4-aminopyrimidine compound targeting EGFR and AXL targets and preparation method and application thereof

Abstract

The invention discloses a 4-aminopyrimidine compound targeting EGFR and AXL targets as well as a preparation method and application thereof, and belongs to the technical field of medicines. The 4-aminopyrimidine compound targeting EGFR and AXL targets, provided by the invention, has good ability of inhibiting EGFR and AXL kinase, so that the 4-aminopyrimidine compound can be used as an active component for preparing a medicine for treating diseases caused by abnormal activation of EGFR and / or AXL kinase; in addition, potential application value is also shown in preparation of medicines for treating and / or preventing pathological hyperplasia diseases (such as cancers).

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a 4-aminopyrimidine compound targeting EGFR and AXL targets, and a preparation method and application thereof. Background Art

[0002] Small molecule targeted therapies have improved the treatment of non-small cell lung cancer (NSCLC) by specifically inhibiting cancer cell growth signaling pathways or angiogenesis. In recent years, the epidermal growth factor receptor (EGFR) has become an important target for the treatment of NSCLC. EGFR, a transmembrane protein with a molecular weight of approximately 170 kDa, is located on the cell membrane. Also known as HER1 or ErbB1, it is a core member of the ErbB protein kinase family and a transmembrane receptor tyrosine kinase (RTK). It plays a key role in physiological processes such as cell growth, proliferation, differentiation, migration, and apoptosis. Abnormal activation of EGFR is closely associated with the occurrence and progression of various tumors. In particular, in NSCLC, EGFR mutations (such as L858R, T790M, and C797S) often lead to abnormal tumor cell proliferation and drug resistance. Therefore, EGFR has become an important target in cancer treatment, and EGFR inhibitors have achieved significant clinical success. Unfortunately, targeted drug use is prone to drug resistance, with EGFR mutations accounting for 50% of resistance mechanisms. Therefore, research on drug resistance caused by EGFR mutations has gradually become a research hotspot.

[0003] Studies have found that AXL expression is upregulated during the investigation of drug resistance caused by EGFR mutations. Sustained EGFR inhibition ultimately induces upregulation of GAS6, leading to increased AXL activity. Elevated levels of AXL, GAS6, and protein S have been found in over 50% of NSCLC cell lines. Furthermore, EGFR and AXL have significant cross-talk and correlation in downstream signaling, regulating cell survival, proliferation, migration, and immune responses through the synergistic effects of pathways such as PI3K / AKT and MAPK / ERK. In cancer, the synergistic effects of EGFR and AXL can enhance tumor cell survival, invasion, and drug resistance. Therefore, the development of dual-target inhibitors for EGFR and AXL is of great significance for cancer treatment.

[0004] Currently, there are no dual-target EGFR / AXL inhibitors on the market, and most drugs still rely on the combination of EGFR inhibitors and AXL inhibitors. Therefore, the development of dual-target EGFR / AXL inhibitors is of great significance in addressing the problem of drug resistance in NSCLC. Summary of the Invention

[0005] The present invention aims to provide a 4-aminopyrimidine compound targeting EGFR and AXL, as well as its preparation method and application. The present invention designs and synthesizes a series of novel 4-aminopyrimidine compounds that are expected to become anti-tumor drugs that simultaneously target EGFR and AXL kinases.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] One of the technical solutions of the present invention is to provide a 4-aminopyrimidine compound targeting EGFR and AXL, the structural formula of which is shown in Formula I or Formula II:

[0008]

[0009] In formula I, when R2 is H, R1 is

[0010] When R2 is halogen, R1 is

[0011] In formula II, when R4 is H, R3 is

[0012] When R4 is halogen, R3 is

[0013] The halogen in R2 and R4 can be Cl.

[0014] The names of the 4-aminopyrimidine compounds targeting EGFR and AXL targets of the present invention are as follows:

[0015] [1] 4-((2-(4-methylpiperazin-1-yl)pyrimidin-4-yl)amino)-N-(1-(methylsulfonyl)piperidin-4-yl)benzamide;

[0016] [2] N-(1-(methylsulfonyl)piperidin-4-yl)-4-((2-morpholinopyrimidin-4-yl)amino)benzamide;

[0017] [3] (R)-4-((2-(2-methylmorpholinyl)pyrimidin-4-yl)amino)-N-(1-(methylsulfonyl)piperidin-4-yl)benzamide;

[0018] [4] 4-((2-((2S,6R)-2,6-dimethylmorpholinyl)pyrimidin-4-yl)amino)-N-(1-(methylsulfonyl)piperidin-4-yl)benzamide;

[0019] [5] 4-((2-(4,4-difluoropiperidin-1-yl)pyrimidin-4-yl)amino)-N-(1-(methylsulfonyl)piperidin-4-yl)benzamide;

[0020] [6] 4-((2-(6,6-dimethyl-3-azabicyclo[3.1.0]hexan-3-yl)pyrimidin-4-yl)amino)-N-(1-(methylsulfonyl)piperidin-4-yl)benzamide;

[0021] [7] 4-((2-(3-(dimethylamino)azetidin-1-yl)pyrimidin-4-yl)amino)-N-(1-(methylsulfonyl)piperidin-4-yl)benzamide;

[0022] [8] 4-((2-(4-(dimethylamino)piperidin-1-yl)pyrimidin-4-yl)amino)-N-(1-(methylsulfonyl)piperidin-4-yl)benzamide;

[0023] [9] 4-((2-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)pyrimidin-4-yl)amino)-N-(1-(methylsulfonyl)piperidin-4-yl)benzamide;

[0024]

[10] 4-((2-(4-isopropylpiperazin-1-yl)pyrimidin-4-yl)amino)-N-(1-(methylsulfonyl)piperidin-4-yl)benzamide;

[0025]

[11] 4-((2-(4-acetylpiperazin-1-yl)pyrimidin-4-yl)amino)-N-(1-(methylsulfonyl)piperidin-4-yl)benzamide;

[0026]

[12] 4-((2-(4-(cyclopropylcarbonyl)piperazin-1-yl)pyrimidin-4-yl)amino)-N-(1-(methylsulfonyl)piperidin-4-yl)benzamide;

[0027]

[13] N-(1-(methylsulfonyl)piperidin-4-yl)-4-((2-(4-(oxetan-3-yl)piperazin-1-yl)pyrimidin-4-yl)amino)benzamide;

[0028]

[14] N-(1-(methylsulfonyl)piperidin-4-yl)-4-((2-(4-morpholinopiperidin-1-yl)pyrimidin-4-yl)amino)benzamide;

[0029]

[15] N-(1-(methylsulfonyl)piperidin-4-yl)-4-((2-(4-(pyrrolidin-1-yl)piperidin-1-yl)pyrimidin-4-yl)amino)benzamide;

[0030]

[16] (4-((2-(4-methylpiperazin-1-yl)pyrimidin-4-yl)amino)phenyl)(4-(methylsulfonyl)piperazin-1-yl)methanone;

[0031]

[17] (4-(Methylsulfonyl)piperazin-1-yl)(4-((2-morpholinopyrimidin-4-yl)amino)phenyl)methanone;

[0032]

[18] (R)-(4-((2-(2-methylmorpholinyl)pyrimidin-4-yl)amino)phenyl)(4-(methylsulfonyl)piperazin-1-yl)methanone;

[0033]

[19] (4-((2-((2S,6R)-2,6-dimethylmorpholino)pyrimidin-4-yl)amino)phenyl)(4-(methylsulfonyl)piperazin-1-yl)methanone;

[0034]

[20] (4-((2-(4,4-difluoropiperidin-1-yl)pyrimidin-4-yl)amino)phenyl)(4-(methylsulfonyl)piperazin-1-yl)methanone;

[0035]

[21] (4-((2-(6,6-dimethyl-3-azabicyclo[3.1.0]hexan-3-yl)pyrimidin-4-yl)amino)phenyl)(4-(methylsulfonyl)piperazin-1-yl)methanone;

[0036]

[22] (4-((2-(3-(dimethylamino)azetidin-1-yl)pyrimidin-4-yl)amino)phenyl)(4-(methylsulfonyl)piperazin-1-yl)methanone;

[0037]

[23] (4-((2-(4-(dimethylamino)piperidin-1-yl)pyrimidin-4-yl)amino)phenyl)(4-(methylsulfonyl)piperazin-1-yl)methanone;

[0038]

[24] (4-((2-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)pyrimidin-4-yl)amino)phenyl)(4-(methylsulfonyl)piperazin-1-yl)methanone;

[0039]

[25] (4-((2-(4-isopropylpiperazin-1-yl)pyrimidin-4-yl)amino)phenyl)(4-(methylsulfonyl)piperazin-1-yl)methanone;

[0040]

[26] 1-(4-(4-((4-(4-(methylsulfonyl)piperazine-1-carboxyl)phenyl)amino)pyrimidin-2-yl)piperazin-1-yl)ethanone;

[0041]

[27] (4-((2-(4-(cyclopropylcarbonyl)piperazin-1-yl)pyrimidin-4-yl)amino)phenyl)(4-(methylsulfonyl)piperazin-1-yl)methanone;

[0042]

[28] (4-(methylsulfonyl)piperazin-1-yl)(4-((2-(4-(oxetan-3-yl)piperazin-1-yl)pyrimidin-4-yl)amino)phenyl)methanone;

[0043]

[29] (4-(methylsulfonyl)piperazin-1-yl)(4-((2-(4-morpholinylpiperidin-1-yl)pyrimidin-4-yl)amino)phenyl)methanone;

[0044]

[30] (4-(methylsulfonyl)piperazin-1-yl)(4-((2-(4-(pyrrolidin-1-yl)piperidin-1-yl)pyrimidin-4-yl)amino)phenyl)methanone;

[0045]

[31] 4-((2-(4-acetylpiperazin-1-yl)-5-chloropyrimidin-4-yl)amino)-N-(1-(methylsulfonyl)piperidin-4-yl)benzamide;

[0046]

[32] 4-((5-chloro-2-(4-(cyclopropylcarbonyl)piperazin-1-yl)pyrimidin-4-yl)amino)-N-(1-(methylsulfonyl)piperidin-4-yl)benzamide;

[0047]

[33] 4-((5-chloro-2-(4-morpholinylpiperidin-1-yl)pyrimidin-4-yl)amino)-N-(1-(methylsulfonyl)piperidin-4-yl)benzamide;

[0048]

[34] (4-((5-chloro-2-((2S,6R)-2,6-dimethylmorpholinyl)pyrimidin-4-yl)amino)phenyl)(4-(methylsulfonyl)piperazin-1-yl)methanone;

[0049]

[35] (4-((5-chloro-2-(6,6-dimethyl-3-azabicyclo[3.1.0]hexan-3-yl)pyrimidin-4-yl)amino)phenyl)(4-(methylsulfonyl)piperazin-1-yl)methanone;

[0050]

[36] (4-((5-chloro-2-(4-(pyrrolidin-1-yl)piperidin-1-yl)pyrimidin-4-yl)amino)phenyl)(4-(methylsulfonyl)piperazin-1-yl)methanone.

[0051] The second technical solution of the present invention is to provide a method for preparing the above-mentioned 4-aminopyrimidine compounds targeting EGFR and AXL targets.

[0052] The preparation route of the compound shown in Formula I is as follows:

[0053]

[0054] In the above route, compound B is prepared by an aromatic nucleophilic substitution reaction; compound C is prepared by an acid-amine condensation reaction; and compound D is prepared by an aromatic nucleophilic substitution reaction.

[0055] The preparation route of the compound shown in formula II is as follows:

[0056]

[0057] In the above route, compound B is prepared by an aromatic nucleophilic substitution reaction; compound E is prepared by an acid-amine condensation reaction; and compound F is prepared by an aromatic nucleophilic substitution reaction.

[0058] The third technical solution of the present invention is to provide a pharmaceutically acceptable salt of the above-mentioned 4-aminopyrimidine compound targeting EGFR and AXL targets.

[0059] The fourth technical solution of the present invention: a pharmaceutical preparation, the active ingredient of which is the above-mentioned 4-aminopyrimidine compound targeting EGFR and AXL targets or a pharmaceutically acceptable salt of the above-mentioned 4-aminopyrimidine compound targeting EGFR and AXL targets.

[0060] Optionally, the dosage form of the pharmaceutical preparation includes but is not limited to injection, tablet, capsule, aerosol, suppository, film, pellet, external application agent, ointment or enteric-coated tablet.

[0061] The fifth technical solution of the present invention: Use of the above-mentioned 4-aminopyrimidine compound targeting EGFR and AXL in the preparation of drugs for treating and / or preventing pathological proliferative diseases.

[0062] Preferably, the pathological proliferative disease is cancer.

[0063] Technical solution six of the present invention: Use of a pharmaceutically acceptable salt of the above-mentioned 4-aminopyrimidine compound targeting EGFR and AXL targets in the preparation of drugs for treating and / or preventing pathological proliferative diseases.

[0064] Preferably, the pathological proliferative disease is cancer.

[0065] Technical solution seven of the present invention: Use of the above-mentioned pharmaceutical preparation in the preparation of drugs for treating and / or preventing pathological proliferative diseases.

[0066] Preferably, the pathological proliferative disease is cancer.

[0067] The aforementioned cancers include lung cancer, pancreatic cancer, breast cancer, metastatic medullary thyroid cancer, liver cancer or kidney cancer.

[0068] The beneficial technical effects of the present invention are as follows:

[0069] The 4-aminopyrimidine compounds provided by the present invention that target EGFR and AXL targets have excellent ability to inhibit EGFR and AXL kinases. Therefore, they can be used as active ingredients to prepare therapeutic drugs for diseases caused by abnormal activation of EGFR and / or AXL kinases, and also show potential application value in the preparation of drugs for treating and / or preventing pathological proliferative diseases (such as cancer). BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 Annexin V-FITC / PI double staining (A), AO staining (B), and JC-1 staining (C) were used to detect the apoptosis-inducing ability of the compound prepared in Example 36 in H1975 cells.

[0071] Figure 2 Effects of the compound prepared in Example 36 on the cell cycle of H1975 cells were detected by flow cytometry.

[0072] Figure 3 The effect of the compound prepared in Example 36 on the content of reactive oxygen species in H1975 cells was detected by DCFH-DA probe.

[0073] Figure 4 The effect of the compound prepared in Example 36 on the migration ability of H1975 cells was detected by cell scratch test.

[0074] Figure 5 The effect of different concentrations of the compound prepared in Example 36 on the hemolysis rate of sheep red blood cells.

[0075] Figure 6 The mean body weight changes of KM mice in each group during the drug administration period.

[0076] Figure 7 These are the macroscopic images and organ indexes of the five internal organs of KM mice in each group after drug administration. A to E are the macroscopic images of the heart, liver, spleen, lungs, and kidneys, respectively, and F is the organ index calculated after weighing the five internal organs.

[0077] Figure 8 Figure 2 is the blood biochemical analysis results of KM mice in each group after administration, where A is the analysis results of total protein, urea and glucose, B is the analysis results of creatinine, aspartate aminotransferase and alanine aminotransferase, and C is the analysis results of alkaline phosphatase, lactate dehydrogenase and creatine kinase.

[0078] Figure 9 These are H&E staining images of the five internal organs of KM mice in each group after drug administration (scale bar: 100 μm). DETAILED DESCRIPTION

[0079] Various exemplary embodiments of the present invention are now described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention.

[0080] It should be pointed out that the matters not described in detail in the present invention are conventional operating means in this field and are not the focus of the present invention.

[0081] In addition, for numerical ranges in the present invention, it is understood that each intervening value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any stated value or stated range, and any other stated value or intervening value in the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

[0082] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention.

[0083] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0084] The H NMR spectra of the compounds in the examples of the present invention were measured using a Bruker ARX-400, and the mass spectra were measured using an Agilent 1100 LC / MSD; all reagents used were of analytical or chemical purity.

[0085] The reaction route for preparing 4-aminopyrimidine compounds in the embodiment of the present invention is Route 1 or Route 2, which is as follows:

[0086] Route 1

[0087]

[0088] Route 2

[0089]

[0090] R1 to R4 in Route 1 and Route 2 are as listed in the Summary of the Invention and will not be described again here.

[0091] The final product obtained in step 3 of Examples 1 to 36 is a precipitate or is dissolved in the original system. For reactions in which the product after the reaction is a precipitate, the separation method is to perform vacuum filtration and wash it with acetonitrile and then dry it to obtain the final product; for reactions in which the product after the reaction is dissolved in the reaction system, the product is purified by thin layer chromatography and dried to obtain the final product.

[0092] For step 3 of Examples 1 to 36, the stirring reaction time after all the raw materials are added is 8 to 12 hours, which is determined as needed.

[0093] Example 1

[0094] Preparation of 4-((2-(4-methylpiperazin-1-yl)pyrimidin-4-yl)amino)-N-(1-(methylsulfonyl)piperidin-4-yl)benzamide:

[0095] Step 1: Preparation of 4-((2-chloropyrimidin-4-yl)amino)benzoic acid (B)

[0096] Add p-aminobenzoic acid (A) (2.0 g) and 2,4-dichloropyrimidine (2.3 g) to a 500 mL eggplant-shaped flask, add 150 mL of a pre-prepared HCl (0.1 M) aqueous solution, and place in a constant-temperature heated magnetic stirrer filled with silicone oil. The temperature is raised to 100°C. The raw material dissolves from the initial white solid to a colorless transparent solution. When the temperature is raised to 90°C, a white solid begins to precipitate. The reaction is continued at 100°C for 4 hours. After the reaction is completed, cool to room temperature, pour the solid-liquid mixture into a 1000 mL beaker, add 500 mL of tap water, stir for 0.5 hours, and filter under reduced pressure. The filter cake is then dried for later use to obtain the key intermediate 4-((2-chloropyrimidin-4-yl)amino)benzoic acid (B).

[0097] Step 2: Preparation of 4-((2-chloropyrimidin-4-yl)amino)-N-(1-(methylsulfonyl)piperidin-4-yl)benzamide (C)

[0098] 4-((2-chloropyrimidin-4-yl)amino)benzoic acid (B) (1.0 g) was added to a 250 mL eggplant flask, followed by the addition of dichloromethane (80 mL), DMF (5 drops), DIPEA (1.6 g), and HATU (4.6 g). The mixture was reacted at room temperature for 0.5 h. 1-methylsulfonyl-4-aminopiperidine (0.9 g) was then added, and the reaction was continued at room temperature for 7 h. After the reaction, dichloromethane and water were added for extraction. The filtrate was dried over anhydrous sodium sulfate and then dried to dryness. The filtrate was then purified by column chromatography to obtain the intermediate 4-((2-chloropyrimidin-4-yl)amino)-N-(1-(methylsulfonyl)piperidin-4-yl)benzamide (C).

[0099] Step 3: Preparation of 4-((2-(4-methylpiperazin-1-yl)pyrimidin-4-yl)amino)-N-(1-(methylsulfonyl)piperidin-4-yl)benzamide (D)

[0100] Piperazine (0.2 g) was weighed into a 50 mL eggplant-shaped flask, along with acetonitrile (10 mL) and DIPEA (0.3 g). The mixture was reacted at 80°C for 0.5 h. The intermediate 4-((2-chloropyrimidin-4-yl)amino)-N-(1-(methylsulfonyl)piperidin-4-yl)benzamide (C) (0.3 g) was then added and stirring continued for 9 h. After the reaction was complete, the mixture was cooled to room temperature. The final product (D) was obtained after isolation and purification.

[0101] The product is a white solid. 1 H NMR (400MHz, DMSO-d6) δ9.53 (s, 1H), 8.18 (d, J = 7.5Hz, 1H), 7.98 (d, J = 5.6Hz, 1H) ,7.83(d,J=8.5Hz,2H),7.72(d,J=8.5Hz,2H),6.10(d,J=5.6Hz,1H),3.91(d,J=6 .7Hz,1H),3.73(s,4H),3.57(d,J=11.6Hz,2H),2.88(s,3H),2.84(d,J=11.9Hz,2 H), 2.45 (s, 4H), 2.28 (s, 3H), 1.89 (d, J = 12.5Hz, 2H), 1.59 (q, J = 10.3, 9.2Hz, 2H). 13 C NMR(151MHz,DMSO-d6)δ165.28,161.07,160.15,156.47,143.01,128.23(2C),127.11,11 8.02(2C),97.34,54.25(2C),45.82(2C),45.53,44.83(2C),43.25,34.23,30.90(2C).TOF MS ES+(m / z):(M+H) + ,calcd for C 22 H 31 N7O3S:474.2287; found,474.2287.HPLC:t R :15.552min,purity:96.41%.

[0102] Example 2

[0103] Preparation of N-(1-(methylsulfonyl)piperidin-4-yl)-4-((2-morpholinopyrimidin-4-yl)amino)benzamide:

[0104] The method of Example 1 was followed, except that piperazine was replaced with an equal molar amount of morpholine in step 3.

[0105] The product is a white solid. 1 H NMR (400MHz, DMSO-d6) δ9.55(s,1H),8.17(d,J=7.0Hz,1H),8.00(d,J=5.5Hz,1H),7.82(d,J=8.6Hz,2H),7.72(d,J=8.6Hz,2H),6.13(d,J=5.5Hz,1H), 3.91(d,J=7.3Hz,1H),3.67(s,8H),3.57(d,J=11.8Hz,2H),2.88(s,3H),2. 84(d,J=12.3Hz,2H), 1.89(d,J=12.5Hz,2H), 1.60(q,J=12.0,11.4Hz,2H). 13 C NMR(151MHz,DMSO-d6)δ165.26,161.23,160.15,156.40,142.97,128.22(2C),127.12 ,118.05(2C),97.63,66.05(2C),45.81,44.83(2C),44.14(2C),34.23,30.89(2C).TOF MS ES+(m / z):(M+H) + ,calcd for C 21 H 28 N6O4S:461.1971; found,461.1971.HPLC:t R :18.283min,purity:94.67%.

[0106] Example 3

[0107] Preparation of (R)-4-((2-(2-methylmorpholinyl)pyrimidin-4-yl)amino)-N-(1-(methylsulfonyl)piperidin-4-yl)benzamide:

[0108] The method of Example 1 was followed, except that in step 3, piperazine was replaced with an equimolar amount of (R)-2-methylmorpholine.

[0109] The product is a pink solid. 1H NMR(400MHz,DMSO-d6)δ9.56(s,1H),8.19(d,J=7.8Hz,1H),7.99(d,J=5.7Hz,1H),7 .82(d,J=8.5Hz,2H),7.72(d,J=8.5Hz,2H),6.12(d,J=5.9Hz,1H),4.38(dd,J=32.6 ,13.0Hz,2H),3.90(d,J=11.4Hz,2H),3.59-3.46(m,4H),3.32(s,2H),2.88(s,3H), 2.86-2.81(m,2H),1.89(d,J=12.7Hz,2H),1.64-1.54(m,2H),1.16(d,J=6.1Hz,3H). 13 C NMR(151MHz,DMSO-d6)δ165.29,161.04,160.16,156.45,142.98,128.22(2C),127.16,118. 06(2C),97.49,71.07,65.76,49.90,45.84,44.84(2C),43.44,34.24,30.91(2C),18.84.TOF MS ES+(m / z):(M+H) + ,calcd for C 22 H 30 N6O4S:475.2127; found,475.2127.HPLC:t R :18.791min,purity:95.89%.

[0110] Example 4

[0111] Preparation of 4-((2-((2S,6R)-2,6-dimethylmorpholinyl)pyrimidin-4-yl)amino)-N-(1-(methylsulfonyl)piperidin-4-yl)benzamide:

[0112] The method of Example 1 was followed, except that in step 3, piperazine was replaced with an equimolar amount of (2S,6R)-2,6-dimethylmorpholine.

[0113] The product is a purple solid. 1H NMR (400MHz, DMSO-d6) δ9.49(s,1H),8.12(d,J=8.0Hz,1H),7.99(d,J=5.7Hz,1H),7.82(d,J=8.4Hz,2H),7.70(d,J=8.6Hz,2H),6.11(d,J=5.8Hz ,1H),4.45(d,J=12.9Hz,2H),3.92(s,1H),3.57(s,4H),2.88(d,J=4.2H z,5H),1.95-1.87(m,2H),1.61(d,J=12.3Hz,2H),1.17(d,J=6.8Hz,6H). 13 C NMR(151MHz,DMSO-d6)δ165.30,160.85,160.17,156.49,143.00,128.22(2C),127.20,118. 04(2C),97.37,71.01(2C),49.26(2C),45.86,44.85(2C),34.24,30.91(2C),18.86(2C).TOF MS ES+(m / z):(M+H) + ,calcd for C 23 H 32 N6O4S:489.2284; found,489.2284.HPLC:t R :19.045min,purity:96.56%.

[0114] Example 5

[0115] Preparation of 4-((2-(4,4-difluoropiperidin-1-yl)pyrimidin-4-yl)amino)-N-(1-(methylsulfonyl)piperidin-4-yl)benzamide:

[0116] The method of Example 1 was followed, except that in step 3, piperazine was replaced with an equimolar amount of 4,4-difluoropiperidine.

[0117] The product is a white solid. 1H NMR (400MHz, DMSO-d6) δ9.59 (s, 1H), 8.20 (d, J = 7.7Hz, 1H), 8.01 (d, J = 5.7Hz ,1H),7.83(d,J=8.4Hz,2H),7.70(d,J=8.5Hz,2H),6.13(d,J=5.7Hz,1H),3.8 6(d,J=6.1Hz,5H),3.56(d,J=11.8Hz,2H),2.88(s,3H),2.83(d,J=11.7Hz,2H ),1.97(d,J=15.0Hz,4H),1.89(d,J=12.5Hz,2H),1.59(q,J=12.4Hz,2H).TOF MS ES+(m / z):(M+H) + ,calcd for C 22 H 28 F2N6O3S:495.1990; found,495.1987.HPLC:t R :16.807min,purity:96.53%.

[0118] Example 6

[0119] Preparation of 4-((2-(6,6-dimethyl-3-azabicyclo[3.1.0]hexan-3-yl)pyrimidin-4-yl)amino)-N-(1-(methylsulfonyl)piperidin-4-yl)benzamide:

[0120] The method of Example 1 was followed, except that in step 3, piperazine was replaced with an equimolar amount of 6,6-dimethyl-3-azabicyclo[3.1.0]hexane.

[0121] The product is a white solid. 1 H NMR (400MHz, DMSO-d6) δ9.50 (s, 1H), 8.16 (d, J = 7.7Hz, 1H), 7.92 (d, J = 5.6Hz, 1H) ,7.87-7.80(m,4H),6.05(d,J=5.6Hz,1H),3.90(s,1H),3.65(d,J=11.4Hz,1H),3. 59-3.52(m,4H),3.47(d,J=11.8Hz,1H),2.88(s,3H),2.83(d,J=12.2Hz,2H),1.89 (d,J=12.6Hz,2H),1.60(t,J=12.1Hz,2H),1.49(s,2H),1.05(s,3H),0.84(s,3H). 13C NMR(151MHz,DMSO-d6)δ165.31,159.95,158.70,156.36,143.39,128.15(2C),126.79,117.86( 2C),96.54,45.79,44.82(2C),34.23,30.91(2C),26.87,26.04(2C),18.80(2C),12.35(2C).TOF MS ES+(m / z):(M+H) + ,calcd for C 24 H 32 N6O3S:485.2335; found,485.2335.HPLC:t R :17.516min,purity:95.86%.

[0122] Example 7

[0123] Preparation of 4-((2-(3-(dimethylamino)azetidin-1-yl)pyrimidin-4-yl)amino)-N-(1-(methylsulfonyl)piperidin-4-yl)benzamide:

[0124] The method of Example 1 was followed, except that in step 3, piperazine was replaced with an equimolar amount of N,N-dimethylazetidin-3-amine.

[0125] The product is a white solid. 1 H NMR (400MHz, DMSO-d6) δ9.56 (s, 1H), 8.18 (d, J = 7.9Hz, 1H), 7.94 (d, J = 5.7Hz, 1H),7.81(s,4H),6.11(d,J=5.7Hz,1H),4.04(t,J=8.0Hz,2H),3.90(d,J=7.9 Hz,1H),3.80(t,J=7.1Hz,2H),3.56(d,J=11.7Hz,2H),3.14(s,1H),2.88(s,3 H),2.86-2.81(m,2H),2.12(s,6H),1.89(d,J=13.0Hz,2H),1.64-1.54(m,2H). 13 C NMR(151MHz,DMSO-d6)δ165.32,162.54,160.29,156.33,143.27,128.20(2C),126.98,11 7.97(2C),97.49,55.62,53.85,45.81(2C),44.83(2C),41.56(2C),34.23,30.90(2C).TOF MSES+(m / z):(M+H) +,calcd for C 22 H 31 N7O3S:474.2287; found,474.2288.HPLC:t R :15.622min,purity:90.14%.

[0126] Example 8

[0127] Preparation of 4-((2-(4-(dimethylamino)piperidin-1-yl)pyrimidin-4-yl)amino)-N-(1-(methylsulfonyl)piperidin-4-yl)benzamide

[0128] The method of Example 1 is followed, except that in step 3, piperazine is replaced with an equimolar amount of N,N-dimethyl-4-aminopiperidine.

[0129] The product is a white solid. 1 H NMR (400MHz, DMSO-d6) δ9.51(s,1H),8.19(d,J=7.7Hz,1H),7.96(d,J=5.4Hz,1H),7.82(d,J =8.4Hz,2H),7.73(d,J=8.5Hz,2H),6.06(d,J=5.7Hz,1H),4.61(d,J=12.9Hz,2H),3.91(d,J= 10.0Hz,1H),3.57(d,J=11.5Hz,2H),2.88(s,4H),2.34(d,J=10.7Hz,1H),2.18(s,6H),1.89 (d,J=12.3Hz,2H),1.81(d,J=12.3Hz,2H),1.59(dt,J=21.3,10.4Hz,2H),1.35-1.24(m,2H). 13 C NMR(151MHz,DMSO-d6)δ165.27,160.90,160.15,156.54,143.12,128.20(2C),127.00,117.94( 2C),96.83,61.82,45.82,44.83(2C),42.87(2C),41.41(2C),34.23,30.91(2C),27.80(2C).TOF MS ES+(m / z):(M+H) + ,calcd for C 24 H 35 N7O3S:502.2600; found,502.2600.HPLC:t R :16.078min,purity:99.14%.

[0130] Example 9

[0131] Preparation of 4-((2-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)pyrimidin-4-yl)amino)-N-(1-(methylsulfonyl)piperidin-4-yl)benzamide:

[0132] The method of Example 1 was followed, except that in step 3, piperazine was replaced with an equimolar amount of 1-methyl-4-(piperidin-4-yl)piperazine.

[0133] The product is a white solid. 1 H NMR (400MHz, DMSO-d6) δ9.52(s,1H),8.19(d,J=7.8Hz,1H),7.96(d,J=5.6Hz,1H),7.82(d,J=8. 6Hz,2H),7.73(d,J=8.5Hz,2H),6.06(d,J=5.6Hz,1H),4.62(d,J=13.0Hz,2H),3.96-3.85(m,1H ),3.57(d,J=11.6Hz,2H),2.88(s,3H),2.84(d,J=12.4Hz,4H),2.31(s,3H),2.14(s,3H),1.89( d,J=12.6Hz,2H),1.81(d,J=12.2Hz,2H),1.59(td,J=12.8,12.3,8.5Hz,2H),1.37-1.27(m,2H). 13 C NMR(151MHz,DMSO-d6)δ165.28,160.92,160.16,156.55,143.12,128.20(2C),127.00,117.94(2C),96.8 5,61.28,55.16(2C),48.44(2C),45.82(2C),45.73,44.83(2C),43.05,34.23,30.90(2C),27.76(2C).TOF MS ES+(m / z):(M+H) + ,calcd for C 27 H 40 N8O3S:557.3022; found,557.3022.HPLC:t R :15.902min,purity:94.81%.

[0134] Example 10

[0135] Preparation of 4-((2-(4-isopropylpiperazin-1-yl)pyrimidin-4-yl)amino)-N-(1-(methylsulfonyl)piperidin-4-yl)benzamide:

[0136] The method of Example 1 was followed, except that in step 3, piperazine was replaced with an equimolar amount of 1-isopropylpiperazine.

[0137] The product is a white solid. 1 H NMR(400MHz,DMSO-d6)δ9.54(s,1H),8.19(d,J=7.6Hz,1H),7.97(d,J=5.7Hz,1H),7 .82(d,J=8.8Hz,2H),7.72(d,J=8.7Hz,2H),6.08(d,J=5.5Hz,1H),3.90(d,J=7.1Hz, 1H),3.68(s,4H),3.56(d,J=11.3Hz,2H),2.88(s,3H),2.83(dd,J=12.0,2.8Hz,2H) ,2.69(d,J=16.0Hz,1H),1.89(d,J=12.7Hz,2H),1.64-1.54(m,2H),1.00(s,6H).TOF MS ES+(m / z):(M+H) + ,calcd for C 24 H 35 N7O3S:502.2600; found,502.2600.HPLC:t R :15.763min,purity:95.28%.

[0138] Example 11

[0139] Preparation of 4-((2-(4-acetylpiperazin-1-yl)pyrimidin-4-yl)amino)-N-(1-(methylsulfonyl)piperidin-4-yl)benzamide:

[0140] The method of Example 1 was followed, except that in step 3, piperazine was replaced with an equimolar amount of 1-(piperazin-1-yl)ethan-1-one.

[0141] The product is a white solid. 1H NMR(400MHz,DMSO-d6)δ9.58(s,1H),8.20(d,J=7.7Hz,1H),8.00(d,J=5.7Hz,1 H),7.83(d,J=8.7Hz,2H),7.73(d,J=8.9Hz,2H),6.12(d,J=5.7Hz,1H),3.91(d ,J=9.6Hz,1H),3.76(s,2H),3.69(s,2H),3.59-3.51(m,6H),2.88(s,3H),2.84 (d,J=11.8Hz,2H),2.05(s,3H),1.89(d,J=12.7Hz,2H),1.59(q,J=11.4Hz,2H). 13 C NMR(151MHz,DMSO-d6)δ168.46,165.30,161.00,160.15,156.48,142.94,128.24(2C),127.21, 118.14(2C),97.49,45.81(2C),45.44,44.83(2C),43.77,43.40,34.23,30.90(2C),21.37.TOF MS ES+(m / z):(M+H) + ,calcd for C 23 H 31 N7O4S:502.2236; found,502.2237.HPLC:t R :17.818min,purity:98.13%.

[0142] Example 12

[0143] Preparation of 4-((2-(4-(cyclopropylcarbonyl)piperazin-1-yl)pyrimidin-4-yl)amino)-N-(1-(methylsulfonyl)piperidin-4-yl)benzamide:

[0144] According to the method of Example 1, in step 3, piperazine is replaced with an equimolar amount of cyclopropyl (piperazin-1-yl) ketone.

[0145] The product is a white solid. 1H NMR (400MHz, DMSO-d6) δ9.59(s,1H),8.20(d,J=7.7Hz,1H),8.00(d,J=5.7Hz,1H),7.83(d,J =8.8Hz,2H),7.73(d,J=7.1Hz,2H),6.12(d,J=5.7Hz,1H),3.91(dq,J=14.5,6.8,6.1Hz,1H) ,3.78(s,4H),3.69(s,2H),3.56(d,J=9.5Hz,4H),2.88(s,3H),2.83(d,J=11.8Hz,2H),2.02 (t,J=10.2Hz,1H),1.89(d,J=12.6Hz,2H),1.59(q,J=11.5,10.4Hz,2H),0.78-0.70(m,4H). 13 C NMR(151MHz,DMSO-d6)δ171.27,165.31,161.00,160.16,156.49,142.95,128.25(2C),127.21,118. 14(2C),97.49,45.81(2C),44.82(2C),43.92,43.42,41.39,34.23,30.90(2C),10.44,7.11(2C).TOF MS ES+(m / z):(M+H) + ,calcd for C 25 H 33 N7O4S:528.2393; found,528.2393.

[0146] Example 13

[0147] Preparation of N-(1-(methylsulfonyl)piperidin-4-yl)-4-((2-(4-(oxetan-3-yl)piperazin-1-yl)pyrimidin-4-yl)amino)benzoyl:

[0148] The method of Example 1 was followed, except that in step 3, piperazine was replaced with an equimolar amount of 1-(oxetane-3-yl)piperazine.

[0149] The product is a white solid. 1H NMR (400MHz, DMSO-d6) δ9.55(s,1H),8.18(d,J=7.6Hz,1H),7.98(d,J=5.7Hz,1H),7.81(d,J=7.7Hz, 2H),7.72(d,J=7.1Hz,2H),6.09(d,J=5.7Hz,1H),4.55(t,J=6.4Hz,2H),4.47(t,J=6.0Hz,2H),3.91( d,J=11.7Hz,1H),3.76-3.70(m,4H),3.56(d,J=11.6Hz,2H),3.41(t,J=6.3Hz,1H),2.88(d,J=1.5Hz, 3H), 2.83 (d, J = 12.2Hz, 2H), 2.34-2.29 (m, 4H), 1.88 (d, J = 12.7Hz, 2H), 1.58 (q, J = 12.6, 11.4Hz, 2H). 13 CNMR(151MHz,DMSO-d6)δ165.27,161.06,160.12,156.48,143.00,128.20(2C),127.11,118.05( 2C),97.26,74.41(2C),58.48,48.95(2C),45.81,44.83(2C),43.34(2C),34.23,30.90(2C).TOF MS ES+(m / z):(M+H) + ,calcd for C 24 H 33 N7O4S:516.2393; found,516.2390.HPLC:t R :15.793min,purity:95.70%.

[0150] Example 14

[0151] Preparation of N-(1-(methylsulfonyl)piperidin-4-yl)-4-((2-(4-morpholinopiperidin-1-yl)pyrimidin-4-yl)amino)benzamide:

[0152] The method of Example 1 was followed, except that in step 3, piperazine was replaced with an equimolar amount of 4-(piperidin-4-yl)morpholine.

[0153] The product is a white solid. 1H NMR (400MHz, DMSO-d6) δ9.51 (s, 1H), 8.19 (d, J = 7.7Hz, 1H), 7.97 (d, J = 5.5Hz, 1H), 7. 82(d,J=8.5Hz,2H),7.72(d,J=9.1Hz,2H),6.06(d,J=5.7Hz,1H),4.62(d,J=12.9Hz,2 H),3.96-3.87(m,1H),3.55(t,J=4.3Hz,6H),2.88(s,6H),2.85-2.81(m,1H),2.49-2 .37(m,5H),1.86(dd,J=23.3,12.5Hz,4H),1.65-1.55(m,2H),1.32(d,J=11.9Hz,2H). 13 C NMR(151MHz,DMSO-d6)δ165.27,160.92,160.15,156.54,143.11,128.20(2C),127.00,117.94(2C),9 6.86,66.59(2C),61.55,49.40(2C),45.82,44.83(2C),42.92(2C),34.22,30.91(2C),27.66(2C).TOF MS ES+(m / z):(M+H) + ,calcd forC 26 H 37 N7O4S:544.2706; found,544.2706.HPLC:t R :15.679min,purity:94.03%.

[0154] Example 15

[0155] Preparation of N-(1-(methylsulfonyl)piperidin-4-yl)-4-((2-(4-(pyrrolidin-1-yl)piperidin-1-yl)pyrimidin-4-yl)amino)benzamide:

[0156] The method of Example 1 was followed, except that in step 3, piperazine was replaced with an equimolar amount of 4-(pyrrolidin-1-yl)piperidine.

[0157] The product is a white solid. 1H NMR (400MHz, DMSO-d6) δ9.51(s,1H),8.19(d,J=7.8Hz,1H),7.97(d,J=5.6Hz,1H),7.8 2(d,J=8.4Hz,2H),7.73(d,J=8.7Hz,2H),6.06(d,J=5.7Hz,1H),4.46(d,J=12.8Hz,2H ),3.97-3.85(m,1H),3.57(d,J=11.3Hz,2H),3.00(t,J=11.9Hz,2H),2.88(s,3H),2.8 6-2.80(m,2H),2.23(s,1H),1.94-1.85(m,4H),1.72-1.54(m,6H),1.40-1.28(m,2H). 13 C NMR(151MHz,DMSO-d6)δ165.28,161.02,160.14,156.52,143.14,128.20(2C),126.99,117.94(2C),9 6.84,61.20,50.83(2C),45.82,44.83(2C),42.23(2C),34.22,30.91(2C),30.81(2C),22.96(2C).TOF MS ES+(m / z):(M+H)+,calcdfor C26H37N7O3S:528.2757; found,528.2757.HPLC:tR:15.820min,purity:99.36%.

[0158] Example 16

[0159] Preparation of (4-((2-(4-methylpiperazin-1-yl)pyrimidin-4-yl)amino)phenyl)(4-(methylsulfonyl)piperazin-1-yl)methanone:

[0160] According to the method of Example 1, in step 2, 1-(methylsulfonyl)piperidin-4-amine was replaced with an equimolar amount of 1-(methylsulfonyl)piperazine.

[0161] The product is a white solid. 1 H NMR (400MHz, DMSO-d6) δ9.50(s,1H),7.97(d,J=5.6Hz,1H),7.71(d,J=8.7Hz,2H),7.40(d,J=8.7Hz,2H),6.09( d,J=5.6Hz,1H),3.71(s,4H),3.61(s,4H),3.18-3.14(m,4H),2.91(d,J=2.0Hz,3H),2.41(s,4H),2.25(s,3H).13 C NMR(151MHz,DMSO-d6)δ169.21,161.10,160.18,156.43,141.90,128.38(2C),127.85, 118.45(2C),97.14,59.80,54.35(2C),48.63,45.65(2C),45.46(2C),43.35,34.04.TOF MS ES+(m / z):(M+H) + ,calcd for C 21 H 29 N7O3S:460.2131; found,460.2131.

[0162] Example 17

[0163] Preparation of (4-(methylsulfonyl)piperazin-1-yl)(4-((2-morpholinylpyrimidin-4-yl)amino)phenyl)methanone:

[0164] Follow the method of Example 16, except that in step 3, piperazine is replaced with an equal molar amount of morpholine.

[0165] The product is a white solid. 1 H NMR (400MHz, DMSO-d6) δ9.53(s,1H),7.99(d,J=5.6Hz,1H),7.71(d,J=8.9Hz,2H),7.40(d,J=8.9Hz,2 H),6.12(d,J=5.7Hz,1H),3.66(s,8H),3.61(s,4H),3.16(t,J=4.7Hz,4H),2.91(d,J=1.5Hz,3H).TOF MS ES+(m / z):(M+H) + ,calcdfor C 20 H 26 N6O4S:447.1814; found,447.1813.HPLC:t R :17.877min,purity:98.53%.

[0166] Example 18

[0167] Preparation of (R)-(4-((2-(2-methylmorpholinyl)pyrimidin-4-yl)amino)phenyl)(4-(methylsulfonyl)piperazin-1-yl)methanone:

[0168] Follow the method of Example 16, except that in step 3, piperazine is replaced with an equimolar amount of (R)-2-methylmorpholine.

[0169] The product is a pink solid. 1H NMR (400MHz, DMSO-d6) δ9.52(s,1H),7.99(d,J=5.6Hz,1H),7.71(d,J=8.4Hz,2H),7.40(d,J=8.4Hz,2H),6.11(d,J=5.7Hz,1H),4.38( dd,J=31.6,13.1Hz,2H),3.91-3.87(m,1H),3.61(s,4H),3.53-3.45(m,2H),3.16(t,J=5.1Hz,4H),2.90(s,3H),1.15(d,J=6.1Hz,3H). 13 C NMR(151MHz,DMSO-d6)δ169.21,161.05,160.19,156.42,141.84,128.35(2C),127.93, 118.53(2C),97.32,71.08(2C),65.80,49.92(2C),45.45,43.44(2C),34.06,18.80.TOF MS ES+(m / z):(M+H) + ,calcd for C 21 H 28 N6O4S:461.1971; found,461.1973.HPLC:t R :7.935min,purity:97.69%.

[0170] Example 19

[0171] Preparation of (4-((2-((2S,6R)-2,6-dimethylmorpholino)pyrimidin-4-yl)amino)phenyl)(4-(methylsulfonyl)piperazin-1-yl)methanone:

[0172] Follow the method of Example 16, except that in step 3, piperazine is replaced with an equimolar amount of (2S,6R)-2,6-dimethylmorpholine.

[0173] The product is a purple solid. 1 H NMR (400MHz, DMSO-d6) δ9.52(s,1H),7.98(d,J=3.7Hz,1H),7.70(d,J=6.5Hz,2H),7.40(d,J=6.5Hz,2H),6.10(d ,J=5.6Hz,1H),4.43(d,J=12.9Hz,2H),3.58(d,J=23.5Hz,6H),3.16(s,4H),2.90(s,3H),1.15(d,J=4.1Hz,6H). 13C NMR(151MHz,DMSO-d6)δ169.19,160.83,160.17,156.45,141.82,128.30(2C),127 .97,118.54(2C),97.17,71.01(3C),49.25(2C),46.23,34.07(2C),18.81(3C).TOF MS ES+(m / z):(M+H) + ,calcd for C 22 H 30 N6O4S:475.2127; found,475.2127.HPLC:t R :16.221min,purity:96.75%.

[0174] Example 20

[0175] Preparation of (4-((2-(4,4-difluoropiperidin-1-yl)pyrimidin-4-yl)amino)phenyl)(4-(methylsulfonyl)piperazin-1-yl)methanone:

[0176] The method of Example 16 was followed, except that in step 3, piperazine was replaced with an equimolar amount of 4,4-difluoropiperidine.

[0177] The product is a white solid. 1 H NMR (400MHz, DMSO-d6) δ9.56(s,1H),8.01(d,J=5.6Hz,1H),7.69(d,J=8.5Hz,2H),7.41(d,J=8.5Hz,2H),6.14( d,J=5.7Hz,1H),3.86(t,J=5.5Hz,4H),3.61(s,4H),3.19-3.12(m,4H),2.91(s,3H),1.97(d,J=15.1Hz,4H).TOF MS ES+(m / z):(M+H) + ,calcd for C 21 H 26 F2N6O3S:481.1833; found,481.1833.HPLC:t R :16.504min,purity:98.19%.

[0178] Example 21

[0179] Preparation of (4-((2-(6,6-dimethyl-3-azabicyclo[3.1.0]hexan-3-yl)pyrimidin-4-yl)amino)phenyl)(4-(methylsulfonyl)piperazin-1-yl)methanone:

[0180] The method of Example 16 was followed, except that in step 3, piperazine was replaced with an equimolar amount of 6,6-dimethyl-3-azabicyclo[3.1.0]hexane.

[0181] The product is a white solid. 1 H NMR (400MHz, DMSO-d6) δ9.45(s,1H),7.92(d,J=5.7Hz,1H),7.83(d,J=8.3Hz,2H),7.39(d,J=8.2Hz,2H),6.05( d,J=5.7Hz,1H),3.67-3.46(m,8H),3.16(t,J=5.0Hz,4H),2.91(s,3H),1.49(s,2H),1.05(s,3H),0.84(s,3H). 13 C NMR(151MHz,DMSO-d6)δ169.26,160.00,158.75,156.31,142.30,128.35(2C),127.50,118.13 (2C),96.43,46.45,46.27,45.47,34.01,27.10,26.95,26.03(2C),18.78(2C),12.37(2C).TOF MS ES+(m / z):(M+H) + ,calcd for C 23 H 30 N6O3S:471.2178; found,471.2177.HPLC:t R :17.390min,purity:97.40%.

[0182] Example 22

[0183] Preparation of (4-((2-(3-(dimethylamino)azetidin-1-yl)pyrimidin-4-yl)amino)phenyl)(4-(methylsulfonyl)piperazin-1-yl)methanone:

[0184] Follow the method of Example 16, except that in step 3, piperazine is replaced with an equimolar amount of N,N-dimethylazetidin-3-amine.

[0185] The product is a white solid. 1H NMR (400MHz, DMSO-d6) δ9.53(s,1H),7.94(d,J=5.6Hz,1H),7.80(d,J=8.3Hz,2H),7.39(d,J=8.3Hz,2H),6.12(d,J=5.7Hz, 1H), 4.04 (t, J = 8.0Hz, 2H), 3.81 (dd, J = 9.0, 5.1Hz, 2H), 3.61 (s, 4H), 3.17 (q, J = 8.1, 4.7Hz, 5H), 2.91 (s, 3H), 2.15 (s, 6H). 13 C NMR(5151MHz,DMSO-d6)δ169.24,162.56,160.32,156.27,142.13,128.33(2C),127 .72,118.36(2C),97.38,55.58(2C),53.77(2C),45.70,41.49(3C),34.03(2C).TOF MS ES+(m / z):(M+H) + ,calcd for C 21 H 29 N7O3S:460.2131; found,460.2131.

[0186] Example 23

[0187] Preparation of (4-((2-(4-(dimethylamino)piperidin-1-yl)pyrimidin-4-yl)amino)phenyl)(4-(methylsulfonyl)piperazin-1-yl)methanone:

[0188] The method of Example 16 was followed, except that in step 3, piperazine was replaced with an equimolar amount of N,N-dimethyl-4-aminopiperidine.

[0189] The product is a white solid. 1 H NMR (400MHz, DMSO-d6) δ9.47(s,1H),7.96(d,J=5.5Hz,1H),7.71(d,J=8.2Hz,2H),7.39(d,J=8.2Hz,2H),6.05(d,J=5.7Hz,1H),4.61(d,J=13 .1Hz,2H),3.61(s,4H),3.20-3.13(m,4H),2.90(s,4H),2.85(d,J=12.8Hz,2H),2.20(s,6H),1.81(d,J=12.4Hz,2H),1.31(t,J=12.2Hz,2H). 13C NMR(151MHz,DMSO-d6)δ169.21,160.91,160.18,156.50,142.00,128.35(2C),127.75 ,118.36(2C),96.67,61.86(2C),45.46,42.89(2C),41.39(4C),34.03,27.81(2C).TOF MS ES+(m / z):(M+H) + ,calcd forC 23 H 33 N7O3S:488.2444; found,488.2443.

[0190] Example 24

[0191] Preparation of (4-((2-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)pyrimidin-4-yl)amino)phenyl)(4-(methylsulfonyl)piperazin-1-yl)methanone:

[0192] The method of Example 16 was followed, except that in step 3, piperazine was replaced with an equimolar amount of 1-methyl-4-(piperidin-4-yl)piperazine.

[0193] The product is a white solid. 1 H NMR (400MHz, DMSO-d6) δ9.47(s,1H),7.96(d,J=5.6Hz,1H),7.71(d,J=8.4H z,2H),7.39(d,J=8.3Hz,2H),6.06(d,J=5.7Hz,1H),4.62(d,J=13.0Hz,2H), 3.61(s,4H),3.16(t,J=5.0Hz,4H),2.91(s,3H),2.86(t,J=12.3Hz,2H),2.5 7(d,J=11.1Hz,4H),2.29(s,3H),1.82(d,J=12.1Hz,2H),1.39-1.28(m,2H). 13 C NMR(151MHz,DMSO-d6)δ168.57,160.27,159.56,155.88,141.35,127.71(2C),127.14,117.75(2C ),96.11,60.60(2C),53.84,47.15(2C),44.82(2C),44.18,42.34(2C),33.40(3C),27.00(2C).TOF MS ES+(m / z):(M+H) + ,calcd for C 26 H 38N8O3S:543.2866; found,543.2867.

[0194] Example 25

[0195] Preparation of (4-((2-(4-isopropylpiperazin-1-yl)pyrimidin-4-yl)amino)phenyl)(4-(methylsulfonyl)piperazin-1-yl)methanone:

[0196] Follow the method of Example 16, except that in step 3, piperazine is replaced with an equimolar amount of 1-isopropylpiperazine.

[0197] The product is a white solid. 1 H NMR (400MHz, DMSO-d6) δ9.50(s,1H),7.97(d,J=5.6Hz,1H),7.72(d,J=8.4Hz,2H),7.40(d,J=8.4Hz,2H),6.08(d, J=5.6Hz,1H),3.74-3.57(m,8H),3.16(t,J=4.9Hz,4H),2.91(s,3H),2.72(d,J=9.0Hz,1H),1.00(d,J=6.5Hz,6H). 13 C NMR(151MHz,DMSO-d6)δ168.58,160.44,159.52,155.78,141.31,127.74(2C),127.17,11 7.80(2C),96.40,59.16,47.39(2C),44.83(2C),33.41(3C),20.17,17.46(2C),13.49.TOF MSES+(m / z):(M+H) + ,calcd for C 23 H 33 N7O3S:488.2444; found,488.2444.

[0198] Example 26

[0199] Preparation of 1-(4-(4-((4-(4-(methylsulfonyl)piperazine-1-formyl)phenyl)amino)pyrimidin-2-yl)piperazin-1-yl)ethanone:

[0200] The method of Example 16 was followed, except that in step 3, piperazine was replaced with an equimolar amount of 1-(piperazin-1-yl)ethan-1-one.

[0201] The product is a white solid. 1H NMR (400MHz, DMSO-d6) δ9.55(s,1H),7.99(d,J=5.6Hz,1H),7.72(d,J=8.3Hz,2H),7.41(d,J=8.4Hz,2H),6.12(d,J=5.7Hz,1H ),3.75(t,J=5.1Hz,2H),3.69-3.66(m,2H),3.61(s,4H),3.52(d,J=6.1Hz,4H),3.18-3.14(m,4H),2.91(s,3H),2.05(s,3H). 13 C NMR(151MHz,DMSO-d6)δ169.20,168.43,161.00,160.19,156.43,141.83,128.40(2C),127 .90,118.52(2C),97.34,45.46(2C),43.77(2C),43.43(2C),40.67(2C),34.04,21.36.TOF MS ES+(m / z):(M+H) + ,calcd for C 22 H 29 N7O4S:488.2080; found,488.2081.HPLC:t R :17.443min,purity:94.73%.

[0202] Example 27

[0203] Preparation of (4-((2-(4-(cyclopropylcarbonyl)piperazin-1-yl)pyrimidin-4-yl)amino)phenyl)(4-(methylsulfonyl)piperazin-1-yl)methanone:

[0204] According to the method of Example 16, in step 3, piperazine is replaced with an equimolar amount of cyclopropyl (piperazin-1-yl) ketone.

[0205] The product is a white solid. 1 H NMR (400MHz, DMSO-d6) δ9.55(s,1H),8.00(d,J=5.7Hz,1H),7.72(d,J=8.6Hz,2H),7.41(d,J=8.6Hz,2H),6.12(d,J=5.7 Hz,1H),3.78(s,4H),3.62(t,J=25.9Hz,8H),3.16(t,J=5.0Hz,4H),2.91(s,3H),2.05-1.98(m,1H),0.78-0.70(m,4H). 13C NMR(151MHz,DMSO-d6)δ171.25,169.20,160.93,160.20,141.81,128.41(4C),127.94,118 .55(2C),97.37,45.46,44.70,43.92,43.45,41.38,34.04(2C),10.42(2C),7.12(3C).TOF MS ES+(m / z):(M+H) + ,calcd for C 24 H 31 N7O4S:514.2236; found,514.2236.HPLC:t R :15.891min,purity:94.57%.

[0206] Example 28

[0207] Preparation of (4-(methylsulfonyl)piperazin-1-yl)(4-((2-(4-(oxetan-3-yl)piperazin-1-yl)pyrimidin-4-yl)amino)phenyl)methanone:

[0208] Follow the method of Example 16, except that in step 3, piperazine is replaced with an equimolar amount of 1-(oxetane-3-yl)piperazine.

[0209] The product is a white solid. 1 H NMR (400MHz, DMSO-d6) δ9.51(s,1H),7.97(d,J=5.7Hz,1H),7.71(d,J=8.7Hz,2H),7.39(d,J=8.7Hz,2H),6.09(d,J=5.7Hz,1H),4.55(t,J= 6.4Hz,2H),4.47(t,J=6.0Hz,2H),3.72(t,J=4.8Hz,4H),3.61(s,4H),3.42(s,1H),3.18-3.13(m,4H),2.90(s,3H),2.30(t,J=4.9Hz,4H). 13 C NMR(151MHz,DMSO-d6)δ169.19,161.08,160.16,156.42,141.90,128.37(2C),127.84,118. 46(2C),97.11,74.42(2C),58.47(2C),48.98(2C),45.47,43.34(2C),40.06,34.03(2C).TOF MS ES+(m / z):(M+H) + ,calcd forC 23 H31 N7O4S:502.2236; found,502.2237.HPLC:t R :15.428min,purity:94.71%.

[0210] Example 29

[0211] Preparation of (4-(methylsulfonyl)piperazin-1-yl)(4-((2-(4-morpholinylpiperidin-1-yl)pyrimidin-4-yl)amino)phenyl)methanone:

[0212] The method of Example 16 was followed, except that in step 3, piperazine was replaced with an equimolar amount of 4-(piperidin-4-yl)morpholine.

[0213] The product is a white solid. 1 H NMR (400MHz, DMSO-d6) δ9.47 (s, 1H), 7.96 (d, J = 5.7Hz, 1H), 7.71 (d, J = 8.3Hz, 2H),7.40(d,J=8.3Hz,2H),6.06(d,J=5.6Hz,1H),4.61(d,J=12.9Hz,2H),3.6 6-3.53(m,8H),3.16(t,J=4.9Hz,4H),2.91(d,J=1.2Hz,3H),2.85(d,J=12.6H z,2H),2.48-2.37(m,5H),1.83(d,J=12.3Hz,2H),1.31(q,J=11.5,9.9Hz,2H). 13 C NMR(151MHz,DMSO-d6)δ169.20,160.92,160.18,156.50,141.99,128.34(2C),127.76,118.3 7(2C),96.69,66.58(3C),61.57(2C),49.40(3C),45.45,42.93(2C),34.03,27.68(2C).TOFMS ES+(m / z):(M+H) + ,calcd for C 25 H 35 N7O4S:530.2549; found,530.2549.

[0214] Example 30

[0215] Preparation of (4-(methylsulfonyl)piperazin-1-yl)(4-((2-(4-(pyrrolidin-1-yl)piperidin-1-yl)pyrimidin-4-yl)amino)phenyl)methanone:

[0216] The method of Example 16 was followed, except that in step 3, piperazine was replaced with an equimolar amount of 4-(pyrrolidin-1-yl)piperidine.

[0217] The product is a white solid. 1 H NMR (400MHz, DMSO-d6) δ9.47(s,1H),7.96(d,J=5.6Hz,1H),7.72(d,J=8.3Hz,2H),7.40(d,J=8.3Hz,2H),6.06(d,J=5.7Hz,1H),4.46(d,J=13.2Hz,2H ),3.61(s,4H),3.16(t,J=5.0Hz,4H),3.03-2.95(m,2H),2.91(s,3H),2.2 2(s,1H),1.92-1.85(m,2H),1.66(q,J=3.7,3.2Hz,4H),1.39-1.25(m,2H). 13 C NMR(151MHz,DMSO-d6)δ169.21,161.02,160.17,156.47,142.02,128.35(2C),127.73,118.35 (2C),96.67,61.23(2C),50.83(3C),45.46,42.26(2C),34.03(2C),30.85(2C),22.95(2C).TOF MS ES+(m / z):(M+H) + ,calcd for C 25 H 35 N7O3S:514.2600; found,514.2600.

[0218] Example 31

[0219] Preparation of 4-((2-(4-acetylpiperazin-1-yl)-5-chloropyrimidin-4-yl)amino)-N-(1-(methylsulfonyl)piperidin-4-yl)benzamide:

[0220] The method of Example 1 was followed, except that in step 1, 2,4-dichloropyrimidine was replaced with an equimolar amount of 2,4,5-trichloropyrimidine, step 2 remained unchanged, and in step 3, piperazine was replaced with an equimolar amount of 1-(piperazin-1-yl)ethan-1-one.

[0221] The product is a white solid. 1H NMR (400MHz, DMSO-d6) δ8.95 (s, 1H), 8.24 (d, J = 7.7Hz, 1H), 8.13 (s, 1H), 7.8 5(d,J=8.5Hz,2H),7.78(d,J=8.6Hz,2H),3.92(dt,J=15.6,5.6Hz,1H),3.69 (t,J=5.1Hz,2H),3.65-3.55(m,4H),3.50(t,J=5.2Hz,4H),2.88(s,3H),2.8 7-2.81(m,2H),2.03(s,3H),1.94-1.87(m,2H),1.61(tt,J=11.9,6.1Hz,2H). 13 C NMR(151MHz,DMSO-d6)δ168.44,165.22,159.06,155.16,155.06,141.56,128.77,127.71(2C),12 0.76(2C),102.85,45.87,45.27,44.81(2C),43.94,43.57,40.51,34.24,30.87(2C),21.34.TOFMS ES+(m / z):(M+H) + ,calcd for C 23 H 30 ClN7O4S:536.1847; found,536.1838.HPLC:t R :16.755min,purity:95.78%.

[0222] Example 32

[0223] Preparation of 4-((5-chloro-2-(4-(cyclopropylcarbonyl)piperazin-1-yl)pyrimidin-4-yl)amino)-N-(1-(methylsulfonyl)piperidin-4-yl)benzamide:

[0224] According to the method of Example 31, in step three, 1-(piperazin-1-yl)ethan-1-one is replaced with an equimolar amount of cyclopropyl(piperazin-1-yl)methanone.

[0225] The product is a white solid. 1H NMR (400MHz, DMSO-d6) δ8.95 (s, 1H), 8.24 (d, J = 7.7Hz, 1H), 8.13 (s, 1H), 7.85 (d,J=8.5Hz,2H),7.79(d,J=8.8Hz,2H),3.92(d,J=6.1Hz,1H),3.74(s,4H),3. 64(s,2H),3.57(d,J=13.2Hz,4H),2.88(s,3H),2.87-2.82(m,2H),2.01(t,J=5 .7Hz,1H),1.93-1.87(m,2H),1.60(td,J=11.8,7.9Hz,2H),0.77-0.70(m,4H). 13 C NMR(151MHz,DMSO-d6)δ171.27,165.24,159.07,155.17,155.07,141.58,128.78,127.73(2C),120.76 (2C),102.87,45.87,44.81(2C),44.54,44.10,43.60,41.24,34.24,30.87(2C),10.39,7.12(2C).TOF MS ES+(m / z):(M+H) + ,calcd for C 25 H 32 ClN7O4S:562.2003; found,562.2003.HPLC:t R :17.037min,purity:94.98%.

[0226] Example 33

[0227] Preparation of 4-((5-chloro-2-(4-morpholinylpiperidin-1-yl)pyrimidin-4-yl)amino)-N-(1-(methylsulfonyl)piperidin-4-yl)benzamide:

[0228] According to the method of Example 31, in step 3, 1-(piperazin-1-yl)ethan-1-one is replaced with an equimolar amount of cyclo-4-(piperidin-4-yl)morpholine.

[0229] White solid, 1H NMR (400MHz, DMSO-d6) δ8.86 (s, 1H), 8.23 ​​(d, J = 7.7Hz, 1H), 8.08 (s, 1H), 7.83 ( d,J=8.2Hz,2H),7.79(d,J=8.6Hz,2H),4.50(d,J=12.9Hz,2H),3.96-3.88(m,1H ),3.57(d,J=14.1Hz,6H),2.87(d,J=10.2Hz,8H),2.45(s,4H),1.90(d,J=12.7 Hz, 2H), 1.82 (d, J = 12.0Hz, 2H), 1.60 (d, J = 12.1Hz, 2H), 1.31 (d, J = 12.3Hz, 2H). 13 C NMR(151MHz,DMSO-d6)δ165.19,158.96,155.11,155.08,141.73,128.58,127.67(2C),120.51(2C),10 2.21,66.57(2C),61.32,49.38(2C),45.87,44.82(2C),43.11(2C),34.24,30.88(2C),27.56(2C).TOF MS ES+(m / z):(M+H) + ,calcd for C 26 H 36 ClN7O4S:578.2316; found,578.2318.

[0230] Example 34

[0231] Preparation of (4-((5-chloro-2-((2S,6R)-2,6-dimethylmorpholinyl)pyrimidin-4-yl)amino)phenyl)(4-(methylsulfonyl)piperazin-1-yl)methanone:

[0232] According to the method of Example 31, in step 2, 1-(methylsulfonyl)piperidin-4-amine is replaced with an equimolar amount of 1-(methylsulfonyl)piperazine, and in step 3, 1-(piperazin-1-yl)ethan-1-one is replaced with an equimolar amount of (2S,6R)-2,6-dimethylmorpholine.

[0233] White solid, 1H NMR (400MHz, DMSO-d6) δ8.93(s,1H),8.10(s,1H),7.77(d,J=8.2Hz,2H),7.42(d,J=8.3Hz,2H), 4.32(d,J=13.1Hz,2H),3.57(d,J=27.4Hz,6H),3.17(s,4H),2.90(s,3H),1.12(d,J=6.1Hz,6H). 13 C NMR(151MHz,DMSO-d6)δ169.05,158.86,155.20,155.01,140.39,129.68,127.60(2 C),121.23(2C),102.62,70.88(3C),49.34(2C),45.39,34.08(2C),18.72(3C).TOF MS ES+(m / z):(M+H) + ,calcd for C 22 H 29 ClN6O4S:509.1738; found,509.1727.HPLC:t R :11.301min,purity:97.78%.

[0234] Example 35

[0235] Preparation of (4-((5-chloro-2-(6,6-dimethyl-3-azabicyclo[3.1.0]hexan-3-yl)pyrimidin-4-yl)amino)phenyl)(4-(methylsulfonyl)piperazin-1-yl)methanone:

[0236] According to the method of Example 34, in step 3, (2S,6R)-2,6-dimethylmorpholine is replaced with an equimolar amount of 6,6-dimethyl-3-azabicyclo[3.1.0]hexane.

[0237] White solid, 1 H NMR (400MHz, DMSO-d6) δ8.76(s,1H),8.05(s,1H),7.94(d,J=8.3Hz,2H),7.41(d,J=8.3Hz,2H),3.57(d,J=30.7Hz ,6H),3.45(d,J=11.8Hz,2H),3.18(d,J=5.3Hz,4H),2.91(s,3H),1.49(t,J=2.3Hz,2H),1.04(s,3H),0.83(s,3H). 13C NMR(151MHz,DMSO-d6)δ169.10,156.89,154.94,154.88,140.83,129.16,127.77(2C),120.44(2 C),102.17,46.80,46.48,45.44,34.04,27.12,26.98,25.97(2C),18.81(2C),12.33(2C).TOFMS ES+(m / z):(M+H) + ,calcd for C 23 H 29 ClN6O3S:505.1789; found,505.1781.HPLC:t R :13.336min,purity:95.09%.

[0238] Example 36

[0239] Preparation of (4-((5-chloro-2-(4-(pyrrolidin-1-yl)piperidin-1-yl)pyrimidin-4-yl)amino)phenyl)(4-(methylsulfonyl)piperazin-1-yl)methanone:

[0240] According to the method of Example 34, in step 3, (2S,6R)-2,6-dimethylmorpholine is replaced with an equimolar amount of 4-(pyrrolidin-1-yl)piperidine.

[0241] White solid, 1 H NMR (400MHz, DMSO-d6) δ8.95(s,1H),8.12(s,1H),7.76(d,J=8.5Hz,2H),7.42(d,J=8.5Hz,2H),4.57(d,J=13.3Hz,2H),3.68-3.56(m,4 H),3.17(t,J=5.0Hz,6H),2.91(d,J=3.0Hz,3H),2.86(d,J=12.8Hz,2H),2.08(d,J=12.1Hz,2H),1.98-1.81(m,4H),1.57-1.46(m,2H). 13 C NMR(151MHz,DMSO-d6)δ169.04,158.86,155.30,155.14,140.40,129.68,127.74(2C),12 1.24(2C),102.82,61.14(2C),50.71(3C),45.43,42.27(2C),34.08(2C),22.57(4C).TOF MS ES+(m / z):(M+H) + ,calcd for C 25H 34 ClN7O3S:548.2211; found,548.2205.HPLC:t R :17.672min,purity:96.48%.

[0242] The structural formulas of the 4-aminopyrimidine compounds prepared in Examples 1 to 36 are shown in Table 1.

[0243] Table 1 Structural formula of 4-aminopyrimidine compounds prepared in Examples 1 to 36

[0244]

[0245]

[0246]

[0247]

[0248] The in vitro anti-tumor cell activity of each 4-aminopyrimidine compound prepared in Examples 1 to 36 was determined.

[0249] The selected cancer cells were lung cancer cells H1975, lung cancer cells A549 and lung cancer cells H460, and the control substances were osimertinib (AZD9291) and bemcentinib. The method was as follows:

[0250] (1) After the cells have recovered and been passaged for 3 times and stabilized, trypsin solution (0.25%) is used to digest them from the bottom of the culture flask. The cell digestion solution is aspirated into a centrifuge tube and culture medium is added to terminate the digestion. The centrifuge tube is centrifuged at 1000 r / min for 3 minutes, the supernatant is discarded and 3 mL of culture medium is added. The cells are pipetted to mix well, 10 μL of the cell suspension is aspirated and added to a cell counting plate for counting, and the cell concentration is adjusted to 10 4 180 μL of cell suspension was added to all wells of the 96-well plate, except for the top, bottom, and leftmost wells, which were blank wells. The 96-well plate was placed in an incubator and incubated for 24 h.

[0251] (2) Dissolve the test sample in 20 μL of dimethyl sulfoxide, then add 980 μL of culture medium to dissolve the sample into a 1 mg / mL solution. Then dilute the sample in an EP tube to 1, 0.333, 0.111, 0.037, and 0.012 μg / mL. Add 20 μL of each concentration to 3 wells. The cells in the two rows and two columns around the well are greatly affected by the environment, so only the blank wells are used. Place the 96-well plate in an incubator and culture for 72 hours.

[0252] (3) Discard the drug-containing culture medium in the 96-well plate, rinse the cells twice with phosphate buffered saline (PBS), add 100 μL of 0.5 mg / mL MTT (thiazolyl blue) solution to each well, place in an incubator for 4 hours, discard the MTT solution, and add 100 μL of dimethyl sulfoxide. Oscillate on a magnetic oscillator to fully dissolve the surviving cells and the MTT reaction product, formazan, and place in a microplate reader to measure the results at a wavelength of 492 nM. The drug IC can be calculated using the Bliss method. 50 value.

[0253] The antiproliferative activities of the 4-aminopyrimidine compounds prepared in Examples 1 to 36 against three lung cancer cell lines are shown in Table 2, where NA represents compound IC 50 >100.00μM, ND means not detected.

[0254] Table 2 Anti-cell proliferation activity of 4-aminopyrimidine compounds

[0255]

[0256] The EGFR and AXL kinase activities (IC 50 and inhibition rate determination)

[0257] Methods: Prepare a 384-well plate with wells for test compounds, blank controls, and positive drug controls. 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 drug control wells. Add 2.5 μL of compound at varying concentrations to the test compound wells and 2.5 μL of kinase buffer to the blank control wells. Add 10 μL of EGFR kinase antibody and EDTA reagent to each well (the same procedure for the AXL kinase assay), centrifuge to mix, and equilibrate at room temperature for 60 minutes. The final antibody concentration is 2 nM, and the final EDTA concentration is 8 mM. Fluorescence values ​​are read using Envision. Calculate the percent inhibition of the compound.

[0258] Percent inhibition rate = (Lance signal value - Min) / (Max - Min) × 100%

[0259] Min: Lance signal value in the absence of enzyme; Max: Lance signal value of DMSO control. Data were analyzed using SPSS, MS Excel, and Graphpad 5.0 to obtain IC 50 .

[0260] AZD9291 and Bemcentinib were used as positive controls, and Kinase-Glo and The inhibitory activity of each compound on EGFR and AXL kinases was tested by Ultra enzyme activity evaluation method. The results are shown in Table 3, where ND means not detected.

[0261] Table 3 Activity of 4-aminopyrimidine compounds against EGFR and AXL kinases

[0262]

[0263] In addition, the present invention also tested the IC values ​​of the compounds prepared in Examples 26 and 36 for AXL. 50 The results showed that the IC of AXL in Examples 26 and 36 was 50 9.77 and 8.23 ​​μM respectively.

[0264] From the above test results, it can be seen that most of the compounds of formula I and formula II to be protected by the present invention have good in vitro anti-cell proliferation activity, such as the compounds prepared in Examples 11, 12, 14, 19, 21, 30, 32, and 36. However, for EGFR T790M / C797S In terms of kinase activity, most of the examples had poor activity, and only the compounds prepared in Examples 26 and 36 had both kinase activity against EGFR T790M / C797S , AXL also showed inhibitory activity, among which the compound prepared in Example 36 had an inhibitory effect on EGFR T790M / C797S The inhibitory activity of the kinase is comparable to that of the positive drug AZD9291. T790M / C797S , inhibitors of AXL kinase, particularly the compound prepared in Example 36.

[0265] Study on the anti-tumor mechanism of 4-aminopyrimidine compounds in vitro

[0266] In order to explore the anti-tumor mechanism of the embodiment, the present invention takes the compound prepared in Example 36 as an example and explores its anti-tumor mechanism on H1975 cells through multiple in vitro pharmacological experiments.

[0267] 1. The compound prepared in Example 36 can induce apoptosis in H1975 cells

[0268] 1.1 Annexin V-FITC / PI double staining assay for apoptosis

[0269] The specific steps are as follows:

[0270] (1) H1975 cells were digested and centrifuged using the passage method, and the number of cells was counted. 2.0×10 5The cells were seeded into 6-well plates at a density of 100 cells / well. Subsequently, the cells were cultured in a 37°C, 5% CO2 incubator for 24 h to allow the cells to adhere.

[0271] (2) After 24 h, 3 mL of serum-free culture medium was added to each well, followed by 300 μL of the compound prepared in Example 36 (2, 4, and 6 μM) and the positive drug (AZD9291, 4 μM) at the prepared concentrations. The blank control group was used and the cells were placed in a 37°C, 5% CO2 incubator for 24 h of drug action.

[0272] (3) Place the 6-well plate in a clean bench, aspirate the original culture medium, and wash twice with PBS. Add 1 mL of EDTA-free trypsin to each well for digestion. After the cells are completely detached, add 1 mL of culture medium to terminate the digestion. Rinse once, transfer to a 5 mL centrifuge tube, and centrifuge. Discard the upper layer of centrifuge liquid, add 1 mL of PBS to the centrifuge tube, and slowly pipette the cells to make them uniform. Repeat this step twice.

[0273] (4) Protect from light and wrap the centrifuge tubes with tin foil. Add 500 μL of buffer to each centrifuge tube and pipette slowly for 8-10 strokes. Add 5 μL of Aximnex V and 5 μL of PI dye to the blank control group. Repeat the blank control procedure for the compound prepared in Example 36 and the positive wells. Incubate in the dark for 30 min.

[0274] (5) The cell-dye mixture was counted by flow cytometry at a rate of 1.0 × 10 4 cells and record the data.

[0275] In order to explore the effect of the compound on cell apoptosis, the Annexin V-FITC / PI double staining method was used to detect the effect of the compound prepared in Example 36 on cell apoptosis. The results are shown in FIG. Figure 1 Middle A.

[0276] Depend on Figure 1 As shown in Figure A, the total apoptosis rate in the blank control group was 2.60%. The compound prepared in Example 36 induced apoptosis in H1975 cells at 2 μM, 4 μM, and 6 μM, ranging from 4.08%, 4.76%, and 9.82%. In late apoptosis, the apoptosis rate increased from 2.47% to 5.71%. The apoptosis results indicate that the compound prepared in Example 36 induces apoptosis in H1975 cells in a dose-dependent manner.

[0277] 1.2 AO staining assay to determine cell morphology

[0278] The specific steps are as follows:

[0279] (1) The cell density was 4×10 4 1 mL of H1975 cell suspension was added to each well of a 24-well plate and cultured in a cell culture incubator for 24 h.

[0280] (2) The original culture medium was aspirated and washed twice with PBS. 1 mg of the compound prepared in Example 36 was dissolved in 20 μL of DMSO and added to serum-free medium to achieve concentrations of 2, 4, and 6 μM. 1 mL of the solution was added to each well, gently shaken until uniform, and incubated in a cell culture incubator for 12 h.

[0281] (3) Aspirate the culture medium and add 1 mL PBS to wash twice. Rinse once with 500 μL 1× buffer, add 200 μL AO dye solution (acridine orange: 1× buffer = 1:20), and incubate in a cell culture incubator for 15 minutes.

[0282] (4) Aspirate the buffer, wash each well three times with PBS, add 500 μL of PBS, and observe under a microscope. Turn the filter to 4 / 5, adjust the laser to blue light, take a picture, and save it.

[0283] In order to detect the effect of the compound on cell apoptosis, the compound prepared in Example 36 was used to detect the staining of H1975 cells by AO staining. The results are shown in FIG. Figure 1 Middle B.

[0284] from Figure 1 As can be clearly seen in Figure B, the compound prepared in Example 36 exhibits distinct orange-red fluorescence at 2 μM, indicating the onset of apoptotic bodies. At 4 μM, the orange-red fluorescence increases, reaching an intensity comparable to or even superior to that of a positive-acting drug. At 6 μM, the orange-red fluorescence intensity continues to increase, and blurred cell edges appear on H1975 cells. These results demonstrate that the ability of the compound prepared in Example 36 to induce apoptosis in H1975 cells gradually increases with increasing concentration.

[0285] 1.3 JC-1 assay for measuring cell MPP

[0286] The specific steps are as follows:

[0287] (1) The cell inoculation and drug addition methods are the same as those in 1.2 above.

[0288] (2) Aspirate the culture medium, add 1 mL of PBS to wash twice, add 1 mL of JC-1 working solution (50 μL of JC-1 (200×): 8 mL of ultrapure water), mix thoroughly, place in a cell culture incubator, and incubate for 20 min.

[0289] (3) Aspirate the working solution and wash twice with 1× JC-1 staining buffer.

[0290] (4) Add 2 mL of 200× JC-1 staining buffer, place under a fluorescence microscope, observe and take pictures.

[0291] In order to detect the ability of the compound to induce cell apoptosis, the JC-1 assay was used to detect the effect of the compound prepared in Example 36 on the mitochondrial membrane potential of H1975 cells. The results are shown in Figure 1 Middle C.

[0292] like Figure 1 As shown in Figure C, compared to the blank control, the compound prepared in Example 36 exhibited significant green fluorescence at 2 μM. The amount and intensity of green fluorescence increased with increasing concentration, demonstrating that the compound prepared in Example 36 was able to induce a decrease in mitochondrial membrane potential in a dose-dependent manner, thereby inducing cell apoptosis. Furthermore, the apoptosis-inducing ability of the compound prepared in Example 36 at a concentration of 4 μM was comparable to that of the positive drug at the same concentration. These results demonstrate that the compound prepared in Example 36 induced apoptosis in H1975 cells in a dose-dependent manner.

[0293] 2. The compound prepared in Example 36 can arrest the H1975 cell cycle at the G2 / M phase

[0294] The specific steps are as follows:

[0295] (1) The cell inoculation and drug addition methods are the same as those in 1.1 above.

[0296] (2) H1975 cells were digested with EDTA-free trypsin, placed in a 5 mL centrifuge tube, and centrifuged. The tube was washed twice with 1 mL of PBS (centrifuged), and 500 μL of PBS was added. The cells were then blown evenly. 500 μL of 70% cold ethanol (ethanol + PBS) was slowly added, and the cells were fixed in a 4°C refrigerator.

[0297] (3) After 2 hours, remove the tube from the refrigerator and centrifuge directly. Wash twice with PBS. Add 500 μL of PBS and blow evenly. Add 200 μL of dye (Rnase A:PI = 1:9) to each tube and place in the dark for 30 minutes.

[0298] (4) The cell-dye mixture was counted by flow cytometry at a rate of 1.0 × 10 4 cells and record the data.

[0299] In order to detect the effect of the compound on the cell cycle, the effect of the compound prepared in Example 36 on the H1975 cell cycle was detected by flow cytometry. The results are shown in Figure 2 .

[0300] Depend on Figure 2As shown, the compound prepared in Example 36 was able to arrest the H1975 cell cycle at the G2 / M phase. The G2 / M arrest rate of the compound prepared in Example 36 increased from 20.62%, 21.1%, and 24.65% at 2 μM, 4 μM, and 6 μM, respectively. These results demonstrate that the compound prepared in Example 36 arrests H1975 cells at the G2 / M phase in a dose-dependent manner, preventing mitosis.

[0301] 3. The compound prepared in Example 36 can increase the content of ROS in H1975 cells

[0302] The specific steps are as follows:

[0303] (1) Cells in the logarithmic proliferation phase were plated at 2.0×10 5 The cells were seeded into 6-well plates at a density of 100 cells / mL and cultured in an incubator for 24 h.

[0304] (2) 3 mL of serum-free culture medium was added to each well, followed by 300 μL of the compound prepared in Example 36 (2, 4, and 6 μM) and the positive drug (AZD9291, 4 μM) at the prepared concentrations. A blank control group was used with no drug solution added. After incubation for the specified time, the old culture medium was discarded, and the cells were washed twice with PBS before adding the staining solution.

[0305] (3) After washing with PBS three times, 1 mL of culture medium containing DCFH-DA was added to each well.

[0306] (4) Remove excess formaldehyde and wash twice with PBS.

[0307] (5) Finally, add 1 mL / well of crystal violet dye and let it stand for 15 minutes.

[0308] (6) After the final two PBS washes, the cells were observed and photographed using a fluorescence microscope.

[0309] In order to detect the effect of the compound on the ROS content in tumor cells, the DCFH-DA probe was used to detect the changes in the reactive oxygen species in H1975 cells caused by the compound prepared in Example 36. The results are shown in Figure 3 .

[0310] Depend on Figure 3 As can be seen, the H1975 cells in the blank group produced no green fluorescence, indicating no ROS generation. However, low concentrations of the compound prepared in Example 36 produced almost no green fluorescence. As the concentration of the compound prepared in Example 36 increased, the intensity of green fluorescence increased, reaching a level comparable to that of a positive drug at 6 μM. These results demonstrate that the compound prepared in Example 36 increased the level of ROS in H1975 cells in a dose-dependent manner.

[0311] 4. The compound prepared in Example 36 can inhibit the migration of H1975 cells

[0312] The specific steps are as follows:

[0313] (1) H1975 cells in the logarithmic growth phase were cultured at 5.0×10 5 Cells were seeded in 6-well plates.

[0314] (2) When the cells adhered and proliferated to 80% to 90%, the six-well plate was scratched vertically with a pipette tip, washed twice with PBS, and photographed under a microscope. Then, 3 mL of serum-free culture medium was added to each well, followed by 300 μL of the compound prepared in Example 36 (2, 4, and 6 μM) and the positive drug (AZD9291, 4 μM) at the adjusted concentrations. A blank control group was used with no drug added, and the cells were placed in an incubator and continued to be cultured.

[0315] (3) After incubation for 24 h, the upper floating cells were washed with PBS and observed and photographed under a microscope.

[0316] In order to detect the effect of the compound on cell migration ability, the cell scratch test was performed to detect the migration of H1975 cells by the compound prepared in Example 36. The results are shown in FIG. Figure 4 .

[0317] pass Figure 4 As can be seen, after 24 hours of H1975 cell culture, cells in the control group and the 2 μM dosing group significantly migrated toward the center, while the positive group and the 4 μM and 6 μM dosing groups significantly inhibited H1975 cell migration. Furthermore, the higher the concentration of the compound prepared in Example 36, the fewer cells migrated. These results demonstrate that the compound prepared in Example 36 inhibited H1975 cell migration in a dose-dependent manner.

[0318] In vitro and in vivo toxicity evaluation of 4-aminopyrimidine compounds

[0319] In order to ensure the biological safety of the examples, the present invention conducted in vivo and in vitro toxicity evaluations on the compound prepared in Example 36 by hemolysis test and KM mouse toxicity test.

[0320] 1. Hemolysis test

[0321] The specific steps are as follows:

[0322] (1) Take 1 mL of 4% sheep red blood cells and perform refrigerated centrifugation and washing. Repeat the operation twice to extract the red blood cells.

[0323] (2) 0.9% physiological saline was added to the red blood cells extracted above, and after pipetting, different concentrations of the compound prepared in Example 36 were added.

[0324] (3) Incubate the above red blood cell suspension at 37°C for 1 hour, measure the absorbance using a UV spectrophotometer, and calculate the hemolysis rate:

[0325] Hemolysis rate (%) = (A experimental group - A negative control group) / (A positive control group - A negative control group)

[0326] The present invention conducted a hemolysis experiment using sheep red blood cells to simulate the effect of the compound prepared in Example 36 in blood and explore whether the compound would affect blood safety. Using 1% Triton as a positive control, the effect of the compound prepared in Example 36 on red blood cell hemolysis at 8, 16, 32, 64, 128, and 256 μg / mL was explored. The hemolysis rate was used as a safety indicator to evaluate the safety of the compound on blood. The results are shown in FIG. Figure 5 .

[0327] Depend on Figure 5 As can be seen, the hemolysis rate gradually increased with increasing concentration of the compound prepared in Example 36. However, the hemolysis rate at 256 μg / mL was 4.46%, which is within the safe range. This indicates that the compound prepared in Example 36 has good biosafety and does not cause hemolytic anemia. These results indicate that the hemolysis rate of the compound prepared in Example 36 on erythrocytes is within a safe range and warrants further study.

[0328] 2. KM mouse toxicity experiment

[0329] 2.1 Experimental methods:

[0330] (1) Grouping: KM mice with ear tags were divided into 4 groups, with 5 mice in each group: a blank group (0.9% saline), a positive group (75 mg / kg), a low-dose group of the compound prepared in Example 36 (25 mg / kg), and a high-dose group of the compound prepared in Example 36 (75 mg / kg).

[0331] (2) Oral administration: Dissolve the compound in saline, add Tween 80 to aid solubilization, and sonicate. The blank group receives an equal amount of saline, while the positive group and the treatment group receive the prescribed dose. Mice are divided into groups and administered once every 24 hours for a total of 7 days.

[0332] (3) Sampling: After 7 days, the four groups of mice were sacrificed by eye bleeding. Blood (>0.5 mL) was collected from each mouse, labeled, and placed in a 4°C refrigerator for later use. Subsequently, each group of mice was dissected, and the heart, liver, spleen, lungs, and kidneys were collected and photographed. In addition, five internal organs of one mouse in each group were collected and fixed with 4% paraformaldehyde for subsequent H&E staining experiments.

[0333] 2.2 Mouse blood sample analysis:

[0334] (1) Place the mouse blood sample in a 4°C centrifuge and centrifuge at 4000 rpm for 5 minutes. Take the supernatant and centrifuge again to take about 150 μL of the supernatant into a clean EP tube.

[0335] (2) Prepare 11 preoperative analysis kits, take 100 μL of serum and add it along the injection hole of the reagent disk, place it in the automatic multifunctional biochemical analyzer, perform blood testing, export the data and perform data analysis using GraphPad Prism 8.

[0336] 2.3 HE staining:

[0337] (1) Tissue processing: Wash the organs with physiological saline and dry them. Add the tissues to 4% formaldehyde solution and fix them for 24 hours. Dehydrate the fixed tissues in 50%, 70%, 80%, and 90% alcohol solutions in sequence, soaking them for 1 hour at each alcohol concentration.

[0338] (2) Sample clearing and wax immersion: Place the dehydrated tissue in a xylene solution and clear it twice, each time for 15 minutes. Place the cleared tissue in a prepared paraffin solution and immerse it twice, each time for 1 hour.

[0339] (3) Dewaxing of paraffin sections: Place the sections 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 then wash with tap water.

[0340] (4) Hematoxylin staining: Place the sections in hematoxylin staining solution for 3-5 minutes, wash with tap water, differentiate with differentiation solution, wash with tap water, reblue with bluing solution, and rinse with running water.

[0341] (5) Eosin staining: The sections were dehydrated in 85% and 95% graded alcohol for 5 min each, and then stained in eosin solution for 5 min.

[0342] (6) Dehydration and sealing: The sections were sequentially placed in anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, xylene I for 5 min, and xylene II for 5 min to make them transparent, and then sealed with neutral gum.

[0343] (7) Observe the sample under a microscope and save the photos, and analyze the sample staining results.

[0344] During the administration period, KM mice maintained normal activities and were in good spirits. The mice were weighed and recorded daily. Figure 6 .

[0345] like Figure 6As shown, the body weight of mice showed an overall upward trend during the administration period. The increase in the low-dose group and the high-dose group of the compound prepared in Example 36 was higher than that in the positive group, and was equivalent to the increase in the blank group, indicating that the compound prepared in Example 36 had no obvious toxicity to mice.

[0346] After 7 consecutive days of administration, blood was collected from the eyeballs of each mouse for subsequent biochemical blood sample analysis. The mice were then dissected, and the five internal organs were weighed and photographed. The effects of the compound prepared in Example 36 on the mouse organs were observed macroscopically. The results are shown in FIG. Figure 7 , where A to E are macroscopic images of the heart, liver, spleen, lungs, and kidneys, respectively, and F is the organ index calculated after weighing the five internal organs separately.

[0347] like Figure 7 As shown in Figures A to E, it can be clearly seen that the compound prepared in Example 36 has no obvious toxic side effects on the five internal organs of mice. Figure 7 As shown in Figure F, compared to the blank group, there were no significant differences in the organ indices of the positive group, the low-dose group of the compound prepared in Example 36, and the high-dose group of the compound prepared in Example 36, indicating that the compound prepared in Example 36 had no significant effect on mouse organs. Relatively speaking, the liver index in the positive group was slightly higher. Therefore, macroscopically, the toxicity of the groups receiving the compound prepared in Example 36 was lower than that of the positive group.

[0348] Mouse biochemical blood sample analysis Figure 8 As shown, A is the analysis results of total protein (TP), urea (UREA) and glucose (GLU), B is the analysis results of creatinine (CREA), aspartate aminotransferase (AST) and alanine aminotransferase (ALT), and C is the analysis results of alkaline phosphatase (ALP), lactate dehydrogenase (LDH) and creatine kinase (CK).

[0349] like Figure 8As shown, in the detection of total protein (TP), creatinine (CREA), alkaline phosphatase (ALP), lactate dehydrogenase (LDH) and creatine kinase (CK) levels, there was no significant difference between the low-dose group and the high-dose group of the compound prepared in Example 36 and the blank group (0.9% saline). In the detection of urea (UREA) and glucose (GLU) levels, the drug-treated group was slightly higher than the blank group (0.9% saline) and the positive group (AZD9291), indicating that there may be some impact on the kidneys, but both were within the normal range. The aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels in the two drug-treated groups were lower than those in the blank group. Overall, the compound prepared in Example 36 had no obvious toxicity. However, alkaline phosphatase (ALP) and creatine kinase (CK) were almost all elevated in the drug-treated group, the blank group, and the positive group, which may be due to the mice themselves. H&E staining of the five internal organs will be performed later to observe whether inflammation occurs.

[0350] Subsequently, the five internal organs of one mouse from each of the four groups of KM mice were taken for H&E staining and the morphology of cells and structures was observed under a microscope to evaluate the effects of the compounds on tissues and cells. The results are shown in Figure 9 .

[0351] like Figure 9 As shown in the results of H&E staining, the compound prepared in Example 36 did not cause significant damage to mouse organs or inflammation, and was safe at a dose of 75 mg / kg. The experimental results indicate that the compound prepared in Example 36 has good in vivo biosafety.

[0352] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A 4-aminopyrimidine compound targeting EGFR and AXL, characterized in that: The structural formula of the 4-aminopyrimidine compound is shown in Formula I or Formula II: In formula I, when R2 is H, R1 is When R2 is halogen, R1 is In formula II, when R4 is H, R3 is When R4 is halogen, R3 is 2. A method for preparing the 4-aminopyrimidine compound targeting EGFR and AXL according to claim 1, characterized in that: The preparation route of the compound shown in Formula I is as follows: In the above route, compound B is prepared by an aromatic nucleophilic substitution reaction; compound C is prepared by an acid-amine condensation reaction; and compound D is prepared by an aromatic nucleophilic substitution reaction. The preparation route of the compound shown in formula II is as follows: In the above route, compound B is prepared by an aromatic nucleophilic substitution reaction; compound E is prepared by an acid-amine condensation reaction; and compound F is prepared by an aromatic nucleophilic substitution reaction.

3. A pharmaceutically acceptable salt of the 4-aminopyrimidine compound targeting EGFR and AXL according to claim 1.

4. A pharmaceutical preparation, characterized in that The active ingredient is the 4-aminopyrimidine compound targeting EGFR and AXL targets according to claim 1 or a pharmaceutically acceptable salt of the 4-aminopyrimidine compound targeting EGFR and AXL targets according to claim 3.

5. Use of the 4-aminopyrimidine compound targeting EGFR and AXL as claimed in claim 1 in the preparation of a drug for treating and / or preventing pathological proliferative diseases.

6. The use according to claim 5, characterized in that The pathological proliferative disease is cancer.

7. Use of a pharmaceutically acceptable salt of a 4-aminopyrimidine compound targeting EGFR and AXL as claimed in claim 3 in the preparation of a medicament for treating and / or preventing pathological proliferative diseases.

8. The use according to claim 7, characterized in that The pathological proliferative disease is cancer.

9. Use of the pharmaceutical preparation according to claim 4 in the preparation of a medicament for treating and / or preventing pathological proliferative diseases.

10. The use according to claim 9, characterized in that The pathological proliferative disease is cancer.

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