Aminopyrimidine compound containing toluene structure as well as preparation method and application of aminopyrimidine compound

Abstract

The invention relates to an aminopyrimidine compound containing a toluene structure as well as a preparation method and application thereof. The aminopyrimidine compound has a structural formula as shown in a general formula (I). Pharmacological activity results of the aminopyrimidine compound containing the toluene structure show that the aminopyrimidine compound has excellent proliferation inhibition activity on human lymphoma cells Daudi, human non-small cell lung cancer cells A549, human non-small cell lung cancer cells H1975 and human chronic myelogenous leukemia cells K562. The invention also provides a preparation method of the compounds, a pharmaceutical composition containing the compounds and application of the pharmaceutical composition. The compound is especially used for preparing drugs for treating and / or preventing lymphoma, lung cancer and leukemia, and has good antitumor drug development and application prospects. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the technical field of compound synthesis, and particularly relates to aminopyrimidine compounds containing a toluene structure, their preparation methods and applications. Background Art

[0002] Cancer, a disease that generally refers to all malignant tumors, has long been a major threat to human health. According to statistics from the World Health Organization (WHO), the number of people who died from cancer globally in 2020 reached as high as 10 million. Among them, what is even more distressing is that approximately 400,000 children are diagnosed with cancer every year. The data shows that about one-fifth of people may develop cancer in their lifetime, and its mortality rate has exceeded that of cardiovascular and cerebrovascular diseases, ranking first among all diseases. It is estimated that by 2030, the number of newly diagnosed cancer cases globally will reach 21.4 million, and the number of deaths will exceed 13 million. Among them, lymphoma, lung cancer, and leukemia are malignant tumor types with high incidence, poor prognosis, and pose a serious threat to human health. Therefore, the research on anti-tumor drugs, especially highly efficient and low-toxic molecular targeted anti-tumor drugs, is of extremely important significance.

[0003] Lymphoma is a group of malignant tumors originating from the lymphohematopoietic system, mainly including Hodgkin lymphoma and non-Hodgkin lymphoma. In recent years, the incidence of lymphoma has shown an upward trend and has high heterogeneity. The treatment regimens and prognoses of different subtypes vary significantly. Although the application of targeted therapy and immunotherapy has improved the survival of some patients, the treatment of relapsed and refractory lymphoma remains a huge challenge in clinical practice.

[0004] Lung cancer is the main cause of cancer-related deaths globally, and non-small cell lung cancer (NSCLC) accounts for the vast majority. Although early diagnosis and precision treatment have improved the survival rate of some patients, the prognosis of advanced NSCLC patients remains poor. Targeted therapy and immunotherapy have brought new hope to NSCLC patients, but problems such as drug resistance and treatment-related adverse reactions still need to be solved.

[0005] Leukemia is a group of malignant clonal diseases originating from hematopoietic stem cells and can be divided into acute leukemia and chronic leukemia according to the course of the disease and cell type. In recent years, with the optimization of chemotherapy regimens and the progress of hematopoietic stem cell transplantation technology, the treatment effect of leukemia has been significantly improved. However, some patients still face challenges such as relapse and refractoriness, and treatment-related toxic side effects.

[0006] The present invention relates to aminopyrimidine compounds containing a toluene structure and their pharmaceutically acceptable salts, their preparation methods, and pharmaceutical compositions containing the compounds, which can be used for the preparation of drugs for treating and / or preventing lymphoma, lung cancer, leukemia, etc., and have good prospects for the development and application of anti-tumor drugs. Summary of the Invention

[0007] The object of the present invention is to design and synthesize a series of novel aminopyrimidine compounds containing a toluene structure. Through in vitro activity screening, it is shown that such compounds have anti-tumor activity.

[0008] The present invention provides an aminopyrimidine compound containing a toluene structure represented by the general formula (I),

[0009]

[0010] wherein,

[0011] Ar is selected from a 6- to 10-membered aryl group or a 5- to 10-membered heteroaryl group, wherein the heteroaryl group contains 1 to 3 heteroatoms selected from N, O or S, and Ar is optionally substituted by 1 to 5 identical or different R1;

[0012] R1 is selected from hydrogen, hydroxyl, halogen, amino, cyano, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, C1-C6 alkylthio, C1-C6 alkyl substituted by hydroxyl or amino or halogen, C1-C6 alkoxy substituted by hydroxyl or amino or halogen, amino substituted by mono- or di-C1-C6 alkyl, C1-C6 alkylcarbonylamino, ester group, free or salt-forming or esterified or amidated carboxyl group, C1-C6 alkylsulfinyl, C1-C6 alkylsulfonyl, C1-C6 alkylcarbonyl, carbamoyl.

[0013] L is selected from

[0014] R is selected from a 6- to 10-membered aryl group or a 5- to 10-membered heteroaryl group or a 4- to 8-membered aliphatic ring or a 4- to 8-membered aliphatic heterocycle. Wherein, the heteroaryl group and the aliphatic heterocycle contain 1 to 3 heteroatoms or groups selected from N, O, S, SO, SO2, and R is optionally substituted by 1 to 5 identical or different R2;

[0015] R2 is selected from hydrogen, hydroxyl, halogen, amino, cyano, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, C1-C6 alkylthio, C1-C6 alkyl substituted by hydroxyl or amino or halogen, C1-C6 alkoxy substituted by hydroxyl or amino or halogen, amino substituted by mono- or di-C1-C6 alkyl, C1-C6 alkylcarbonylamino, ester group, free or salt-forming or esterified or amidated carboxyl group, C1-C6 alkylsulfinyl, C1-C6 alkylsulfonyl, C1-C6 alkylcarbonyl, carbamoyl.

[0016] Furthermore, the above-mentioned aminopyrimidine compound containing a toluene structure,

[0017] Ar is selected from benzene or a 5- or 6-membered heteroaryl, wherein the heteroaryl contains 1 to 3 heteroatoms selected from N, O or S, and Ar is optionally substituted by 1 to 3 identical or different R1;

[0018] R1 is selected from hydrogen, hydroxy, halogen, amino, C1-C5 alkyl, C1-C5 alkoxy.

[0019] L is selected from

[0020] R is selected from a benzene ring, a 5- or 6-membered heteroaryl or a 4- to 6-membered aliphatic heterocycle. Wherein the heteroaryl and the aliphatic heterocycle contain 1 to 3 heteroatoms or groups selected from N, O, S, SO2, and R is optionally substituted by 1 to 3 identical or different R2;

[0021] R2 is selected from hydrogen, hydroxy, halogen, amino, C1-C5 alkyl, C1-C5 alkoxy, C1-C5 alkyl acyl, carbamoyl.

[0022] Furthermore, the above-mentioned aminopyrimidine compounds containing a toluene structure,

[0023] Ar is selected from a 5- or 6-membered heteroaryl, wherein the heteroaryl contains 1 to 2 N atoms, and Ar is optionally substituted by 1 to 3 identical or different R1;

[0024] R1 is selected from hydrogen, methyl, ethyl, methoxy.

[0025] L is selected from

[0026] R is selected from a benzene ring or a 4- to 6-membered aliphatic heterocycle. Wherein the aliphatic heterocycle contains 1 to 2 heteroatoms or groups selected from N, SO2, and R is optionally substituted by 1 to 3 identical or different R2;

[0027] R2 is selected from hydrogen, chlorine, fluorine, methyl, methoxy, isopropoxy, formyl.

[0028] Furthermore, the above-mentioned aminopyrimidine compounds containing a toluene structure and their pharmaceutically acceptable salts have the following structural formula:

[0029]

[0030]

[0031]

[0032] A pharmaceutical composition comprising the above-mentioned aminopyrimidine compounds containing a toluene structure and their pharmaceutically acceptable salts as active ingredients and pharmaceutically acceptable excipients.

[0033] Preferably, for the above-mentioned pharmaceutical composition, the pharmaceutically acceptable carrier is selected from one or more of fillers, disintegrants, binders, and lubricants.

[0034] Preferably, for the above-mentioned pharmaceutical composition, the pharmaceutical composition is made into dosage forms such as tablets, capsules, granules, sprays, or injections.

[0035] Use of the aminopyrimidine compound containing a toluene structure and its pharmaceutically acceptable salt as described in any one of the above, or the pharmaceutical composition as described in any one of the above, in the preparation of a drug for treating and / or preventing proliferative diseases.

[0036] Use of the aminopyrimidine compound containing a toluene structure and its pharmaceutically acceptable salt as described in any one of the above, or the pharmaceutical composition as described in any one of the above, in the preparation of a drug for treating and / or preventing cancer.

[0037] Use of the aminopyrimidine compound containing a toluene structure and its pharmaceutically acceptable salt as described in any one of the above, or the pharmaceutical composition as described in any one of the above, in the preparation of a drug for treating and / or preventing lymphoma, lung cancer, and leukemia.

[0038] According to some common methods in the field to which the present invention pertains, the aminopyrimidine compound containing a toluene structure represented by the general formula (I) in the present invention can form pharmaceutically acceptable salts with acids. Pharmaceutically acceptable addition salts include inorganic acid and organic acid addition salts. Salts formed by addition with the following acids are particularly preferred: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalenedisulfonic acid, acetic acid, propionic acid, lactic acid, trifluoroacetic acid, maleic acid, citric acid, fumaric acid, oxalic acid, tartaric acid, benzoic acid.

[0039] In addition, the present invention also includes prodrugs of the compounds of the present invention. The prodrugs of the compounds of the present invention are aminopyrimidine compounds containing a toluene structure of the general formula (I). They may have weak activity or even no activity by themselves, but after administration, they are converted into the corresponding bioactive forms under physiological conditions (such as through metabolism, solvolysis, or other means).

[0040] The beneficial effects of the present invention are:

[0041] The aminopyrimidine compound containing a toluene structure and its pharmaceutically acceptable salt obtained in the present invention have excellent anti-tumor activity and safety. Through in vitro inhibition tests on human lymphoma cell Daudi, human non-small cell lung cancer cell A549, human non-small cell lung cancer cell H1975, and human chronic myeloid leukemia cell K562, it is proved that the compounds of the present invention have significant inhibitory effects on human lymphoma cells, human lung cancer cells, and human leukemia cells. The compounds described in the present invention are chemical drug raw materials, and are particularly used for the preparation of drugs for treating and / or preventing lymphoma, lung cancer, and leukemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 It is the Hoechst staining experiment of compound 4 on H1975, where (a) is the blank control group, (b) is the group with a concentration of 1.0 μM of compound 4, and (c) is the group with a concentration of 10 μM of compound 4.

[0043] Figure 2 It is the migration inhibition experiment of compound 4 on H1975. DETAILED DESCRIPTION OF THE INVENTION

[0044] The examples and preparation examples provided below further illustrate and exemplify the compounds of the present invention and their preparation methods. It should be understood that the scope of the following examples does not limit the scope of the present invention in any way.

[0045] The following synthetic route describes the preparation method of the aminopyrimidine compounds containing toluene structure of general formula (I) of the present invention.

[0046] All raw materials are prepared by the methods described in the following synthetic route or by methods well-known to those of ordinary skill in the art of organic chemistry or are commercially available. All the final compounds of the present invention are prepared by the methods described in the following synthetic route or by methods similar thereto, which are well-known to those of ordinary skill in the art of organic chemistry. All the variable factors used in the following synthetic route are defined as below or as defined in the claims.

[0047] The examples are intended to illustrate rather than limit the scope of the present invention. The 1H NMR spectra of the compounds were measured using a Bruker ARX-400 or ARX-600, and the mass spectra were measured using an Agilent 1100 LC / MSD; all reagents used were of analytical grade or chemical pure grade.

[0048] The synthetic routes of compounds 1-24 of general formula (I) according to the present invention are as follows:

[0049] 1) As shown in Route 1, using 2-methyl-4-bromoaniline and di-tert-butyl dicarbonate as raw materials, a substitution reaction occurs in toluene to obtain intermediate 2a; under the condition of nitrogen protection, intermediate 2a and bis(pinacolato)diboron undergo a coupling reaction in 1,4-dioxane under the action of Pd(dppf)Cl2 and KOAc to obtain intermediate 3a; under the condition of nitrogen protection, intermediate 3a and 2,4-dichloropyrimidine undergo a coupling reaction in 1,4-dioxane / water (4:1) under the action of Pd(dppf)Cl2 and K2CO3 to obtain intermediate 4a; under the condition of nitrogen protection, intermediate 4a and different nitrogen-containing five-membered rings undergo a substitution reaction in 1,4-dioxane under the action of Pd2(dba)3, S-Phos, and Cs2CO3 to obtain intermediates 5a-b; intermediates 5a-b are deprotected by Boc groups in trifluoroacetic acid and dichloromethane solution to obtain 6a-b; intermediates 6a-b and 1,1-cyclopropanedicarboxylic acid undergo an acylation reaction in thionyl chloride and tetrahydrofuran to obtain key intermediate 7a-b; key intermediate 7a-b and different substituted aromatic aniline compounds undergo an acylation reaction in DMF under the action of HATU and TEA to obtain compounds 1 - 15.

[0050]

[0051] Among them, (i) Toluene, 110 °C, 3 h; (ii) PinB - BPin, Pd(dppf)Cl2, KOAc, 1,4-dioxane, 100 °C, 2 h; (iii) Pd(dppf)Cl2, K2CO3, 1,4-dioxane / H2O(4:1), 90 °C, 2 h; (iv) Pd2(dba)3, S-Phos, Cs2CO3, 1,4-dioxane, 120 °C, 2 h; (v) HCl / MeOH, rt, 6 h; (vi) TEA, SOCl2, THF, -5 °C, 2 h; (vii) HATU, TEA, DMF, rt, 3 h.

[0052] 2) The synthesis of compounds 16 - 24 is shown in Route 2. Intermediate 6a-b and chloroacetyl chloride undergo a substitution reaction in potassium carbonate and tetrahydrofuran to obtain key intermediate 8a-b; the key intermediate undergoes a substitution reaction with different nitrogen-containing heterocycles to obtain the target end products compounds 16 - 24.

[0053]

[0054] Among them, (i) K2CO3, THF, rt, 2 h; (ii) K2CO3, DMF, 55 °C, 3 h.

[0055] For all intermediates and compounds in the above two routes, the substituents R2 and Ar are as follows:

[0056] Ar is selected from 6- to 10-membered aryl or 5- to 10-membered heteroaryl, wherein the heteroaryl contains 1 to 3 heteroatoms selected from N, O or S, and Ar is optionally substituted by 1 to 5 identical or different R1;

[0057] R1 is selected from hydrogen, hydroxy, halogen, amino, cyano, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, C1-C6 alkylthio, C1-C6 alkyl substituted by hydroxy or amino or halogen, C1-C6 alkoxy substituted by hydroxy or amino or halogen, amino substituted by mono- or di-C1-C6 alkyl, C1-C6 alkylcarbonylamino, ester group, free or salt-forming or esterified or amidated carboxyl, C1-C6 alkylsulfinyl, C1-C6 alkylsulfonyl, C1-C6 alkylcarbonyl, carbamoyl;

[0058] L is selected from

[0059] R is selected from 6- to 10-membered aryl or 5- to 10-membered heteroaryl or 4- to 8-membered alicyclic ring or 4- to 8-membered heteroalicyclic ring, wherein the heteroaryl and heteroalicyclic ring contain 1 to 3 heteroatoms or groups selected from N, O, S, SO, SO2, and R is optionally substituted by 1 to 5 identical or different R2;

[0060] R2 is selected from hydrogen, hydroxy, halogen, amino, cyano, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, C1-C6 alkylthio, C1-C6 alkyl substituted by hydroxy or amino or halogen, C1-C6 alkoxy substituted by hydroxy or amino or halogen, amino substituted by mono- or di-C1-C6 alkyl, C1-C6 alkylcarbonylamino, ester group, free or salt-forming or esterified or amidated carboxyl, C1-C6 alkylsulfinyl, C1-C6 alkylsulfonyl, C1-C6 alkylcarbonyl, carbamoyl.

[0061] General preparation method:

[0062] Step A tert-Butyl (4-bromo-2-methylphenyl)carbamate (2a)

[0063]

[0064] 4-Bromo-2-methylaniline (5.00 g) was placed in a 100 mL eggplant-shaped flask, toluene (20 mL) was added, and the mixture was stirred in an oil bath at 110 °C. Di-tert-butyl dicarbonate (6.45 g) was dissolved in toluene (5 mL) and slowly added dropwise. After reacting for about 2 h, the reaction was monitored by TLC and was complete with no remaining raw materials. It was extracted 3 times with ethyl acetate, the organic phases were combined, washed once with saturated NaCl solution, dried over anhydrous Na2SO4 to remove water, and the organic phase was evaporated under reduced pressure to obtain 7.57 g of a pale yellow solid 2a.

[0065] tert-Butyl (2-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)carbamate (3a)

[0066]

[0067] The intermediate 2a (5.00 g), bis-3,3-dimethyl-2-butanonediboronate (6.66 g) and potassium acetate (5.15 g) were placed in a 250 mL three-necked flask, 1,4-dioxane (30 mL) was added, and the mixture was stirred at room temperature with N2 bubbling for 0.5 h. Bis(triphenylphosphine)palladium dichloride (1.22 g) was added, the air was evacuated and nitrogen was flushed in for protection, and the mixture was stirred in an oil bath at 100 °C for reaction. After reacting for about 3 h, the reaction was monitored by TLC and was complete with no remaining raw materials. Diatomaceous earth was added and the mixture was filtered while hot, and the filter cake was rinsed with dioxane until no product remained. The filtrate was dried under reduced pressure, extracted 3 times with ethyl acetate, the organic phases were combined, washed once with saturated NaCl aqueous solution, dried over anhydrous Na2SO4, and the organic phase was evaporated under reduced pressure to obtain a black oily crude product, which was separated and purified by silica gel column chromatography to obtain 5.52 g of a pale yellow solid product intermediate 3a.

[0068] tert-Butyl (4-(2-chloropyrimidin-4-yl)-2-methylphenyl)carbamate (4a)

[0069]

[0070] Take intermediate 3a (5.00 g), 2,4-dichloropyrimidine (2.68 g) and cesium carbonate (14.67 g), place them in a 250 mL three-necked flask, add 1,4-dioxane (30 mL), stir at room temperature with N2 bubbling for 0.5 h, add bis(triphenylphosphine)palladium(II) dichloride (1.05 g), evacuate the air and flush with nitrogen for protection, and place in an oil bath at 90 °C and stir for reaction. After reacting for about 3 h, monitor the reaction by TLC until it is complete and there is no remaining raw material. Add diatomaceous earth and filter while hot, rinse the filter cake with dioxane until there is no product residue. Rotate the filtrate to dryness under reduced pressure, extract with ethyl acetate 3 times, combine the organic phases, wash once with saturated NaCl aqueous solution, dry over anhydrous Na2SO4, evaporate the organic phase under reduced pressure by rotation to obtain a black oily crude product, and separate and purify it by silica gel column chromatography to obtain 2.53 g of a pale yellow solid product, intermediate 4a.

[0071] Step D tert-Butyl (2-methyl-4-(2-((1-methyl-1H-pyrazol-3-yl)amino)pyrimidin-4-yl)phenyl)carbamate (5a)

[0072]

[0073] Take intermediate 4a (3.00 g), 1-methyl-1H-pyrazol-3-amine (1.18 g) and cesium carbonate (6.11 g) and place them in a 100 mL three-necked flask, add 1,4-dioxane (30 mL), stir at room temperature with N2 bubbling for 0.5 h, add tris(dibenzylideneacetone)dipalladium(0) (0.86 g) and 2-dicyclohexylphosphino-2′,6′-dimethoxybiphenyl (0.77 g), evacuate the air and flush with nitrogen for protection, and place in an oil bath at 90 °C and stir for reaction. After reacting for about 3 h, monitor the reaction by TLC until it is complete and there is no remaining raw material. Add diatomaceous earth and filter while hot, rinse the filter cake with dioxane until there is no product residue. Rotate the filtrate to dryness under reduced pressure, extract with ethyl acetate 3 times, combine the organic phases, wash once with saturated NaCl aqueous solution, dry over anhydrous Na2SO4, evaporate the organic phase under reduced pressure by rotation to obtain a black oily crude product, and separate and purify it by silica gel column chromatography to obtain a pale yellow solid product, intermediate 5a, with a product weight of 2.12 g and a yield of 59.6%.

[0074] Step E tert-Butyl (2-methyl-4-(2-((1-methyl-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)phenyl)carbamate (5b)

[0075]

[0076] Intermediate 4a (3.00 g), 1-methyl-1H-pyrazol-4-amine (1.18 g), and cesium carbonate (6.11 g) were placed in a 100 mL three-necked flask. 1,4-dioxane (30 mL) was added, and the mixture was stirred at room temperature with N2 bubbling for 0.5 h. Tris(dibenzylideneacetone)dipalladium(0) (0.86 g) and 2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl (0.77 g) were added. The air was evacuated and nitrogen was flushed in for protection, and the mixture was stirred at 90 °C in an oil bath for reaction. After reacting for about 3 h, the reaction was monitored by TLC and was complete with no remaining raw materials. Diatomaceous earth was added and the mixture was filtered while hot, and the filter cake was rinsed with dioxane until no product residue remained. The filtrate was concentrated under reduced pressure, extracted 3 times with ethyl acetate, the organic phases were combined, washed once with saturated NaCl aqueous solution, dried over anhydrous Na2SO4, and the organic phase was evaporated under reduced pressure to obtain a crude product as a black oil. It was separated and purified by silica gel column chromatography to obtain 2.08 g of a pale yellow solid product, intermediate 5b.

[0077] Step F 4-(4-Amino-3-methylphenyl)-N-(1-methyl-1H-pyrazol-3-yl)pyrimidin-2-amine (6a)

[0078]

[0079] Intermediate 5a (2.00 g) was placed in a 100 mL eggplant-shaped flask. Dichloromethane (10 mL) was added, and trifluoroacetic acid (2.98 g) was added dropwise, and the mixture was stirred at room temperature for reaction. After reacting for about 1 h, the reaction was monitored by TLC and was complete with no remaining raw materials. Excess dichloromethane and trifluoroacetic acid were removed by rotary evaporation under reduced pressure. Saturated Na2CO3 solution was added dropwise at room temperature with stirring until pH = 9 - 10, and a pale yellow solid precipitated. It was filtered with a funnel and rinsed three times with water. The filter cake was transferred to a vacuum drying oven at 50 °C and dried for 24 h to obtain 1.31 g of a pale yellow solid product, 6a.

[0080] Step G 4-(4-Amino-3-methylphenyl)-N-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-amine (6b)

[0081]

[0082] Intermediate 5b (2.00 g) was placed in a 100 mL eggplant-shaped flask. Dichloromethane (10 mL) was added, and trifluoroacetic acid (2.98 g) was added dropwise, and the mixture was stirred at room temperature for reaction. After reacting for about 1 h, the reaction was monitored by TLC and was complete with no remaining raw materials. Excess dichloromethane and trifluoroacetic acid were removed by rotary evaporation under reduced pressure. Saturated Na2CO3 solution was added dropwise at room temperature with stirring until pH = 9 - 10, and it was extracted 3 times with ethyl acetate. The organic phases were combined, washed once with saturated NaCl aqueous solution, dried over anhydrous Na2SO4, and the organic phase was evaporated under reduced pressure to obtain a crude product as a yellow solid. It was slurried with petroleum ether for 5 h and then filtered to obtain 1.20 g of a pale yellow solid pure product, 6b.

[0083] Step H1 - ((2-Methyl-4-(2-((1-methyl-1H-pyrazol-3-yl)amino)pyrimidin-4-yl)phenyl)carbamoyl)cyclopropane-1-carboxylic acid (7a)

[0084]

[0085] Take 1,1-cyclopropanedicarboxylic acid (0.88 g) and place it in a 100 mL eggplant-shaped flask. Add tetrahydrofuran (20 mL) and stir until completely dissolved. Place it in an ice bath and cool to -5°C. Dropwise add triethylamine (0.73 g), controlling the temperature not to exceed -5°C throughout the process. After the addition is complete, continue stirring and reacting for 30 min. Dropwise add 6a (2.00 g) dissolved in tetrahydrofuran (5 mL), controlling the temperature not to exceed 0°C throughout the process. After the addition is complete, continue reacting at -5°C for 2 h. Monitor the reaction by TLC until it is complete with no raw materials remaining. Slowly pour the reaction solution into sodium hydroxide solution (2 mol / L, 20 mL) and stir for 30 min. Extract the resulting solution with dichloromethane 3 times, collect the aqueous phase, adjust the pH of the aqueous phase to 1 with concentrated hydrochloric acid, extract with dichloromethane 3 times, combine the organic phases, wash with saturated NaCl solution once, and rotary evaporate the organic phase under reduced pressure to obtain 1.96 g of a pale yellow solid product 7a.

[0086] Step I1 - ((2-Methyl-4-(2-((1-methyl-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)phenyl)carbamoyl)cyclopropane-1-carboxylic acid (7b)

[0087]

[0088] Take 1,1-cyclopropanedicarboxylic acid (0.88 g) and place it in a 100 mL eggplant-shaped flask. Add tetrahydrofuran (20 mL) and stir until completely dissolved. Place it in an ice bath and cool to -5°C. Dropwise add triethylamine (0.73 g), controlling the temperature not to exceed -5°C throughout the process. After the addition is complete, continue stirring and reacting for 30 min. Dropwise add 6b (2.00 g) dissolved in tetrahydrofuran (5 mL), controlling the temperature not to exceed 0°C throughout the process. After the addition is complete, continue reacting at -5°C for 2 h. Monitor the reaction by TLC until it is complete with no raw materials remaining. Slowly pour the reaction solution into sodium hydroxide solution (2 mol / L, 20 mL) and stir for 30 min. Extract the resulting solution with dichloromethane 3 times, collect the aqueous phase, adjust the pH of the aqueous phase to 1 with concentrated hydrochloric acid, extract with dichloromethane 3 times, combine the organic phases, wash with saturated NaCl solution once, and rotary evaporate the organic phase under reduced pressure to obtain 2.18 g of a pale yellow solid product 7b.

[0089] Step J 2-Chloro-N-(2-methyl-4-(2-((1-methyl-1H-pyrazol-3-yl)amino)pyrimidin-4-yl)phenyl)acetamide (8a)

[0090]

[0091] Take the key intermediate 7a (1.00 g) and potassium carbonate (0.74 g) and place them in a 50 mL eggplant-shaped flask. Add ultra-dry THF (20 mL). Take chloroacetyl chloride (0.48 g), dilute it with THF (2 mL), and slowly add it dropwise at 0 °C. After the addition is complete, transfer it to room temperature and stir to react. After reacting for 2 h, monitor the reaction by TLC and the reaction is complete with no raw materials remaining. Extract with DCM 3 times, combine the organic phases, wash once with saturated NaCl solution, dry over anhydrous Na2SO4, and rotary evaporate under reduced pressure to obtain the yellow solid product 8a (1.20 g).

[0092] Step K 2-chloro-N-(2-methyl-4-(2-((1-methyl-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)phenyl)acetamide (8b)

[0093]

[0094] Take the key intermediate 7b (1.00 g) and potassium carbonate (0.74 g) and place them in a 50 mL eggplant-shaped flask. Add ultra-dry THF (20 mL). Take chloroacetyl chloride (0.48 g), dilute it with THF (2 mL), and slowly add it dropwise at 0 °C. After the addition is complete, transfer it to room temperature and stir to react. After reacting for 2 h, monitor the reaction by TLC and the reaction is complete with no raw materials remaining. Extract with DCM 3 times, combine the organic phases, wash once with saturated NaCl solution, dry over anhydrous Na2SO4, and rotary evaporate under reduced pressure to obtain 1.12 g of the yellow solid product 8b.

[0095] General preparation method for Examples 1-15:

[0096] Take the key intermediate 7a or 7b (100 mg, 1.0 equivalent) and different amines (1.2 equivalents) and place them in a 25 mL eggplant-shaped flask. Add DMF (5 mL), dropwise add triethylamine (3.0 equivalents), then add HATU (2.0 equivalents), and place it at room temperature to stir and react. After reacting for 3 h, monitor the reaction by TLC and the reaction is complete with no raw materials remaining. Add saturated Na2CO3 solution to adjust the pH to about 10, extract with DCM 3 times, combine the organic phases, and wash 3 times with saturated NaCl solution to remove the excess DMF. Dry over anhydrous Na2SO4 to remove water, rotary evaporate the organic phase under reduced pressure to obtain a yellow crude product. Obtain the white solid compounds 1-15 by trituration with methanol.

[0097] General preparation method for Examples 16-24:

[0098] The key intermediate 8a or 8b (100 mg, 1.0 equiv), different amines (1.2 equiv) and potassium carbonate (2.5 equiv) were placed in a 25 mL eggplant-shaped flask, and DMF (5 mL) dried with molecular sieve was added. The mixture was stirred at 55 °C in an oil bath. After reacting for 3 h, the reaction was monitored by TLC and was complete without remaining raw materials. It was extracted 3 times with ethyl acetate, the organic phases were combined, washed 3 times with saturated NaCl aqueous solution to remove excess DMF, dried over anhydrous Na2SO4, and the organic phase was evaporated under reduced pressure to obtain a solid crude product. The light yellow solid compounds 16 - 24 were obtained by separation on a TLC silica gel plate and trituration with methyl tert-butyl ether.

[0099] According to the general preparation method of the examples, the compounds of Examples 1 - 24 were prepared respectively, as shown in Table 1.

[0100] Table 1:

[0101]

[0102]

[0103]

[0104]

[0105]

[0106] In vitro antitumor cell proliferation inhibitory activities of the compounds 1 - 24 in Example 25

[0107] Inhibitory experiments on the proliferation activities of compounds 1 - 24 against human lymphoma cells Daudi, human non-small cell lung cancer cells A549, human non-small cell lung cancer cells H1975, and human chronic myeloid leukemia cells K562 were carried out in vitro.

[0108] 1) Seeding operation: Tumor cells in the logarithmic growth phase were selected, the supernatant was discarded after centrifugation, and the cells were resuspended with 1 mL of medium. 100 μL of the cell suspension was taken, diluted 10-fold with 900 μL of medium, and accurately counted using a cell counting plate. Subsequently, 100 μL of the cell suspension was inoculated into each well of a 96-well plate so that the cell density in each well was 1×10 4 cells. After inoculation, the 96-well plate was placed in an incubator and cultured for 24 h under standard conditions.

[0109] 2) Weigh the test compound accurately. First, dissolve the sample with 100 μL of DMSO, then add 50 μL of Tween 80 to assist dissolution, and finally make up the volume to 2 mL with RPMI-1640 medium. After thorough mixing, a clear and transparent 1000 μmol / L mother liquor was obtained.

[0110] 3) Drug treatment: The stock solution was diluted by 10-fold serial dilution to prepare working solutions at five concentrations of 100, 10, 1, 0.1, and 0.01 μmol / L in sequence. 100 μL of the working solution was added to each well of a 96-well plate and mixed with the medium in the well at a ratio of 1:1 to obtain five final concentrations of 50, 5, 0.5, 0.05, and 0.005 μmol / L. After adding the drug, the cells were cultured for another 48 h for subsequent detection.

[0111] 4) MTT assay: Under light-proof conditions, 20 μL of 0.5% MTT solution was added to each well and incubated at 37 °C for 4 h. After centrifugation at 1500 rpm for 5 min, the supernatant was discarded. 150 μL of DMSO was added to each well and shaken for 5 min until the formazan crystals were completely dissolved. The OD value of each well was measured at a wavelength of 490 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and the cell inhibition rate was calculated and the IC 50 value was determined.

[0112] The results of the inhibitory activities of the compounds against the proliferation of human lymphoma cell line Daudi, human non-small cell lung cancer cell line A549, human non-small cell lung cancer cell line H1975, and human chronic myeloid leukemia cell line K562 are shown in Table 2. The IC 50 ≤1 μM is denoted as A, 10 μM ≥ IC 50 >1 μM is denoted as B, and IC 50 >10 μM is denoted as C.

[0113] Table 2 Results of the in vitro anti-tumor cell activities of the compounds

[0114]

[0115]

[0116] It can be clearly seen from Table 2 that the aminopyrimidine compounds containing a toluene structure among Compounds 1-24 have good inhibitory activities against human lymphoma cell line Daudi, human non-small cell lung cancer cell line A549, human non-small cell lung cancer cell line H1975, and human chronic myeloid leukemia cell line K562 in vitro. This type of compound has good prospects for development and application as anti-tumor drugs.

[0117] Example 26 Apoptosis-inducing activity of Compound 4 on tumor cells

[0118] Hoechst staining is a type of fluorescence staining technique and belongs to the family of lipophilic fluorescent dyes. They have the property of being able to specifically interact with the base pairs in DNA molecules. During the staining process, Hoechst dyes penetrate into the cells and bind to the base pairs (usually adenine-thymine base pairs) in the DNA double helix structure, forming a stable fluorescent complex. This binding enables DNA to emit strong fluorescent signals under ultraviolet light of a specific wavelength, thus being clearly observable under a fluorescence microscope. Compound 4 was selected for the Hoechst staining experiment to observe its effect on inducing apoptosis of tumor cells at different concentrations.

[0119] Cells in the logarithmic growth phase were seeded in 6-well plates at a cell density of 2×10 5 cells / well. The next day, compound 4 (DMSO < 0.1%) at the specified concentrations (0.1 μM, 0.3 μM, 1 μM) prepared with the culture medium was added, and the control group was added with the culture medium containing 0.1% DMSO, and then continued to be cultured in an incubator for 24 h.

[0120] The culture medium was discarded, and 1.5 mL of 4% paraformaldehyde was added to each well for fixation. The cells were washed with PBS, and 0.5 ml / well of Hoechst 33258 staining solution was added and stained for 15 minutes. The cells were washed with PBS twice to remove the residual staining solution. Observation and photography were carried out under an inverted fluorescence microscope.

[0121] Through the observation of the results of the Hoechst staining experiment, as Figure 1 shown, the H1975 cell line in the blank control group showed blue color and the cell morphology was full and intact after staining. When compound 4 acted on the H1975 cell line, obvious morphological changes occurred in the cell morphology of the drug-administered group along with the increase in the drug administration concentration. The number of cells decreased significantly, the number of apoptotic cells gradually increased, and the blue brightness of the remaining cells increased, showing a concentration-dependent trend.

[0122] Example 27 Inhibitory Activity of Compound 4 on Tumor Cell Migration in Vitro

[0123] The scratch assay is a simple, highly reproducible, and economical in vitro cell biology experiment method, aiming to study cell migration, adhesion, proliferation, and responses to external stimuli. This experiment creates an artificial blank area (i.e., "scratch") on the surface of a cell culture dish to simulate the healing process after in vivo cell injury, so as to observe and analyze the ability of cells to migrate and fill the scratch. The faster the cell migration speed in the scratched area, the stronger the migration ability of the cells; at the same time, the degree of scratch healing can also be used as an indicator to evaluate cell proliferation ability. This experiment is mainly used to study the effects of different drugs or compounds on cell migration and proliferation, providing important basis for drug screening and disease treatment. This experiment aims to explore the anti-migration effect of representative compound 4 on the H1975 cell line, and the experimental operations are as follows:

[0124] (1) Cell seeding: Seed approximately 5×10 5 cells in a well plate, and then culture them for 24 hours in a suitable environment to promote cell attachment and growth.

[0125] (2) Cell scratching: After culturing the cells for 24 hours, take out the six-well plate, remove the cover, and gently and evenly create a scratch on the cell monolayer using a sterile pipette tip (1.0 mL) after aligning the pipette tip with the scale line in the well plate.

[0126] (3) Cell washing and observation: Wash the floating cells with phosphate buffer and repeat the rinsing three times to ensure effective removal. Subsequently, use an optical microscope to observe and record the cell status after scratch treatment, and save the cell morphology and distribution information

[0127] (4) Cell treatment and migration observation: Select compound 4 at concentrations of 0.1 μM and 1.0 μM to treat the cells. At 0 hours, 24 hours, 48 hours, and 72 hours, use a microscope to capture the healing images of the scratched area.

[0128] The experimental results are as Figure 2 shown, and compound 4 can inhibit the migration of H1975 cells in a concentration-dependent manner.

[0129] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various equivalent transformations can be made to the technical solutions of the present invention. To avoid unnecessary repetition, the present invention will not separately describe various possible combinations. Any modification, equivalent replacement, or improvement made within the technical concept of the present invention is included within the protection scope of the present invention.

Claims

1. The aminopyrimidine compound containing toluene structure is characterized in that: Having a structure as shown in general formula (I), in, Ar is selected from 6-10 membered aryl or 5-10 membered heteroaryl, wherein the heteroaryl contains 1-3 heteroatoms selected from N, O or S, and Ar is optionally substituted by 1-5 identical or different R1; R1 is selected from hydrogen, hydroxy, halogen, amino, cyano, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, C1-C6 alkylthio, C1-C6 alkyl substituted by hydroxy or amino or halogenated, C1-C6 alkoxy substituted by hydroxy or amino or halogenated, amino substituted by mono- or di-C1-C6 alkyl, C1-C6 alkylamido, ester, free or salified or esterified or amidated carboxyl, C1-C6 alkylsulfinyl, C1-C6 alkylsulfonyl, C1-C6 alkylacyl, carbamoyl; L is selected from R is selected from 6-10 membered aryl or 5-10 membered heteroaryl or 4-8 membered aliphatic ring or 4-8 membered aliphatic heterocyclic ring, wherein the heteroaryl and aliphatic heterocyclic ring contain 1-3 heteroatoms or groups selected from N, O, S, SO, SO2, and R is optionally substituted by 1-5 identical or different R2; R2 is selected from hydrogen, hydroxy, halogen, amino, cyano, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, C1-C6 alkylthio, C1-C6 alkyl substituted by hydroxy or amino or halogenated, C1-C6 alkoxy substituted by hydroxy or amino or halogenated, amino substituted by mono- or di-C1-C6 alkyl, C1-C6 alkylamido, ester, free or salified or esterified or amidated carboxyl, C1-C6 alkylsulfinyl, C1-C6 alkylsulfonyl, C1-C6 alkylacyl, carbamoyl.

2. The aminopyrimidine compound containing a toluene structure according to claim 1, characterized in that: Ar is selected from benzene or a 5-6 membered heteroaryl group, wherein the heteroaryl group contains 1-3 heteroatoms selected from N, O or S, and Ar is optionally substituted by 1-3 identical or different R1; R1 is selected from hydrogen, hydroxy, halogen, amino, C1-C5 alkyl, C1-C5 alkoxy; L is selected from R is selected from a benzene ring or a 5-6 membered heteroaryl or a 4-6 membered aliphatic heterocycle, wherein the heteroaryl and aliphatic heterocycle contain 1-3 heteroatoms or groups selected from N, O, S, SO2, and R is optionally substituted by 1-3 identical or different R2; R2 is selected from hydrogen, hydroxy, halogen, amino, C1-C5 alkyl, C1-C5 alkoxy, C1-C5 alkyl acyl, carbamoyl.

3. The aminopyrimidine compound containing a toluene structure according to claim 2, characterized in that: Ar is selected from 5-6 membered heteroaryl, wherein the heteroaryl contains 1-2 N atoms, and Ar is optionally substituted by 1-3 identical or different R1; R1 is selected from hydrogen, methyl, ethyl, methoxy, L is selected from R is selected from a benzene ring or a 4-6 membered aliphatic heterocycle, wherein the aliphatic heterocycle contains 1-2 heteroatoms or groups selected from N and SO2, and R is optionally substituted by 1-3 identical or different R2; R2 is selected from hydrogen, chlorine, fluorine, methyl, methoxy, isopropoxy, formyl.

4. The aminopyrimidine compound containing a toluene structure according to claim 3, characterized in that: It has the following structural formula:

5. A pharmaceutical composition, characterized in that The invention is prepared by combining the aminopyrimidine compound containing a toluene structure and a pharmaceutically acceptable salt thereof as claimed in any one of claims 1 to 4 as an active ingredient with a pharmaceutically acceptable carrier.

6. A pharmaceutical composition according to claim 5, characterized in that: The pharmaceutically acceptable carrier is selected from one or more of a filler, a disintegrant, a binder and a lubricant.

7. A pharmaceutical composition according to claim 5, characterized in that: The pharmaceutical composition is prepared into the dosage form of tablets, capsules, granules, sprays or injections.

8. Use of the aminopyrimidine compound containing a toluene structure according to any one of claims 1 to 4 or the pharmaceutical composition according to any one of claims 5 to 7 in the preparation of a drug for treating and / or preventing a proliferative disease.

9. Use of the aminopyrimidine compound containing a toluene structure according to any one of claims 1 to 4 or the pharmaceutical composition according to any one of claims 5 to 7 in the preparation of a drug for treating and / or preventing cancer.

10. The use according to claim 9, characterized in that: The tumors are: lymphoma, lung cancer, leukemia.

Images