s,2H),2.45–2.29(m,10H),0.98(t,J=7.1Hz,3H).

[0158] Example 31

[0159] The preparation of 2-(4'-chloro-[1,1'-biphenyl]-4-yl)-N-(5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)thieno[3,2-d]pyrimidine-4-amine was performed in the same manner as in Example 1, except that in step three, 2,4-dichloropyrimidine was replaced with an equimolar amount of 2,4-dichlorothieno[3,2-d]pyrimidine, and in step four, 4-methanesulfonylphenylboronic acid was replaced with an equimolar amount of 4'-chloro-[1,1'-biphenyl]-4-boronic acid.

[0160] TOF MS ES+(m / z):(M+H)+,calcd for C 25 H 31 FN6O2S: 541.1941, found, 541.1921, HPLC (CHCN / KHPO: = 1:1): tR: 15.040min, puity: 94.7376%, 1 H NMR(400MHz, DMSO-d6)δ10.56(s,1H),8.52(dd,J=8.5,1.8Hz,2H),8.31–8.28(m,2H),8.23(d,J=8.5H z,1H),7.87–7.80(m,5H),7.58–7.55(m,3H),3.50(s,2H),2.48–2.25(m,10H),0.98(t,J=7.1Hz,3H). 13C NMR(151MHz,DMSO-d6)δ161.40,158.28,153.98,150.92,147.10,139.65,138.00,137.73,136.77,135.38,132.08,128.37,1 27.85(2C),127.81(2C),126.16,123.68,114.39,113.99,58.22,54.32,51.93,51.74(2C),50.99(2C),39.44,39.30,11.39.

[0161] Example 32

[0162] The preparation of N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-2-(4'-methyl-[1,1'-biphenyl]-4-yl)thieno[3,2-d]pyrimidine-4-amine was performed in the same manner as in Example 1, except that in step three, 2,4-dichloropyrimidine was replaced with an equimolar amount of 2,4-dichlorothieno[3,2-d]pyrimidine, and in step four, 4-methanesulfonylphenylboronic acid was replaced with an equimolar amount of 4'-methyl-[1,1'-biphenyl]-4-boronic acid.

[0163] TOF MS ES+(m / z):(M+H)+,calcd for C 31 H 32 N6S:521.2487,found,531.2440,HPLC(CHCN / KHPO:=1:1):tR:14.613min,puity:96.7902%, 1 HNMR (400MHz, DMSO-d6) δ10.54(s,1H),8.50(d,J=8.4Hz,2H),8.28(d,J=5.6Hz,2H),8.24(d,J=8.9Hz,1H),7.83(d,J=1.9Hz,2H),7.81(d,J=1.8 Hz,1H),7.67(d,J=8.2Hz,2H),7.55(d,J=5.4Hz,1H),7.31(d,J=7.3Hz,2 H), 5.76 (s, 1H), 3.49 (s, 2H), 2.48–2.27 (m, 13H), 0.98 (t, J = 7.2Hz, 3H).

[0164] Example 33

[0165] The preparation of N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-2-(4-morpholinophenyl)thieno[3,2-d]pyrimidine-4-amine was performed in the same manner as in Example 1, except that in step three, 2,4-dichloropyrimidine was replaced with an equimolar amount of 2,4-dichlorothieno[3,2-d]pyrimidine, and in step four, 4-methanesulfonylphenylboronic acid was replaced with an equimolar amount of 4-morpholinophenylboronic acid.

[0166] TOF MS ES+(m / z):(M+H)+,calcd for C 25 H 31 FN6O2S: 516.2546, found, 516.2548, HPLC (CHCN / KHPO: = 1:1): tR: 8.978min, puity: 96.3620%, 1 HNMR (400MHz, DMSO-d6) δ10.40(s,1H),8.30(d,J=8.7Hz,3H),8.22(d,J=5.4Hz,2H),7.79(d,J=8.5Hz,1H),7.48(d,J=5.4Hz,1H),7.06(d, J=8.8Hz,2H),3.78–3.74(m,4H),3.48(s,2H),3.25–3.22(m,4H),2.39(q,J=12.2,6.1Hz,6H),2.30(q,J=7.0Hz,4H),0.97(t,J=7.1Hz,4H). 13 C NMR(151MHz,DMSO-d6)δ162.55,159.99,154.83,152.82,152.13,148.19,139.01,135.98,129.31(2C),128.72,1 28.65,124.64,114.93,114.57(2C),114.35,66.47(2C),59.30,53.02,52.83(2C),52.07(2C),48.08(2C),12.50.

[0167] Example 34

[0168] The preparation of N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-(4-fluorophenyl)pyrazolo[1,5-a]pyrimidine-7-amine was carried out in the same manner as in Example 1, except that 2,4-dichloropyrimidine in step three was replaced with an equimolar amount of 5,7-dichloropyrazolopyrimidine, and 4-methanesulfonylphenylboronic acid in step four was replaced with an equimolar amount of 4-fluorophenylboronic acid.

[0169] TOF MS ES+(m / z):(M+H)+,calcd for C 24 H 26 FN7: 432.2312, found, 432.2319, HPLC (CHCN / KHPO: = 1:1): tR: 11.148min, puity: 96.1276%, 1 H NMR (400MHz, Chloroform-d) δ10.75(s,1H),8.35(s,1H),8.28(d,J=2.1Hz,1H),8.00(s,2H),7.75(d,J=10.4 Hz,2H),7.64(d,J=8.4Hz,2H),6.61(d,J=2.1Hz,1H),3.47(s,2H),2.40–2.27(m,10H),0.97(t,J=7.1Hz,3H).

[0170] Example 35

[0171] The preparation of N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-(4-(trifluoromethyl)phenyl)pyrazolo[1,5-a]pyrimidine-7-amine was performed in the same manner as in Example 1, except that in step three, 2,4-dichloropyrimidine was replaced with an equimolar amount of 5,7-dichloropyrazolopyrimidine, and in step four, 4-methanesulfonylphenylboronic acid was replaced with an equimolar amount of 4-trifluoromethylphenylboronic acid.

[0172] TOF MS ES+(m / z):(M+H)+,calcd for C 25 H 26 F3N7:482.2280,found,482.2273,HPLC(CHCN / KHPO:=1:1):tR:9.541min,puity:96.0446%, 1 HNMR (400MHz, DMSO-d6) δ10.76(s,1H),8.35(d,J=7.9Hz,2H),8.28(s,1H),7.98(d,J=12.6Hz,3H),7.75(d,J=8.4Hz,1H),7 .69(d,J=8.1Hz,1H),7.64(d,J=8.4Hz,1H),6.61(d,J=2.2Hz,1H),3.47(s,2H),2.47–2.24(m,10H),0.97(t,J=7.1Hz,3H). 13C NMR(151MHz, Acetonitrile-d3)δ151.88,151.63,148.46(2C),148.00,144.52(2C),143.52, 139.81(2C),114.37,96.49,91.69,59.43,53.12,53.06(2C),52.45(2C),11.75,1.31,1.17.

[0173] Example 36

[0174] The preparation of N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-(p-tolyl)pyrazolo[1,5-a]pyrimidine-7-amine was performed in the same manner as in Example 1, except that in step three, 2,4-dichloropyrimidine was replaced with an equimolar amount of 5,7-dichloropyrazolopyrimidine, and in step four, 4-methanesulfonylphenylboronic acid was replaced with an equimolar amount of 4-methylphenylboronic acid.

[0175] TOF MS ES+(m / z):(M+H)+,calcd for C 25 H 29 N7: 428.2563, found, 428.2542, HPLC (CHCN / KHPO: = 1:1): tR: 10.604min, puity: 96.1434%, 1 H NMR (400MHz, Acetonitrile-d3) δ8.41(d,J=8.1Hz,3H),8.27(s,2H),8.06(d,J=5.5Hz,1H),7.83(dd,J=8.4,2. 3Hz,1H),7.49(d,J=5.3Hz,1H),7.37(d,J=7.9Hz,2H),3.51(s,2H),2.58–2.28(m,13H),1.03(t,J=7.2Hz,3H). 13 C NMR(151MHz,DMSO-d6)δ162.48,159.84,154.98,152.03,148.15,140.26,139.07,136.28,135.78,129.62(2 C),128.81,128.20(2C),124.70,115.15,114.96,59.28,52.99,52.81(2C),52.05(2C),21.47,12.47,0.57.

[0176] Example 37

[0177] The preparation of N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-(4-isopropoxyphenyl)pyrazolo[1,5-a]pyrimidine-7-amine was performed in the same manner as in Example 1, except that in step three, 2,4-dichloropyrimidine was replaced with an equimolar amount of 5,7-dichloropyrazolopyrimidine, and in step four, 4-methanesulfonylphenylboronic acid was replaced with an equimolar amount of 4-isopropoxyphenylboronic acid.

[0178] TOF MS ES+(m / z):(M+H)+,calcd for C 27 H 33 N7O: 491.2825, found, 491.2798, HPLC (CHCN / KHPO: = 1:1): tR: 12.058min, puity: 97.1030%, 1 H NMR (400MHz, Acetonitrile-d3) δ8.09(d,J=18.1Hz,1H),7.80(d,J=8.5Hz,2H),7.52(s,1H),7.05(d,J=8.1Hz,1H),6.88(d,J=8.4Hz,2H),6. 80(d,J=7.3Hz,1H),6.25(d,J=14.5Hz,1H),4.68–4.60(m,1H),3.29(s,2H),2.49–2.26(m,10H),1.30(d,J=6.3Hz,6H),1.04(t,J=7.3Hz,3H).

[0179] Example 38

[0180] The preparation of 4-(7-((5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)pyrazolo[1,5-a]pyrimidin-5-yl)benzonitrile was carried out in the same manner as in Example 1, except that 2,4-dichloropyrimidine in step 3 was replaced with an equimolar amount of 5,7-dichloropyrazolopyrimidine, and 4-methanesulfonylphenylboronic acid in step 4 was replaced with an equimolar amount of 4-formonitrilephenylboronic acid.

[0181] TOF MS ES+(m / z):(M+H)+,calcd for C 25 H 26 N8: 439.2346, found, 439.2359, HPLC (CHCN / KHPO: = 1:1): tR: 9.142min, puity: 96.7321%, 1H NMR(400MHz, Acetonitrile-d3)δ9.14(s,1H),8.35(d,J=1.8Hz,1H),8.13(d,J=2.2Hz,2H),8.02(s,2H),7.75(d,J =10.7Hz,3H),7.30(d,J=8.4Hz,1H),6.53(d,J=2.2Hz,1H),3.52(s,2H),2.59–2.46(m,10H),1.08(t,J=7.2Hz,3H).

[0182] Example 39

[0183] The preparation of methyl 4-(7-((5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)pyrazolo[1,5-a]pyrimidin-5-yl)benzoate was carried out in the same manner as in Example 1, except that 2,4-dichloropyrimidine in step three was replaced with an equimolar amount of 5,7-dichloropyrazolopyrimidine, and 4-methanesulfonylphenylboronic acid in step four was replaced with an equimolar amount of methyl 4-carboxylate phenylboronic acid.

[0184] TOF MS ES+(m / z):(M+H)+,calcd for C 26 H 29 N7O2: 472.2461, found, 472.2487, HPLC (CHCN / KHPO: = 1:1): tR: 9.142min, puity: 95.9879%, 1 H NMR (400MHz, DMSO-d6) δ10.55(s,1H),8.55(d,J=8.0Hz,1H),8.07(d,J=7.1Hz,1H),7.92(d,J=7.2Hz,4H),7.74(d,J=7.9Hz,1H ),7.56(d,J=5.4Hz,1H),7.43(s,1H),6.65(d,J=37.5Hz,1H),3.89(s,3H),3.50(s,2H),2.48–2.30(m,10H),1.01–0.96(m,3H).

[0185] The structural formulas of the compounds prepared in Examples 1-39 are shown in Table 1.

[0186] Table 1 Structural Formulas of Examples 1-39

[0187]

[0188]

[0189]

[0190] Performance testing

[0191] I. Validation of in vitro anti-tumor cell activity

[0192] (1) The heterocyclic pyrimidine compounds prepared in Examples 1-39 were tested for their in vitro inhibitory activity against breast cancer cells MCF-7, MDA-MB-231, and 4T1. The control was Abemaciclib, and a blank group without the drug was also set up (the same below).

[0193] The testing steps are as follows:

[0194] (a) Plating: When the cell line density in the culture flask reaches 80%, digestion and centrifugation are performed. The supernatant after centrifugation is removed, and the cells at the bottom are retained. Then, 1 mL of culture medium is added to a 5 mL centrifuge tube, and the cell suspension is injected into a counting chamber. Cells are then observed and counted under a microscope. Based on the counting results, the cell suspension concentration is adjusted to 20,000-25,000 cells / mL. Next, 180 μL of PBS is added to rows A and H and column 1 of a 96-well plate, and 180 μL of the well-swept cell suspension is added to rows B and G. Finally, the 96-well plate is incubated at 37°C in a 5% CO2 incubator for 24 h. Drug administration can be performed when the cell density reaches half capacity.

[0195] (b) Drug Addition: There are 5 drug concentrations available. One drug is prepared at 5 concentrations. First, dissolve 1 mg of drug in a 1.5 mL EP tube, then add 20 μL of DMSO (specifically for cell cryopreservation). Use a pipette to mix the drug thoroughly to promote dissolution. Then add 980 μL of serum-free culture medium and mix thoroughly to form a stock solution. Next, prepare the daughter solutions: Take 200 μL of the higher concentration drug solution from a 1.5 mL EP tube and add it to 400 μL of serum-free culture medium, diluting four times in total, each time by a 3-fold ratio. Add the drug sequentially from low to high concentration, adding 20 μL of each concentration to two replicate wells. The seventh well is left untreated as a control. Each 96-well plate can test four drugs. Finally, incubate the 96-well plate at 37°C and 5% CO2 for 72 hours.

[0196] (c) Preparation of MTT solution: Under light-protected conditions, add commercially available MTT powder to a 50 mL aliquot tube and add PBS to bring the volume to 50 mL. Then, filter the solution through a 0.22 μM filter membrane into a centrifuge tube, wrap it with aluminum foil, and store it in a refrigerator at 4°C.

[0197] (d) Adding MTT solution: After 72 hours of drug treatment, remove the liquid from the 96-well plate and add 20 μL of the prepared MTT solution to each well containing cells. Then, incubate the 96-well plate in an incubator for 4 hours. After incubation, remove the MTT solution, add DMSO to each well, mix by magnetic oscillation, and then measure the OD value of each well using an enzyme-linked immunosorbent assay (ELISA) reader.

[0198] (e) Formula for calculating inhibition rate (IR%):

[0199] IR% = (OD) 对照 -OD 样品 ) / (OD 对照 -OD 空白 )×100%

[0200] Table 2 shows the inhibitory activity of heterocyclic pyrimidine compounds prepared in Examples 1-39 on breast cancer cells MCF-7, MDA-MB-231, and 4T1, where NA represents IC50. 50 If the value is greater than 1000, ND indicates that no detection was performed.

[0201] Table 2 Antiproliferative activity (IC50) 50 (Measurement)

[0202]

[0203]

[0204] (2) CDK4 / 6 kinase activity (IC50) 50 (and inhibition rate measurement)

[0205] Prepare a 384-well plate with wells containing the test compound (heterocyclic pyrimidine compounds prepared in Examples 1-39), blank control wells, and positive control wells (Abemaciclib). Add 2.5 μL of kinase solution to the test compound wells, 2.5 μL of kinase buffer to the blank control wells, and 2.5 μL of kinase solution to the positive control wells. Add 2.5 μL of different concentrations of the compound to the test compound wells, 2.5 μL of kinase buffer to the blank control wells, and 10 μL of ALK kinase antibody and EDTA reagent to each well. Centrifuge to mix and incubate at room temperature for 60 min. The final antibody concentration is 2 nM, and the final EDTA concentration is 8 mM. Read the fluorescence values ​​using Envision. Calculate the percentage inhibition rate of the compound.

[0206] Percentage inhibition rate = (Lance signal value - Min) / (Max - Min) × 100;

[0207] In the formula, Min is the Lance signal value without enzyme; Max is the Lance signal value when the enzyme is fully active without inhibitor; the Lance signal value is the Lance signal value of the Max-DMSO control; the data were analyzed by SPSS, MSExcel and Graphpad 5.0 to obtain IC50. 50 .

[0208] Kinase-Glo and The inhibitory activity of the compounds against ALK kinase was tested using the Ultra enzyme activity evaluation method. The results are shown in Table 3. The data in the left column are the CDK4 inhibition rate (%), and the data in the right column are the CDK6 inhibition rate (%).

[0209] Table 3 CDK4 / 6 kinase activity (IC50) 50 (Measurement)

[0210]

[0211]

[0212] As can be seen from the data in Tables 2 and 3, the compounds prepared in the embodiments of the present invention have good in vitro anti-cell proliferation activity and anti-CDK4 / 6 kinase activity. Among them, Examples 28, 36, 37, and 38 showed excellent inhibition rates against CDK4 / 6 kinase. Further kinase IC50 assays were performed on the compounds with higher inhibition rates. 50 The assays revealed that Example 37 exhibited excellent toxic activity against all selected cell lines, comparable to that of positive control drugs. This demonstrates that the compounds prepared in these embodiments of the invention show promise as potential inhibitors of CDK4 / 6 kinases.

[0213] II. Evaluation of Hemolytic Toxicity

[0214] The hemolytic toxicity of the compound prepared in Example 37 was evaluated. Hemolytic toxicity testing is a key indicator for evaluating the stability of a compound on erythrocyte membranes and its blood compatibility. The hemolytic toxicity of Example 37 at different concentrations was assessed using an in vitro hemolysis assay system to provide a safety basis for subsequent in vivo experiments and clinical applications. Hemolytic toxicity evaluation experimental procedures:

[0215] (1) Take 1.5 mL of red blood cells from 2% sheep blood red blood cells for red blood cell extraction.

[0216] (2) Add 0.9% physiological saline to the extracted red blood cells, and then add the test compound to ensure that it is fully dispersed.

[0217] (3) Incubate the treated red blood cells at 37°C for 1 hour. Measure the absorbance A using a UV spectrophotometer and calculate the hemolysis rate using the following formula:

[0218] Hemolysis rate (%) = (A 实验组 -A 阴性对照组 ) / (A 阳性对照组 -A 阴性对照组 )

[0219] Figure 1 The results of the hemolytic safety evaluation of the compound in Example 37 are shown (where 16 represents the concentration of Compound 37 in Example 37 at 16 μg / mL, and so on; the inset shows photographs of the hemolytic experiment results, corresponding to the horizontal axis from left to right). The results show that the hemolysis rate of the compound in Example 37 at 16 μg / mL was 6.61%. The hemolysis rate gradually increased with increasing compound concentration, but the overall hemolytic toxicity remained at a low level. Notably, the hemolysis rate at all tested concentrations was below 17%. This indicates that the compound in Example 37 has a weak destructive effect on the erythrocyte membrane within the set concentration range, exhibiting good blood compatibility. The experimental results suggest that the polar and hydrophobic groups of Example 37 may reduce the destructive effect on the erythrocyte membrane. Furthermore, the low hemolytic toxicity also indicates that Example 37 may have high safety in in vivo applications, and the hemolytic toxicity of Example 37 against cancer is safe.

[0220] III. KM Mouse Toxicity Test

[0221] The KM mouse toxicity test is an important part of drug safety evaluation. The specific procedures are as follows:

[0222] (1) Experimental grouping: KM mice were ear-tagged and then divided into 4 groups of 3 mice each: blank control group, positive Abemaciclib group (Abemaciclib-120mg / kg), low-dose administration group (HXL37-60mg / kg) and high-dose administration group (HXL37-120mg / kg). The compound used in all administration groups was the compound of Example 37.

[0223] (2) Gavage administration: The administration frequency was 24 hours / time. The dosage was 60 mg / kg for the positive control group and the low-dose group, and 120 mg / kg for the high-dose group. The administration solvent was physiological saline. The blank group was given an equal amount of physiological saline.

[0224] (3) Sampling: Seven days after the experiment, no less than 0.5 mL of blood was taken from the eyeballs of various mice and placed in a 1.5 mL EP tube. The tube was then labeled and stored at 4°C for later use. Then, one mouse was randomly selected from each group for dissection. Organs such as heart, liver, spleen, lung, and kidney were collected, soaked in 4% paraformaldehyde fixative, and stored at 4°C for later use.

[0225] Mouse blood sample analysis:

[0226] (1) Centrifuge mouse blood samples at 15000r / min for 10min using a 4℃ centrifuge, take the supernatant, and repeat the centrifugation until there is about 200μL of serum before stopping the centrifugation.

[0227] (2) Take 150 μL of serum and inject it into the blood sample analysis reagent tray. Place it into the automatic multifunctional biochemical analyzer and perform ten preoperative tests. Record the data and analyze it.

[0228] HE staining experiment:

[0229] (1) Tissue treatment: After washing the mouse organs with physiological saline, the water was absorbed and then soaked in 4% formaldehyde solution for 24 hours. After soaking, the organs were dehydrated by 50%, 70%, 80%, and 90% alcohol for 1 hour in sequence.

[0230] (2) Sample clearing and paraffin treatment: The dehydrated tissue was cleared twice in xylene solution, 15 min each time. Then the tissue was soaked in paraffin solution for 2 h.

[0231] (3) Dewaxing of paraffin sections: The paraffin sections were placed in xylene I, xylene II, anhydrous ethanol I, anhydrous ethanol II and 75% alcohol in sequence for 20, 20, 5, 5 and 5 minutes respectively, and finally rinsed with tap water.

[0232] (4) Hematoxylin staining: stain with hematoxylin solution for 3-5 minutes, rinse with tap water after staining, differentiate with differentiation solution, rinse with tap water again, then reflect with blue solution, and rinse with running water.

[0233] (5) Eosin staining: The sections were dehydrated in 85% and 95% alcohol for 5 minutes each, and then stained in eosin staining solution for 5 minutes.

[0234] (6) Dehydration and mounting: The sections were placed in EtOHI-EtOHII-EtOHIII-XyleneI-XyleneII5 for 5 min each, and then mounted with neutral resin.

[0235] (7) Observe the slides under a microscope, take pictures, save the pictures, and analyze the experimental results.

[0236] The results of the in vivo toxicity test of each group in KM mice are shown below. Figure 2 In this study, A represents the biochemical indicators of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and serum creatinine (CREA); B represents the biochemical indicators of total protein (TP), urea (UREA), and blood glucose (GLU); and C represents the biochemical indicators of lactate dehydrogenase (LDH), alkaline phosphatase (ALP), and creatine kinase (CK). HE staining results of various organs and tissues in each group of Kunming mice to assess the biotoxicity of these treatments are shown below. Figure 3As can be seen, the liver function indicators—alanine aminotransferase (ALT) and aspartate aminotransferase (AST)—showed relatively stable and normal levels in both the blank control group and the treatment group of Example 37 before surgery. Under high-dose treatment with Example 37 (120 mg / kg), ALT and AST levels in the treatment group slightly increased, but remained within the safe range. Blood glucose levels were similar in the treatment and control groups before surgery. High-dose treatment with Example 37 slightly increased blood glucose levels, but did not cause significant metabolic disturbances. There was no significant difference in total protein (TP) levels between the Example 37 treatment group and the blank control group. Overall, Example 37 had minimal impact on the physiological indicators of KM mice, with no significant toxic reactions. Although some liver and kidney function indicators, such as ALT, AST, and CK, showed slight increases at high doses (120 mg / kg), these levels did not exceed the normal physiological range and had no significant effect on blood glucose, lipid metabolism, or electrolyte levels. Overall, Example 37 did not cause significant adverse effects on the main biochemical indicators of mice within the normal dose range, further demonstrating its good safety profile in preclinical studies. To assess the toxic effects of Example 37 on the organs of Kunming mice, histological examination of the main organs (liver, kidney, spleen, heart, and lungs) was performed using hematoxylin-eosin (HE) staining. Histological examination of each organ using hematoxylin-eosin staining showed that Example 37 had a relatively mild effect on the mouse organs. At high doses, the liver and kidneys showed very slight cellular degeneration and edema, but none reached the level of serious damage; the heart and lungs showed no obvious damage or lesions, and the overall organ tissue structure remained intact. These results indicate that Example 37 did not cause serious organ damage at high doses and has good safety, providing strong support for further preclinical studies.

[0237] IV. Time Dependency Test

[0238] (1) MCF-7 cells were cultured and seeded according to the aforementioned method.

[0239] (2) Following the method for measuring the MTT of the compound, the samples were prepared at concentrations of 100, 33.33, 11.11, 3.70 and 1.23 μM / ml, respectively, and added to a 96-well plate for three parallel experiments.

[0240] (3) Incubate at 37℃ for 24h, 48h and 72h respectively, and use an enzyme-linked immunosorbent assay reader to test the OD values ​​at 490nm wavelength for 24h, 48h and 72h respectively.

[0241] (4) Calculate the cell inhibition rate based on the absorbance value measured by the MTT method.

[0242] The time-dependent test results of the compound in Example 37 on MCF-7 are shown in [the original text]. Figure 4 As can be seen, the inhibitory effect of Example 37 on the proliferation of MCF-7 cells exhibits significant dose- and time-dependent characteristics. The inhibitory effect on MCF-7 cells becomes more pronounced with increasing concentration. At the maximum concentration of 100 μg / ml, the inhibitory effect on cells becomes more significant with increasing treatment time. At 24 h, the inhibition rate of Example 37 on MCF-7 cells is 58.95%, which is relatively low. After 48 h of treatment, the inhibitory effect significantly increases with increasing concentration, reaching an inhibition rate of 83.64%. Cell viability significantly decreases after 72 h of treatment, demonstrating strong anti-tumor activity. After 72 h of treatment, at a concentration of 33.33 μg / ml, the cell inhibition rate of Example 37 reaches 93.03%, and at the highest concentration of 100 μg / ml, the cell inhibition rate is as high as 94.68%. This indicates that Example 37 has a strong inhibitory effect on MCF-7 cells. It is evident that the inhibitory effect of Example 37 on MCF-7 cells exhibits a significant time-dose dependence, especially under conditions of higher concentrations (≥50 μg / ml) and longer treatment times (≥48 h), where its anti-proliferative effect is more pronounced.

[0243] V. Scratch test to assess cell migration ability

[0244] To evaluate the effect of Example 37 on the migration ability of MCF-7 breast cancer cells, a scratch assay was used to investigate its effect on cell migration. In the experiment, MCF-7 cells were scratched and then treated with different concentrations (1 μm, 2 μm) of the compound of Example 37 (HXL37). The healing of the scratched areas was observed at 0 h and 24 h to determine the effect of Example 37 on MCF-7 wound healing. A blank control group (Control) and a positive Abemaciclib group (Abemaciclib-120 mg / kg) were also set up.

[0245] (1) After digesting and centrifuging the MCF-7 cells that have grown to 80% or more, reselect them and seed them into 6-well plates at 3 ml of cell solution per well. Incubate them in a 37°C incubator for 24 h.

[0246] (2) Use a sterile pipette tip to draw lines in the holes, making 3 lines in each hole, and mark the positions with a marker.

[0247] (3) After washing the cells, take a 0h photo, then add drugs to the cells and incubate them in an incubator for 24h.

[0248] (4) After 24 hours, remove the 6-well plate, wash the cells, and then take pictures of the cell scratches at the corresponding locations based on the locations marked in the 0-hour photo.

[0249] The results of the wound healing experiment of each group treated with MCF-7 are shown in the figure. Figure 5 The results showed that the scratched area of ​​the blank control group (Control) MCF-7 cells gradually shrank after 24 hours, indicating strong cell migration ability. However, in the treatment group of Example 37, cell migration was significantly inhibited, and the degree of scratch healing was significantly reduced, showing a concentration-dependent effect: after low concentration (1 μM) treatment, cell migration ability was inhibited after 24 hours, but there was still a certain healing trend. After high concentration (2 μM) treatment, almost no obvious healing was observed in the scratched area, indicating that Example 37 can effectively inhibit the migration ability of MCF-7 cells. After positive Abemaciclib (2 μM) treatment, no significant cell migration was observed, which can effectively inhibit cell migration. In summary, Example 37 can significantly inhibit the migration ability of MCF-7 cells in a concentration-dependent manner. This result indicates that Example 37, in addition to inhibiting the proliferation of MCF-7 cells, can also effectively inhibit their migration, suggesting that it may have potential application value in inhibiting the invasion and metastasis of breast cancer cells.

[0250] VI. Methods for assessing cell cloning capacity

[0251] (1) Cell culture and drug administration are as described above.

[0252] (2) 48 hours after drug administration, remove the original culture medium, wash the cells with PBS, replace with new culture medium and culture for 7 days, replacing with new culture medium every other day.

[0253] (3) After the blank reaches the required density, the cells are processed by adding 2 mL of 4% paraformaldehyde solution to each well for about 15 min.

[0254] (4) Remove the above solution, wash the cells with PBS, repeat twice, add 1 mL of crystal violet staining solution to each well, and incubate at room temperature for 15 min.

[0255] (5) Remove the crystal violet staining solution, gently rinse each well with distilled water to remove residual dye, and air dry at room temperature. Then use a digital camera to photograph and record the formation of cell clones.

[0256] The cloning experiment results for each group acting on MCF-7 are shown below. Figure 6As can be seen, the cells in the blank control group (Control) formed a large number of clones after 14 days of plate culture. Crystal violet staining clearly showed that the number and size of the clones in the blank control group were both significant. However, in the different concentrations of the treatment group in Example 37, the clone-forming ability of cells gradually decreased from low to high concentrations. Especially in the high-concentration treatment group and the positive control group, the number of clones was significantly reduced, and many clones exhibited irregular morphology and varying sizes, indicating that cell proliferation was inhibited. Statistical analysis revealed that with increasing concentration of the treatment group in Example 37, the number of clones formed showed a concentration-dependent inhibitory effect. This suggests that the compound in Example 37 inhibited the proliferation and clone formation of MCF-7 cells to some extent, possibly by affecting the cell proliferation mechanism or by inducing cell cycle arrest.

[0257] VII. AO Acridine Orange Staining Experiment

[0258] To investigate the inhibitory effect of Example 37 on MCF-7 cells, AO (Acridine Orange) staining was used to assess its ability to induce apoptosis. The drug administration details for each group were as described previously. Specific procedures are as follows:

[0259] (1) Add 1 mL of cells at a density of 4 × 10⁶ cells to each well of a 24-well plate. 4 The cell suspension was placed in a 24-well plate and cultured at 37°C in a 5% CO2 cell culture incubator for 24 hours.

[0260] (2) Aspirate the original culture medium and wash twice with PBS. Add 1 mL of the pre-prepared sample of the required concentration to each well, mix gently, and then place the 24-well plate in a cell culture incubator for 12 h.

[0261] (3) Remove the culture medium from the well plate and wash twice with 1 mL PBS. After rinsing once with 500 μL 1× buffer, add 200 μL of acridine orange: 1× buffer = 1:20 staining solution and incubate in an incubator for 15 min.

[0262] (4) Remove the staining solution from the well plate, wash each well three times with PBS, and add 500 μL of PBS. Observe under a microscope, adjust the filter to 4 / 5, set the laser to blue light, take a picture and save the image.

[0263] The results of the AO staining experiment on MCF-7 in each group are shown below. Figure 7Under a fluorescence microscope, the blank control group (Control) emitted round green fluorescence with clear nuclear structure, indicating a normal cell state. In contrast, the MCF-7 cells treated in Example 37 exhibited typical apoptotic characteristics. After treatment with Example 37, MCF-7 cells showed obvious dose- and time-dependent apoptotic features. With increasing concentration, nuclear condensation, chromatin condensation, and the formation of apoptotic bodies became more pronounced, further demonstrating that Example 37 exerts its anti-tumor effect by inducing apoptosis.

[0264] 8. Mitochondrial membrane potential detection: JC-1 staining method

[0265] To further investigate the effect of Example 37 on the mitochondrial membrane potential (ΔΨ) of MCF-7 cells m The effects of [drug name] were detected using the JC-1 fluorescent probe method. Changes in mitochondrial functional status can be observed through the JC-1 experiment. The drug administration details for each group are as described above, and the specific procedures are as follows:

[0266] (1) Add 1 mL of cells at a density of 4 × 10⁶ cells to each well of a 24-well plate. 4 The cell suspension was cultured in a 37°C, 5% CO2 cell culture incubator for 24 hours.

[0267] (2) Remove the culture medium, wash the cells twice with 1 mL PBS, add 1 mL of JC-1 working solution prepared by mixing 50 μL JC-1 (200×) with 8 mL of ultrapure water, and then incubate in an incubator for 20 min.

[0268] (3) After the time is up, aspirate the JC-1 working solution, wash the cells with 1×JC-1 staining buffer, and repeat the operation twice.

[0269] (4) Add 2 mL of 200×JC-1 staining buffer, place the cells under a fluorescence microscope to observe and photograph the experimental results.

[0270] The results of JC-1 staining experiments on MCF-7 in each group are shown below. Figure 8In the blank control group (Control), the mitochondria of MCF-7 cells mainly exhibited red fluorescence, indicating a high mitochondrial membrane potential and that the cells were in a normal state. However, in the Example 37 treatment group, as the concentration increased from 1 μM to 2 μM, it was clearly observed that the red fluorescence emitted by the cells decreased progressively, while the green fluorescence increased sequentially. Especially at the high dose of Example 37 (2 μM), the red fluorescence was almost completely obscured. The experimental results indicate a significant decrease in mitochondrial membrane potential, further intensifying the apoptosis process, and suggesting that the cells may have entered late-stage apoptosis. The positive Abemaciclib (2 μM) treatment also showed the same experimental phenomenon, indicating that most cells entered the early apoptosis stage. Therefore, Example 37 can significantly reduce the mitochondrial membrane potential of MCF-7 cells in a dose- and time-dependent manner. The decrease in mitochondrial membrane potential occurs in the early stages of apoptosis, suggesting that Example 37 may reduce the mitochondrial membrane potential, thereby inducing apoptosis in MCF-7 cells.

[0271] IX. Annexin V / PI staining method for detecting cell apoptosis

[0272] To evaluate the apoptosis-inducing effect of Example 37 on MCF-7 cells, flow cytometry was performed using Annexin V-FITC / PI double staining. This experiment assessed whether Example 37 could induce MCF-7 cell apoptosis by detecting the proportion of early apoptotic (Annexin V+ / PI-) and late apoptotic / necrotic (Annexin V+ / PI+) cells. The drug administration details for each group were as described above, and the specific procedures are as follows:

[0273] (1) After digesting and centrifuging the cells according to the passage method, count them and add 2.0 × 10⁻⁶ cells to each well of a 6-well plate. 5 Cells were collected and then the 6-well plate was placed in a 37°C, 5% CO2 incubator for 24 hours.

[0274] After the culture was completed, serum-free culture medium, sample and positive drug were added to the wells respectively, and then the 6-well plate was placed in a 37°C, 5% CO2 incubator for 24 hours.

[0275] (2) Remove the 6-well plate, remove the culture medium, and wash twice with PBS. Add 1 mL of EDTA-free trypsin to each well to digest the cells. After the cells have completely detached, add 1 mL of culture medium to stop the digestion, rinse once, and transfer to a 5 mL centrifuge tube. Centrifuge at 1000 rpm for 3 min. Remove the supernatant, add 1 mL of PBS, and gently mix by pipetting. Repeat the washing process twice.

[0276] (3) Under light-protected conditions, wrap the centrifuge tube with aluminum foil, then add 500 μL of buffer solution to the centrifuge tube and gently pipette 8-10 times. Add 5 μL of Annexin V dye to blank 1, 5 μL of PI dye to blank 2, 5 μL of Annexin V and 5 μL of PI dye to blank 3, and add 5 μL of Annexin V and 5 μL of PI dye to the remaining sample wells and positive wells. Incubate in the dark for 15-30 min.

[0277] (4) The cell staining mixture was analyzed using flow cytometry, and the cell count was 1.0 × 10⁻⁶. 4 Each cell was analyzed, and data was recorded.

[0278] The results of the apoptosis experiments on MCF-7 cells in each group are shown below. Figure 9 In the control group, Q1 represented mechanical damage, Q2 late apoptosis, Q3 early apoptosis, and Q4 surviving cells. Most cells in the control group were viable. Annexin V-FITC / PI double staining showed cells primarily distributed in the lower left quadrant (Annexin V- / PI-), indicating normal cell growth and a very low proportion of apoptotic cells. In the low-concentration (1 μM) treatment group, treatment with Example 37 showed that the low concentration of Example 37 slightly induced early apoptosis in MCF-7 cells, with an overall apoptosis rate of 11.91%. In the high-concentration (2 μM) treatment group, after treatment with the high concentration of Example 37, cells clearly entered the apoptosis process, with an early apoptotic cell rate of 7.24%, a late apoptotic cell rate of 5.99%, and a total apoptotic cell rate of 13.23%. In the positive control group (Abemaciclib), early apoptosis accounted for 6.93%, and late apoptosis accounted for 6.37%. The high concentration of Example 37 induced MCF7 cells to enter the apoptosis process, possibly by activating the apoptosis pathway and inhibiting cell proliferation. Flow cytometry analysis showed that Example 37 significantly influenced MCF-7 cell apoptosis in a concentration-dependent manner. The proportion of early apoptotic cells increased with increasing concentration. This result indicates that Example 37 can effectively induce apoptosis in MCF-7 cells, and this process is more pronounced at higher concentrations. The antitumor effect of Example 37 may be partly exerted through the induction of apoptosis, thereby inhibiting the proliferation and growth of tumor cells.

[0279] 10. Flow cytometry for cell cycle detection

[0280] To investigate the effect of Example 37 on the cell cycle of MCF-7 cells, flow cytometry was used to detect changes in cell cycle distribution. In the experiment, MCF-7 cells were treated with different concentrations (1 and 2 μM) of Example 37 for 24 h. PI staining was then used to stain the cells, and the distribution of different cell cycle stages was analyzed. The drug administration details for each group are as described above, and the specific procedures are as follows:

[0281] (1) After digesting, centrifuging and resuspending MCF-7 cells with a cell density of about 80%, add 1×10 cells per well. 6 One cell was seeded into a 6-well plate, 3 mL of cell culture was added to each well, and the plate was incubated in a cell culture incubator for 24 h.

[0282] (2) After 24 hours, the cells were treated with drugs and placed in a cell culture incubator for 24 hours.

[0283] (3) Digest the cells with trypsin without EDTA and then transfer them to 5 mL centrifuge tubes for centrifugation. After centrifugation, wash the cells with 1 mL PBS, then repeat the centrifugation and washing process. Then add 500 μL PBS and 500 μL pre-cooled 70% ethanol solution (ethanol and PBS mixed), and finally place in a 4°C refrigerator for 2 h.

[0284] (4) Take the centrifuge tube out of the refrigerator and centrifuge directly. After centrifugation, wash twice with PBS, then add 500μL PBS and 200μL dye (Rnase A:PI = 1:9) and incubate for 30 min in the dark.

[0285] (5) After incubation, count the cells using flow cytometry at a density of 1.0 × 10⁻⁶. 4 Each cell records data.

[0286] The cell cycle assay results of each group acting on MCF-7 are shown below. Figure 10(Where A: Control group; B: Positive group (Abemaciclib 2μM); C: HXL37 (1μM); D: HXL37 (2μM); E: Proportion of each group at each stage), the results showed that Example 37 had a significant effect on the cell cycle distribution of MCF-7 cells in a concentration-dependent manner. Cell cycle distribution in the control group: most cells were in G1 phase, approximately 35.94% were in S phase, and approximately 18.95% were in G2 / M phase, indicating that cells normally transition from G1 to S and G2 / M phases, maintaining normal cell proliferation. After treatment with a low concentration (1μM), cell cycle analysis showed a slight increase in the proportion of cells in G1 phase (56.69%), a slight decrease in the proportion of cells in S phase to 26.26%, and a decrease in the proportion of cells in G2 / M phase to 17.05%. This change suggests that Example 37 at low concentrations may slightly inhibit cell entry into S phase, delaying cell proliferation. After high-concentration (2 μM) treatment, the proportion of cells in G1 phase further increased (approximately 65.13%), while the proportions of cells in S phase and G2 / M phase decreased significantly, with S phase cells accounting for only 22.95% and G2 / M phase cells accounting for only 11.92%. At this point, the cell cycle inhibition effect of Example 37 was most significant, preventing the transition of MCF-7 cells from G1 phase to subsequent phases, indicating that Example 37 strongly inhibited cell proliferation. After positive Abemaciclib (1 μM) treatment, the proportion of cells in G1 phase significantly increased (approximately 62.49%), the proportion in S phase significantly decreased to 21.58%, and the proportion in G2 / M phase decreased to 15.93%. This suggests that Example 37 may significantly prevent cell proliferation by inhibiting the transition of cells from G1 phase to S phase, exhibiting a strong cell cycle arrest effect. Flow cytometry analysis showed that Example 37 exhibited a concentration-dependent cell cycle arrest effect on MCF-7 cells. Particularly at higher concentrations, cells were significantly arrested in the G1 phase and unable to normally enter the S and G2 / M phases. This suggests that Example 37 may exert its anti-tumor effect by interfering with normal cell cycle regulation and inhibiting the proliferation of MCF-7 cells.

[0287] XI. Detection Methods for Reactive Oxygen Species (ROS) in Cells

[0288] In the experiment evaluating the regulatory effect of Example 37 on oxidative stress in MCF-7 cells, DCFH-DA probe detection showed that the compound exhibited a concentration-dependent ROS-inducing effect. The dosing procedures for each group were as described above, and the specific operations are as follows:

[0289] (1) After digesting, centrifuging and resuspending MCF-7 cells with a cell density of about 80%, add 1×10 cells per well. 5 One cell line was seeded into a 24-well plate, 1 mL of cell culture was added to each well, and the plate was incubated in a cell culture incubator for 24 hours.

[0290] (2) After preparing the sample to the required concentration, add it to the 24-well plate and let it stand for 24 hours.

[0291] (3) After 24 hours, the cells were washed and stained with reactive oxygen species (ROS). DCF-DA dye was added to the cells and incubated in an incubator for 30 minutes.

[0292] (4) After 30 minutes, remove the plate, wash off the staining solution in the cells with PBS, repeat 1-2 times, and then take pictures with a fluorescence microscope.

[0293] The DCF-DA dye was replaced with H33342 dye as a blank control group.

[0294] The results of ROS staining experiments on MCF-7 in each group are shown below. Figure 11 In the control group, green fluorescence was almost undetectable. The green fluorescence gradually increased from the low-dose to the high-dose group and the positive control group of Example 37, with comparable intensity in the high-dose and positive control groups. Quantitative analysis of fluorescence intensity revealed that the ROS level in the high-dose group of Example 37 was approximately two times higher than in the control group, indicating that Example 37 significantly induced oxidative stress in MCF-7 cells. This result may suggest that Example 37 exerts its anti-tumor effect by interfering with cellular redox balance through promoting ROS generation.

[0295] Application Example 1: Tablets

[0296] 10g of the compound prepared in Example 1 was mixed with 20g of excipients according to the general pharmaceutical tableting method and then compressed into 100 tablets, each weighing 300mg.

[0297] Application Example 2: Capsules

[0298] 5g of the compound prepared in Example 10 was mixed with 10g of excipients according to the requirements of pharmaceutical capsules and then filled into empty capsules, each weighing 300mg.

[0299] Application Example 3: Ointment

[0300] The compound prepared in Example 12 was finely ground and then mixed with 500g of an oily matrix such as petrolatum.

[0301] Application Example 4: Aerosol

[0302] Dissolve 10g of the compound prepared in Example 15 in an appropriate amount of propylene glycol, add distilled water and other excipients, and prepare a 500mL clear solution.

[0303] Application Example 5: Suppositories

[0304] After grinding 10g of the compound prepared in Example 20 into a fine powder, an appropriate amount of glycerin was added and ground evenly. Then, melted glycerin gelatin was added and ground evenly. The mixture was poured into a mold coated with lubricant to obtain 50 suppositories.

[0305] Application Example 6: Droplets

[0306] After heating and melting 5g of the compound prepared in Example 22 and 25g of a matrix such as gelatin, the mixture was added dropwise into low-temperature liquid paraffin to obtain 1000 pellets.

[0307] Application Example 7: Topical Liniments

[0308] 10g of the compound prepared in Example 23 was mixed and ground with 2.5g of excipients such as emulsifiers according to conventional pharmaceutical methods, and then distilled water was added to 200mL to obtain the final product.

[0309] Application Example 8: Injectables

[0310] Using 6g of the compound prepared in Example 26, the compound was adsorbed onto activated carbon according to conventional pharmaceutical methods, filtered through a 0.65μm microporous membrane, and then filled into a nitrogen tank to prepare an aqueous injection formulation, with each vial containing 2mL, for a total of 100 vials.

[0311] Application Example 9: Film Formulation

[0312] Polyvinyl alcohol, pharmaceutical glycerin, water, etc. are stirred and expanded, then heated and dissolved. The mixture is filtered through an 80-mesh sieve. The compound prepared in Example 15 is then added to the filtrate and stirred and dissolved. 100 membranes are made by coating the membrane.

[0313] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A compound containing heterocyclic pyrimidines, characterized in that, The structural formula is shown in Formula I or Formula II: 、 ， In Equations I and II, R1 is independently selected from... , , , , , , , , , , , , , , , , , , , , , , , and One of them; In Equation II, the fused ring containing the X ring is selected from... One of them; When the condensed ring containing the X ring is selected When R2 is -N, R3 is -CH; When the condensed ring containing the X ring is selected When R2 is -CH and R3 is -N.

2. The heterocyclic pyrimidine compound according to claim 1, characterized in that, The heterocyclic pyrimidine compounds include: N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-2-(4-(methanesulfonyl)phenyl)pyrimidin-4-amine, N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-2-(4-(isopropylsulfonyl)phenyl)pyrimidin-4-amine, 4-(4-((5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidin-2-yl)-N,N-dimethylbenzenesulfonamide, N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidin-2-yl)-N,N-dimethylbenzenesulfonamide, N-(5-((4-ethylpiperazin-1-yl)) N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-2-(4-(trifluoromethyl)phenyl)pyrimidin-4-amine, N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-2-(4-(trifluoromethoxy)phenyl)pyrimidin-4-amine, N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-2-(4-(trifluoromethoxy)phenyl)pyrimidin-4-amine, N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-2-(4-(methoxymethyl)phenyl) 4-Pyrimidine-4-amine, N-(5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-2-(4-(methoxymethoxy)phenyl)pyrimidine-4-amine, 4-(4-((5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidine-2-yl)benzaldehyde, 1-(4-(4-((5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidine-2-yl)phenyl)acetone, N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidine-2-yl)phenyl)acetone 2-(4-ethoxyphenyl)-N-(5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoropyrimidin-4-amine, N-(5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoropyrimidin-4-amine, 4'-(4-((5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-2-(4-methoxyphenyl)pyrimidin-4-amine, 4'-(4-((5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidin-2-yl)-[1,Methyl 1'-biphenyl]-4-carboxylate, N-(5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-2-(p-tolyl)pyrimidin-4-amine, 2-(4-ethylphenyl)-N-(5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoropyrimidin-4-amine, N-(5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-2-(4-isopropylphenyl)pyrimidin-4-amine, N-(5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-2-(4-propylphenyl)pyrimidin-4-amine, 4-(4-((5-(((4-ethylpiperazin-1-yl)methyl)pyridin)pyridine -2-yl)amino)-5-fluoropyrimidin-2-yl)benzonitrile, 4-(4-((5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidin-2-yl)-N,N-dimethylbenzamide, 2-([1,1'-biphenyl]-4-yl)-N-(5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoropyrimidin-4-amine, 2-(4'-chloro-[1,1'-biphenyl]-4-yl)-N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoropyrimidin-4-amine, N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-2-(4'-methyl) -[1,1'-biphenyl]-4-yl)pyrimidin-4-amine, (4-(4-(((5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidin-2-yl)phenyl)(phenyl)methyl ketone, N-(5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-2-(4-morpholinophenyl)pyrimidin-4-amine, N-(5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-2-(4-fluorophenyl)thieno[3,2-d]pyrimidin-4-amine, N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-2-(4-methoxyphenyl)thieno[3,2-d]pyrimidin-4-amine , N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-2-(p-tolyl)thieno[3,2-d]pyrimidin-4-amine, N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-2-(4-isopropylphenyl)thieno[3,2-d]pyrimidin-4-amine, 4-(4-((5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)thieno[3,2-d]pyrimidin-2-yl)benzonitrile, 2-([1,1'-biphenyl]-4-yl)-N-(5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)thieno[3,2-d]pyrimidin-4-amine, 2-(4'-chloro-[1,1'-Biphenyl]-4-yl)-N-(5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)thieno[3,2-d]pyrimidin-4-amine, N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-2-(4'-methyl-[1,1'-biphenyl]-4-yl)thieno[3,2-d]pyrimidin-4-amine, N-(5 -((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-2-(4-morpholinophenyl)thieno[3,2-d]pyrimidin-4-amine, N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-(4-fluorophenyl)pyrazolo[1,5-a]pyrimidin-7-amine, N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-(4-(trifluoromethyl)phenyl)pyrazolo[1,5-a]pyrimidin-7-amine, N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-(p-tolyl)pyrazolo[1,5-a]pyrimidin-7-amine, N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-(4-isopropoxyphenyl) 4-(7-((5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)pyrazolo[1,5-a]pyrimidin-5-yl)benzonitrile or methyl 4-(7-((5-(((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)pyrazolo[1,5-a]pyrimidin-5-yl)benzoate.

3. A method for preparing a heterocyclic pyrimidine compound as described in any one of claims 1-2, characterized in that, Starting with 2-chloro-5-chloromethylpyridine, 2-amino-5-chloromethylpyridine was obtained via a Buchwald-Hartwig coupling reaction; then, the intermediate 5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-amine was prepared via a nucleophilic substitution reaction; 2-chloro-N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)pyrimidin-4-amine, 5-chloro-N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)pyrazolopyrimidin-7-amine and 2-chloro-N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)thieno[3,2-d]pyrimidin-4-amine were obtained via a Suzuki-Miyaura coupling reaction.

4. A pharmaceutical composition, characterized in that, The heterocyclic pyrimidine compound as described in any one of claims 1-2 is used as the active ingredient.

5. The use of a heterocyclic pyrimidine compound as described in any one of claims 1-2 or a pharmaceutical composition as described in claim 3 in the preparation of a CDK4 / 6 inhibitor.

6. The application according to claim 5, characterized in that, The CDK4 / 6 inhibitors also include pharmaceutically acceptable carriers or excipients.

7. The application according to claim 5, characterized in that, The dosage forms of the CDK4 / 6 inhibitors include injections, tablets, capsules, aerosols, suppositories, films, pellets, topical liniments, or ointments.

8. The use of a heterocyclic pyrimidine compound as described in any one of claims 1-2 or a pharmaceutical composition as described in claim 3 in the preparation of a medicament for the treatment and / or prevention of breast cancer.

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