8-phenyl quinazoline compound and application thereof

Abstract

The application discloses an 8-phenyl quinazoline compound and application thereof, and a structural formula of the compound is shown in the figure.The application shows significant inhibitory activity on a PD-1 / PD-L1 signal path, and can be applied to treating related diseases including tumor diseases mediated by the PD-1 / PD-L1 signal path.

Description

Technical Field

[0001] This invention belongs to the field of organic synthesis technology, specifically relating to an 8-phenylquinazoline compound and its applications. Background Technology

[0002] PD-1 (programmed death 1, CD279) is a type I transmembrane protein primarily expressed on activated T cells, B cells, NK cells, monocytes, and macrophages. PD-1 ligands include PD-L1 (programmed cell death-Ligand 1, CD274) and PD-L2. PD-L1 is also a type I transmembrane glycoprotein belonging to the B7 family. PD-L1 is mainly expressed on antigen-presenting cells, B cells, T cells, epithelial cells, myocytes, endothelial cells, and tumor cells, and participates in tumor-associated immune responses. PD-1 and PD-L1 together form the PD-1 / PD-L1 signaling pathway, inhibiting cytokine production and cell proliferation, and playing a crucial role in T cell activation and immune response regulation. PD-1 and PD-L1 interact, inducing phosphorylation of ITIMs and ITSMs within their domains. This recruits the phosphatase SHP-2, leading to dephosphorylation of several key proteins in the T cell antigen receptor (TCR) signaling pathway, inhibiting downstream signaling pathways, thereby suppressing cytokine production and T cell proliferation and differentiation, ultimately resulting in T cell loss of immune function. During the body's immune response to tumor cells, PD-1 overexpression on T cells in the tumor microenvironment or PD-L1 overexpression on tumor cells interact to some extent, suppressing the body's cellular immune response and allowing tumors to evade the immune system's surveillance and killing mechanism. PD-1 / PD-L1 inhibitors work by blocking the PD-1 / PD-L1 signaling pathway, inhibiting their interaction, restoring the T cell's immune killing function, and ultimately achieving the goal of killing tumor cells.

[0003] PD-1 / PD-L1 inhibitors can be divided into antibody inhibitors and small molecule inhibitors. Antibody inhibitors are particularly effective and specific, exhibiting high affinity for PD-1 / PD-L1 proteins. In recent years, several antibody-based PD-1 / PD-L1 inhibitors have been launched both domestically and internationally, such as durvalumab, atezolizumab, and avelumab. However, inherent limitations of antibody inhibitors restrict their use. For example, their large molecular weight results in poor tissue and tumor penetration, even within tumor tissues, where they may be unable to reach certain areas. Furthermore, immune-exempt sites further restrict their application. In contrast, small molecule inhibitors have smaller molecular weights, stronger penetration, and can reach tissues where antibody therapy is limited. They also possess well-controllable pharmacokinetic properties, better oral bioavailability, and a reasonable half-life. Furthermore, small molecule drugs have simple manufacturing processes, low costs, and diverse dosage forms, increasing treatment accessibility and effectively compensating for the inherent shortcomings of antibody-based inhibitors. Therefore, developing small molecule inhibitors targeting the PD-1 / PD-L1 immune checkpoint has unique advantages and enormous development potential, and has become a very active research area in recent years. In the past few years, several classes of PD-L1 small molecule inhibitors have been reported. Currently, these small molecule compounds are in different stages of preclinical or clinical research, such as Bristol-Myers Squibb's BMS-200 and BMS-1166, Reigen Pharmaceuticals' MAX-10181, Hongri Pharmaceuticals' IMMH-010, Gilead Sciences' GS4224, and Incyte's INCB086550. Summary of the Invention

[0004] The purpose of this invention is to provide an 8-phenylquinazoline compound.

[0005] Another object of the present invention is to provide the use of the above-mentioned 8-phenylquinazoline compounds and their pharmaceutically acceptable salts in the preparation of pharmaceutical compositions for treating diseases related to the PD-1 / PD-L1 signaling pathway.

[0006] Another object of the present invention is to provide a pharmaceutical composition for treating diseases related to the PD-1 / PD-L1 signaling pathway.

[0007] The technical solution of the present invention is as follows:

[0008] An 8-phenylquinazoline compound, the structural formula of which is in,

[0009] R1 represents H and C. 1-5 Alkyl or C 3-7Cycloalkyl, R2 is H or C 1-5 Alkyl or C 3-7 Cycloalkyl; C in R1 and R2 1-5 Alkyl and C 3-7 The cycloalkyl group has 1-3 identical or different amino, hydroxyl, amide, carboxyl or ester groups; or R1, R2 and the nitrogen atom attached thereto form a 4-7 membered heterocyclic group, and the heterocyclic group contains 1-3 identical or different amino, hydroxyl, amide, carboxyl or ester groups;

[0010] R3 represents H, halogen, or C. 1-3 Alkyl, C 1-3 Alkoxy or C 1-3 Alkylamine group;

[0011] R4 is H, halogen, or methyl;

[0012] X is C or N.

[0013] In a preferred embodiment of the present invention, the for

[0014] Further preferably, R3 is a halogen.

[0015] More preferably, R4 is H or methyl.

[0016] The use of the above-mentioned 8-phenylquinazoline compounds and their pharmaceutically acceptable salts in the preparation of pharmaceutical compositions for treating diseases related to the PD-1 / PD-L1 signaling pathway.

[0017] In a preferred embodiment of the present invention, the PD-1 / PD-L1 signaling pathway-related disease is a tumor disease.

[0018] Further preferably, the tumor diseases include lung cancer, liver cancer, kidney cancer, non-small cell lung cancer, prostate cancer, thyroid cancer, skin cancer, pancreatic cancer, ovarian cancer, breast cancer, bladder cancer, myelodysplastic syndrome, lymphoma, esophageal cancer, gastrointestinal cancer, and tumors of the central and peripheral nervous systems.

[0019] A pharmaceutical composition for treating diseases related to the PD-1 / PD-L1 signaling pathway, wherein the active ingredient comprises the above-mentioned 8-phenylquinazoline compound and / or a pharmaceutically acceptable salt.

[0020] In a preferred embodiment of the invention, the pharmaceutically acceptable salt is selected from at least one of hydrogen sulfate, hydrochloride, hydrobromide, sulfate, oxalate, lactate, gluconate, tartrate, fumarate, methanesulfonate, ethanesulfonate, benzenesulfonate, acetate, citrate, and p-toluenesulfonate.

[0021] Further preferably, the PD-1 / PD-L1 signaling pathway-related diseases are tumor diseases, including lung cancer, liver cancer, kidney cancer, non-small cell lung cancer, prostate cancer, thyroid cancer, skin cancer, pancreatic cancer, ovarian cancer, breast cancer, bladder cancer, myelodysplastic syndrome, lymphoma, esophageal cancer, gastrointestinal cancer, and tumors of the central and peripheral nervous systems.

[0022] The beneficial effects of this invention are: this invention exhibits significant inhibitory activity on the PD-1 / PD-L1 signaling pathway and can be applied to the treatment of PD-1 / PD-L1 signaling pathway-mediated diseases, including tumors. Detailed Implementation

[0023] The technical solution of the present invention will be further explained and described below through specific embodiments.

[0024] All raw materials and reagents used in the following examples are commercially available analytical grade or chemically pure pharmaceutical products. The proton NMR spectra of the compounds were determined using a Bruker ARX-400, and the high-resolution mass spectrometry was determined using an ultra-high performance liquid chromatography / quadrupole time-of-flight mass spectrometer (UHPLC-QTOF).

[0025] The synthetic routes for Examples 1-25 are as follows:

[0026]

[0027] The preparation of intermediate i-1 included: at room temperature, 12 g (0.087 mol) of 2,4-dihydroxybenzaldehyde, 30 mL of chloroform, and 1.2 mL of concentrated hydrochloric acid were added to a 250 mL round-bottom flask. The mixture was heated to 60 °C and stirred. NCS (12.76 g, 0.096 mol) was added in four batches, and the reaction was allowed to proceed for 3 h. The reaction was terminated by TLC monitoring. The mixture was filtered while hot, and after the filtrate was allowed to stand to precipitate crystals, it was further filtered to obtain 4.5 g of white needle-like crystals, i.e., intermediate i-1, with a yield of 40.0%. The characterization data of intermediate i-1 are as follows: 1 H NMR (400MHz, DMSO-d6) δ11.40 (s, 1H), 10.88 (s, 1H), 9.98 (s, 1H), 7.60 (s, 1H), 6.59 (s, 1H).

[0028] The preparation of intermediate i-2 included: at room temperature, intermediate i-1 (9.00 g, 52.16 mmol) was added to 50 mL of MeCN, followed by the sequential addition of 2-methyl-3-bromobenzyl chloride (12.02 g, 54.76 mmol), potassium iodide (4.33 g, 26.08 mmol), and sodium bicarbonate (5.71 g, 67.80 mmol). After the addition was complete, the temperature was raised to 60 °C, and the reaction was stirred for approximately 50 h. After the reaction was confirmed to be complete by TLC, the mixture was cooled to room temperature, filtered, the filter cake was washed with water, and dried to obtain 13.40 g of white solid, i.e., intermediate i-2, with a yield of 72.21%. The characterization data of intermediate i-2 are as follows: 1 H NMR (400MHz, CDCl3) δ11.45 (s, 1H), 9.72 (s, 1H), 7.61 (d, J=8.0Hz, 1H), 7.57 (s, 1H), 7.43 (d, J=7.5Hz, 1H), 7.13 (t, J=7.8Hz, 1H), 6.60 (s, 1H), 5.18 (s, 2H), 2.47 (s, 3H).

[0029] The preparation of intermediate i-3 included: at room temperature, intermediate i-2 (2.46 g, 6.92 mmol) was added to 25 mL of 1,4-dioxane, followed by the sequential addition of dried pinacol diboron ester (2.11 g, 8.30 mmol), dried potassium acetate (2.87 g, 20.75 mmol), and palladium dichloride bis(triphenylphosphine) (0.49 g, 0.69 mmol). After the addition was complete, the mixture was immediately purged three times with nitrogen to remove oxygen from the apparatus. Under nitrogen protection, the mixture was heated to 80 °C and stirred for 15 h. After the reaction was confirmed to be complete by TLC, the mixture was allowed to cool to room temperature, filtered with diatomaceous earth, and the filtrate was concentrated under reduced pressure. Then, the filtrate was added to 35 mL of ice water and stirred for 30 min. The mixture was then filtered, the filter cake was dried, and purified by column chromatography using petroleum ether:ethyl acetate (15:1) to obtain 1.55 g of white solid, i.e., intermediate i-3, with a yield of 55.76%. The characterization data of intermediate i-3 are as follows: 1 H NMR (400MHz, CDCl3) δ11.45 (s, 1H), 9.71 (s, 1H), 7.80 (d, J=7.1Hz, 1H), 7.59-7.5 2(m, 2H), 7.26(s, 1H), 6.61(s, 1H), 5.19(s, 2H), 2.59(s, 3H), 1.43-1.33(m, 12H).

[0030] The preparation of intermediate i-4-1 included: at room temperature, intermediate i-3 (2.00 g, 4.97 mmol) was added to 20 mL of DMF, followed by the sequential addition of m-cyanobenzyl bromide (1.07 g, 5.46 mmol), cesium carbonate (2.43 g, 7.45 mmol), and [other ingredients]. The mixture was stirred at rt for approximately 3 h. After the reaction was confirmed to be complete by TLC, approximately 60 mL of ice water was added, and the mixture was stirred for approximately 30 min. During stirring, the pH was adjusted to neutral with dilute HCl. The mixture was then filtered, and the filter cake was dried to obtain 2.33 g of white solid, which was intermediate i-4-1, with a yield of 90.51%. The characterization data of intermediate i-4-1 are as follows: 1 H NMR (400MHz, CDCl3) δ10.33 (s, 1H), 7.92 (s, 1H), 7.80 (d, J=7.4Hz, 1H), 7.72 (s, 1H), 7.71-7.65 (m, 2H), 7.56 (t, J=7.8Hz, 1H), 7.46 (d, J=7.7Hz, 1H), 7.24 (t, J=7.5Hz, 1H), 6.55 (s, 1H), 5.20 (s, 2H), 5.16 (s, 2H), 2.59 (s, 3H), 1.42-1.36 (m, 12H).

[0031] The preparation of intermediate i-4-2 included: at room temperature, intermediate i-3 (2.00 g, 4.97 mmol) was added to 20 mL of DMF, followed by the sequential addition of 5-(bromomethyl)nicotinonitrile (1.07 g, 5.46 mmol), cesium carbonate (2.43 g, 7.45 mmol), and [other ingredients]. The mixture was stirred at room temperature for approximately 3 h. After the reaction was confirmed to be complete by TLC, approximately 60 mL of ice water was added, and the mixture was stirred for approximately 30 min. During stirring, the pH was adjusted to neutral with dilute HCl. The mixture was then filtered, and the filter cake was dried to obtain 2.18 g of white solid, i.e., intermediate i-4, with a yield of 85.00%. The characterization data of intermediate i-4 are as follows: 1 H NMR (400MHz, DMSO) δ10.38 (s, 1H), 9.04 (s, 1H), 9.00 (s, 1H), 8.88 (s, 1H), 8.54 (s, 1H) , 8.47 (t, J=7.5Hz, 2H), 7.93 (s, 1H), 7.78 (d, J=7.8Hz, 1H), 7.75 (s, 1H), 5.38 (s, 2H).

[0032] The preparation of intermediate i-5 included: weighing 20 g (0.093 mol) of 3-bromo-2-aminobenzoic acid into a 250 mL flask, adding 80 mL of formamide as a solvent, reacting at 150 °C with stirring for 6 h, and observing the reaction completion by TLC. The reaction solution was allowed to cool to room temperature, then ice water was added and stirred for 30 min. The mixture was then filtered to obtain 18.85 g of a brown solid, i.e., intermediate i-5, with a yield of 90.6%. The characterization data of intermediate i-5 are as follows: 1 H NMR (400MHz, DMSO-d6) δ12.52 (s, 1H), 8.23 ​​(d, J=3.6Hz, 1H), 8.17-8.10 (m, 2H), 7.43 (t, J=7.8Hz, 1H).

[0033] The preparation of intermediate i-6 included: at room temperature, intermediate i-5 (8.00 g, 34.19 mmol) was added to 45 mL of thionyl chloride, followed by 2 drops of DMF. After the addition was complete, the mixture was stirred and heated to 80 °C, reacted for 9 h, and the reaction was monitored by TLC until complete. The mixture was then cooled to room temperature, the solvent was concentrated under reduced pressure, and 120 mL of dichloromethane was added to dissolve the solid. After complete dissolution, the pH was adjusted to neutral with saturated sodium bicarbonate solution. The solution was transferred to a separatory funnel, separated, and allowed to stand. The dichloromethane layer was collected, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated to obtain 6.50 g of a brownish-yellow solid, i.e., intermediate i-6, with a yield of 75.50%. The characterization data of intermediate i-6 are as follows: 1 HNMR (400MHz, CDCl3) δ9.20 (s, 1H), 8.34-8.28 (m, 2H), 7.64 (t, J=8.0Hz, 1H).

[0034] The preparation of intermediate i-7 included: At room temperature under nitrogen atmosphere, intermediate i-6 (5.00 g, 20.53 mmol) was added to 35 mL of MeCN, followed by intermediate i-1 (3.72 g, 21.55 mmol), potassium iodide (1.70 g, 10.27 mmol), and sodium bicarbonate (2.24 g, 26.69 mmol). After the addition was complete, the mixture was heated to 60 °C and stirred for approximately 50 h. After TLC detection of complete reaction, the mixture was allowed to cool to room temperature and filtered using a Buchner funnel. The filter cake was transferred to a 50 mL round-bottom flask, and 20 mL of ice water was added and stirred for approximately 30 min. The mixture was then filtered again, and the filter cake was dried to obtain 4.26 g of white solid, i.e., intermediate i-7, with a yield of 54.90%. The characterization data of intermediate i-7 are as follows: 1H NMR (400MHz, DMSO-d6) δ11.42 (s, 1H), 10.27 (s, 1H), 8.90 (s, 1H), 8.47-8.42 (m, 2H), 7.84 (s, 1H), 7.75 (t, J=7.9Hz, 1H), 7.20 (s, 1H).

[0035] The preparation of intermediate i-8-1 included: At room temperature under nitrogen atmosphere, intermediate i-7 (2.79 g, 7.35 mmol) was added to 20 mL of DMF, followed by the sequential addition of m-cyanobenzyl bromide (1.73 g, 8.82 mmol) and cesium carbonate (3.59 g, 11.03 mmol). After the addition was complete, the mixture was stirred at room temperature for 3 h. After TLC detection of complete reaction, 60 mL of ice water was added, and the mixture was stirred for approximately 30 min. During stirring, the pH was adjusted to neutral with dilute HCl. The mixture was then filtered, and the filter cake was dried to obtain 2.20 g of white solid, i.e., intermediate i-8-1, with a yield of 60.12%. The characterization data of intermediate i-8 are as follows: 1 H NMR (400MHz, DMSO-d6) δ10.31 (s, 1H), 8.82 (s, 1H), 8.44-8.37 (m, 2H), 7.96 (s, 1H), 7.85 (s, 1 H), 7.81-7.76 (m, 2H), 7.70 (t, J=8.0Hz, 1H), 7.65 (s, 1H), 7.57 (t, J=7.8Hz, 1H), 5.27 (s, 2H).

[0036] The preparation of intermediate i-8-2 included: At room temperature under nitrogen atmosphere, intermediate i-7 (2.79 g, 7.35 mmol) was added to 20 mL of DMF, followed by the sequential addition of 5-(bromomethyl)nicotinonitrile (1.74 g, 8.82 mmol) and cesium carbonate (3.59 g, 11.03 mmol). After the addition was complete, the mixture was stirred at rest for 3 h. After TLC detection of complete reaction, 60 mL of ice water was added, and the mixture was stirred for approximately 30 min. During stirring, the pH was adjusted to neutral with dilute HCl. The mixture was then filtered, and the filter cake was dried to obtain 2.02 g of white solid, i.e., intermediate i-8-2, with a yield of 55.13%. The characterization data of intermediate i-8-2 are as follows: 1 H NMR (400MHz, CDCl3) δ10.26 (s, 1H), 8.89 (d, J = 4.5Hz, 2H), 8.06 (s, 1H), 7.89 (s, 1H), 7.77 (s, 1H), 7.45 (d , J=6.2Hz, 1H), 7.22(t, J=7.5Hz, 1H), 6.55(s, 1H), 5.21(s, 2H), 5.17(s, 2H), 2.58(s, 3H), 1.37(s, 12H).

[0037] The preparation of intermediate i-9-1 included: at room temperature, intermediate i-4-1 (1.03 g, 2.01 mmol) was added to 8 mL of THF, followed by 1 mL of water. Then, intermediate i-8-1 (0.99 g, 2.02 mmol), tripotassium phosphate (1.28 g, 6.03 mmol), and XPhos-Pd(G2) (0.16 g, 0.20 mmol) were added sequentially. After the addition was complete, the mixture was purged with nitrogen three times, heated to 70 °C, and reacted for approximately 16 h. After TLC detection of complete reaction, the solvent was concentrated under reduced pressure, and 15 mL of ice water was added and stirred for approximately 30 min. The mixture was then filtered, and the filter cake was dried. Column chromatography purification was performed using dichloromethane / methanol (150:1) as the eluent, yielding 0.30 g of a yellow solid, i.e., intermediate i-9-1, with a yield of 18.58%. The characterization data of intermediate i-9-1 are as follows: 1 H NMR (400MHz, DMSO-d6) δ10.39 (s, 1H), 10.24 (s, 1H), 8.68 (s, 1H), 8.49 (s, 1H), 8.06 (s, 1H), 8.03 (s, 1H), 7.99 (s, 1H), 7.96-7.91 (m, 2H) , 7.90-7.82(m, 4H), 7.75(s, 1H), 7.73(s, 1H), 7.70-7.52(m, 4H), 7.36(s, 1H), 7.30(s, 2H), 5.52-5.40(m, 4H), 5.35(s, 2H), 2.04(s, 3H).

[0038] The preparation of intermediate i-9-2 included: at room temperature, intermediate i-4-2 (1.04 g, 2.01 mmol) was added to 8 mL of THF, followed by 1 mL of water. Then, intermediate i-8-2 (0.99 g, 2.02 mmol), tripotassium phosphate (1.28 g, 6.03 mmol), and XPos-Pd(G2) (0.16 g, 0.20 mmol) were added sequentially. After the addition was complete, the mixture was purged with nitrogen three times, heated to 70 °C, and reacted for approximately 16 h. After TLC detection to confirm complete reaction, the solvent was concentrated under reduced pressure, and 15 mL of ice water was added and stirred for approximately 30 min. The mixture was then filtered, and the filter cake was dried. Column chromatography purification was performed using dichloromethane / methanol (150:1) as the eluent, yielding 0.24 g of a yellow solid, i.e., intermediate i-9-2, with a yield of 15.29%. The characterization data of intermediate i-9-2 are as follows: 1H NMR (400MHz, CDCl3) δ10.43 (s, 1H), 10.28 (s, 1H), 8.92 (s, 2H), 8.89 (s, 2H), 8.69 (s, 1H), 8.48 (d, J=6.8Hz, 1H), 8.06 (s, 2H), 7.91(s, 2H), 7.84-7.69(m, 2H), 7.38-7.31(m, 3H), 7.18(s, 1H), 6.65(s, 2H), 5.30(s, 2H), 5.27(s, 4H), 2.11(s, 3H).

[0039] Example 1

[0040] At room temperature, intermediate i-9-1 (50.00 mg, 62.06 μmol) was added to a 25 mL pear-shaped reaction flask, followed by 3 mL of dichloromethane and 2 mL of methanol. After complete dissolution, N-acetylethylenediamine (50.71 mg, 496.48 μmol) was added, along with 2 drops of glacial acetic acid. The mixture was stirred at room temperature for 12 h. Then, STAB (103.72 mg, 496.48 μmol) was added in approximately three batches, with each batch added 15 min apart. After all additions were complete, the mixture was stirred at room temperature for 4 h. After the reaction was confirmed to be complete by TLC, the solvent was concentrated under reduced pressure, and 15 mL of ice water was added. The mixture was stirred for approximately 30 min, during which time the pH was adjusted to approximately 8 with a saturated sodium bicarbonate aqueous solution. The mixture was then filtered, the filter cake was dried, and the crude product was purified by preparative thin-layer chromatography to obtain 15 mg of a pale yellow solid product 1, with a yield of 24.72%. The characterization data of this yellow solid product 1 are as follows: HRMS (ESI) for C 54 H 50 Cl2N8O6[M+H] + .Calcd:977.3309,found:977.3307; 1H NMR (400MHz, CDCl3) δ8.71 (s, 1H), 8.50 (dd, J=8.2, 1.6Hz, 1H), 7.87 (dd, J=7.2, 1.4Hz, 1H), 7.82-7.7 7(m, 1H), 7.74(s, 1H), 7.72(s, 1H), 7.70-7.62(m, 4H), 7.59-7.49(m, 4H), 7.40-7.29(m, 3H), 6.96(s, 1 H), 6.62(s, 1H), 6.28(s, 1H), 6.16(s, 1H), 5.23-5.14(m, 4H), 5.13(s, 2H), 3.90(s, 2H), 3.80(s, 2H), 3 .43-3.31(m, 4H), 2.84(t, J=5.8Hz, 2H), 2.78(t, J=5.7Hz, 2H), 2.10(s, 4H), 2.00(s, 3H), 1.97(s, 3H).

[0041] Example 2

[0042] Using intermediate i-9-1 (50.00 mg, 62.06 μmol) as the starting material and ethanolamine (30.3 mg, 496.48 μmol) as the small molecule amine, 13 mg of white solid product 2 was obtained according to the preparation method in Example 1, with a yield of 23.62%. The characterization data of this white solid product 2 are as follows: HRMS (ESI) for C 50 H 44 Cl2N6O6[M+H] + .Calcd: 895.2778, found: 895.2774; 1 HNMR (400MHz, DMSO-d6) δ8.66 (s, 1H), 8.49 (dd, 1H), 8.03 (s, 1H), 7.99-7.94 (m, 2H), 7.9 4-7.88(m, 2H), 7.88-7.81(m, 3H), 7.72(s, 1H), 7.68-7.61(m, 2H), 7.60-7.54(m, 2H), 7.4 4(s, 1H), 7.34(t, J=7.6Hz, 1H), 7.27(d, J=7.4Hz, 1H), 7.18(s, 1H), 5.34(s, 2H), 5.33(s, 2H), 5.22(s, 2H), 3.97(s, 2H), 3.96(s, 2H), 3.60(s, 4H), 2.86-2.73(m, 4H), 2.04(s, 3H).

[0043] Example 3

[0044] Using intermediate i-9-1 (50.00 mg, 62.06 μmol) as the starting material and serine alcohol (45.2 mg, 496.48 μmol) as the small molecule amine, the preparation procedure was the same as in Example 1, yielding 13 mg of beige solid product 3, with a yield of 21.0%. The characterization data of this beige solid product 3 are as follows: HRMS (ESI) for C 52 H 48 Cl2N6O8[M+H] + .Calcd:955.2989,found:955.2985; 1 H NMR (400MHz, DMSO-d6) δ8.65 (s, 1H), 8.49 (d, J=7.8Hz, 1H), 7.99 (s, 1H), 7.97-7.92 (m, 2H), 7.90 (d, J=10.0Hz, 1H), 7.87-7.81(m, 3H), 7.69(s, 1H), 7.67-7.60(m, 2H), 7.57(d, J=7.3Hz, 1H), 7.47(s, 1H) , 7.39 (s, 1H), 7.33 (t, J=7.4Hz, 1H), 7.26 (d, J=7.5Hz, 1H), 7.13 (s, 1H), 5.31 (s, 2H), 5.29 (s, 2H), 5. 21(s, 2H), 4.72-4.51(m, 2H), 3.89(s, 2H), 3.82(s, 2H), 3.54-3.38(m, 8H), 2.64(s, 2H), 2.04(s, 3H).

[0045] Example 4

[0046] Using intermediate i-9-1 (50.00 mg, 62.06 μmol) as the starting material and 2-amino-2-methyl-1,3-propanediol (52.1 mg, 496.48 μmol) as the small molecule amine, the preparation procedure was the same as in Example 1, yielding 14 mg of white solid product 4, with a yield of 21.84%. The characterization data of this white solid product 4 are as follows: HRMS (ESI) for C 54 H 52 Cl2N6O8[M+H] + .Calcd: 983.3302, found: 983.3305; 1H NMR (400MHz, DMSO-d6) δ8.64 (s, 1H), 8.49 (d, J=7.9Hz, 1H), 7.97 (s, 1H), 7.96-7.89 (m, 3H), 7.88- 7.80 (m, 4H), 7.69-7.59 (m, 3H), 7.56 (d, J=7.4Hz, 1H), 7.42 (s, 1H), 7.36 (s, 1H), 7.33 (t, J=7.7Hz , 1H), 7.25 (d, J=7.5Hz, 1H), 7.09 (s, 1H), 5.34-5.24 (m, 4H), 5.21 (s, 2H), 4.51-4.34 (m, 4H), 3.76 (s, 2H), 3.64 (s, 2H), 3.34-3.31 (m, 5H), 3.31-3.27 (m, 4H), 2.03 (s, 3H), 0.96 (s, 3H), 0.93 (s, 3H).

[0047] Example 5

[0048] Using intermediate i-9-1 (50.00 mg, 62.06 μmol) as a starting material and 3-hydroxyaminomethane (60.1 mg, 496.48 μmol) as a small molecule amine, the preparation procedure was the same as in Example 1, yielding 12 mg of a light yellow solid product 5, with a yield of 19.56%. The characterization data of this light yellow solid product 5 are as follows: HRMS (ESI) for C 54 H 52 Cl2N6O 10 [M+H] + .Calcd: 1015.3200, found: 1015.3205; 1 H NMR (400MHz, DMSO-d6) δ8.64 (s, 1H), 8.49 (d, J=7.8Hz, 1H), 7.99-7.94 (m, 2H), 7.94-7.90 (m, 2H), 7.89- 7.84 (m, 2H), 7.83 (s, 1H), 7.81 (s, 1H), 7.69 (s, 1H), 7.66-7.59 (m, 2H), 7.55 (d, J=7.4Hz, 1H), 7.44 (s, 1H ), 7.36 (s, 1H), 7.32 (t, J=7.6Hz, 1H), 7.25 (d, J=7.4Hz, 1H), 7.09 (s, 1H), 5.30 (s, 2H), 5.27 (s, 2H), 5.22 (s, 2H), 4.54-4.17 (m, 6H), 3.86 (s, 2H), 3.74 (s, 2H), 3.47-3.42 (m, 6H), 3.42-3.39 (m, 6H), 2.03 (s, 3H).

[0049] Example 6

[0050] Using intermediate i-9-1 (50.00 mg, 62.06 μmol) as the starting material and (R)-3-amino-1,2-propanediol (45.2 mg, 496.48 μmol) as the small molecule amine, the preparation procedure was the same as in Example 1, yielding 13 mg of a light yellow solid product 6, with a yield of 21.67%. The characterization data of this light yellow solid product 6 are as follows: HRMS (ESI) for C 52 H 48 Cl2N6O8[M+H] + .Calcd:955.2989,found:955.2984; 1 H NMR (400MHz, DMSO-d6) δ8.66 (s, 1H), 8.49 (d, J=7.8Hz, 1H), 7.96 (s, 2H), 7.92 (s, 2H), 7.88-7.80 (m , 4H), 7.65 (s, 1H), 7.63 (s, 1H), 7.61 (s, 1H), 7.57 (d, J=7.4Hz, 1H), 7.42-7.36 (m, 2H), 7.33 (t, J=7. 5Hz, 1H), 7.26 (d, J=7.4Hz, 1H), 7.11 (s, 1H), 5.29 (s, 2H), 5.28 (s, 2H), 5.20 (s, 2H), 3.79 (s, 2H), 3. 68(s, 2H), 3.62-3.52(m, 2H), 3.37-3.34(m, 4H), 2.70-2.55(m, 2H), 2.48-2.40(m, 2H), 2.04(s, 3H).

[0051] Example 7

[0052] Using intermediate i-9-1 (50.00 mg, 62.06 μmol) as the starting material and (S)-3-amino-1,2-propanediol (45.2 mg, 496.48 μmol) as the small molecule amine, the preparation procedure was the same as in Example 1, yielding 9 mg of a light yellow solid product 7, with a yield of 15.80%. The characterization data of this light yellow solid product 7 are as follows: HRMS (ESI) for C 52 H 48 Cl2N6O8[M+H] + .Calcd:955.2989,found:955.2990; 1H NMR (400MHz, DMSO-d6) δ8.66 (s, 1H), 8.49 (d, J=7.7Hz, 1H), 8.04-7.94 (m, 2H), 7.92 (s, 2H), 7.88-7. 83 (m, 2H), 7.83-7.73 (m, 2H), 7.70-7.59 (m, 3H), 7.57 (d, J=7.4Hz, 1H), 7.44-7.36 (m, 2H), 7.33 (t, J= 7.4Hz, 1H), 7.26 (d, J=7.4Hz, 1H), 7.12 (s, 1H), 5.30 (s, 2H), 5.28 (s, 2H), 5.20 (s, 2H), 3.81 (s, 2H), 3 .71(s, 2H), 3.63-3.53(m, 2H), 3.37-3.36(m, 2H), 3.35-3.34(m, 2H), 2.73-2.57(m, 2H), 2.04(s, 3H).

[0053] Example 8

[0054] Using intermediate i-9-1 (50.00 mg, 62.06 μmol) as the starting material and ethylamine (22.4 mg, 496.48 μmol) as the small molecule amine, the preparation procedure was the same as in Example 1, yielding 10 mg of a light yellow solid product 8 with a yield of 19.99%. The characterization data for this light yellow solid product 8 are as follows: HRMS (ESI) for C 50 H 44 Cl2N6O4[M+H] + .Calcd: 863.2879, found: 863.2882; 1 H NMR (400MHz, DMSO-d6) δ8.66 (s, 1H), 8.49 (d, J=7.7Hz, 1H), 8.04 (s, 1H), 7.97 (s, 1H), 7.95 (d, J=6.6Hz, 1H), 7.93-7.87 (m, 2H), 7.87-7.81 (m, 3H), 7.72 (s, 1H), 7.68-7.61 (m, 2H), 7.60-7.52 (m, 2H), 7.45 (s, 1H), 7.34 (t, J=7.3Hz, 1H), 7.26 (d, J=7.3Hz, 1H), 7.18 (s, 1H), 5.38-5.31 (m, 4H), 5.22(s, 2H), 3.93(s, 2H), 3.91(s, 2H), 2.82-2.70(m, 4H), 2.04(s, 3H), 1.18-1.11(m, 6H).

[0055] Example 9

[0056] Using intermediate i-9-1 (50.00 mg, 62.06 μmol) as a starting material and isopropylamine (29.3 mg, 496.48 μmol) as a small molecule amine, the preparation procedure was the same as in Example 1, yielding 14 mg of yellow solid product 9, with a yield of 26.65%. The characterization data for this yellow solid product 9 are as follows: HRMS (ESI) for C 52 H 48 Cl2N6O4[M+H] + .Calcd: 891.3192, found: 891.3197; 1 H NMR (400MHz, DMSO-d6) δ8.65 (s, 1H), 8.49 (d, J = 7.6Hz, 1H), 8.02 (s, 1H), 8.00-7.89 (m, 3 H), 7.89-7.78 (m, 4H), 7.70-7.61 (m, 3H), 7.58 (d, J=7.1Hz, 1H), 7.48 (s, 1H), 7.42 (s, 1H ), 7.34 (t, J=7.3Hz, 1H), 7.27 (d, J=7.3Hz, 1H), 7.17 (s, 1H), 5.37-5.28 (m, 4H), 5.25-5. 18(m, 2H), 3.91-3.79(m, 4H), 2.96(s, 1H), 2.89(s, 1H), 2.04(s, 3H), 1.13-1.03(m, 12H).

[0057] Example 10

[0058] Using intermediate i-9-1 (50.00 mg, 62.06 μmol) as a starting material and 4-hydroxypiperidine (57.144 mg, 496.48 μmol) as a small molecule amine, the preparation procedure was the same as in Example 1, yielding 10 mg of a light yellow solid product 10, with a yield of 17.69%. The characterization data for this light yellow solid product 10 are as follows: HRMS (ESI) for C 56 H 52 Cl2N6O6[M+H] + .Calcd: 975.3404, found: 975.3407; 1H NMR (400MHz, DMSO-d6) δ8.66 (s, 1H), 8.49 (d, J=7.9Hz, 1H), 8.03-7.95 (m, 2H), 7.95-7.89 (m, 2H), 7.89-7.83(m, 3H), 7.83(s, 1H), 7.68-7.55(m, 4H), 7.54-7.39(m, 2H), 7.35(t, J=7.5Hz, 1H), 7.27(d, J=7.5Hz, 1H), 7.19(s, 1H), 5.38-5.28(m, 4H), 5.26-5.18(m, 2H), 3.75-3.45(m, 4H), 3.21-2.58(m, 6H), 2.35-2.09(m, 2H), 2.04(s, 3H), 1.92-1.64(m, 5H), 1.65-1.33(m, 5H).

[0059] Example 11

[0060] Using intermediate i-9-1 (50.00 mg, 62.06 μmol) as a starting material and 2-methoxyethylamine (37.27 mg, 496.48 μmol) as a small molecule amine, the preparation procedure was the same as in Example 1, yielding 12 mg of a yellowish-white solid product 11, with a yield of 22.42%. The characterization data of this yellow solid product 11 are as follows: HRMS (ESI) for C 52 H 48 Cl2N6O6[M+H] + .Calcd: 923.3091, found: 923.3095; 1 H NMR (400MHz, DMSO-d6) δ8.65 (s, 1H), 8.52-8.45 (m, 1H), 7.99-7.94 (m, 2H), 7.94-7.88 (m, 2H), 7 .86-7.79(m, 4H), 7.63(m, 2H), 7.61-7.55(m, 2H), 7.41-7.37(m, 2H), 7.33(t, J=7.6Hz, 1H), 7.26 (d, J=7.4Hz, 1H), 7.12 (s, 1H), 5.33-5.25 (m, 4H), 5.20 (s, 2H), 3.80 (s, 2H), 3.70 (s, 2H), 3.48-3 .37(m, 6H), 3.25(s, 3H), 3.22(s, 3H), 2.71(t, J=5.5Hz, 2H), 2.66(t, J=5.4Hz, 2H), 2.03(s, 3H).

[0061] Example 12

[0062] Using intermediate i-9-1 (50.00 mg, 62.06 μmol) as a starting material and N-methyl-2-hydroxyethylamine (37.27 mg, 496.48 μmol) as a small molecule amine, the preparation procedure was the same as in Example 1, yielding 12 mg of off-white solid product 12, with a yield of 22.42%. The characterization data of this off-white solid product 12 are as follows: HRMS (ESI) for C 52 H 48 Cl2N6O6[M+H] + .Calcd: 923.3091, found: 923.3096; 1 H NMR (400MHz, DMSO-d6) δ8.66 (s, 1H), 8.48 (d, J=8.0Hz, 1H), 8.01-7.88 (m, 4H), 7.88-7.79 (m, 4H), 7.68-7.49 (m, 4H), 7.40 (s, 1H), 7.39 (s, 1H), 7.34 (t, J=7.6Hz, 1H), 7.26 (d, J=7.5Hz, 1H) , 7.12(s, 1H), 5.30(s, 2H), 5.28(s, 2H), 5.22-5.17(m, 2H), 3.60(s, 2H), 3.58-3.53(m, 2H), 3. 53-3.45(m, 4H), 2.54-2.51(m, 2H), 2.49-2.41(m, 2H), 2.25(s, 3H), 2.18(s, 3H), 2.04(s, 3H).

[0063] Example 13

[0064] Using intermediate i-9-1 (50.00 mg, 62.06 μmol) as a starting material and 4-aminotetrahydropyran (50.2 mg, 496.48 μmol) as a small molecule amine, the preparation procedure was the same as in Example 1, yielding 15 mg of white solid product 13, with a yield of 24.92%. The characterization data of this white solid product 13 are as follows: HRMS (ESI) for C 56 H 52 Cl2N6O6[M+H] + .Calcd: 975.3404, found: 975.3409; 1H NMR (400MHz, DMSO-d6) δ8.65 (s, 1H), 8.49 (d, J=7.9Hz, 1H), 8.00-7.87 (m, 4H), 7.86-7. 78 (m, 4H), 7.67-7.60 (m, 3H), 7.57 (d, J = 7.4Hz, 1H), 7.43-7.37 (m, 2H), 7.33 (t, J = 7.6Hz , 1H), 7.26 (d, J=7.4Hz, 1H), 7.12 (s, 1H), 5.29 (s, 4H), 5.20 (s, 2H), 3.89-3.76 (m, 6H), 3 .74-3.63(m, 2H), 3.34-3.18(m, 6H), 2.04(s, 3H), 1.84-1.71(m, 4H), 1.35-1.19(m, 6H).

[0065] Example 14

[0066] Using intermediate i-9-1 (50.00 mg, 62.06 μmol) as a starting material and cyclopropylamine (28.3 mg, 496.48 μmol) as a small molecule amine, the preparation procedure was the same as in Example 1, yielding 14 mg of a yellowish-white solid product 14, with a yield of 25.93%. The characterization data for this yellow solid product 14 are as follows: HRMS (ESI) for C 52 H 44 Cl2N6O4[M+H] + .Calcd: 887.2879, found: 887.2882; 1 H NMR (400MHz, DMSO-d6) δ8.65 (s, 1H), 8.49 (d, J=8.2Hz, 1H), 7.98 (s, 1H), 7.97-7.93 (m, 2H), 7.90(t, 1H), 7.87-7.79(m, 4H), 7.68-7.60(m, 2H), 7.60-7.54(m, 2H), 7.39(s, 2H), 7.33(t, J =7.5Hz, 1H), 7.26 (d, J = 7.4Hz, 1H), 7.12 (s, 1H), 5.30 (s, 2H), 5.29 (s, 2H), 5.21 (s, 2H), 3.8 4(s, 2H), 3.77(s, 2H), 2.19-2.10(m, 2H), 2.03(s, 3H), 0.45-0.36(m, 4H), 0.36-0.26(m, 4H).

[0067] Example 15

[0068] Using intermediate i-9-1 (50.00 mg, 62.06 μmol) as the starting material and cyclopentylamine (42.2 mg, 496.48 μmol) as the small molecule amine, the preparation procedure was the same as in Example 1, yielding 12 mg of a light yellow solid product 15, with a yield of 21.95%. The characterization data of this light yellow solid product 15 are as follows: HRMS (ESI) for C 56 H 52 Cl2N6O4[M+H] + .Calcd: 943.3505, found: 943.3510; 1 H NMR (400MHz, DMSO-d6) δ8.65 (s, 1H), 8.49 (dd, J=7.9, 1.5Hz, 1H), 8.06 (s, 1H), 7.99 (s, 1H), 7.98-7.94 (m, 1H), 7.94-7.87 (m, 2H), 7.87-7.80 (m, 3H), 7.72 (s, 1H), 7.68-7.61 (m, 2H), 7.59 (d, J=7.4Hz, 1H), 7.55 (s, 1H), 7.45 (s, 1H), 7.34 (t, J=7.5Hz, 1H), 7.27 (d, J=7.3Hz, 1H), 7.19 (s, 1H), 5.34 (s, 2H), 5.32 (s, 2H), 5.21 (s, 2H), 3.89 (s, 2H), 3.88 (s, 2H), 3.29-3.19 (m, 2H), 2.04 (s, 3H), 1.88-1.78 (m, 4H), 1.69-1.59 (m, 4H), 1.54-1.43 (m, 8H).

[0069] Example 16

[0070] Using intermediate i-9-1 (50.00 mg, 62.06 μmol) as the starting material and (R)-3-pyrrolidone (50.2 mg, 496.48 μmol) as the small molecule amine, the preparation procedure was the same as in Example 1, yielding 12 mg of a light yellow solid product 16, with a yield of 21.86%. The characterization data of this light yellow solid product 16 are as follows: HRMS (ESI) for C 54 H 48 Cl2N6O6[M+H] + .Calcd: 947.3091, found: 947.3092; 1H NMR (400MHz, DMSO-d6) δ8.67 (s, 1H), 8.48 (d, J=7.9Hz, 1H), 8.00-7.94 (m, 2H), 7.94-7.88 (m, 2H), 7.8 8-7.80 (m, 4H), 7.67-7.61 (m, 2H), 7.60-7.55 (m, 2H), 7.40 (s, 1H), 7.37 (s, 1H), 7.33 (d, J=7.5Hz, 1H), 7.26(d, J=7.4Hz, 1H), 7.13(s, 1H), 5.31(s, 2H), 5.29(s, 2H), 5.21(s, 2H), 4.78(s, 2H), 4.23(s, 2H), 3.79-3.54(m, 5H), 3.47-3.36(m, 2H), 2.82-2.63(m, 4H), 2.48-2.40(m, 2H), 2.04(s, 3H), 1.59(s, 2H).

[0071] Example 17

[0072] Using intermediate i-9-1 (50.00 mg, 62.06 μmol) as a starting material and (S)-3-pyrrolidone (50.2 mg, 496.48 μmol) as a small molecule amine, the preparation procedure was the same as in Example 1, yielding 11 mg of a light yellow solid product 17, with a yield of 19.43%. The characterization data for this light yellow solid product 17 are as follows: HRMS (ESI) for C 54 H 48 Cl2N6O6[M+H] + .Calcd: 947.3091, found: 947.3088; 1H NMR (400MHz, DMSO-d6) δ8.66 (s, 1H), 8.48 (d, J = 8.0Hz, 1H), 7.97 (s, 1H), 7.95 (s, 1H), 7.94-7.90 (m, 2H), 7.88 (d, J = 10 .0Hz, 1H), 7.85-7.83(m, 2H), 7.83-7.78(m, 2H), 7.66-7.61(m, 2H), 7.58(s, 1H), 7.57(s, 1H), 7.40(s, 1H), 7.38(s, 1H) , 7.34 (t, J=7.6Hz, 1H), 7.26 (d, J=7.4Hz, 1H), 7.13 (s, 1H), 5.31 (s, 2H), 5.29 (s, 2H), 5.21 (s, 2H), 4.92-4.75 (m, 2H), 4.29-4.17 (m, 2H), 3.78-3.60 (m, 4H), 2.74 (dd, J=15.8, 7.0Hz, 4H), 2.51-2.35 (m, 4H), 2.04 (s, 3H), 1.65-1.56 (m, 2H).

[0073] Example 18

[0074] Using intermediate i-9-1 (50.00 mg, 62.06 μmol) as a starting material and cyclohexylamine (49.2 mg, 496.48 μmol) as a small molecule amine, the preparation procedure was the same as in Example 1, yielding 10 mg of yellow solid product 18 with a yield of 17.76%. The characterization data for this yellow solid product 18 are as follows: HRMS (ESI) for C 58 H 56 Cl2N6O4[M+H] + .Calcd: 971.3818, found: 971.3819; 1H NMR (400MHz, DMSO-d6) δ8.65 (s, 1H), 8.49 (dd, J=7.9, 1.7Hz, 1H), 8.03 (s, 1H), 8.01-7.94 (m, 2H), 7.94-7.89 (m, 1 H), 7.88-7.80 (m, 4H), 7.68 (s, 1H), 7.65 (d, J=2.9Hz, 1H), 7.64-7.61 (m, 1H), 7.59 (d, J=7.7Hz, 1H), 7.50 (s, 1H), 7.42 (s, 1H), 7.34 (t, J=7.5Hz, 1H), 7.26 (d, J=7.4Hz, 1H), 7.17 (s, 1H), 5.33 (s, 2H), 5.30 (s, 2H), 5.20 (s, 2H), 3. 88(s, 2H), 3.85(s, 2H), 2.04(s, 3H), 1.95-1.85(m, 4H), 1.72-1.63(m, 4H), 1.60-1.43(m, 4H), 1.21-1.10(m, 10H).

[0075] Example 19

[0076] Using intermediate i-9-1 (50.00 mg, 62.06 μmol) as a starting material and cyclobutylamine (35.3 mg, 496.48 μmol) as a small molecule amine, the preparation procedure was the same as in Example 1, yielding 12 mg of yellow solid product 19, with a yield of 21.36%. The characterization data of this yellow solid product 19 are as follows: HRMS (ESI) for C 54 H 48 Cl2N6O4[M+H] + .Calcd: 915.3192, found: 915.3194; 1 H NMR (400MHz, DMSO-d6) δ8.65 (s, 1H), 8.49 (d, J=7.8Hz, 1H), 8.01 (s, 1H), 7.98-7.93 (m, 2H) , 7.94-7.88 (m, 1H), 7.88-7.84 (m, 2H), 7.84-7.79 (m, 2H), 7.68-7.60 (m, 3H), 7.57 (d, J=7.4 Hz, 1H), 7.42 (s, 1H), 7.39 (s, 1H), 7.33 (t, J=7.5Hz, 1H), 7.26 (d, J=7.4Hz, 1H), 7.14 (s, 1H) , 5.31(s, 4H), 5.21(s, 2H), 3.71(s, 2H), 3.66(s, 2H), 2.15-1.96(m, 8H), 1.81-1.53(m, 9H).

[0077] Example 20

[0078] Using intermediate i-9-2 (50.00 mg, 62.03 μmol) as the starting material and ethanolamine (30.2 mg, 496.24 μmol) as the small molecule amine, 11.3 mg of white solid product 20 was obtained according to the preparation method in Example 1, with a yield of 20.31%. The characterization data of this white solid product 20 are as follows: HRMS (ESI) for C 48 H 43 Cl2N8O6[M+H] + .Calcd: 897.2683, found: 897.2679.

[0079] Example 21

[0080] Using intermediate i-9-2 (50.00 mg, 62.03 μmol) as the starting material and serine alcohol (45.1 mg, 496.24 μmol) as the small molecule amine, the preparation procedure was the same as in Example 1, yielding 16.8 mg of beige solid product 21, with a yield of 28.5%. The characterization data for this beige solid product 21 are as follows: HRMS (ESI) for C 50 H 47 Cl2N8O8[M+H] + .Calcd: 957.2894, found: 957.2890.

[0081] Example 22

[0082] Using intermediate i-9-2 (50.00 mg, 62.03 μmol) as the starting material and 2-amino-2-methyl-1,3-propanediol (52.0 mg, 496.24 μmol) as the small molecule amine, the preparation procedure was the same as in Example 1, yielding 20.3 mg of white solid product 22, with a yield of 33.21%. The characterization data of this white solid product 22 are as follows: HRMS (ESI) for C 52 H 51 Cl2N8O8[M+H] + .Calcd: 985.3207, found: 985.3211.

[0083] Example 23

[0084] Using intermediate i-9-2 (50.00 mg, 62.03 μmol) as a starting material and 3-hydroxyaminomethane (60.0 mg, 496.24 μmol) as a small molecule amine, the preparation procedure was the same as in Example 1, yielding 19.8 mg of a light yellow solid product 23, with a yield of 31.50%. The characterization data for this light yellow solid product 23 are as follows: HRMS (ESI) for C 52H 50 Cl2N8O 10 [M+H] + .Calcd: 1017.3105, found: 1017.3110.

[0085] Example 24

[0086] Using intermediate i-9-2 (50.00 mg, 62.03 μmol) as the starting material and (S)-3-amino-1,2-propanediol (45.0 mg, 496.24 μmol) as the small molecule amine, the preparation procedure was the same as in Example 1, yielding 15.7 mg of a light yellow solid product 24, with a yield of 26.4%. The characterization data of this light yellow solid product 24 are as follows: HRMS (ESI) for C 50 H 46 Cl2N8O8[M+H] + .Calcd: 957.2894, found: 957.2889.

[0087] Example 25

[0088] Using intermediate i-9-1 (50.00 mg, 62.03 μmol) as the starting material and (R)-3-amino-1,2-propanediol (45.0 mg, 496.24 μmol) as the small molecule amine, the preparation procedure was the same as in Example 1, yielding 13.6 mg of a light yellow solid product 25, with a yield of 22.9%. The characterization data of this light yellow solid product 25 are as follows: HRMS (ESI) for C 50 H 46 Cl2N8O8[M+H] + .Calcd: 957.2894, found: 957.2890.

[0089] The structural formulas of the 8-phenylquinazoline compounds obtained in Examples 1-25 above are shown in Table 1.

[0090] Table 1 Chemical names and structural formulas of each embodiment

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098] Example 26 PD-1 / PD-L1 Inhibitory Activity Test

[0099] To verify whether the 8-phenylquinazoline compounds prepared in Examples 1-25 have a significant inhibitory effect on PD-1 / PD-L1 protein, this example uses the Csibio PD-1 / PD-L1 binding affinity assay kit (Cat. No. 64PD1PEH) and employs HTRF (homogeneous time-resolved fluorescence) to test the PD-1 / PD-L1 inhibitory activity of the 8-phenylquinazoline compounds prepared in Examples 1-25, including the following steps:

[0100] (1) Preparation of compounds: Dissolve and mix 25 compounds and positive control compound (BMS-202) with DMSO to prepare a 10mM stock solution. Then, use buffer to serially dilute each compound to the corresponding concentration: 100μM, 25μM, 6.3μM, 1.6μM, 0.39μM, and 0.1μM.

[0101] (2) Prepare test reagents: Thaw the materials in the PD-1 / PD-L1 binding force test kit at room temperature and shake well;

[0102] (3) Dilute Tag1-PD-L1 protein and Tag2-PD-1 protein 40 times with buffer; dilute Anti-Tag1 Eu Cryptate Reagent and Anti-Tag2 XL665 antibody 50 times with buffer, and then mix Anti-Tag1 Eu Cryptate Reagent and Anti-Tag2 XL665 antibody 1:1.

[0103] (4) Add 100 nL of the diluted test compound or positive control compound to a 384-well plate using an Echo 550 Liquid Handler (Labcyte), then add 5 uL of diluted Tag1-PD-L1 protein and 2.5 uL of diluted Tag2-PD-1 protein in sequence, and mix by pipetting (3 replicates per group).

[0104] (5) Add 5 μL of the mixture of Anti-Tag1 Eu Cryptate Reagent and Anti-Tag2 XL665 antibody to each well. The final concentration of DMSO is 1%, and the final concentrations of the compounds are 1000 nM, 250 nM, 63 nM, 16 nM, 3.9 nM, and 1.0 nM. Mix well by blowing, seal and incubate at room temperature in the dark for 1 h.

[0105] (6) The fluorescence signal was detected using an EnVision multi-functional microplate reader (Perkin Elmer) (excitation at 320nm, emission at 665nm and 615nm);

[0106] (7) Emission Ratio and Suppression Ratio are calculated using the following formulas: Emission Ratio (ER) = 665nm Emission signal / 615nm Emission signal, Suppression Ratio = (Max - Signal) / (Max - Min) × 100%. The three IC values ​​are calculated using Graphpadprism 5 software. 50 Value, then based on IC 50 Calculate the mean and standard deviation (SD).

[0107] Inhibition rate = (ER) positive -ER sample ) / (ER positive -ER negative )×100%

[0108] The experimental results are shown in Table 2. A: IC 50 <50nM; B: 50nM <IC 50 <100nM; C: 100nM <IC 50 <1000nM; D: 1000nM <IC 50 ;

[0109] Table 2. Results of the inhibitory activity of 8-phenylquinazoline compounds prepared in Examples 1-25 against PD-1 / PD-L1.

[0110] Example 1 B Example 14 C Example 2 A Example 15 C Example 3 A Example 16 46.17 Example 4 A Example 17 B Example 5 A Example 18 C Example 6 A Example 19 C Example 7 A Example 20 A Example 8 A Example 21 A Example 9 51.49 Example 22 A Example 10 B Example 23 A Example 11 C Example 24 A Example 12 44.78 Example 25 A Example 13 B BMS-202 87.20

[0111] As can be seen from the results in Table 2, the 8-phenylquinazoline compounds prepared in Examples 1-25 all exhibited outstanding inhibitory activity against PD-1 / PD-L1, and most of the 8-phenylquinazoline compounds showed high IC50 values. 50 The value is below 100 nM.

[0112] In summary, the activity test results in Table 2 show that the 8-phenylquinazoline compounds prepared in this invention exhibit significant inhibitory activity against the PD-1 / PD-L1 signaling pathway and have the potential to treat related diseases mediated by the PD-1 / PD-L1 signaling pathway.

[0113] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. An 8-phenylquinazoline compound, characterized in that: Its structural formula is ,in, for , , , , , , , , , , , , , , , , , or ; R3 represents H, halogen, or C. 1-3 Alkyl, C 1-3 Alkoxy or C 1-3 Alkylamine group; R4 is H, halogen, or methyl; X is CH or N.

2. The 8-phenylquinazoline compound as described in claim 1, characterized in that: R3 is a halogen.

3. An 8-phenylquinazoline compound as described in claim 2, characterized in that: R4 is H or methyl.

4. The use of the 8-phenylquinazoline compound of any one of claims 1 to 3 and its pharmaceutically acceptable salt in the preparation of a pharmaceutical composition for treating diseases related to the PD-1 / PD-L1 signaling pathway, characterized in that: The diseases related to the PD-1 / PD-L1 signaling pathway are tumor diseases, including lung cancer, liver cancer, kidney cancer, prostate cancer, thyroid cancer, skin cancer, pancreatic cancer, ovarian cancer, breast cancer, bladder cancer, myelodysplastic syndrome, lymphoma, esophageal cancer, gastrointestinal cancer, and tumors of the central and peripheral nervous systems.

5. A pharmaceutical composition for treating diseases related to the PD-1 / PD-L1 signaling pathway, characterized in that: Its active ingredients include the 8-phenylquinazoline compound as described in any one of claims 1 to 3 and / or a pharmaceutically acceptable salt, wherein the PD-1 / PD-L1 signaling pathway-related disease is an oncological disease, including lung cancer, liver cancer, kidney cancer, prostate cancer, thyroid cancer, skin cancer, pancreatic cancer, ovarian cancer, breast cancer, bladder cancer, myelodysplastic syndrome, lymphoma, esophageal cancer, gastrointestinal cancer, and tumors of the central and peripheral nervous systems.

6. The pharmaceutical composition according to claim 5, characterized in that: The pharmaceutically acceptable salt is selected from at least one of the following: hydrogen sulfate, hydrochloride, hydrobromide, sulfate, oxalate, lactate, gluconate, tartrate, fumarate, methanesulfonate, ethanesulfonate, benzenesulfonate, acetate, citrate, and p-toluenesulfonate.

Images