EGFR (epidermal growth factor receptor) and PD-1 / PD-L1 double-target inhibitor as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and relates to an EGFR (epidermal growth factor receptor) and PD-1 / PD-L1 double-target inhibitor as well as a preparation method and application thereof. The compound is novel in structure, can act on EGFR (epidermal growth factor receptor) and PD-1 / PD-L1, and shows good EGFR kinase inhibitory activity and PD-1 / PD-L1 protein inhibitory activity; in addition, the compound has certain in-vitro anti-tumor activity, and tumor growth can be obviously delayed in vivo; the preparation is high in bioavailability, stable in medicine, low in toxicity and capable of being taken orally; in addition, the compound is convenient to prepare and low in production cost; the compound can be widely applied to preparation of EGFR / PD-L1 dual inhibitors and preparation of drugs for treating and / or preventing cancers, and the compound has further development and research values as a dual-target antitumor drug based on EGFR and PD-1 / PD-L1 reported for the first time.

Description

Technical Field

[0001] This invention relates to a dual-target inhibitor of EGFR and PD-1 / PD-L1, its preparation method, and its uses. Background Technology

[0002] Malignant tumors pose a serious threat to human health, and the search for highly effective, low-toxicity, and minimally toxic anti-tumor molecular targets is a key area of ​​current drug development. Protein tyrosine kinases are the largest known protein superfamily, and their abnormal activation is closely related to tumor development and progression, making them one of the most important and successful drug targets for current cancer treatment.

[0003] EGFR is a transmembrane protein tyrosine kinase member of the ERBB receptor family. When bound to growth factor ligands, such as epidermal growth factor (EGF), the receptor can homodimerize with the attached EGFR molecule or heterodimerize with another family member, such as ERBB2 (HER2), ERBB3 (HER3), or ERBB4 (HER4). Homodimerization or heterodimerization of the ERBB receptor leads to phosphorylation of key tyrosine residues in the intracellular domain and stimulates many intracellular signaling pathways involved in cell proliferation and survival. Currently, small molecule inhibitors targeting EGFR (such as gefitinib, erlotinib, and osimertinib) have been approved for the treatment of non-small cell lung cancer, but single-target EGFR inhibitors are prone to significant side effects and mutational resistance.

[0004] Multi-target inhibition of tumor signal transduction is a new direction for tumor treatment and drug development. Studies have shown that multi-target single-entity drugs have better therapeutic effects than single-target drugs and have fewer side effects.

[0005] PD-L1 (also known as B7-H1 or CD274) is one of the most extensively studied immune checkpoints in clinical practice. The extracellular domain of PD-L1 consists of immunoglobulin V-like and C-like domains, which are involved in recognizing PD-1. PD-L1 is expressed in tumor cells, epithelial cells, immune cells, and endothelial cells. PD-L1 acts as a pro-tumorigenic factor in cancer cells by binding to its receptor and activating proliferation and survival signaling pathways.

[0006] Studies have shown that PD-1 activation and downstream signaling of EGFR are interdependent and both are present in lung tumors. EGFR activity increases PD-L1 expression. Glioblastoma mouse model experiments demonstrated the interaction between PTEN-deficient EGFR proto-oncogenes. PTEN removal promoted PD-L1 overexpression, and PTEN deficiency led to increased PD-L1 expression. Furthermore, tumor invasiveness and PD-L1 expression are directly related to downstream signaling in tumor cells via the PI3K / Akt / mTORS6K1 pathway. These studies indicate that both PD-L1 and EGFR are involved in the development and progression of gliomas and are closely related. Other studies have shown that the combined use of EGFR inhibitors and PD-1 / PD-L1 inhibitors has a synergistic anti-tumor effect.

[0007] Based on the above research, we designed and synthesized a class of dual-target inhibitors of EGFR and PD-1 / PD-L1 in order to overcome the shortcomings of single-target inhibitors, and conducted in-depth research on the application of this class of dual-target inhibitors. Summary of the Invention

[0008] To overcome the problems existing in the prior art, one objective of the present invention is to provide a dual-target inhibitor of EGFR and PD-1 / PD-L1; a second objective of the present invention is to provide a method for preparing such a compound; a third objective of the present invention is to provide a pharmaceutical composition; and a fourth objective of the present invention is to provide the use of such a compound.

[0009] The technical solution adopted in this invention is:

[0010] Compounds having the structure shown in Formula I, or pharmaceutically acceptable salts, hydrates, solvates, polymorphs, tautomers, or prodrugs thereof; Formula I is:

[0011] In the formula,

[0012] R1 is selected from H, CH3, halogen, CH3O, or Z is selected from CH2 or CH2O, and R4 is selected from H, halogen, CN, NO2, NH2, N(CH3)2, NHCH3, OH, OCH3, OC2H5, OCF3, CF3, cyclopropyl, C 1-6 Alkyl, sulfonyl, sulfonylalkyl;

[0013] n = 0 to 2;

[0014] X is selected from CH or N;

[0015] Y is selected from CH2O, CH2NH, or a single bond;

[0016] R2 is selected from R5 is selected from H, halogen, methoxy, cyano, and m = 1 to 3;

[0017] R3 is selected from H, halogens, and CH3O;

[0018] L1 is selected from C 0-4 alkyl or Where A1 is C 1-2 alkyl;

[0019] L2 is selected from C 0-6 Alkyl or amide bonds;

[0020] L3 is C 1-8 Alkyl group, or single bond;

[0021] L4 is C 1-4 Alkyl group, or H (where AE does not form bonds).

[0022] Preferably, the compound comprises the structure shown below;

[0023] N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(4-((2-methoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)butoxy)quinazoline-4-amine (C1)

[0024] N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(4-((3-methyl-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)butoxy)quinazoline-4-amine (C2)

[0025] N-(3-chloro-4-fluorophenyl)-6-(4-((2,6-dimethoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)butoxy)-7-methoxyquinazoline-4-amine (C3)

[0026] 3-((4-chloro-2-(((4-(((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-yl)oxy)butyl)amino)methyl)-5-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)phenoxy)methyl)benzylnitrile (C4)

[0027] N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-((2-methoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)propoxy)quinazoline-4-amine (C5)

[0028] N-(3-chloro-4-fluorophenyl)-6-(2-((2,6-dimethoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)ethoxy)-7-methoxyquinazoline-4-amine (C6)

[0029] N-(3-chloro-4-fluorophenyl)-6-(3-((2,6-dimethoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)propoxy)-7-methoxyquinazoline-4-amine (C7)

[0030] N-(3-chloro-4-fluorophenyl)-6-((5-((2,6-dimethoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)pentyl)oxy)-7-methoxyquinazoline-4-amine (C8)

[0031] N-(3-chloro-4-fluorophenyl)-6-((6-((2,6-dimethoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)hexyl)oxy)-7-methoxyquinazoline-4-amine (C9)

[0032] 3-((4-chloro-2-(((3-((4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-yl)oxy)propyl)amino)methyl)-5-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)phenoxy)methyl)benzonitrile (C10)

[0033] 4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-yl4-(2,6-dimethoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)piperazine-1-carboxylate (C11)

[0034] N-(3-chloro-2-fluorophenyl)-7-methoxy-6-(4-((2-methoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)butoxy)quinazoline-4-amine (C12)

[0035] N-(3-chloro-2-fluorophenyl)-7-methoxy-6-(4-((2-methoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)butoxy)quinazoline-4-amine (C13)

[0036] N-(3-chloro-2-fluorophenyl)-6-(4-((2,6-dimethoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)butoxy)-7-methoxyquinazoline-4-amine (C14)

[0037] N-(3-chloro-4-methoxyphenyl)-6-(4-((2,6-dimethoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)butoxy)-7-methoxyquinazoline-4-amine (C15)

[0038] 3-((6-(4-(2,6-dimethoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)butoxy)-7-methoxyquinazoline-4-yl)amino)benzylnitrile (C16)

[0039] 6-(4-((2,6-dimethoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)butoxy)-7-methoxy-N-(quinolin-6-yl)quinazolin-4-amine (C17)

[0040] N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(4-(((2-methoxy-6-(2-methyl-[1,1'-biphenyl]-3-yl)pyridin-3-yl)methyl)amino)butoxy)quinazoline-4-amine (C18)

[0041] N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(2-((2-methoxy-6-(2-methyl-[1,1'-biphenyl]-3-yl)pyridin-3-yl)methyl)amino)ethoxy)quinazoline-4-amine (C19)

[0042] N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-((2-methoxy-6-(2-methyl-[1,1'-biphenyl]-3-yl)pyridin-3-yl]methyl)amino)propoxy)quinazoline-4-amine (C20)

[0043] N-(3-chloro-4-fluorophenyl)-6-((5-((2,6-dimethoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)pentyl)oxy)-7-methoxyquinazoline-4-amine (C21)

[0044] N-(3-chloro-4-fluorophenyl)-7-methoxy-6-((6-((2-methoxy-6-(2-methyl-[1,1'-biphenyl]-3-yl)pyridin-3-yl]methyl)amino)hexyl)oxy)quinazoline-4-amine (C22)

[0045] 4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-yl-4-((2-methoxy-6-(2-methyl-[1,1'-biphenyl]-3-yl)pyridin-3-yl]methyl)piperazine-1-carboxylate (C23)

[0046] N-(3-chloro-4-fluorophenyl)-7-methoxy-6-((8-((2-methoxy-6-(2-methyl-[1,1'-biphenyl]-3-yl)pyridin-3-yl]methyl)amino)octyl)oxy)quinazoline-4-amine (C24)

[0047] N-(5-((4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-yl)oxy)pentyl)-1-(2,6-dimethoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)piperidine-2-carboxamide (C25)

[0048] N-(6-((4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-yl)oxy)hexyl)-1-(2,6-dimethoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)piperidine-2-carboxamide (C26)

[0049] N-(5-((4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-yl)oxy)pentyl)-2-((2,6-dimethoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)(methyl)amino)acetamide (C27)

[0050] N-(8-((4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-yl)oxy)octyl)-2-((2,6-dimethoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)(methyl)amino)acetamide (C28)

[0051] N-(6-((4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-yl)oxy)hexyl)-1-(3-methyl-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)piperidine-2-carboxamide (C29)

[0052] (S)-N-(6-((4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-yl)oxy)hexyl)-1-(3-methyl-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)piperidine-2-carboxamide (C30)

[0053] 1-(5-chloro-2-((3-cyanobenzyl)oxy)-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)-N-(4-((4-(3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-yl)oxy)butyl)piperidine-2-carboxamide (C31)

[0054] N-(4-((4-(3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-yl)oxy)butyl)-1-((2-methoxy-6-(2-methyl-[1,1'-biphenyl]-3-yl)pyridin-3-yl]methyl)piperidine-2-carboxamide (C32)

[0055] N-(5-((4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-yl)oxy)pentyl)-1-((2-methoxy-6-(2-methyl-[1,1'-biphenyl]-3-yl)pyridin-3-yl]methyl)piperidine-2-carboxamide (C33)

[0056] N-(6-((4-(((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-yl)oxy)hexyl)-3-((2-methoxy-6-(2-methyl-[1,1'-biphenyl]-3-yl)pyridin-3-yl]methyl)amino)propionamide (C34)

[0057] The present invention provides a pharmaceutical composition comprising at least one of the following active ingredients: a) a compound, b) a pharmaceutically acceptable salt of the compound, c) a hydrate of the compound, d) a solvate of the compound, e) a polymorph of the compound, f) a tautomer of the compound, and g) a prodrug of the compound; wherein the compound is a compound having the structure shown in general formula I as described above.

[0058] The drug applications described in this invention include tumors related to the EGFR signaling pathway or tumors related to the PD-1 / PD-L1 signaling pathway.

[0059] Preferably, the tumor diseases are lung cancer, liver cancer, kidney cancer, non-small cell lung cancer, prostate cancer, thyroid cancer, skin cancer, pancreatic cancer, ovarian cancer, breast cancer, bladder cancer, myelodysplastic syndrome, lymphoma, esophageal cancer, gastrointestinal cancer, and tumors of the central or peripheral nervous system.

[0060] The beneficial effects of this invention are:

[0061] 1. The compound disclosed in this invention has a novel structure and can act on EGFR and PD-1 / PD-L1, exhibiting good EGFR kinase inhibitory activity and PD-1 / PD-L1 protein inhibitory activity; it also has certain in vitro antitumor activity and can significantly delay tumor growth in vivo; this invention has high bioavailability, drug stability, low toxicity, and can be administered orally; in addition, the compound is easy to prepare and has low production cost; this compound can be widely used in the preparation of EGFR / PD-L1 dual inhibitors, as well as in the preparation of drugs for the treatment and / or prevention of cancer.

[0062] 2. As the first reported dual-target antitumor drugs based on EGFR and PD-1 / PD-L1, these compounds have further development and research value. Attached Figure Description

[0063] Figure 1 Example 14 (C14) shows the inhibitory effect on the growth of subcutaneous glioma tumors in C57BL / 6 mice. Detailed Implementation

[0064] The following examples further illustrate specific implementations of the present invention, but the implementation and protection of the present invention are not limited thereto. It should be noted that any processes not specifically described below are those that can be implemented or understood by those skilled in the art by referring to existing technology. Reagents or instruments whose manufacturers are not specified are considered to be conventional products that can be purchased commercially.

[0065] The reaction routes for the preparation of compounds 1-17 (C1-C17) in Examples are as follows:

[0066]

[0067] Example 1:

[0068] N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-((2-methoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)propoxy)quinazoline-4-amine (C1)

[0069] 1) Synthesis of intermediate compound 3a

[0070]

[0071] 3-(bromomethyl)-2-methyl-1,1'-biphenyl (compound 2) (2.0 g, 7.7 mmol), 4-hydroxy-2-methoxy-benzaldehyde (1.4 g, 9.2 mmol), and potassium carbonate (2.1 g, 15.3 mmol) were dissolved in 15 mL of N,N-dimethylformamide. The mixture was stirred at 60 °C for 4 h. After the reaction was complete, 75 mL of water was added, and the mixture was stirred for another 6 h. The mixture was then filtered and concentrated to give 2.4 g of a white solid, with a yield of 94%. ESI-MS m / z: 333.15 [M+H] + ;

[0072] 2) Synthesis of intermediate compound 8a-1

[0073]

[0074] 2.2 g (8.5 mmol) of 4-chloro-7-methoxyquinazoline-6-acetate and 1.2 g (8.5 mmol) of 3-chloro-4-fluoroaniline were dissolved in 80 mL of isopropanol and heated at 80 °C for 3 h. After the reaction was complete, the solvent was evaporated to dryness, and 20 mL of methanol was added to precipitate the solid. The precipitate was filtered, washed three times with methanol, and dried to give 3.0 g of a pale yellow solid (compound 5a), with a yield of 97%. ESI-MS m / z: 362.07 [M+H] + ;

[0075] Compound 5a (2.2 g, 6.1 mmol) was dissolved in 20 mL of methanol, then 3 mL of ammonia was added. The mixture was stirred at room temperature for 1 h. After the reaction was complete, the reaction solution was concentrated and passed through a gradient rapid column chromatography (methanol / dichloromethane = 1%–4%) to give 1.8 g of a white solid (compound 6a), with a yield of 94%. ESI-MS m / z: 320.06 [M+H] + ;

[0076] Compound 6a (291 mg, 0.91 mmol), N-(4-bromobutyl)phthalimide (308 mg, 1.09 mmol), and potassium carbonate (252 mg, 1.82 mmol) were dissolved in 7 mL of DMF and heated at 80 °C for 2 h. After the reaction was complete, 40 mL of water was added, and the mixture was extracted three times with ethyl acetate. The combined organic phases were dried over anhydrous magnesium sulfate, filtered, concentrated, and passed through a gradient rapid column chromatography (methanol / dichloromethane = 5%–10%) to give 289 mg of compound (7a-1), with a yield of 61%. ESI-MS m / z: 521.13 [M+H] + ;

[0077] Compound 7a-1 (389 mg, 0.75 mmol) was dissolved in 10 mL of anhydrous ethanol, and 0.4 mL of hydrazine hydrate was added. The mixture was heated under reflux for 6 h. After the reaction was complete as monitored by TLC, 1 M hydrochloric acid was added to adjust the pH to 3, and impurities were washed three times with dichloromethane. Then, saturated sodium bicarbonate solution was added to the aqueous phase to adjust the pH to 8, and the mixture was extracted three times with dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate and evaporated under reduced pressure to obtain 250 mg of compound (8a-1), with a yield of 86%. ESI-MS m / z: 391.12 [M+H] + ;

[0078] 3) Target compound N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(4-((2-methoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)butoxy)quinazoline-4-amine (C1)

[0079]

[0080] Compound 8a-1 (63 mg, 0.16 mmol) and compound 3a (45 mg, 0.13 mmol) were dissolved in 8 mL of a 1:1 mixture of dichloromethane and methanol. NaBH3CN (42 mg, 0.67 mmol) and acetic acid (0.02 mL) were added, and the reaction was carried out at room temperature for 12 h. The reaction solution was concentrated and passed through a gradient rapid column chromatography (methanol / dichloromethane = 4%–8%) to give 33 mg of the product, with a yield of 61%. 1 HNMR(400MHz,DMSO)δ9.58(s,1H),8.51(s,1H),8.13(dd,J=6.7,2.4Hz,1H),7.84(s,1H),7.81(dd,J=8.3,3.4Hz,1H),7.47 (s,1H),7.46(s,1H),7.45(s,1H),7.43(d,J=6.0Hz,1H),7.39(d,J=7.2Hz,1H),7.35(d,J=9.1Hz,1H),7.32(s,1H),7.30(s ,1H),7.27(d,J=7.5Hz,1H),7.23(s,1H),7.20(d,J=7.3Hz,1H),6.77(s,1H),6.70(d,J=8.3Hz,1H),5.16(s,2H),4.18(d,J =5.2Hz,2H),4.02(s,2H),3.94(s,3H),3.83(s,3H),2.99(d,J=7.0Hz,2H),2.19(s,3H),1.89(d,J=5.7Hz,2H),1.85(s,2H). ESI-MS m / z:707.25[M+H] + .

[0081] Example 2:

[0082] N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(4-((3-methyl-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)butoxy)quinazoline-4-amine (C2)

[0083]

[0084] After synthesizing compound 3b according to Example 1, the target compound C2 was synthesized according to the method for synthesizing target compound C1 in Example 1, with a yield of 52%. 1H NMR (400MHz, CDCl3) δ8.49 (s, 1H), 7.99 (s, 1H), 7.61 (d, J = 2.4Hz, 1H), 7.42 (s, 5 H),7.36–7.28(m,3H),7.25(s,2H),7.15(t,J=20.4Hz,3H),7.08–7.02(m,1H),6. 96(d,J=15.9Hz,1H),6.87(d,J=5.6Hz,1H),5.01(s,2H),4.04(s,2H),3.93(s,2H ),3.81(s,3H),2.95(s,2H),2.26(s,2H),2.21(s,3H),1.98(s,2H),1.90(s,3H). ESI-MS m / z: 691.28 [M+H] + .

[0085] Example 3:

[0086] N-(3-chloro-4-fluorophenyl)-6-(4-((2,6-dimethoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)butoxy)-7-methoxyquinazoline-4-amine (C3)

[0087]

[0088] After synthesizing compound 3c according to Example 1, the synthesis of target compound C3 was carried out according to the synthesis method of target compound C1 in Example 1, with a yield of 53%. 1 H NMR (400MHz, CDCl3) δ8.80(s,1H),8.48(s,1H),8.04(d,J=6.5Hz,1H),7.66(d,J=7.4H z,1H),7.56(s,1H),7.43–7.35(m,4H),7.29(d,J=7.1Hz,2H),7.26(d,J=3.2Hz,2H),7. 03(dd,J=17.1,8.3Hz,2H),6.17(s,2H),5.03(s,2H),4.16(s,3H),3.75(s,2H),3.74( s, 6H), 3.03 (s, 2H), 2.23 (s, 3H), 1.98 (d, J = 12.4Hz, 4H), 1.25 (dd, J = 18.7, 8.9Hz, 2H). ESI-MS m / z:737.22[M+H] + .

[0089] Example 4:

[0090] 3-((4-chloro-2-(((4-(((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-yl)oxy)butyl)amino)methyl)-5-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)phenoxy)methyl)benzylnitrile (C4)

[0091] 1) Synthesis of intermediate compound 4d

[0092]

[0093] Compound 4d was synthesized according to Example 1. 3d (0.4 g, 1.1 mmol), 3-(bromomethyl)benzyl nitrile (431.8 mg, 2.2 mmol), and K₂CO₃ (303.6 mg, 2.2 mmol) were dissolved in 10 mL of DMF and reacted at 60 °C for 6 h with stirring. After the reaction was completed by TLC, appropriate amounts of water and ethyl acetate were added for extraction. The solvent in the organic phase was removed under reduced pressure. The crude product was dissolved in 10 mL of DCM, and 200 mL of petroleum ether was added. The mixture was stirred for 2 h, and the precipitate was collected by filtration and washed with petroleum ether to give a white solid 4d (395.5 mg, 77%). 1 H NMR (400MHz, CDCl3) δ10.34(s,1H),7.94(s,1H),7.75(s,1H),7.70(s,2H),7.57(t,J=7.7Hz,1H),7.49–7.42(m, 3H), 7.40 (d, J = 6.4Hz, 1H), 7.34 (s, 1H), 7.31 (d, J = 4.5Hz, 3H), 6.65 (s, 1H), 5.23 (d, J = 6.6Hz, 4H), 2.29 (s, 3H). ESI-MS m / z:467.14[M+H] + .

[0094] 2) Synthesis of 3-((4-chloro-2-(((4-(((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-yl)oxy)butyl)amino)methyl)-5-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)phenoxy)methyl)benzylnitrile (C4):

[0095]

[0096] The target compound C4 was synthesized according to the method for synthesizing target compound C1 in Example 1, with a yield of 59%. 1H NMR (400MHz, CDCl3) δ8.59(s,1H),8.27(s,1H),7.99(d,J=2.6Hz,1H),7.79(s,1H),7.71–7.65(m,1H),7.59(t,J=8.8Hz,2H), 7.49(d,J=7.7Hz,1H),7.47–7.44(m,1H),7.44(s,1H),7.42(d,J=2.8Hz,1H),7.40(s,1H),7.39(s,1H),7.36(d,J=7.6Hz,1H), 7.33–7.31(m,2H),7.30(s,1H),7.26(d,J=4.5Hz,2H),7.14(s,1H),7.11(t,J=8.8Hz,1H),6.60(s,1H),5.11(s,2H),5.06(s,2 H),4.12(t,J=6.5Hz,2H),3.90(s,3H),3.86(s,2H),2.87(t,J=7.0Hz,2H),2.26(s,3H),1.98–1.89(m,2H),1.85–1.74(m,2H). ESI-MS m / z:842.16[M+H] + .

[0097] Example 5:

[0098] N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-((2-methoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)propoxy)quinazoline-4-amine (C5)

[0099]

[0100] After synthesizing compound 8a-2 according to Example 1, the synthesis of target compound C5 was carried out according to the method for synthesizing target compound C1 in Example 1, with a yield of 51%. 1H NMR (400MHz, CDCl3) δ8.97(s,1H),8.62(s,1H),8.13(s,1H),7.82(s,1H),7.77(s,1H),7.44( s,1H),7.42(s,1H),7.42–7.41(m,1H),7.38(s,1H),7.37–7.36(m,1H),7.32(s,1H),7.31–7. 30(m,1H),7.26–7.23(m,1H),7.20(s,1H),7.08(s,1H),7.07–7.04(m,1H),6.55(s,2H),5.02 (s,2H),4.25(s,2H),4.08(s,2H),3.76(s,6H),3.10(s,2H),2.24(s,2H),2.23–2.21(m,3H). ESI-MS m / z:693.77[M+H] + .

[0101] Example 6:

[0102] N-(3-chloro-4-fluorophenyl)-6-(2-((2,6-dimethoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)ethoxy)-7-methoxyquinazoline-4-amine (C6)

[0103]

[0104] The target compound C6 was synthesized according to the method for synthesizing target compound C1 in Example 1, with a yield of 44%. 1 H NMR(400MHz, CDCl3)δ8.76(s,1H),8.44(s,1H),8.05(dd,J=6.6,2.4Hz,1H),7.63–7.57(m,1H ),7.52(s,1H),7.44(s,1H),7.41(s,1H),7.41(s,1H),7.37(d,J=7.2Hz,1H),7.32(s,1H),7. 30(s,1H),7.28(d,J=3.6Hz,1H),7.27(s,1H),7.05(t,J=8.8Hz,1H),6.85(s,1H),6.28(s,2H ),5.11(s,2H),4.53(s,2H),4.37(s,2H),3.88(s,6H),3.78(s,3H),3.44(s,2H),2.27(s,3H). ESI-MS m / z:709.23[M+H] + .

[0105] Example 7:

[0106] N-(3-chloro-4-fluorophenyl)-6-(3-((2,6-dimethoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)propoxy)-7-methoxyquinazoline-4-amine (C7)

[0107]

[0108] After synthesizing compound 8a-3 according to Example 1, the target compound C7 was synthesized according to the method for synthesizing target compound C1 in Example 1, with a yield of 42%. 1 H NMR(400MHz, CDCl3)δ9.60(s,1H),8.40(s,1H),7.83–7.78(m,1H),7.60(s,1H),7.53(d,J= 8.3Hz,1H),7.44(s,1H),7.42(s,1H),7.40(s,1H),7.36(d,J=7.1Hz,1H),7.31(d,J=1.2Hz ,1H),7.29(s,1H),7.28(d,J=3.7Hz,1H),7.26(s,1H),7.01(t,J=8.6Hz,2H),6.27(s,2H), 5.11(s,2H),4.38(s,2H),4.31(s,2H),3.83(s,9H),3.31(s,2H),2.42(s,2H),2.27(s,3H). ESI-MS m / z:723.22[M+H] + .

[0109] Example 8:

[0110] N-(3-chloro-4-fluorophenyl)-6-((5-((2,6-dimethoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)pentyl)oxy)-7-methoxyquinazoline-4-amine (C8)

[0111]

[0112] After synthesizing compound 8a-4 according to Example 1, the target compound C8 was synthesized according to the method for synthesizing target compound C1 in Example 1, with a yield of 56%. 1H NMR(400MHz, CDCl3)δ9.01(s,1H),8.57(s,1H),8.06(dd,J=6.6,2.4Hz,1H),7.77–7.71(m,1H),7.57(s,1H),7.45( d,J=7.1Hz,1H),7.42(d,J=4.6Hz,1H),7.39(s,1H),7.37(d,J=7.4Hz,1H),7.32(d,J=7.1Hz,2H),7.28(d,J=4.3Hz ,2H),7.09(s,1H),7.06(d,J=8.8Hz,1H),6.19(s,2H),5.04(s,2H),4.18(s,2H),4.00(t,J=6.8Hz,2H),3.88(s,3H ),3.78(s,6H),2.92(t,J=7.0Hz,2H),2.26(s,3H),1.86(d,J=6.4Hz,2H),1.83(d,J=6.3Hz,2H),1.59–1.48(m,2H). ESI-MS m / z:751.42[M+H] + .

[0113] Example 9:

[0114] N-(3-chloro-4-fluorophenyl)-6-((6-((2,6-dimethoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)hexyl)oxy)-7-methoxyquinazoline-4-amine (C9)

[0115]

[0116] After synthesizing compound 8a-5 according to Example 1, the target compound C9 was synthesized according to the method for synthesizing target compound C1 in Example 1, with a yield of 60%. 1H NMR (400MHz, CDCl3) δ8.98 (s, 1H), 8.51 (s, 1H), 8.08 (dd, J = 6.6, 2.4Hz, 1H), 7.71–7.65 (m, 1H), 7.50 (s, 1H), 7. 45(d,J=7.2Hz,1H),7.41(d,J=4.2Hz,1H),7.39(s,1H),7.36(d,J=7.3Hz,1H),7.33(s,1H),7.31(s,1H),7.28( s,1H),7.27(s,1H),7.10(t,J=8.8Hz,1H),6.99(s,1H),6.16(s,2H),5.03(s,2H),4.14(s,2H),4.02(t,J=6.7H z,2H),3.83(s,3H),3.75(s,6H),2.96(t,J=6.7Hz,2H),2.25(s,3H),1.83(dd,J=12.3,6.2Hz,4H),1.49(s,4H). ESI-MS m / z:765.27[M+H] + .

[0117] Example 10:

[0118] 3-((4-chloro-2-(((3-((4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-yl)oxy)propyl)amino)methyl)-5-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)phenoxy)methyl)benzonitrile (C10)

[0119]

[0120] After synthesizing compound 4e according to Example 4, the target compound C10 was synthesized according to the method for synthesizing target compound C1 in Example 1, with a yield of 59%. 1H NMR (400MHz, DMSO) δ9.56 (s, 1H), 8.52 (s, 1H), 8.14 (d, J = 6.7Hz, 1H), 7.98 (s, 1H), 7.85–7.78(m,4H),7.63–7.57(m,2H),7.47(t,J=8.6Hz,4H),7.40(dd,J=13.7,6.8H z,2H),7.33–7.28(m,3H),7.23(d,J=6.4Hz,2H),7.19(s,1H),5.34(s,2H),5.32(s, 2H),4.25(s,2H),4.19(s,2H),3.88(s,3H),3.17(s,2H),2.25(s,3H),2.21(s,2H). ESI-MS m / z:878.24[M+H] + .

[0121] Example 11:

[0122] 4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-yl4-(2,6-dimethoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)piperazine-1-carboxylate (C11)

[0123] 1) Synthesis of intermediate compound (8a-6)

[0124]

[0125] Intermediates 4-Boc-1-piperazincarbamoyl chloride (1.5 g, 6.03 mmol), 6a (1.93 g, 6.03 mmol), and K₂CO₃ (1.67 g, 12.06 mmol) were dissolved in 10 mL of dry DMF and stirred overnight at room temperature. Five times the volume of water was added to the reaction mixture, and the mixture was filtered. The filter cake was dried to give a yellow solid 7a-6 (2.8 g, 87%). ESI-MS m / z: 532.18 [M+H] + .

[0126] Compound 7a-6 (250 mg, 0.47 mmol) was dissolved in 5 mL of methanol, and 1 mL of 4 M 1,4-dioxane hydrochloride was added. The mixture was stirred at room temperature for 1 h, and the reaction was monitored by TLC. The solvent was evaporated under reduced pressure, reconstituted with 30 mL of DCM, and washed three times with water. The organic layer was concentrated to give a yellow solid 8a-6 (150 mg, 74%). ESI-MS m / z: 432.12 [M+H] + .

[0127] 2) 4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-yl4-(2,6-dimethoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)piperazine-1-carboxylate (C11)

[0128]

[0129] The target compound C11 was synthesized according to the method of Example 1, with a yield of 58%. 1 H NMR (400MHz, CDCl3) δ8.47(s,1H),8.12(s,1H),7.82(dd,J=6.5,2.2Hz,1H),7.77(dd,J=8.7,3.3Hz ,1H),7.68(s,1H),7.46(s,1H),7.44(s,1H),7.42(s,1H),7.37(d,J=6.9Hz,1H),7.34(s,1H),7.32( s,1H),7.29(d,J=6.6Hz,2H),7.08(t,J=8.8Hz,1H),6.79(s,1H),6.31(s,2H),5.12(s,2H),3.84(s, 6H), 3.80 (s, 2H), 3.78 (s, 2H), 3.78–3.74 (m, 2H), 3.37 (s, 3H), 2.71 (d, J = 16.2Hz, 4H), 2.29 (s, 3H). ESI-MS m / z:778.27[M+H] + .

[0130] Example 12:

[0131] N-(3-chloro-2-fluorophenyl)-7-methoxy-6-(4-((2-methoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)butoxy)quinazoline-4-amine (C12)

[0132]

[0133] After synthesizing compounds 8a-7 according to Example 1, the target compound C12 was synthesized according to the method for synthesizing target compound C1 in Example 1, with a yield of 59%. 1H NMR (400MHz, CDCl3) δ8.49(s,1H),7.73(t,J=7.2Hz,1H),7.42(s,1H),7.41(s,1H),7.39(s,1H),7.37(d,J=6. 4Hz,1H),7.33(d,J=7.3Hz,1H),7.30(s,1H),7.28(s,1H),7.26(s,1H),7.24(d,J=1.8Hz,1H),7.23(s,1H),7. 20(d,J=8.3Hz,1H),7.15(t,J=7.2Hz,1H),7.05(s,1H),7.02(d,J=4.2Hz,1H),6.54(d,J=8.4Hz,1H),6.50(s, 1H),5.02(s,2H),4.08(s,2H),4.04(s,2H),3.73(s,3H),3.70(s,3H),3.03(s,2H),2.21(s,3H),1.95(s,4H). ESI-MS m / z:707.25[M+H] + .

[0134] Example 13:

[0135] N-(3-chloro-2-fluorophenyl)-7-methoxy-6-(4-((2-methoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)butoxy)quinazoline-4-amine (C13)

[0136]

[0137] After synthesizing compound 8b-1 according to Example 1, the synthesis of target compound C13 was carried out according to the synthesis method of target compound C1 in Example 1, with a yield of 59%. 1H NMR (400MHz, CDCl3) δ8.49(s,1H),7.73(t,J=7.2Hz,1H),7.42(s,1H),7.41(s,1H),7.39(s,1H),7.37(d,J=6. 4Hz,1H),7.33(d,J=7.3Hz,1H),7.30(s,1H),7.28(s,1H),7.26(s,1H),7.24(d,J=1.8Hz,1H),7.23(s,1H),7. 20(d,J=8.3Hz,1H),7.15(t,J=7.2Hz,1H),7.05(s,1H),7.02(d,J=4.2Hz,1H),6.54(d,J=8.4Hz,1H),6.50(s, 1H),5.02(s,2H),4.08(s,2H),4.04(s,2H),3.73(s,3H),3.70(s,3H),3.03(s,2H),2.21(s,3H),1.95(s,4H). ESI-MS m / z:707.25[M+H] + .

[0138] Example 14:

[0139] N-(3-chloro-2-fluorophenyl)-6-(4-((2,6-dimethoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)butoxy)-7-methoxyquinazoline-4-amine (C14)

[0140]

[0141] After synthesizing compound 8b-1 according to Example 1, the target compound C14 was synthesized according to the method for synthesizing target compound C1 in Example 1, with a yield of 59%. 1H NMR (400MHz, CDCl3) δ8.50(s,1H),7.72(t,J=7.3Hz,1H),7.51(s,1H),7.44(d,J=7.3Hz,1H),7. 42(s,1H),7.41(d,J=4.8Hz,1H),7.37(d,J=7.1Hz,1H),7.34(d,J=8.9Hz,2H),7.31(s,1H),7.2 8–7.24(m,2H),7.17(t,J=7.2Hz,1H),7.07(s,1H),7.04(d,J=8.5Hz,1H),6.18(s,2H),5.06(s, 2H),4.15(s,2H),4.14(s,2H),3.78(s,3H),3.75(s,6H),3.05(s,2H),2.26(s,3H),1.99(s,4H). ESI-MS m / z:737.22[M+H] + .

[0142] Example 15:

[0143] N-(3-chloro-4-methoxyphenyl)-6-(4-((2,6-dimethoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)butoxy)-7-methoxyquinazoline-4-amine (C15)

[0144]

[0145] After synthesizing compound 8c-1 according to Example 1, the synthesis of target compound C15 was carried out according to the synthesis method of target compound C1 in Example 1, with a yield of 65%. 1 H NMR (400MHz, CDCl3) δ8.84 (s, 1H), 8.50 (s, 1H), 7.92 (d, J = 2.1Hz, 1H), 7.62 (d ,J=11.5Hz,2H),7.43–7.35(m,4H),7.30(d,J=7.4Hz,2H),7.25(s,1H),7.04(s ,1H),6.87(d,J=8.9Hz,1H),6.16(s,2H),5.02(s,2H),4.13(d,J=9.7Hz,4H), 3.85(s,3H),3.79(s,3H),3.73(s,6H),3.01(s,2H),2.23(s,3H),1.95(s,4H). ESI-MS m / z: 749.26 [M+H] + .

[0146] Example 16:

[0147] 3-((6-(4-(2,6-dimethoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)butoxy)-7-methoxyquinazoline-4-yl)amino)benzylnitrile (C16)

[0148]

[0149] After synthesizing compound 8d-1 according to Example 1, the target compound C16 was synthesized according to the method for synthesizing target compound C1 in Example 1, with a yield of 73%. 1 H NMR (400MHz, CDCl3) δ8.47(s,1H),8.30(s,1H),8.08(d,J=8.3Hz,1H),7.54(s,1H),7.40(dd,J=13.6,6.2Hz,3H),7.36–7.32(m,2H),7.31–7.28( m,2H),7.25(d,J=3.0Hz,3H),7.01(s,1H),6.18(s,2H),5.03(s,2H),4. 19(s,4H),3.76(d,J=3.2Hz,9H),3.07(s,2H),2.23(s,3H),2.01(s,4H). ESI-MS m / z:710.30[M+H] + .

[0150] Example 17:

[0151] 6-(4-((2,6-dimethoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)butoxy)-7-methoxy-N-(quinolin-6-yl)quinazolin-4-amine (C17)

[0152]

[0153] After synthesizing compound 8e-1 according to Example 1, the synthesis of target compound C17 followed the method for synthesizing target compound C1 in Example 1, with a yield of 61%. 1H NMR (400MHz, CDCl3) δ8.95 (s, 1H), 8.61 (d, J = 3.9Hz, 1H), 8.51 (s, 1H), 8.37 (s, 1H), 8. 09(d,J=9.1Hz,1H),7.97(d,J=8.2Hz,1H),7.85(d,J=9.1Hz,1H),7.51(s,1H),7.41–7 .32(m,4H),7.27(d,J=6.0Hz,2H),7.21(t,J=4.0Hz,3H),6.90(s,1H),6.12(s,2H),4. 99(s,2H),4.11(s,4H),3.70(d,J=9.3Hz,9H),3.01(s,2H),2.20(s,3H),1.92(s,4H). ESI-MS m / z:736.34[M+H] + .

[0154] Synthetic routes for Examples 18-24 (C18-C24):

[0155]

[0156] Example 18:

[0157] N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(4-(((2-methoxy-6-(2-methyl-[1,1'-biphenyl]-3-yl)pyridin-3-yl)methyl)amino)butoxy)quinazoline-4-amine (C18)

[0158] 1) Synthesis of intermediate compound 14

[0159] 1-Bromo-3-iodo-2-toluene (compound 11) (1.2 g, 4.03 mmol), phenylboronic acid (590 mg, 4.84 mmol), and potassium carbonate (1.11 g, 8.06 mmol) were dissolved in 48 mL of dioxane / water solution (mixing ratio 8:1). Tetraphenylphosphine palladium (233 mg, 0.20 mmol) was added, and the mixture was heated and stirred at 92 °C for 12 h under nitrogen protection. The reaction solution was filtered, the filtrate was concentrated, and the solution was passed through a gradient rapid column chromatography (ethyl acetate / petroleum ether = 0%–5%) to give 800 mg of a colorless oily liquid (compound 12), with a yield of 80%. ESI-MS m / z: 247.01 [M+H] + .

[0160] Compound 12 (2.0 g, 8.1 mmol), sodium borate ester (2.5 g, 9.7 mmol), and cesium carbonate (2.4 g, 24.3 mmol) were dissolved in 40 mL of dioxane. 1,1'-bis(di-tert-butylphosphino)ferrocene palladium dichloride (296.0 mg, 0.4 mmol) was added, and the mixture was heated and stirred at 80 °C for 12 h under nitrogen protection. The reaction solution was filtered, the filtrate was concentrated, and the solution was passed through a gradient rapid column chromatography (ethyl acetate / petroleum ether = 0%–5%) to give 850 mg of a colorless solid (compound 13), with a yield of 35%. ESI-MS m / z: 295.19 [M+H] + .

[0161] Compound 13 (790.0 mg, 2.7 mmol), methyl 6-chloro-2-methoxynicotinic acid (650.0 mg, 3.2 mmol), and potassium carbonate (742.0 mg, 5.4 mmol) were dissolved in 18 mL of dioxane / water solution (mixing ratio 8:1). Tetraphenylphosphine palladium (156.0 mg, 0.1 mmol) was added, and the mixture was heated and stirred overnight at 92 °C under nitrogen protection. The reaction solution was filtered, the filtrate was concentrated, and the solution was passed through a gradient rapid column chromatography column (ethyl acetate / petroleum ether = 5%–15%) to give 426 mg of a white solid (compound 14), in a yield of 48%. ESI-MS m / z: 334.14 [M+H] + .

[0162] 2) Synthesis of intermediate compound 15

[0163] Compound 14 (2.0 g, 6.0 mmol) was dissolved in 20 mL of anhydrous tetrahydrofuran. Lithium aluminum hydride (683.0 mg, 18.0 mmol) was added with stirring in an ice bath, and the mixture was then stirred at room temperature for 3 h. The reaction solution was quenched with ethanol, the solvent was evaporated to dryness under reduced pressure, 80 mL of water was added, and the mixture was extracted three times with ethyl acetate. The combined organic phases were dried over anhydrous magnesium sulfate and evaporated to dryness under reduced pressure to give a white solid.

[0164] The white solid was dissolved in 20 mL of dichloromethane. Desmartin reagent (2.5 g, 5.9 mmol) was added under ice bath conditions, followed by stirring at room temperature for 1 h. The reaction was quenched with saturated sodium thiosulfate solution after completion. The mixture was concentrated under vacuum, then 80 mL of water was added, and the mixture was extracted three times with ethyl acetate. The combined organic phases were dried over anhydrous magnesium sulfate, evaporated to dryness under reduced pressure, and passed through a gradient rapid chromatography column (ethyl acetate / petroleum ether = 5%–15%) to give 1.2 g of a pale yellow solid (compound 15), in 80% yield. ESI-MS m / z: 304.13 [M+H] + .

[0165] 3) N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(4-(((2-methoxy-6-(2-methyl-[1,1'-biphenyl]-3-yl)pyridin-3-yl)methyl)amino)butoxy)quinazoline-4-amine (C18)

[0166]

[0167] Using compounds 8a-1 and 15, target compound C18 was obtained by referring to the synthesis method of target compound C1 in Example 1, with a yield of 51%. 1 H NMR(400MHz, CDCl3)δ8.59(s,1H),7.84(d,J=7.6Hz,2H),7.55–7.49(m,1H),7.41(t,J=7 .2Hz,3H),7.35(d,J=7.1Hz,1H),7.32(s,2H),7.30(s,1H),7.28(s,1H),7.26(d,J=3.8Hz ,2H),7.22(d,J=6.9Hz,2H),7.10(d,J=8.6Hz,1H),7.00(s,1H),4.03(s,2H),3.99(s,3H) ,3.96(s,3H),3.87(s,2H),2.84(s,2H),2.19(s,3H),2.00(d,J=6.0Hz,2H),1.91(s,2H). ESI-MS m / z:678.28[M+H] + .

[0168] Example 19:

[0169] N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(2-((2-methoxy-6-(2-methyl-[1,1'-biphenyl]-3-yl)pyridin-3-yl)methyl)amino)ethoxy)quinazoline-4-amine (C19)

[0170]

[0171] Using compounds 8a-2 and 15, target compound C19 was obtained by referring to the synthesis method of target compound C1 in Example 1, with a yield of 47%. 1H NMR (400MHz, CDCl3) δ8.59(s,1H),7.96(dd,J=6.6,2.0Hz,1H),7.67(d,J=7.4Hz,1H) ,7.64–7.59(m,1H),7.48(s,1H),7.44–7.40(m,2H),7.35(d,J=7.5Hz,5H),7.30(d,J =8.0Hz,2H),7.16(s,1H),7.11(t,J=8.7Hz,1H),7.04(d,J=7.4Hz,1H),4.42(t,J=5. 3Hz, 2H), 3.99 (s, 3H), 3.95 (s, 3H), 3.24 (t, J = 5.4Hz, 2H), 2.37 (s, 2H), 2.22 (s, 3H). ESI-MS m / z:650.16[M+H] + .

[0172] Example 20:

[0173] N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-((2-methoxy-6-(2-methyl-[1,1'-biphenyl]-3-yl)pyridin-3-yl]methyl)amino)propoxy)quinazoline-4-amine (C20)

[0174]

[0175] The target compound C20 was obtained using compounds 8a-1 and 14 according to the synthetic method of target compound C1 in Example 1, with a yield of 44%. 1 H NMR (400MHz, CDCl3) δ8.55(s,1H),7.99(d,J=2.1Hz,1H),7.71(d,J=4.8Hz,1H),7.63(d,J=3.6Hz,1H),7.45(s,2H),7.43(s,1H),7.37( s,4H),7.30(d,J=9.4Hz,2H),7.06(s,3H),4.25(s,2H),4.09(s,2H),3.94(s,3H),3.82(s,3H),3.18(s,2H),2.28(s,2H),2.23(s,3H). ESI-MS m / z:664.22[M+H] + .

[0176] Example 21:

[0177] N-(3-chloro-4-fluorophenyl)-6-((5-((2,6-dimethoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)pentyl)oxy)-7-methoxyquinazoline-4-amine (C21)

[0178]

[0179] The target compound C21 was obtained using compounds 8a-4 and 14 according to the synthetic method of target compound C11 in Example 11, with a yield of 48%. 1 H NMR (400MHz, DMSO) δ9.53(s,1H),8.49(s,1H),8.10(dd,J=6.7,2.5Hz,1H),7.87(d,J=7.6Hz,1H),7.81(s,1H) ,7.80–7.76(m,1H),7.46(d,J=7.0Hz,1H),7.43(s,1H),7.42(d,J=9.1Hz,1H),7.39(s,1H),7.36(d,J=4.0Hz, 2H),7.34(s,1H),7.25(d,J=7.2Hz,1H),7.20(t,J=3.6Hz,2H),4.15(t,J=6.2Hz,2H),4.06(s,1H),3.93(s,3H ), 3.92 (s, 2H), 2.95 (s, 2H), 2.16 (s, 3H), 1.85 (d, J = 6.8Hz, 2H), 1.72 (d, J = 8.7Hz, 2H), 1.55 (d, J = 6.4Hz, 2H). ESI-MS m / z:692.44[M+H] + .

[0180] Example 22:

[0181] N-(3-chloro-4-fluorophenyl)-7-methoxy-6-((6-((2-methoxy-6-(2-methyl-[1,1'-biphenyl]-3-yl)pyridin-3-yl]methyl)amino)hexyl)oxy)quinazoline-4-amine (C22)

[0182]

[0183] The target compound C22 was obtained using compounds 8a-5 and 14 according to the synthetic method of target compound C11 in Example 11, with a yield of 49%. 1H NMR (400MHz, CDCl3) δ9.50(s,1H),8.57(s,1H),8.02(dd,J=6.6,2.4Hz,1H),7.69(d,J=7.3Hz,2H),7.67(s, 1H),7.44(t,J=7.2Hz,2H),7.40–7.33(m,3H),7.30(d,J=3.4Hz,2H),7.17(s,1H),7.08(t,J=8.8Hz,1H),7.0 4(d,J=7.5Hz,1H),6.09(s,2H),4.10(s,2H),4.00(s,3H),3.97(d,J=7.3Hz,2H),3.91(s,3H),3.01(t,J=6.6 Hz,2H),2.20(s,3H),1.88(dt,J=13.1,6.5Hz,2H),1.83–1.75(m,2H),1.53–1.45(m,2H),1.44–1.38(m,2H). ESI-MS m / z:706.29[M+H] + .

[0184] Example 23:

[0185] 4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-yl-4-((2-methoxy-6-(2-methyl-[1,1'-biphenyl]-3-yl)pyridin-3-yl]methyl)piperazine-1-carboxylate (C23)

[0186]

[0187] The target compound C23 was obtained using compounds 8a-6 and 14 according to the synthetic method of target compound C11 in Example 11, with a yield of 58%. 1H NMR (400MHz, CDCl3) δ8.49 (s, 1H), 8.02 (s, 1H), 7.85–7.81 (m, 1H), 7.77 (dd, J = 10.4, 4.9Hz, 2H), 7. 68(s,1H),7.45(s,1H),7.43(s,1H),7.42(s,1H),7.39(s,1H),7.38(s,1H),7.36(d,J=3.5Hz,1H),7 .32(d,J=7.4Hz,1H),7.30–7.27(m,1H),7.13(d,J=8.8Hz,1H),7.08(d,J=7.4Hz,1H),6.85(s,1H), 4.02(s,3H),3.86(d,J=16.9Hz,4H),3.71(s,2H),3.47(s,3H),2.73(d,J=17.2Hz,4H),2.28(s,3H). ESI-MS m / z:719.26[M+H] + .

[0188] Example 24:

[0189] N-(3-chloro-4-fluorophenyl)-7-methoxy-6-((8-((2-methoxy-6-(2-methyl-[1,1'-biphenyl]-3-yl)pyridin-3-yl]methyl)amino)octyl)oxy)quinazoline-4-amine (C24)

[0190]

[0191] After synthesizing compounds 8a-7 according to Example 1, the target compound C24 was synthesized according to the method for synthesizing target compound C11 in Example 11, with a yield of 58%. 1H NMR(400MHz, CDCl3)δ8.58(s,1H),7.98(dd,J=6.6,2.6Hz,1H),7.69–7.65(m,2H),7.64(s,1H),7.44(t, J=7.1Hz,2H),7.37(dt,J=7.3,2.4Hz,3H),7.34(d,J=5.0Hz,1H),7.31(t,J=5.2Hz,2H),7.21(s,1H),7. 09(t,J=8.8Hz,1H),7.05(d,J=7.5Hz,1H),4.11(s,2H),4.03(d,J=7.2Hz,2H),4.01(s,3H),3.94(s,3H) ,2.95(t,J=7.4Hz,2H),2.21(s,3H),1.88–1.82(m,2H),1.79–1.72(m,2H),1.37(dd,J=11.6,4.7Hz,6H). ESI-MS m / z:734.31[M+H] + .

[0192] Preparation of series III compounds (C25-C34):

[0193]

[0194] Example 25

[0195] N-(5-((4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-yl)oxy)pentyl)-1-(2,6-dimethoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)piperidine-2-carboxamide (C25)

[0196] 1) 3-Bromo-2-methyl-1,1'-biphenyl (compound 4c-1)

[0197]

[0198] Compound 3c (120 mg, 0.33 mmol) and piperidine-2-carboxylic acid (130 mg, 1.0 mmol) were dissolved in a mixture of DCM (2 mL) and MeOH (2 mL) and stirred at room temperature for 1 h. Then, NaBH3CN (108.1 mg, 1.7 mmol) and acetic acid (0.02 mL) were added to the reaction solution, and stirring was continued for 12 h. The solvent was evaporated under reduced pressure, redissolved with DCM, and washed three times with water. The organic layer was concentrated to give the crude product, which was purified by silica gel column chromatography (dichloromethane / methanol = 20:1) to give a white solid 4c-1 (85.6 mg, yield 54%). ESI-MS m / z: 476.24 [M+H] + ;

[0199] Compound 4c-1 (42 mg, 0.10 mmol) was dissolved in 3 mL of ultradry DMSO, and HATU (63 mg, 0.17 mmol) and DIPEA (0.071 mL, 0.40 mmol) were added. The mixture was stirred at room temperature for 3 h. Then, compound 8a-4 (36 mg, 0.09 mmol) was added, and the mixture was stirred at room temperature for another 24 h. 15 mL of water was added to the reaction mixture, and DCM was added for extraction. The organic layer was concentrated to obtain the crude product, which was purified by silica gel column chromatography (dichloromethane / methanol = 50:1) to give 15 mg of a white solid, in 20% yield. 1 H NMR (400MHz, CDCl3) δ9.55(s,1H),8.62(s,1H),7.87(s,1H),7.85(s,1H),7.63(s,1H),7.46(s,1H),7.45(s,1H),7.43(s,1H),7.39(d,J= 7.1Hz,1H),7.35(s,1H),7.33(s,1H),7.31(s,1H),7.28(d,J=5.3Hz,1H),7.25(s,1H),7.15(t,J=8.8Hz,1H),6.28(s,2H),5.11(s,2H),4 .24(t,J=7.8Hz,2H),4.02(s,3H),3.84(s,6H),3.72(d,J=43.8Hz,2H),3.36–3.17(m,2H),2.69(dd,J=65.8,5.8Hz,2H),2.30(s,3H),2.2 8–2.20(m,1H),1.98(s,2H),1.95–1.82(m,2H),1.70(s,2H),1.66(dd,J=13.8,9.2Hz,2H),1.53(d,J=3.0Hz,2H),1.42(d,J=19.7Hz,2H). ESI-MS m / z:862.23[M+H] + .

[0200] Example 26:

[0201] N-(6-((4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-yl)oxy)hexyl)-1-(2,6-dimethoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)piperidine-2-carboxamide (C26)

[0202]

[0203] The target compound C26 was obtained using compounds 4c-1 and 8a-5 according to the synthetic method of target compound C25 in Example 25, with a yield of 65%. 1 H NMR (400MHz, CDCl3) δ9.52(s,1H),8.64(s,1H),7.96(d,J=4.7Hz,1H),7.74(d,J=40.5Hz,3H),7.43( d,J=6.9Hz,2H),7.40(s,1H),7.36(d,J=7.2Hz,1H),7.32(s,1H),7.30(s,1H),7.28(s,1H),7.22(s,1 H),7.11(t,J=8.4Hz,1H),6.24(s,2H),5.08(s,2H),4.04(s,1H),3.97(s,3H),3.79(s,6H),3.61(s,2 H),3.16(s,2H),2.86–2.44(m,4H),2.27(s,3H),1.94(s,2H),1.87(s,4H),1.62(s,4H),1.46(s,4H). ESI-MS m / z:876.38[M+H] + .

[0204] Example 27:

[0205] N-(5-((4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-yl)oxy)pentyl)-2-((2,6-dimethoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)(methyl)amino)acetamide (C27)

[0206]

[0207] After synthesizing compound 4c-2 according to Examples 1 and 18, the synthesis of target compound C27 was carried out according to the method for synthesizing target compound C25 in Example 25, with a yield of 45%. 1H NMR (400MHz, CDCl3) δ9.38(s,1H),8.59(s,1H),7.99(s,1H),7.79(dd,J=6.5,2.3Hz,1H),7.75(s,1H),7.64(dd,J=8.3,3.1H z,1H),7.44(d,J=2.0Hz,1H),7.43(s,1H),7.41(d,J=3.3Hz,1H),7.37(d,J=7.3Hz,1H),7.32(s,1H),7.30(s,1H),7.28(s,1 H),7.26(s,1H),7.22(s,1H),7.12(t,J=8.8Hz,1H),6.24(s,2H),5.08(s,2H),4.22–4.14(m,2H),4.00(s,3H),3.79(s,6H), 3.55(s,2H),3.44(dd,J=12.3,6.3Hz,2H),3.05(s,2H),2.27(s,3H),2.22(s,3H),2.04–1.97(m,2H),1.62(d,J=6.4Hz,4H). ESI-MS m / z:822.60[M+H] + .

[0208] Example 28:

[0209] N-(8-((4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-yl)oxy)octyl)-2-((2,6-dimethoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)(methyl)amino)acetamide (C28)

[0210]

[0211] The target compound C28 was obtained using compounds 4c-2 and 8a-7 according to the synthetic method of target compound C25 in Example 25, with a yield of 48%. 1H NMR (400MHz, CDCl3) δ8.94(s,1H),8.64(s,1H),7.85(dd,J=6.6,2.5Hz,1H),7.82(t,J=5.9Hz,1H),7.64–7.59(m, 2H),7.42(dd,J=8.1,4.7Hz,3H),7.36(d,J=7.2Hz,1H),7.30(dd,J=12.9,6.0Hz,3H),7.21(s,1H),7.09(t,J=8.8 Hz,1H),6.24(s,2H),5.08(s,2H),4.01(t,J=7.1Hz,2H),3.96(s,3H),3.77(s,6H),3.52(s,2H),3.28(dd,J=13.1 ,6.6Hz,2H),3.02(s,2H),2.27(s,3H),2.18(s,3H),1.85(dd,J=13.7,6.9Hz,4H),1.55–1.48(m,2H),1.30(s,6H). ESI-MS m / z:864.27[M+H] + .

[0212] Example 29:

[0213] N-(6-((4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-yl)oxy)hexyl)-1-(3-methyl-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)piperidine-2-carboxamide (C29)

[0214]

[0215] Following the synthesis of compound 4b-1 in Example 25, the synthesis of target compound C29 followed the method described in Example 25 for the synthesis of target compound C25, with a yield of 44%. 1H NMR(400MHz, CDCl3)δ9.23(s,1H),8.64(s,1H),7.90(dd,J=6.6,2.4Hz,1H),7.70–7.65(m,2H),7.45(d,J=7.4Hz,1H),7.41(d,J=7.5Hz,2H) ,7.36(d,J=7.2Hz,1H),7.34–7.27(m,3H),7.24(s,1H),7.20(s,1H),7.10(t,J=8.8Hz,2H),7.01(s,2H),6.87(d,J=8.8Hz,1H),5.06(s,2H) ,3.99(t,J=6.8Hz,2H),3.94(s,3H),3.72(d,J=13.1Hz,1H),3.48(s,1H),3.34(dd,J=13.2,6.6Hz,2H),2.96(d,J=12.5Hz,1H),2.89(d,J=1 1.9Hz,1H),2.72(s,1H),2.27(s,3H),2.24(s,3H),1.86(dd,J=23.3,9.7Hz,4H),1.64(d,J=12.9Hz,2H),1.56(d,J=6.9Hz,4H),1.37(s,4H). ESI-MS m / z:830.28[M+H] + .

[0216] Example 30:

[0217] (S)-N-(6-((4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-yl)oxy)hexyl)-1-(3-methyl-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)piperidine-2-carboxamide (C30)

[0218]

[0219] Following the synthesis of compound 4b-2 in Example 25, the synthesis of target compound C30 followed the method for synthesizing target compound C25 in Example 25, with a yield of 40%. 1H NMR(400MHz, CDCl3)δ9.09(s,1H),8.64(s,1H),7.89(dd,J=6.6,2.5Hz,1H),7.70–7.60(m,2H),7.45(d,J=7.8Hz,1H),7.41(d,J =7.5Hz,2H),7.36(d,J=7.2Hz,1H),7.32(d,J=1.3Hz,1H),7.30(s,1H),7.28(s,1H),7.24(d,J=2.3Hz,1H),7.22(s,1H),7.11(t, J=8.4Hz,1H),7.01(s,2H),6.86(d,J=8.7Hz,1H),5.05(s,2H),4.02(s,2H),3.96(s,3H),3.73(d,J=21.6Hz,1H),3.37(dt,J=13 .5, 6.9Hz, 2H), 2.88 (d, J = 12.2Hz, 2H), 2.68 (s, 1H), 2.25 (d, J = 11.2Hz, 6H), 1.86 (s, 4H), 1.69 (s, 2H), 1.57 (s, 4H), 1.44 (s, 4H). ESI-MS m / z:830.38[M+H] + .

[0220] Example 31:

[0221] 1-(5-chloro-2-((3-cyanobenzyl)oxy)-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)-N-(4-((4-(3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-yl)oxy)butyl)piperidine-2-carboxamide (C31)

[0222]

[0223] Five days after the synthesis of compound C31 in Example 25, the synthesis of target compound C25 was carried out in accordance with the method used in Example 25, with a yield of 39%. 1H NMR (400MHz, CDCl3) δ9.34 (s, 1H), 8.61 (s, 1H), 7.93 (dd, J = 6.5, 2.2Hz, 1H), 7.85–7 .79(m,1H),7.71(s,1H),7.63(d,J=11.9Hz,2H),7.57(d,J=7.8Hz,1H),7.46(d,J=7. 8Hz,1H),7.42(s,1H),7.40(s,1H),7.38(d,J=7.0Hz,1H),7.35(d,J=7.4Hz,1H),7. 29(d,J=7.0Hz,3H),7.24(d,J=4.2Hz,2H),7.19(s,1H),7.12(dd,J=17.4,8.3Hz,2H) ,6.53(s,1H),5.06(d,J=12.1Hz,4H),4.42–4.31(m,2H),3.97(s,3H),3.73(d,J=14 .0Hz,1H),3.24(s,2H),3.11(d,J=14.0Hz,1H),2.88(d,J=11.5Hz,1H),2.63(d,J=8. 3Hz,1H),2.24(s,3H),1.93–1.84(m,2H),1.79(d,J=6.3Hz,1H),1.63(dd,J=25.1,1 3.3Hz, 2H), 1.50 (d, J = 4.9Hz, 2H), 1.46–1.38 (m, 2H), 1.28 (dd, J = 17.2, 13.9Hz, 2H). ESI-MS m / z:954.07[M+H] + .

[0224] Example 32:

[0225] N-(4-((4-(3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-yl)oxy)butyl)-1-((2-methoxy-6-(2-methyl-[1,1'-biphenyl]-3-yl)pyridin-3-yl]methyl)piperidine-2-carboxamide (C32)

[0226]

[0227] After synthesizing compound 15a-1 according to Examples 18 and 25, the synthesis of target compound C32 was carried out according to the synthesis method of target compound C25 in Example 25, with a yield of 34%. 1H NMR (400MHz, CDCl3) δ9.61 (s, 1H), 8.63 (s, 1H), 7.97 (dd, J = 6.6, 2.5Hz, 1H), 7. 89–7.83(m,2H),7.70(t,J=6.6Hz,1H),7.51(d,J=7.4Hz,1H),7.44(d,J=8.0Hz ,1H),7.41(s,1H),7.39–7.37(m,2H),7.35(s,1H),7.34–7.30(m,1H),7.30–7. 28(m,1H),7.23(s,1H),7.18(t,J=8.8Hz,1H),7.00(d,J=7.3Hz,1H),4.49(dtd, J=19.3,11.0,8.1Hz,2H),4.00(s,6H),3.91(d,J=13.3Hz,1H),3.61(td,J=13. 1,6.6Hz,1H),3.48–3.39(m,1H),2.92(d,J=13.3Hz,1H),2.86(d,J=11.5Hz,1H ),2.58(dd,J=11.1,2.8Hz,1H),2.24(s,3H),1.94–1.87(m,3H),1.81(d,J=9.1 Hz, 2H), 1.75 (d, J = 5.6 Hz, 2H), 1.64–1.53 (m, 2H), 1.36 (dd, J = 20.5, 7.8 Hz, 2H). ESI-MS m / z: 789.23 [M+H] + .

[0228] Example 33:

[0229] N-(5-((4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-yl)oxy)pentyl)-1-((2-methoxy-6-(2-methyl-[1,1'-biphenyl]-3-yl)pyridin-3-yl]methyl)piperidine-2-carboxamide (C33)

[0230]

[0231] The target compound C33 was obtained using compounds 15a-1 and 8a-4 according to the synthetic method of target compound C25 in Example 25, with a yield of 50%. 1H NMR (400MHz, CDCl3) δ9.42 (s, 1H), 8.63 (s, 1H), 7.83 (dd, J = 6.6, 2.5Hz, 1H), 7. 79(s,1H),7.70–7.65(m,1H),7.60(dd,J=14.3,7.0Hz,2H),7.48–7.42(m,3H),7 .39(d,J=7.1Hz,3H),7.34–7.29(m,2H),7.18(t,J=8.8Hz,1H),7.06(d,J=7.3Hz ,1H),4.19(dt,J=16.1,6.1Hz,2H),4.05(s,3H),4.02(s,3H),3.92(d,J=13.4Hz ,1H),3.70(td,J=13.7,8.1Hz,1H),3.31(dq,J=11.0,5.4Hz,1H),3.03–2.96(m ,1H),2.94(d,J=11.5Hz,1H),2.70(dd,J=11.0,3.0Hz,1H),2.28(s,3H),2.20–2 .11(m,2H),2.01(d,J=12.2Hz,1H),1.93–1.77(m,2H),1.75–1.68(m,2H),1.65( dd,J=13.6,9.2Hz,2H),1.52–1.45(m,2H),1.37(ddd,J=14.3,12.3,7.0Hz,2H). ESI-MS m / z: 803.40 [M+H] + .

[0232] Example 34:

[0233] N-(6-((4-(((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-yl)oxy)hexyl)-3-((2-methoxy-6-(2-methyl-[1,1'-biphenyl]-3-yl)pyridin-3-yl]methyl)amino)propionamide (C34)

[0234]

[0235] After synthesizing compound 15a-2 according to Examples 18 and 25, the synthesis of target compound C34 was carried out according to the synthesis method of target compound C25 in Example 25, with a yield of 59%. 1H NMR (400MHz, CDCl3) δ9.26(s,1H),8.64(s,1H),8.61(s,1H),7.95(dd,J=6.6,2.5Hz,1H),7.71–7.66(m,1H),7.64(s,1H),7.50(d,J=7 .4Hz,1H),7.43(d,J=8.1Hz,1H),7.40(d,J=3.1Hz,2H),7.36(s,1H),7.35(s,2H),7.32(d,J=7.9Hz,1H),7.28(d,J=2.8Hz,1H),7.21(s ,1H),7.11(t,J=8.8Hz,1H),7.02(d,J=7.3Hz,1H),4.07–3.97(m,5H),3.95(s,3H),3.54(s,2H),3.27(dd,J=12.7,6.4Hz,2H),2.67(d, J=5.4Hz,2H),2.42–2.32(m,2H),2.25(d,J=3.3Hz,6H),1.84(dd,J=13.8,6.9Hz,4H),1.48(dd,J=13.1,6.5Hz,2H),1.44–1.39(m,2H). ESI-MS m / z:791.48[M+H] + .

[0236] Test Example 1:

[0237] Inhibit EGFR activity (IC) 50 )

[0238] EGFR kinase activity was detected using homogeneous time-resolved fluorescence (HTRF) technology. The HTRF tyrosine kinase activity assay kit (…) KinEASE-TK Kit (62TK0PEC); EGFR kinase (catalog number 08-115) was purchased from Carna Biosciences, Japan; 384-well white flat-bottom reaction plate (catalog number 3572) was purchased from Corning.

[0239] 1) Assay of the inhibitory activity of the compound against EGFR kinase

[0240] Prepare kinase buffer solutions (containing 1 mM DTT, 5 mM MgCl2 and 1 mM MnCl2), 5 μM tyrosine kinase substrate solution, 30 μM ATP solution, 0.037 ng / μL EGFR kinase solution, 0.25 μM Streptavidin-XL665 solution and 4×TK-Antibody-Cryptate solution; prepare target compound solutions with seven concentration gradients using kinase buffer solutions containing 2.5% DMSO.

[0241] A 384-well microplate was divided into experimental, positive control, and negative control wells. 4 μL of the target compound solution at different concentrations was added to all experimental wells, and 4 μL of kinase buffer solution containing 2.5% DMSO was added to all control wells. 2 μL of EGFR kinase solution was added to the experimental and positive control wells, and 2 μL of kinase buffer solution was added to the negative control wells. Subsequently, 2 μL of TK Substrate-Biotin solution and 2 μL of ATP solution were added to all wells, and the mixture was incubated at 37°C for 30 min. The reaction was then terminated by adding 5 μL of Streptavidin-XL665 solution and 5 μL of TK-Antibody-Cryptate solution, and incubated for another 1 h at 37°C. Fluorescence intensities at 665 nm and 620 nm were detected using a microplate reader under excitation light at 317 nm. The signal-to-weight ratio was calculated as (665 nm fluorescence intensity / 620 nm fluorescence intensity). Each well was prepared in duplicate. The inhibition rate of the corresponding compound concentration against EGFR kinase was calculated using the following formula.

[0242] Single-point inhibition rate = [1 - ((signal ratio of experimental wells - signal ratio of negative wells) / (signal ratio of positive wells - signal ratio of negative wells))] × 100%.

[0243] Using GraphPad Prism 7 software for data analysis and processing computational IC 50 The experimental results are shown in Table 1.

[0244] Table 1. IC50 of compounds inhibiting EGFR kinase activity 50 Value (unit: nM)

[0245]

[0246] Test Example 2:

[0247] Inhibit PD-1 / PD-L1 activity

[0248] The PD-1 / PD-L1 activity assay was performed using homogeneous time-resolved fluorescence (HTRF) technology. The PD-1 / PD-L1 binding assay kit (Cat#64ICP01PEG) was purchased from Cisbio.

[0249] The procedure is as follows: First, add 2 μL of compound dilution buffer to each well of a 384-well plate and centrifuge at 800 rpm for 1 min (add 6 μL buffer to negative control wells and 2 μL buffer to positive control wells). Then, add 4 μL of (2.5X) PD-L1 mixture to each well and centrifuge at 800 rpm for 1 min. Next, add 4 μL of (2.5X) PD-L1 mixture to each well (except negative control wells), centrifuge at 800 rpm for 1 min, and incubate at room temperature for 15 min. Subsequently, add 10 μL of (2X) test mixture to each well and centrifuge at 800 rpm for 1 min. Incubate at room temperature for 60 min, and use a microplate reader to detect the fluorescence intensity at 665 nm and 620 nm under excitation light at 320 nm. Calculate the signal-to-weight ratio as (665 nm fluorescence intensity / 620 nm fluorescence intensity). Each well is configured with two replicates. The inhibition rate of the corresponding compound concentration against EGFR kinase is calculated using the following formula.

[0250] Single-point inhibition rate = [1 - ((signal ratio of experimental wells - signal ratio of negative wells) / (signal ratio of positive wells - signal ratio of negative wells))] × 100%.

[0251] Table 2. Inhibition rates of target compounds on PD-1 / PD-L1 activity (10 μM and 1 μM)

[0252]

[0253] d This indicates that no signal value can be detected.

[0254] Test Example 3:

[0255] Inhibits the proliferation activity of tumor cell lines (IC50) 50 ):

[0256] The inhibitory activity of the compounds of this invention on the proliferation of glioma cell lines U87, U251, U87VⅢ mutant, and GL261 was investigated using the MTT assay.

[0257] 1) Select cells in the logarithmic growth phase, wash twice with phosphate-buffered saline (PBS), add an appropriate amount of digestion solution containing 0.25% trypsin and 0.02% EDTA, digest at 37°C for 1-2 min, then add 2-3 times the amount of RPMI-1640 culture medium containing 10% FBS and double antibiotics (penicillin 1000 U / mL, streptomycin 100 mg / mL), gently pipette, centrifuge, and then prepare a cell suspension;

[0258] 2) Add the cell suspension to a 96-well plate. The 96-well plate is divided into experimental group, control group and blank group. Add 100 μL of cell suspension (about 2000 cells) to each well of the experimental group and control group. After culturing for 12 h, aspirate the culture medium.

[0259] 3) For the experimental group, different concentrations of the test compound were added to three replicates for each concentration, with 100 μL added to each well. For the control group and blank group, 100 μL of culture medium was added to each well, and the mixture was incubated for 72 h.

[0260] 4) Add 10 μL of MTT (5 mg / mL) to each well of the experimental group, control group and blank group, incubate for 4 h, then aspirate the liquid from the well, add 100 μL of DMSO to each well and shake for 10 min.

[0261] 5) The OD value was measured at a wavelength of 570 nm, and the cell inhibition rate was calculated: Inhibition rate (%) = [1 - (OD value)] 实验组 -OD value 空白组 ) / (OD value) 对照组 -OD value 空白组 )]×100%;

[0262] 6) Calculate the compound concentration (IC50) at which the inhibition rate is 50% using GraphPad Prism 5 software. 50 The experimental results are shown in Table 1.

[0263] Table 3. IC50 of the target compound in inhibiting tumor cell proliferation. 50 Value (unit: μM)

[0264]

[0265]

[0266] Test Example 4:

[0267] Pharmacokinetic Properties of Compounds

[0268] The pharmacokinetic properties described in this test section were assessed by administering compound C7 of this invention via single intravenous injection and gavage to SD rats, determining the concentration of the test substance in SD rat plasma using LC-MS / MS, and calculating relevant parameters to examine the pharmacokinetic characteristics of the test substance in vivo.

[0269] The experimental procedure is as follows:

[0270] 1) Prepare a 4 mg / mL solution of compound C14 (solvent ratio: DMSO: polyoxyethylene castor oil: physiological saline).

[0271] =5:25:75 (volume ratio).

[0272] 2) Ten male SD rats (200-250g) were selected and divided into two groups (for intravenous administration and gavage administration). The rats were fasted for about 12 hours before administration and resumed feeding about 4 hours after administration.

[0273] 3) The dosage for the gavage group (20 mg / kg) and the intravenous group (4 mg / kg) was calculated based on the actual weight of the rats and administered in a single dose.

[0274] 4) Blood samples were collected within 0.033 hours after administration (intravenous group only), 0.083 hours, 0.167 hours, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 48 ​​hours and 72 hours after administration (gavage group only). The samples were centrifuged at 4000 rpm for 10 minutes at 4°C, and the supernatant was collected and stored at -80°C.

[0275] 5) Plasma drug concentration was detected using UPLC-MS / MS. An ACQUITY-UPLC BEHC18 column (1.7 mm, 2.1 x 50 mm) was used; mobile phase A was 0.1% ammonium acetate aqueous solution, and mobile phase B was methanol. The flow rate was 0.30 m / min. Quantification was performed using the internal standard method, with Gefitinib as the internal standard. Mass spectrometry was performed using a positive ion mode electrospray ionization (ESI) source.

[0276] 6) Plot plasma drug concentration-time curves and analyze pharmacokinetic parameters (MRT, C) using DAS software. max ,T max ,T 1 / 2 ,CL,V z The bioavailability of the drug was calculated using the pharmacokinetic parameters (and AUC). Table 4 shows the relevant parameters for pharmacokinetic properties.

[0277] Table 4. Pharmacokinetic parameters of compound C14 in SD rats (n=5, Mean±SD)

[0278]

[0279] Under the conditions of this experiment, the average C14 level in SD rats after intravenous administration of 4 mg / kg of compound C14 was... max It is 325.6 ug / L, with an average half-life t. 1 / 2 The mean residence time (MRT) is 6.8 hours.(0-∞) The average AUC is 12.5h. (0-∞) It is 2906.6 ug / L*h;

[0280] Under the conditions of this experiment, the average T in SD rats after gavage administration of 20 mg / kg compound C14 was... max It is 10.7h, C max It is 255.3 ug / L, and the half-life is t. 1 / 2 The mean residence time (MRT) is 13.0 hours. (0-∞) The mean AUC is 17.2h. (0-∞) The concentration was 3238.3 ug / L*h; the average bioavailability of compound C14 in rats was 22%.

[0281] Pharmacokinetic studies showed that the compound described in this invention exhibited good pharmacokinetic characteristics, high oral bioavailability (F = 22%), good stability, and a reasonable half-life, providing important evidence for further preclinical studies.

[0282] Test Example 5:

[0283] In vivo antitumor activity test of compounds

[0284] The in vivo antitumor activity test of the compounds described in this test section was conducted using a mouse glioblastoma subcutaneous tumor transplantation model to preliminarily evaluate the therapeutic effect of compound C14 on glioma.

[0285] The experimental procedure is as follows:

[0286] 1) Take 25 male C57BL / 6 mice (20-25g) and divide them into five groups (blank group, C14 50mg / kg dose group, C14 100mg / kg dose group, Gefitinib 100mg / kg dose group, NP19 100mg / kg dose group), with 5 mice in each group.

[0287] 2) Take GL261 glioma cells from mice in the logarithmic growth phase, digest them, centrifuge (800 rpm, 5 minutes), resuspend in PBS to obtain a cell suspension (1.25 × 10⁻⁶). 7 The cell suspension was injected subcutaneously into the left axillary region of C57 mice (2.5 × 10⁹ cells per mouse). 6 indivual).

[0288] 3) Prepare solutions of compounds C14, Gefitinib, and NP19 to a specific concentration using a solvent with a solvent ratio of DMSO:polyoxyethylene castor oil:physiological saline = 5:25:75 (volume ratio).

[0289] 4) Once the tumors in the mice were clearly visible, the mice were weighed, the dosage was calculated, and the medication was administered via gavage (approximately 1 mL per mouse) once daily. Changes in mouse weight and tumor size were recorded. After 21 days of treatment, the mice were euthanized, and the tumors were dissected, weighed, and photographed. The drug's inhibitory effect on mouse tumor growth was then analyzed.

[0290] In vivo anti-tumor effect Figure 1 The experimental results showed that no significant weight loss or adverse reactions were observed in any group during the treatment period. Furthermore, compound C14 exhibited significant dose-dependent antitumor activity. Figure 1 (BD). At a dose of 100 mg / kg, C14 reduced tumor weight and tumor volume by 92.0% and 89.7% (compared to the blank control group), respectively, which was superior to the gefitinib group (77.2% and 71.1%, respectively) and the NP-19 group (82.8% and 79.0%, respectively). At a lower dose of 50 mg / kg, C14 also significantly inhibited the growth of glioblastoma, with a TGI of 61.4% (tumor weight) and 55.8% (tumor volume).

[0291] 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 shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A compound, characterized in that: The structure of the compound is shown in formula (Ⅰ); 2. The compound according to claim 1, characterized in that: R1 is selected from H, CH3, halogen, CH3O, or Z is selected from CH2 or CH2O, and R4 is selected from H, halogen, CN, NO2, NH2, N(CH3)2, NHCH3, OH, OCH3, OC2H5, OCF3, CF3, cyclopropyl, C 1-6 Alkyl, sulfonyl, sulfonylalkyl; n＝0～2； X is selected from CH or N; Y is selected from CH2O, CH2NH, or a single bond; R2 is selected from R5 is selected from H, halogen, methoxy, cyano, and m = 1 to 3; R3 is selected from H, halogens, and CH3O; L1 is selected from C 0-4 alkyl or Where A1 is C 1-2 alkyl; L2 is selected from C 0-6 Alkyl or amide bonds; L3 is C 1-8 Alkyl or single bond; L4 is C 1-4 Alkyl group, or H (where AE does not form bonds).

3. The compound according to claim 1, characterized in that: The compound is preferably selected from: N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(4-((2-methoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)butoxy)quinazolin-4-amine (C1); N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(4-((3-methyl-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)butoxy)quinazolin-4-amine (C2); N-(3-chloro-4-fluorophenyl)-6-(4-((2,6-dimethoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)butoxy)-7-methoxyquinazoline-4-amine (C3); 3-((4-chloro-2-(((4-(((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-yl)oxy)butyl)amino)methyl)-5-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)phenoxy)methyl)benzylnitrile (C4); N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-((2-methoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)propoxy)quinazolin-4-amine (C5); N-(3-chloro-4-fluorophenyl)-6-(2-((2,6-dimethoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)ethoxy)-7-methoxyquinazoline-4-amine (C6); N-(3-chloro-4-fluorophenyl)-6-(3-((2,6-dimethoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)propoxy)-7-methoxyquinazoline-4-amine (C7); N-(3-chloro-4-fluorophenyl)-6-((5-((2,6-dimethoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)pentyl)oxy)-7-methoxyquinazoline-4-amine (C8); N-(3-chloro-4-fluorophenyl)-6-((6-((2,6-dimethoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)hexyl)oxy)-7-methoxyquinazoline-4-amine (C9); 3-((4-chloro-2-(((3-((4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-yl)oxy)propyl)amino)methyl)-5-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)phenoxy)methyl)benzonitrile (C10); 4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-yl4-(2,6-dimethoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)piperazine-1-carboxylate (C11); N-(3-chloro-2-fluorophenyl)-7-methoxy-6-(4-((2-methoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)butoxy)quinazolin-4-amine (C12); N-(3-chloro-2-fluorophenyl)-7-methoxy-6-(4-((2-methoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)butoxy)quinazolin-4-amine (C13); N-(3-chloro-2-fluorophenyl)-6-(4-((2,6-dimethoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)butoxy)-7-methoxyquinazoline-4-amine (C14); N-(3-chloro-4-methoxyphenyl)-6-(4-((2,6-dimethoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)butoxy)-7-methoxyquinazoline-4-amine (C15); 3-((6-(4-(2,6-dimethoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)butoxy)-7-methoxyquinazoline-4-yl)amino)benzylnitrile (C16); 6-(4-((2,6-dimethoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)butoxy)-7-methoxy-N-(quinolin-6-yl)quinazolin-4-amine (C17); N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(4-(((2-methoxy-6-(2-methyl-[1,1'-biphenyl]-3-yl)pyridin-3-yl)methyl)amino)butoxy)quinazoline-4-amine (C18); N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(2-((2-methoxy-6-(2-methyl-[1,1'-biphenyl]-3-yl)pyridin-3-yl)methyl)amino)ethoxy)quinazoline-4-amine (C19); N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-((2-methoxy-6-(2-methyl-[1,1'-biphenyl]-3-yl)pyridin-3-yl]methyl)amino)propoxy)quinazoline-4-amine (C20); N-(3-chloro-4-fluorophenyl)-6-((5-((2,6-dimethoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)pentyl)oxy)-7-methoxyquinazoline-4-amine (C21); N-(3-chloro-4-fluorophenyl)-7-methoxy-6-((6-((2-methoxy-6-(2-methyl-[1,1'-biphenyl]-3-yl)pyridin-3-yl]methyl)amino)hexyl)oxy)quinazoline-4-amine (C22); 4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-yl-4-((2-methoxy-6-(2-methyl-[1,1'-biphenyl]-3-yl)pyridin-3-yl]methyl)piperazine-1-carboxylate (C23); N-(3-chloro-4-fluorophenyl)-7-methoxy-6-((8-((2-methoxy-6-(2-methyl-[1,1'-biphenyl]-3-yl)pyridin-3-yl]methyl)amino)octyl)oxy)quinazoline-4-amine (C24); N-(5-((4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-yl)oxy)pentyl)-1-(2,6-dimethoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)piperidine-2-carboxamide (C25); N-(6-((4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-yl)oxy)hexyl)-1-(2,6-dimethoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)piperidine-2-carboxamide (C26); N-(5-((4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-yl)oxy)pentyl)-2-((2,6-dimethoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)(methyl)amino)acetamide (C27); N-(8-((4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-yl)oxy)octyl)-2-((2,6-dimethoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)(methyl)amino)acetamide (C28); N-(6-((4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-yl)oxy)hexyl)-1-(3-methyl-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)piperidine-2-carboxamide (C29); (S)-N-(6-((4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-yl)oxy)hexyl)-1-(3-methyl-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)piperidine-2-carboxamide (C30); 1-(5-chloro-2-((3-cyanobenzyl)oxy)-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)-N-(4-((4-(3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-yl)oxy)butyl)piperidine-2-carboxamide (C31); N-(4-((4-(3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-yl)oxy)butyl)-1-((2-methoxy-6-(2-methyl-[1,1'-biphenyl]-3-yl)pyridin-3-yl]methyl)piperidine-2-carboxamide (C32); N-(5-((4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-yl)oxy)pentyl)-1-((2-methoxy-6-(2-methyl-[1,1'-biphenyl]-3-yl)pyridin-3-yl]methyl)piperidine-2-carboxamide (C33); Alternatively, N-(6-((4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-yl)oxy)hexyl)-3-((2-methoxy-6-(2-methyl-[1,1'-biphenyl]-3-yl)pyridin-3-yl]methyl)amino)propionamide (C34).

4. A pharmaceutical composition, characterized in that: The compound comprises at least one of the following active ingredients: a) a compound, b) a pharmaceutically acceptable salt of the compound, c) a hydrate of the compound, d) a solvate of the compound, e) a polymorph of the compound, f) a tautomer of the compound, g) a prodrug of the compound; wherein the compound is the compound having the structure shown in general formula I as described in claim 1.

5. The use of the compound or pharmaceutical composition according to any one of claims 1 to 4 in the preparation of a medicament for inhibiting the PD-1 / PD-L1 signaling pathway and / or EGFR-mediated related diseases.

6. The application as described in claim 5, characterized in that, This includes tumors associated with the EGFR signaling pathway or tumors associated with the PD-1 / PD-L1 signaling pathway. Preferably, the tumor diseases are lung cancer, liver cancer, kidney cancer, non-small cell lung cancer, prostate cancer, thyroid cancer, skin cancer, pancreatic cancer, ovarian cancer, breast cancer, bladder cancer, myelodysplastic syndrome, lymphoma, esophageal cancer, gastrointestinal cancer, and tumors of the central or peripheral nervous system.

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