Aminopyrimidine compounds containing a toluene structure, and methods of making and using the same

Abstract

The present application relates to toluene-containing aminopyrimidine compounds, and a preparation method and application thereof, the compounds have a structural formula shown in general formula (I). The pharmacological activity results of the toluene-containing aminopyrimidine compounds of the present application show that excellent proliferation inhibition activity is shown in 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. The present application also provides a preparation method of the compounds, and a pharmaceutical composition and use containing the compounds. In particular, the compounds are used for preparing medicines for treating and / or preventing lymphoma, lung cancer and leukemia, and have a good application prospect in the development of anti-tumor medicines.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of compound synthesis, and particularly relates to an amino pyrimidine compound containing a toluene structure and a preparation method and application thereof. BACKGROUND

[0002] Cancer, a disease that refers to all malignant tumors, has long been a major threat to human health. According to statistics of the World Health Organization (WHO), the number of people who died of cancer worldwide in 2020 reached 10 million, and more heartbreakingly, about 400,000 children were diagnosed with cancer every year. Data shows that about one in five people may suffer from 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 new cancer cases worldwide will reach 21.4 million, and the number of deaths will exceed 13 million, among which lymphoma, lung cancer and leukemia are the types of malignant tumors with high incidence, poor prognosis and serious threat to human health. Therefore, the research of anti-tumor drugs, especially the highly efficient and low-toxicity molecular targeted anti-tumor drugs, has extremely important significance.

[0003] Lymphoma is a group of malignant tumors originating from the lymphoid hematopoietic system, mainly including Hodgkin's lymphoma and non-Hodgkin's lymphoma. In recent years, the incidence of lymphoma has shown an upward trend, and it is highly heterogeneous, with significant differences in treatment options and prognosis of different subtypes. Although the application of targeted therapy and immunotherapy has improved the survival period of some patients, the treatment of refractory lymphoma is still a great challenge for clinicians.

[0004] Lung cancer is the leading cause of cancer-related deaths worldwide, among which non-small cell lung cancer (NSCLC) accounts for the majority. Although early diagnosis and precise treatment have improved the survival rate of some patients, the prognosis of patients with advanced NSCLC is still poor. Targeted therapy and immunotherapy have brought new hope for 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, which can be divided into acute leukemia and chronic leukemia according to the course 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 application relates to amino pyrimidine compounds containing toluene structure and pharmaceutically acceptable salts thereof, their preparation methods and pharmaceutical compositions containing the compounds, which can be used for the preparation of treatment and / or prevention of lymphoma, lung cancer, leukemia, etc., and have good anti-tumor drug development and application prospect. SUMMARY

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

[0008] The present application provides an aminopyrimidine compound containing toluene structure with general formula (I),

[0009]

[0010] Among them,

[0011] 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 with 1-5 same 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 with hydroxyl or amino or halogen, C1-C6 alkoxy substituted with hydroxyl or amino or halogen, amino substituted with mono or di C1-C6 alkyl, C1-C6 alkyl amido, ester group, free or salt or esterified or amidated carboxyl, C1-C6 alkyl sulfinyl, C1-C6 alkyl sulfonyl, C1-C6 alkyl acyl, carbamoyl.

[0013] L is selected from

[0014] R is selected from 6-10 membered aryl or 5-10 membered heteroaryl or 4-8 membered aliphatic ring or 4-8 membered aliphatic heterocycle. Among them, the heteroaryl and aliphatic heterocycle contain 1-3 heteroatoms or groups selected from N, O, S, SO, SO2, and R is optionally substituted with 1-5 same 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 with hydroxyl or amino or halogen, C1-C6 alkoxy substituted with hydroxyl or amino or halogen, amino substituted with mono or di C1-C6 alkyl, C1-C6 alkyl amido, ester group, free or salt or esterified or amidated carboxyl, C1-C6 alkyl sulfinyl, C1-C6 alkyl sulfonyl, C1-C6 alkyl acyl, carbamoyl.

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

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

[0018] R1is selected from hydrogen, hydroxyl, halogen, amino, C1-C5alkyl, C1-C5alkoxyl.

[0019] L is selected from

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

[0021] R2is selected from hydrogen, hydroxyl, halogen, amino, C1-C5alkyl, C1-C5alkoxyl, C1-C5alkylacyl, carbamoyl.

[0022] Further, the above-mentioned aminopyrimidine compound containing toluene structure,

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

[0024] R1is selected from hydrogen, methyl, ethyl, methoxyl.

[0025] L is selected from

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

[0027] R2is selected from hydrogen, chloro, fluoro, methyl, methoxyl, isopropoxyl, formyl.

[0028] Further, the above-mentioned aminopyrimidine compound containing toluene structure and pharmaceutically acceptable salts thereof, have the following structural formula:

[0029]

[0030]

[0031]

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

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

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

[0035] The use of any of the above-described aminopyrimidine compounds containing a toluene structure, their pharmaceutically acceptable salts, or any of the above-described pharmaceutical compositions in the preparation of medicaments for the treatment and / or prevention of proliferative diseases.

[0036] Use in the preparation of medicaments for treating and / or preventing cancer by any of the above-described aminopyrimidine compounds containing a toluene structure, their pharmaceutically acceptable salts, or any of the above-described pharmaceutical compositions.

[0037] The use of any of the above-described aminopyrimidine compounds containing a toluene structure and their pharmaceutically acceptable salts, or any of the above-described pharmaceutical compositions, in the preparation of remedies for the treatment and / or prevention of lymphoma, lung cancer, and leukemia.

[0038] According to some common methods in the field to which this invention pertains, the aminopyrimidine compounds containing a toluene structure represented by general formula (I) of this invention can react with acids to form pharmaceutically acceptable salts. Pharmaceutically acceptable addition salts include inorganic acid and organic acid addition salts, with salts that react with the following acids being 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, and benzoic acid.

[0039] Furthermore, 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 of general formula (I) containing a toluene structure. They may have weak or no activity on their own, but after administration, they are converted into the corresponding biologically active form under physiological conditions (e.g., through metabolism, solvation, or other means).

[0040] The beneficial effects of this invention are:

[0041] The aminopyrimidine compounds containing a toluene structure and their pharmaceutically acceptable salts obtained in this invention possess excellent antitumor activity and safety. In vitro inhibition assays of 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) demonstrate that the compounds of this invention have significant inhibitory effects on human lymphoma cells, human lung cancer cells, and human leukemia cells. The compounds described in this invention are chemical pharmaceutical raw materials, particularly for the preparation of drugs for the treatment and / or prevention of lymphoma, lung cancer, and leukemia. Attached Figure Description

[0042] Figure 1 The results are Hurst staining experiments of compound 4 on H1975, where (a) is the blank control group, (b) is compound 4 at a concentration of 1.0 μM, and (c) is compound 4 at a concentration of 10 μM.

[0043] Figure 2 This is an experiment on the migration inhibition of H1975 by compound 4. Detailed Implementation

[0044] The examples and preparation methods provided below further illustrate and demonstrate 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 compound containing the toluene structure of general formula (I) of this invention.

[0046] All raw materials were prepared by methods well known to those skilled in the art of organic chemistry, or were commercially available, as described in the synthetic routes below. All final compounds of this invention were prepared by methods described in the synthetic routes below or by similar methods well known to those skilled in the art of organic chemistry. All variable factors used in the synthetic routes below are as defined below or as defined in the claims.

[0047] The examples are intended to illustrate, and not limit, the scope of the invention. The proton NMR spectra of the compounds were determined using a Bruker ARX-400 or ARX-600, and the mass spectrometry was performed using an Agilent 1100 LC / MSD; all reagents used were analytical grade or chemically pure.

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

[0049] 1) As shown in Route 1, 2-methyl-4-bromoaniline, di-tert-butyl dicarbonate as raw material, substitution reaction in toluene to obtain intermediate 2a; under the condition of nitrogen protection, intermediate 2a and pinacol diboron in 1,4-dioxane, coupling reaction in the presence of Pd(dppf)Cl2 and KOAc to obtain intermediate 3a; under the condition of nitrogen protection, intermediate 3a and 2,4-dichloropyrimidine in 1,4-dioxane / water (4:1), coupling reaction in the presence 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 in 1,4-dioxane, substitution reaction in the presence of Pd2(dba)3, S-Phos and Cs2CO3 to obtain intermediate 5a-b; intermediate 5a-b is removed from the Boc group in a solution of trifluoroacetic acid and dichloromethane to obtain 6a-b; acylation reaction of intermediate 6a-b and 1,1-cyclopropanedicarboxylic acid in chlorosulfoxide and tetrahydrofuran to obtain key intermediate 7a-b; acylation reaction of key intermediate 7a-b and different substituted aromatic aniline compounds in DMF by the action of HATU and TEA to obtain compounds 1-15.

[0050]

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

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

[0053]

[0054] Wherein, (i) K2CO3, THF, rt, 2h; (ii) K2CO3, DMF, 55℃, 3h.

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

[0056] Ar is selected from a 6-10 membered aryl group or a 5-10 membered heteroaryl group, wherein the heteroaryl group contains 1-3 heteroatoms selected from N, O or S, and Ar is optionally substituted with 1-5 R1groups which are the same or different;

[0057] R1is selected from hydrogen, hydroxy, halogen, amino, cyano, C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C1-C6alkoxy, C1-C6alkylsulfanyl, C1-C6alkyl substituted with hydroxy or amino or halogen, C1-C6alkoxy substituted with hydroxy or amino or halogen, amino substituted with mono or di C1-C6alkyl, C1-C6alkylamido, ester, free or salified or esterified or amidated carboxy, C1-C6alkylsulfinyl, C1-C6alkylsulfonyl, C1-C6alkylacyl, carbamoyl;

[0058] L is selected from

[0059] R is selected from a 6-10 membered aryl group or a 5-10 membered heteroaryl group or a 4-8 membered aliphatic ring or a 4-8 membered aliphatic heterocyclic ring, wherein the heteroaryl and aliphatic heterocyclic ring contains 1-3 heteroatoms or groups selected from N, O, S, SO, SO2, and R is optionally substituted with 1-5 R2groups which are the same or different;

[0060] R2is selected from hydrogen, hydroxy, halogen, amino, cyano, C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C1-C6alkoxy, C1-C6alkylsulfanyl, C1-C6alkyl substituted with hydroxy or amino or halogen, C1-C6alkoxy substituted with hydroxy or amino or halogen, amino substituted with mono or di C1-C6alkyl, C1-C6alkylamido, ester, free or salified or esterified or amidated carboxy, C1-C6alkylsulfinyl, C1-C6alkylsulfonyl, C1-C6alkylacyl, carbamoyl.

[0061] General procedure for the preparation:

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

[0063]

[0064] To a 100 mL round bottom flask, was added 4-bromo-2-methylaniline (5.00 g), toluene (20 mL), and the flask was placed in an oil bath at 110 °C and stirred. Di-tert-butyl dicarbonate (6.45 g) was dissolved in toluene (5 mL) and added dropwise. The reaction was monitored by TLC after about 2 h and was complete with no starting material remaining. The reaction was extracted with ethyl acetate (3x), the organic layers were combined and washed with saturated NaCl solution (1x), dried over anhydrous Na2SO4, and the organic phase was removed by rotary evaporation under reduced pressure to give 7.57 g of 2a as a light yellow solid.

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

[0066]

[0067] To a 250 mL three necked flask was added intermediate 2a (5.00 g), bis-3,3- dimethyl-2-butanone dioxaborinane (6.66 g), and potassium acetate (5.15 g), 1,4- dioxane (30 mL), and the flask was stirred at room temperature while bubbling N2for 0.5 h. Palladium tetrakis(triphenylphosphine) (1.22 g) was added and the flask was evacuated and backfilled with N2. The flask was placed in an oil bath at 100 °C and stirred. The reaction was monitored by TLC after about 3 h and was complete with no starting material remaining. Celite was added and the mixture was hot filtered and the filter cake was rinsed with dioxane until no product was present. The filtrate was removed by rotary evaporation under reduced pressure and the product was extracted with ethyl acetate (3x), the organic layers were combined and washed with saturated NaCl solution (1x), dried over anhydrous Na2SO4, and the organic phase was removed by rotary evaporation under reduced pressure to give 5.52 g of intermediate 3a as a light yellow solid.

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

[0069]

[0070] Intermediate 3a (5.00 g), 2,4-dichloropyrimidine (2.68 g) and cesium carbonate (14.67 g) were taken in a 250 mL three necked flask, added 1,4-dioxane (30 mL), stirred at room temperature for 0.5 h under N2bubbling, added bis(triphenylphosphine)palladium dichloride (1.05 g), evacuated air and flushed with nitrogen for protection, placed in an oil bath at 90 °C and stirred for reaction. The reaction was monitored by TLC after about 3 h and the reaction was complete as there was no starting material left. Filtered hot by adding celite and washed the filter cake with dioxane until there was no product left. The filtrate was evaporated under reduced pressure, extracted with ethyl acetate three times, combined the organic layer, washed with saturated NaCl aqueous solution once, dried over anhydrous Na2S04, evaporated the organic layer under reduced pressure and the crude product was obtained as a black colored oil. The product was purified by column chromatography on silica gel to obtain 2.53 g of the product intermediate 4a as a light yellow colored solid.

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

[0072]

[0073] Intermediate 4a (3.00 g), 1-methyl-1H-pyrazol-3-amine (1.18 g) and cesium carbonate (6.11 g) were taken in a 100 mL three necked flask, added 1,4-dioxane (30 mL), stirred at room temperature for 0.5 h under N2bubbling, added tris(dibenzylideneacetone)dipalladium(0) (0.86 g) and 2-dicyclohexylphosphino-2',6'-dimehtoxybiphenyl (0.77 g), evacuated air and flushed with nitrogen for protection, placed in an oil bath at 90 °C and stirred for reaction. The reaction was monitored by TLC after about 3 h and the reaction was complete as there was no starting material left. Filtered hot by adding celite and washed the filter cake with dioxane until there was no product left. The filtrate was evaporated under reduced pressure, extracted with ethyl acetate three times, combined the organic layer, washed with saturated NaCl aqueous solution once, dried over anhydrous Na2S04, evaporated the organic layer under reduced pressure and the crude product was obtained as a black colored oil. The product was purified by column chromatography on silica gel to obtain the product intermediate 5a as a light yellow colored solid, 2.12 g, 59.6 % yield.

[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 taken in a 100 mL three necked flask, 1,4-dioxane (30 mL) was added, stirred at room temperature under N2bubbling for 0.5 h, tris(dibenzylideneacetone)dipalladium(0) (0.86 g) and 2-dicyclohexylphosphino-2',6'-dimehtoxybiphenyl (0.77 g) were added, evacuated air and filled with nitrogen, placed in an oil bath at 90 °C and stirred. The reaction was monitored by TLC after about 3 h, no starting material was left. Celite was added and filtered hot, the filter cake was washed with dioxane until no product was left. The filtrate was concentrated under reduced pressure, extracted with ethyl acetate three times, the organic phase was combined, washed with saturated NaCl solution once, dried over anhydrous Na2SO4, evaporated under reduced pressure to get the crude product as black oil, which was purified by silica gel column chromatography to get 2.08 g of the product intermediate 5b as light yellow solid.

[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 taken in a 100 mL flask, dichloromethane (10 mL) was added, trifluoroacetic acid (2.98 g) was added dropwise, stirred at room temperature. The reaction was monitored by TLC after about 1 h, no starting material was left. Excess dichloromethane and trifluoroacetic acid were removed by rotary evaporation under reduced pressure, saturated Na2CO3 solution was added at room temperature until pH = 9-10, light yellow solid was precipitated, filtered through a Buchner funnel and washed with water three times, the filter cake was transferred to a vacuum oven at 50 °C and dried for 24 h to get 1.31 g of the product 6a as light yellow solid.

[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 taken in a 100 mL flask, dichloromethane (10 mL) was added, trifluoroacetic acid (2.98 g) was added dropwise, stirred at room temperature. The reaction was monitored by TLC after about 1 h, no starting material was left. Excess dichloromethane and trifluoroacetic acid were removed by rotary evaporation under reduced pressure, saturated Na2CO3 solution was added at room temperature until pH = 9-10, extracted with ethyl acetate three times, the organic phase was combined, washed with saturated NaCl solution once, dried over anhydrous Na2SO4, evaporated under reduced pressure to get the crude product as yellow solid, which was slurried in petroleum ether for 5 h and filtered to get 1.20 g of the product 6b as light yellow solid.

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

[0084]

[0085] Into a 100 mL flask, 1,1-cyclopropanedicarboxylic acid (0.88 g) was placed, tetrahydrofuran (20 mL) was added, stirred until completely dissolved, placed in an ice bath to cool to -5 °C, triethylamine (0.73 g) was added dropwise, the temperature was controlled not to exceed -5 °C throughout the process, after the completion of the dropwise addition, the reaction was continued to stir for 30 min, 6a (2.00 g) dissolved in tetrahydrofuran (5 mL) was added dropwise, the temperature was controlled not to exceed 0 °C throughout the process, after the completion of the dropwise addition, the reaction was continued to stir for 2 h at -5 °C. TLC monitoring reaction was complete, no raw material left. The reaction solution was slowly poured into sodium hydroxide solution (2 mol / L, 20 mL), stirred for 30 min. The resulting solution was extracted with dichloromethane 3 times, the aqueous phase was collected, the pH of the aqueous phase was adjusted to 1 with concentrated hydrochloric acid, extracted with dichloromethane 3 times, the organic phase was combined, washed with saturated NaCl solution once, the organic phase was rotary evaporated under reduced pressure to obtain 1.96 g of light yellow solid product 7a.

[0086] Step H 1 -((2-methyl-4-(2-((1 -methyl- 1 H-pyrazol-3 -yl)amino)pyrimidin-4-yl)phenyl)carbamoyl)cyclopropane- 1 - carboxylic acid (7a)

[0087]

[0088] Into a 100 mL flask, 1,1-cyclopropanedicarboxylic acid (0.88 g) was placed, tetrahydrofuran (20 mL) was added, stirred until completely dissolved, placed in an ice bath to cool to -5 °C, triethylamine (0.73 g) was added dropwise, the temperature was controlled not to exceed -5 °C throughout the process, after the completion of the dropwise addition, the reaction was continued to stir for 30 min, 6a (2.00 g) dissolved in tetrahydrofuran (5 mL) was added dropwise, the temperature was controlled not to exceed 0 °C throughout the process, after the completion of the dropwise addition, the reaction was continued to stir for 2 h at -5 °C. TLC monitoring reaction was complete, no raw material left. The reaction solution was slowly poured into sodium hydroxide solution (2 mol / L, 20 mL), stirred for 30 min. The resulting solution was extracted with dichloromethane 3 times, the aqueous phase was collected, the pH of the aqueous phase was adjusted to 1 with concentrated hydrochloric acid, extracted with dichloromethane 3 times, the organic phase was combined, washed with saturated NaCl solution once, the organic phase was rotary evaporated under reduced pressure to obtain 1.96 g of light yellow solid product 7a.

[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), potassium carbonate (0.74 g) in a 50 mL tomato bottle, add super dry THF (20 mL), take the chloroacetyl chloride (0.48 g) diluted with THF (2 mL) slowly drop at 0 ℃, after drop completion, transfer to room temperature, stir the reaction. After 2 h of reaction, TLC monitoring reaction is complete, no raw material is left. Extracted with DCM 3 times, combined organic phase, washed with saturated NaCl solution 1 time, dried with anhydrous Na2SO4, rotary evaporation under reduced pressure to get 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), potassium carbonate (0.74 g) in a 50 mL tomato bottle, add super dry THF (20 mL), take the chloroacetyl chloride (0.48 g) diluted with THF (2 mL) slowly drop at 0 ℃, after drop completion, transfer to room temperature, stir the reaction. After 2 h of reaction, TLC monitoring reaction is complete, no raw material is left. Extracted with DCM 3 times, combined organic phase, washed with saturated NaCl solution 1 time, dried with anhydrous Na2SO4, rotary evaporation under reduced pressure to get 1.12 g yellow solid product 8b.

[0095] General procedure for the preparation of examples 1-15:

[0096] Take the key intermediate 7a or 7b (100 mg, 1.0 eq) and different amines (1.2 eq) in a 25 mL tomato bottle, add DMF (5 mL), drop triethylamine (3.0 eq), then add HATU (2.0 eq), stir the reaction at room temperature. After 3 h of reaction, TLC monitoring reaction is complete, no raw material is left. Add saturated Na2CO3 solution to adjust pH to about 10, extract with DCM 3 times, combine the organic phase, wash with saturated NaCl solution 3 times to remove excess DMF. Dry with anhydrous Na2SO4, rotary evaporation of the organic phase under reduced pressure to get yellow crude product. By methanol slurry to get white solid compound 1-15.

[0097] General procedure for the preparation of examples 16-24:

[0098] Take the key intermediate 8a or 8b (100 mg, 1.0 equivalent), different amine (1.2 equivalent) and potassium carbonate (2.5 equivalent) in 25 mL of ajar, add dry DMF (5 mL) with molecular sieve, and place in an oil bath pot at 55°C and stir the reaction. After 3 h of reaction, TLC monitoring shows that the reaction is complete, and there is no starting material left. Extract with ethyl acetate 3 times, wash the combined organic phase with saturated NaCl aqueous solution 3 times to remove excess DMF, dry with anhydrous Na2SO4, evaporate the organic phase under reduced pressure, and obtain the solid crude product. Separate by thin layer chromatography on silica gel plate and wash with methyl tert-butyl ether to obtain light yellow solid compound 16-24.

[0099] According to the preparation method, 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 tumor cell proliferation inhibition activity of Example 25 compound 1-24

[0107] In vitro proliferation activity inhibition experiments of compounds 1-24 on 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.

[0108] 1) Plating operation: select tumor cells in the logarithmic growth phase, centrifuge and discard the supernatant, add 1 mL of medium to resuspend the cells. Take 100 μL of cell suspension, dilute 10 times with 900 μL of medium, and use a cell counting plate to accurately count. Then, inoculate 100 μL of cell suspension in each well of a 96-well plate, so that the cell density in each well is 1×10 4 After inoculation, place the 96-well plate in an incubator and culture under standard conditions for 24 h.

[0109] 2) Accurately weigh the test compound. First dissolve the sample with 100 μL of DMSO, then add 50 μL of Tween 80 to aid dissolution, and finally dilute to 2 mL with RPMI-1640 medium. After thorough mixing, a clear and transparent 1000 μmol / L stock solution is obtained.

[0110] 3) Drug treatment: The mother liquor was diluted by 10 times gradient, and working solutions of 100, 10, 1, 0.1, 0.01 μmol / L were prepared in turn. 100 μL working solution was added to each well of a 96-well plate, and after mixing with the medium in the well at a ratio of 1:1, five final concentrations of 50, 5, 0.5, 0.05, 0.005 μmol / L were obtained. After drug addition, the cells were incubated for 48 h for subsequent detection.

[0111] 4) MTT detection: 20 μL of 0.5% MTT solution was added to each well under light-proof conditions, and the cells were 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 the mixture was shaken for 5 min until the formazan crystals were completely dissolved. The OD value of each well was determined using a microplate reader at a wavelength of 490 nm, the cell inhibition rate was calculated, and the IC 50 value was determined.

[0112] The results of the proliferation inhibition activity of the compounds 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 are shown in Table 2. In Table 2, IC 50 ≤ 1 μM is represented by A, 10 μM ≥ IC 50 > 1 μM is represented by B, and IC 50 > 10 μM is represented by C.

[0113] Table 2: Results of in vitro anti-tumor cell activity of compounds

[0114]

[0115]

[0116] As can be clearly seen from Table 2, the compounds 1-24, which are toluene-structure-containing aminopyrimidine compounds, have good inhibition activity 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 in vitro. Such compounds have good prospects for development and application as anti-tumor drugs.

[0117] Example 26: In vitro apoptosis induction activity of compound 4

[0118] Hoechst staining is a type of fluorescent staining technique, belonging to the lipophilic fluorescent dye family, which has the property of specific interaction with base pairs in DNA molecules. During the staining process, Hoechst dye penetrates into cells and binds to base pairs (usually adenine-thymine base pairs) in the DNA double helix structure to form stable fluorescent complexes. This binding allows DNA to emit strong fluorescent signals under the irradiation of ultraviolet light of a specific wavelength, so that it can be clearly observed under a fluorescence microscope. Compound 4 was selected for Hoechst staining experiments to observe its effect on tumor cell apoptosis at different concentrations.

[0119] Cells in the logarithmic growth phase were inoculated in a 6-well plate with a cell density of 2x10 5 On the next day, compound 4 (DMSO <0.1%) at the specified concentration (0.1 μM, 0.3 μM, 1 μM) prepared in culture medium was added, and the control group was added with culture medium containing 0.1% DMSO, and then placed in an incubator for continuous culture 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 of Hoechst 33258 staining solution was added to each well for staining for 15 minutes. The cells were washed with PBS twice to remove residual staining solution. Then, the cells were observed and photographed under an inverted fluorescence microscope.

[0121] Through observation of the results of the Hoechst staining experiment, as shown in Figure 1 The H1975 cell line in the blank control group showed blue color and full and complete cell morphology after staining. When compound 4 was used on the H1975 cell line, the cell morphology of the dosing group changed significantly with the increase of the dosing concentration, the number of cells decreased significantly, the number of apoptotic cells increased gradually, and the remaining cells showed increased blue brightness, showing a concentration-dependent trend.

[0122] Example 27 In vitro inhibitory activity of compound 4 on tumor cell migration

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

[0124] (1) Cell inoculation: inoculate about 5x10 5 cells in the hole plate, and then culture for 24 hours in a suitable environment to promote cell adhesion and growth.

[0125] (2) Cell scratch: after 24 hours of cell culture, take out the six-hole plate, uncover the cover plate, and ensure that the pipette tip (1.0 mL) is aligned with the scale line in the hole plate before creating a scratch on the cell monolayer with a sterile pipette tip.

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

[0127] (4) Cell treatment and migration observation: select 0.1 μM and 1.0 μM concentrations of compound 4 for cell treatment. At 0 hours, 24 hours, 48 hours and 72 hours, use a microscope to capture the healing images of the scratch area.

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

[0129] The above detailed the preferred embodiments of the present application, but the present application is not limited to the specific details in the above embodiments, and various equivalent transformations can be made to the technical solutions of the present application within the technical concept of the present application. In order to avoid unnecessary repetition, the present application does not further describe various possible combination modes. Any modification, equivalent replacement or improvement made within the technical concept of the present application is included in the protection scope of the present application.

Claims

1. Aminopyrimidines containing a toluene structure, characterized in that, having the following structural formula: 。 2. A pharmaceutical composition, characterized by comprising: The toluene-containing aminopyrimidine compound and pharmaceutically acceptable salt thereof according to claim 1 are combined with a pharmaceutically acceptable carrier to form a pharmaceutical composition.

3. A pharmaceutical composition according to claim 2, wherein The pharmaceutically acceptable carrier is selected from one or more of a filler, a disintegrant, a binder, and a lubricant.

4. A pharmaceutical composition according to claim 2, wherein The pharmaceutical composition is prepared in the form of a tablet, a capsule, a granule, a spray, or an injection.

5. Use of the toluene-containing aminopyrimidine compound according to claim 1 or the pharmaceutical composition according to any one of claims 2 to 4 in the manufacture of a medicament for the treatment and / or prevention of cancer, wherein the cancer is lymphoma, lung cancer, leukemia.

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