A substituted aniline pyrimidine compound, its preparation method and uses

Abstract

This invention relates to a novel substituted phenylamine pyrimidine compound that exhibits strong inhibitory activity against both Mer and c-Met kinases, as well as specificity and selectivity against tumor cells and normal cells. It also has low potential cardiotoxicity and minimal side effects. The invention further discloses the application of this compound as an antitumor drug.

Description

Technical Field

[0001] This invention relates to a substituted aniline pyrimidine compound, its preparation method, and its uses. Background Technology

[0002] Mer belongs to the TAM family of receptor tyrosine kinases, with Gas6 as its ligand. When Gas6 binds to Mer, it induces phosphorylation of the Mer receptor tyrosine kinase, activating downstream signaling pathways. Phosphatidylserine on the cell membrane surface also enhances Gas6-mediated activation, inducing cell proliferation, apoptosis, invasion, and metastasis. Abnormal activation of Mer is associated with the development and progression of many human cancers and is frequently linked to poor prognosis, such as non-small cell lung cancer, gastric cancer, and liver cancer. Therefore, ongoing research into the Gas / Mer signaling pathway provides new ideas and directions for cancer treatment. Currently, nearly 20 small-molecule Mer inhibitors have been reported, such as UNC569, UNC1062, UNC2250, and UNC2025, which are pyrrolopyrimidine inhibitors. Mer inhibitor 9, obtained by modifying the structure of UNC1062, exhibits excellent pharmacokinetic (PK) properties.

[0003]

[0004] c-Met is located on human chromosomes 7q21-q31. Its encoded protein product is a single-chain precursor that cleaves and rearranges into α and β subunits, which are linked by disulfide bonds to form a heterodimer. The α subunit is located in the extracellular domain, while the β subunit is divided into extracellular, transmembrane, and intracellular domains. The extracellular domain includes a signaling domain responsible for binding to the SPH domain of the ligand HGF, while the intracellular domain includes a PTK region and an autophosphorylation site, exhibiting tyrosine kinase activity. Hepatocyte growth factor (HGF) is mainly composed of an N-terminal region and four keratin domains (K1-K4) on the α chain. Upon binding to HGF, the c-Met receptor dimers and autophosphorylates, thereby activating downstream signaling molecules such as Ras, PI3K, and MAPK to regulate cellular biological functions. Studies have confirmed that abnormalities in the HGF / Met complex are a driving factor in tumorigenesis; HGF stimulates cell motility and invasion through various signaling pathways, promoting tumor metastasis.

[0005] Based on their structural characteristics and binding modes with c-Met kinases, small molecule c-Met inhibitors are mainly classified into: type Ia ATP-competitive inhibitors, type Ib ATP-competitive inhibitors, and type II multi-target ATP-competitive inhibitors. Crizotinib, developed by Pfizer, is a type Ia inhibitor targeting both c-Met and ALK, primarily used to treat advanced ALK-positive NSCLC. Cabozantinib is a type II MET inhibitor with strong activity against tyrosine kinases such as MET, VEGFR2, FLT3, AXL, KIT, and RET; its indication is medullary thyroid carcinoma. AMG-337 is a type Ib inhibitor; among more than 400 different kinases, AMG-337 exhibits strong selectivity for c-Met, with an IC50 concentration of [missing information]. 50 The value is less than 5nM.

[0006]

[0007] Analysis of the amino acid sequences of the kinase-binding domains of human protein tyrosine kinases revealed that Mer and c-Met kinases exhibit similar evolutionary sequences and high homology. Furthermore, Mer and c-Met receptors activate common signaling molecules and possess similar functions. Exelixis's patent (WO2019148043A1) reports that the multi-target kinase inhibitor Cabozantinib and its analogues also possess strong inhibitory activity against the TAM family, with an IC50 of [missing information - likely referring to an IC50 value] against Mer kinase. 50 The value is less than 10 nM.

[0008] The research and development of Mer / c-Met dual-target inhibitors is at the forefront of international drug research, but the following major problems still exist: the research foundation for Mer / c-Met dual-target inhibitors is weak both domestically and internationally, especially in China where there is a lack of independently developed novel Mer / c-Met dual-target inhibitors; the structure-activity relationship of existing Mer / c-Met dual-target inhibitors is incomplete, and the structural types are limited; there is a lack of effective pharmacophore models for compound docking and predicting the inhibitory activity of compounds. Therefore, there is an urgent need to obtain novel compounds with high efficiency and low toxicity to study the mechanism of action, applicability, and inhibitory action of the target, laying the foundation for the discovery of Mer / c-Met dual-target inhibitor anti-tumor drug candidates. Summary of the Invention

[0009] The purpose of this invention is to provide a Mer / c-Met dual-target inhibitor antitumor compound with a novel structural type.

[0010] The present invention adopts the following technical solution:

[0011] A substituted phenylamine pyrimidine compound with the general formula:

[0012]

[0013] R 1 Selected from H, halogen groups, methyl, halogen-substituted alkyl, saturated heterocyclic group, substituted saturated heterocyclic group, carbamoyl group, substituted acetamido group, substituted saturated heterocyclic group;

[0014] R 2 Selected from H, halogen groups, saturated heterocyclic groups, substituted saturated heterocyclic groups, substituted acetamido groups, and alkoxyamino groups;

[0015] R 3 Selected from H, C 1-3 Alkyl groups and saturated heterocyclic groups.

[0016] Furthermore, R 1 Selected from H, F, Cl, methyl, trifluoromethyl, morpholino, piperazinyl, N-methylpiperazinyl, 2-(4-methylpiperazin-1-yl)acetamido, 2-(4-methylpiperazin-1-yl)propamido, N-methyl-2-(4-methylpiperazin-1-yl)acetamido, N-methylpiperidinoxy;

[0017] R 2 Selected from the following groups: H, F, Cl, morpholino, piperazine, N-methylpiperazine, N-ethylpiperazine, N-acetylpiperazine, 2-(4-methylpiperazin-1-yl)acetamido, 2-(4-ethylpiperazin-1-yl)acetamido, methoxyethylamine, ethoxyethylamine;

[0018] R 3 Selected from the following groups: H, methyl, ethyl, isopropyl, tetrahydropyran-4-methyl, tetrahydrofuran-2-methyl.

[0019] Preferred, R 1 Selected from H,

[0020] R 2 Selected from H,

[0021] R 3 Selected from H,

[0022] More preferably, the following specific compounds are preferred from the compounds with the structure of formula (I):

[0023] I-1: 5-(4-fluorophenyl)-N-(4-(2-(3-morpholinophenyl)amino)pyrimidine-4-oxy)phenyl)-4-oxo-1,4-dihydropyridine-3-carboxamide;

[0024] I-2: 5-(4-fluorophenyl)-N-(4-(2-(3-(2-(4-methylpiperazin-1-yl)acetamido)phenyl)amino)pyrimidin-4-oxy)phenyl)-4-oxo-1,4-dihydropyridine-3-carboxamide;

[0025] I-3: N-(4-(2-(3-(4-acetylpiperazin-1-yl)phenyl)amino)pyrimidin-4-oxy)phenyl)-5-(4-fluorophenyl)-4-oxy-1,4-dihydropyridine-3-carboxamide;

[0026] I-4: 5-(4-fluorophenyl)-N-(4-(2-(4-(2-(4-(4-(4-methylpiperazin-1-yl)acetamido)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)-4-oxo-1,4-dihydropyridine-3-carboxamide;

[0027] I-5: 5-(4-fluorophenyl)-N-(4-(2-(4-(2-(4-(4-(4-methylpiperazin-1-yl)propaneamide)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)-4-oxo-1,4-dihydropyridine-3-carboxamide;

[0028] I-6: 5-(4-fluorophenyl)-N-(4-(2-(4-(4-(N-methyl-2-(4-methyl-2-(4-methylpiperazin-1-yl)acetamido)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)-4-oxo-1,4-dihydropyridine-3-carboxamide;

[0029] I-7: 5-(4-fluorophenyl)-N-(4-(2-((3-(2-methoxyethyl)amino)phenyl)amino)pyrimidine-4-oxyphenyl)-4-oxo-1,4-dihydropyridine-3-carboxamide;

[0030] I-8: 5-(4-fluorophenyl)-N-(4-(2-(4-(4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-4-oxy)phenyl)-4-oxo-1,4-dihydropyridine-3-carboxamide;

[0031] I-9: 5-(4-fluorophenyl)-N-(4-(2-(3-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-4-oxy)phenyl)-4-oxo-1,4-dihydropyridine-3-carboxamide;

[0032] I-10: 5-(4-fluorophenyl)-N-(4-(2-(4-(2-(4-(4-(4-methylpiperazin-1-yl)acetamido)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)-4-oxo-1,4-dihydropyridine-3-carboxamide;

[0033] I-11: 5-(4-fluorophenyl)-N-(4-(2-(4-morpholinophenyl)amino)pyrimidine-4-oxy)phenyl)-4-oxo-1,4-dihydropyridine-3-carboxamide;

[0034] I-12: 5-(4-fluorophenyl)-4-oxo-N-(4-(2-(4-(trifluoromethylphenyl)phenyl)amino)pyrimidin-4-oxy)phenyl)-1,4-dihydropyridine-3-carboxamide;

[0035] I-13: 5-(4-fluorophenyl)-N-(4-(2-(4-(1-methylpiperidin-4-oxy)phenyl)amino)pyrimidin-4-oxy)phenyl)-4-oxo-1,4-dihydropyridine-3-carboxamide;

[0036] I-14: 5-(4-fluorophenyl)-N-(4-(2-(3-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-4-yl)phenyl)-4-oxo-1-(tetrahydro-2H-pyran-4-yl)methyl)-1,4-dihydropyridine-3-carboxamide;

[0037] I-15: 5-(4-fluorophenyl)-1-isopropyl-N-(4-(2-(3-(4-methylpiperazin-1-phenyl)amino)pyrimidin-4-oxy)phenyl)-4-oxo-1,4-dihydropyridine-3-carboxamide;

[0038] I-16: 5-(4-fluorophenyl)-N-(4-(2-(3-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-4-yl)phenyl)-4-oxo-1-(tetrahydrofuran-2-yl)methyl)-1,4-dihydropyridine-3-carboxamide.

[0039] The structural formulas of the above compounds are shown in Table 1.

[0040] Table 1. Preferred compound numbers and corresponding structural formulas

[0041]

[0042] A method for preparing the above-mentioned substituted aniline pyrimidine compounds, the synthetic route is as follows:

[0043]

[0044] It includes the following steps:

[0045] 1) Cycloisopropyl malonate and 4-fluorophenylacetyl chloride were dissolved in a polar aprotic solvent, and compound II was obtained by nucleophilic addition elimination, ketal hydrolysis and decarboxylation under heating after the addition of a base catalyst.

[0046] 2) Compounds II, III and N,N-dimethylformamide dimethyl acetal were subjected to acetal hydrolysis, Knoevenagel reaction and cyclization reaction to give compound IV;

[0047] 3) Compound IV undergoes alkaline hydrolysis to yield compound V;

[0048] 4) Dissolve p-nitrophenol and 2,4-dichloropyrimidine in a polar aprotic solvent, add a base catalyst, and then undergo a substitution reaction to obtain compound VI;

[0049] 5) Compounds VI and VII undergo a substitution reaction in a polar aprotic solvent in the presence of an acid catalyst to yield compound VIII;

[0050] 6) Compound VIII is reduced to give compound IX;

[0051] 7) Compound IX and Compound V undergo a condensation reaction in a polar aprotic solvent in the presence of a base catalyst to obtain target compound I.

[0052] In step 1) of the preparation method, the polar aprotic solvent is selected from dichloromethane (DCM), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), and dimethyl sulfoxide (DMSO); the base catalyst is selected from N,N-diisopropylethylamine, triethylamine, diisopropylethylamine, or pyridine. The reaction temperature is from 0°C to reflux.

[0053] In step 2) of the preparation method, the reaction solvent is selected from toluene, ethanol, methanol, etc.; the reaction temperature is 60℃ to 100℃.

[0054] In step 3) of the preparation method, the alkaline reagent is selected from sodium hydroxide, potassium hydroxide, and lithium hydroxide; the reaction solvent is selected from methanol and water; and the reaction temperature is room temperature or 100℃.

[0055] In step 4) of the preparation method, the polar aprotic solvent is selected from N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), and dimethyl sulfoxide (DMSO); the alkaline catalyst is potassium carbonate, sodium hydroxide, potassium hydroxide, or potassium tert-butoxide, etc.; the reaction temperature is 80-100℃.

[0056] In step 5) of the preparation method, the reaction solvent is selected from N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), and dimethyl sulfoxide (DMSO); the acid catalyst is p-toluenesulfonic acid (PTSA), hydrochloric acid, or trifluoroacetic acid, etc.; and the reaction temperature is 90-110℃.

[0057] In step 6) of the preparation method, the reaction solvent is anhydrous ethanol; the reducing agent is SnCl2·2H2O; and the reaction temperature is 90–110℃.

[0058] In step 7) of the preparation method, the polar aprotic solvent is N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), dimethyl sulfoxide (DMSO), etc.; the alkaline catalyst is potassium carbonate, sodium carbonate, sodium hydroxide, potassium hydroxide or potassium tert-butoxide, etc.; the reaction temperature is 80-100℃.

[0059] The application of one of the above-mentioned substituted phenylamine pyrimidine compounds in the preparation of receptor tyrosine kinase inhibitors.

[0060] Specifically, the receptor tyrosine kinase inhibitor is a Mer / c-Met dual-target inhibitor.

[0061] The application of one of the above-mentioned substituted phenylamine pyrimidine compounds in the preparation of antitumor drugs.

[0062] Furthermore, the indications for the aforementioned antitumor drugs include one or more of the following: acute lymphoblastic leukemia, acute myeloid leukemia, glioblastoma, non-small cell lung cancer, gastric cancer, and hepatocellular carcinoma.

[0063] The compounds described in this invention can be used in combination via oral, injectable, or other routes for the treatment of tumors in mammals (including humans), with oral administration being the most effective. The dosage ranges from 0.0001 mg / kg to 200 mg / kg body weight daily. The optimal dosage depends on the individual, and typically a lower initial dose is followed by a gradual increase.

[0064] The antitumor drug comprises a therapeutically effective dose of the compound represented by formula (I) and a pharmaceutically acceptable carrier.

[0065] The carrier mentioned refers to a carrier commonly used in the pharmaceutical field, such as: diluents, excipients such as water; binders such as cellulose derivatives, gelatin, polyvinylpyrrolidone, etc.; fillers such as starch; disintegrants such as calcium carbonate and sodium bicarbonate; in addition, other excipients such as flavoring agents and sweeteners may be added to the composition.

[0066] This invention can also be prepared into conventional solid dosage forms, such as tablets and capsules, for oral administration, or into injectable formulations for injection. The various dosage forms described above can be prepared using conventional methods in the pharmaceutical field, wherein the content of active ingredient I is 0.1% to 99.5% (by weight) of the compound.

[0067] The beneficial effects of this invention are as follows:

[0068] 1) The compounds described in this invention possess excellent Mer / c-Met enzyme inhibitory activity and exhibit good inhibitory effects on various human tumor cells, demonstrating broad-spectrum and high efficacy. They may also show good therapeutic effects for diseases caused by abnormal gene expression, such as endocrine disorders, immune system diseases, genetic diseases, and nervous system diseases.

[0069] 2) Compared with marketed positive control drugs, the compound of the present invention has a weaker inhibitory effect on normal cells while effectively inhibiting tumor cells, showing better selective inhibitory activity and has good prospects for clinical application in tumors.

[0070] 3) Compared with marketed positive control drugs, the compounds described in this invention have better safety and physicochemical properties, and are worthy of in-depth research.

[0071] In summary, this invention designs compounds targeting the binding sites of Mer and c-Met kinases, using a pyrimidine ring structure as the parent nucleus and retaining the parent nucleus, aminophenoxy linker, hydrogen bond acceptor segment, and hydrophobic group to synthesize a series of novel substituted aniline pyrimidine compounds. In the structure of these compounds, the pyrimidine ring structure can theoretically form hydrogen bond interactions with the acceptor, making it more suitable for binding to Mer and c-Met kinases. Furthermore, the novel structural compounds of this invention exhibit strong enzyme inhibitory activity while also showing specificity and selectivity against tumor cells and normal cells. Simultaneously, the novel structural compounds have high safety and fewer toxic side effects, making them easier to use as anti-tumor drugs. Detailed Implementation

[0072] The present invention will be further described in detail below with reference to embodiments, but the implementation of the invention is not limited thereto.

[0073] Example 1: Synthesis of intermediate 5-(4-fluorophenyl)-4-oxo-1,4-dihydropyridine-3-carboxylic acid (V-1)

[0074]

[0075] Cycloisopropyl malonate (5.00 g, 34.70 mmol) and N,N-diisopropylethylamine (8.98 g, 69.41 mmol) were dissolved in dichloromethane. A dichloromethane solution of 4-fluorophenylacetamide (6.00 g, 34.70 mmol) was added dropwise at constant pressure at 0 °C. The reaction was carried out at low temperature for 1 h, then at room temperature for 5 h. The reaction solution was diluted with dichloromethane, acid-washed with 2 mol / L HCl solution, washed with saturated brine, dried, filtered, concentrated, and refluxed for 2 h with 40 mL of ethanol. The reaction solution was concentrated to give a yellow solid. The solid was purified by column chromatography (PE:EA = 20:1-10:1) to give 6.38 g of ethyl 4-(4-fluorophenyl)-3-oxobutyrate, with a yield of 81.77%.

[0076]

[0077] Ethyl 4-(4-fluorophenyl)-3-oxobutyrate (6.38 g, 28.40 mmol) and 1,1-dimethoxy-N,N-dimethylmethylamine (10.14 g, 85.10 mmol) were dissolved in toluene (25 mL), and the mixture was heated to 100 °C and reacted for 12 h. The reaction solution was concentrated, and ammonium acetate (10.97 g, 142.30 mmol) and ethanol (20 mL) were added. The mixture was heated to 60 °C and reacted for 15 h. The reaction solution was filtered and dried to give 1.92 g of ethyl 5-(4-fluorophenyl)-4-oxo-1,4-dihydropyridine-3-carboxylic acid, with a yield of 25.84%.

[0078]

[0079] Ethyl 5-(4-fluorophenyl)-4-oxo-1,4-dihydropyridine-3-carboxylate (1.92 g, 7.35 mmol) was suspended in 3 mol / L NaOH solution and reacted at 100 °C for 4 h. 100 mL of water and 1 mol / L HCl solution were added to adjust the pH to 1.0, causing a white solid to precipitate. The precipitate was filtered and dried to obtain 1.43 g of white solid, with a yield of 83.62%. HRMS: m / z C 12 H9FNO3[M+H] + 234.0488.

[0080] Example 2 Synthesis of intermediate 5-(4-fluorophenyl)-4-oxo-1-(tetrahydro-2H-pyran-4-yl)methyl)-1,4-dihydropyridine-3-carboxylic acid (V-2)

[0081]

[0082] Ethyl 4-(4-fluorophenyl)-3-oxobutyrate (5.68 g, 25.35 mmol) and 1,1-dimethoxy-N,N-dimethylmethylamino (9.06 g, 76.05 mmol) from Example 1 were dissolved in toluene (20 mL), and the mixture was heated to 100 °C and reacted for 12 h. The reaction solution was concentrated, and 4-nitromethyltetrahydropyran (3.21 g, 27.88 mmol) and ethanol (20 mL) were added. The mixture was heated to 60 °C and reacted for 11 h. The reaction solution was filtered and dried to give 6.34 g of ethyl 5-(4-fluorophenyl)-4-oxo-1-(tetrahydro-2H-pyran-4-yl)methyl)-1,4-dihydropyridine-3-carboxylic acid, with a yield of 69.65%.

[0083]

[0084] Ethyl 5-(4-fluorophenyl)-4-oxo-1-(tetrahydro-2H-pyran-4-yl)methyl)-1,4-dihydropyridine-3-carboxylic acid (1.72 g, 4.79 mmol) was dissolved in 10 mL of methanol, and 1 mol / L NaOH solution (14.37 mL) was added. The reaction mixture was allowed to react at room temperature for 2 h. The reaction solution was concentrated, and 50 mL of water was added. The pH was adjusted to 1.0 with 1 mol / L HCl solution, resulting in the precipitation of a yellow solid. The solid was filtered, and the filter cake was slurried with methanol to give 1.31 g of a white solid, with a yield of 82.60%. HRMS: m / z C 18 H 19 FNO4[M+H] + 332.1120.0488.

[0085] Example 3: Synthesis of intermediate 5-(4-fluorophenyl)-1-isopropyl-4-oxo-1,4-dihydropyridine-3-carboxylic acid (V-3)

[0086]

[0087] Ethyl 4-(4-fluorophenyl)-3-oxobutyrate (5.68 g, 25.35 mmol) and 1,1-dimethoxy-N,N-dimethylmethylamine (9.06 g, 76.05 mmol) from Example 1 were dissolved in toluene (20 mL), and the mixture was heated to 100 °C and reacted for 12 h. The reaction solution was concentrated, and isopropylamine (1.65 g, 27.88 mmol) and ethanol (20 mL) were added. The mixture was heated to 60 °C and reacted for 12 h. The reaction solution was filtered and dried to obtain 5.43 g of ethyl 5-(4-fluorophenyl)-1-isopropyl-4-oxo-1,4-dihydropyridine-3-carboxylic acid, with a yield of 70.64%.

[0088]

[0089] Ethyl 5-(4-fluorophenyl)-1-isopropyl-4-oxo-1,4-dihydropyridine-3-carboxylate (0.72 g, 2.36 mmol) was dissolved in 10 mL of methanol, and 7.09 mL of 1 mol / L NaOH solution was added. The reaction mixture was allowed to react at room temperature for 2 h. The reaction solution was concentrated, and 50 mL of water was added. The pH was adjusted to 1.0 with 1 mol / L HCl solution, resulting in the precipitation of a yellow solid. The solid was filtered, and the filter cake was slurried with methanol to give 0.44 g of a white solid, with a yield of 67.10%. HRMS: m / z C 15 H 15 FNO3[M+H] + 276.0958.

[0090] Example 4: Synthesis of intermediate 5-(4-fluorophenyl)-4-oxo-1-(tetrahydrofuran-2-yl)methyl)-1,4-dihydropyridine-3-carboxylic acid (V-4)

[0091]

[0092] Ethyl 4-(4-fluorophenyl)-3-oxobutyrate (5.68 g, 25.35 mmol) and 1,1-dimethoxy-N,N-dimethylmethylamine (9.06 g, 76.05 mmol) from Example 1 were dissolved in toluene (20 mL), and the mixture was heated to 100 °C and reacted for 12 h. The reaction solution was concentrated, and tetrahydrofurfurylamide (2.82 g, 27.88 mmol) and ethanol (20 mL) were added. The mixture was heated to 60 °C and reacted for 13 h. The reaction solution was filtered and dried to obtain 5.62 g of ethyl 5-(4-fluorophenyl)-4-oxo-1-(tetrahydrofuran-2-yl)methyl)-1,4-dihydropyridine-3-carboxylic acid, with a yield of 64.23%.

[0093]

[0094] Ethyl 5-(4-fluorophenyl)-4-oxo-1-(tetrahydrofuran-2-yl)methyl)-1,4-dihydropyridine-3-carboxylic acid (0.45 g, 1.30 mmol) was dissolved in 10 mL of methanol, and 3.90 mL of 1 mol / L NaOH solution was added. The reaction mixture was allowed to react at room temperature for 2 h. The reaction solution was concentrated, and 50 mL of water was added. The pH was adjusted to 1.0 with 1 mol / L HCl solution, resulting in the precipitation of a yellow solid. The solid was filtered, and the filter cake was slurried with methanol to give 0.19 g of a white solid, with a yield of 47.33%. HRMS: m / z C 17 H 17 FNO4[M+H] + 318.1063.

[0095] Example 5 Synthesis of intermediate 2-chloro-4-(4-nitrophenoxy)pyrimidine (VI)

[0096]

[0097] p-Aminophenol (3.00 g, 21.56 mmol), 2,4-dichloropyrimidine (4.20 g, 28.00 mmol), and potassium carbonate (3.30 g, 23.70 mmol) were dissolved in N,N-dimethylformamide (DMF, 20 mL), and the mixture was heated to 80 °C and reacted for 5.5 h. After cooling to room temperature, the reaction solution was added to 200 mL of ice water, resulting in the precipitation of a white solid. The solid was filtered, dried, and yielded 5.24 g of a white solid, with a yield of 96.90%. HRMS: m / z C 10 H7ClN3O3[M+H] + 252.0098.

[0098] Example 6 Synthesis of intermediate N-(3-morpholinophenyl)-4-(4-nitrophenoxy)pyrimidine-2-amine (VIII-1)

[0099]

[0100] 3-morpholinoaniline (0.50 g, 2.81 mmol), the intermediate 2-chloro-4-(4-nitrophenoxy)pyrimidine (0.59 g, 2.34 mmol), and p-toluenesulfonic acid (0.60 g, 3.51 mmol) were dissolved in N,N-dimethylformamide (DMF, 8 mL), and the mixture was heated to 90 °C and reacted for 4 h. After cooling to room temperature, the reaction solution was added to 150 mL of ice water. The resulting aqueous solution was adjusted to pH 8.0 with Na₂CO₃ solution, extracted with ethyl acetate (15 mL × 4), dried, filtered, and concentrated to obtain a brown solid. The solid was purified by column chromatography (DCM:MeOH = 50:1-40:1) to give 1.09 g of a yellow solid, yield 68.30%. HRMS: m / z C 20 H 20 N5O4[M+H] + 394.1437.

[0101] Example 7 Synthesis of intermediate N-(3-morpholinophenyl)-4-(4-aminophenoxy)pyrimidine-2-amine (IX-1)

[0102]

[0103] Intermediate N-(3-morpholinophenyl)-4-(4-nitrophenoxy)pyrimidin-2-amine (0.88 g, 2.24 mmol) and stannous chloride dihydrate (2.52 g, 11.19 mmol) were dissolved in anhydrous ethanol (8 mL), one drop of concentrated hydrochloric acid was added, and the mixture was heated to 90 °C for 2 h under N2 protection. The reaction solution was slowly added to 200 mL of ice water, and the pH was adjusted to 8.0 with Na2CO3 aqueous solution. The mixture was extracted with ethyl acetate (15 mL × 5), dried, filtered, and concentrated to obtain a brown solid. The solid was purified by column chromatography (DCM:MeOH = 40:1-20:1) to give 0.44 g of a yellow solid, yield 54.30%. HRMS: m / z C 20 H 22 N5O2[M+H] + 364.1695.

[0104] Example 8 Synthesis of 5-(4-fluorophenyl)-N-(4-(2-(3-morpholinophenyl)amino)pyrimidine-4-oxy)phenyl)-4-oxo-1,4-dihydropyridine-3-carboxamide (target compound I-1)

[0105]

[0106] Intermediate N-(3-morpholinophenyl)-4-(4-aminophenoxy)pyrimidin-2-amine (0.09 g, 0.26 mmol), intermediate 5-(4-fluorophenyl)-4-oxo-1,4-dihydropyridine-3-carboxylic acid (0.05 g, 0.21 mmol), and TEA (0.06 g, 0.64 mmol) were dissolved in N,N-dimethylformamide (3 mL). After stirring for 10 min, HBTU (0.10 g, 0.26 mmol) was added, and the reaction was carried out at room temperature for 3 h. The reaction solution was added to 100 mL of ice water, extracted with ethyl acetate (15 mL × 4), dried over anhydrous MgSO4, filtered, and concentrated to give a white solid. The solid was purified by thin-layer chromatography (DCM:MeOH = 20:1-15:1) to give 16.4 mg of white solid, yield 13.25%. HRMS: m / z C 32 H 28 FN6O4[M+H] + 579.2078.

[0107] 1H NMR (600MHz, DMSO-d6) δ13.03(s,1H),9.41(s,1H),8.64(d,J=1.4Hz,1H),8.35(d,J=5. 6Hz,1H),8.09(d,J=1.4Hz,1H),7.79(d,J=8.9Hz,2H),7.72(dd,J=8.7,5.7Hz,2H),7.2 7(t,J=8.9Hz,2H),7.22(d,J=8.9Hz,2H),7.17(s,1H),7.06(d,J=7.3Hz,1H),6.95(t,J =8.1Hz,1H),6.49-6.46(m,1H),6.42(d,J=5.6Hz,1H),3.67-3.64(m,4H),2.90(s,4H).

[0108] Example 9 Synthesis of 5-(4-fluorophenyl)-N-(4-(2-(3-(2-(4-methylpiperazin-1-yl)acetamido)phenyl)amino)pyrimidin-4-oxy)phenyl)-4-oxo-1,4-dihydropyridine-3-carboxamide (target compound I-2)

[0109]

[0110] Following the preparation method for target compound I-1 in Example 8, target compound I-2 was prepared with a yield of 22.03%. HRMS: m / z C 35 H 34 FN8O4[M+H] + 649.2609.

[0111] 1 H NMR (500MHz, DMSO-d6) δ13.03 (s, 1H), 9.54 (d, J = 13.5Hz, 2H), 8.65-8.61 (m, 1H), 8. 35(d,J=5.6Hz,1H),8.11-8.08(m,1H),7.79(d,J=8.8Hz,2H),7.75-7.70(m,3H),7. 32-7.27(m,3H),7.27-7.22(m,3H),7.20(d,J=8.1Hz,1H),7.03(t,J=8.0Hz,1H),6. 41(d,J=5.6Hz,1H),3.13(s,2H),2.57(s,4H),2.27(s,3H),1.25(d,J=13.6Hz,4H).

[0112] Example 10 Synthesis of N-(4-(2-(3-(4-acetylpiperazin-1-yl)phenyl)amino)pyrimidine-4-oxy)phenyl)-5-(4-fluorophenyl)-4-oxy-1,4-dihydropyridine-3-carboxamide (target compound I-3)

[0113]

[0114] Following the preparation method for target compound I-1 in Example 8, target compound I-3 was prepared with a yield of 32.91%. HRMS: m / z C 34 H 31 FN7O4[M+H] + 620.2343.

[0115] 1 H NMR (500MHz, DMSO-d6) δ13.03(s,1H),12.65(s,1H),9.40(s,1H),8.63(s,1H),8.35(d,J=5.6Hz,1H),8.13-8.06(m,1H),7.80(d,J=8.8Hz 2H),7.75-7.70(m,2H),7.28(t,J=8.9Hz,2H),7.23(d,J=8.8Hz 2H),7.19(s,1H),7.09(d,J=8.1Hz,1H),6.96(t,J=8.1Hz,1H),6.50(dd,J=8.2,2.4Hz,1H ), 6.42 (d, J = 5.6 Hz, 1H), 3.51 (dt, J = 10.6, 5.1 Hz, 4H), 2.96 (q, J = 5.1 Hz, 4H), 2.00 (s, 3H).

[0116] Example 11 Synthesis of 5-(4-fluorophenyl)-N-(4-(2-(4-(2-(4-(4-(4-methylpiperazin-1-yl)acetamido)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)-4-oxo-1,4-dihydropyridine-3-carboxamide (target compound I-4)

[0117]

[0118] Following the preparation method for target compound I-1 in Example 8, target compound I-4 was prepared in a yield of 20.55%. HRMS: m / z C 35 H 34 FN8O4[M+H] + 649.2609.

[0119] 1H NMR(500MHz,DMSO-d6)δ13.05(s,1H),9.49(d,J=11.1Hz,2H),8.63(s,1H),8 .33(d,J=5.4Hz,1H),8.10(s,1H),7.81(d,J=8.3Hz,2H),7.76-7.70(m,2H), 7.48(d,J=7.2Hz,2H),7.35(d,J=7.9Hz,2H),7.31-7.20(m,5H),6.39(d,J=5 .4Hz,1H),3.03(s,2H),2.48(s,2H),2.38(s,4H),2.17(s,3H),1.23(s,2H).

[0120] Example 12 Synthesis of 5-(4-fluorophenyl)-N-(4-(2-(4-(2-(4-(4-methylpiperazin-1-yl)propaneamide)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)-4-oxo-1,4-dihydropyridine-3-carboxamide (target compound I-5)

[0121]

[0122] Following the preparation method for target compound I-1 in Example 8, target compound I-5 was prepared in a yield of 10.17%. HRMS: m / z C 36 H 36 FN8O4[M+H] + 663.2765.

[0123] 1 H NMR(500MHz,DMSO-d6)δ13.06(s,1H),9.52(d,J=16.5Hz,2H),8.63(s,1H),8.33(d, J=5.4Hz,1H),8.10(s,1H),7.81(d,J=8.4Hz,2H),7.76-7.69(m,2H),7.47(d,J=6.7 Hz,2H),7.35(d,J=7.6Hz,2H),7.31-7.15(m,5H),6.39(d,J=5.4Hz,1H),3.15(d,J= 6.7Hz, 1H), 2.41 (d, J = 40.9Hz, 6H), 2.16 (s, 3H), 1.23 (s, 2H), 1.11 (d, J = 6.3Hz, 3H).

[0124] Example 13 Synthesis of 5-(4-fluorophenyl)-N-(4-(2-(4-(4-(N-methyl-2-(4-methyl-2-(4-methylpiperazin-1-yl)acetamido)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)-4-oxo-1,4-dihydropyridine-3-carboxamide (target compound I-6)

[0125]

[0126] Following the preparation method for target compound I-1 in Example 8, target compound I-6 was prepared with a yield of 17.93%. HRMS: m / z C 36 H 36 FN8O4[M+H] + 663.2765.

[0127] 1 H NMR (500MHz, DMSO-d6) δ13.04(s,1H),9.72(s,1H),8.62(s,1H),8.37(d,J=5.5Hz,1H),8.10(s,1H),7.80(d,J=8.8Hz,2H),7.72(dd,J=8.4,5.8Hz ,2H),7.53(d,J=6.8Hz,2H),7.31-7.19(m,5H),7.03(d,J=7.1Hz,2H),6. 48(d,J=5.4Hz,1H),3.04(s,3H),2.80(s,2H),2.30(s,8H),2.15(s,3H).

[0128] Example 14 Synthesis of 5-(4-fluorophenyl)-N-(4-(2-((3-(2-methoxyethyl)amino)phenyl)amino)pyrimidine-4-oxyphenyl)-4-oxo-1,4-dihydropyridine-3-carboxamide (target compound I-7)

[0129]

[0130] Following the preparation method for target compound I-1 in Example 8, target compound I-7 was prepared with a yield of 24.27%. HRMS: m / z C 31 H 28 FN6O4[M+H] + 567.2078.

[0131] 1H NMR (500MHz, DMSO-d6) δ13.02(s,1H),12.65(s,1H),9.28(s,1H),8.64(s,1H),8.32(d,J=5. 5Hz,1H),8.09(s,1H),7.80(d,J=8.8Hz,2H),7.73(dd,J=8.4,5.8Hz,2H),7.28(t,J=8.9Hz,2 H),7.23(d,J=8.8Hz,2H),6.85(s,1H),6.79(dt,J=13.1,7.6Hz,2H),6.38(d,J=5.5Hz,1H),6 .15(d,J=7.4Hz,1H),5.09(s,1H),3.41(t,J=5.8Hz,2H),3.23(s,3H),3.03(q,J=5.3Hz,2H).

[0132] Example 15 Synthesis of 5-(4-fluorophenyl)-N-(4-(2-(4-(4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-4-oxy)phenyl)-4-oxo-1,4-dihydropyridine-3-carboxamide (target compound I-8)

[0133]

[0134] Following the preparation method for target compound I-1 in Example 8, target compound I-8 was prepared in a yield of 33.06%. HRMS: m / z C 33 H 31 FN7O3[M+H] + 592.2394.

[0135] 1 H NMR (500MHz, DMSO-d6) δ13.04(s,1H),9.34(s,1H),8.65(s,1H),8.28(d,J=5.6Hz,1H),8.12(s,1H),7.81(d,J=8.8Hz,2H),7.73(dd,J=8.4 ,5.8Hz,2H),7.28(t,J=8.9Hz,3H),7.20(d,J=8.8Hz,2H),6.70-6.63(m,2H),6.38(d,J=5.5Hz,1H),3.01(s,4H),2.56(s,4H),2.28(s,3H).

[0136] Example 16 Synthesis of 5-(4-fluorophenyl)-N-(4-(2-(3-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-4-oxy)phenyl)-4-oxo-1,4-dihydropyridine-3-carboxamide (target compound I-9)

[0137]

[0138] Following the preparation method for target compound I-1 in Example 8, target compound I-9 was prepared with a yield of 20.88%. HRMS: m / z C 33 H 31 FN7O3[M+H] + 592.2394.

[0139] 1 H NMR(500MHz,DMSO-d6)δ13.05(s,1H),9.37(s,1H),8.63(s,1H),8.35(d,J=5.6Hz,1H ),8.09(s,1H),7.79(d,J=8.4Hz,2H),7.72(dd,J=8.5,5.6Hz,2H),7.27(t,J=8.8Hz, 2H),7.22(d,J=8.5Hz,2H),7.17(s,1H),7.03(d,J=8.1Hz,1H),6.94(t,J=8.1Hz,1H) ,6.47(d,J=7.6Hz,1H),6.42(d,J=5.5Hz,1H),2.94(s,4H),2.42(s,4H),2.18(s,3H).

[0140] Example 17 Synthesis of N-(4-(2-(2-(3-fluoro-4-(2-(2-(4-methylpiperazin-1-yl)acetamido)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)-5-(4-fluorophenyl)-4-oxo-1,4-dihydropyridine-3-carboxamide (target compound I-10)

[0141]

[0142] Following the preparation method for target compound I-1 in Example 8, target compound I-10 was prepared with a yield of 19.86%. HRMS: m / z C 35 H 33 F2N8O4[M+H] + 667.2515.

[0143] 1H NMR(500MHz,DMSO-d6)δ13.06(s,1H),9.78(s,1H),9.31(s,1H),8.63(s,1H),8.3 8(d,J=5.6Hz,1H),8.10(s,1H),7.82(d,J=8.8Hz,2H),7.72(dd,J=8.4,5.8Hz,2H) ,7.65(t,J=8.8Hz,1H),7.59(d,J=13.2Hz,1H),7.31-7.21(m,5H),7.19(d,J=7.6H z, 1H), 6.50 (d, J = 5.6Hz, 1H), 3.06 (s, 2H), 2.48 (s, 4H), 2.30 (s, 4H), 2.08 (s, 3H).

[0144] Example 18 Synthesis of 5-(4-fluorophenyl)-N-(4-(2-(4-morpholinophenyl)amino)pyrimidine-4-oxy)phenyl)-4-oxo-1,4-dihydropyridine-3-carboxamide (target compound I-11)

[0145]

[0146] Following the preparation method for target compound I-1 in Example 8, target compound I-11 was prepared with a yield of 35.89%. HRMS: m / z C 32 H 28 FN6O4[M+H] + 579.2078.

[0147] 1 H NMR (500MHz, DMSO-d6) δ13.04(s,1H),12.67(s,1H),9.34(s,1H),8.64(s,1H),8.28(d,J=5.5Hz,1H),8.12(s,1H),7.81(d,J=8.6Hz,2H ),7.75-7.71(m,2H),7.28(t,J=8.8Hz,4H),7.20(d,J=8.7Hz,2H),6.68-6.60(m,2H),6.39(d,J=5.5Hz,1H),3.56(s,4H),2.89(s,4H).

[0148] Example 19 Synthesis of 5-(4-fluorophenyl)-4-oxo-N-(4-(2-(4-(trifluoromethylphenyl)phenyl)amino)pyrimidin-4-oxy)phenyl)-1,4-dihydropyridine-3-carboxamide (target compound I-12)

[0149]

[0150] Following the preparation method for target compound I-1 in Example 8, target compound I-12 was prepared in a yield of 14.22%. HRMS: m / z C 29 H 20 F4N5O3[M+H] + 562.1424.

[0151] 1 H NMR(500MHz,DMSO-d6)δ13.03(s,1H),12.64(s,1H),10.00(s,1H),8.64(s,1H),8.42(d,J=5.6Hz,1H),8.10(s, 1H),7.83(d,J=8.8Hz,2H),7.75-7.70(m,4H),7.45(d,J=8.5Hz,2H),7.30-7.24(m,4H),6.56(d,J=5.6Hz,1H).

[0152] Example 20 Synthesis of 5-(4-fluorophenyl)-N-(4-(2-(4-(1-methylpiperidin-4-oxy)phenyl)amino)pyrimidin-4-oxy)phenyl)-4-oxo-1,4-dihydropyridine-3-carboxamide (target compound I-13)

[0153]

[0154] Following the preparation method for target compound I-1 in Example 8, target compound I-13 was prepared in a yield of 8.87%. HRMS: m / z C 34 H 32 FN6O4[M+H] + 607.2351.

[0155] 1H NMR(500MHz,DMSO-d6)δ13.06(s,1H),9.50(s,1H),8.63(s,1H),8.36(d,J=5.6Hz,1H),8.09(s, 1H),7.80(d,J=8.5Hz,2H),7.73(dd,J=8.5,5.5Hz,2H),7.28(t,J=8.8Hz,2H),7.23(d,J=8.3Hz ,3H),7.12(d,J=8.2Hz,1H),6.99(t,J=8.1Hz,1H),6.47(d,J=2.5Hz,1H),6.43(d,J=5.6Hz,1H) ,4.18(s,1H),2.67(s,2H),2.26(s,2H),2.21(s,3H),1.90(d,J=12.5Hz,2H),1.66-1.57(m,2H).

[0156] Example 21 Synthesis of 5-(4-fluorophenyl)-N-(4-(2-(3-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-4-yl)phenyl)-4-oxo-1-(tetrahydro-2H-pyran-4-yl)methyl)-1,4-dihydropyridine-3-carboxamide (target compound I-14)

[0157]

[0158] Following the preparation method for target compound I-1 in Example 8, target compound I-14 was prepared in a yield of 20.45%. HRMS: m / z C 39 H 41 FN7O4[M+H] + 690.3126.

[0159] 1H NMR (500MHz, DMSO-d6) δ13.03(s,1H),9.38(s,1H),8.74(s,1H),8.35(d,J=5.2Hz,1H),8.23(s,1H),7.79(d,J=8.2Hz,2H),7.74(t,J=5.6Hz 2H),7.30(t,J=8.5Hz,2H),7.23(d,J=8.2Hz,2H),7.16(s,1H),7.04-6.98(m,1H ),6.94(t,J=7.7Hz,1H),6.46(d,J=7.4Hz,1H),6.42(d,J=5.2Hz,1H),4.12(d,J =6.4Hz,2H),3.87(d,J=9.1Hz,2H),3.26(d,J=11.1Hz,2H),2.93(s,4H),2.40(s ,4H),2.16(s,3H),1.46(d,J=11.7Hz,2H),1.32(d,J=10.2Hz,2H),1.24(s,1H).

[0160] Example 22 Synthesis of 5-(4-fluorophenyl)-1-isopropyl-N-(4-(2-(3-(4-methylpiperazin-1-phenyl)amino)pyrimidin-4-oxy)phenyl)-4-oxo-1,4-dihydropyridine-3-carboxamide (target compound I-15)

[0161]

[0162] Following the preparation method for target compound I-1 in Example 8, target compound I-15 was prepared in a yield of 15.49%. HRMS: m / z C 36 H 37 FN7O3[M+H] + 634.2864.

[0163] 1H NMR (400MHz, DMSO-d6) δ13.04(s,1H),9.34(s,1H),8.76(s,1H),8.34(d,J=5.2Hz,1H),8.26( s,1H),7.79(d,J=8.4Hz,2H),7.77-7.70(m,2H),7.28(t,J=8.5Hz,2H),7.22(d,J=8.3Hz,2H) ,7.16(s,1H),7.03(d,J=7.5Hz,1H),6.93(t,J=7.9Hz,1H),6.46(d,J=7.6Hz,1H),6.41(d,J= 5.3Hz,1H),4.71-4.58(m,1H),2.95(s,4H),2.41(s,4H),2.17(s,3H),1.52(d,J=6.4Hz,6H).

[0164] Example 23 Synthesis of 5-(4-fluorophenyl)-N-(4-(2-(3-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-4-yl)phenyl)-4-oxo-1-(tetrahydrofuran-2-yl)methyl)-1,4-dihydropyridine-3-carboxamide (target compound I-16)

[0165]

[0166] Following the preparation method for target compound I-1 in Example 8, target compound I-16 was prepared in a yield of 30.05%. HRMS: m / z C 38 H 39 FN7O4[M+H] + 676.2969.

[0167] 1H NMR (400MHz, DMSO-d6) δ13.00(s,1H),9.35(s,1H),8.72(s,1H),8.34(d,J=5.4Hz,1H),8.18(s,1H),7.79(d,J=8.5Hz,2H),7.76 -7.69(m,2H),7.29(t,J=8.7Hz,2H),7.22(d,J=8.5Hz,2H),7.16(s,1H),7.02(d,J=7.6Hz,1H),6.93(t,J=8.0Hz,1H),6.46(d,J =7.8Hz,1H),6.41(d,J=5.5Hz,1H),4.34(d,J=11.7Hz,1H),4.17(q,J=8.6,8.1Hz,2H),3.81(q,J=6.8Hz,1H),3.69(q,J=7.0Hz, 1H),2.93(s,4H),2.39(s,4H),2.16(s,3H),2.02(dt,J=12.4,6.6Hz,1H),1.89-1.79(m,2H),1.57(dq,J=14.3,7.6,6.8Hz,1H).

[0168] Example 24: In vitro inhibitory activity assay of the compounds of the present invention against Mer and c-Met kinases.

[0169] A kit was selected to test the inhibitory activity of the compounds against Mer and c-Met kinases. The experimental procedures were performed according to the kit instructions. The IC50 of the compounds was tested. 50 Testing. Cabozantinib was selected as the control drug, and the trial procedure was as follows:

[0170] (1) Compound preparation

[0171] Prepare a 10 mM DMSO solution of the compound and store it in the dark for later use.

[0172] (2) Reaction process of Mer and c-Met

[0173] a. Prepare a 1× reaction solution.

[0174] b. Compound Preparation: The final concentration of the compound to be tested was initially 10 μM, diluted 3-fold, resulting in 8 concentrations, and a single-well assay was set up. The positive control compound Cabozantinib was tested at a final concentration of 3 μM, diluted 3-fold, resulting in 10 concentrations, and a replicate assay was set up. Solutions were serially diluted to the corresponding 100-fold final concentration in a 384-well Source plate, and then 250 nmol was transferred to the 384-well reaction plate using an Echo 550. 250 nmol of 100% DMSO was transferred to both the Max and Min wells.

[0175] c. Prepare a 1.67× enzyme solution using a 1× reaction solution.

[0176] d. Add 15 μL of 1.67× enzyme solution to each well; add 15 μL of 1.6× reaction solution to the Min well. Incubate at room temperature for 15 minutes.

[0177] e. Prepare a 2.5× substrate mixture using 1× reaction solution.

[0178] f. Add 10 μL of 2.5× substrate mixture to each well of the reaction plate to initiate the reaction.

[0179] g. Use Synergy to continuously read fluorescence signals.

[0180] (3) Data Analysis

[0181] The slope is obtained by selecting the linear response segment, and the percentage inhibition rate is calculated using the following formula:

[0182]

[0183] Where: Mean(Max) is the mean slope value of each Max well (containing DMSO and enzyme); Mean(Min) is the mean slope value of each Min well (without enzyme); Sample Signal is the slope value of the compound well.

[0184] Fitting dose-response curves: Using the log value of compound concentration as the X-axis and the corresponding percentage inhibition rate as the Y-axis, the dose-response curves were fitted using the log(inhibitor) vs. response-variable slope function of GraphPad Prism 5 to obtain the IC50 of each compound inhibiting enzyme activity. 50 Values ​​(Table 2).

[0185] Table 2 shows the results of the enzyme inhibition assays of the compounds against Mer and c-Met kinases.

[0186]

[0187] As shown in Table 1 above, the compounds of the present invention exhibit strong inhibitory activity against Mer and c-Met. Among them, compounds I-1, I-2, I-3, I-5, I-7, I-9, I-10, I-14, I-15, and I-16 show better inhibitory activity against both Mer and c-Met kinases than the positive control drug Cabozantinib, demonstrating good dual-target inhibitory activity against both Mer and c-Met. Compounds I-4, I-6, I-8, and I-13 show better inhibitory activity against Mer than the positive control drug Cabozantinib, but slightly weaker inhibitory activity against c-Met. Compounds I-11 and I-12 show weaker inhibitory activity against both Mer and c-Met kinases than the positive control drug Cabozantinib.

[0188] Example 25: In vitro tumor cell inhibitory activity assay of the compounds of the present invention.

[0189] Compounds with good inhibitory activity against Mer and c-Met were selected, and their activities against human hepatocellular carcinoma cell line HepG2, human prostate cancer cell line PC-3, human colorectal cancer cell line HCT116, and non-small cell lung cancer cell line A549 were tested. Their IC50 values ​​were... 50 The values ​​were determined using the CCK-8 assay. Cabozantinib was selected as the control drug. The experimental procedure was as follows:

[0190] (1) Cell seeding: Take cells in the logarithmic growth phase and seed them into 96-well plates, 10,000 cells per well. Incubate for 12 hours to allow the cells to adhere properly.

[0191] (2) Drug preparation: Weigh the drug and dissolve it in the appropriate solvent to prepare a stock solution with a concentration of 1 mM. For testing, perform serial dilutions of 2-fold using complete culture medium.

[0192] (3) Drug treatment: The culture medium in the 96-well plate was removed, and the cells were washed once with PBS. 100 μL of culture medium containing different drug concentrations was added to each well, with 3 replicates for each concentration; DMSO control wells and blank culture medium control wells were also set up. The drug was allowed to act for 12 hours.

[0193] (4) CCK8 detection: Add 10 μL of CCK8 to each well and continue culturing the cells for 1-2 hours. Use dual wavelengths for detection: 450 nm for detection and 600-650 nm for reference.

[0194] (5) Results statistics: Cell viability: [(As-Ab) / (Ac-Ab)]×100%

[0195] Where: As: absorbance of experimental wells (containing cell culture medium, CCK8, and test drug); Ab: absorbance of blank wells (containing no cells and test drug, and CCK8); Ac: absorbance of control wells (containing cell culture medium, CCK8, and no test drug).

[0196] (6) Graphpad Prism 9.5.1 software was used to calculate the IC50 of the above three different cell types. 50 Values. The experimental results are shown in Table 3 (unit: μM).

[0197] Table 3. Antiproliferative activity of compounds against tumor cells (IC50) 50 / μM)

[0198]

[0199] As shown in Table 3 above, compared with the positive control drug Cabozantinib, the compounds of this invention exhibited good in vitro anti-tumor cell proliferation activity against various tumor cell types, and some compounds showed superior anti-tumor cell proliferation activity compared to the positive control drug Cabozantinib. Specifically, compound I-1 showed superior inhibitory activity against HepG2 and HCT116 compared to the positive control drug Cabozantinib, while its inhibitory activity against PC-3 and A549 was comparable to that of Cabozantinib; compound I-2 showed superior inhibitory activity against PC-3 and HCT116 compared to the positive control drug Cabozantinib, while its inhibitory activity against HepG2 and A549 was comparable to that of Cabozantinib; compound I-3 showed superior inhibitory activity against HepG2 and A549 compared to the positive control drug. Compound I-5 showed comparable inhibitory activity against PC-3 and HCT116 to the positive control, Cabozantinib; compound I-5 exhibited superior inhibitory activity against HepG2 and A549 compared to Cabozantinib, and comparable inhibitory activity against PC-3 and HCT116 compared to Cabozantinib; compound I-7 showed superior inhibitory activity against HepG2, PC-3, and A549 compared to Cabozantinib, and exhibited superior inhibitory activity against HCT116. The inhibitory activity of compound I-9 was comparable to that of the positive control drug Cabozantinib; compound I-9 showed superior inhibitory activity against HepG2, A549, PC-3, and HCT116 compared to Cabozantinib; compound I-10 showed superior inhibitory activity against A549, PC-3, and HCT116 compared to Cabozantinib, and its inhibitory activity against HepG2 was comparable to that of Cabozantinib; compound I-14 showed superior inhibitory activity against HepG2 and PC-3 compared to the positive control. The inhibitory activity of compound I-15 against A549 and HCT116 was comparable to that of the positive control compound I-15; the inhibitory activity of compound I-15 against HepG2, PC-3 and HCT116 was superior to that of the positive control compound I-15, and its inhibitory activity against A549 was comparable to that of the positive control compound I-15; the inhibitory activity of compound I-16 against HepG2, A549, PC-3 and HCT116 was superior to that of the positive control compound I-15.

[0200] Example 26: In vitro inhibitory activity assay of the compound of the present invention against normal cells.

[0201] The activity of the compound of this invention against MRC-5 human embryonic lung fibroblasts was determined, IC50. 50The IC50 value was determined using the CCK-8 assay. Cabozantinib was selected as the control drug for in vitro inhibitory activity against normal cell lines. 50 The test and experimental procedures are as follows:

[0202] (1) Cell seeding: Take cells in the logarithmic growth phase and seed them into 96-well plates, 10,000 cells per well. Incubate for 12 hours to allow the cells to adhere properly.

[0203] (2) Drug preparation: Weigh the drug and dissolve it in the appropriate solvent to prepare a stock solution with a concentration of 1 mM. For testing, perform serial dilutions of 2-fold using complete culture medium.

[0204] (3) Drug treatment: The culture medium in the 96-well plate was removed, and the cells were washed once with PBS. 100 μL of culture medium containing different drug concentrations was added to each well, with 3 replicates for each concentration; DMSO control wells and blank culture medium control wells were also set up. The drug was allowed to act for 12 hours.

[0205] (4) CCK8 detection: Add 10 μL of CCK8 to each well and continue culturing the cells for 1-2 hours. Use dual wavelengths for detection: 450 nm for detection and 600-650 nm for reference.

[0206] (5) Results statistics: Cell viability: [(As-Ab) / (Ac-Ab)]×100%

[0207] Where: As: absorbance of experimental wells (containing cell culture medium, CCK8, and test drug); Ab: absorbance of blank wells (containing no cells and test drug, and CCK8); Ac: absorbance of control wells (containing cell culture medium, CCK8, and no test drug).

[0208] (6) The IC50 of different drugs on MRC-5 human embryonic lung fibroblasts was calculated using Graphpad Prism 9.5.1 software. 50 Values. The experimental results are shown in Table 4 (unit: μM).

[0209] Table 4. In vitro inhibitory activity of compounds against normal cells (IC50) 50 / μM)

[0210]

[0211] As can be seen from Table 4 above, the compounds of the present invention tested showed weak inhibitory activity against normal cells and lower toxicity compared to the control drug Cabozantinib. This reveals that the novel substituted phenylamine pyrimidine Mer / c-Met dual-target inhibitor of the present invention has better selectivity in inhibiting the proliferation of tumor cells and normal cells, suggesting that it will have lower toxicity when used as an anti-tumor drug.

[0212] Example 27: hERG potassium channel activity assay of the compounds of the present invention

[0213] The potential cardiotoxic side effects of the compounds of this invention were preliminarily investigated in vitro using an hERG potassium channel inhibition assay. The experimental procedure is as follows:

[0214] (1) Cell preparation: CHO-hERG cells were cultured at 175 cm⁻¹ 2 In the culture flask, once the cell density reaches 60-80%, remove the culture medium, wash once with 7 mL of PBS (Phosphate Buffered Saline), and then add 3 mL of L-Detachin for digestion. After complete digestion, add 7 mL of culture medium to neutralize, then centrifuge, aspirate the supernatant, and resuspend in 5 mL of culture medium to ensure a cell density of 2-5 × 10⁶ cells / year. 6 / mL.

[0215] (2) Electrophysiological recording process: The single-cell high-impedance sealing and whole-cell pattern formation were all automatically completed by the Qpatch instrument. After obtaining the whole-cell recording pattern, the cells were clamped at -80 mV. Before applying a 5-second +40 mV depolarization stimulus, a 50-millisecond -50 mV pre-voltage was applied, followed by repolarization to -50 mV for 5 seconds, and then back to -80 mV. This voltage stimulus was applied every 15 seconds. After recording for 2 minutes, extracellular fluid was applied for 5 minutes of recording, and then the drug administration process began. The compound concentration started from the lowest test concentration, and each test concentration was administered for 2.5 minutes. After all concentrations were administered, the positive control compound 3 μM Cabozantinib was administered. At least 3 cells (n≥3) were tested for each concentration.

[0216] (3) Compound preparation: The stock solution of the compound was diluted with DMSO. 10 μL of the stock solution was added to 20 μL of DMSO solution and serially diluted 3-fold to 6 DMSO concentrations. 4 μL of each of the 6 DMSO concentrations was added to 396 μL of extracellular fluid and diluted 100-fold to 6 intermediate concentrations. Then, 80 μL of each of the 6 intermediate concentrations was added to 320 μL of extracellular fluid and diluted 5-fold to the final concentration to be tested. The highest test concentration was 40.00 μM, with the following concentrations: 40.00, 13.33, 4.44, 1.48, 0.49, and 0.16 μM. The DMSO content in the final test concentration did not exceed 0.2%, as this concentration of DMSO had no effect on the hERG potassium channel. The entire dilution process was performed using a Bravo instrument.

[0217] (4) Data analysis: Experimental data were analyzed using GraphPad Prism 5.0 software.

[0218] Table 5 Results of hERG potassium channel activity assays for the compounds (IC50). 50 / μM)

[0219]

[0220] hERG assay results showed that the compounds of this invention exhibited inhibitory activity against hERG potassium ion channels greater than 40 μM, indicating that the compounds of this invention have low potential cardiotoxicity.

[0221] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention should be considered equivalent substitutions and are included within the scope of the present invention.

Claims

1. A substituted aniline pyrimidine compound, characterized in that, The general formula is: Selected from:

2. A method for preparing the substituted aniline pyrimidine compound as described in claim 1, characterized in that, The synthetic route is as follows: It includes the following steps: 1) Cycloisopropyl malonate and 4-fluorophenylacetyl chloride were dissolved in a polar aprotic solvent, and compound II was obtained by nucleophilic addition elimination, ketal hydrolysis and decarboxylation under heating after the addition of a base catalyst. 2) Compounds II, III and N,N-dimethylformamide dimethyl acetal were subjected to acetal hydrolysis, Knoevenagel reaction and cyclization reaction to give compound IV; 3) Compound IV undergoes alkaline hydrolysis to yield compound V; 4) Dissolve p-nitrophenol and 2,4-dichloropyrimidine in a polar aprotic solvent, add a base catalyst, and then undergo a substitution reaction to obtain compound VI; 5) Compounds VI and VII undergo a substitution reaction in a polar aprotic solvent in the presence of an acid catalyst to give compound VIII; 6) Compound VIII is reduced to give compound IX; 7) Compound IX and compound V undergo a condensation reaction in a polar aprotic solvent in the presence of a base catalyst to obtain target compound I.

3. The preparation method according to claim 2, characterized in that, In steps 1), 4), 5), and 7), the polar aprotic solvent is selected from dichloromethane, N,N-dimethylformamide, N,N-dimethylacetamide, or dimethyl sulfoxide.

4. The preparation method according to claim 2, characterized in that, In step 1), the base catalyst is selected from N,N-diisopropylethylamine, triethylamine, diisopropylethylamine, or pyridine; in steps 4) and 7), the base catalyst is selected from potassium carbonate, sodium carbonate, sodium hydroxide, potassium hydroxide, or potassium tert-butoxide; in step 5), the acid catalyst is selected from p-toluenesulfonic acid, hydrochloric acid, or trifluoroacetic acid.

5. The use of a substituted phenylamine pyrimidine compound as described in claim 1 in the preparation of a dual-target inhibitor of the receptor tyrosine kinase Mer / c-Met.

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