2, 4-disubstituted quinazoline derivative as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicinal chemistry, and particularly relates to a 2, 4-disubstituted quinazoline derivative as well as a preparation method and application thereof. According to the invention, a series of 2, 4-disubstituted quinazoline derivative micromolecular ligands capable of acting on telomere G4 are developed on the basis of a quinazoline skeleton. The 2, 4-disubstituted quinazoline derivative disclosed by the invention has certain telomere G4 stability and telomere G4 binding capacity, better anti-tumor cell proliferation activity, low toxicity, higher cell membrane permeability and certain oral bioavailability, in addition, the 2, 4-disubstituted quinazoline derivative disclosed by the invention can activate congenital immune response of tumor cells, and can be used for preparing anti-tumor drugs. The compound has a wide prospect when being applied to preparation of anti-tumor drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicinal chemistry. More specifically, it relates to a 2,4-disubstituted quinazoline derivative, a preparation method thereof, and an application thereof. Background Art

[0002] Cancer is one of the important diseases threatening human life and health. Telomeres of tumor cells are infinitely extended through the telomerase-mediated telomere elongation or through the alternative lengthening of telomeres (ALT) mechanism, enabling tumor cells to acquire immortality. Therefore, disrupting the structural integrity of telomeres and interfering with the normal function of telomeres are one of the important treatment methods for tumors. G-quadruplex (G4) is a special secondary structure formed by guanine-rich nucleic acid sequences and is ubiquitous in the telomeric TTAGGG repeat sequence. It has been confirmed by research that small molecule ligands targeting telomeric G4 often induce DNA damage in tumor cells and induce tumor cell death. Currently, developing small molecule G4 ligands targeting the telomeric G4 structure has been proven to be an important means in the research of anti-tumor drugs. Patent Publication No. CN104130200A reported a class of G4 ligands targeting the reduction of c-myc expression or VEGFA expression. However, the anti-tumor cell proliferation activity of these molecules is limited, the physicochemical properties of the drugs are poor, and it is difficult to apply clinically. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to overcome the defects and deficiencies of the prior art and provide a 2,4-disubstituted quinazoline derivative or a pharmaceutically acceptable salt thereof.

[0004] The object of the present invention is to provide a preparation method of the 2,4-disubstituted quinazoline derivative or a pharmaceutically acceptable salt thereof.

[0005] Another object of the present invention is to provide the application of the 2,4-disubstituted quinazoline derivative or a pharmaceutically acceptable salt thereof or the pharmaceutical composition in the preparation of a drug for treating cancer.

[0006] Another object of the present invention is to provide the application of the 2,4-disubstituted quinazoline derivative or a pharmaceutically acceptable salt thereof or the pharmaceutical composition in the preparation of a drug for activating the innate immune response.

[0007] The above objects of the present invention are achieved by the following technical solutions:

[0008] The present invention provides a 1,2,4-disubstituted quinazoline derivative or a pharmaceutically acceptable salt thereof, and the structure of the 2,4-disubstituted quinazoline derivative is shown in formula (I):

[0009]

[0010] R1 is selected from C 3-8 heterocyclic group, amino group, C1-6 Alkoxy, -O-(CH2CH2O) n -CH3; wherein, n = 1 - 3; C 3-8 Heterocyclic group, amino group, C 1-6 The alkoxy is unsubstituted or substituted by one or more C 1-6 Alkyl group, hydroxyl group, C 3-8 Heterocyclic group, C 1-6 Alkyl C 3-8 Heterocyclic group, the substituents of -NR6R7; wherein, C 1-6 The alkyl is unsubstituted or substituted by one or more hydroxyl groups, C 3-8 Heterocyclic group, C 1-6 Alkyl C 3-8 Heterocyclic group, -NR6R7, halogenated phenyl substituents;

[0011] R6 and R7 are each independently selected from hydrogen, C 1-6 Alkyl group.

[0012] Preferably, R1 is selected from C 5-7 Heterocyclic group, amino group, C 1-4 Alkoxy, -O-(CH2CH2O) n -CH3; wherein, n = 1 - 3; C 5-7 Heterocyclic group, amino group and C 1-4 The alkoxy is unsubstituted or substituted by one or more C 1-4 Alkyl group, hydroxyl group, C 5-7 Heterocyclic group, C 1-4 Alkyl C 5-7 Heterocyclic group, the substituents of -NR6R7; wherein, C 1-4 The alkyl is unsubstituted or substituted by one or more hydroxyl groups, C 5-7 Heterocyclic group, C 1-4 Alkyl C 5-7 Heterocyclic group, -NR6R7, chlorinated phenyl substituents;

[0013] R6 and R7 are each independently selected from hydrogen, C 1-4 Alkyl group.

[0014] Preferably, R1 is selected from a five-membered heterocycle containing a nitrogen atom, a six-membered heterocycle containing a nitrogen atom, amino group, C 1-4 Alkoxy;

[0015] Wherein, the five-membered heterocycle containing a nitrogen atom and the six-membered heterocycle containing a nitrogen atom are unsubstituted or substituted by one or more C 1-4 Substituents of alkyl groups; wherein, the C 1-4 The alkyl is unsubstituted or substituted by one or more hydroxyl groups;

[0016] The amino group is unsubstituted or substituted by one or more C 1-4Substituted by a substituent of an alkyl group; wherein, C 1-4 The alkyl group is unsubstituted or substituted by one or more -NR6R7; R6 and R7 are each independently selected from hydrogen, C 1-4 alkyl group.

[0017] Preferably, the R1 is selected from one of methylpiperazinyl, morpholinyl, methyl homopiperazinyl, pyrrolyl, hydroxyethylamino, pyrrolylethylamino, N-(N,N-diethylaminopropyl)amino, hydroxymethylpyrrolyl, hydroxymethylpiperidyl, N-(N-methylpiperidylmethyl)amino, N-(N,N-diethylaminoethyl)amino, pyrrolylethoxy, N,N-dimethylaminoethoxy, N,N-diethylaminoethoxy, N,N-diethylaminopropoxy, N,N-dimethylaminopropoxy, pyrrolylpropyloxy, thiophenylpropyloxy, N-methylpiperazinylethoxy, piperidylethoxy, morpholinylethoxy, thiophenylmethoxy, -O-(CH2CH2O)2-CH3, pyridylpropyloxy, fluorophenylpropyloxy, methylfurylpropyloxy.

[0018] Preferably, the structure of the 2,4-disubstituted quinazoline derivative is as follows:

[0019]

[0020] Preferably, the 2,4-disubstituted quinazoline derivative is selected from any one of the following structures:

[0021]

[0022] Furthermore, the present invention protects a preparation method of the above 2,4-disubstituted quinazoline derivative, and the synthetic route of the preparation method is as follows:

[0023]

[0024] S1. The compound 1 undergoes an amidation reaction to obtain the compound 2;

[0025] S2. The compound 2 undergoes an addition reaction to obtain the compound 3;

[0026] S3. The compound 3 undergoes a condensation reaction to obtain the compound shown in formula (I).

[0027] In the steps S1 to S3 of the present invention, the amidation reaction, addition reaction and condensation reaction are conventional technical means in the art.

[0028] As one specific embodiment of the present invention, the preparation method of the 2,4-disubstituted quinazoline derivative includes the following operations:

[0029] S1. Compound 1, acryloyl chloride, and a base are subjected to an amidation reaction at -20 to 0 °C to obtain Compound 2;

[0030] S2. Compound 2, diethylamine, and a catalyst are subjected to a Michael addition reaction at 75 to 85 °C to obtain Compound 3;

[0031] S3. Compound 3, an R1-H compound, a basic reagent, and a catalyst are subjected to a condensation reaction at 65 to 135 °C to obtain the compound shown in formula (I).

[0032] Preferably, in step S1, the base is an organic base conventionally used in the amidation reaction in the field of organic synthesis. More specifically, the organic base includes, but is not limited to, triethylamine, etc.

[0033] Preferably, in step S2, the catalyst is a catalyst conventionally used in the Michael addition reaction in the field of organic synthesis. More specifically, the catalyst includes, but is not limited to, p-toluenesulfonic acid (p-TsOH), etc.

[0034] Preferably, in step S3, the basic reagent is a basic reagent conventionally used in the condensation reaction in the field of organic synthesis. More specifically, the basic reagent includes, but is not limited to, K2CO3, Cs2CO3, etc. In step S3, the catalyst is a catalyst conventionally used in the condensation reaction in the field of organic synthesis. More specifically, the catalyst includes, but is not limited to, one or more of KI, a palladium catalyst, NaH, 1,1'-binaphthalene-2,2'-bis(diphenylphosphine) (BINAP).

[0035] Furthermore, the present invention claims protection for a pharmaceutical composition containing the 2,4-disubstituted quinazoline derivative or a pharmaceutically acceptable salt thereof of the present invention.

[0036] Furthermore, the present invention protects the use of the 2,4-disubstituted quinazoline derivative or a pharmaceutically acceptable salt thereof or the pharmaceutical composition in the preparation of a drug for treating cancer.

[0037] Preferably, the cancer is one or more of lung cancer, cervical cancer, osteosarcoma, and colon cancer.

[0038] Furthermore, the present invention claims protection for the use of the 2,4-disubstituted quinazoline derivative or a pharmaceutically acceptable salt thereof or the pharmaceutical composition in the preparation of a drug for activating the innate immune response.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] The present invention has developed a series of 2,4-disubstituted quinazoline derivatives or pharmaceutically acceptable salts thereof based on the quinazoline skeleton. The 2,4-disubstituted quinazoline derivatives provided by the present invention have more excellent anti-tumor cell proliferation activity, and at the same time have lower cytotoxicity to normal cells. The 2,4-disubstituted quinazoline derivatives provided by the present invention have certain telomeric G4 stabilizing ability and telomeric G4 binding ability, good anti-tumor cell proliferation activity, high cell membrane permeability and certain oral bioavailability, and have broad prospects in the application of preparing anti-tumor drugs. At the same time, the said 2,4-disubstituted quinazoline derivatives of the present invention can also activate the innate immune response of tumor cells. Description of the Drawings

[0041] Figure 1 It is a diagram showing the telomeric DNA damage effect of compound A4 on HCT116 cells. Among them, Figure 1 Figure A in it is a fluorescence micrograph of the co-localization of BG4 and TRF2, and BG4 / TRF2 in HCT116 cells after treatment with compound A4, Figure 1 Figure B in it is a fluorescence micrograph of the co-localization of γ-H2AX and TRF2, and γ-H2AX / TRF2 in HCT116 cells after treatment with compound A4, Figure 1 Figure C in it is a diagram showing the mRNA expression levels of G4-related genes in cells after treatment with compound A4, and ns indicates no significant difference.

[0042] Figure 2 It is a diagram showing the effect of compound A4 on the innate immunity of tumor cells. Among them, Figure 2 Figure A in it is a diagram showing the mRNA expression levels of innate immunity-related genes in HCT116 cells after treatment with compound A4, Figure 2 Figure B in it is a fluorescence micrograph of IFN-γ in HCT116 cells after treatment with compound A4.

[0043] Figure 3 It is a diagram showing the results of the pharmacodynamic studies of oral and intraperitoneal injection of compound A4 in mice. Among them, Figure 3 Figure A in it is a diagram showing the change in tumor volume during drug administration, Figure 3 Figure B in it is a real photo of the tumor volume on the 26th day, Figure 3 Figure C in it is a diagram showing the change in tumor weight obtained by dissection after the last drug administration, Figure 3 Figure D in it is a diagram showing the weights of the heart, liver, spleen, lung, and kidney of mice, Figure 3 Figure E in it is a diagram showing the change in the body weight of mice during drug administration. Detailed Embodiments

[0044] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments. However, the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0045] Example 1 Synthesis of Compounds A1 - A5

[0046] Synthesis of Compound 3:

[0047]

[0048] a represents: Compound 1, acryloyl chloride, and Et3N (triethylamine) are mixed and dissolved in dichloromethane, and reacted at 0 °C for 3 h to obtain Compound 2.

[0049] b represents: Compound 2, diethylamine, and p-toluenesulfonic acid are mixed and dissolved in tetrahydrofuran, and reacted at 80 °C for 12 h to obtain Compound 3.

[0050] Synthesis of Compound A1: Compound 3 (0.5 mmol, 1.0 equivalent), N-methylpiperazine (7.5 mmol, 15 equivalents), cesium carbonate (1.5 mmol, 3 equivalents), and potassium iodide (0.5 mmol, 1 equivalent) are mixed in DMF (8 mL) to obtain a mixture, stirred and reacted at 130 °C for 12 h and monitored by TLC. After cooling to room temperature, water (100 mL) is added to the reaction mixture, and the aqueous phase is extracted with ethyl acetate (30 mL × 6). The combined organic phases are washed with saturated NaCl (30 mL × 3) and dried over anhydrous Na2SO4. Then, the organic phase is concentrated in vacuo, adsorbed on silica gel, and purified by flash chromatography. Finally, Compound A1 is obtained, yield: 83%, yellow solid.

[0051] The NMR data are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 10.21 (s, 1H), 9.52 (s, 1H), 8.30 (d, J = 8.3 Hz, 1H), 7.70 (d, J = 8.6 Hz, 2H), 7.58 (dd, J = 11.6, 7.9 Hz, 3H), 7.34 (d, J = 8.4 Hz, 1H), 7.16 (t, J = 7.6 Hz, 1H), 3.75 (d, J = 6.2 Hz, 4H), 2.92 (s, 2H), 2.69 (s, 4H), 2.40 (d, J = 5.3 Hz, 4H), 2.23 (s, 3H), 1.06 (t, J = 7.1 Hz, 6H). 13 C NMR (126 MHz, DMSO-d6) 1313C NMR (126 MHz, DMSO) δ 169.97, 158.67, 158.38, 152.68, 135.26, 135.02, 133.24, 125.73, 123.54, 122.98 (2C), 121.40, 119.46 (2C), 111.06, 54.94 (2C), 48.40, 46.65 (2C), 46.17, 43.86 (2C), 33.47, 22.97, 11.32. ESI-HRMS [M+H] + m / z: 462.2990, calculated molecular formula: C 26 H 35 N7O. The purity measured by HPLC was: 97.4%, retention time: 5.7 min, HPLC mobile phase was 60% methanol / 40% water (containing 0.1% trifluoroacetic acid).

[0052] The synthetic method of A2 is different from that of A1 in that N-methylpiperazine is replaced by morpholine, and the remaining experimental steps and operations are the same as those of A1. Finally, the yield of compound A2 was 85%, a yellow solid.

[0053] The NMR data of compound A2 are as follows: 1 1H NMR (400 MHz, DMSO-d6) δ 10.27 (s, 1H), 9.57 (s, 1H), 8.33 (d, J = 8.3 Hz, 1H), 7.71 (d, J = 8.3 Hz, 2H), 7.65 - 7.57 (m, 3H), 7.36 (d, J = 8.3 Hz, 1H), 7.18 (t, J = 7.5 Hz, 1H), 3.76 - 3.69 (m, 4H), 3.66 (d, J = 5.2 Hz, 4H), 3.06 - 2.98 (m, 2H), 2.79 (s, 4H), 2.65 (d, J = 8.2 Hz, 2H), 1.10 (t, J = 7.1 Hz, 6H). 13 13C NMR (101 MHz, DMSO) δ 158.81, 158.45, 152.57, 135.24, 135.03, 133.28, 125.81, 123.57, 123.08 (2C), 121.58, 119.53 (2C), 111.22, 66.62, 48.20, 46.75, 44.71, 10.81. ESI-HRMS [M+H] + m / z: 462.2990, calculated molecular formula: C 26 H 35 N7. The purity measured by HPLC was: 95.9%, retention time: 5.9 min, HPLC mobile phase was 80% methanol / 20% water (containing 0.1% trifluoroacetic acid).

[0054] The synthesis method of A3 is different from that of A1 in that N-methylpiperazine is replaced by N-methylhomopiperazine, and the remaining experimental steps and operations are the same as those of A1. Finally, the yield of compound A3 is 76%, a yellow solid.

[0055] The NMR data of compound A3 are as follows: 1 H NMR(400MHz,CDCl3-d6)δ11.23(s,1H),7.80(d,J=8.2Hz,1H),7.74(s,1H),7.66(d,J=8.5Hz,2H),7.09(m,1H),4.06-3.97(m,2H),3.90(t,J=6.4Hz,2H),2.80(t,J=5.9Hz,4H),2.67(dd,J=15.2,8.4Hz,6H),2.53(t,J=5.9Hz,2H),2.41(s,3H),2.08(m,2H),1.14(t,J=7.1Hz,6H). 13 CNMR(126MHz,CDCl3)δ170.92,158.26,157.47,152.35,134.72,134.53,132.78,125.74,121.99,121.15,121.11,119.90(2C),110.36,77.30,58.56,57.10,48.87,46.32,45.98(2C),45.87,45.32,32.99,27.06,11.42(2C).ESI-HRMS[M+H] + m / z:476.3138, and the calculated molecular formula is: C 27 H 37 N7O. The purity measured by HPLC is: 99.1%, the retention time: 8.5 min, and the HPLC mobile phase is 40% methanol / 60% water (containing 0.1% trifluoroacetic acid).

[0056] The synthesis method of A4 is different from that of A1 in that N-methylpiperazine is replaced by pyrrolidine, and the remaining experimental steps and operations are the same as those of A1. Finally, the yield of compound A4 is 69%, a yellow solid.

[0057] The NMR data of compound A4 are as follows: 11H NMR (400 MHz, MeOD) δ 8.24 (d, J = 8.3 Hz, 1H), 7.79 (d, J = 8.6 Hz, 2H), 7.71 (t, J = 7.7 Hz, 1H), 7.64 (d, J = 8.6 Hz, 2H), 7.59 (d, J = 8.4 Hz, 1H), 7.33 (t, J = 7.7 Hz, 1H), 3.62 (q, J = 6.7 Hz, 4H), 3.38 (t, J = 6.9 Hz, 2H), 3.15 (q, J = 7.3 Hz, 4H), 2.87 (t, J = 6.8 Hz, 2H), 2.03 (s, 4H), 1.32 (t, J = 7.2 Hz, 6H). 13 13C NMR (126 MHz, MeOD) δ 171.13, 157.82, 156.61, 150.52, 135.49, 134.13, 132.85, 122.89, 122.19, 121.98 (2C), 121.23, 119.82 (2C), 110.38, 48.10, 46.36 (2C), 42.20, 32.48, 25.02, 10.42, 9.94 (2C). ESI-HRMS [M+H] + m / z: 462.2990, the calculated molecular formula is: C 26 H 35 N7O. The purity measured by HPLC is: 95.9%, retention time: 5.9 min, HPLC mobile phase is 40% methanol / 60% water (containing 0.1% trifluoroacetic acid).

[0058] The synthesis method of A5 is different from that of A1 in that N-methylpiperazine is replaced by aminoethanol, and the remaining experimental steps and operations are the same as those of A1. Finally, the yield of compound A5 prepared is 52%, a yellow solid.

[0059] The NMR data of compound A5 are as follows: 1 1H NMR (400 MHz, CDCl3-d6) δ 11.32 (s, 1H), 7.78 (dd, J = 8.2, 1.3 Hz, 1H), 7.64 - 7.57 (m, 2H), 7.56 - 7.51 (m, 1H), 7.51 - 7.47 (m, 2H), 7.43 (dd, J = 8.4, 1.2 Hz, 1H), 7.13 (t, J = 1.3 Hz, 1H), 5.51 (s, 1H), 3.88 - 3.76 (m, 2H), 3.60 (d, J = 12.9 Hz, 2H), 2.76 (dd, J = 6.6, 5.0 Hz, 2H), 2.65 (q, J = 7.1 Hz, 4H), 2.50 (dd, J = 6.6, 5.0 Hz, 2H), 1.12 (t, J = 7.1 Hz, 6H). 1313C NMR (126 MHz, CDCl3) δ 171.03, 158.21, 135.25, 133.87, 133.20, 122.80 (2C), 122.01, 120.98, 120.05 (2C), 111.13, 48.94, 45.94 (2C), 45.30, 33.01, 23.75, 22.99, 11.57 (2C). ESI-HRMS [M+Na] + m / z: 445.2304, calculated molecular formula: C 23 H 30 N6O2. Purity measured by HPLC: 98.9%, retention time: 5.9 min, HPLC mobile phase: 80% methanol / 20% water (containing 0.1% trifluoroacetic acid).

[0060] Synthesis of Compounds A6 - A11 in Example 2

[0061] Synthesis method of Compound A6:

[0062] At 110 °C, under N2 atmosphere, Compound 3 (0.38 mmol, 1.0 equivalent), 1-(2-aminoethyl)pyrrolidine (3.8 mmol, 10.0 equivalents, CAS No.: 7154-73-6), cesium carbonate (1.1 mmol, 3.0 equivalents), palladium acetate (0.04 mmol, 0.1 equivalent) and 2,2'-bis(diphenylphosphino)-1,1'-binaphthyl (BINAP, 0.08 mmol, 0.2 equivalent) were mixed with toluene (8 mL) and stirred for reaction for 6 h. After completion of the reaction, the mixture was filtered through diatomaceous earth and rinsed with ethyl acetate and methanol. The filtrate was concentrated in vacuo and adsorbed on silica gel, and purified by flash chromatography. The yield of the prepared Compound A6 was 23%, a yellow solid.

[0063] NMR data of Compound A6: 1 1H NMR (400 MHz, DMSO) δ 10.19 (s, 1H), 9.40 (s, 1H), 8.30 (d, J = 8.2 Hz, 1H), 7.84 (d, J = 8.5 Hz, 2H), 7.56 (dd, J = 17.4, 8.2 Hz, 3H), 7.30 (d, J = 8.3 Hz, 1H), 7.12 (t, J = 7.6 Hz, 1H), 6.66 (s, 1H), 3.72 (s, 4H), 2.79 (s, 2H), 2.73 - 2.62 (m, 4H), 2.57 (d, J = 7.0 Hz, 2H), 2.52 (d, J = 7.4 Hz, 2H), 2.47 (d, J = 7.0 Hz, 2H), 1.81 - 1.66 (m, 4H), 1.01 (t, J = 7.1 Hz, 6H). 1313C NMR (126 MHz, DMSO) δ 170.49, 135.27, 133.05, 123.52, 122.85 (2C), 120.86, 119.45 (2C), 55.49, 54.04 (2C), 48.76, 46.57 (2C), 34.27, 23.49 (2C), 12.05 (2C). ESI-HRMS [M+H] + m / z: 476.3141, calculated molecular formula: C 27 H 37 N7O. HPLC purity: 98.3%, retention time: 9.0 min, HPLC mobile phase: 40% methanol / 60% water (containing 0.1% trifluoroacetic acid).

[0064] The synthesis method of A7 is different from that of A6 in that 1-(2-aminoethyl)pyrrolidine is replaced by 3-diethylaminopropylamine (CAS No.: 203-236-4), and the yield of the prepared compound A7 is 39%, a yellow solid.

[0065] 1H NMR data of compound A7: 1 1H NMR (500 MHz, CDCl3) δ 11.30 (s, 1H), 7.92 (s, 1H), 7.70 (d, J = 8.4 Hz, 2H), 7.52 (d, J = 8.5 Hz, 3H), 7.46 (s, 1H), 7.14 (t, J = 7.6 Hz, 1H), 3.51 (t, J = 6.9 Hz, 2H), 2.83 - 2.77 (m, 2H), 2.67 (m, 8H), 2.56 - 2.49 (m, 2H), 1.85 (p, J = 6.6 Hz, 2H), 1.21 - 1.05 (m, 12H). 13 13C NMR (126 MHz, MeOD) δ 171.38, 159.47, 158.91, 151.26, 135.21, 134.62, 132.92, 123.48, 122.88 (2C), 122.25, 121.39, 119.89 (2C), 49.63, 48.17, 46.48 (2C), 46.27 (2C), 38.65, 32.78, 25.43, 10.17 (2C), 10.11, 8.92. ESI-HRMS [M+H] + m / z: 492.3451, calculated molecular formula: C 28 H 41 N7O. HPLC purity: 98.5%, retention time: 6.0 min, HPLC mobile phase: 80% methanol / 20% water (containing 0.1% trifluoroacetic acid).

[0066] The synthesis method of A8 is different from that of A6 in that 1-(2-aminoethyl)pyrrolidine is replaced by pyrrolidine-2-methanol (CAS No.: 498-63-5), and the yield of the prepared compound A8 is 27%, a yellow solid.

[0067] 1H NMR data of compound A8: 1 H NMR(400MHz,CDCl3)δ10.99(s,1H),8.20(s,1H),7.92(d,J=8.5Hz,1H),7.76(d,J=8.5Hz,2H),7.57(d,J=8.6Hz,2H),7.51(d,J=6.5Hz,2H),7.14(t,J=7.7Hz,1H),4.40(s,1H),3.81(q,J=9.3Hz,2H),3.72(d,J=9.3Hz,1H),3.69-3.56(m,2H),2.95(d,J=6.4Hz,2H),2.82(q,J=7.2Hz,4H),2.69(s,2H),2.10(d,J=9.8Hz,1H),1.92(m,2H),1.75(m,1H),1.22(t,J=7.1Hz,6H).ESI-HRMS[M+H] + m / z:492.3451, calculated molecular formula: C 26 H 34 N6O2. The purity measured by HPLC is: 98.8%, the retention time is: 7.0 min, and the HPLC mobile phase is 80% methanol / 20% water (containing 0.1% trifluoroacetic acid).

[0068] The synthesis method of A9 is different from that of A6 in that 1-(2-aminoethyl)pyrrolidine is replaced by 2-piperidinemethanol (CAS No.: 3433-37-2), and the yield of the prepared compound A9 is 24%, a yellow solid.

[0069] 1H NMR data of compound A9: 1 H NMR(400MHz,DMSO)δ10.35(s,1H),9.50(s,1H),8.30(s,1H),7.75(s,2H),7.60(s,3H),7.32(s,1H),7.13(s,1H),4.89(s,1H),4.85-4.60(m,2H),3.67(s,1H),3.49(s,2H),3.08(s,4H),2.85(s,2H),1.91(s,1H),1.73-1.51(m,4H),1.44(s,4H),1.22(s,6H).ESI-HRMS[M+Na] +m / z: 499.2770, Calculated molecular formula: C 27 H 36 N6O2. The purity measured by HPLC is: 97.1%, the retention time is: 6.0 min, and the HPLC mobile phase is 80% methanol / 20% water (containing 0.1% trifluoroacetic acid).

[0070] The difference in the synthesis method of A10 from A6 is that 1-(2-aminoethyl)pyrrolidine is replaced with (1-methyl-4-piperidyl-)methylamine (CAS No.: 7149-42-0), and the yield of the prepared compound A10 is 46%, a yellow solid.

[0071] 1H NMR data of compound A10: 1 H NMR(500MHz,DMSO)δ10.16(s,1H),9.44 - 9.20(m,1H),8.27(d,J = 8.2Hz,1H),7.95 - 7.80(m,2H),7.60 - 7.48(m,3H),7.28(s,1H),7.10(t,J = 7.6Hz,1H),6.93 - 6.66(m,1H),3.20(s,2H),2.88 - 2.79(m,2H),2.77(t,J = 7.0Hz,2H),2.57 - 2.51(m,4H),2.44(t,J = 7.1Hz,2H),2.22(s,3H),1.99(d,J = 17.4Hz,2H),1.71(s,2H),1.60(s,1H),1.00(t,J = 7.1Hz,6H). 13 CNMR(126MHz,MeOD)δ171.31,159.3,158.87,151.31,135.28,134.56,132.84,122.85(2C),122.19,121.20,119.85(2C),54.83,46.29(2C),44.54,34.92,32.69,28.97,10.12(2C).ESI-HRMS[M + H] + m / z: 490.3307, Calculated molecular formula: C 28 H 39 N7O. The purity measured by HPLC is: 94.3%, the retention time is: 5.9 min, and the HPLC mobile phase is 80% methanol / 20% water (containing 0.1% trifluoroacetic acid).

[0072] The difference in the synthesis method of A11 from A6 is that 1-(2-aminoethyl)pyrrolidine is replaced with N,N-diethylethylenediamine (CAS No.: 100-36-7), and the yield of the prepared compound A11 is 83%, a yellow solid.

[0073] 1H NMR data of compound A11: 1 H NMR(500MHz,DMSO)δ10.30(s,1H),9.50(s,1H),8.34(d,J=8.3Hz,1H),7.83(s,2H),7.59(dd,J=17.6,8.3Hz,3H),7.32(s,1H),7.15(t,J=7.9Hz,1H),6.82(s,1H),3.51(s,4H),2.90(s,6H),2.68(s,4H),2.57(s,2H),1.25 - 1.03(m,12H). 13 C NMR(126MHz,MeOD)δ168.56,160.22,153.53,139.54,137.20,135.75,132.51,125.57,125.56,125.19,123.92,120.15(2C),116.82,110.26,50.14,35.63,30.09(2C),29.43,28.93,22.34,7.85(2C),7.80,7.67(2C).ESI - HRMS[M + H] + m / z:478.3307, calculated molecular formula: C 28 H 39 N7O. The purity measured by HPLC is: 99.1%, the retention time is: 8.9 min, and the HPLC mobile phase is 40% methanol / 60% water (containing 0.1% trifluoroacetic acid).

[0074] Synthesis of compounds A12 - A26 in Example 3

[0075] Synthesis method of compound A12: At 0 °C, N-(2 - hydroxyethyl)-pyrrolidine (0.75 mmol, 2.0 equiv, CAS No.: 2955 - 88 - 6) was mixed and stirred with sodium hydride (5.6 mmol, 15.0 equiv) in anhydrous THF (5 mL) for 15 min, then compound 3 (0.38 mmol, 1.0 equiv) was added, and the mixture was reacted at 70 °C for 24 h. After the reaction was completed, ice water was added to the mixture at 0 °C until no more bubbles were generated. The aqueous phase was extracted with ethyl acetate (30 mL × 6). The combined organic phases were washed with saturated NaCl (30 mL × 3) and dried over anhydrous Na2SO4. Then, the organic phase was concentrated in vacuo, adsorbed on silica gel, and purified by flash chromatography. The yield of the prepared compound A12 was 76%, a yellow solid.

[0076] 1H NMR data of compound A12: 11H NMR (400 MHz, MeOD) δ 8.33 (d, J = 8.3 Hz, 1H), 7.81 - 7.76 (m, 1H), 7.74 - 7.65 (m, 4H), 7.58 (d, J = 8.3 Hz, 1H), 7.45 (t, J = 7.7 Hz, 1H), 4.74 - 4.70 (m, 2H), 3.68 - 3.62 (m, 2H), 3.55 (d, J = 6.9 Hz, 2H), 3.52 - 3.36 (m, 4H), 2.98 (t, J = 6.8 Hz, 2H), 2.14 - 2.04 (m, 4H), 1.39 (t, J = 7.3 Hz, 6H). 13 13C NMR (126 MHz, MeOD) δ 168.40, 161.07, 160.86, 150.70, 135.30, 134.36, 133.66, 125.21, 124.26, 123.69, 122.62, 120.04, 119.87, 112.50, 61.87, 56.52, 56.28, 54.26(2C), 53.83, 53.76, 30.02, 22.54(2C), 22.48, 7.87(2C). ESI - HRMS [M + Na] + m / z: 499.2777, calculated molecular formula: C 27 H 36 N6O2. The purity measured by HPLC was: 98.4%, retention time was: 5.9 min, and the HPLC mobile phase was 80% methanol / 20% water (containing 0.1% trifluoroacetic acid).

[0077] The synthesis method of A13 is different from that of A12 in that N-(2 - hydroxyethyl)-pyrrolidine is replaced with N,N - dimethylethanolamine (CAS No.: 108 - 01 - 0). The yield of the prepared compound A13 was 76%, a yellow solid.

[0078] 1H NMR data of compound A13: 1 1H NMR (400 MHz, CDCl3) δ 11.14 (s, 1H), 7.95 (d, J = 8.7 Hz, 1H), 7.66 (d, J = 6.8 Hz, 4H), 7.53 (d, J = 8.2 Hz, 2H), 7.32 (s, 1H), 4.55 (d, J = 6.3 Hz, 2H), 2.84 (td, J = 9.5, 3.7 Hz, 4H), 2.74 (q, J = 7.3 Hz, 4H), 2.59 (t, J = 6.1 Hz, 2H), 2.39 (d, J = 2.0 Hz, 6H), 1.17 (dt, J = 8.2, 4.1 Hz, 6H). 1313C NMR (126 MHz, CDCl3) δ 170.74, 162.00, 159.68, 151.98, 135.28, 133.81, 133.14, 127.22, 123.68, 122.64 (2C), 121.05, 120.18 (2C), 112.38, 64.41, 57.87, 48.92, 46.03 (2C), 45.63 (2C), 32.97, 11.32 (2C). ESI-HRMS [M+Na] + m / z: 473.2615, calculated molecular formula: C 25 H 34 N6O2. The purity measured by HPLC was: 94.3%, retention time was: 5.9 min, and the HPLC mobile phase was 80% methanol / 20% water (containing 0.1% trifluoroacetic acid).

[0079] The synthetic method of A14 is different from that of A12 in that N-(2-hydroxyethyl)-pyrrolidine is replaced with diethylaminoethanol (CAS No.: 100-37-8). The yield of the prepared compound A14 was 63%, a yellow solid.

[0080] 1H NMR data of compound A14: 1 1H NMR (400 MHz, CDCl3) δ 11.21 (s, 1H), 7.93 (d, J = 8.2 Hz, 1H), 7.84 (s, 1H), 7.67 (d, J = 7.2 Hz, 4H), 7.55 (d, J = 8.4 Hz, 2H), 7.33 (t, J = 7.0 Hz, 1H), 4.59 (t, J = 6.6 Hz, 2H), 3.04 (t, J = 6.7 Hz, 2H), 2.85 (d, J = 6.2 Hz, 2H), 2.78 (d, J = 7.3 Hz, 4H), 2.72 (d, J = 7.4 Hz, 4H), 2.57 (t, J = 6.1 Hz, 2H), 1.14 (m, 12H). 13 13C NMR (126 MHz, CDCl3) δ 170.74, 161.86, 159.76, 151.96, 135.27, 133.83, 133.16, 127.16, 123.73, 122.72 (2C), 121.23, 120.20 (2C), 112.40, 64.01, 51.07, 48.90, 47.61 (2C), 46.03 (2C), 32.93, 11.28 (2C). ESI-HRMS [M+Na] + m / z: 501.2945, calculated molecular formula: C 27 H 38N6O2. The purity measured by HPLC is: 98.1%, the retention time is: 5.9 min, and the HPLC mobile phase is 80% methanol / 20% water (containing 0.1% trifluoroacetic acid).

[0081] The synthesis method of A15 is different from that of A12 in that N-(2-hydroxyethyl)-pyrrolidine is replaced by 3-diethylamino-1-propanol (CAS No.: 622-93-5). The yield of the prepared compound A15 is 71%, a yellow solid.

[0082] The NMR data of compound A15: 1 H NMR (400 MHz, CDCl3) δ 11.29 (s, 1H), 7.88 (d, J = 8.3 Hz, 1H), 7.72 (s, 1H), 7.67 (d, J = 6.6 Hz, 4H), 7.58 (d, J = 8.4 Hz, 2H), 7.36 (d, J = 7.6 Hz, 1H), 4.47 (t, J = 6.2 Hz, 2H), 2.94 (t, J = 7.9 Hz, 2H), 2.83 (q, J = 7.2 Hz, 6H), 2.72 (t, J = 7.3 Hz, 4H), 2.55 (t, J = 5.9 Hz, 2H), 2.21 - 2.13 (m, 2H), 1.17 (dd, J = 16.3, 9.1 Hz, 12H). 13 C NMR (126 MHz, CDCl3) δ (126 MHz, CDCl3) δ 170.82, 161.94, 159.65, 152.01, 135.48, 133.64, 133.30, 127.24, 123.84, 122.58 (2C), 121.02, 120.17 (2C), 112.25, 64.79, 49.62, 48.88, 46.60 (2C), 46.03 (2C), 33.00, 29.72, 24.79, 11.37 (2C), 9.77. ESI-HRMS [M+Na] + m / z: 515.3046, the calculated molecular formula: C 28 H 40 N6O2. The purity measured by HPLC is: 94.8%, the retention time is: 8.8 min, and the HPLC mobile phase is 40% methanol / 60% water (containing 0.1% trifluoroacetic acid).

[0083] The synthesis method of A16 is different from that of A12 in that N-(2-hydroxyethyl)-pyrrolidine is replaced by 3-dimethylamino-1-propanol (CAS No.: 3179-63-3). The yield of the prepared compound A16 is 66%, a yellow solid.

[0084] The NMR data of compound A16:1 1H NMR (500 MHz, CDCl3) δ 11.24 (s, 1H), 7.96 (d, J = 8.2 Hz, 1H), 7.91 (s, 1H), 7.70 - 7.64 (m, 4H), 7.56 (d, J = 8.6 Hz, 2H), 7.33 (ddd, J = 8.2, 5.9, 2.2 Hz, 1H), 4.46 (t, J = 6.2 Hz, 2H), 2.86 (t, J = 6.0 Hz, 2H), 2.73 (q, J = 6.8 Hz, 6H), 2.58 (t, J = 5.9 Hz, 2H), 2.45 (s, 6H), 2.12 (p, J = 6.4 Hz, 2H), 1.17 (t, J = 7.1 Hz, 6H). 13 13C NMR (101 MHz, CDCl3) δ 170.61, 162.04, 159.70, 152.03, 135.32, 133.82, 133.23, 127.17, 123.72, 122.67 (2C), 121.20, 120.20 (2C), 112.30, 77.25, 64.91, 56.35, 48.88, 46.10 (2C), 44.63 (2C), 32.88, 26.32, 11.24 (2C). ESI-HRMS [M+Na] + m / z: 487.2792, calculated molecular formula: C 26 H 36 N6O2. The purity measured by HPLC was: 95.3%, the retention time was: 8.5 min, and the HPLC mobile phase was 40% methanol / 60% water (containing 0.1% trifluoroacetic acid).

[0085] The synthetic method of A17 is different from that of A12 in that N-(2-hydroxyethyl)-pyrrolidine is replaced by 1-(3-hydroxypropyl)pyrrolidine (CAS No.: 19748-66-4). The yield of the prepared compound A17 was 79%, a yellow solid.

[0086] 1H NMR data of compound A17: 11H NMR (500 MHz, CDCl3) δ 11.35 (s, 1H), 8.07 - 7.93 (m, 2H), 7.71 - 7.62 (m, 4H), 7.55 (d, J = 8.5 Hz, 2H), 7.33 (ddd, J = 8.3, 6.1, 2.0 Hz, 1H), 4.45 (t, J = 6.0 Hz, 2H), 3.00 - 2.94 (m, 2H), 2.93 - 2.85 (m, 4H), 2.80 (t, J = 5.9 Hz, 2H), 2.69 (q, J = 7.1 Hz, 4H), 2.54 (t, J = 5.8 Hz, 2H), 2.23 (dq, J = 12.3, 6.2 Hz, 2H), 1.94 (dd, J = 10.4, 3.3 Hz, 4H), 1.15 (t, J = 7.1 Hz, 6H). 13 13C NMR (126 MHz, CDCl3) δ 170.94, 161.97, 159.77, 151.98, 135.32, 133.83, 133.24, 127.07, 123.73, 122.74 (2C), 121.42, 120.18 (2C), 112.35, 64.72, 55.57, 54.08, 53.90, 53.34, 48.87, 45.99, 32.98, 28.87, 27.07, 23.44, 23.35, 11.47. ESI - HRMS [M + Na] + m / z: 513.2915, calculated molecular formula: C 28 H 38 N6O2. The purity measured by HPLC was: 95.6%, retention time was: 8.7 min, and the HPLC mobile phase was 40% methanol / 60% water (containing 0.1% trifluoroacetic acid).

[0087] The synthesis method of A18 is different from that of A12 in that N-(2-hydroxyethyl)-pyrrolidine is replaced by 3-(2-thienyl)-1-propanol (CAS No.: 19498 - 72 - 7). The yield of the prepared compound A18 was 56%, a yellow solid.

[0088] 1H NMR data of compound A18: 11H NMR (400 MHz, CDCl3) δ 11.17 (s, 1H), 7.81 (d, J = 8.2 Hz, 1H), 7.71 - 7.66 (m, 4H), 7.59 (d, J = 8.9 Hz, 2H), 7.52 (s, 1H), 7.35 (dt, J = 8.3, 4.1 Hz, 1H), 7.12 (dd, J = 5.1, 1.2 Hz, 1H), 6.92 (dd, J = 5.1, 3.4 Hz, 1H), 6.87 - 6.82 (m, 1H), 4.48 (t, J = 6.3 Hz, 2H), 3.07 (t, J = 7.7 Hz, 2H), 2.87 (s, 2H), 2.75 (q, J = 6.8 Hz, 4H), 2.61 (s, 2H), 2.25 - 2.18 (m, 2H), 1.18 (t, J = 7.2 Hz, 6H). 13 13C NMR (126 MHz, CDCl3) δ 159.43, 156.41, 152.19, 144.65, 133.20, 127.41, 126.76, 124.43, 123.68, 123.04, 122.28 (2C), 120.60, 120.11 (2C), 112.14, 66.13, 49.03, 46.18, 33.03, 31.13, 26.57, 11.14. ESI - HRMS [M + Na] + m / z: 526.2240, calculated molecular formula: C 28 H 33 N5O2S. The purity measured by HPLC was: 98.1%, the retention time was: 6.0 min, and the HPLC mobile phase was 80% methanol / 20% water (containing 0.1% trifluoroacetic acid).

[0089] The synthetic method of A19 is different from that of A12 in that N-(2-hydroxyethyl)-pyrrolidine is replaced by 1-hydroxyethyl-4-methylpiperazine (CAS No.: 5464 - 12 - 0). The yield of the prepared compound A19 was 78%, a yellow solid.

[0090] NMR data of compound A19: 11H NMR (500 MHz, CDCl3) δ 11.30 (s, 1H), 7.93 (d, J = 8.2 Hz, 1H), 7.86 (s, 1H), 7.68 - 7.64 (m, 4H), 7.55 - 7.51 (m, 2H), 7.32 (ddd, J = 8.2, 5.2, 3.0 Hz, 1H), 4.55 (t, J = 6.0 Hz, 2H), 2.82 (dt, J = 11.5, 6.0 Hz, 6H), 2.70 (q, J = 7.1 Hz, 6H), 2.63 - 2.52 (m, 6H), 2.30 (s, 3H), 1.15 (t, J = 7.1 Hz, 6H). 13 13C NMR (126 MHz, CDCl3) δ 170.83, 162.02, 159.68, 152.04, 135.29, 133.79, 133.15, 127.20, 123.64, 122.66 (2C), 121.10, 120.16 (2C), 112.34, 64.93, 64.39, 57.83, 56.91, 54.91, 53.32, 53.25, 48.92, 45.99, 45.85, 32.98, 11.42 (2C). ESI - HRMS [M+Na] + m / z: 528.3036, calculated molecular formula: C 28 H 39 N7O2. The purity measured by HPLC was: 94.2%, the retention time was: 8.3 min, and the HPLC mobile phase was 60% methanol / 40% water (containing 0.1% trifluoroacetic acid).

[0091] The synthesis method of A20 is different from that of A12 in that N-(2-hydroxyethyl)-pyrrolidine is replaced by N-hydroxyethylpiperidine (CAS No.: 3040 - 44 - 6). The yield of the prepared compound A20 was 62%, a yellow solid.

[0092] 1H NMR data of compound A20: 11H NMR (500 MHz, CDCl3) δ 11.36 (s, 1H), 7.90 (d, J = 8.2 Hz, 1H), 7.78 (s, 1H), 7.69 - 7.66 (m, 4H), 7.53 (d, J = 8.8 Hz, 2H), 7.32 (dt, J = 8.2, 4.1 Hz, 1H), 4.58 (t, J = 6.3 Hz, 2H), 2.83 (t, J = 6.3 Hz, 2H), 2.80 - 2.76 (m, 2H), 2.68 (q, J = 7.1 Hz, 4H), 2.53 (dd, J = 12.5, 6.5 Hz, 6H), 1.60 (d, J = 17.1 Hz, 4H), 1.43 (p, J = 6.0 Hz, 2H), 1.14 (t, J = 7.1 Hz, 6H). 13 13C NMR (126 MHz, CDCl3) δ 170.96, 162.04, 159.62, 152.10, 135.29, 133.79, 133.12, 127.26, 123.60, 122.56 (2C), 121.01, 120.15 (2C), 112.31, 64.42, 57.61, 54.88 (2C), 48.94, 45.95 (2C), 33.01, 25.79 (2C), 24.14, 11.56 (2C). ESI - HRMS [M + Na] + m / z: 513.2922, calculated molecular formula: C 28 H 38 N6O2. The purity measured by HPLC was: 99.9%, the retention time was: 5.9 min, and the HPLC mobile phase was 80% methanol / 20% water (containing 0.1% trifluoroacetic acid).

[0093] The synthetic method of A21 is different from that of A12 in that N-(2-hydroxyethyl)-pyrrolidine is replaced by 2-morpholinoethanol (CAS No.: 622 - 40 - 2). The yield of the prepared compound A21 was 88%, a yellow solid.

[0094] 1H NMR data of compound A21: 11H NMR (400 MHz, CDCl3) δ 11.33 (s, 1H), 7.99 - 7.93 (m, 2H), 7.69 - 7.64 (m, 4H), 7.51 (d, J = 8.7 Hz, 2H), 7.32 (dq, J = 8.3, 3.6 Hz, 1H), 4.56 (t, J = 6.0 Hz, 2H), 3.69 (t, J = 4.6 Hz, 4H), 2.81 (td, J = 6.0, 2.6 Hz, 4H), 2.69 (q, J = 7.1 Hz, 4H), 2.55 (dt, J = 11.4, 5.0 Hz, 6H), 1.15 (t, J = 7.1 Hz, 6H). 13 13C NMR (101 MHz, CDCl3) δ 170.89, 162.05, 159.75, 152.04, 135.24, 133.85, 133.16, 127.14, 123.62, 122.74 (2C), 121.24, 120.17 (2C), 112.38, 66.96, 64.24 (2C), 57.47, 54.01 (2C), 48.92, 45.97 (2C), 32.96, 11.44 (2C). ESI - HRMS [M + Na] + m / z: 515.2716, calculated molecular formula: C 27 H 36 N6O3. The purity measured by HPLC was: 99.8%, retention time was: 5.7 min, and the HPLC mobile phase was 80% methanol / 20% water (containing 0.1% trifluoroacetic acid).

[0095] The synthesis method of A22 is different from that of A12 in that N-(2 - hydroxyethyl)-pyrrolidine is replaced by 2 - thiopheneethanol (CAS No.: 5402 - 55 - 1). The yield of the prepared compound A22 was 54%, a yellow solid.

[0096] 1H NMR data of compound A22: 1 1H NMR (500 MHz, CDCl3) δ 11.27 (s, 1H), 7.88 (d, J = 7.7 Hz, 1H), 7.72 - 7.66 (m, 4H), 7.55 (d, J = 8.4 Hz, 2H), 7.38 - 7.32 (m, 1H), 7.26 (s, 1H), 7.14 (d, J = 5.1 Hz, 1H), 7.00 - 6.88 (m, 2H), 4.75 - 4.59 (m, 2H), 3.43 - 3.30 (m, 2H), 2.88 - 2.81 (m, 2H), 2.71 (q, J = 7.2 Hz, 4H), 2.60 - 2.52 (m, 2H), 1.22 - 1.11 (m, 6H). 1313C NMR (126 MHz, CDCl3) δ 170.70, 161.96, 159.60, 152.09, 140.47, 135.29, 133.78, 133.17, 127.35, 126.85, 125.47, 123.82, 123.53, 122.44 (2C), 120.87, 120.19 (2C), 112.32, 67.30, 48.98, 46.04 (2C), 32.97, 29.69, 11.37 (2C). ESI-HRMS [M+Na] + m / z: 512.2065, calculated molecular formula: C 27 H 37 N5O2S. HPLC purity: 96.7%, retention time: 6.0 min, HPLC mobile phase: 80% methanol / 20% water (containing 0.1% trifluoroacetic acid).

[0097] The synthesis method of A23 is different from that of A12 in that N-(2-hydroxyethyl)-pyrrolidine is replaced with diethylene glycol monomethyl ether (CAS No.: 111-77-3). The yield of the obtained compound A23 is 83%, yellow solid.

[0098] 1H NMR data of compound A23: 1 1H NMR (500 MHz, CDCl3) δ 11.31 (s, 1H), 7.91 (d, J = 8.2 Hz, 1H), 7.79 (s, 1H), 7.70 - 7.64 (m, 4H), 7.55 - 7.51 (m, 2H), 7.32 (ddd, J = 8.2, 5.6, 2.5 Hz, 1H), 4.59 (t, J = 5.1 Hz, 2H), 3.89 (t, J = 5.1 Hz, 2H), 3.75 - 3.68 (m, 2H), 3.60 - 3.54 (m, 2H), 3.37 (s, 3H), 2.81 (t, J = 5.8 Hz, 2H), 2.70 (q, J = 7.1 Hz, 4H), 2.56 - 2.52 (m, 2H), 1.15 (t, J = 7.1 Hz, 6H). 13 13C NMR (126 MHz, CDCl3) δ 170.83, 162.06, 159.59, 152.02, 135.21, 133.84, 133.09, 127.25, 123.62, 122.51 (2C), 121.02, 120.15 (2C), 112.34, 71.97, 70.57, 69.61, 66.20, 59.05, 48.95, 45.99 (2C), 32.99, 11.45 (2C). ESI-HRMS [M+Na] +m / z: 504.2570, Calculated molecular formula: C 26 H 35 N5O4. The purity measured by HPLC is: 94.9%, the retention time is: 6.0 min, and the HPLC mobile phase is 80% methanol / 20% water (containing 0.1% trifluoroacetic acid).

[0099] The difference in the synthesis method of A24 from that of A12 is that N-(2-hydroxyethyl)-pyrrolidine is replaced by 4-pyridinepropanol (CAS No.: 2629-72-3). The yield of the prepared compound A24 is 80%, and it is a yellow solid.

[0100] 1H NMR data of compound A24: 1 H NMR(400MHz,CDCl3)δ11.33(s,1H),8.53(d,J=4.9Hz,1H),7.89(d,J=8.2Hz,1H),7.76(s,1H),7.68(d,J=7.9Hz,4H),7.56(q,J=8.2Hz,3H),7.32(s,1H),7.19(d,J=7.8Hz,1H),7.13 - 7.06(m,1H),4.48(t,J=6.1Hz,2H),3.01(t,J=7.8Hz,2H),2.79(t,J=5.7Hz,2H),2.69(q,J=7.1Hz,4H),2.58 - 2.50(m,2H),2.28(q,J=7.0Hz,2H),1.15(t,J=7.0Hz,6H). 13 C NMR(126MHz,CDCl3)δ170.93,162.31,161.55,159.56,152.15,149.25,136.37,135.20,133.90,133.10,127.22,123.53,123.05,122.42(2C),121.07,121.00,120.15(2C),112.29,66.47,48.94,45.98(2C),34.86,33.01,29.10,11.53(2C).ESI-HRMS[M+Na] + m / z: 521.2612, Calculated molecular formula: C 29 H 34 N6O. The purity measured by HPLC is: 95.8%, the retention time is: 9.0 min, and the HPLC mobile phase is 60% methanol / 40% water (containing 0.1% trifluoroacetic acid).

[0101] The synthesis method of A25 is different from that of A12 in that N-(2-hydroxyethyl)-pyrrolidine is replaced by 4-fluorophenylpropanol (CAS No.: 702-15-8). The yield of the prepared compound A25 is 85%, and it is a yellow solid.

[0102] The NMR data of compound A25: 1 H NMR(500MHz,CDCl3)δ11.38(s,1H),7.92(d,J=8.2Hz,1H),7.83(d,J=3.9Hz,1H),7.71-7.65(m,4H),7.53(d,J=8.5Hz,2H),7.32(dq,J=8.2,4.5Hz,1H),7.16(dd,J=8.3,5.4Hz,2H),6.95(t,J=8.5Hz,2H),4.42(t,J=6.4Hz,2H),2.82-2.75(m,4H),2.67(q,J=7.1Hz,4H),2.55-2.49(m,2H),2.15-2.06(m,2H),1.13(t,J=7.1Hz,6H). 13 C NMR(126MHz,DMSO)δ170.77,162.29,162.05,160.45,160.13,151.90,138.03,138.00,136.02,134.37,133.62,130.53,130.46,126.63,123.76,123.71,123.62,122.50,119.47,119.29,115.49,115.33,112.92,65.95,48.85,46.54,34.56,31.22,30.70,12.28.ESI-HRMS[M+Na] + m / z:538.2569, the calculated molecular formula: C 30 H 34 N5O2F. The purity measured by HPLC is: 95.5%, the retention time is: 6.0 min, and the HPLC mobile phase is 80% methanol / 20% water (containing 0.1% trifluoroacetic acid).

[0103] The synthesis method of A26 is different from that of A12 in that N-(2-hydroxyethyl)-pyrrolidine is replaced by 3-(5-methyl-furan-2-yl)-propan-1-ol (CAS No.: 17162-95-7). The yield of the prepared compound A26 is 45%, and it is a yellow solid.

[0104] The NMR data of compound A26: 11H NMR (500 MHz, CDCl3) δ 11.24 (s, 1H), 7.90 (d, J = 8.2 Hz, 1H), 7.76 (s, 1H), 7.70 - 7.65 (m, 4H), 7.57 - 7.51 (m, 2H), 7.35 - 7.30 (m, 1H), 5.89 (d, J = 3.0 Hz, 1H), 5.86 - 5.80 (m, 1H), 4.46 (t, J = 6.5 Hz, 2H), 2.83 (t, J = 5.8 Hz, 2H), 2.78 (t, J = 7.6 Hz, 2H), 2.71 (q, J = 7.1 Hz, 4H), 2.59 - 2.53 (m, 2H), 2.24 (s, 3H), 2.14 (d, J = 14.7 Hz, 2H), 1.15 (t, J = 7.1 Hz, 6H). 13 13C NMR (101 MHz, MeOD) δ 171.08, 162.17, 160.30, 153.13, 151.41, 150.09, 134.92, 134.63, 133.15, 125.13, 123.40, 122.91 (2C), 122.30, 119.84, 112.10, 105.53 (2C), 66.01, 48.11, 46.34 (2C), 32.50, 27.58, 24.08, 12.12, 9.98 (2C). ESI - HRMS [M + H] + m / z: 501.2740, calculated molecular formula: C 29 H 34 N6O2. The purity measured by HPLC was: 96.5%, the retention time was: 6.0 min, and the HPLC mobile phase was 80% methanol / 20% water (containing 0.1% trifluoroacetic acid).

[0105] Study on the stability of telomeric G4 by the compound in Example 4

[0106] The in vitro stabilizing ability of the compound on telomeric G4 and double - stranded DNA was evaluated by fluorescence resonance energy transfer (FRET) experiments. The specific test results are shown in Table 1 below. The specific experimental method is as follows:

[0107] (1) Prepare buffer solutions for telomeric G4 and double - stranded DNA: an aqueous solution with a pH of 7.4, containing 100 mM KCl and 10 mM Tris - HCl.

[0108] (2) Preparation of nucleic acid sample with FAM fluorescent label at the 5'-end and TAMRA label at the 3'-end: Dilute the FAM- and TAMRA-labeled DNA sequence (5'-FAM-GGGTTAGGGTTAGGG TTAGGG-TAMRA-3') to 800 nM with buffer solution, anneal at 95 °C for 10 min, and then slowly cool to room temperature.

[0109] (3) Mix the nucleic acid sample with the compound to make the DNA concentration 400 nM and the compound concentration 2 μM, with a total volume of 20 μL. After incubating at 37 °C for 1 hour, perform the experiment using a Roche LightCycler 2 real-time fluorescence quantitative PCR instrument. The experimental condition parameters are set as follows: the excitation wavelength is set to 470 nm, and the emission fluorescence intensity at 530 nm is collected; the temperature range is set to 37 - 99 °C, the temperature increase interval is 1 °C, and sampling is performed after equilibration for 30 seconds. After the experiment, use Prism software to fit the T m value. The difference between the T m value of the compound and the T m value of DNA gives ΔT m (°C). The larger the difference, the stronger the ability of the compound to stabilize the telomeric G4.

[0110] Table 1 Experimental results of the compound on the stability of telomeric G4

[0111] Compound number Telomeric G4 ΔTm (°C) Double-stranded DNA ΔTm (°C) A1 5.6 0.4 A2 6.1 0.3 A3 9.4 1.3 A4 12.9 1.5 A5 6.1 0.1 A6 9.5 0.3 A7 13.7 1.3 A8 5.3 0.5 A9 2.7 0 A10 18.1 2.3 A11 8.5 0.1 A12 0.8 -0.1 A13 2.7 0.4 A14 3.0 0.5 A15 2.3 0.1 A16 6.6 0.5 A17 3.1 0.2 A18 3.6 -0.5 A19 7.5 0.1 A20 6.0 0.3 A21 1.2 -0.1 A22 2.5 0 A23 0.8 -0.2 A24 2.0 -0.3 A25 2.6 0.8 A26 2.6 -0.5

[0112] As shown in Table 1, compounds A3, A4, A6, A7, and A10 can all significantly stabilize the telomeric G4 structure (ΔT m > 9 °C), and have a certain selectivity for double-stranded DNA (ΔT m < 3 °C). This indicates that the 2,4-disubstituted quinazoline derivatives provided by the present invention are effective telomeric G4 ligands.

[0113] Example 5 Study on the antitumor cell proliferation activity of the compound

[0114] The anti-tumor cell proliferation activity of the compound was determined by the PI method, and the test results are shown in Table 2 below. The specific experimental method is as follows: Tumor cells in the logarithmic growth phase (U2OS osteosarcoma cells, Hela cervical cancer cells, HCT116 colorectal cancer cells) and NCM460 human normal intestinal epithelial cells were seeded in a 96-well plate at a density of 4000 cells per well and allowed to adhere and grow overnight to ensure the stability of the cells in subsequent experiments. The next day, the original medium was removed, and the drug was diluted to a predetermined concentration range (the highest concentration was 50 μM, and 8 different concentration gradients were obtained by the serial dilution method) with fresh medium. 100 μL of each concentration was added to the corresponding wells, and the cells were cultured for another 48 h to observe the effect of the drug on the cells. After 48 h, the medium containing the drug was removed, and 70% ethanol pre-cooled at -20 °C was added to fix the cells overnight at -20 °C. Subsequently, the ethanol was removed, and the cells were washed once with phosphate buffer (PBS) to remove residual ethanol and other impurities. Then, a PBS solution containing 0.5 μg / mL DAPI was added to stain the cell nuclei for observing the morphology and quantity of the cell nuclei in subsequent analysis. Finally, the stained cells were photographed using a high-content imaging system to record the cell morphology and distribution information, the cell nuclei were counted using the analysis system built into the high-content instrument, and the IC 50 value was fitted using Prism software. The selectivity ratio was the IC 50 of NCM460 divided by the IC 50 of tumor cells. The selectivity ratio indicated the toxicity selectivity ratio of the compound. The larger the selectivity ratio value, the lower the toxicity of the compound.

[0115] The compound B-5-3 prepared in Example 21 of Patent Publication No. CN104130200A was used as a control.

[0116]

[0117] Table 2 IC 50

[0118]

[0119]

[0120] As can be seen from Table 2 above, for U2OS osteosarcoma cells, the 2,4-disubstituted quinazoline derivatives provided by the present invention had an inhibition rate similar to or better than that of compound B-5-3; among them, the inhibition of compounds A3, A4, and A8 on osteosarcoma cells was significantly higher than that of compound B-5-3. Compounds A4, A5, and A8 also had excellent inhibition rates against cervical cancer and colorectal cancer.

[0121] As can be seen from Table 2 above, the 2,4-disubstituted quinazoline derivatives provided by the present invention have a higher selectivity multiple than compound B-5-3, indicating that the 2,4-disubstituted quinazoline derivatives provided by the present invention have lower cytotoxicity. Among them, the cytotoxicity of compounds A1, A3, A4, A8, and A14 is significantly lower than that of compound B-5-3.

[0122] Study on the Antitumor Cell Proliferation Activity and Cellular Uptake of Compound A4 in Example 6

[0123] The experimental method for anti-proliferation activity was the same as in Example 5, and the highest concentration of the compound was set to 5 μM. The operation of the cellular uptake experiment is as follows:

[0124] (1) Sample preparation: Cells in the logarithmic growth phase (U2OS human osteosarcoma cells, G292 human osteosarcoma cells, HCT116 human colorectal cancer cells, HELA human cervical cancer cells, MC38 mouse colon cancer cells) were seeded in 6-well plates at a density of approximately 150,000 cells / well and incubated overnight under culture conditions to promote cell adhesion and growth. Subsequently, the old medium was removed, and fresh medium containing a specific concentration of the compound (starting from 5 μM, serially diluted 8-fold) was added, and the non-drug-administered group was set as a control. The cells were cultured at 37 °C for another 48 h to evaluate the effect of the compound on the cells.

[0125] (2) Sample treatment and analysis: After removing the medium, the cells were washed twice with 1×PBS buffer to remove residual medium and impurities. Subsequently, the adherent cells were gently scraped off in 1×PBS and transferred to a 1.5 ml centrifuge tube. At room temperature, the cells were centrifuged at 1000 rpm for 5 min to precipitate the cells, and the supernatant was discarded. The cells were counted, and 100,000 cells from each drug-administered group were centrifuged again at 1000 rpm for 5 min to precipitate the cells, and the supernatant was discarded. Then, an appropriate amount of cell lysis solution was added, and the cells were lysed at room temperature for 30 min to release the intracellular components. Subsequently, the cells were centrifuged at 1000 rpm for 5 min, and the supernatant was collected. At the same time, the compound was diluted with the cell lysis solution according to a concentration gradient to prepare the corresponding standard curve for subsequent quantitative analysis.

[0126] (3) UV determination: After completing the sample treatment, the samples were measured using a UV spectrophotometer.

[0127] The 48 h IC of compound A4 against different tumor cells 50 And the cellular uptake rate at a concentration of 2 μM is shown in Table 3 below.

[0128] Table 3 The 48 h IC of compound A4 against different tumor cells 50and the cellular uptake rate at a concentration of 2 μM

[0129]

[0130] Example 7 Compound A4 selectively acts on telomeric G4 in tumor cells and induces telomeric DNA damage effect

[0131] The present invention uses the G4-specific antibody BG4 for immunofluorescence experiments to visualize the G4 level in HCT116 cells, and different concentrations of Compound A4 are used for drug administration treatment for 48 h.

[0132] The experimental results are as Figure 1 shown in Figure A of. Compared with the control group (Ctrl), Compound A4 significantly increased the number of BG4 fluorescent dots in the nucleus of HCT116 cells, indicating that it can induce and stabilize G4 in cells. To further determine whether the increased BG4 fluorescent dots are located in the telomeric region, an immunofluorescence co-localization experiment was carried out, and BG4 and TRF2 antibodies were used to visualize G4 and telomeres respectively. The results showed that after treatment with A4, the number of BG4 / TRF2 co-localized fluorescent dots in HCT116 cells increased significantly, indicating that Compound A4 promoted the formation of telomeric G4.

[0133] The present invention uses the specific antibody γ-H2AX for DNA double-strand damage for immunofluorescence experiments to visualize the DNA damage level in HCT116 cells, and different concentrations of Compound A4 are used for drug administration treatment for 48 h. The experimental results are as Figure 1 shown in Figure B of. Compared with the control group (Ctrl), Compound A4 significantly increased the number of γ-H2AX fluorescent dots in the nucleus of HCT116 cells, indicating that it can induce DNA damage in HCT116 cells. To further determine whether the increased γ-H2AX fluorescent dots are located in the telomeric region, an immunofluorescence co-localization experiment was carried out, and γ-H2AX and TRF2 antibodies were used to visualize G4 and telomeres respectively. The results showed that after treatment with A4, the number of γ-H2AX / TRF2 co-localized fluorescent dots in HCT116 cells increased significantly, indicating that Compound A4 promoted the formation of DNA damage at the telomeric position.

[0134] The present invention further verified the effect of Compound A4 on the mRNA levels of a series of G4-related genes (including BCL-2, c-MYC, HRAS, and HSP90) using RT-qPCR technology. After treatment at a concentration of 1 μM for 48 h, it was found that Compound A4 had no obvious effect on the transcription of these classical G4-related genes ( Figure 1 Figure C of), which indicates that this compound does not affect the transcription of G4-rich oncogenes in HCT116 cells, but mainly affects DNA damage in the telomeric region.

[0135] Example 8: Transcription Activation of Key Genes in Innate Immune Response by Compound A4 in Tumor Cells

[0136] The present invention further verified the effect of Compound A4 on the mRNA levels of a series of innate immune-related genes (including IFNB1, CXCL10, and ISG15) using RT-qPCR technology. After treatment at a concentration of 1 μM for 48 h, it was found that Compound A4 upregulated these innate immune-related genes ( Figure 2 Figure A in). This indicates that A4 can effectively activate the innate immunity in tumor cells.

[0137] The present invention used a specific antibody against IFN-γ to perform immunofluorescence experiments to visualize the IFN-γ damage level in HCT116 cells, and different concentrations of Compound A4 were used for drug administration treatment for 48 h. The experimental results are shown in Figure 2 Figure B in. Compared with the control group (Ctrl), Compound A4 significantly increased the number of IFN-γ fluorescent dots in the nuclei of HCT116 cells. This indicates that A4 can effectively activate the innate immunity in tumor cells.

[0138] Example 9: Pharmacokinetic Study of Compound A4 in Rats

[0139] To evaluate the pharmacokinetic properties of Compound A4, the pharmacokinetic parameters of Compound A4 were evaluated in SD rats. The animal experimental protocols in this study have all been reviewed by the Experimental Animal Ethics Committee of Sun Yat-sen University and comply with the principles of animal protection, animal welfare, and ethics, as well as the relevant regulations of national experimental animal welfare ethics. The specific experimental operations are as follows:

[0140] (1) Animal preparation: SPF-grade SD rats were purchased from the Experimental Animal Center of Sun Yat-sen University and randomly divided into 2 groups for intravenous injection (i.v.) and oral administration (p.o.) respectively, with each group containing 3 male rats and 3 female rats.

[0141] (2) Compound preparation: Compound A4 was dissolved in physiological saline containing 0.5% Tween 80; the dose used for the i.v. group was 1 mg / kg, and the dose used for the p.o. group was 6 mg / kg.

[0142] (3) Blood sampling: Rats were bled from the orbital cavity at time points of 0 h, 0.083 h, 0.25 h, 0.5 h, 1 h, 1.5 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h after drug administration, placed in heparinized centrifuge tubes, centrifuged at 3000 rpm for 15 min, and the upper plasma was taken and stored at -20 °C for later measurement.

[0143] (4) Detection: The plasma samples were extracted with methyl tert-butyl ether, and the content of the compound was analyzed by LC / MS using a Zobax C8 column. The PK-related parameters were calculated with Phoenix WinNonlin software.

[0144] The results are shown in Table 4. After intravenous injection, compound A4 had a long half-life (t1 / 2 = 4.4 ± 3.1 h) and a rapid time to peak (T max = 0.1 ± 0.0 h). When administered orally, the time to peak of compound A4 (T max = 2.1 ± 1.9 h) was significantly prolonged, and at the same time, the peak concentration (C max = 14.6 ± 4.7 h) and the area under the concentration-time curve (AUC 0→t = 59.4 ± 27.5 h) were both significantly decreased, indicating that under oral conditions, the absorption rate and absorption extent of compound A4 were relatively low. It should be noted that although the absorption efficiency of oral administration was low, the oral half-life of compound A4 was relatively long (t1 / 2 = 12.2 ± 10.1 h), suggesting a slow clearance rate in vivo and potentially prolonged residence time of the drug in the body. In addition, compound A4 also had a certain oral bioavailability.

[0145] Table 4 Pharmacokinetics of compound A4 with different administration routes

[0146]

[0147] Example 10 Pharmacodynamic study of compound A4 by oral and intraperitoneal injection in mice

[0148] Experimental method: An MC38 xenograft tumor mouse model was established. C57BL / 6J male mice at 5 weeks of age with a body weight of 15 - 20 g were selected and cultured in a pathogen-free environment for about one week to adapt. Subsequently, MC38 cells in the logarithmic growth phase were subcutaneously injected at a dose of 5 × 10 5 cells / mouse into the anterior axillary region of C57 mice. When the tumor volume reached approximately 50 mm 3 , the mice were randomly divided into 6 groups (6 mice per group) and administered drugs daily for 26 days. For the intraperitoneal injection route (i.p.), the doses of compound A4 were selected as 15 mg / kg and 10 mg / kg; for the oral route (p.o.), the doses of compound A4 were set as 40 mg / kg and 20 mg / kg. Meanwhile, Oxaliplatin was used as a positive control and administered by intraperitoneal injection at a dose of 5 mg / kg; the control group (vehicle) was intraperitoneally injected with normal saline. During the experiment, the growth of xenograft tumors and the body weight changes of C57 mice were continuously recorded.

[0149] The results are as Figure 3As shown in Figures A and B, during the drug administration period, the tumor volume of the control group (vehicle) continued to increase, while the growth of the tumor volume in the drug administration groups slowed down. As the experimental time elapsed, the gap in tumor volume between the drug administration groups and the control group also increased. Moreover, Compound A4 showed significant tumor growth inhibitory effects under both intraperitoneal injection and gavage administration methods. There was no significant difference in the efficacy between the gavage route and the intraperitoneal injection route, and the efficacy was comparable to that of Oxaliplatin. This result confirmed the excellent anti-tumor performance of Compound A4 as an oral anti-tumor drug. In addition, no obvious change in the body weight of the mice was observed in the A4 treatment group.

[0150] 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 other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A 2,4-disubstituted quinazoline derivative or a pharmaceutically acceptable salt thereof, characterized in that, The structure of the 2,4-disubstituted quinazoline derivative is shown in formula (I): R1 is selected from C 3-8 heterocyclic group, amino group, C 1-6 alkoxy group, -O-(CH2CH2O) n -CH3; wherein, n = 1 - 3; C 3-8 heterocyclic group, amino group, C 1-6 alkoxy group is unsubstituted or substituted by one or more C 1-6 alkyl groups, hydroxyl groups, C 3-8 heterocyclic groups, C 1-6 alkyl C 3-8 heterocyclic groups, substituents of -NR6R7; wherein, C 1-6 alkyl group is unsubstituted or substituted by one or more hydroxyl groups, C 3-8 heterocyclic groups, C 1-6 alkyl C 3-8 heterocyclic groups, -NR6R7, substituents of halogenated phenyl; R6 and R7 are each independently selected from hydrogen, C 1-6 alkyl groups.

2. The 2,4-disubstituted quinazoline derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein R1 is selected from C 5-7 heterocyclic group, amino group, C 1-4 alkoxy group, -O-(CH2CH2O) n -CH3; wherein, n = 1 - 3; C 5-7 heterocyclic group, amino group and C 1-4 alkoxy group is unsubstituted or substituted by one or more C 1-4 alkyl group, hydroxyl group, C 5-7 heterocyclic group, C 1-4 alkyl C 5-7 heterocyclic group, substituent of -NR6R7; wherein, C 1-4 alkyl group is unsubstituted or substituted by one or more hydroxyl groups, C 5-7 heterocyclic group, C 1-4 alkyl C 5-7 heterocyclic group, -NR6R7, substituent of chlorophenyl; R6 and R7 are each independently selected from hydrogen, C 1-4 alkyl groups.

3. The 2,4-disubstituted quinazoline derivative or a pharmaceutically acceptable salt thereof according to claim 2, wherein R1 is selected from a five-membered heterocycle containing a nitrogen atom, a six-membered heterocycle containing a nitrogen atom, an amino group, C 1-4 alkoxy group; Among them, the five-membered heterocycle containing a nitrogen atom and the six-membered heterocycle containing a nitrogen atom are unsubstituted or substituted by one or more substituents of C 1-4 alkyl; wherein, the C 1-4 alkyl is unsubstituted or substituted by one or more hydroxyl groups; The amino group is unsubstituted or substituted by one or more substituents of C 1-4 alkyl; wherein, C 1-4 alkyl is unsubstituted or substituted by one or more -NR6R7; R6 and R7 are each independently selected from hydrogen, C 1-4 alkyl.

4. The 2,4-disubstituted quinazoline derivative or a pharmaceutically acceptable salt thereof according to claim 3, wherein, It is selected from any of the following structures:

5. A method for preparing the 2,4-disubstituted quinazoline derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, characterized in that, It includes the following preparation steps: S1. The compound 1 undergoes an amidation reaction to obtain the compound 2; S2. The compound 2 undergoes an addition reaction to obtain the compound 3; S3. The compound 3 undergoes a condensation reaction to obtain the compound shown in formula (I); The synthetic route is as follows:

6. The preparation method according to claim 5, characterized in that, The specific preparation method includes the following preparation steps: S1. The compound 1, acryloyl chloride, and a base undergo an amidation reaction at -20 to 0 °C to obtain the compound 2; S2. The compound 2, diethylamine, and a catalyst undergo a Michael addition reaction at 75 to 85 °C to obtain the compound 3; S3. The compound 3, the R1-H compound, a basic reagent, and a catalyst undergo a condensation reaction at 65 to 135 °C to obtain the compound shown in formula (I).

7. A pharmaceutical composition, characterized in that, It contains the 2,4-disubstituted quinazoline derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1-4.

8. Use of the 2,4-disubstituted quinazoline derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1-4 in the preparation of a medicament for treating cancer.

9. The application according to claim 8, characterized in that The cancer is one or more of lung cancer, cervical cancer, osteosarcoma, and colon cancer.

10. Use of the 2,4-disubstituted quinazoline derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1-4 in the preparation of a medicament for activating the innate immune response.

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