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

Abstract

The application discloses a 4-aminopyrimidine compound targeting EGFR and AXL targets and 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 application has good abilities of inhibiting EGFR and AXL kinases, and therefore can be used as an active ingredient for preparing a therapeutic drug for diseases caused by abnormal activation of EGFR and / or AXL kinases, and also shows potential application values in preparation of a drug for treating and / or preventing a pathological hyperplasia disease (such as cancer).

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to a 4-aminopyrimidine compound targeting EGFR and AXL target points and a preparation method and application thereof. BACKGROUND

[0002] Small molecule targeted therapeutic drugs improve the treatment effect of non-small cell lung cancer (NSCLC) by specifically inhibiting the growth signal pathway or angiogenesis of cancer cells. In recent years, the epidermal growth factor receptor (EGFR) has become an important target for treating NSCLC. EGFR is a transmembrane protein with a molecular weight of about 170 kDa, located on the surface of the cell membrane, also known as HER1 or ErbB1, and is a core member of the ErbB protein kinase family, belonging to the transmembrane receptor tyrosine kinase (RTK), and plays a key role in cell growth, proliferation, differentiation, migration and apoptosis. Abnormal activation of EGFR is closely related to the occurrence and development of various tumors, especially in non-small cell lung cancer (NSCLC), mutations of EGFR (such as L858R, T790M and C797S) often lead to abnormal proliferation and drug resistance of tumor cells. Therefore, EGFR has become an important target for cancer treatment, and inhibitors targeting EGFR have achieved remarkable results in clinical treatment. Unfortunately, targeted drugs are prone to drug resistance after use, of which EGFR mutations account for 50% of drug resistance mechanisms. Therefore, research on drug resistance caused by EGFR mutations has gradually become a research hotspot.

[0003] Research has found that during the research on drug resistance caused by EGFR mutations, AXL expression is up-regulated. Continuous EGFR inhibition eventually induces GAS6 up-regulation, thereby increasing AXL activity, and AXL, GAS6 and protein S levels are found to be elevated in more than 50% of NSCLC cell lines. In addition, EGFR and AXL have significant cross-talk and correlation in downstream signaling, and through the synergistic action of PI3K / AKT, MAPK / ERK and other pathways, they regulate cell survival, proliferation, migration and immune response. In cancer, the synergistic effect of EGFR and AXL can enhance the survival, invasion and drug resistance of tumor cells. Therefore, the development of dual-target inhibitors targeting EGFR and AXL is of great significance for the treatment of cancer.

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

[0005] The application aims to provide a 4-aminopyrimidine compound targeting EGFR and AXL targets, and a preparation method and application thereof.

[0006] To achieve the above-mentioned object, the application provides the following technical scheme.

[0007] One of the technical schemes of the application provides a 4-aminopyrimidine compound targeting EGFR and AXL targets, and the structural formula 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 4-aminopyrimidine compound targeting EGFR and AXL targets in the application has the following name:

[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-l-yl)pyrimidin-4-yl)amino)-N-(l- (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-(l- (methylsulfonyl)piperidin-4-yl)benzamide;

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

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

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

[0024]

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

[0025]

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

[0026]

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

[0027]

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

[0028]

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

[0029]

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

[0048]

[34] (4-((5-chloro-2-((2S,6R)-2,6-dimethylmorpholino)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 scheme of the present application provides a preparation method of the 4-aminopyrimidine compound targeting EGFR and AXL,

[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 scheme of the present application provides a pharmaceutically acceptable salt of the 4-aminopyrimidine compound targeting EGFR and AXL.

[0059] The fourth technical scheme of the present application provides a pharmaceutical preparation, wherein the active ingredient is the 4-aminopyrimidine compound targeting EGFR and AXL or the pharmaceutically acceptable salt of the 4-aminopyrimidine compound targeting EGFR and AXL.

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

[0061] The fifth technical scheme of the present application provides an application of the 4-aminopyrimidine compound targeting EGFR and AXL in the preparation of a drug for treating and / or preventing a pathological hyperplasia disease.

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

[0063] The sixth technical scheme of the present application provides an application of the pharmaceutically acceptable salt of the 4-aminopyrimidine compound targeting EGFR and AXL in the preparation of a drug for treating and / or preventing a pathological hyperplasia disease.

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

[0065] The seventh technical scheme of the present application provides an application of the pharmaceutical preparation in the preparation of a drug for treating and / or preventing a pathological hyperplasia disease.

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

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

[0068] The present application has the following beneficial technical effects:

[0069] The 4-aminopyrimidine compound targeting EGFR and AXL targets provided by the application has good ability to inhibit EGFR and AXL kinases, and can be used as an active ingredient for preparing a therapeutic drug for diseases caused by abnormal activation of EGFR and / or AXL kinases, and also shows potential application value in preparing a drug for treating and / or preventing a pathological hyperplasia disease (such as cancer). BRIEF DESCRIPTION OF DRAWINGS

[0070] Figure 1 Apoptosis induction ability of the compound prepared in Example 36 on H1975 cells detected by Annexin V-FITC / PI double staining method (A), AO staining (B) and JC-1 staining (C) experiments.

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

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

[0073] Figure 4 Effect of the compound prepared in Example 36 on the migration ability of H1975 cells detected by cell scratch experiment.

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

[0075] Figure 6 Average body weight change of KM mice in each group during administration.

[0076] Figure 7 Macrographs of five organs of KM mice in each group after administration and organ indexes, wherein, A-E are macrographs of heart, liver, spleen, lung and kidney respectively, and F is the organ index calculated after weighing the five organs respectively.

[0077] Figure 8 Blood biochemical analysis results of KM mice in each group after administration, wherein, 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 H&E staining diagrams of five organs of KM mice in each group after administration (scale bar is 100 μm). DETAILED DESCRIPTION

[0079] The following detailed description of various example embodiments of the application should not be considered to be limiting of the application, but merely illustrative in nature. The description of the application is intended to provide a functional description, and is not intended to limit the aspects, characteristics, or implementations of the application described herein.

[0080] It should be noted that the present application does not describe in detail the conventional operation means in the art, and is not the focus of the present application.

[0081] In addition, for the numerical range in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or stated range of intermediate values, and any other stated value or intermediate value in the stated range, is also encompassed within the present application. The upper and lower limits of these smaller ranges can be independently included or excluded from 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 this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, preferred methods and materials are described. All publications mentioned herein are incorporated by reference for the material described in them.

[0083] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" or the like are open-ended and do not exclude additional, unrecited elements or method steps.

[0084] The nuclear magnetic resonance hydrogen spectrum of the compound in the embodiment of the present application is determined by using a Bruker ARX-400, and the mass spectrum is determined by using an Agilent 1100 LC / MSD; the reagents used are all of analytical purity or chemical purity.

[0085] The reaction route for preparing the 4-aminopyrimidine compound in the embodiment of the present application is route 1 or route 2, and is specifically as follows:

[0086] Route 1

[0087]

[0088] Route 2

[0089]

[0090] R1-R4 in route 1 and route 2 are as listed in the summary, and are not described here again.

[0091] The final product obtained in Step 3 of Examples 1-36 is precipitated or dissolved in the original system. For the reaction product that is precipitated after the reaction, the separation method is to perform vacuum filtration and wash with acetonitrile until clean, dry, and the final product is obtained. For the reaction product that 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 1-36, the reaction time after all the raw materials are added is 8-12 h, 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] P-aminobenzoic acid (A) (2.0 g) and 2,4-dichloropyrimidine (2.3 g) were added to a 500 mL eggplant flask, 150 mL of previously prepared HCl (0.1 M) aqueous solution was added, and it was placed in a constant temperature heating magnetic stirrer with silicone oil, the temperature was raised to 100°C, the raw materials were dissolved from white solid to colorless transparent solution, when the temperature was raised to 90°C, white solid began to precipitate, and the reaction was carried out at 100°C for 4 h. After the reaction was completed, it was cooled to room temperature, the solid-liquid mixture was poured into a 1000 mL beaker, 500 mL of tap water was added and stirred for 0.5 h, after vacuum filtration, the filter cake was dried 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, dichloromethane (80 mL), DMF (5 drops), DIPEA (1.6 g), and HATU (4.6 g) were added in sequence, the reaction was carried out at room temperature for 0.5 h, 1-methylsulfonyl-4-aminopiperidine (0.9 g) was added, and the reaction was continued at room temperature for 7 h. After the reaction was completed, dichloromethane and water were added for extraction, the filtrate was dried over anhydrous sodium sulfate and rotary evaporated, and then the intermediate 4-((2-chloropyrimidin-4-yl)amino)-N-(1- (methylsulfonyl)piperidin-4-yl)benzamide (C) was purified by column chromatography.

[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 in a 50 mL flask, acetonitrile (10 mL) and DIPEA (0.3 g) were added and reacted at 80 °C for 0.5 h, intermediate 4-((2-chloropyrimidin-4-yl)amino)-N-(1- (methylsulfonyl)piperidin-4-yl)benzamide (C) (0.3 g) was added and stirring was continued for 9 h. After the reaction was completed, it was cooled to room temperature. The final product (D) was obtained after separation and purification.

[0101] The product was a white solid, 1 H NMR (400 MHz, DMSO-d6) δ 9.53 (s, 1H), 8.18 (d, J = 7.5 Hz, 1H), 7.98 (d, J = 5.6 Hz, 1H), 7.83 (d, J = 8.5 Hz, 2H), 7.72 (d, J = 8.5 Hz, 2H), 6.10 (d, J = 5.6 Hz, 1H), 3.91 (d, J = 6.7 Hz, 1H), 3.73 (s, 4H), 3.57 (d, J = 11.6 Hz, 2H), 2.88 (s, 3H), 2.84 (d, J = 11.9 Hz, 2H), 2.45 (s, 4H), 2.28 (s, 3H), 1.89 (d, J = 12.5 Hz, 2H), 1.59 (q, J = 10.3, 9.2 Hz, 2H). 13 C NMR (151 MHz, DMSO-d6) δ 165.28, 161.07, 160.15, 156.47, 143.01, 128.23 (2C), 127.11, 118.02 (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 : 15.552 min, purity: 96.41%.

[0102] Example 2

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

[0104] Following the procedure of Example 1, step three, replace piperazine with an equimolar amount of morpholine.

[0105] The product was a white solid, 1 H NMR (400 MHz, DMSO-d6) δ 9.55 (s, 1H), 8.17 (d, J = 7.0 Hz, 1H), 8.00 (d, J = 5.5 Hz, 1H), 7.82 (d, J = 8.6 Hz, 2H), 7.72 (d, J = 8.6 Hz, 2H), 6.13 (d, J = 5.5 Hz, 1H), 3.91 (d, J = 7.3 Hz, 1H), 3.67 (s, 8H), 3.57 (d, J = 11.8 Hz, 2H), 2.88 (s, 3H), 2.84 (d, J = 12.3 Hz, 2H), 1.89 (d, J = 12.5 Hz, 2H), 1.60 (q, J = 12.0, 11.4 Hz, 2H). 13 C NMR (151 MHz, DMSO-d6) δ 165.26, 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 : 18.283 min, purity: 94.67%.

[0106] Example 3

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

[0108] Following the procedure of Example 1, step three, replace piperazine with an equimolar amount of (R)-2-methylmorpholine.

[0109] The product was a white solid, 1H NMR (400 MHz, DMSO-d6) δ 9.56 (s, 1H), 8.19 (d, J = 7.8 Hz, 1H), 7.99 (d, J = 5.7 Hz, 1H), 7.82 (d, J = 8.5 Hz, 2H), 7.72 (d, J = 8.5 Hz, 2H), 6.12 (d, J = 5.9 Hz, 1H), 4.38 (dd, J = 32.6, 13.0 Hz, 2H), 3.90 (d, J = 11.4 Hz, 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 = 12.7 Hz, 2H), 1.64 - 1.54 (m, 2H), 1.16 (d, J = 6.1 Hz, 3H). 13 C NMR (151 MHz, DMSO-d6) δ 165.29, 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 : 18.791 min, purity: 95.89%.

[0110] Example 4

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

[0112] Following the procedure of Example 1, step three, replace piperazine with an equimolar amount of (2S,6R)-2,6-dimethylmorpholine.

[0113] The product was a purple solid, 1H NMR (400 MHz, DMSO-d6) δ 9.49 (s, 1H), 8.12 (d, J = 8.0 Hz, 1H), 7.99 (d, J = 5.7 Hz, 1H), 7.82 (d, J = 8.4 Hz, 2H), 7.70 (d, J = 8.6 Hz, 2H), 6.11 (d, J = 5.8 Hz, 1H), 4.45 (d, J = 12.9 Hz, 2H), 3.92 (s, 1H), 3.57 (s, 4H), 2.88 (d, J = 4.2 Hz, 5H), 1.95 - 1.87 (m, 2H), 1.61 (d, J = 12.3 Hz, 2H), 1.17 (d, J = 6.8 Hz, 6H). 13 C NMR (151 MHz, DMSO-d6) δ 165.30, 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 : 19.045 min, purity: 96.56%.

[0114] Example 5

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

[0116] Following the procedure of Example 1, Step three, replace piperazine with an equimolar amount of 4,4-difluoropiperidine.

[0117] The product was a white solid, 1H NMR (400 MHz, DMSO-d6) δ 9.59 (s, 1H), 8.20 (d, J = 7.7 Hz, 1H), 8.01 (d, J = 5.7 Hz, 1H), 7.83 (d, J = 8.4 Hz, 2H), 7.70 (d, J = 8.5 Hz, 2H), 6.13 (d, J = 5.7 Hz, 1H), 3.86 (d, J = 6.1 Hz, 5H), 3.56 (d, J = 11.8 Hz, 2H), 2.88 (s, 3H), 2.83 (d, J = 11.7 Hz, 2H), 1.97 (d, J = 15.0 Hz, 4H), 1.89 (d, J = 12.5 Hz, 2H), 1.59 (q, J = 12.4 Hz, 2H). TOF MS ES+(m / z): (M+H) + , calcd for C 22 H 28 F2N6O3S: 495.1990; found, 495.1987. HPLC: t R : 16.807 min, 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] Following the procedure of Example 1, step three, replace piperazine with an equimolar amount of 6,6-dimethyl-3-azabicyclo[3.1.0]hexane.

[0121] The product was a white solid, 1 H NMR (400 MHz, DMSO-d6) δ 9.59 (s, 1H), 8.20 (d, J = 7.7 Hz, 1H), 8.01 (d, J = 5.7 Hz, 1H), 7.83 (d, J = 8.4 Hz, 2H), 7.70 (d, J = 8.5 Hz, 2H), 6.13 (d, J = 5.7 Hz, 1H), 3.86 (d, J = 6.1 Hz, 5H), 3.56 (d, J = 11.8 Hz, 2H), 2.88 (s, 3H), 2.83 (d, J = 11.7 Hz, 2H), 1.97 (d, J = 15.0 Hz, 4H), 1.89 (d, J = 12.5 Hz, 2H), 1.59 (q, J = 12.4 Hz, 2H). TOF MS ES+(m / z): (M+H) 13C NMR (151 MHz, DMSO-d6) δ 165.31, 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 : 17.516 min, purity: 95.86%.

[0122] Example 7

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

[0124] Following the procedure of Example 1, step three, replace piperazine with an equimolar amount of N,N-dimethylazetidin-3-amine.

[0125] The product was a white solid, 1 H NMR (400 MHz, DMSO-d6) δ 9.56 (s, 1H), 8.18 (d, J = 7.9 Hz, 1H), 7.94 (d, J = 5.7 Hz, 1H), 7.81 (s, 4H), 6.11 (d, J = 5.7 Hz, 1H), 4.04 (t, J = 8.0 Hz, 2H), 3.90 (d, J = 7.9 Hz, 1H), 3.80 (t, J = 7.1 Hz, 2H), 3.56 (d, J = 11.7 Hz, 2H), 3.14 (s, 1H), 2.88 (s, 3H), 2.86 - 2.81 (m, 2H), 2.12 (s, 6H), 1.89 (d, J = 13.0 Hz, 2H), 1.64 - 1.54 (m, 2H). 13 C NMR (151 MHz, DMSO-d6) δ 165.32, 162.54, 160.29, 156.33, 143.27, 128.20 (2C), 126.98, 117.97 (2C), 97.49, 55.62, 53.85, 45.81 (2C), 44.83 (2C), 41.56 (2C), 34.23, 30.90 (2C). TOF MS ES+(m / z): (M+H) +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] Following the procedure of Example 1, step three, replace piperazine with an equimolar amount of N,N-dimethyl-4-aminopiperidine.

[0129] The product was a white solid, 1 H NMR (400 MHz, DMSO-d6) δ 9.51 (s, 1H), 8.19 (d, J = 7.7 Hz, 1H), 7.96 (d, J = 5.4 Hz, 1H), 7.82 (d, J = 8.4 Hz, 2H), 7.73 (d, J = 8.5 Hz, 2H), 6.06 (d, J = 5.7 Hz, 1H), 4.61 (d, J = 12.9 Hz, 2H), 3.91 (d, J = 10.0 Hz, 1H), 3.57 (d, J = 11.5 Hz, 2H), 2.88 (s, 4H), 2.34 (d, J = 10.7 Hz, 1H), 2.18 (s, 6H), 1.89 (d, J = 12.3 Hz, 2H), 1.81 (d, J = 12.3 Hz, 2H), 1.59 (dt, J = 21.3, 10.4 Hz, 2H), 1.35 - 1.24 (m, 2H). 13 C NMR (151 MHz, DMSO-d6) δ 165.27, 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 :16.078 min, 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] Following the procedure of Example 1, Step three, replace piperazine with an equimolar amount of 1-methyl-4-(piperidin-4-yl)piperazine.

[0133] The product was a white solid, 1 H NMR (400 MHz, DMSO-d6) δ 9.52 (s, 1H), 8.19 (d, J = 7.8 Hz, 1H), 7.96 (d, J = 5.6 Hz, 1H), 7.82 (d, J = 8.6 Hz, 2H), 7.73 (d, J = 8.5 Hz, 2H), 6.06 (d, J = 5.6 Hz, 1H), 4.62 (d, J = 13.0 Hz, 2H), 3.96 - 3.85 (m, 1H), 3.57 (d, J = 11.6 Hz, 2H), 2.88 (s, 3H), 2.84 (d, J = 12.4 Hz, 4H), 2.31 (s, 3H), 2.14 (s, 3H), 1.89 (d, J = 12.6 Hz, 2H), 1.81 (d, J = 12.2 Hz, 2H), 1.59 (td, J = 12.8, 12.3, 8.5 Hz, 2H), 1.37 - 1.27 (m, 2H). 13 C NMR (151 MHz, DMSO-d6) δ 165.28, 160.92, 160.16, 156.55, 143.12, 128.20 (2C), 127.00, 117.94 (2C), 96.85, 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 : 15.902 min, 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 procedure of Example 1, step three is followed by replacing piperazine with an equimolar amount of 1 -isopropylpiperazine.

[0137] The product is a white solid, 1 H NMR (400 MHz, DMSO-d6) δ 9.54 (s, 1H), 8.19 (d, J = 7.6 Hz, 1H), 7.97 (d, J = 5.7 Hz, 1H), 7.82 (d, J = 8.8 Hz, 2H), 7.72 (d, J = 8.7 Hz, 2H), 6.08 (d, J = 5.5 Hz, 1H), 3.90 (d, J = 7.1 Hz, 1H), 3.68 (s, 4H), 3.56 (d, J = 11.3 Hz, 2H), 2.88 (s, 3H), 2.83 (dd, J = 12.0, 2.8 Hz, 2H), 2.69 (d, J = 16.0 Hz, 1H), 1.89 (d, J = 12.7 Hz, 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 : 15.763 min, purity: 95.28 %.

[0138] Example 11

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

[0140] The procedure of Example 1, step three is followed by replacing piperazine with an equimolar amount of 1 -(piperazin- 1 -yl)ethan- 1 -one.

[0141] The product is a white solid, 1H NMR (400 MHz, DMSO-d6) δ 9.58 (s, 1H), 8.20 (d, J = 7.7 Hz, 1H), 8.00 (d, J = 5.7 Hz, 1H), 7.83 (d, J = 8.7 Hz, 2H), 7.73 (d, J = 8.9 Hz, 2H), 6.12 (d, J = 5.7 Hz, 1H), 3.91 (d, J = 9.6 Hz, 1H), 3.76 (s, 2H), 3.69 (s, 2H), 3.59 - 3.51 (m, 6H), 2.88 (s, 3H), 2.84 (d, J = 11.8 Hz, 2H), 2.05 (s, 3H), 1.89 (d, J = 12.7 Hz, 2H), 1.59 (q, J = 11.4 Hz, 2H). 13 C NMR (151 MHz, DMSO-d6) δ 168.46, 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 : 17.818 min, purity: 98.13%.

[0142] Example 12

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

[0144] Following the procedure of Example 1, step three, replace piperazine with an equimolar amount of cyclopropyl(piperazin-l-yl)methanone.

[0145] The product was a white solid, 1H NMR (400 MHz, DMSO-d6) δ 9.59 (s, 1H), 8.20 (d, J = 7.7 Hz, 1H), 8.00 (d, J = 5.7 Hz, 1H), 7.83 (d, J = 8.8 Hz, 2H), 7.73 (d, J = 7.1 Hz, 2H), 6.12 (d, J = 5.7 Hz, 1H), 3.91 (dq, J = 14.5, 6.8, 6.1 Hz, 1H), 3.78 (s, 4H), 3.69 (s, 2H), 3.56 (d, J = 9.5 Hz, 4H), 2.88 (s, 3H), 2.83 (d, J = 11.8 Hz, 2H), 2.02 (t, J = 10.2 Hz, 1H), 1.89 (d, J = 12.6 Hz, 2H), 1.59 (q, J = 11.5, 10.4 Hz, 2H), 0.78 - 0.70 (m, 4H). 13 C NMR (151 MHz, DMSO-d6) δ 171.27, 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-(l-(methylsulfonyl)piperidin-4-yl)-4-((2-(4-(oxetan-3-yl)piperazin-l- yl)pyrimidin-4-yl)amino)benzamide:

[0148] Following the procedure of Example 1, Step three, replace piperazine with an equimolar amount of l-(oxetan-3-yl)piperazine.

[0149] The product was a white solid, 1H NMR (400 MHz, DMSO-d6) δ 9.55 (s, 1H), 8.18 (d, J = 7.6 Hz, 1H), 7.98 (d, J = 5.7 Hz, 1H), 7.81 (d, J = 7.7 Hz, 2H), 7.72 (d, J = 7.1 Hz, 2H), 6.09 (d, J = 5.7 Hz, 1H), 4.55 (t, J = 6.4 Hz, 2H), 4.47 (t, J = 6.0 Hz, 2H), 3.91 (d, J = 11.7 Hz, 1H), 3.76 - 3.70 (m, 4H), 3.56 (d, J = 11.6 Hz, 2H), 3.41 (t, J = 6.3 Hz, 1H), 2.88 (d, J = 1.5 Hz, 3H), 2.83 (d, J = 12.2 Hz, 2H), 2.34 - 2.29 (m, 4H), 1.88 (d, J = 12.7 Hz, 2H), 1.58 (q, J = 12.6, 11.4 Hz, 2H). 13 CNMR (151 MHz, DMSO-d6) δ 165.27, 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 : 15.793 min, purity: 95.70%.

[0150] Example 14

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

[0152] Following the procedure of Example 1, step three, replace piperazine with an equimolar amount of 4-(piperidin-4-yl)morpholine.

[0153] The product was a white solid, 1H NMR (400 MHz, DMSO-d6) δ 9.51 (s, 1H), 8.19 (d, J = 7.7 Hz, 1H), 7.97 (d, J = 5.5 Hz, 1H), 7.82 (d, J = 8.5 Hz, 2H), 7.72 (d, J = 9.1 Hz, 2H), 6.06 (d, J = 5.7 Hz, 1H), 4.62 (d, J = 12.9 Hz, 2H), 3.96 - 3.87 (m, 1H), 3.55 (t, J = 4.3 Hz, 6H), 2.88 (s, 6H), 2.85 - 2.81 (m, 1H), 2.49 - 2.37 (m, 5H), 1.86 (dd, J = 23.3, 12.5 Hz, 4H), 1.65 - 1.55 (m, 2H), 1.32 (d, J = 11.9 Hz, 2H). 13 C NMR (151 MHz, DMSO-d6) δ 165.27, 160.92, 160.15, 156.54, 143.11, 128.20 (2C), 127.00, 117.94 (2C), 96.86, 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 for C 26 H 37 N7O4S: 544.2706; found, 544.2706. HPLC: t R : 15.679 min, purity: 94.03%.

[0154] Example 15

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

[0156] Following the procedure of Example 1, Step three, replace piperazine with an equimolar amount of 4-(pyrrolidin-l-yl)piperidine.

[0157] The product was a white solid, 1H NMR (400 MHz, DMSO-d6) δ 9.51 (s, 1H), 8.19 (d, J = 7.8 Hz, 1H), 7.97 (d, J = 5.6 Hz, 1H), 7.82 (d, J = 8.4 Hz, 2H), 7.73 (d, J = 8.7 Hz, 2H), 6.06 (d, J = 5.7 Hz, 1H), 4.46 (d, J = 12.8 Hz, 2H), 3.97 - 3.85 (m, 1H), 3.57 (d, J = 11.3 Hz, 2H), 3.00 (t, J = 11.9 Hz, 2H), 2.88 (s, 3H), 2.86 - 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 (151 MHz, DMSO-d6) δ 165.28, 161.02, 160.14, 156.52, 143.14, 128.20 (2C), 126.99, 117.94 (2C), 96.84, 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)+, calcd for C26H37N7O3S: 528.2757; found, 528.2757. HPLC: tR: 15.820 min, purity: 99.36%.

[0158] Example 16

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

[0160] Following the procedure of Example 1, Step two, replace 1-(methylsulfonyl)piperidin-4-amine with an equimolar amount of 1-(methylsulfonyl)piperazin.

[0161] The product was a white solid, 1 H NMR (400 MHz, DMSO-d6) δ 9.50 (s, 1H), 7.97 (d, J = 5.6 Hz, 1H), 7.71 (d, J = 8.7 Hz, 2H), 7.40 (d, J = 8.7 Hz, 2H), 6.09 (d, J = 5.6 Hz, 1H), 3.71 (s, 4H), 3.61 (s, 4H), 3.18 - 3.14 (m, 4H), 2.91 (d, J = 2.0 Hz, 3H), 2.41 (s, 4H), 2.25 (s, 3H).13 C NMR (151 MHz, DMSO-d6) δ 169.21, 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-l-yl)(4-((2-morpholino pyrimidin-4- yl)amino)phenyl)methanone:

[0164] Follow the procedure of Example 16, step three, substituting piperazine with an equimolar amount of morpholine.

[0165] The product was a white solid, 0.030 g, 0.067 mmol, 48.9 % yield. 1 H NMR (400 MHz, DMSO-d6) δ 9.53 (s, 1H), 7.99 (d, J = 5.6 Hz, 1H), 7.71 (d, J = 8.9 Hz, 2H), 7.40 (d, J = 8.9 Hz, 2H), 6.12 (d, J = 5.7 Hz, 1H), 3.66 (s, 8H), 3.61 (s, 4H), 3.16 (t, J = 4.7 Hz, 4H), 2.91 (d, J = 1.5 Hz, 3H). TOF MS ES+(m / z): (M+H) + , calcd for C 20 H 26 N6O4S: 447.1814; found, 447.1813. HPLC: t R : 17.877 min, purity: 98.53 %.

[0166] Example 18

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

[0168] Follow the procedure of Example 16, step three, substituting piperazine with an equimolar amount of (R)-2-methylmorpholine.

[0169] The product was a pink solid, 0.030 g, 0.067 mmol, 48.9 % yield. 1H NMR (400 MHz, DMSO-d6) δ 9.52 (s, 1H), 7.99 (d, J = 5.6 Hz, 1H), 7.71 (d, J = 8.4 Hz, 2H), 7.40 (d, J = 8.4 Hz, 2H), 6.11 (d, J = 5.7 Hz, 1H), 4.38 (dd, J = 31.6, 13.1 Hz, 2H), 3.91 - 3.87 (m, 1H), 3.61 (s, 4H), 3.53 - 3.45 (m, 2H), 3.16 (t, J = 5.1 Hz, 4H), 2.90 (s, 3H), 1.15 (d, J = 6.1 Hz, 3H). 13 C NMR (151 MHz, DMSO-d6) δ 169.21, 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 : 7.935 min, 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] Following the procedure of Example 16, step three, replace piperazine with an equimolar amount of (2S,6R)-2,6-dimethylmorpholine.

[0173] The product was a purple solid, 1 H NMR (400 MHz, DMSO-d6) δ 9.52 (s, 1H), 7.99 (d, J = 3.7 Hz, 1H), 7.70 (d, J = 6.5 Hz, 2H), 7.40 (d, J = 6.5 Hz, 2H), 6.10 (d, J = 5.6 Hz, 1H), 4.43 (d, J = 12.9 Hz, 2H), 3.58 (d, J = 23.5 Hz, 6H), 3.16 (s, 4H), 2.90 (s, 3H), 1.15 (d, J = 4.1 Hz, 6H). 13C NMR (151 MHz, DMSO-d6) δ 169.19, 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 : 16.221 min, purity: 96.75%.

[0174] Example 20

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

[0176] Following the procedure of Example 16, Step three, replace piperazine with an equimolar amount of 4,4-difluoropiperidine.

[0177] The product was a white solid, 1 H NMR (400 MHz, DMSO-d6) δ 9.56 (s, 1H), 8.01 (d, J = 5.6 Hz, 1H), 7.69 (d, J = 8.5 Hz, 2H), 7.41 (d, J = 8.5 Hz, 2H), 6.14 (d, J = 5.7 Hz, 1H), 3.86 (t, J = 5.5 Hz, 4H), 3.61 (s, 4H), 3.19 - 3.12 (m, 4H), 2.91 (s, 3H), 1.97 (d, J = 15.1 Hz, 4H). TOF MS ES+(m / z): (M+H) + , calcd for C 21 H 26 F2N6O3S: 481.1833; found, 481.1833. HPLC: t R : 16.504 min, 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-l-yl) methanone:

[0180] The procedure of Example 16, step three is followed by replacing piperazine with an equimolar amount of 6,6-dimethyl-3-azabicyclo[3.1.0]hexane.

[0181] The product is a white solid, 1 H NMR (400 MHz, DMSO-d6) δ 9.45 (s, 1H), 7.92 (d, J = 5.7 Hz, 1H), 7.83 (d, J = 8.3 Hz, 2H), 7.39 (d, J = 8.2 Hz, 2H), 6.05 (d, J = 5.7 Hz, 1H), 3.67 - 3.46 (m, 8H), 3.16 (t, J = 5.0 Hz, 4H), 2.91 (s, 3H), 1.49 (s, 2H), 1.05 (s, 3H), 0.84 (s, 3H). 13 C NMR (151 MHz, DMSO-d6) δ 169.26, 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 : 17.390 min, purity: 97.40%.

[0182] Example 22

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

[0184] The procedure of Example 16, step three is followed by replacing piperazine with an equimolar amount of N,N-dimethylazetidin-3-amine.

[0185] The product is a white solid, 1H NMR (400 MHz, DMSO-d6) δ 9.53 (s, 1H), 7.94 (d, J = 5.6 Hz, 1H), 7.80 (d, J = 8.3 Hz, 2H), 7.39 (d, J = 8.3 Hz, 2H), 6.12 (d, J = 5.7 Hz, 1H), 4.04 (t, J = 8.0 Hz, 2H), 3.81 (dd, J = 9.0, 5.1 Hz, 2H), 3.61 (s, 4H), 3.17 (q, J = 8.1, 4.7 Hz, 5H), 2.91 (s, 3H), 2.15 (s, 6H). 13 C NMR (5151 MHz, DMSO-d6) δ 169.24, 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-l-yl)pyrimidin-4-yl)amino)phenyl)(4- (methylsulfonyl)piperazin-l-yl)methanone:

[0188] Following the procedure of Example 16, Step three, replace piperazine with an equimolar amount of N,N-dimethyl-4-aminopiperidine.

[0189] The product was a white solid, 0.030 g (0.058 mmol, 20% yield). 1 H NMR (400 MHz, DMSO-d6) δ 9.53 (s, 1H), 7.94 (d, J = 5.6 Hz, 1H), 7.80 (d, J = 8.3 Hz, 2H), 7.39 (d, J = 8.3 Hz, 2H), 6.12 (d, J = 5.7 Hz, 1H), 4.04 (t, J = 8.0 Hz, 2H), 3.81 (dd, J = 9.0, 5.1 Hz, 2H), 3.61 (s, 4H), 3.17 (q, J = 8.1, 4.7 Hz, 5H), 2.91 (s, 3H), 2.15 (s, 6H). 13C NMR (151 MHz, DMSO-d6) δ 169.21, 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 for C 23 H 33 N7O3S: 488.2444; found, 488.2443.

[0190] Example 24

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

[0192] Following the procedure of Example 16, Step three, replace piperazine with an equimolar amount of l-methyl-4-(piperidin-4-yl)piperazine.

[0193] The product was a white solid, 0.030 g, 0.059 mmol, 48.2 % yield. 1 H NMR (400 MHz, DMSO-d6) δ 9.47 (s, 1H), 7.96 (d, J = 5.6 Hz, 1H), 7.71 (d, J = 8.4 Hz, 2H), 7.39 (d, J = 8.3 Hz, 2H), 6.06 (d, J = 5.7 Hz, 1H), 4.62 (d, J = 13.0 Hz, 2H), 3.61 (s, 4H), 3.16 (t, J = 5.0 Hz, 4H), 2.91 (s, 3H), 2.86 (t, J = 12.3 Hz, 2H), 2.57 (d, J = 11.1 Hz, 4H), 2.29 (s, 3H), 1.82 (d, J = 12.1 Hz, 2H), 1.39 - 1.28 (m, 2H). 13 C NMR (151 MHz, DMSO-d6) δ 168.57, 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] Following the procedure of Example 16, Step three, replace piperazine with an equimolar amount of 1 -isopropylpiperazine.

[0197] The product was a white solid, 1 H NMR (400 MHz, DMSO-d6) δ 9.50 (s, 1H), 7.97 (d, J = 5.6 Hz, 1H), 7.72 (d, J = 8.4 Hz, 2H), 7.40 (d, J = 8.4 Hz, 2H), 6.08 (d, J = 5.6 Hz, 1H), 3.74 - 3.57 (m, 8H), 3.16 (t, J = 4.9 Hz, 4H), 2.91 (s, 3H), 2.72 (d, J = 9.0 Hz, 1H), 1.00 (d, J = 6.5 Hz, 6H). 13 C NMR (151 MHz, DMSO-d6) δ 168.58, 160.44, 159.52, 155.78, 141.31, 127.74 (2C), 127.17, 117.80 (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 -carbonyl)phenyl)amino)pyrimidin- 2-yl)piperazin-1-yl)ethanone:

[0200] Following the procedure of Example 16, Step three, replace piperazine with an equimolar amount of 1 -(piperazin-1 -yl)ethan-1 -one.

[0201] The product was a white solid, 1H NMR (400 MHz, DMSO-d6) δ 9.55 (s, 1H), 7.99 (d, J = 5.6 Hz, 1H), 7.72 (d, J = 8.3 Hz, 2H), 7.41 (d, J = 8.4 Hz, 2H), 6.12 (d, J = 5.7 Hz, 1H), 3.75 (t, J = 5.1 Hz, 2H), 3.69-3.66 (m, 2H), 3.61 (s, 4H), 3.52 (d, J = 6.1 Hz, 4H), 3.18-3.14 (m, 4H), 2.91 (s, 3H), 2.05 (s, 3H). 13 C NMR (151 MHz, DMSO-d6) δ 169.20, 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 : 17.443 min, purity: 94.73%.

[0202] Example 27

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

[0204] Following the procedure of Example 16, step three, replace piperazine with an equimolar amount of cyclopropyl(piperazin-l-yl)methanone.

[0205] The product was a white solid, 0.030 g, 0.066 mmol, 48.2% yield. 1 H NMR (400 MHz, DMSO-d6) δ 9.55 (s, 1H), 7.99 (d, J = 5.6 Hz, 1H), 7.72 (d, J = 8.3 Hz, 2H), 7.41 (d, J = 8.4 Hz, 2H), 6.12 (d, J = 5.7 Hz, 1H), 3.75 (t, J = 5.1 Hz, 2H), 3.69-3.66 (m, 2H), 3.61 (s, 4H), 3.52 (d, J = 6.1 Hz, 4H), 3.18-3.14 (m, 4H), 2.91 (s, 3H), 2.05 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 171.25, 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 : 15.891 min, purity: 94.57%.

[0206] Example 28

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

[0208] Following the procedure of Example 16, step three, replace piperazine with an equimolar amount of l-(oxetan-3-yl)piperazine.

[0209] The product was a white solid, 1 H NMR (400 MHz, DMSO-d6) δ 9.51 (s, 1H), 7.97 (d, J = 5.7 Hz, 1H), 7.71 (d, J = 8.7 Hz, 2H), 7.39 (d, J = 8.7 Hz, 2H), 6.09 (d, J = 5.7 Hz, 1H), 4.55 (t, J = 6.4 Hz, 2H), 4.47 (t, J = 6.0 Hz, 2H), 3.72 (t, J = 4.8 Hz, 4H), 3.61 (s, 4H), 3.42 (s, 1H), 3.18 - 3.13 (m, 4H), 2.90 (s, 3H), 2.30 (t, J = 4.9 Hz, 4H). 13 C NMR (151 MHz, DMSO-d6) δ 169.19, 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 for C 23 H31 N7O4S: 502.2236; found, 502.2237. HPLC: t R : 15.428 min, purity: 94.71 %.

[0210] Example 29

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

[0212] Following the procedure of Example 16, step three, replace piperazine with an equimolar amount of 4-(piperidin-4-yl)morpholine.

[0213] The product was a white solid, 1 H NMR (400 MHz, DMSO-d6) δ 9.47 (s, 1H), 7.96 (d, J = 5.7 Hz, 1H), 7.71 (d, J = 8.3 Hz, 2H), 7.40 (d, J = 8.3 Hz, 2H), 6.06 (d, J = 5.6 Hz, 1H), 4.61 (d, J = 12.9 Hz, 2H), 3.66 - 3.53 (m, 8H), 3.16 (t, J = 4.9 Hz, 4H), 2.91 (d, J = 1.2 Hz, 3H), 2.85 (d, J = 12.6 Hz, 2H), 2.48 - 2.37 (m, 5H), 1.83 (d, J = 12.3 Hz, 2H), 1.31 (q, J = 11.5, 9.9 Hz, 2H). 13 C NMR (151 MHz, DMSO-d6) δ 169.20, 160.92, 160.18, 156.50, 141.99, 128.34 (2C), 127.76, 118.37 (2C), 96.69, 66.58 (3C), 61.57 (2C), 49.40 (3C), 45.45, 42.93 (2C), 34.03, 27.68 (2C). TOF MS 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- morpholinopiperidin-1-yl)pyrimidin-4-yl)amino)phenyl)methanone:

[0216] The procedure of Example 16, step three, is followed using 4-(pyrrolidin-l- yl)piperidine in place of piperazine.

[0217] The product is a white solid, 1 H NMR (400 MHz, DMSO-d6) δ 9.47 (s, 1H), 7.96 (d, J = 5.6 Hz, 1H), 7.72 (d, J = 8.3 Hz, 2H), 7.40 (d, J = 8.3 Hz, 2H), 6.06 (d, J = 5.7 Hz, 1H), 4.46 (d, J = 13.2 Hz, 2H), 3.61 (s, 4H), 3.16 (t, J = 5.0 Hz, 4H), 3.03 - 2.95 (m, 2H), 2.91 (s, 3H), 2.22 (s, 1H), 1.92 - 1.85 (m, 2H), 1.66 (q, J = 3.7, 3.2 Hz, 4H), 1.39 - 1.25 (m, 2H). 13 C NMR (151 MHz, DMSO-d6) δ 169.21, 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-l-yl)-5-chloropyrimidin-4-yl)amino)-N-(l- (methylsulfonyl)piperidin-4-yl)benzamide:

[0220] The procedure of Example 1, step one, is followed using 2,4-dichloropyrimidine in place of 2,4,5-trichloropyrimidine, step two is unchanged, and step three is followed using l-(piperazin-l-yl)ethan-l-one in place of piperazine.

[0221] The product is a white solid, 1H NMR (400 MHz, DMSO-d6) δ 8.95 (s, 1H), 8.24 (d, J = 7.7 Hz, 1H), 8.13 (s, 1H), 7.85 (d, J = 8.5 Hz, 2H), 7.78 (d, J = 8.6 Hz, 2H), 3.92 (dt, J = 15.6, 5.6 Hz, 1H), 3.69 (t, J = 5.1 Hz, 2H), 3.65 - 3.55 (m, 4H), 3.50 (t, J = 5.2 Hz, 4H), 2.88 (s, 3H), 2.87 - 2.81 (m, 2H), 2.03 (s, 3H), 1.94 - 1.87 (m, 2H), 1.61 (tt, J = 11.9, 6.1 Hz, 2H). 13 C NMR (151 MHz, DMSO-d6) δ 168.44, 165.22, 159.06, 155.16, 155.06, 141.56, 128.77, 127.71 (2C), 120.76 (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 : 16.755 min, purity: 95.78%.

[0222] Example 32

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

[0224] Following the procedure of Example 31, Step three, replace 1-(piperazin-l- yl)ethan-l-one with an equimolar amount of cyclopropyl(piperazin-l-yl)methanone.

[0225] The product was a white solid, 1H NMR (400 MHz, DMSO-d6) δ 8.95 (s, 1H), 8.24 (d, J = 7.7 Hz, 1H), 8.13 (s, 1H), 7.85 (d, J = 8.5 Hz, 2H), 7.79 (d, J = 8.8 Hz, 2H), 3.92 (d, J = 6.1 Hz, 1H), 3.74 (s, 4H), 3.64 (s, 2H), 3.57 (d, J = 13.2 Hz, 4H), 2.88 (s, 3H), 2.87 - 2.82 (m, 2H), 2.01 (t, J = 5.7 Hz, 1H), 1.93 - 1.87 (m, 2H), 1.60 (td, J = 11.8, 7.9 Hz, 2H), 0.77 - 0.70 (m, 4H). 13 C NMR (151 MHz, DMSO-d6) δ 171.27, 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 : 17.037 min, purity: 94.98 %.

[0226] Example 33

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

[0228] Following the procedure of Example 31, Step three, replace 1-(piperazin-l- yl)ethan-l-one with an equimolar amount of ring 4-(piperidin-4-yl)morpholine.

[0229] White solid, 1H NMR (400 MHz, DMSO-d6) δ 8.86 (s, 1H), 8.23 (d, J = 7.7 Hz, 1H), 8.08 (s, 1H), 7.83 (d, J = 8.2 Hz, 2H), 7.79 (d, J = 8.6 Hz, 2H), 4.50 (d, J = 12.9 Hz, 2H), 3.96 - 3.88 (m, 1H), 3.57 (d, J = 14.1 Hz, 6H), 2.87 (d, J = 10.2 Hz, 8H), 2.45 (s, 4H), 1.90 (d, J = 12.7 Hz, 2H), 1.82 (d, J = 12.0 Hz, 2H), 1.60 (d, J = 12.1 Hz, 2H), 1.31 (d, J = 12.3 Hz, 2H). 13 C NMR (151 MHz, DMSO-d6) δ 165.19, 158.96, 155.11, 155.08, 141.73, 128.58, 127.67 (2C), 120.51 (2C), 102.21, 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-dimethylmorpholino)pyrimidin-4- yl)amino)phenyl)(4-(methylsulfonyl)piperazin-l-yl)methanone:

[0232] Following the procedure of Example 31, Step two, replace 1-(methylsulfonyl)piperidin-4-amine with an equimolar amount of 1-(methylsulfonyl)piperazine, and Step three, replace 1-(piperazin-l-yl)ethan-l-one with an equimolar amount of (2S,6R)-2,6-dimethylmorpholine.

[0233] White solid, 1H NMR (400 MHz, DMSO-d6) δ 8.93 (s, 1H), 8.10 (s, 1H), 7.77 (d, J = 8.2 Hz, 2H), 7.42 (d, J = 8.3 Hz, 2H), 4.32 (d, J = 13.1 Hz, 2H), 3.57 (d, J = 27.4 Hz, 6H), 3.17 (s, 4H), 2.90 (s, 3H), 1.12 (d, J = 6.1 Hz, 6H). 13 C NMR (151 MHz, DMSO-d6) δ 169.05, 158.86, 155.20, 155.01, 140.39, 129.68, 127.60 (2C), 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 : 11.301 min, 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] Following the procedure of Example 34, step three, replace (2S,6R)-2,6- dimethylmorpholine with an equimolar amount of 6,6-dimethyl-3-azabicyclo[3.1.0]hexane.

[0237] White solid, 1 H NMR (400 MHz, DMSO-d6) δ 8.93 (s, 1H), 8.10 (s, 1H), 7.77 (d, J = 8.2 Hz, 2H), 7.42 (d, J = 8.3 Hz, 2H), 4.32 (d, J = 13.1 Hz, 2H), 3.57 (d, J = 27.4 Hz, 6H), 3.17 (s, 4H), 2.90 (s, 3H), 1.12 (d, J = 6.1 Hz, 6H). 13C NMR (151 MHz, DMSO-d6) d 169.10, 156.89, 154.94, 154.88, 140.83, 129.16, 127.77 (2C), 120.44 (2C), 102.17, 46.80, 46.48, 45.44, 34.04, 27.12, 26.98, 25.97 (2C), 18.81 (2C), 12.33 (2C). TOF MS ES+(m / z): (M+H) + , calcd for C 23 H 29 ClN6O3S: 505.1789; found, 505.1781. HPLC: t R : 13.336 min, 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] Following the procedure of Example 34, step three, replace (2S,6R)-2,6- dimethylmorpholine with an equimolar amount of 4-(pyrrolidin-1-yl)piperidine.

[0241] White solid, 1 H NMR (400 MHz, DMSO-d6) d 8.95 (s, 1H), 8.12 (s, 1H), 7.76 (d, J = 8.5 Hz, 2H), 7.42 (d, J = 8.5 Hz, 2H), 4.57 (d, J = 13.3 Hz, 2H), 3.68 - 3.56 (m, 4H), 3.17 (t, J = 5.0 Hz, 6H), 2.91 (d, J = 3.0 Hz, 3H), 2.86 (d, J = 12.8 Hz, 2H), 2.08 (d, J = 12.1 Hz, 2H), 1.98 - 1.81 (m, 4H), 1.57 - 1.46 (m, 2H). 13 C NMR (151 MHz, DMSO-d6) d 169.04, 158.86, 155.30, 155.14, 140.40, 129.68, 127.74 (2C), 121.24 (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 CIN7O3S: 548.2211; found, 548.2205. HPLC: t R : 17.672 min, purity: 96.48%.

[0242] The structural formula of each 4-aminopyrimidine compound prepared in Examples 1-36 is shown in Table 1.

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

[0244]

[0245]

[0246]

[0247]

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

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

[0250] (1) After the cells were recovered and subcultured for 3 times, they were digested from the bottom of the culture bottle with trypsin solution (0.25%). After the cell digestion solution was sucked into a centrifuge tube, culture solution was added to stop the digestion. The centrifuge tube was centrifuged at 1000 r / min for 3 min, and after the supernatant was discarded, 3 mL of culture solution was added, the cells were mixed by blowing, 10 μL of cell suspension was taken and added to a cell counting plate for counting, and the cell concentration was adjusted to 10 4 cells / well. Except for the uppermost, lowermost and leftmost blank wells, 180 μL of cell suspension was added to the remaining wells. The 96-well plate was placed in an incubator for 24 h.

[0251] (2) The test sample was dissolved in 20 μL of dimethyl sulfoxide, then 980 μL of culture solution was added to dissolve the sample into a 1 mg / mL drug solution, and then the sample was diluted to 1, 0.333, 0.111, 0.037 and 0.012 μg / mL in an EP tube. Each concentration was added to 3 wells, 20 μL per well, and the surrounding two rows and two columns of cells were affected by the environment, and were only used as blank cell wells. The 96-well plate was placed in an incubator for 72 h.

[0252] (3) Discard the drug-containing culture solution in the 96-well plate, rinse the cells twice with phosphate buffer solution (PBS), add 100 μL of 0.5 mg / mL MTT (thiazolyl blue) solution to each well, and place in the incubator for 4 h. Discard the MTT solution, and add 100 μL of dimethyl sulfoxide. Shake on a magnetic shaker to dissolve the MTT reaction product formazan in the viable cells, and place in an enzyme label meter to measure the results at a wavelength of 492 nm. The IC 50 values of the drugs can be calculated by the Bliss method.

[0253] The anti-proliferative activities of each 4-aminopyrimidine compound prepared in Examples 1 to 36 against three lung cancer cell lines are shown in Table 2, where NA indicates that the IC 50 > 100.00 μM, and ND indicates not detected.

[0254] Table 2 Anti-cell proliferation activities of each 4-aminopyrimidine compound

[0255]

[0256] The EGFR, AXL kinase activities (IC 50 and inhibition rates) of each 4-aminopyrimidine compound prepared in Examples 1 to 36 were measured.

[0257] Method: Take a 384-well plate, and set up test compound wells, blank control wells, and positive drug control wells on the plate. 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 different 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 (the same for AXL kinase testing) to each well, centrifuge, and mix well. Place at room temperature for 60 min to equilibrate. The final concentration of the antibody is 2 nM, and the final concentration of EDTA is 8 mM. Read the fluorescence values using Envision. Calculate the percentage inhibition of the compound.

[0258] Percentage inhibition = (Lance signal value - Min) / (Max - Min) x 100%

[0259] Min: Lance signal value without enzyme; Max: Lance signal value of the DMSO control. The data was analyzed by SPSS, MS Excel, and Graphpad 5.0 to obtain the IC 50 .

[0260] AZD9291 and Bemcentinib were used as positive controls, and Kinase-Glo and Ultra enzyme activity evaluation method was used to test the inhibitory activity of each compound on EGFR and AXL kinase, and the results are shown in Table 3, wherein ND represents not detected.

[0261] Table 3 Activity of each 4-aminopyrimidine compound on EGFR and AXL kinase

[0262]

[0263] In addition, the IC50 of the compound prepared in Example 26 and 36 on AXL was also tested respectively, and the results showed that the IC50 of Example 26 and 36 on AXL was 9.77 and 8.23 μM respectively. 50 50

[0264] From the above test results, it can be seen that the compounds of formula I and formula II to be protected by the present application have good in vitro anti-cell proliferation activity, such as the compounds prepared in Examples 11, 12, 14, 19, 21, 30, 32, 36. However, for EGFR T790M / C797S , AXL kinase activity, the activity of most examples is poor, only the compounds prepared in Examples 26 and 36 show inhibitory activity on EGFR T790M / C797S , AXL at the same time, wherein the inhibitory activity of the compound prepared in Example 36 on EGFR T790M / C797S kinase is equivalent to that of the positive drug AZD9291. It can be seen that the compounds of formula I and formula II of the present application are expected to become a potential inhibitor of EGFR T790M / C797S , AXL kinase, especially the compound prepared in Example 36.

[0265] In vitro anti-tumor mechanism of 4-aminopyrimidine compounds

[0266] In order to explore the anti-tumor mechanism of the examples, the present application takes the compound prepared in Example 36 as an example to explore the anti-tumor mechanism of H1975 cells through various in vitro pharmacological experiments.

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

[0268] 1.1 AnneXIN V-FITC / PI double staining method for detecting cell apoptosis

[0269] The specific operation steps are as follows:

[0270] (1) After the H1975 cells were digested and centrifuged by the subculture method, the number was counted, and 2.0 x 10 5 ​​Cells were seeded at a density of 100 cells per well in 6-well plates. The cells were then incubated at 37°C and 5% CO2 for 24 hours to allow them to adhere.

[0271] (2) After 24 hours, 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, 6 μM) and positive control (AZD9291, 4 μM) at the prepared concentrations. The control group without drug was placed in a 37°C, 5% CO2 incubator for 24 hours.

[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 have completely detached, add 1 mL of culture medium to stop the digestion, rinse once, transfer to a 5 mL centrifuge tube, and centrifuge. Discard the supernatant, add 1 mL of PBS to the centrifuge tube, and gently pipette the cells to make them homogeneous. Repeat this step twice.

[0273] (4) Protect from light and wrap the centrifuge tubes with aluminum foil. Add 500 μL of buffer to each centrifuge tube and gently pipette 8-10 times. Add 5 μL of Laximnex V and 5 μL of LPI dye to the blank control group. Repeat the blank control group procedure for the compound prepared in Example 36 and the positive wells, and incubate in the dark for 30 min.

[0274] (5) The cell staining mixture was counted by flow cytometry at a concentration of 1.0 × 10⁻⁶. 4 Each cell records data.

[0275] To investigate the effect of the compound on apoptosis, the AnneXIN V-FITC / PI double staining method was used to detect the effect of the compound prepared in Example 36 on apoptosis. The results are shown in [Figure 1]. Figure 1 A.

[0276] Depend on Figure 1 As shown in Figure A, the total apoptosis rate of 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% and 4.76% to 9.82%, respectively. In particular, during late apoptosis, the apoptosis rate increased from 2.47% to 5.71%. The apoptosis results indicate that the compound prepared in Example 36 induced apoptosis in H1975 cells in a dose-dependent manner.

[0277] 1.2 AO staining assay for cell morphology determination

[0278] The specific operating steps are as follows:

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

[0280] (2) The original culture solution was sucked out and washed with PBS twice. 1 mg of the compound prepared in Example 36 was dissolved in 20 μL of DMSO, and then added to serum-free medium to prepare a solution with a concentration of 2, 4, or 6 μM. The solution was added to each well at a volume of 1 mL, and then incubated in a cell incubator for 12 h.

[0281] (3) The culture medium was sucked out and washed with 1 mL of PBS twice. After washing with 500 μL of 1x buffer once, 200 μL of AO staining solution (acridine orange: 1x buffer = 1:20) was added, and then incubated in a cell incubator for 15 min.

[0282] (4) The buffer was sucked out, and each well was washed with PBS three times. 500 μL of PBS was added, and then observed under a microscope. The filter was rotated to 4 / 5, the laser was adjusted to blue light, and then photographed and saved.

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

[0284] As can be clearly seen from Figure 1 B, the compound prepared in Example 36 showed obvious orange-red fluorescence at a concentration of 2 μM, indicating that apoptotic bodies had begun to appear. At a concentration of 4 μM, the orange-red fluorescence increased, and the fluorescence intensity was comparable to or even better than that of a positive drug. When the concentration was increased to 6 μM, the orange-red fluorescence intensity continued to increase, and the H1975 cells showed a blurred cell edge. The results show that the ability of the compound prepared in Example 36 to induce H1975 cell apoptosis gradually increased with increasing concentration.

[0285] 1.3 JC-1 experiment for detecting MPP of cells

[0286] The specific operation steps are as follows:

[0287] (1) The cell inoculation and drug treatment method were the same as described above in 1.2.

[0288] (2) The culture medium was sucked out, and then washed with 1 mL of PBS twice. 1 mL of JC-1 working solution (50 uL of JC-1 (200x): 8 mL of ultrapure water) was added, mixed thoroughly, and then incubated in a cell incubator for 20 min.

[0289] (3) The working solution was sucked out, and then washed with 1x JC-1 staining buffer twice.

[0290] (4) 2 mL of 200x JC-1 staining buffer was added, and then observed and photographed under a fluorescence microscope.

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

[0292] like Figure 1 As shown in Figure C, compared to the control group, the compound prepared in Example 36 exhibited significant green fluorescence at 2 μM. The amount and intensity of the green fluorescence increased with increasing concentration, indicating that the compound prepared in Example 36 could induce a dose-dependent decrease in mitochondrial membrane potential, thereby inducing 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 control at the same concentration. These results demonstrate that the compound prepared in Example 36 induces apoptosis in H1975 cells in a dose-dependent manner.

[0293] 2. The compound prepared in Example 36 was able to arrest the cell cycle of H1975 cells in the G2 / M phase.

[0294] The specific operating steps are as follows:

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

[0296] (2) Digest H1975 cells with trypsin without EDTA, centrifuge in a 5mL centrifuge tube, wash twice with 1mL PBS (centrifuge), add 500μL PBS, and mix the cells. Slowly add 500μL of 70% cold ethanol (ethanol + PBS), and fix the cells at 4℃.

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

[0298] (4) The cell staining mixture was counted by flow cytometry at a concentration of 1.0 × 10⁻⁶. 4 Each cell records data.

[0299] To investigate the effect of the compounds on the cell cycle, flow cytometry was used to examine the effect of the compounds prepared in Example 36 on the H1975 cell cycle. The results are shown in [Figure 1]. Figure 2 .

[0300] Depend on Figure 2It was found that the compound prepared in Example 36 could arrest the cell cycle of H1975 cells at the G2 / M phase. The arrest rate of the compound in the G2 / M phase increased from 20.62% and 21.1% to 24.65% at 2 μM, 4 μM, and 6 μM. The results indicate that the compound prepared in Example 36 arrested 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 ROS content in H1975 cells.

[0302] The specific operating steps are as follows:

[0303] (1) Cells in the logarithmic growth phase were divided into groups of 2.0 × 10⁶ cells per well. 5 Cells were seeded at a density of 1,000 cells per well in a 6-well plate and incubated for 24 hours.

[0304] (2) Add 3 mL of serum-free culture medium to each well, and then add 300 μL of the compound (2, 4, 6 μM) prepared in Example 36 and the positive control (AZD9291, 4 μM) at the prepared concentrations. Use the control group without the drug solution as a blank control group. After the specified time of culture, discard the old culture medium, wash twice with PBS, and then add the staining solution.

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

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

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

[0308] (6) After two final PBS washes, observe and photograph using a fluorescence microscope.

[0309] To investigate the effect of the compound on intracellular ROS levels in tumor cells, the changes in reactive oxygen species in H1975 cells caused by the compound prepared in Example 36 were detected using the DCFH-DA probe. The results are shown in [Figure 1]. Figure 3 .

[0310] Depend on Figure 3 It was found that the H1975 cells in the blank group did not produce green fluorescence, indicating that no ROS were generated. The low concentration of the compound prepared in Example 36 showed almost no green fluorescence. As the concentration of the compound prepared in Example 36 increased, the intensity of the green fluorescence increased, with the intensity at 6 μM comparable to that of the positive control. The results indicate that the compound prepared in Example 36 increased the intracellular ROS content of H1975 cells in a dose-dependent manner.

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

[0312] The specific operating steps are as follows:

[0313] (1) H1975 cells in the logarithmic growth phase were divided into 5.0 × 10⁶ cells per well. 5 Cells were seeded in 6-well plates.

[0314] (2) When the cells adhered to the plate and proliferated to 80%–90%, the plates were scored vertically with a pipette tip. The cells were 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 control (AZD9291, 4 μM) at the prepared concentrations. The control group without the added drug was used as a blank control group. The plates were then placed in an incubator for further culture.

[0315] (3) After incubation for 24 hours, wash away the floating cells in the upper layer with PBS, observe and photograph under a microscope.

[0316] To investigate the effect of the compound on cell migration ability, a cell scratch assay was performed to examine the migration of H1975 cells by the compound prepared in Example 36. The results are shown in […]. Figure 4 .

[0317] pass Figure 4 It can be seen that after culturing H1975 cells for 24 hours, cells in the control group and the 2 μM drug-treated group significantly migrated towards the center, while the positive group, the 4 μM drug-treated group, and the 6 μM drug-treated group significantly inhibited the migration of H1975 cells. Moreover, the higher the concentration of the compound prepared in Example 36, the fewer cells migrated. The results indicate that the compound prepared in Example 36 inhibited the migration of H1975 cells in a dose-dependent manner.

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

[0319] To ensure the biosafety of the embodiments, the compounds prepared in Example 36 were evaluated for in vivo and in vitro toxicity using hemolysis and KM mouse toxicity tests.

[0320] 1. Hemolysis test

[0321] The specific operating steps are as follows:

[0322] (1) Take 1 mL of 4% sheep blood red blood cells, freeze, centrifuge, wash, and repeat the operation twice to extract red blood cells.

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

[0324] (3) The above red blood cell suspension was incubated at 37°C for 1 hour. The absorbance was measured by ultraviolet spectrophotometer, and the hemolysis rate was calculated.

[0325] Hemolysis rate (%) = (Experimental group A - Negative control group A) / (Positive control group A - Negative control group A)

[0326] This invention conducted hemolysis experiments using sheep red blood cells to simulate the effect of the compound prepared in Example 36 in blood, exploring whether the compound would affect blood safety. Using 1% Triton as a positive control, the effects of the compound prepared in Example 36 on red blood cell hemolysis were investigated at concentrations of 8, 16, 32, 64, 128, and 256 μg / mL. The hemolysis rate was used as a safety indicator to evaluate the compound's safety to blood. The results are shown in [Figure number missing]. Figure 5 .

[0327] Depend on Figure 5 It can be seen that the hemolysis rate gradually increased with the increase of the concentration of the compound prepared in Example 36, but 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 biocompatibility and will not cause hemolytic anemia. The results show that the hemolysis rate of the compound prepared in Example 36 on erythrocytes is within the safe range and warrants further investigation.

[0328] 2. KM mouse toxicity test

[0329] 2.1 Experimental Methods:

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

[0331] (2) Oral administration: The compound was dissolved in physiological saline, Tween 80 was added to aid dissolution, and the solution was dissolved by sonication. The blank group was given an equal volume of physiological saline, while the positive control group and the drug administration group were given the prescribed doses. The mice were administered the drugs in groups. The drugs were administered once every 24 hours for a total of 7 days.

[0332] (3) Sampling: Seven days later, the mice in the four groups were euthanized by blood collection from their eyeballs. More than 0.5 mL of blood was collected from each mouse, labeled, and stored in a 4°C refrigerator for later use. Then, each group of mice was dissected, and the heart, liver, spleen, lungs, kidneys, and other organs obtained from the dissection were collected and photographed. In addition, the five internal organs of one mouse in each group were taken and soaked in 4% paraformaldehyde fixative for subsequent H&E staining experiments.

[0333] 2.2 Analysis of mouse blood samples:

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

[0335] (2) Prepare a preoperative 11-item analysis kit, add 100 μL of serum through the injection hole of the reagent tray, put it into the automated multifunctional biochemical analyzer, perform blood testing, export the data and perform data analysis through GraphPad Prism 8.

[0336] 2.3 HE staining:

[0337] (1) Tissue treatment: Clean the organs with physiological saline and dry them. Add the tissue to 4% formaldehyde solution and fix for 24 hours. Place the fixed tissue in 50%, 70%, 80%, and 90% alcohol solutions in sequence for dehydration, and soak for 1 hour in each alcohol concentration.

[0338] (2) Sample clearing and paraffin impregnation: Place the dehydrated tissue in xylene solution and repeat the clearing process twice, 15 min each time. Place the cleared tissue in the prepared paraffin solution and impregnate it twice, 1 h each time.

[0339] (3) Dewaxing paraffin sections to water: 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 min respectively, and then wash with tap water.

[0340] (4) Hematoxylin staining: Place the slide in hematoxylin staining solution and let it act for 3-5 minutes. Wash with tap water, differentiate with differentiation solution, wash with tap water, and then re-blue with blue solution. 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 staining solution for 5 min.

[0342] (6) Dehydration and mounting: 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, xylene II for 5 min, and then cleared and mounted with neutral resin.

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

[0344] During the drug administration period, the KM mice maintained normal activity and good mental state daily. The mice were weighed and their weight recorded daily; results are shown below. Figure 6 .

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

[0346] After 7 days of continuous administration, each mouse was subjected to eye blood sampling, and the blood was used for subsequent biochemical blood sample analysis, and then the mouse was dissected, and the five internal organs were weighed and arranged for photography to observe the influence of the compound prepared in Example 36 on the internal organs of the mouse from a macroscopic point of view, and the results are shown in Figure 7 , wherein A-E are the macroscopic images of the heart, liver, spleen, lung and kidney, respectively, and F is the organ index calculated after weighing the five internal organs.

[0347] As shown in Figure 7 A-E, it can be seen that the compound prepared in Example 36 has no obvious toxic side effects on the five internal organs of the mouse. As shown in Figure 7 F, compared with the blank group, there is no significant difference in the organ index 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 has no significant effect on the internal organs of the mouse. Relatively, the liver index of the positive group is slightly higher. Therefore, macroscopically, the toxicity of the compound prepared in Example 36 is lower than that of the positive administration group.

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

[0349] As shown in Figure 8As shown in the total protein (TP), creatinine (CREA), alkaline phosphatase (ALP), lactate dehydrogenase (LDH) and creatine kinase (CK) level detection, the low-dose group and the high-dose group of the compound prepared in Example 36 of the administration group had no significant difference with the blank group (0.9% normal saline). In the urea (UREA) and glucose (GLU) level detection, the administration group was slightly higher than the blank group (0.9% normal saline) and the positive group (AZD9291), indicating that it may have an impact on the kidney, but it is within the normal range, and the two administration groups of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels are lower than the blank group. Overall, the compound prepared in Example 36 has no obvious toxicity. However, the administration group, the blank group and the positive group almost all have the phenomenon of high alkaline phosphatase (ALP) and creatine kinase (CK), which may be due to the mouse itself, and the five organ slices will be stained with H&E to observe whether there is inflammation.

[0350] Subsequently, the five organs of one mouse from each of the four groups of KM mice were observed under a microscope after H&E staining to observe the morphology of cells and structures, and the effects of the compound on tissues and cells were evaluated, and the results are shown in Figure 9 .

[0351] As shown in Figure 9 , the H&E staining results show that the compound prepared in Example 36 has no obvious damage to the mouse organs and no inflammation occurs, and it is safe at a dose of 75 mg / kg. The experimental results show that the in vivo biological safety of the compound prepared in Example 36 is good.

[0352] The above-described examples only describe the preferred modes of the present application and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. A 4-aminopyrimidine compound targeting EGFR, AXL target point, characterized in that, The 4-aminopyrimidine compound has the following structural formula: 。 2. A pharmaceutically acceptable salt of the 4-aminopyrimidine compound targeting EGFR and AXL targets according to claim 1.

3. A pharmaceutical preparation, characterized by, The active ingredient is the 4-aminopyrimidine compound targeting EGFR and AXL targets according to claim 1 or the pharmaceutically acceptable salt of the 4-aminopyrimidine compound targeting EGFR and AXL targets according to claim 2.

4. Use of the 4-aminopyrimidine compound targeting EGFR and AXL targets according to claim 1 in the preparation of a medicament for treating and / or preventing lung cancer.

5. Use of the pharmaceutically acceptable salt of the 4-aminopyrimidine compound targeting EGFR and AXL targets according to claim 2 in the preparation of a medicament for treating and / or preventing lung cancer.

6. Use of the pharmaceutical preparation according to claim 3 in the preparation of a medicament for treating and / or preventing lung cancer.

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