A quinazoline compound with anti-colorectal cancer activity and a preparation method and application thereof

Abstract

The application provides a quinazoline compound with anti-colorectal cancer activity and a preparation method and application thereof. The quinazoline compound with the structure shown in formula (I) is prepared by selecting a specific quinazoline mother nucleus and substituting and modifying at specific 2, 4, 6 and 7 positions, and selecting a specific group for modification. As a result, it is found that the quinazoline compound provided by the application has good activity when applied to preparation of a drug inhibitor for resisting colorectal cancer. The preparation method of the quinazoline compound provided by the application has the advantages of mild reaction condition, simple operation, green efficiency, wide substrate applicability and the like, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, and in particular to a quinazoline compound with anti-colorectal cancer activity, its preparation method, and its application. Background Technology

[0002] Colorectal cancer (CRC) is the most common malignant tumor of the digestive system. Its prevalence is becoming increasingly severe, and the disease burden continues to rise, drawing significant attention from the global cancer prevention and control field.

[0003] Currently, commonly used small molecule inhibitors for colorectal cancer include larotrectinib, fruquintinib, and Regorafenib. Larotrectinib was approved by the US FDA in 2018 for the treatment of metastatic colorectal cancer patients with neurotrophic factor receptor tyrosine kinase (NTRK) gene fusions. It achieved an objective response rate of 47% in patients with NTRK fusion-positive locally advanced or metastatic colorectal cancer, indicating significant lesion shrinkage in nearly half of the patients. Furthermore, larotrectinib achieved a disease control rate of up to 89%. However, after a period of treatment with larotrectinib, many patients develop drug resistance, with major adverse reactions including nausea, cough, and vomiting. Fruquintinib is a VEGF inhibitor independently developed in my country and was approved in my country in 2018 for the treatment of advanced colorectal cancer patients. Fruquintinib can significantly prolong the median overall survival and median progression-free survival of patients with metastatic colorectal cancer, and its overall safety profile is good. Common adverse reactions include hypertension, proteinuria, and elevated transaminase levels. Regorafenib is a small-molecule tyrosine kinase inhibitor with multi-target inhibitory activity. Approved for marketing in China in 2017, it has become a standard-recommended drug for third-line treatment of colorectal cancer. Multiple phase III clinical trials have confirmed its ability to improve overall survival in patients with metastatic colorectal cancer. However, patients may experience mild to moderate adverse reactions during treatment, such as mild to moderate liver dysfunction, redness and swelling of the hands and feet, hypertension, and diarrhea. Furthermore, approximately 50% of patients develop drug resistance after 6 months of treatment, severely limiting its long-term clinical application. In summary, existing targeted inhibitors still face challenges such as drug resistance and significant toxic side effects, necessitating the development of novel targeted drugs for colorectal cancer.

[0004] Kinases are key functional proteins that regulate cell signaling, cell cycle progression, gene transcription, cell proliferation, and apoptosis. In colorectal cancer, various kinases exhibit gene mutations, abnormal overexpression, or persistent hyperactivation, broadly participating in the entire process of tumorigenesis and development by activating multiple core oncogenic signaling pathways, including MAPK, PI3K / AKT / mTOR. Specifically, abnormalities in kinases such as EGFR, BRAF, MEK, and CDK4 / 6 can drive tumor cell cycle disorder and unlimited proliferation; kinases such as VEGFR and PDGFR can promote tumor angiogenesis, providing nutritional support for tumor growth and distant metastasis; kinases such as c-MET, SRC, and FGFR can enhance tumor cell invasion and migration, accelerating lesion invasion and metastasis; and abnormal activation of PI3K, AKT, and mTOR can significantly inhibit tumor cell apoptosis, maintaining the malignant survival state of the tumor. CDK9, as a key kinase regulating gene transcription elongation, is abnormally highly expressed in colorectal cancer, regulating oncogene transcription, mediating DNA damage repair, inhibiting apoptosis, and participating in the formation of targeted therapy and chemotherapy resistance. Furthermore, kinases such as JAK can regulate the inflammatory response and immunosuppressive state of the tumor microenvironment, further promoting the malignant progression and treatment resistance of colorectal cancer. Targeted intervention against novel kinase targets such as CDK9 provides an important research direction for overcoming the bottlenecks in colorectal cancer treatment and developing next-generation anti-tumor drugs. Therefore, targeting and inhibiting abnormally activated kinases is an important research and development direction for precision targeted therapy drugs for colorectal cancer. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a quinazoline compound with anti-colorectal cancer activity, its preparation method, and its application. The quinazoline compound provided by this invention exhibits excellent anti-colorectal cancer activity as an inhibitor of anti-colorectal cancer drugs.

[0006] Compared with existing technologies, this invention provides a quinazoline compound with anti-colorectal cancer activity, its preparation method, and its application. The quinazoline compound with the structure shown in formula (I) provided by this invention, through selective substitution modification of a specific quinazoline core at positions 2, 4, 6, and 7, and by selecting specific groups for modification, demonstrates through experimental results that the quinazoline compound provided by this invention exhibits excellent activity as a drug inhibitor for colorectal cancer. Furthermore, the preparation method of the quinazoline compound provided by this invention has advantages such as mild reaction conditions, simple operation, green and efficient operation, and wide substrate applicability, showing great application prospects. Attached Figure Description

[0007] Figure 1 Compound 22 prepared in Example 22 1 H NMR spectrum;

[0008] Figure 2Compound 22 prepared in Example 22 13 C NMR spectrum;

[0009] Figure 3 Figure showing the clonal formation results of compound 22 prepared in Example 22;

[0010] Figure 4 Figure showing the apoptosis experiment results of compound 22 prepared in Example 22. Detailed Implementation

[0011] This invention provides a quinazoline compound having the structure shown in formula (Ⅰ).

[0012] Formula (I)

[0013] Where X is O, S, CH2 or NH;

[0014] R 1 It is hydrogen, substituted phenyl, substituted or unsubstituted C3~C6 cycloalkyl;

[0015] R 2 It is one or more of the following: hydrogen, halogen, C1-C4 alkyl, C3-C7 cycloalkyl, C1-C4 haloalkyl, C1-C5 alkoxy, C1-C5 haloalkoxy, hydroxyl, cyano, nitro, amino, mercapto, formyl, acetyl, methanesulfonyl, trifluoromethanesulfonyl, hydroxymethyl, sulfonyl, 5-membered heterocycle containing one or two nitrogen atoms, 6-membered heterocycle containing one or two nitrogen atoms, 5-membered heterocycle containing one or two oxygen atoms, 6-membered heterocycle containing one or two oxygen atoms, tri- or more heteroalkyl groups containing one or more nitrogen atoms, and tri- or more heteroalkyl groups containing one or more oxygen atoms;

[0016] R 3 It is hydrogen or methoxy;

[0017] R 4 It is hydrogen or methoxy.

[0018] In this invention, the R 1 In the substituted phenyl group, the substituent is preferably one or more of fluorine, chlorine, bromine, and iodine; the substituent in the substituted C3-C6 cycloalkyl group is preferably one or more of fluorine, chlorine, bromine, iodine, methyl, ethyl, n-propyl, and isopropyl; specifically, the R 1 Preferably, it is hydrogen, phenyl containing a halogen substituent, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, more preferably hydrogen, 3-chloro-4-fluorophenyl, 3-chlorophenyl, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. The R... 2Preferably, it is one or more of the following: fluorine, chlorine, bromine, iodine, methyl, ethyl, n-propyl, isopropyl, n-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, trifluoromethyl, trifluoroethyl, trifluoromethoxy, methoxy, ethoxy, propoxy, isopropoxy, tert-butoxy, piperazine, piperidinyl, N-methylpiperazine, N-methylpiperridinyl, morpholinyl, hydroxy, cyano, nitro, amino, mercapto, formyl, acetyl, methanesulfonyl, trifluoromethanesulfonyl, hydroxymethyl, sulfonyl, and 2-methoxyethoxy; it should be noted that R 2 It can be at any position on the benzene ring, and can be monosubstituted or polysubstituted.

[0019] More specifically, the quinazoline compounds have the following structures

[0020] This invention also provides a method for preparing a quinazoline compound with the structure shown in formula (I) having anti-colorectal cancer activity, comprising:

[0021] 1) The compound with the structure of formula (II) is combined with R 1 The XH2 reaction yields a compound with the structure shown in formula (III);

[0022] Formula (II), Equation (III)

[0023] 2) The compound with structure (III) is mixed and reacted with the compound with structure (IV) to obtain the compound with structure (I);

[0024] Formula (IV), Formula (I)

[0025] Where X is O, S, CH2 or NH;

[0026] R 1 It is hydrogen, substituted phenyl, substituted or unsubstituted C3-C6 cycloalkyl;

[0027] R 2 It is one or more of the following: hydrogen, halogen, C1-C4 alkyl, C3-C7 cycloalkyl, C1-C4 haloalkyl, C1-C5 alkoxy, C1-C5 haloalkoxy, hydroxyl, cyano, nitro, amino, mercapto, formyl, acetyl, methanesulfonyl, trifluoromethanesulfonyl, hydroxymethyl, sulfonyl, 5-membered heterocycle containing one or two nitrogen atoms, 6-membered heterocycle containing one or two nitrogen atoms, 5-membered heterocycle containing one or two oxygen atoms, 6-membered heterocycle containing one or two oxygen atoms, tri- or more heteroalkyl groups containing one or more nitrogen atoms, and tri- or more heteroalkyl groups containing one or more oxygen atoms;

[0028] R3 It is hydrogen or methoxy;

[0029] R 4 It is hydrogen or methoxy.

[0030] According to the present invention, the compound of formula (II) is first reacted with R 1 The XH reaction yields a compound with the structure shown in formula (III). This invention does not require special reaction conditions; reaction conditions known in the art for this type of reaction are acceptable. Preferably, K₂CO₃ is used as the base, DMF as the solvent, and the reaction is carried out at room temperature. This invention also reacts a mixture of the compound with the compound with the structure of formula (IV) to obtain a compound with the structure of formula (I). This invention does not require special reaction conditions; reaction conditions known in the art for this type of reaction are acceptable. Preferably, isopropanol or 1,4-dioxane is used as the solvent, and the reaction catalyst is preferably a hydrochloric acid solution or no catalyst is added.

[0031] The present invention also provides the use of the quinazoline compounds described herein and their pharmaceutically acceptable salts in the preparation of medicaments with anti-colorectal cancer activity.

[0032] The present invention also provides a pharmaceutical composition comprising: a quinazoline compound of the present invention or a pharmaceutically acceptable salt thereof, and an excipient; wherein the excipient is a pharmaceutically acceptable carrier or excipient. The quinazoline compound may be a stereoisomer or an optical isomer thereof. The pharmaceutical composition may be suitable for oral dosage forms, including but not limited to tablets, solutions, suspensions, capsules, granules, and powders.

[0033] This invention provides a quinazoline compound with anti-colorectal cancer activity, its preparation method, and its application. The quinazoline compound with the structure shown in formula (I) provided by this invention, through selective substitution modification of a specific quinazoline core at positions 2, 4, 6, and 7, and by selecting specific groups for modification, has been found to exhibit excellent activity in the preparation of anti-colorectal cancer drug inhibitors. Furthermore, the preparation method of the quinazoline compound provided by this invention has advantages such as mild reaction conditions, simple operation, green and efficient operation, and wide substrate applicability, and has good application prospects.

[0034] The technical solutions of the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] Example 1: 2-Chloro-N-cyclopentyl-6,7-dimethoxyquinazoline-4-amine (Intermediate 1a)

[0036] 1a

[0037] 350 mg (1.35 mmol) of 2,4-dichloro-6,7-dimethoxyquinazoline was weighed into a 250 mL single-necked round-bottom flask. Approximately 20 mL of N,N-dimethylformamide was added, followed by cyclopentanamine (126 mg, 1.48 mmol) and potassium carbonate (279 mg, 2.02 mmol). The mixture was reacted at room temperature for 1 h, and TLC was monitored until the reaction of 2,4-dichloroquinazoline was complete. 15 mL of water was slowly added to the reaction solution, resulting in the precipitation of a white solid. The solid was filtered, washed with water, and dried to obtain 384 mg of a white solid, with a yield of 92.6%. 1 HNMR (400 MHz, DMSO-d6) δ 8.02 (d, J = 6.8 Hz, 1H), 7.66 (s, 1H), 7.05 (s,1H), 4.52 – 4.42 (m, 1H), 3.89 (s, 3H), 3.88 (s, 3H), 2.04 (d, J = 3.4 Hz, 2H), 1.75 (s, 2H), 1.61 (d, J = 9.5 Hz, 4H).

[0038] 4-((4-(cyclopentylamino)-6,7-dimethoxyquinazoline-2-yl)amino)phenol

[0039] 1

[0040] Weigh intermediate 1a (216 mg, 0.7 mmol) and p-hydroxyaniline (153 mg, 1.4 mmol) into a 25 mL microwave-safe reaction flask, dissolve in 10 mL of isopropanol, and then place the reaction flask at 160°C. o The reaction was carried out in a microwave reactor for 50 min. TLC monitoring continued until the reaction was complete. The pH was adjusted to 8-9 with saturated sodium bicarbonate solution. The product was extracted with dichloromethane, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The crude product was separated by silica gel column chromatography (dichloromethane / methanol = 20:1) to give 91 mg of a purple solid, yield 34.2%, melting point: 145-147°C. o C. 1H NMR (400 MHz, DMSO-d6) δ 9.01 (s, 1H), 8.76 (s,1H), 7.73 (d, J = 17.3 Hz, 1H), 7.63 – 7.51 (m, 3H), 6.84 (s, 1H), 6.70 (d, J= 8.8 Hz, 1H), 4.60 – 4.49 (m, 1H), 3.85 (d, J = 1.6 Hz, 6H), 2.15 – 1.96 (m,2H), 1.77 (s, 2H), 1.67 – 1.52 (m, 4H), 1.22 (s, 1H). 13 C NMR (101 MHz, DMSO-d6) δ 158.72, 154.44, 154.27, 145.66, 122.02, 114.97, 104.10, 103.56, 102.93,56.29, 55.56, 52.50, 31.86, 23.68. HRMS (ESI) (m / z): [M+H] + calcd forC 21 H 24 N4O3, 381.1848, found 381.1922. HPLC purity: 99.48%.

[0041] Example 2: 2-Chloro-N-cyclopropylquinazoline-4-amine (Intermediate 1b)

[0042] 1b

[0043] 1 g (5 mmol) of 2,4-dichloroquinazoline was weighed into a 250 mL single-necked round-bottom flask, and about 15 mL of N,N-dimethylformamide was added. Cyclopropylamine (0.314 g, 5.5 mmol) and potassium carbonate (1.035 g, 7.5 mmol) were then added. The mixture was reacted at room temperature for 1 h, and TLC was monitored until the reaction of 2,4-dichloroquinazoline was complete. 15 mL of water was slowly added to the reaction solution, and a white solid precipitated. The precipitate was filtered, washed with water, and dried to give 0.905 g of a white solid, with a yield of 82.6%. 1H NMR (400 MHz, DMSO-d6) δ 8.66 (d, J = 2.4 Hz, 1H), 8.24 (d, J = 8.1 Hz, 1H), 7.82 – 7.74(m, 1H), 7.61 (d, J = 8.2 Hz, 1H), 7.54 – 7.47 (m, 1H), 3.08 – 3.00 (m, 1H), 0.85 – 0.80 (m, 2H), 0.73 – 0.66 (m, 2H).

[0044] 4-((4-(cyclopropylamino)quinazolin-2-yl)amino)phenol

[0045] 2

[0046] Weigh intermediate 1b (300 mg, 1.37 mmol) and p-hydroxyaniline (224 mg, 2.05 mmol) into a 50 mL round-bottom flask, add 10 mL of 1,4-dioxane and 0.5 mL of 4 mol / L HCl, heat to reflux under nitrogen protection, monitor by TLC until the reactants have reacted completely, cool to room temperature, filter, wash with 1,4-dioxane, adjust the pH of the filter cake to 8-9 with saturated sodium bicarbonate aqueous solution, stir at room temperature for 30 min, then filter and dry to obtain 390 mg of yellow solid, yield 97.4%, melting point: 248-250 °C. o C. 1 H NMR (400 MHz, DMSO-d6) δ 8.93 (d, J = 28.5 Hz, 2H), 8.19 – 7.98 (m,2H), 7.71 (d, J = 8.8 Hz, 2H), 7.58 – 7.52 (m, 1H), 7.35 (d, J = 8.2 Hz, 1H), 7.11 (t, J = 7.3 Hz, 1H), 6.69 (d, J = 8.9 Hz, 2H), 3.08 – 2.98 (m, 1H), 0.85– 0.79 (m, 2H), 0.73 – 0.66 (m, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 161.22,156.63, 151.78, 150.11, 132.74, 132.48, 124.18, 122.87, 120.87, 120.77,114.74, 111.17, 24.26, 6.21. HRMS (ESI) (m / z): [M+H]+ calcd for C 17 H 16 N4O,293.1324, found 293.1401. HPLC purity: 99.64%.

[0047] Example 3: 2-Chloro-N-cyclobutylquinazoline-4-amine (Intermediate 1c)

[0048] 1c

[0049] 1 g (5 mmol) of 2,4-dichloroquinazoline was weighed into a 250 mL single-necked round-bottom flask, and about 15 mL of N,N-dimethylformamide was added. Cyclobutylamine (0.391 g, 5.5 mmol) and potassium carbonate (1.035 g, 7.5 mmol) were then added. The mixture was reacted at room temperature for 1 h, and TLC was monitored until the reaction of 2,4-dichloroquinazoline was complete. 15 mL of water was slowly added to the reaction solution, resulting in the precipitation of a white solid. The solid was filtered, washed with water, and dried to obtain 1.067 g of a white solid, with a yield of 91.6%. 1 H NMR (400 MHz, DMSO-d6) δ 8.76 (d, J = 6.7 Hz, 1H), 8.33 (d, J = 8.2 Hz, 1H), 7.78 (t, J =7.5 Hz, 1H), 7.59 (d, J = 8.2 Hz, 1H), 7.52 (t, J = 7.5 Hz, 1H), 4.69 – 4.58 (m, 1H), 2.37 – 2.26 (m, 2H), 2.20 – 2.08 (m, 2H), 1.80 – 1.68 (m, 2H).

[0050] 4-((4-(cyclobutylamino)quinazolin-2-yl)amino)phenol

[0051] 3

[0052] Weigh intermediate 1c (300 mg, 1.29 mmol) and p-hydroxyaniline (281 mg, 2.57 mmol) into a 25 mL microwave-safe reaction flask, dissolve in 10 mL of isopropanol, and then place the reaction flask at 160°C. o The reaction was carried out in a microwave reactor for 50 min. A brown solid was formed. The reaction was monitored by TLC until complete. The mixture was cooled to room temperature, filtered, washed with isopropanol, and dried. The pH was adjusted to 8-9 with NaOH aqueous solution, and the mixture was stirred at room temperature for 30 min. It was then filtered and dried to obtain 358 mg of brown solid, with a yield of 90.9% and a melting point of 151-153°C.o C. 1 H NMR (500 MHz, DMSO-d6) δ 9.49 (s, 1H), 9.28 (s, 1H), 8.87 (s,1H), 8.27 (d, J = 8.0 Hz, 1H), 7.65 (t, J = 7.5 Hz, 1H), 7.52 – 7.39 (m, 3H),7.26 (t, J = 7.4 Hz, 1H), 6.76 (d, J = 8.7 Hz, 2H), 4.64 (d, J = 6.7 Hz, 1H),2.37 – 2.27 (m, 2H), 2.24 – 2.15 (m, 2H), 1.81 – 1.67 (m, 2H). 13 C NMR (126MHz, DMSO-d6) δ 159.00, 154.34, 153.57, 133.77, 130.24, 123.71, 122.89,122.58, 121.03, 115.16, 110.60, 46.36, 29.68, 15.02. HRMS (ESI) (m / z): [M+H] + calcd for C 18 H 19 N4O, 307.1481, found 307.1552. HPLC purity: 99.43%.

[0053] Example 4: 2-Chloro-N-cyclohexylquinazoline-4-amine (Intermediate 1d)

[0054] 1d

[0055] 1 g of 2,4-dichloroquinazoline (5 mmol) was weighed into a 250 mL single-necked round-bottom flask, and about 15 mL of N,N-dimethylformamide was added. Cyclohexylamine (0.545 g, 5.5 mmol) and potassium carbonate (1.035 g, 7.5 mmol) were then added. The mixture was reacted at room temperature for 1 h, and TLC was monitored until the reaction of 2,4-dichloroquinazoline was complete. 15 mL of water was slowly added to the reaction solution, resulting in the precipitation of a white solid. The solid was filtered, washed with water, and dried to obtain 1.286 g of a white solid, with a yield of 98.5%. 1H NMR (400 MHz, DMSO-d6) δ 8.34 (d, J = 8.2 Hz, 2H), 7.80 – 7.73 (m, 1H), 7.59 (d, J = 8.2Hz, 1H), 7.50 (t, J = 7.6 Hz, 1H), 4.16 – 4.02 (m, 1H), 1.93 (d, J = 10.2 Hz, 2H), 1.77 (d, J = 12.0 Hz, 2H), 1.64 (d, J = 12.8 Hz, 1H), 1.47 – 1.28 (m,4H), 1.24 – 1.09 (m, 1H).

[0056] 4-((4-(cyclohexylamino)quinazolin-2-yl)amino)phenol

[0057] 4

[0058] Weigh intermediate 1d (300 mg, 1.29 mmol) and p-hydroxyaniline (225 mg, 2.07 mmol) into a 25 mL microwave-safe reaction flask, add 10 mL of isopropanol, and then place the reaction flask at 160°C. o The reaction was carried out in a microwave reactor for 50 min. TLC monitoring continued until the reaction was complete. The pH was adjusted to 8-9 with saturated sodium bicarbonate solution. The product was extracted with dichloromethane, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The crude product was separated by silica gel column chromatography (dichloromethane / methanol = 30:1) to give 166 mg of a black solid, yield 43.2%, melting point: 138-140 °C. o C. 1 H NMR (400 MHz, DMSO-d6) δ 9.01 (s, 1H), 8.75 (s,1H), 8.10 (d, J = 8.1 Hz, 1H), 7.75 (d, J = 6.1 Hz, 1H), 7.62 – 7.49 (m, 3H),7.31 (d, J = 8.3 Hz, 1H), 7.10 (t, J = 7.4 Hz, 1H), 6.67 (d, J = 8.8 Hz, 2H), 4.17 – 4.06 (m, 1H), 2.03 – 1.92 (m, 2H), 1.82 – 1.77 (m, 1H), 1.71 – 1.61(m, 1H), 1.46 – 1.09 (m, 6H). 13C NMR (101 MHz, DMSO-d6) δ 159.11, 156.69,151.89, 132.72, 132.55, 125.88, 124.22, 123.12, 122.88, 120.95, 120.84,115.78, 115.48, 115.44, 114.77, 111.28, 49.61, 48.61, 32.15, 25.39, 25.27.HRMS (ESI) (m / z): [M+H] + calcd for C 20 H 22 N4O, 335.1794, found 335.1868. HPLC purity: 96.18%.

[0059] Example 5: 2-Chloro-N-(3-Chlorophenyl)quinazoline-4-amine (Intermediate 1e)

[0060] 1e

[0061] 1 g of 2,4-dichloroquinazoline (5 mmol) was weighed into a 250 mL single-necked round-bottom flask, and about 15 mL of N,N-dimethylformamide was added. Then, 0.699 g of m-chloroaniline (5.5 mmol) and 1.035 g of potassium carbonate (7.5 mmol) were added. The mixture was reacted at room temperature for 1 h, and TLC was monitored until the 2,4-dichloroquinazoline reaction was complete. 15 mL of water was slowly added to the reaction solution, precipitating a white solid. The precipitate was filtered, washed with water, and dried to give 0.931 g of a white solid, with a yield of 64.4%. 1 H NMR (400 MHz, DMSO-d6) δ 8.74 (d, J = 8.2 Hz, 1H), 7.82 – 7.77 (m, 2H), 7.59 (d, J = 8.2Hz, 1H), 7.50 (t, J = 8.0 Hz, 1H), 7.45 – 7.27 (m, 4H).

[0062] 4-((4-((3-chlorophenyl)amino)quinazolin-2-yl)amino)phenol

[0063] 5

[0064] Weigh intermediate 1e (300 mg, 1.04 mmol) and p-hydroxyaniline (227 mg, 2.08 mmol) into a 25 mL microwave-safe reaction flask, add 10 mL of isopropanol, and then place the reaction flask at 160°C.o The reaction was carried out in a microwave reactor for 50 min, and TLC was monitored until the reaction was complete. The mixture was then cooled to room temperature, filtered, washed with isopropanol, and dried. The pH was adjusted to 8-9 with NaOH aqueous solution, and the mixture was stirred at room temperature for 30 min. It was then filtered and dried to obtain 105 mg of a yellow solid, with a yield of 27.9% and a melting point of 246-248 °C. o C. 1 H NMR(400 MHz, DMSO-d6) δ 10.42 (s, 1H), 9.66 (s, 1H), 9.33 (s, 1H), 8.58 (d, J =7.9 Hz, 1H), 7.96 (s, 1H), 7.86 (s, 1H), 7.75 (t, J = 7.5 Hz, 1H), 7.51 (d, J= 8.3 Hz, 1H), 7.37 (dd, J = 15.1, 7.2 Hz, 4H), 7.22 (d, J = 7.7 Hz, 1H), 6.75 (d, J = 8.6 Hz, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 158.52, 154.70, 153.60,140.08, 134.08, 132.82, 129.94, 123.92, 123.76, 122.99, 122.67, 122.28,121.21, 115.18, 110.99. HRMS (ESI) (m / z): [M+H] + calcd for C 20 H 15 ClN4O,363.0934, found 363.1008. HPLC purity: 99.01%.

[0065] Example 6: 2-Chloro-N-cyclopentylquinazoline-4-amine (Intermediate 1f)

[0066] 1f

[0067] 3 g (15 mmol) of 2,4-dichloroquinazoline was weighed into a 250 mL single-necked round-bottom flask, and approximately 20 mL of N,N-dimethylformamide was added. Cyclopentanylamine (1.4 g, 16 mmol) and potassium carbonate (3.1 g, 32 mmol) were then added. The mixture was reacted at room temperature for 1 h, and TLC was monitored until the reaction of 2,4-dichloroquinazoline was complete. After the reaction was complete, 15 mL of water was slowly added to the reaction solution, resulting in the precipitation of a white solid. The solid was filtered, washed with water, and dried to obtain 3.399 g of a white solid, with a yield of 91.7%. 1 H NMR (400MHz, DMSO-d6) δ 8.43 (d, J = 7.0 Hz,1H), 8.35 (d, J = 8.1 Hz,1H), 7.77 (t, J= 7.7 Hz,1H), 7.59 (d, J = 8.2 Hz,1H), 7.51 (t, J = 7.6 Hz,1H), 4.55 – 4.45(m,1H), 2.06 – 1.95 (m,2H), 1.78 – 1.69 (m,2H), 1.67 – 1.54 (m,4H).

[0068] N4-Cyclopentyl-N2-phenylquinazoline-2,4-diamine

[0069] 6

[0070] Weigh intermediate 1f (200 mg, 0.81 mmol) and aniline (151 mg, 1.62 mmol) into a 25 mL microwave-safe reaction flask, dissolve in 10 mL of isopropanol, and then place the reaction flask at 160°C. o The reaction was carried out in a microwave reactor for 50 min, and TLC was monitored until the reaction was complete. The mixture was then cooled to room temperature, filtered, washed twice with isopropanol, and dried. The pH was adjusted to 8-9 with NaOH aqueous solution, and the mixture was stirred at room temperature for 30 min. It was then filtered and dried to obtain 195 mg of a brown solid, with a yield of 79.1% and a melting point of 154-156 °C. o C. 1H NMR (500 MHz, DMSO-d6) δ 9.92 (s, 1H), 8.81 (s, 1H), 8.39 (d, J = 8.0 Hz, 1H), 7.76 – 7.67 (m, 3H), 7.47 (d, J = 8.2 Hz, 1H), 7.38 – 7.28 (m, 3H), 7.07 (t,J = 7.2 Hz, 1H), 4.58 – 4.48 (m, 1H), 2.02 (d, J = 8.0 Hz, 2H), 1.78 – 1.69(m, 4H), 1.61 – 1.55 (m, 2H). 13 C NMR (126 MHz, DMSO-d6) δ 159.73, 153.54,138.77, 134.14, 128.68, 124.09, 123.21, 120.64, 110.81, 53.21, 31.71, 23.81.HRMS (ESI) (m / z): [M+H] + calcd for C 19 H 20 N4O3, 305.1688; found 305.1762. HPLCpurity: 100.00%.

[0071] Example 7 N2-(4-aminophenyl)-N4-cyclopentylquinazoline-2,4-diamine

[0072] 7

[0073] Weigh 300 mg (1.21 mmol) of intermediate 1f and 196 mg (1.81 mmol) of p-phenylenediamine into a 50 mL round-bottom flask. Add 10 mL of 1,4-dioxane and 0.5 mL of 4 mol / L HCl. Heat under reflux for 2 h under nitrogen protection. Monitor the reaction by TLC until the reactants have reacted completely. Cool to room temperature, filter, wash with 1,4-dioxane, adjust the pH of the filter cake to 8-9 with saturated sodium bicarbonate aqueous solution, then extract with dichloromethane, dry with anhydrous sodium sulfate, remove the solvent by rotary evaporation, and separate the crude product by silica gel column chromatography (dichloromethane / methanol = 15:1) to obtain 171 mg of brown solid, yield 43.4%, melting point: 84-86°C. o C. 1H NMR(400 MHz, DMSO-d6) δ 8.61 (s, 1H), 8.12 (d, J = 7.1 Hz, 1H), 7.75 (s, 1H),7.61 – 7.40 (m, 3H), 7.30 (d, J = 7.5 Hz, 1H), 7.08 (s, 1H), 6.51 (d, J = 7.3Hz, 2H), 5.01 (s, 2H), 4.55 (s, 1H), 2.02 (s, 2H), 1.68 (d, J = 66.3 Hz, 6H). 13 C NMR (101 MHz, DMSO-d6) δ 159.54, 156.87, 150.65, 142.98, 132.24, 130.48,124.22, 123.00, 122.64, 120.96, 120.41, 114.23, 113.88, 111.27, 52.11, 31.89,23.62. HRMS (ESI) (m / z): [M+H] + calcd for C 19 H 21 N5, 320.1797, found 320.1871.HPLC purity: 100.00%.

[0074] Example 8 4-((4-(cyclopentylamino)quinazolin-2-yl)amino)benzylthiol

[0075] 8

[0076] Weigh intermediate 1f (300 mg, 1.21 mmol) and 4-aminothiophenol (272 mg, 2.18 mmol) into a 25 mL microwave-safe reaction flask, dissolve in 10 mL of isopropanol, and then place the reaction flask at 160°C. o The reaction was carried out in a microwave reactor for 50 min, and TLC was monitored until the reaction was complete. The pH was adjusted to 8-9 with saturated sodium bicarbonate aqueous solution, followed by extraction with dichloromethane, drying with anhydrous sodium sulfate, and removing the solvent by rotary evaporation. The crude product was separated by silica gel column chromatography (dichloromethane / methanol = 50:1) to give 143 mg of brown solid, yield 35.2%, melting point: 185-187°C. o C. 1H NMR (400 MHz, DMSO-d6) δ 9.86 (s, 1H), 8.64(d, J = 8.0 Hz, 1H), 7.88 (t, J = 7.5 Hz, 1H), 7.75 (d, J = 8.2 Hz, 1H), 7.55(t, J = 7.5 Hz, 1H), 7.29 (d, J = 8.2 Hz, 2H), 6.74 (d, J = 8.2 Hz, 2H), 4.02(dd, J = 13.9, 6.9 Hz, 1H), 3.50 (s, 1H), 1.72 – 1.39 (m, 9H). 13 C NMR (101MHz, DMSO-d6) δ 166.05, 157.78, 149.66, 137.26, 135.10, 126.29, 124.74,118.95, 114.78, 111.34, 53.91, 30.90, 23.18. HRMS (ESI) (m / z): [M+H] + calcdfor C 19 H 20 N4S, 337.1409, found 337.1485. HPLC purity: 100.00%.

[0077] Example 9 (4-((4-(cyclopentylamino)quinazolin-2-yl)amino)phenyl)methanol

[0078] 9

[0079] Weigh 300 mg (1.21 mmol) of intermediate 1f and 145 mg (1.18 mmol) of 4-aminobenzyl alcohol into a 50 mL round-bottom flask. Add 10 mL of 1,4-dioxane and 0.5 mL of 4 mol / L HCl. Heat under reflux for 2 h under nitrogen protection. Monitor the reaction by TLC until the reactants have reacted completely. Cool to room temperature, filter, wash with 1,4-dioxane, adjust the pH of the filter cake to 8-9 with saturated sodium bicarbonate aqueous solution, extract with dichloromethane, dry with anhydrous sodium sulfate, remove the solvent by rotary evaporation, and separate the crude product by silica gel column chromatography (dichloromethane / methanol = 20:1) to obtain 38 mg of yellow solid, yield 9.4%, melting point: 90-92°C. o C. 1H NMR(400 MHz, DMSO-d6) δ 9.15 (s, 1H), 8.20 (d, J = 7.8 Hz, 1H), 7.99 (d, J = 5.9Hz, 1H), 7.82 (d, J = 8.4 Hz, 2H), 7.62 – 7.56 (m, 1H), 7.40 (d, J = 7.9 Hz,1H), 7.23 – 7.16 (m, 3H), 5.06 (s, 1H), 4.66 – 4.53 (m, 1H), 4.44 (s, 2H),2.10 – 2.02 (m, 2H), 1.81 – 1.72 (m, 2H), 1.69 – 1.56 (m, 4H). 13 C NMR (101MHz, DMSO-d6) δ 159.76, 156.64, 150.41, 139.96, 134.71, 132.53, 126.81,124.64, 123.16, 121.23, 118.51, 111.55, 62.87, 52.31, 31.96, 23.71. HRMS(ESI) (m / z): [M+H] + calcd for C 20 H 22 N4O, 335.1794, found 335.1866. HPLC purity:100.00%.

[0080] Example 10 N4-cyclopentyl-N2-(4-fluorophenyl)quinazoline-2,4-diamine

[0081] 10

[0082] Weigh 300 mg (1.21 mmol) of intermediate 1f and 201 mg (1.81 mmol) of p-fluoroaniline into a 50 mL round-bottom flask. Add 10 mL of 1,4-dioxane and 0.5 mL of 4 mol / L HCl. Heat under reflux for 2 h under nitrogen protection. Monitor the reaction by TLC until the reactants have reacted completely. Cool to room temperature, filter, wash with 1,4-dioxane, adjust the pH of the filter cake to 8-9 with saturated sodium bicarbonate aqueous solution, stir at room temperature for 30 min, then filter and dry to obtain 327 mg of yellow solid, yield 83.9%, melting point: 255-257°C. o C. 1H NMR (500 MHz, DMSO-d6) δ 10.18 (s, 1H), 9.10 (s, 1H), 8.44 (d, J= 8.0 Hz, 1H), 7.77 (t, J = 7.6 Hz, 1H), 7.69 (s, 2H), 7.52 (d, J = 8.2 Hz,1H), 7.39 (t, J = 7.5 Hz, 1H), 7.24 (t, J = 8.7 Hz, 2H), 4.58 – 4.41 (m, 1H), 2.02 – 1.95 (m, 2H), 1.79 – 1.68 (m, 4H), 1.61 – 1.53 (m, 2H). 13 C NMR (126MHz, DMSO-d6) δ 159.68, 157.83, 152.57, 134.65, 134.12, 124.34, 123.85,119.01, 115.54, 115.36, 110.59, 53.43, 31.63, 23.84. HRMS (ESI) (m / z): [M+H] + calcd for C 19 H 19 FN4, 323.1594, found 323.1669. HPLC purity: 99.63%.

[0083] Example 11 N2-(4-chlorophenyl)-N4-cyclopentylquinazoline-2,4-diamine

[0084] 11

[0085] Weigh intermediate 1f (300 mg, 1.21 mmol) and p-chloroaniline (307 mg, 2.42 mmol) into a 25 mL microwave-safe reaction flask, dissolve in 10 mL of isopropanol, and then place the reaction flask at 160°C. o The reaction was carried out in a microwave reactor for 50 min, and TLC was monitored until the reaction was complete. The mixture was then cooled to room temperature, filtered, washed with isopropanol, and dried. The pH was adjusted to 8-9 with NaOH aqueous solution, and the mixture was stirred at room temperature for 30 min. It was then filtered and dried to obtain 364 mg of a brown solid, with a yield of 89.0% and a melting point of 168-170°C. o C. 1H NMR (400 MHz, DMSO-d6) δ 10.23 (s, 1H), 9.03 (s, 1H), 8.43 (d, J = 8.2 Hz, 1H), 7.81 – 7.66 (m, 3H), 7.50 (d, J = 8.2 Hz, 1H), 7.46 – 7.32 (m, 3H), 4.57 –4.47 (m, 1H), 2.09 – 1.95 (m, 2H), 1.82 – 1.67 (m, 4H), 1.65 – 1.51 (m, 2H). 13 C NMR (101 MHz, MeOD-d4) δ 161.77, 155.06, 144.39, 138.21, 135.50, 130.47,129.87, 125.29, 124.57, 124.54, 121.08, 112.36, 55.09, 33.15, 25.05. HRMS(ESI) (m / z): [M+H] + calcd for C 19 H 19 ClN4, 339.1298, found 339.1370. HPLCpurity: 100.00%.

[0086] Example 12 N4-cyclopentyl-N2-(p-tolyl)quinazolin-2,4-diamine

[0087] 12

[0088] Weigh intermediate 1f (300 mg, 1.21 mmol) and p-toluidine (259 mg, 2.42 mmol) into a 25 mL microwave-safe reaction flask, dissolve in 10 mL of isopropanol, and then place the reaction flask at 160°C. o The reaction was carried out in a microwave reactor for 50 min, and TLC was monitored until the reaction was complete. The mixture was then cooled to room temperature, filtered, washed with isopropanol, and dried. The pH was adjusted to 8-9 with NaOH aqueous solution, and the mixture was stirred at room temperature for 30 min. It was then filtered and dried to give 194 mg of a white solid, with a yield of 49.8% and a melting point of 153-155°C. o C. 1H NMR(400 MHz, DMSO-d6) δ 10.45 (s, 1H), 9.44 (d, J = 5.7 Hz, 1H), 8.54 (d, J =8.1 Hz, 1H), 7.79 (t, J = 7.7 Hz, 1H), 7.52 – 7.46 (m, 3H), 7.40 (t, J = 7.6Hz, 1H), 7.20 (d, J = 8.2 Hz, 2H), 4.50 (dd, J = 13.1, 6.6 Hz, 1H), 2.29 (s,3H), 2.04 – 1.94 (m, 2H), 1.81 – 1.70 (m, 4H), 1.62 – 1.53 (m, 2H). 13 C NMR(101 MHz, DMSO-d6) δ 159.71, 156.59, 138.68, 132.53, 129.38, 128.70, 124.50,123.19, 121.16, 118.87, 111.50, 52.28, 31.94, 23.71, 20.37. HRMS (ESI) (m / z):[M+H] + calcd for C 20 H 22 N4, 319.1844, found 319.1914. HPLC purity: 100.00%.

[0089] Example 13 N4-cyclopentyl-N2-(4-methoxyphenyl)quinazoline-2,4-diamine

[0090] 13

[0091] Weigh intermediate 1f (300 mg, 1.21 mmol) and p-methoxyaniline (298 mg, 2.42 mmol) into a 25 mL microwave-safe reaction flask, dissolve in 10 mL of isopropanol, and then place the reaction flask at 160°C. o The reaction was carried out in a microwave reactor for 50 min. A yellow solid was formed. The reaction was monitored by TLC until complete. The mixture was cooled to room temperature, filtered, washed with isopropanol, and dried. The pH was adjusted to 8-9 with NaOH aqueous solution, and the mixture was stirred at room temperature for 30 min. It was then filtered and dried to give 231 mg of a white solid, with a yield of 57.1% and a melting point of 127-129°C. o C. 1H NMR (400 MHz, DMSO-d6) δ 10.37 (s, 1H), 9.39 (s, 1H), 8.54 (d,J = 8.2 Hz, 1H), 7.78 (t, J = 7.7 Hz, 1H), 7.53 – 7.47 (m, 3H), 7.40 (t, J =7.7 Hz, 1H), 6.98 (d, J = 8.9 Hz, 2H), 4.48 (d, J = 5.7 Hz, 1H), 3.77 (s,3H), 2.03 – 1.93 (m, 2H), 1.81 – 1.71 (m, 4H), 1.61 – 1.52 (m, 2H). 13 C NMR(101 MHz, DMSO-d6) δ 159.67, 157.04, 153.50, 151.29, 134.85, 132.29, 125.01,123.04, 120.73, 120.05, 113.50, 111.60, 55.13, 52.12, 31.99, 23.70. HRMS(ESI) (m / z): [M+H] + calcd for C 20 H 22 N4O, 335.1794, found 335.1864. HPLC purity:100.00%.

[0092] Example 14 N4-cyclopentyl-N2-(2-fluorophenyl)quinazoline-2,4-diamine

[0093] 14

[0094] Weigh intermediate 1f (300 mg, 1.21 mmol) and o-fluoroaniline (268 mg, 2.42 mmol) into a 25 mL microwave-safe reaction flask, dissolve in 10 mL of isopropanol, and then place the reaction flask at 160°C. o The reaction was carried out in a microwave reactor for 50 min. A brown solid was formed. The reaction was monitored by TLC until complete. The mixture was cooled to room temperature, filtered, washed with isopropanol, and dried. The pH was adjusted to 8-9 with NaOH aqueous solution, and the mixture was stirred at room temperature for 30 min. It was then filtered and dried to give 216 mg of a white solid, with a yield of 55.4% and a melting point of 113-115°C. o C. 1H NMR (400 MHz, DMSO-d6) δ 8.29 – 8.22 (m, 1H), 8.20 – 8.11 (m,2H), 7.82 (d, J = 6.9 Hz, 1H), 7.60 – 7.52 (m, 1H), 7.36 (d, J = 7.7 Hz, 1H),7.23 – 7.11 (m, 3H), 7.05 – 6.97 (m, 1H), 4.55 – 4.44 (m, 1H), 2.05 – 1.94(m, 2H), 1.80 – 1.68 (m, 2H), 1.67 – 1.49 (m, 4H). 13 C NMR (101 MHz, DMSO-d6)δ 159.88, 156.88, 154.85, 152.43, 151.02, 132.44, 128.74, 128.64, 125.22,123.92, 123.88, 123.43, 123.12, 122.67, 122.59, 121.38, 115.03, 114.83,111.84, 52.19, 31.90, 23.70. HRMS (ESI) (m / z): [M+H] + calcd for C 19 H 19 FN4,323.1594, found 323.1663. HPLC purity: 100.00%.

[0095] Example 15 N4-cyclopentyl-N2-(3-fluorophenyl)quinazolin-2,4-diamine

[0096] 15

[0097] Weigh intermediate 1f (300 mg, 1.21 mmol) and 3-fluoroaniline (268 mg, 2.42 mmol) into a 25 mL microwave-safe reaction flask, dissolve in 10 mL of isopropanol, and then place the reaction flask at 160°C. o The reaction was carried out in a microwave reactor for 50 min. A white solid was formed. TLC monitoring continued until the reaction was complete. The mixture was cooled to room temperature, filtered, washed with isopropanol, and dried. The pH was adjusted to 8-9 with NaOH aqueous solution, and the mixture was stirred at room temperature for 30 min. Filtering and drying were then performed to obtain 313 mg of a white solid, with a yield of 80.3% and a melting point of 230-232°C. o C. 1H NMR (400 MHz, DMSO-d6) δ 10.64 (s, 1H), 9.46 (s, 1H), 8.51 (d, J= 8.2 Hz, 1H), 7.83 (t, J = 7.6 Hz, 1H), 7.70 (d, J = 11.5 Hz, 1H), 7.56 (d,J = 8.3 Hz, 1H), 7.49 – 7.40 (m, 2H), 7.34 (d, J = 8.1 Hz, 1H), 7.01 (t, J =7.3 Hz, 1H), 4.52 (d, J = 6.4 Hz, 1H), 2.03 (d, J = 6.6 Hz, 2H), 1.78 (s,4H), 1.60 (s, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 163.33, 160.93, 159.77, 151.32,139.10, 135.27, 130.57, 130.47, 124.66, 117.82, 117.31, 110.86, 110.37,108.66, 108.40, 53.97, 31.50, 23.77. HRMS (ESI) (m / z): [M+H] + calcd forC 19 H 19 FN4, 323.1594, found 323.1662. HPLC purity: 97.95%.

[0098] Example 16 N2-(2-chlorophenyl)-N4-cyclopentylquinazoline-2,4-diamine

[0099] 16

[0100] Weigh intermediate 1f (300 mg, 1.21 mmol) and o-chloroaniline (307 mg, 2.42 mmol) into a 25 mL microwave-safe reaction flask, dissolve in 10 mL of isopropanol, and then place the reaction flask at 160°C. o The reaction was carried out in a microwave reactor for 50 min. TLC monitoring continued until the reaction was complete. The pH was adjusted to 8-9 with saturated sodium bicarbonate solution, and the product was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The crude product was separated by silica gel column chromatography (petroleum ether / ethyl acetate = 10:1) to give 119 mg of a white solid, yield 29.1%, melting point: 160-162°C. o C. 1H NMR (400 MHz, DMSO-d6) δ 8.52 (d, J = 6.6 Hz, 1H), 8.20 (d, J = 6.1 Hz, 1H), 7.89 (s, 1H), 7.77 (s, 1H), 7.59 (s, 1H), 7.48 –7.38 (m, 2H), 7.32 (s, 1H), 7.20 (s, 1H), 7.01 (s, 1H), 4.56 – 4.42 (m, 1H), 2.01 (s, 2H), 1.78 – 1.52 (m, 6H). 13 C NMR (101 MHz, ) δ 159.94, 156.50,150.83, 137.08, 132.58, 128.97, 127.27, 125.41, 123.15, 123.05, 122.78,122.33, 121.79, 112.01, 52.26, 31.94, 23.75. HRMS (ESI) (m / z): [M+H] + calcdfor C 19 H 19 ClN4, 339.1298, found 339.1368. HPLC purity: 100.00%.

[0101] Example 17 N2-(3-chlorophenyl)-N4-cyclopentylquinazoline-2,4-diamine

[0102] 17

[0103] Weigh intermediate 1f (300 mg, 1.21 mmol) and m-chloroaniline (307 mg, 2.42 mmol) into a 25 mL microwave-safe reaction flask, dissolve in 10 mL of isopropanol, and then place the reaction flask at 160°C. o The reaction was carried out in a microwave reactor for 50 min. TLC monitoring continued until the reaction was complete. The pH was adjusted to 8-9 with saturated sodium bicarbonate aqueous solution, and the product was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The crude product was separated by silica gel column chromatography (dichloromethane / methanol = 50:1) to give 189 mg of a brown solid, yield 67.6%, melting point: 116-118 °C. o C. 1H NMR (400 MHz, DMSO-d6) δ 9.30 (s, 1H), 8.32 (t, J =7.1, 5.2 Hz, 1H), 8.20 (d, J = 8.0 Hz, 1H), 7.90 (d, J = 6.9 Hz, 1H), 7.68(d, J = 8.2 Hz, 1H), 7.60 (t, J = 7.6 Hz, 1H), 7.41 (d, J = 8.2 Hz, 1H), 7.28– 7.16 (m, 2H), 6.90 (dd, J = 7.8, 1.5 Hz, 1H), 4.65 – 4.53 (m, 1H), 2.14 –2.02 (m, 2H), 1.84 – 1.73 (m, 2H), 1.71 – 1.56 (m, 4H). 13 C NMR (101 MHz, DMSO-d6) δ 159.89, 156.72, 150.89, 143.19, 132.88, 132.60, 129.79, 125.19,123.18, 121.56, 119.75, 117.67, 116.77, 111.72, 52.45, 31.93, 23.67. HRMS(ESI) (m / z): [M+H] + calcd for C 19 H 19 ClN4, 339.1298, found 339.1368. HPLCpurity: 96.44%.

[0104] Example 18 N4-cyclopentyl-N2-(o-tolyl)quinazolin-2,4-diamine

[0105] 18

[0106] Weigh intermediate 1f (300 mg, 1.21 mmol) and o-methylaniline (259 mg, 2.42 mmol) into a 25 mL microwave-safe reaction flask, dissolve in 10 mL of isopropanol, and then place the reaction flask at 160°C. o The reaction was carried out in a microwave reactor for 50 min. TLC monitoring was performed until the reaction was complete. 15 mL of water was slowly added to the reaction solution. During the water addition, a brown solid precipitated. The pH was adjusted to 8-9 with NaOH aqueous solution, and the mixture was stirred at room temperature for 30 min. The solution was then filtered and dried to obtain 304 mg of brown solid, with a yield of 79.0% and a melting point of 116-118 °C. o C.1 H NMR (400 MHz, DMSO-d6) δ 9.58 (s, 1H), 9.08 (d, J= 5.3 Hz, 1H), 8.46 (d, J = 8.1 Hz, 1H), 7.73 (t, J = 7.7 Hz, 1H), 7.65 (d, J= 7.8 Hz, 1H), 7.46 (d, J = 8.3 Hz, 1H), 7.35 (t, J = 7.6 Hz, 1H), 7.30 –7.18 (m, 2H), 7.15 (t, J = 7.4 Hz, 1H), 4.42 – 4.30 (m, 1H), 2.29 (s, 3H),1.95 – 1.81 (m, 2H), 1.75 – 1.61 (m, 4H), 1.49 (d, J = 4.2 Hz, 2H). 13 C NMR(101 MHz, DMSO-d6) δ 159.63, 153.29, 135.64, 134.49, 130.44, 126.10, 125.60,125.33, 124.39, 123.58, 118.79, 110.49, 53.21, 31.56, 23.80, 18.00. HRMS(ESI) (m / z): [M+H] + calcd for C 20 H 20 N4, 319.1844, found 319.1915. HPLC purity:100.00%.

[0107] Example 19 N4-cyclopentyl-N2-(m-tolyl)quinazolin-2,4-diamine

[0108] 19

[0109] Weigh intermediate 1f (300 mg, 1.21 mmol) and 3-methylaniline (259 mg, 2.42 mmol) into a 25 mL microwave-safe reaction flask, dissolve in 10 mL of isopropanol, and then place the reaction flask at 160°C. o The reaction was carried out in a microwave reactor for 50 min. TLC monitoring was performed until the reaction was complete. The mixture was cooled to room temperature, filtered, washed with isopropanol, and dried. The pH was adjusted to 8-9 with NaOH aqueous solution, and the mixture was stirred at room temperature for 30 min. Filtering and drying were then performed to give 295 mg of a white solid (76.6% yield), with a melting point of 212-214°C. o C.1 HNMR (400 MHz, DMSO-d6) δ 10.44 (s, 1H), 9.41 (s, 1H), 8.51 (d, J = 8.1 Hz,1H), 7.81 (t, J = 7.3 Hz, 1H), 7.55 (s, 1H), 7.50 (d, J = 8.2 Hz, 1H), 7.43(t, J = 7.6 Hz, 1H), 7.36 (d, J = 8.2 Hz, 1H), 7.29 (t, J = 7.8 Hz, 1H), 7.00(d, J = 7.4 Hz, 1H), 4.58 – 4.48 (m, 1H), 2.33 (s, 3H), 2.08 – 1.96 (m, 2H),1.84 – 1.72 (m, 4H), 1.60 (d, J = 9.6 Hz, 2H). 13 C NMR (101 MHz, DMSO-d6) δ159.69, 151.48, 138.18, 137.12, 135.14, 130.83, 128.74, 125.16, 124.68,124.33, 121.94, 118.53, 117.57, 110.20, 54.49, 53.85, 31.49, 23.79, 21.13.HRMS (ESI) (m / z): [M+H] + calcd for C 20 H 22 N4, 319.1844, found 319.1914. HPLCpurity: 100.00%.

[0110] Example 20 N4-cyclopentyl-N2-(2-methoxyphenyl)quinazolin-2,4-diamine

[0111] 20

[0112] Weigh 300 mg (1.21 mmol) of intermediate 1f and 298 mg (2.42 mmol) of o-methoxyaniline into a 25 mL microwave-safe reaction flask, dissolve in 10 mL of isopropanol, and then place the flask in a microwave-safe reactor at 160 °C for 50 min. Monitor the reaction by TLC until complete. Cool to room temperature, filter, wash with isopropanol, and dry. Adjust the pH to 8-9 with NaOH aqueous solution, stir at room temperature for 30 min, then filter and dry to obtain 228 mg of white solid (yield 56.4%), melting point: 135-137 °C.o C. 1 HNMR (400 MHz, DMSO-d6) δ 8.73 (d, J = 7.5 Hz, 1H), 8.20 (d, J = 8.0 Hz, 1H), 7.84 (d, J = 6.6 Hz, 1H), 7.59 (t, J = 7.4 Hz, 1H), 7.48 – 7.35 (m, 2H), 7.18(t, J = 7.4 Hz, 1H), 7.04 – 6.88 (m, 3H), 4.55 (dd, J = 13.3, 6.6 Hz, 1H), 3.89 (s, 3H), 2.05 (d, J = 11.1 Hz, 2H), 1.79 – 1.55 (m, 6H). 13 C NMR (101MHz, DMSO-d6) δ 159.78, 156.40, 150.89, 147.33, 132.38, 129.73, 125.36,123.00, 121.44, 120.74, 120.35, 118.18, 111.82, 110.20, 55.69, 52.15, 31.89,23.66. HRMS (ESI) (m / z): [M+H] + calcd for C 20 H 22 N4O, 335.1794, found 335.1871.HPLC purity: 100.00%.

[0113] Example 21 N4-cyclopentyl-N2-(3-methoxyphenyl)quinazolin-2,4-diamine

[0114] twenty one

[0115] Weigh intermediate 1f (300 mg, 1.21 mmol) and 3-methoxyaniline (298 mg, 2.42 mmol) into a 25 mL microwave-safe reaction flask, dissolve in 10 mL of isopropanol, and then place the reaction flask at 160°C. o The reaction was carried out in a microwave reactor for 50 min, and TLC was monitored until the reaction was complete. The mixture was then cooled to room temperature, filtered, washed with isopropanol, and dried. The pH was adjusted to 8-9 with NaOH aqueous solution, and the mixture was stirred at room temperature for 30 min. It was then filtered and dried to obtain 361 mg of a white solid, with a yield of 89.3% and a melting point of 63-65°C. o C. 1HNMR (400 MHz, DMSO-d6) δ 9.02 (s, 1H), 8.18 (d, J = 7.6 Hz, 1H), 7.84 (d, J =7.0 Hz, 1H), 7.77 (t, J = 2.0 Hz, 1H), 7.61 – 7.54 (m, 1H), 7.39 (d, J = 8.2Hz, 2H), 7.19 – 7.09 (m, 2H), 6.47 (dd, J = 8.0, 2.2 Hz, 1H), 4.67 – 4.56 (m,1H), 3.75 (s, 3H), 2.12 – 2.00 (m, 2H), 1.84 – 1.72 (m, 2H), 1.70 – 1.52 (m, 4H). 13 C NMR (101 MHz, DMSO-d6) δ 159.89, 156.72, 150.89, 143.19, 132.88,132.60, 129.79, 125.19, 123.18, 121.56, 119.75, 117.67, 116.77, 111.72,52.45, 31.93, 23.67. HRMS (ESI) (m / z): [M+H] + calcd for C 20 H 22 N4O, 335.1794, found 335.1862. HPLC purity: 100.00%.

[0116] Example 22: Synthesis of Compound 22

[0117] 1-Methyl-4-(4-nitrophenyl)piperazine (intermediate 2a)

[0118] 2a

[0119] 3 g (21.3 mmol) of 4-fluoronitrobenzene was weighed into a 250 mL single-necked round-bottom flask and dissolved in 30 mL of N,N-dimethylformamide. Then, 3.192 g (31.9 mmol) of 1-methylpiperazine and 4.401 g (21 mmol) of potassium carbonate were added. The mixture was heated to reflux and monitored by TLC until the reaction was complete. The mixture was then quenched with water, resulting in the formation of an orange solid. The solid was filtered, washed with deionized water, and dried to obtain 4.231 g of orange solid, with a yield of 89.8%. This solid was used directly in the next reaction.

[0120] 4-(4-methylpiperazin-1-yl)aniline (intermediate 2b)

[0121] 2b

[0122] Intermediate 2a (500 mg, 2.26 mmol) was weighed into a 100 mL round-bottom flask, and dissolved in a mixture of 15 mL ethanol and water (v:v = 4:1). Ammonium chloride (1.209 g, 22.6 mmol) and iron powder (633 mg, 11.3 mmol) were added. The mixture was refluxed under nitrogen protection for 2 h. The reaction was monitored by TLC until the end of the reaction. The remaining iron powder was removed by hot filtration, and the solvent was removed by rotary evaporation. The pH was adjusted to 8-9 with saturated sodium bicarbonate aqueous solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain 394 mg of brown solid, with a yield of 91.2%.

[0123] Synthesis of N4-cyclopentyl-N2-(4-(4-methylpiperazin-1-yl)phenyl)quinazoline-2,4-diamine

[0124] twenty two

[0125] Intermediates 1f (243 mg, 0.98 mmol) and 2b (282 mg, 1.48 mmol) were weighed into a 50 mL round-bottom flask. 10 mL of 1,4-dioxane and 0.5 mL of 4 mol / L HCl were added. The mixture was heated to reflux under nitrogen protection, and TLC was monitored until the reaction was complete. After cooling to room temperature, the mixture was filtered and washed with 1,4-dioxane. The filter cake was adjusted to pH 8-9 with saturated sodium bicarbonate aqueous solution, then extracted with dichloromethane, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The crude product was separated by silica gel column chromatography (dichloromethane / methanol = 15:1) to give 171 mg of a yellow solid, yield 43.4%. Melting point: 80-82°C o C. 1H NMR (400 MHz, DMSO-d6) δ 8.69 (s, 1H), 8.12 (d, J = 7.9 Hz, 1H), 7.74 (d, J = 9.0 Hz, 2H), 7.66(d, J = 7.0 Hz, 1H), 7.56 – 7.48 (m, 1H), 7.32 (d, J = 8.2 Hz, 1H), 7.09 (t,J = 7.2 Hz, 1H), 6.85 (d, J = 9.0 Hz, 2H), 4.63 – 4.51 (m, 1H), 3.11 – 2.96(m, 4H), 2.48 – 2.40 (m, 4H), 2.22 (s, 3H), 2.09 – 1.97 (m, 2H), 1.83 – 1.72 (m, 2H), 1.69 – 1.55 (m, 4H). 13 C NMR (101 MHz, DMSO-d6) δ 159.64, 157.08,151.39, 145.40, 133.97, 132.25, 124.97, 123.03, 120.59, 119.68, 115.86,111.55, 54.74, 52.10, 49.06, 45.75, 31.99, 23.70. HRMS (ESI) (m / z): [M+H] + calcd for C 24 H 30 N6, 403.2532, found 403.2605. HPLC purity: 100.00%.

[0126] Example 23 Synthesis of Compound 23

[0127] 4-(4-Nitrophenyl)morpholine (intermediate 3a)

[0128] 3a

[0129] 1 g of 4-fluoronitrobenzene (7.1 mmol) was weighed into a 100 mL single-necked round-bottom flask, and 15 mL of N,N-dimethylformamide was added to dissolve it. Then, morpholine (0.680 g, 7.8 mmol) and potassium carbonate (1.468 g, 10.6 mmol) were added, and the resulting mixture was refluxed for 2 h. The reaction was monitored by TLC until the reactants were completely reacted. The reaction was then quenched with water, resulting in the formation of an orange solid. The solid was filtered, washed with deionized water, and dried to obtain 1.408 g of orange solid, with a yield of 95.3%.

[0130] 4-Morphyrin-aniline (Intermediate 3b)

[0131] 3b

[0132] Intermediate 3a (400 mg, 1.92 mmol) was weighed into a 100 mL round-bottom flask, and dissolved in a mixture of 15 mL of ethanol and water (v:v = 4:1). Ammonium chloride (1.207 g, 19.2 mmol) and iron powder (538 mg, 9.6 mmol) were added, and the mixture was refluxed under nitrogen for 2 h. After the reaction was completed by TLC, the remaining iron powder was removed by hot filtration with diatomaceous earth, and the solvent was removed by rotary evaporation. The pH was adjusted to 8-9 with saturated sodium bicarbonate aqueous solution, and the mixture was extracted with dichloromethane, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain 326 mg of brown solid, with a yield of 95.3%.

[0133] N4-Cyclopentyl-N2-(4-morpholinophenyl)quinazolin-2,4-diamine

[0134] twenty three

[0135] Weigh intermediate 1f (180 mg, 1.22 mmol) and intermediate 3b (326 mg, 1.83 mmol) into a 50 mL round-bottom flask, add 10 mL of 1,4-dioxane and 0.5 mL of 4 mol / L HCl, heat to reflux under nitrogen protection, monitor by TLC until the reactants have reacted completely, cool to room temperature, filter, wash with 1,4-dioxane, adjust the pH of the filter cake to 8-9 with saturated sodium bicarbonate aqueous solution, then extract with dichloromethane, dry to anhydrous sodium sulfate, remove solvent by rotary evaporation, separate the crude product by silica gel column chromatography (dichloromethane / methanol = 30:1), give 165 mg of white solid, yield 34.7%, melting point: 258-260°C. o C. 1H NMR (400MHz, DMSO-d6) δ 8.75 (s, 1H), 8.13 (d, J = 7.6 Hz, 1H), 7.77 (d, J = 9.0 Hz, 2H), 7.70 (d, J = 7.0 Hz, 1H), 7.56 – 7.50 (m, 1H), 7.33 (d, J = 7.9 Hz, 1H),7.13 – 7.07 (m, 1H), 6.87 (d, J = 9.1 Hz, 2H), 4.63 – 4.52 (m, 1H), 3.75 –3.70 (m, 4H), 3.04 – 2.98 (m, 4H), 2.11 – 1.98 (m, 2H), 1.81 – 1.72 (m, 2H),1.66 – 1.56 (m, 4H). 13 C NMR (101 MHz, DMSO-d6) δ 159.61, 157.03, 151.34,145.35, 134.21, 132.14, 124.93, 122.92, 120.52, 119.66, 115.55, 111.50,66.14, 52.05, 49.46, 31.92, 23.61. HRMS (ESI) (m / z): [M+H] + calcd forC 23 H 27 N5O, 390.2216, found 390.2290. HPLC purity: 99.50%.

[0136] Example 24 Synthesis of Compound 24

[0137] 1-(2-Methoxyethoxy)-4-nitrobenzene (Intermediate 4a)

[0138] 4a

[0139] 2 g (14 mmol) of 4-fluoronitrobenzene was weighed into a 250 mL single-necked round-bottom flask, and 30 mL of N,N-dimethylformamide was added to dissolve it. Then, 2.6 g (34 mmol) of ethylene glycol monomethyl ether and 2.90 g (21 mmol) of potassium carbonate were added, and the mixture was heated under reflux for 2 h. The reaction was monitored by TLC until the reactants were completely reacted. The reaction was then quenched with water, resulting in the formation of an orange solid. The solid was filtered, washed with deionized water, and dried to give 1.235 g of a yellow solid, with a yield of 44.8%.

[0140] 4-(2-Methoxyethoxy)aniline (intermediate 4b)

[0141] 4b

[0142] Intermediate 4a (400 mg, 2.03 mmol) was weighed into a 100 mL round-bottom flask, and dissolved in a mixture of 15 mL of ethanol and water (v:v = 4:1). Ammonium chloride (1.086 g, 20.3 mmol) and iron powder (568 mg, 10.15 mmol) were added. The mixture was refluxed under nitrogen. After the reaction was completed by TLC, the remaining iron powder was removed by hot filtration with diatomaceous earth. The solvent was removed by rotary evaporation. The pH was adjusted to 8-9 with saturated sodium bicarbonate aqueous solution. The mixture was extracted with dichloromethane, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain 280 mg of brown solid, with a yield of 82.5%.

[0143] N4-Cyclopentyl-N2-(4-(2-methoxyethoxy)phenyl)quinazolin-2,4-diamine

[0144] twenty four

[0145] Weigh intermediate 1f (319 mg, 1.29 mmol) and intermediate 4b (324 mg, 1.94 mmol) into a 50 mL round-bottom flask, add 10 mL of 1,4-dioxane and 0.5 mL of 4 mol / L HCl, heat to reflux under nitrogen protection, monitor by TLC until the reactants have reacted completely, cool to room temperature, filter, wash with 1,4-dioxane, adjust the pH of the filter cake to 8-9 with saturated sodium bicarbonate aqueous solution, then extract with dichloromethane, dry with anhydrous sodium sulfate, remove solvent by rotary evaporation, separate the crude product by silica gel column chromatography (dichloromethane / methanol = 30:1), recrystallize from methanol to give 135 mg of yellow solid, yield 27.7%, melting point: 124-126 °C. o C. 1H NMR (400 MHz, DMSO-d6) δ 8.80 (s, 1H), 8.14 (d, J = 7.6 Hz, 1H), 7.81 (d, J= 9.0 Hz, 2H), 7.70 (d, J = 7.0 Hz, 1H), 7.56 – 7.50 (m, 1H), 7.34 (d, J =7.8 Hz, 1H), 7.14 – 7.07 (m, 1H), 6.87 – 6.82 (m, 2H), 4.65 – 4.51 (m, 1H), 4.06 – 4.00 (m, 2H), 3.66 – 3.61 (m, 2H), 3.31 (s, 3H), 2.09 – 2.00 (m, 2H),1.81 – 1.71 (m, 2H), 1.68 – 1.55 (m, 4H). 13 C NMR (101 MHz, DMSO-d6) δ 159.64,157.02, 152.65, 151.30, 134.93, 132.17, 124.99, 122.94, 120.63, 120.01,114.16, 111.56, 70.50, 67.04, 58.07, 52.07, 31.92, 23.61. HRMS (ESI) (m / z):[M+H] + calcd for C 22 H 26 N4O2, 379.2056, found 379.2129. HPLC purity: 100.00%.

[0146] Example 25 N4-cyclobutyl-N2-(4-(4-methylpiperazin-1-yl)phenyl)quinazolin-2,4-diamine

[0147] 25

[0148] Weigh intermediate 1c (343 mg, 1.48 mmol) and intermediate 2b (188 mg, 0.98 mmol) into a 50 mL round-bottom flask. Add 10 mL of 1,4-dioxane and 0.5 mL of 4 mol / L HCl. Heat under reflux under nitrogen protection. Monitor the reaction by TLC until the reactants have reacted completely. Adjust the pH to 8-9 with saturated sodium bicarbonate solution, then extract with dichloromethane. Dry the product on anhydrous sodium sulfate, remove the solvent by rotary evaporation, and separate the crude product by silica gel column chromatography (dichloromethane / methanol = 15:1) to obtain 70 mg of a yellow solid, yield 12.2%, melting point: 86-88°C.o C. 1 H NMR (400 MHz, DMSO-d6) δ 8.73 (s, 1H), 8.10 (d, J =7.8 Hz, 1H), 8.04 (d, J = 7.0 Hz, 1H), 7.75 (d, J = 8.9 Hz, 2H), 7.56 – 7.50(m, 1H), 7.33 (d, J = 8.1 Hz, 1H), 7.11 (t, J = 7.5 Hz, 1H), 6.86 (d, J = 9.1Hz, 2H), 4.79 – 4.68 (m, 1H), 3.07 – 3.00 (m, 4H), 2.47 – 2.43 (m, 4H), 2.40– 2.32 (m, 2H), 2.21 (s, 3H), 2.18 – 2.10 (m, 2H), 1.73 (m, J = 10.2, 8.4 Hz, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 159.01, 157.05, 151.52, 145.46, 133.93,132.37, 125.08, 122.93, 120.76, 119.70, 115.89, 111.33, 54.77, 49.08, 45.79,45.76, 30.20, 14.95. HRMS (ESI) (m / z): [M+H] + calcd for C 23 H 28 N6, 389.2375, found 389.2450. HPLC purity: 96.85%.

[0149] Example 26 N4-Cyclobutyl-N2-(4-morpholinophenyl)quinazoline-2,4-diamine

[0150] 26

[0151] Weigh intermediate 1c (338 mg, 1.3 mmol) and intermediate 3b (324 mg, 1.94 mmol) into a 50 mL round-bottom flask. Add 10 mL of 1,4-dioxane and 0.5 mL of 4 mol / L HCl. Heat under reflux under nitrogen protection. Monitor the reaction by TLC until the reactants have reacted completely. Cool to room temperature, filter, wash with 1,4-dioxane, adjust the pH of the filter cake to 8-9 with saturated sodium bicarbonate aqueous solution, then extract with dichloromethane, dry with anhydrous sodium sulfate, remove the solvent by rotary evaporation, and separate the crude product by silica gel column chromatography (dichloromethane / methanol = 30:1) to obtain 240 mg of brown solid, yield 85.5%, melting point: 235-237°C. o C. 1 H NMR (400MHz, DMSO-d6) δ 8.97 (s, 1H), 8.29 (s, 1H), 8.16 (d, J = 7.9 Hz, 1H), 7.71(d, J = 8.9 Hz, 2H), 7.61 – 7.55 (m, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.19 –7.14 (m, 1H), 6.90 (d, J = 9.0 Hz, 2H), 4.77 – 4.64 (m, 1H), 3.75 – 3.72 (m,4H), 3.06 – 3.00 (m, 4H), 2.40 – 2.30 (m, 2H), 2.23 – 2.11 (m, 2H), 1.82 –1.65 (m, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 158.96, 156.10, 145.95, 133.12,132.68, 123.74, 123.08, 121.24, 120.37, 115.52, 111.06, 66.12, 49.30, 45.93,29.92, 14.88. HRMS (ESI) (m / z): [M+H] + calcd for C 22 H 25 N5O, 376.2059, found376.2133. HPLC purity: 100.00%.

[0152] Example 27 N4-cyclobutyl-N2-(4-(2-methoxyethoxy)phenyl)quinazolin-2,4-diamine

[0153] 27

[0154] Weigh intermediate 1c (260 mg, 1.12 mmol) and intermediate 4b (280 mg, 1.68 mmol) into a 50 mL round-bottom flask, add 10 mL of 1,4-dioxane and 0.5 mL of 4 mol / L HCl, heat to reflux under nitrogen protection, monitor by TLC until the reactants have reacted completely, cool to room temperature, filter, wash with 1,4-dioxane, adjust the pH of the filter cake to 8-9 with saturated sodium bicarbonate aqueous solution, stir at room temperature for 30 min, then filter and dry to obtain 353 mg of gray solid, yield 86.5%, melting point: 148-150 °C. o C. 1 H NMR (400 MHz, DMSO-d6) δ 9.88 (s, 1H), 9.27 (s, 1H), 8.40 (d, J = 8.1Hz, 1H), 7.77 – 7.66 (m, 1H), 7.57 (d, J = 8.8 Hz, 2H), 7.46 (d, J = 8.2 Hz,1H), 7.36 – 7.29 (m, 1H), 6.97 (d, J = 9.0 Hz, 2H), 4.71 – 4.55 (m, 1H), 4.15 – 4.03 (m, 2H), 3.70 – 3.61 (m, 2H), 3.31 (s, 3H), 2.35 – 2.18 (m, 4H), 1.82–1.66 (m, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 158.98, 154.96, 134.22, 124.06,123.20, 122.91, 114.55, 110.33, 70.38, 67.11, 58.09, 46.63, 29.33, 14.96.HRMS (ESI) (m / z): [M+H] + calcd for C 21 H 24 N4O2, 365.1899, found 365.1974. HPLC purity: 97.37%.

[0155] Example 28 N4-cyclohexyl-N2-(4-(4-methylpiperazin-1-yl)phenyl)quinazolin-2,4-diamine

[0156] 28

[0157] Weigh intermediate 1d (223 mg, 0.85 mmol) and intermediate 2b (245 mg, 1.28 mmol) into a 50 mL round-bottom flask. Add 10 mL of 1,4-dioxane and 0.5 mL of 4 mol / L HCl. Heat under reflux under nitrogen protection. Monitor the reaction by TLC until the reactants have reacted completely. Cool to room temperature, filter, wash with 1,4-dioxane, adjust the pH of the filter cake to 8-9 with saturated sodium bicarbonate aqueous solution, then extract with dichloromethane, dry with anhydrous sodium sulfate, remove the solvent by rotary evaporation, and separate the crude product by silica gel column chromatography (dichloromethane / methanol = 30:1) to obtain 105 mg of brown solid, yield 29.7%, melting point: 89-91°C. o C. 1 H NMR (400MHz, DMSO-d6) δ 8.71 (s, 1H), 8.11 (d, J = 8.1 Hz, 1H), 7.72 (d, J = 8.7 Hz, 2H), 7.61 (d, J = 7.6 Hz, 1H), 7.52 (t, J = 7.5 Hz, 1H), 7.31 (d, J = 8.3 Hz,1H), 7.09 (t, J = 7.4 Hz, 1H), 6.85 (d, J = 8.8 Hz, 2H), 4.17 (s, 1H), 3.05(s, 4H), 2.24 (s, 3H), 1.99 (s, 2H), 1.82 (s, 2H), 1.69 (d, J = 11.8 Hz, 1H), 1.39 (d, J = 8.5 Hz, 3H), 1.32 (d, J = 16.4 Hz, 2H), 1.24 (d, J = 12.2 Hz, 4H). 13 C NMR (101 MHz, DMSO-d6) δ 159.13, 157.11, 151.45, 145.39, 134.00,132.29, 124.84, 123.00, 120.55, 119.70, 115.90, 111.42, 54.67, 49.03, 45.65,32.20, 25.42, 25.23. HRMS (ESI) (m / z): [M+H] + calcd for C 25 H 32 N6, 417.2688, found 417.2761. HPLC purity: 98.39%.

[0158] Example 29 N4-cyclohexyl-N2-(4-morpholinophenyl)quinazolin-2,4-diamine

[0159] 29

[0160] Intermediate 1d (322 mg, 1.23 mmol) and intermediate 3b (330 mg, 1.85 mmol) were weighed into a 50 mL round-bottom flask. 10 mL of 1,4-dioxane and 0.5 mL of 4 mol / L HCl were added. The mixture was heated to reflux under nitrogen protection, and TLC was monitored until the reaction was complete. After cooling to room temperature, the mixture was filtered and washed with 1,4-dioxane. The filter cake was adjusted to pH 8-9 with saturated sodium bicarbonate aqueous solution. The mixture was stirred at room temperature for 30 min, then filtered and dried to obtain 409 mg of a gray solid, yield 82.5%, melting point: 254-256 °C. o C. 1 H NMR (400 MHz, MeOD-d4) δ 8.21 (dd, J = 8.2, 0.9 Hz, 1H), 7.80 – 7.74(m, 1H), 7.50 (d, J = 8.2 Hz, 1H), 7.46 – 7.36 (m, 3H), 7.05 (d, J = 9.0 Hz, 2H), 4.20 (s, 1H), 3.88 – 3.82 (m, 4H), 3.21 – 3.14 (m, 4H), 2.05 (d, J =10.6 Hz, 2H), 1.87 (d, J = 12.8 Hz, 2H), 1.73 (d, J = 12.3 Hz, 1H), 1.53 –1.20 (m, 5H). 13 C NMR (101 MHz, MeOD-d4) δ 161.10, 154.23, 151.35, 141.22,136.13, 126.29, 125.90, 124.81, 118.83, 117.40, 111.91, 67.93, 53.06, 50.71,32.94, 26.53. HRMS (ESI) (m / z): [M+H] + calcd for C 24 H 29 N5O, 404.2372, found404.2448. HPLC purity: 99.63%.

[0161] Example 30 N4-cyclohexyl-N2-(4-(2-methoxyethoxy)phenyl)quinazolin-2,4-diamine

[0162] 30

[0163] Intermediate 1d (338 mg, 1.3 mmol) and intermediate 4b (324 mg, 1.94 mmol) were weighed into a 50 mL round-bottom flask. 10 mL of 1,4-dioxane and 0.5 mL of 4 mol / L HCl were added. The mixture was heated under reflux for 2 h under nitrogen protection, resulting in the formation of a brown solid. TLC monitoring continued until the reaction was complete. The mixture was cooled to room temperature, filtered, and washed with 1,4-dioxane. The filter cake was adjusted to pH 8-9 with saturated sodium bicarbonate aqueous solution, followed by extraction with dichloromethane. The product was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The crude product was separated by silica gel column chromatography (dichloromethane / methanol = 40:1) to obtain 240 mg of a brown solid, yield 47.1%, melting point: 158-160 °C. o C. 1 H NMR (400 MHz, DMSO-d6) δ 8.82 (s, 1H), 8.12 (d, J = 8.0 Hz, 1H), 7.77 (d, J = 8.9 Hz, 2H), 7.67 (d, J = 7.5 Hz, 1H), 7.54 (t, J = 7.4 Hz, 1H),7.34 (d, J = 8.2 Hz, 1H), 7.11 (t, J = 7.4 Hz, 1H), 6.85 (d, J = 9.0 Hz, 2H),4.17 (d, J = 3.3 Hz, 1H), 4.07 – 4.01 (m, 2H), 3.67 – 3.61 (m, 2H), 1.99 (d,J = 9.2 Hz, 2H), 1.81 (d, J = 9.7 Hz, 2H), 1.68 (d, J = 12.3 Hz, 1H), 1.47 –1.15 (m, 8H). 13 C NMR (101 MHz, DMSO-d6) δ 159.15, 156.93, 152.77, 151.06,134.80, 132.39, 124.70, 123.04, 120.77, 120.17, 114.16, 111.44, 70.53, 67.04,58.14, 49.45, 32.17, 25.40, 25.23. HRMS (ESI) (m / z): [M+H] + calcd forC 23 H 28N4O2, 393.2212, found 393.2285. HPLC purity: 100.00%.

[0164] Bioevaluation methods:

[0165] (1) Anti-proliferative activity against colorectal cancer cells. Colorectal cancer cells: HCT116, SW480

[0166] Assay for anti-proliferative activity against colorectal cancer cells:

[0167] The in vitro antiproliferative activity of quinazoline derivatives against human colorectal cancer cells HCT116 and SW480 was detected using the CCK-8 assay, and their half-maximal inhibitory concentration (IC50) was calculated. 50 ).

[0168] Experimental principle:

[0169] Intracellular dehydrogenases can reduce CCK-8 in the culture medium to a water-soluble formazan product, which has a characteristic absorption peak at 450 nm, and its absorbance value is positively correlated with the number of viable cells. Under the action of the test compound, tumor cell proliferation was inhibited, the number of viable cells decreased, and the absorbance value decreased accordingly. By detecting the absorbance values ​​of cells treated with different concentrations of the compound, the cell proliferation inhibition rate was calculated, and the half-maximal inhibitory concentration (IC50) was further fitted to obtain the result. 50 This study aimed to evaluate the in vitro antiproliferative activity of the compound against human colorectal cancer cells HCT116 and SW480.

[0170] Specific experimental steps:

[0171] ① The cell lines used were human colorectal cancer cells SW480 and HCT116, both purchased from ATCC.

[0172] ② Take cells in the logarithmic growth phase and in good growth condition, and seed them in 96-well plates at a density of 5000 cells per well, and add 100 μL of culture medium to each well; at the same time, set up wells containing only an equal volume of complete culture medium as blank control group.

[0173] ③ Place the culture plate at 37°C o C. Pre-culture in a cell culture incubator with 5% CO2 and saturated humidity for 24 h; add 100 μL of sterile PBS to the outer wells of the culture plate to reduce liquid evaporation.

[0174] ④ Dilute the test compound serially with culture medium to a concentration of 100 μM, 20 μM, 4 μM, 0.8 μM and 0.16 μM. Add 100 μL of drug-containing culture medium to each well and set up 3 replicates for each concentration.

[0175] ⑤ In 37 oC. Continue culturing for 48 h under conditions of 5% CO2 and saturated humidity.

[0176] ⑥ After incubation, precisely add 10 μL of CCK-8 reagent to each well and incubate at 37°C. o Continue incubation in a constant temperature incubator for 2 hours.

[0177] ⑦ After incubation, the absorbance of each well was measured at a wavelength of 450 nm using a multi-functional microplate reader.

[0178] ⑧ The cell proliferation inhibition rate was calculated based on the absorbance values, and the IC was calculated using nonlinear regression analysis with GraphPad Prism 8.0 software. 50 Values. The results of the anti-tumor cell proliferation activity test are shown in Table 1.

[0179] Table 1. Results of antitumor cell proliferation activity tests of compounds 1-30 prepared in the examples.

[0180]

[0181]

[0182] (2) Cloning experiment

[0183] Colony formation assays were conducted to detect the inhibitory effect of the compound on the long-term proliferation and colony formation ability of HCT116 colorectal cancer cells.

[0184] Experimental principle:

[0185] The clonogenic assay is based on the biological characteristic that a single tumor cell can proliferate to form cell colonies (clones), reflecting the cell's independent proliferative capacity, survival ability, and tumorigenic potential. After drug treatment, if the compound can inhibit tumor cell proliferation or induce cell death, the number of cell clones decreases and the clone volume shrinks. Visualization of the clones using crystal violet staining, counting, and calculation of the clonogenic rate allows for a quantitative evaluation of the long-term inhibitory effect of the test compound on tumor cell proliferation.

[0186] The specific experimental steps are as follows:

[0187] ① Cell Culture and Seeding: HCT116 cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-drug antibiotics at 37°C. o C. Culture in 5% CO2 saturated humidity until the logarithmic growth phase; seed 2000 cells / well in 6-well plates, add 2 mL of culture medium per well, and incubate overnight until the cells adhere.

[0188] ② Grouping and treatment: The experiment was divided into a solvent control group and a drug administration group, with 3 replicates in each group; Solvent control group: an equal volume of DMSO (final concentration ≤0.1%) was added.

[0189] Drug administration groups: final concentrations of 0.4 μM, 2 μM, 4 μM, and 8 μM; continuous drug treatment for 24 h.

[0190] ③Cloning and medium change: Discard the culture medium, wash once with pre-cooled PBS; add 2 mL of complete culture medium to each well, continue culturing for 10 days, and replace with fresh culture medium every 2 days.

[0191] ④ Fixation and staining: Discard the culture medium and wash twice with PBS; add 1 mL of 4% paraformaldehyde to each well and fix at room temperature for 15 min; after washing with PBS, add 1 mL of 1% crystal violet staining solution and stain at room temperature in the dark for 10 min; gently rinse several times with deionized water until excess staining solution is removed, invert the culture plate and place it in a ventilated place to air dry naturally.

[0192] ⑤ Imaging and Statistical Analysis: Clonal morphology was photographed using an inverted microscope (10× objective lens); ImageJ software was used for quantitative analysis of clone formation. Data are expressed as mean ± standard deviation (mean ± SD). GraphPad Prism 8.0 software was used for statistical processing and analysis of experimental data. Differences between groups were assessed using one-way ANOVA, combined with Tukey's multiple comparison test for pairwise analysis. P < 0.05 was considered statistically significant.

[0193] The results of the clonal formation experiment of compound 22 are attached. Figure 3 . Figure 3 The figure shows the results of the cloning experiment of compound 22 prepared in Example 22. In the figure, the error bars represent the standard deviation of three repeated experiments, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0194] Compound 22 significantly inhibited colony formation in HCT116 cells, and the inhibitory effect gradually increased with increasing concentration, exhibiting a dose-dependent effect. Specifically, compound 22 inhibited colony formation in HCT116 cells at a concentration of 0.4 μM, and the colony formation ability of HCT116 cells was essentially suppressed at a concentration of 8 μM.

[0195] (3) Apoptosis detection

[0196] The study aimed to investigate the apoptosis-inducing effect of a compound on HCT116 colorectal cancer cells, clarifying its pro-apoptotic ability. Quantitative analysis was conducted on the proportions of early apoptosis, late apoptosis, and necrosis at different compound concentrations to determine the dose-response relationship. This study elucidates the mechanism by which the compound inhibits the proliferation of colorectal cancer cells at the apoptosis level, providing a basis for evaluating its antitumor activity.

[0197] Experimental principle:

[0198] Phosphatidylserine (PS) eversion is a hallmark event in the early stages of apoptosis. Normally located on the inner side of the cell membrane, it flips to the outer side during early apoptosis. Annexin V is Ca 2+ Phospholipid-dependent binding proteins (PDCs) can specifically bind to everted phospholipids (PSs) for labeling apoptotic cells. Propidium iodide (PI) is a membrane-impermeable nucleic acid dye that can only stain late-stage apoptotic and necrotic cells whose cell membrane integrity has been disrupted. Using Annexin V-FITC / PI double staining combined with flow cytometry, cells can be classified into four categories—live cells, early-stage apoptotic cells, late-stage apoptotic cells, and necrotic cells—based on fluorescence signals. The total apoptosis rate can then be calculated, allowing for an objective and accurate evaluation of the ability of the test compounds to induce tumor cell apoptosis.

[0199] The specific experimental steps are as follows:

[0200] ① Cell seeding and culture: HCT116 cells were seeded at 5 × 10⁶ cells / year. 5 The cells were seeded at a density of 10 cells / well into 6-well plates. 2 mL of DMEM medium containing 10% FBS and 1% penicillin-streptomycin antibiotics was added to each well. The plates were then incubated overnight at 37°C with 5% CO2. Once the cells had completely adhered to the plate, the cells were treated with the antibiotics.

[0201] ② Grouping and drug administration: The grouping and drug administration methods were the same as in the clonogenic assay. The final concentrations of the compound treatment groups were 0 μM, 0.4 μM, 2 μM, 4 μM, and 8 μM. Each group had 3 biological replicates and was cultured for 48 h.

[0202] ③ Cell collection and processing: After incubation, carefully collect the culture medium from each well into a 15 mL centrifuge tube. Gently wash the cells twice with pre-cooled PBS, avoiding vigorous pipetting to prevent cell damage. Add 1 mL of 0.25% EDTA-trypsin digestion solution to each well and incubate at 37 °C for 2-3 min until the cells are completely detached from the well wall. Then add 2 mL of culture medium containing 10% FBS to terminate the digestion process. Transfer the cell suspension to a 15 mL centrifuge tube in which the supernatant has been collected beforehand, centrifuge at 1000 rpm for 5 min, carefully aspirate the supernatant, and collect the cell pellet.

[0203] ④ Cell apoptosis staining: Add 100 μL of binding buffer to the cell pellet, gently pipette to resuspend the cells and avoid cell aggregation; then add 5 μL of Annexin V-FITC reagent and incubate at room temperature in the dark for 15 min; after incubation, add 5 μL of PI staining solution and continue incubation in the dark for 5 min. The entire process should be performed on ice to avoid strong light exposure affecting the staining effect.

[0204] ⑤ Flow cytometry detection: After staining, immediately add 400 μL of binding buffer to the centrifuge tube to dilute the sample, gently invert and mix, and complete the detection within 1 h using a FACSCanto II flow cytometer; set the excitation wavelength to 488 nm, and detect FITC (FL1 channel) and PI (FL2 channel) fluorescence signals respectively, with 10,000 cells detected for each sample.

[0205] ⑥ Data Processing and Analysis: FlowJo 7.6 software was used to analyze flow cytometry data. Based on the Annexin V-FITC and PI fluorescence intensity quadrant diagrams, cells were divided into live cells (Annexin V⁻ / PI⁻), early apoptotic cells (Annexin V⁺ / PI⁻), late apoptotic cells (Annexin V⁺ / PI⁺), and necrotic cells (Annexin V⁻ / PI⁺). The total apoptosis rate = early apoptosis rate + late apoptosis rate. The statistical analysis methods were the same as those used in the clonogenic assay.

[0206] The results of the apoptosis experiment of compound 22 in Example 22 are shown in the appendix. Figure 4 At concentrations of compound 22 of 0.4 µM, 2 µM, 4 µM, and 8 µM, the sum of early and late apoptosis rates detected by flow cytometry were 6.33%, 11.28%, 17.59%, and 73.6%, respectively. These results indicate that compound 22 can induce apoptosis in HCT116 cells in a concentration-dependent manner.

[0207] The above description of the embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A quinazoline compound having the structure shown in formula (Ⅰ), Equation (I) in, X is O, S, CH2, or NH; R 1 It is hydrogen, substituted phenyl, substituted or unsubstituted C3-C6 cycloalkyl; R 2 It is one or more of the following: hydrogen, halogen, C1-C4 alkyl, C3-C7 cycloalkyl, C1-C4 haloalkyl, C1-C5 alkoxy, C1-C5 haloalkoxy, hydroxyl, cyano, nitro, amino, mercapto, formyl, acetyl, methanesulfonyl, trifluoromethanesulfonyl, hydroxymethyl, sulfonyl, 5-membered heterocycle containing one or two nitrogen atoms, 6-membered heterocycle containing one or two nitrogen atoms, 5-membered heterocycle containing one or two oxygen atoms, 6-membered heterocycle containing one or two oxygen atoms, tri- or more heteroalkyl groups containing one or more nitrogen atoms, and tri- or more heteroalkyl groups containing one or more oxygen atoms; R 3 It is hydrogen or methoxy; R 4 It is hydrogen or methoxy.

2. The quinazoline compound according to claim 1, characterized in that, The R 1 In the substituted phenyl group, the substituents are one or more of fluorine, chlorine, bromine and iodine; The substituents in the substituted C3-C6 cycloalkyl group are one or more of fluorine, chlorine, bromine, iodine, methyl, ethyl, n-propyl and isopropyl.

3. The quinazoline compound according to claim 1, characterized in that, The R 1 It is hydrogen, phenyl containing halogen substituents, cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.

4. The quinazoline compound according to claim 1, characterized in that, The R 2 It is one or more of the following: fluorine, chlorine, bromine, iodine, methyl, ethyl, n-propyl, isopropyl, n-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, trifluoromethyl, trifluoroethyl, trifluoromethoxy, methoxy, ethoxy, propoxy, isopropoxy, tert-butoxy, piperazine, piperidinyl, N-methylpiperazine, N-methylpiperridinyl, morpholinyl, hydroxy, cyano, nitro, amino, mercapto, formyl, acetyl, methanesulfonyl, trifluoromethanesulfonyl, hydroxymethyl, sulfonyl, and 2-methoxyethoxy.

5. The quinazoline compound according to claim 1, characterized in that, The quinazoline compounds have the following structures. .

6. A method for preparing a quinazoline compound with the structure shown in formula (I) having anti-colorectal cancer activity, comprising: 1) The compound with the structure of formula (II) is combined with R 1 The XH2 reaction yields a compound with the structure shown in formula (III); Formula (II), Equation (III) 2) The compound with structure (III) is mixed and reacted with the compound with structure (IV) to obtain the compound with structure (I); Formula (IV), Formula (I) Where X is O, S, CH2 or NH; R 1 It is hydrogen, substituted phenyl, substituted or unsubstituted C3~C6 cycloalkyl; R 2 It is one or more of the following: hydrogen, halogen, C1-C4 alkyl, C3-C7 cycloalkyl, C1-C4 haloalkyl, C1-C5 alkoxy, C1-C5 haloalkoxy, hydroxyl, cyano, nitro, amino, mercapto, formyl, acetyl, methanesulfonyl, trifluoromethanesulfonyl, hydroxymethyl, sulfonyl, 5-membered heterocycle containing one or two nitrogen atoms, 6-membered heterocycle containing one or two nitrogen atoms, 5-membered heterocycle containing one or two oxygen atoms, 6-membered heterocycle containing one or two oxygen atoms, tri- or more heteroalkyl groups containing one or more nitrogen atoms, and tri- or more heteroalkyl groups containing one or more oxygen atoms; R 3 It is hydrogen or methoxy; R 4 It is hydrogen or methoxy.

7. The use of a quinazoline compound according to any one of claims 1 to 5, or a quinazoline compound prepared by the preparation method according to claim 6, and a pharmaceutically acceptable salt thereof, in the preparation of a medicament with anti-colorectal cancer activity.

8. A pharmaceutical composition comprising: The quinazoline compound according to any one of claims 1 to 5 or the quinazoline compound prepared by the preparation method according to claim 6, or a pharmaceutically acceptable salt and adjuvant thereof; The adjuvant is a pharmaceutically acceptable carrier, salt form, or excipient.

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