A novel tyrosine kinase inhibitor and preparation method and use thereof

Abstract

This invention belongs to the field of pharmaceutical technology, specifically relating to a novel tyrosine kinase inhibitor, its preparation method, and its uses. The inhibitor is selected from compounds 56, 57, and 58 and their pharmaceutically acceptable forms, all with quinazoline as the parent nucleus, 4-fluoro-3-chlorophenylamino at the 4-position, N-chloroacetylamino at the 6-position, and 2,2,2-trifluoroethoxy, deuterated ethoxy, and deuterated methoxy at the 7-position, respectively. The preparation involves three steps: S1, reacting 4-chloro-6-nitro-7-fluoroquinazoline with 4-fluoro-3-chloroaniline in isopropanol with the addition of triethylamine to obtain the 4-(4-fluoro-3-chlorophenylamino)-7-fluoro-6-nitroquinazoline intermediate; S2, introducing a substituent at the 7-position; and S3, reductive acylation purification. The parameters for each step are clearly defined. Experiments show that the compound exhibits high tumor-suppressive efficacy even at low concentrations and has good safety profiles, providing a new approach for the development of lung cancer treatment drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a novel tyrosine kinase inhibitor, its preparation method, and its uses. Background Technology

[0002] Tyrosine kinases (TKs), as important signal transduction proteins, participate in various life processes such as cell proliferation, differentiation, apoptosis, and metabolism. Their abnormal activation is closely related to the occurrence and development of various malignant tumors. Especially in non-small cell lung cancer (NSCLC), the sustained activation of the epidermal growth factor receptor (EGFR) signaling pathway is one of the key mechanisms driving tumor progression, and tyrosine kinase inhibitors (TKIs) targeting this pathway have become the main means of clinical treatment for EGFR-mutant lung cancer.

[0003] Currently, the most widely used EGFR-TKI in clinical practice is gefitinib. This drug inhibits the growth and division of tumor cells by competitively binding to the ATP binding site of EGFR kinase and blocking downstream signal transduction. Although it shows significant initial efficacy in patients with EGFR activating mutation-positive lung cancer, its clinical application still has obvious limitations: First, drug resistance is widespread and irreversible, with more than 60% of patients developing resistance after 9-12 months of treatment. Common mechanisms include EGFR T790M secondary mutation, C797S mutation, or reactivation of downstream pathways (such as MET, PI3K / AKT), leading to decreased affinity between the drug and the kinase binding site. Second, it has poor inhibitory effects on multi-mutant strains, exhibiting extremely low inhibitory activity against tumor cells with multiple mutation sites (such as L858R / T790M mutation). Third, its metabolic stability and pharmacokinetics are insufficient, as the methoxy structure in the molecule is prone to O-demethylation metabolism, resulting in a short drug half-life and large fluctuations in blood drug concentration. Fourth, its safety and selectivity need improvement; its strong inhibitory effect on EGFR wild-type often causes adverse reactions such as rash and diarrhea, limiting the tolerated dose and long-term use.

[0004] In response to the problem of gefitinib resistance, researchers at home and abroad have developed a variety of second- and third-generation EGFR-TKIs in recent years, such as the novel quinazoline or pyrimidine tyrosine kinase inhibitor (hereinafter referred to as compound 1) disclosed in patent ZL 2022 1 0197875.X (CN 114394942 B). These compounds enhance the binding selectivity of mutant EGFR by introducing specific substituents, thus improving the inhibitory effect on the T790M mutant strain to some extent. However, this technical approach still has shortcomings: First, its activity is insufficient in multi-mutant strains, and its inhibitory effect on BaF3 cell models containing multiple mutations (such as T790M / L858R) is limited, making it difficult to cover the complex mutation spectrum in clinical practice. Second, the improvement in metabolic stability is limited, as the structure still contains easily metabolized alkoxy fragments, which cannot effectively avoid CYP450-mediated rapid oxidative metabolism, resulting in limited in vivo exposure and duration of inhibition. Third, the target binding conformation is not fully optimized, with 7-position substitution modifications mostly using conventional alkoxy or aryloxy groups, resulting in a rigid molecular conformation that lacks adaptability to the diverse structures of the EGFR ATP binding cavity, and the binding energy and selectivity need to be improved. Fourth, the safety and selectivity are not ideal, as some structural modifications may lead to cross-inhibition of wild-type EGFR, causing toxic reactions in tissues such as skin and liver.

[0005] In summary, while existing EGFR-TKI drugs (including gefitinib and patented compound 1) have made significant progress in the field of anti-tumor therapy, they still suffer from core problems such as insufficient coverage of resistance mutations, poor metabolic stability, insufficient inhibitory persistence, and unsatisfactory selectivity and safety. These problems severely limit their long-term efficacy in multimutant and drug-resistant non-small cell lung cancer. Therefore, there is an urgent need to design and synthesize a class of tyrosine kinase inhibitors with novel structures, more stable target binding, effective ability to overcome multimutant resistance, and good in vivo safety. Summary of the Invention

[0006] To address the aforementioned shortcomings, this invention, based on the quinazoline core, successfully synthesized novel compounds 56, 57, and 58 through systematic optimization and rational design of the 7-position substituent expansion. This achieves targeted improvements to the shortcomings of existing technologies and provides a new solution for developing more efficient and safer next-generation TKI drugs.

[0007] The technical solution of this invention is as follows: On one hand, the present invention provides a tyrosine kinase inhibitor, characterized in that it is selected from at least one of the following compounds, pharmaceutically acceptable salts, stereoisomers, solvates, or prodrugs: Compound 56: A quinazoline derivative with a 4-fluoro-3-chlorophenylamino group at the 4-position and a 2,2,2-trifluoroethoxy group at the 7-position of the quinazoline core; Compound 57: A quinazoline derivative with a 4-fluoro-3-chlorophenylamino group at the 4-position and a deuterated ethoxy group at the 7-position; Compound 58: A quinazoline derivative with a 4-fluoro-3-chlorophenylamino group at the 4-position and a deuterated methoxy group at the 7-position.

[0008] Specifically, in the structural formula of compound 56, the 6-position of the quinazoline core is substituted with N-chloroacetylamino; in the structural formulas of compounds 57 and 58, the 6-position of the quinazoline core is also substituted with N-chloroacetylamino.

[0009] In another aspect, the present invention provides a method for preparing the aforementioned tyrosine kinase inhibitor, comprising the following steps: S1: 4-chloro-6-nitro-7-fluoroquinazoline and 4-fluoro-3-chloroaniline are reacted in isopropanol with triethylamine and at 50-70°C for 5-7 hours to obtain the intermediate 4-(4-fluoro-3-chlorophenylamino)-7-fluoro-6-nitroquinazoline; S2: For compound 56, the intermediate from step S1 was reacted with 2,2,2-trifluoroethanol in a sodium hydride / tetrahydrofuran system at 90-110°C for 7-9 hours; for compound 57, the intermediate from step S1 was reacted with deuterated ethanol in the same system at the same temperature and time; for compound 58, the intermediate from step S1 was reacted with deuterated methanol in the same system at the same temperature and time, respectively, to obtain the corresponding 7-position substituted 6-nitroquinazoline derivatives; S3: The derivatives obtained in step S2 are reacted with reduced iron powder and ammonium chloride in an ethanol-water mixed solvent at 80-100℃ for 5-7 hours to complete the nitro reduction. Then, a tetrahydrofuran solution of chloroacetyl chloride is added dropwise under an ice bath, and the reaction is carried out at room temperature for 3-5 hours. The compounds 56, 57 or 58 are obtained by chromatography purification.

[0010] Preferably, for compound 56, the steps are as follows: S1: 4-chloro-6-nitro-7-fluoroquinazoline was reacted with 4-fluoro-3-chloroaniline in isopropanol, triethylamine was added, the reaction temperature was 60℃, and the reaction time was 6 hours to obtain the intermediate 4-(4-fluoro-3-chlorophenylamino)-7-fluoro-6-nitroquinazoline.

[0011] S2: The intermediate from step S1 was reacted with 2,2,2-trifluoroethanol in a sodium hydride / tetrahydrofuran system at a reaction temperature of 100°C for 8 hours to give a 7-(2,2,2-trifluoroethoxy)-6-nitroquinazoline derivative.

[0012] S3: The derivative from step S2 was reacted with reduced iron powder and ammonium chloride in an ethanol-water mixed solvent at a reduction temperature of 90°C for 6 hours. After adding a tetrahydrofuran solution of chloroacetyl chloride dropwise in an ice bath, the reaction was carried out at room temperature for 4 hours. The compound 56 was obtained by chromatography purification.

[0013] Preferably, for compound 57, the steps are as follows: S1: 4-chloro-6-nitro-7-fluoroquinazoline reacts with 4-fluoro-3-chloroaniline in isopropanol, triethylamine is added, the reaction temperature is 60℃, and the reaction time is 6 hours to give the intermediate 4-(4-fluoro-3-chlorophenylamino)-7-fluoro-6-nitroquinazoline.

[0014] S2: The intermediate from step S1 reacts with deuterated ethanol in a sodium hydride / tetrahydrofuran system at a temperature of 100°C for 8 hours to obtain a 7-deuterated ethoxy-6-nitroquinazoline derivative.

[0015] S3: The derivative from step S2 was reacted with reduced iron powder and ammonium chloride in an ethanol-water mixed solvent at a reduction temperature of 90°C for 6 hours. After adding tetrahydrofuran solution of chloroacetyl chloride dropwise in an ice bath, the reaction was carried out at room temperature for 3.5 hours. The product was purified by chromatography to obtain compound 57.

[0016] Preferably, for compound 58, the steps are as follows: S1: 4-chloro-6-nitro-7-fluoroquinazoline reacts with 4-fluoro-3-chloroaniline in isopropanol, triethylamine is added, the reaction temperature is 60℃, and the reaction time is 6 hours to give the intermediate 4-(4-fluoro-3-chlorophenylamino)-7-fluoro-6-nitroquinazoline.

[0017] S2: The intermediate from step S1 reacts with deuterated methanol in a sodium hydride / tetrahydrofuran system at a reaction temperature of 100°C for 8 hours to obtain a 7-deuterated methoxy-6-nitroquinazoline derivative.

[0018] S3: The derivative from step S2 was reacted with reduced iron powder and ammonium chloride in an ethanol-water mixed solvent at a reduction temperature of 90°C for 6 hours. After adding tetrahydrofuran solution of chloroacetyl chloride dropwise in an ice bath, the reaction was carried out at room temperature for 4.5 hours. The product was purified by chromatography to obtain compound 58.

[0019] Specifically, in step S1, the molar ratio of 4-chloro-6-nitro-7-fluoroquinazoline to 4-fluoro-3-chloroaniline is 1:(1.0~1.2); in step S2, the molar ratio of sodium hydride to the intermediate of step S1 is (1.2~1.5):1, and the molar ratios of 1,2,2-trifluoroethanol, deuterated ethanol, and deuterated methanol to the intermediate of step S1 are (1.1~1.3):1; in step S3, the molar ratio of chloroacetyl chloride to the nitro reduction product is (1.1~1.3):1.

[0020] Preferably, when the preparation method is used to synthesize compound 56: in step S1, the molar ratio of 4-chloro-6-nitro-7-fluoroquinazoline to 4-fluoro-3-chloroaniline is 1:1.1; in step S2, the molar ratio of sodium hydride to the intermediate of step S1 (4-(4-fluoro-3-chlorophenylamino)-7-fluoro-6-nitroquinazoline) is 1.3:1, and the molar ratio of 2,2,2-trifluoroethanol to the intermediate of step S1 is 1.2:1; in step S3, the molar ratio of chloroacetyl chloride to the nitro reduction product (4-(4-fluoro-3-chlorophenylamino)-7-(2,2,2-trifluoroethoxy)-6-aminoquinazoline) is 1.2:1.

[0021] Preferably, when the preparation method is used to synthesize compound 57: in step S1, the molar ratio of 4-chloro-6-nitro-7-fluoroquinazoline to 4-fluoro-3-chloroaniline is 1:1.0; in step S2, the molar ratio of sodium hydride to the intermediate of step S1 (4-(4-fluoro-3-chlorophenylamino)-7-fluoro-6-nitroquinazoline) is 1.2:1, and the molar ratio of deuterated ethanol to the intermediate of step S1 is 1.1:1; in step S3, the molar ratio of chloroacetyl chloride to the nitro reduction product (4-(4-fluoro-3-chlorophenylamino)-7-deuterated ethoxy-6-aminoquinazoline) is 1.1:1.

[0022] Preferably, when the preparation method is used to synthesize compound 58: in step S1, the molar ratio of 4-chloro-6-nitro-7-fluoroquinazoline to 4-fluoro-3-chloroaniline is 1:1.2; in step S2, the molar ratio of sodium hydride to the intermediate of step S1 (4-(4-fluoro-3-chlorophenylamino)-7-fluoro-6-nitroquinazoline) is 1.5:1, and the molar ratio of deuterated methanol to the intermediate of step S1 is 1.3:1; in step S3, the molar ratio of chloroacetyl chloride to the nitro reduction product (4-(4-fluoro-3-chlorophenylamino)-7-deuterated methoxy-6-aminoquinazoline) is 1.3:1.

[0023] In another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of at least one compound of the tyrosine kinase inhibitor of claim 1 or 2.

[0024] Specifically, the pharmaceutical composition further includes a pharmaceutically acceptable carrier.

[0025] Preferably, the pharmaceutically acceptable carrier is selected from at least one of microcrystalline cellulose, lactose, hydroxypropyl methylcellulose, magnesium stearate, water for injection, physiological saline, and polyethylene glycol 400.

[0026] Specifically, the dosage form of the pharmaceutical composition is a tablet, capsule, intravenous injection, or intraperitoneal injection.

[0027] In another aspect, the present invention provides the use of the aforementioned tyrosine kinase inhibitor in the preparation of medicaments for the prevention or treatment of non-small cell lung cancer.

[0028] Specifically, the non-small cell lung cancer includes, but is not limited to: EGFR wild-type non-small cell lung cancer, EGFR mutant non-small cell lung cancer, non-small cell lung cancer resistant to gefitinib, and non-small cell lung cancer carrying EGFR T790M and / or C797S compound mutations.

[0029] Specifically, the EGFR-mutant non-small cell lung cancer includes, but is not limited to: EGFR L858R mutant, EGFR T790M mutant, and EGFR T790M / C797S complex mutant.

[0030] The beneficial effects of this invention are as follows: (1) Achieve efficient inhibition of multidrug resistance models Compounds 56, 57, and 58 of this invention exhibit superior inhibitory effects in a BaF3 engineered cell model (EGFR Del19-T790M-C797S / BaF3) carrying complex multiple mutations (including compound mutations such as T790M / C797S). At extremely low concentrations (20-60 nM), their CCK-8 proliferation inhibition rate and apoptosis induction capacity as detected by flow cytometry were significantly superior to the control drugs gefitinib and compound 1. The molecular mechanism of this breakthrough effect can be attributed to the precise design of the 7-position substituent in the quinazoline nucleus. The deuteroyl group stabilizes the metabolically sensitive site through a kinetic isotope effect, while the strong electron-withdrawing properties and moderate steric volume of the trifluoroethoxy group synergistically optimize the adaptability of the compounds to the binding cavity of the multiple mutant EGFR kinase, enabling them to effectively respond to conformational changes in the binding cavity caused by compound mutations.

[0031] (2) Achieve true broad-spectrum antitumor activity The compounds of this invention exhibited optimal proliferation inhibition in four cell models, including EGFR wild-type (A549), EGFR-sensitive mutant (PC-9), EGFR single-drug-resistant mutant (NCI-H1975), and multidrug-resistant mutant (EGFR Del19-T790M-C797S / BaF3), overcoming the limitation of existing EGFR-TKIs that are only effective against specific mutation types. The chemical essence of this broad-spectrum inhibitory activity lies in the synergistic mechanism of multiple substituents at the 7-position. The deuterated alkoxy group prolongs the duration of target inhibition, while the trifluoroethoxy group, through its unique electronic and spatial properties, guides the molecule to adopt a more optimal binding posture, enabling it to adapt to the diverse structures of the ATP-binding domains of different kinases, thereby achieving effective inhibition of tumor cells with a broad genetic background.

[0032] (3) Effectively induces tumor cell apoptosis In flow cytometry analysis of EGFR Del19-T790M-C797S / BaF3 cells, the compound of this invention induced apoptosis rates significantly higher than the control drug even at low concentrations (20, 60 nM), demonstrating a powerful ability to directly eliminate tumor cells. The core chemical basis of this potent apoptosis-inducing effect is sustained and potent signaling pathway blocking. The metabolic stability and binding affinity enhanced by the 7-position modification enable the compound to inhibit the survival signaling pathways of tumor cells more persistently and powerfully, decisively shifting the cell's pro-survival / pro-apoptosis balance towards apoptosis.

[0033] (4) Effectively inhibits tumor cell migration Cell scratch assays confirmed that the compounds of this invention exhibited superior migration inhibition compared to control drugs in three types of adherent cell lines, suggesting their potential to inhibit tumor metastasis. The molecular regulatory mechanism of this migration inhibition relies on the optimization of a specific substituent at the 7-position (particularly trifluoroethoxy) – this not only enhances the membrane permeability of the compound through structural modification but also strengthens its specific interaction with non-EGFR kinases (such as c-Met, a key node driving migration), thereby enhancing the inhibition of tumor cell motility and invasion.

[0034] (5) Excellent in vivo safety At an effective dose (10 mg / kg / day), the compounds of this invention had no significant adverse effects on the body weight, liver and kidney function biochemical indicators, or major organ tissue structure of experimental animals, exhibiting a broad therapeutic window. The excellent in vivo safety and pharmacokinetic and pharmacodynamic properties are based on precise target selectivity and optimized metabolic pathways. The design of the specific substituent at the 7-position improves selectivity for tumor-associated kinases through structure-activity relationship optimization, reducing off-target effects; simultaneously, deuteration modification guides the compound through a safer metabolic pathway by altering metabolic kinetics, reducing the formation of highly toxic metabolic intermediates. Attached Figure Description

[0035] Figure 1 Results of CCK-8 experiments (A549 and PC9 cell lines).

[0036] Figure 2 Results of CCK-8 experiments (NCI-H1975, EGFR Del19-T790M-C797S / BaF3 cell lines).

[0037] Figure 3 Results of plate colony formation experiment (A549 cell line).

[0038] Figure 4 Results of plate colony formation experiment (PC9 cell line).

[0039] Figure 5 Results of plate colony formation experiment (NCI-H1975 cell line).

[0040] Figure 6 The results of apoptosis were detected by flow cytometry (EGFR Del19-T790M-C797S / BaF3 cell line - control group and compound 56 group).

[0041] Figure 7 The results of apoptosis were detected by flow cytometry (EGFR Del19-T790M-C797S / BaF3 cell line - gefitinib group and compound 57 group).

[0042] Figure 8 The results of apoptosis were detected by flow cytometry (EGFR Del19-T790M-C797S / BaF3 cell line - compound 1 group and compound 58 group).

[0043] Figure 9 A bar chart showing the apoptosis rate of EGFR Del19-T790M-C797S / BaF3 cells.

[0044] Figure 10 The results are from the scratch assay (cell migration inhibition).

[0045] Figure 11 This is the curve showing the change in mouse body weight.

[0046] Figure 12 The results are the biochemical indicators of liver and kidney function in mice.

[0047] Figure 13 HE-stained sections of mouse liver and kidney tissue. Detailed Implementation

[0048] The present invention will be further clearly and completely illustrated below through embodiments. These embodiments are only some examples of the present invention and are not intended to limit the present invention, but are only for illustrating the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are all conventional experiments, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0049] Example 1 The basic reaction process for preparing the quinazoline derivative of the present invention is as follows:

[0050] Preparation of intermediate: 4-(4-fluoro-3-chlorophenylamino)-7-fluoro-6-nitroquinazolino 2.3 g of 4-chloro-6-nitro-7-fluoroquinazoline and 100 mL of isopropanol were added to a 250 mL round-bottom flask. After stirring well, 5 mL of triethylamine and 1.6 g of 4-fluoro-3-chloroaniline were added. The mixture was reacted at 60 °C for 6 hours. After the reaction was completed, the mixture was cooled and filtered to obtain a filter cake. The filter cake was washed with a small amount of methanol-water solution (5%, v / v) to give 3.0 g of the product 4-(4-fluoro-3-chlorophenylamino)-7-fluoro-6-nitroquinazoline. MS (m / z): 337.0 (M+1) + .

[0051] 1.1 Synthesis of Compound 56 (1) Preparation of 4-(4-fluoro-3-chlorophenylamino)-7-(2,2,2-trifluoroethoxy)-6-nitroquinazoline: 1.5 g of 2,2,2-trifluoroethanol, 500 mg of sodium hydride, and 100 mL of tetrahydrofuran were added to a 250 mL round-bottom flask. After stirring evenly, 3.3 g of 4-(4-fluoro-3-chlorophenylamino)-7-fluoro-6-nitroquinazoline was added. The mixture was reacted at 100 °C for 8 hours. After the reaction was completed, the reaction solution was poured into cold water and neutralized with hydrochloric acid aqueous solution (1 mol / L). The filter cake was obtained by suction filtration and washed with a small amount of methanol aqueous solution (5%, v / v) to obtain 3 g of the product 4-(4-fluoro-3-chlorophenylamino)-7-(2,2,2-trifluoroethoxy)-6-nitroquinazoline. MS (m / z): 417.1 (M+1) + .

[0052] (2) Preparation of 4-(4-fluoro-3-chlorophenylamino)-7-(2,2,2-trifluoroethoxy)-6-aminoquinazoline: 4.2 g of 4-(4-fluoro-3-chlorophenylamino)-7-(2,2,2-trifluoroethoxy)-6-nitroquinazoline, 150 mL of ethanol, 10 mL of water, 3 g of reduced iron powder, and 2 g of ammonium chloride were added to a 250 mL round-bottom flask. After stirring evenly, the mixture was slowly heated to 90 °C for 6 hours. After the reaction was complete, excess iron powder was removed by hot filtration. The solvent was concentrated using a rotary evaporator, 100 mL of distilled water was added, and the mixture was extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 3 g of the product 4-(4-fluoro-3-chlorophenylamino)-7-(2,2,2-trifluoroethoxy)-6-aminoquinazoline. MS (m / z): 387.1 (M+1) + .

[0053] (3) Preparation of 4-(4-fluoro-3-chlorophenylamino)-7-(2,2,2-trifluoroethoxy)-6-(N-chloroacetyl)aminoquinazoline: Add 3.9 g of 4-(4-fluoro-3-chlorophenylamino)-7-(2,2,2-trifluoroethoxy)-6-aminoquinazoline, 120 mL of tetrahydrofuran, and 5 mL of triethylamine to a 250 mL round-bottom flask. Place the flask in an ice bath, stir well, and then slowly add 30 mL of a THF solution containing 1.5 g of chloroacetyl chloride using a funnel. The addition is completed over 30 minutes. Continue the reaction at room temperature for 4 hours. After the reaction is complete, pour the reaction solution into cold water. A large amount of solid precipitates out. Filter to obtain the crude product, and further purify by column chromatography (dichloromethane:methanol = 100:0-97:3) to obtain the product 4-(4-fluoro-3-chlorophenylamino)-7-(2,2,2-trifluoroethoxy)-6-(N-chloroacetyl)aminoquinazoline. (Trifluoroethoxy)-6-(N-chloroacetyl)aminoquinazoline 3 g, MS (m / z): 463.0 (M+1) + The structural formula of compound 56 is shown below:

[0054] 1.2 Synthesis of Compound 57 (1) Preparation of 4-(4-fluoro-3-chlorophenylamino)-7-deuterated ethoxy-6-nitroquinazoline: 1 g of deuterated ethanol, 500 mg of sodium hydride, and 100 mL of tetrahydrofuran were added to a 250 mL round-bottom flask. After stirring evenly, 3.3 g of 4-(4-fluoro-3-chlorophenylamino)-7-fluoro-6-nitroquinazoline was added. The mixture was reacted at 100 °C for 8 hours. After the reaction was complete, the reaction solution was poured into cold water and neutralized with hydrochloric acid aqueous solution (1 mol / L). The filter cake was obtained by suction filtration and washed with a small amount of methanol aqueous solution (5%, v / v) to obtain 2.5 g of the product 4-(4-fluoro-3-chlorophenylamino)-7-deuterated ethoxy-6-nitroquinazoline. MS (m / z): 368.1 (M+1) + .

[0055] (2) Preparation of 4-(4-fluoro-3-chlorophenylamino)-7-deuterated ethoxy-6-aminoquinazoline: 3.7 g of 4-(4-fluoro-3-chlorophenylamino)-7-deuterated ethoxy-6-nitroquinazoline, 150 mL of ethanol, 10 mL of water, 3 g of reduced iron powder, and 2 g of ammonium chloride were added to a 250 mL round-bottom flask. After stirring evenly, the mixture was slowly heated to 90 °C for 6 hours. After the reaction was complete, excess iron powder was removed by hot filtration. The solvent was concentrated using a rotary evaporator, 100 mL of distilled water was added, and the mixture was extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 3 g of the product 4-(4-fluoro-3-chlorophenylamino)-7-deuterated ethoxy-6-aminoquinazoline. MS (m / z): 338.1 (M+1) + .

[0056] (3) Preparation of 4-(4-fluoro-3-chlorophenylamino)-7-deuterated ethoxy-6-(N-chloroacetyl)aminoquinazoline: Add 3.4 g of 4-(4-fluoro-3-chlorophenylamino)-7-deuterated ethoxy-6-aminoquinazoline, 120 mL of tetrahydrofuran, and 5 mL of triethylamine to a 250 mL round-bottom flask. Place the flask in an ice bath, stir well, and then slowly add 30 mL of a THF solution containing 1.5 g of chloroacetyl chloride dropwise using a funnel. The addition is completed over 30 minutes. Continue the reaction at room temperature for 4 hours. After the reaction is complete, pour the reaction solution into cold water. A large amount of solid precipitates out. Filter to obtain the crude product, and further purify it by column chromatography (dichloromethane:methanol = 100:0-97:3) to obtain 2.5 g of 4-(4-fluoro-3-chlorophenylamino)-7-deuterated ethoxy-6-(N-chloroacetyl)aminoquinazoline. gram, MS (m / z): 414.1 (M+1) + The structural formula of compound 57 is shown below:

[0057] 1.3 Synthesis of Compound 58 (1) Preparation of 4-(4-fluoro-3-chlorophenylamino)-7-deuterated methoxy-6-nitroquinazoline: 500 mg of deuterated methanol, 500 mg of sodium hydride, and 100 mL of tetrahydrofuran were added to a 250 mL round-bottom flask. After stirring evenly, 3.3 g of 4-(4-fluoro-3-chlorophenylamino)-7-fluoro-6-nitroquinazoline was added. The mixture was reacted at 100 °C for 8 hours. After the reaction was completed, the reaction solution was poured into cold water and neutralized with hydrochloric acid aqueous solution (1 mol / L). The filter cake was obtained by suction filtration and washed with a small amount of methanol aqueous solution (5%, v / v) to obtain 2.5 g of the product 4-(4-fluoro-3-chlorophenylamino)-7-deuterated methoxy-6-nitroquinazoline. MS (m / z): 352.1 (M+1)+ .

[0058] (2) Preparation of 4-(4-fluoro-3-chlorophenylamino)-7-deuterated methoxy-6-aminoquinazoline: 3.5 g of 4-(4-fluoro-3-chlorophenylamino)-7-deuterated methoxy-6-nitroquinazoline, 150 mL of ethanol, 10 mL of water, 3 g of reduced iron powder, and 2 g of ammonium chloride were added to a 250 mL round-bottom flask. After stirring evenly, the mixture was slowly heated to 90 °C for 6 hours. After the reaction was complete, excess iron powder was removed by hot filtration. The solvent was concentrated using a rotary evaporator, 100 mL of distilled water was added, and the mixture was extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 2.8 g of the product 4-(4-fluoro-3-chlorophenylamino)-7-deuterated methoxy-6-aminoquinazoline. MS (m / z): 322.1 (M+1) + .

[0059] (3) Preparation of 4-(4-fluoro-3-chlorophenylamino)-7-deuterated methoxy-6-(N-chloroacetyl)aminoquinazoline: Add 3.2 g of 4-(4-fluoro-3-chlorophenylamino)-7-deuterated methoxy-6-aminoquinazoline, 120 mL of tetrahydrofuran, and 5 mL of triethylamine to a 250 mL round-bottom flask. Place the flask in an ice bath, stir well, and then slowly add 30 mL of a THF solution containing 1.5 g of chloroacetyl chloride dropwise using a funnel. The addition is completed over 30 minutes. Continue the reaction at room temperature for 4 hours. After the reaction is complete, pour the reaction solution into cold water. A large amount of solid precipitates out. Filter to obtain the crude product, and further purify it by column chromatography (dichloromethane:methanol = 100:0-97:3) to obtain 2.5 g of 4-(4-fluoro-3-chlorophenylamino)-7-deuterated methoxy-6-(N-chloroacetyl)aminoquinazoline. gram, MS (m / z): 398.1 (M+1) + The structural formula of compound 58 is shown below:

[0060] The compounds described in this invention include, but are not limited to, the following three specific compounds: (1) Compound 56: a quinazoline derivative with 4-fluoro-3-chlorophenylamino at the 4-position, chloroacetyl at the 6-position, and trifluoroethoxy at the 7-position; (2) Compound 57: a quinazoline derivative with 4-fluoro-3-chlorophenylamino at the 4-position, chloroacetyl at the 6-position, and deuterated ethoxy at the 7-position; (3) Compound 58: a quinazoline derivative with 4-fluoro-3-chlorophenylamino at the 4-position, chloroacetyl at the 6-position, and deuterated methoxy at the 7-position. The molecular design of this series of compounds reflects the synergistic optimization of isotope effect and electronic effect: (1) Deuterated substituents effectively improve metabolic stability by reducing the oxidation metabolic rate of C–H bond; (2) Trifluoroethoxy enhances hydrophobicity and electronic attraction, optimizing the binding conformation and affinity with the cavity of EGFR mutant kinase.

[0061] Example 1: CCK-8 Experiment Lung cancer cells in logarithmic growth phase, including A549 (Shanghai Jinyuan Biotechnology Co., Ltd., JY135), PC-9 (Shanghai Jinyuan Biotechnology Co., Ltd., JY123), NCI-H1975 (Shanghai Jinyuan Biotechnology Co., Ltd., JY102), and EGFRDel19-T790M-C797S / BaF3 (Nanjing Kebai Biotechnology Co., Ltd., CBP73173), were washed three times with sterile 1×PBS. An appropriate amount of 0.25% trypsin was added to digest the cells. When the intercellular spaces widened and the cells became rounded and shrunken, an equal volume of complete cell culture medium containing 10% fetal bovine serum was added to terminate the digestion. The cells were centrifuged at 300 g for 5 minutes, and seeded evenly in each well of a 96-well plate at a count of 5000 cells / well. After cell adhesion, the culture medium was discarded. Add 200 μL of the prepared solutions of gefitinib (Maclean, G828597-1g), compound 1, and compounds 56, 57, and 58 to each well (the drug concentrations used for A549, PC-9, and NCI-H1975 cells were 0, 250 nM, and 500 nM, respectively; the drug concentrations used for EGFR Del19-T790M-C797S / BaF3 cells were 0, 20 nM, and 60 nM, respectively). After treating the cells with each drug for 72 hours, remove the 96-well plates from the cell culture incubator, add CCK8 working solution to each well, incubate for 1 hour, and measure and count the cell absorbance at 450 nm using a full-spectrum microplate reader.

[0062] The results showed that compounds 56, 57, and 58 exhibited significantly superior inhibitory activity compared to the control (gefitinib and compound 1) in all four cell models, particularly in the EGFR Del19-T790M-C797S / BaF3 multimutant strain, demonstrating potent inhibitory effects at low doses (20 nM and 60 nM). No significant difference was observed in ns.* P ≤0.05;** P ≤0.01; *** P ≤0.001; **** P ≤0.0001 (as shown in Figures 1-2).

[0063] Example 2: Plate Cloning Experiment Three human lung cancer cell lines (A549, PC-9, and NCI-H1975) in logarithmic growth phase were digested with trypsin and prepared into single-cell suspensions. The cell suspensions were seeded at a density of 500 cells / well in 6-well plates, with 2 mL of cell suspension added to each well. The plates were gently shaken to ensure even distribution of cells at the bottom, and then incubated at 37°C in a 5% CO2 incubator until cell attachment. After cell attachment, the culture medium was discarded. 2 mL of the corresponding concentrations of drug solutions (0, 250 nM, and 500 nM solutions of gefitinib, afatinib, compound 1, compound 56, compound 57, and compound 58, respectively) were added to the corresponding wells. After 72 hours of drug treatment, the medium was replaced with normal culture medium, and subsequently, the medium was changed every 5 days until visible cell colonies formed. After cell clones have formed, discard the culture supernatant in the 6-well plate, gently wash the plate three times with PBS, add 500 μL of 4% paraformaldehyde to each well, and fix at room temperature for 15 minutes. After fixation, add 500 μL of crystal violet staining solution to each well and stain at room temperature for 15 minutes to ensure thorough staining of the cell clones. After staining, wash the plate repeatedly with PBS until the background is clean, invert to dry, and then photograph and record the data.

[0064] The results showed that, compared with the control group, the number of cell clones gradually decreased with increasing drug concentrations of gefitinib, compound 1, compound 56, compound 57, and compound 58. This indicates that the drugs inhibit cell proliferation and colony formation. Compounds 56, 57, and 58 showed significantly better inhibitory effects on the three types of human lung cancer cells than gefitinib and compound 1. There was no significant difference in ns.* P ≤0.05;** P ≤0.01; *** P ≤0.001; **** P ≤0.0001 (as shown in Figures 3-5).

[0065] Example 3: Flow cytometry detection of apoptosis Log-phase EGFR Del19-T790M-C797S / BaF3 cells were seeded in 6-well plates and cultured overnight. The supernatant was discarded, and the cells were washed once with PBS. The cells were then divided into a control group (complete culture medium without drugs), a gefitinib group, compound 1 group, compound 56 group, compound 57 group, and compound 58 group. 2 mL of the corresponding concentration of drug solution (100 nM for each well) was added to each well. After co-incubation with the drug for 24 hours, the cell suspension was aspirated into centrifuge tubes, centrifuged at 1000 g for 5 minutes, the supernatant was discarded, and the cells were collected. The cells were gently resuspended in 195 μL of Annexin V-FITC binding solution. Then, 2.5 μL of Annexin V-FITC and 5 μL of propidium iodide staining solution were added and gently mixed. The cells were incubated at room temperature in the dark for 10-20 minutes, then placed on ice and protected from light by aluminum foil. The apoptosis rate of each group was then measured and statistically analyzed using flow cytometry.

[0066] The results showed that in EGFR Del19-T790M-C797S / BaF3 cells, compounds 56, 57, and 58 induced apoptosis significantly more strongly than the control group, gefitinib group, and compound 1 group. This suggests that compounds 56, 57, and 58 all possess higher pro-apoptotic activity. No significant difference was observed in ns. P ≤0.001; **** P ≤0.0001 (as shown in Figures 6-9).

[0067] Example 4: Scratch Test Collect cells in the logarithmic growth phase (A549, PC-9, NCI-H1975), wash three times with sterile 1×PBS, add an appropriate amount of 0.25% trypsin for digestion, and when the intercellular spaces widen and the cells become rounded and wrinkled, add an equal volume of complete cell culture medium containing 10% fetal bovine serum to terminate the digestion; collect the cells in sterile centrifuge tubes, centrifuge at 300 g for 5 minutes to collect and count the cells; after counting, divide the cells into 5×10⁻⁶ cells per cell line. 6 Cells were evenly seeded into 6-well plates. When the cell confluence reached 80%, the cells were streaked vertically into the 6-well plates using a 200 μL pipette tip. The cells were washed 2-3 times with sterile 1×PBS to remove detached cells and cell debris. The cells were then cultured in serum-free medium and photographed at 0 hours. Subsequently, photographs were taken at 12-hour intervals, and the data were saved for statistical analysis.

[0068] The experimental results showed that, compared with the control drugs (gefitinib, compound 1), compounds 56, 57, and 58 significantly inhibited cell migration in three types of human lung cancer cells at concentrations of 250 nM and 500 nM, with statistical significance. There was no significant difference in ns. P ≤0.05;** P ≤0.01; *** P ≤0.001; **** P ≤0.0001 (as shown in Figure 10).

[0069] Example 5: In vivo safety experiment Male C57 mice aged 6-8 weeks (Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd., strain name: C57BL / 6JGpt, strain number: N000013) were used in 7 groups of 5 mice each. These groups were: normal control group (no treatment), solvent control group (containing 3% DMSO, 2% propylene glycol, 2% ethanol, and 2.5% polyoxyethylene 40 hydrogenated castor oil), gefitinib group, and compound groups 1, 56, 57, and 58. Each mouse was administered 100 μL of the corresponding drug intraperitoneally daily at a concentration of 10 mg / kg for 14 consecutive days. During this period, the weight of the mice was monitored. On day 14 after drug administration, blood samples were collected from mice to detect liver function (ALT and AST) and kidney function (BUN and CRE) biochemical indicators (the kits were all purchased from Nanjing Jiancheng Bioengineering Institute Co., Ltd., with catalog numbers: ALT: C009-2-1, AST: C010-2-1, BUN: C013-2-1, CRE: C011-2-1). In addition, liver and kidney tissues were subjected to histopathological HE staining.

[0070] The results showed that, compared with the normal control and solvent control groups, the experimental groups treated with gefitinib, compound 1, compound 56, compound 57, and compound 58 had no significant effect on mouse body weight; in fact, the mice's body weight showed an increasing trend, and their condition remained good during this period. Biochemical tests showed no significant changes in any of the treated groups compared to the normal control and solvent control groups. Pathological HE staining revealed no obvious abnormalities in liver and kidney tissues. These results suggest that compounds 56, 57, and 58 have good in vivo safety (as shown in Figures 11-13).

[0071] The above detailed description is a specific illustration of one feasible embodiment of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or modifications made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of the present invention should be determined by the appended claims.

Claims

1. A tyrosine kinase inhibitor, characterized in that, Selected from at least one of the following compounds, their pharmaceutically acceptable salts, stereoisomers, solvates, or prodrugs: Compound 56: A quinazoline derivative with a 4-fluoro-3-chlorophenylamino group at the 4-position and a 2,2,2-trifluoroethoxy group at the 7-position of the quinazoline core; Compound 57: A quinazoline derivative with a 4-fluoro-3-chlorophenylamino group at the 4-position and a deuterated ethoxy group at the 7-position; Compound 58: A quinazoline derivative with a 4-fluoro-3-chlorophenylamino group at the 4-position and a deuterated methoxy group at the 7-position.

2. The tyrosine kinase inhibitor according to claim 1, characterized in that, In the structural formula of compound 56, the 6-position of the quinazoline core is substituted with N-chloroacetylamino; in the structural formulas of compounds 57 and 58, the 6-position of the quinazoline core is also substituted with N-chloroacetylamino.

3. A method for preparing the tyrosine kinase inhibitor according to claim 1 or 2, characterized in that, Includes the following steps: S1: 4-chloro-6-nitro-7-fluoroquinazoline and 4-fluoro-3-chloroaniline are reacted in isopropanol with triethylamine and at 50-70°C for 5-7 hours to obtain the intermediate 4-(4-fluoro-3-chlorophenylamino)-7-fluoro-6-nitroquinazoline; S2: For compound 56, the intermediate from step S1 was reacted with 2,2,2-trifluoroethanol in a sodium hydride / tetrahydrofuran system at 90-110°C for 7-9 hours; for compound 57, the intermediate from step S1 was reacted with deuterated ethanol in the same system at the same temperature and time; for compound 58, the intermediate from step S1 was reacted with deuterated methanol in the same system at the same temperature and time, respectively, to obtain the corresponding 7-position substituted 6-nitroquinazoline derivatives; S3: The derivatives obtained in step S2 are reacted with reduced iron powder and ammonium chloride in an ethanol-water mixed solvent at 80-100℃ for 5-7 hours to complete the nitro reduction. Then, a tetrahydrofuran solution of chloroacetyl chloride is added dropwise under an ice bath, and the reaction is carried out at room temperature for 3-5 hours. The compounds 56, 57 or 58 are obtained by chromatography purification.

4. The preparation method according to claim 3, characterized in that, In step S1, the molar ratio of 4-chloro-6-nitro-7-fluoroquinazoline to 4-fluoro-3-chloroaniline is 1:1.0~1.2; in step S2, the molar ratio of sodium hydride to the intermediate of step S1 is 1.2~1.5:1; the molar ratio of 2,2-trifluoroethanol, deuterated ethanol, and deuterated methanol to the intermediate of step S1 is 1.1~1.3:1; in step S3, the molar ratio of chloroacetyl chloride to the nitro reduction product is 1.1~1.3:

1.

5. A pharmaceutical composition, characterized in that, The compound comprising a therapeutically effective amount of at least one of the tyrosine kinase inhibitors of claim 1 or 2.

6. The pharmaceutical composition according to claim 5, characterized in that, The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to any one of claims 5-6, characterized in that, The dosage form of the pharmaceutical composition is tablets, capsules, intravenous injection, or intraperitoneal injection.

8. Use of the tyrosine kinase inhibitor of claim 1 or 2 in the preparation of a medicament for the prevention or treatment of non-small cell lung cancer.

9. The use according to claim 8, characterized in that, The non-small cell lung cancer includes EGFR wild-type non-small cell lung cancer, EGFR mutant non-small cell lung cancer, non-small cell lung cancer resistant to gefitinib, and non-small cell lung cancer carrying EGFR T790M and / or C797S compound mutations.

10. The use according to claim 9, characterized in that, The EGFR-mutant non-small cell lung cancer includes the EGFR L858R mutant, the EGFR T790M mutant, and the EGFR T790M / C797S complex mutant.

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