Pyrazolopyridine compound linked with nitrogen-containing four-membered ring or pharmaceutically acceptable salt thereof, and use thereof

Abstract

Provided are a pyrazolopyridine compound linked with a nitrogen-containing four-membered ring, or a pharmaceutically acceptable salt thereof, and a use thereof. The compound can act as a highly selective PDE10A inhibitor for the treatment of peripheral tissue disorders, comprising myocardial hypertrophy and cardiac dysfunction (heart failure), diabetes mellitus, hypertension, renal impairment and renal fibrosis, pulmonary inflammation and pulmonary fibrosis, immune regulation disorders, and cancers (colon cancer, ovarian cancer, and lung cancer). Two strategies are employed to achieve high selectivity of the compound for PDE10A, while reducing the inhibitory effect thereof on the central nervous system. 1. Molecular structure modification: the structure of the pyrazolopyridine compound linked with the nitrogen-containing four-membered ring is optimized by, for example, increasing the proportion of N, O and S atoms in the molecular structure, to enhance polar surface area and reduce lipophilicity, or introducing hydrophilic groups to increase water solubility, thereby reducing the permeability of the blood-brain barrier (BBB) to the compound; 2. Targeted pulmonary tissue administration is implemented by means of drug formulation techniques.

Description

A class of pyrazolopyridine compounds or their pharmaceutically acceptable salts linked by a nitrogen-containing four-membered ring and their applications Technical Field

[0001] This invention relates to a pyrazolopyridine compound, particularly to a class of pyrazolopyridine compounds with a nitrogen-containing four-membered ring and their applications, belonging to the technical field of pyrazolopyridine compounds. Background Technology

[0002] Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), as second messengers, are important regulators of intracellular signal transduction, widely involved in physiological processes such as cell proliferation and apoptosis, relaxation of vascular and airway smooth muscle, immune / inflammatory responses, visual transduction, secretion, and skeletal development. Cyclic nucleotide phosphodiesterases (PDEs), a family of superenzymes that specifically hydrolyze cAMP and cGMP in human cells, determine the circulating concentrations of these two second messengers within cells. PDE4 / 7 / 8 specifically hydrolyze cAMP, PDE5 / 6 / 9 specifically hydrolyze cGMP, while PDE1 / 2 / 3 / 10 / 11 can hydrolyze both cAMP and cGMP simultaneously, but to varying degrees.

[0003] Existing technologies show that phosphodiesterase 10A (PDE10A) is highly expressed in the central striatum of the human brain and participates in the regulation of information processing in the corticobasal ganglia circuit, suggesting that it may play a role in the pathogenesis of neuropsychiatric diseases.

[0004] Currently, most small molecule drugs targeting PDE10A are focused on the treatment of central nervous system diseases (schizophrenia, Huntington's disease). Several representative drugs, such as MP-10, AMG-579MK8189, and TAK-063, have entered Phase I / II clinical trials. This requires these drugs to have good blood-brain barrier (BBB) ​​permeability to accumulate in brain tissue and maintain a certain efficacy concentration. However, these PDE10A inhibitors are not suitable for treating peripheral tissue diseases, mainly because their good BBB permeability easily leads to strong inhibition of highly expressed PDE10A in the brain, significantly increasing the incidence of adverse clinical events.

[0005] Therefore, it is extremely challenging to achieve the therapeutic effect of PDE10A inhibitors on peripheral tissue diseases without producing significant central nervous system depression. To address this issue, a class of pyrazolopyridine compounds with a nitrogen-containing four-membered ring and their applications were designed. Summary of the Invention

[0006] The main objective of this invention is to provide a class of pyrazolidine compounds linked by a nitrogen-containing four-membered ring and their applications.

[0007] The objective of this invention can be achieved through the same technical solution as that described in the claims, and will not be repeated here.

[0008] Beneficial technical effects of the present invention:

[0009] This invention provides a class of pyrazolopyridine compounds linked by a nitrogen-containing four-membered rings and their applications, aiming to discover that these compounds can serve as novel, highly selective PDE10A inhibitors for the treatment of peripheral tissue diseases, including: myocardial hypertrophy and cardiac dysfunction (heart failure), diabetes, hypertension, renal impairment and renal fibrosis, pulmonary inflammation and pulmonary fibrosis, immune dysregulation, and cancers (colon cancer, ovarian cancer, and lung cancer). Two strategies are employed to achieve high selectivity for PDE10A inhibition while simultaneously reducing the inhibitory effect on the central nervous system. First, in terms of molecular structure modification: the structure of the pyrazolopyridine compounds linked by nitrogen-containing four-membered rings is optimized, such as by increasing the proportion of N, O, and S atoms in the molecular structure to increase the polar surface area and reduce lipophilicity; and by introducing hydrophilic groups to increase water solubility, thereby reducing the permeability of the compound to the BBB (biologically modified bone lining). Second, targeted drug delivery to lung tissue can be achieved through drug formulation methods (e.g., inhalable dry powder solid particle drug-loaded microspheres), reducing the first-pass effect in vivo caused by oral administration, lowering the dosage and the systemic exposure of drug molecules, thereby reducing the accumulation of such compounds in the brain and the inhibitory effect on the central nervous system, and significantly reducing the potential risk of adverse events. Attached Figure Description

[0010] Figure 1 shows a statistical graph of cardiac specific gravity and cardiac-tibial ratio after administration of a preferred embodiment B16 of a pyrazolopyridine compound with a nitrogen-containing four-membered ring according to the present invention and its application. *P<0.05, **P<0.01, ***p<0.001, ****p<0.0001, ns indicates no statistical difference, n=6.

[0011] Figure 2 is a statistical graph showing the changes in the expression of atrial natriuretic peptide (ANP) and β-myosin heavy chain (β-MHC) mRNA in myocardial tissue after B16 administration intervention according to a preferred embodiment of a class of aza-quaternary ring-linked pyrazolopyridine compounds and their applications according to the present invention. *P<0.05, **P<0.01, ***p<0.001, ****p<0.0001, ns indicates no statistical difference, n=6.

[0012] Figure 3 shows the statistical graphs of left ventricular end-systolic diameter and left ventricular end-diastolic diameter after administration of a preferred embodiment B16 of a pyrazolopyridine compound with a nitrogen-containing four-membered ring according to the present invention and its application. *P<0.05, **P<0.01, ***p<0.001, ****p<0.0001, ns indicates no statistical difference, n=6.

[0013] Figure 4 shows a statistical graph of left ventricular ejection fraction and left ventricular shortening fraction after administration of a preferred embodiment of a pyrazolopyridine compound with a nitrogen-containing four-membered ring according to the present invention and its application, B16. *P<0.05, **P<0.01, ***p<0.001, ****p<0.0001, ns indicates no statistical difference, n=6.

[0014] Figure 5 shows the staining of myocardial tissue sections after B16 administration intervention according to a preferred embodiment of a class of aza-quaternary ring-linked pyrazolopyridine compounds and their applications, where H&E represents hematoxylin staining; WGA represents wheat germ agglutinin staining; picrosiriusred represents Sirius red staining; ISO represents isoproterenol; and propranolol represents propranolol.

[0015] Figure 6 illustrates a preferred embodiment of a class of aza-quaternary ring-linked pyrazolopyridine compounds according to the present invention and their applications. B11 administration intervention improved lung function in mice with pulmonary fibrosis (WBP method). (A) Enhanced pause (Penh); (B) End-expiratory pause (EEP); (C) Relaxation time (RT). Compared with the normal control group, # P<0.05, ## P<0.01; compared with the model group, *P<0.05, **P<0.01, ns indicates no statistical difference, n=6.

[0016] Figure 7 illustrates a preferred embodiment of a class of aza-quaternary ring-linked pyrazolopyridine compounds according to the present invention and their applications. B11 administration intervention improved lung function in mice with pulmonary fibrosis (PFT method). (A) Airway resistance (RI); (B) Dynamic compliance (Cdyn); (C) Total lung capacity (TLC); (D) Inspiratory capacity (IC); (E) Forced vital capacity (FVC); (F) Forced expiratory volume in one second (FEV100); compared with the normal control group, compared with the normal control group, # P<0.05, ## P<0.01; compared with the model group, *P<0.05, **P<0.01, ns indicates no statistical difference, n=6.

[0017] Figure 8 shows a preferred embodiment of a class of aza-quaternary ring-linked pyrazolopyridine compounds according to the present invention, B11, which improves and alleviates bleomycin-induced pulmonary fibrosis in mice. (A) HE and Masson representation; (B) H&E score; (C) Masson score. Scale bar: 100 μm; compared with the normal control group, # P<0.05, ## P<0.01; compared with the model group, *P<0.05, **P<0.01, ns indicates no statistical difference, n=6.

[0018] Figure 9 is a schematic diagram of the preparation of compound B11 nano-suspension and its dry powder inhaler, a preferred embodiment of a type of nitrogen-containing four-membered ring-linked pyrazolopyridine compound and its application according to the present invention.

[0019] Figure 10 shows (A) the physical appearance and nanoparticle size distribution of compound B11 nanosuspension, a preferred embodiment of a type of aza-quaternary ring-linked pyrazolopyridine compound and its application according to the present invention; and (B) the transmission electron microscopy result.

[0020] Figure 11 shows the (A) morphology and (B) particle size distribution of an unloaded dry powder of a preferred embodiment of a nitrogen-containing four-membered ring-linked pyrazolopyridine compound and its application according to the present invention; and the (C) morphology and (D) particle size distribution of the drug-loaded B11 dry powder.

[0021] Figure 12 shows the particle size distribution of a preferred embodiment B11 of a nitrogen-containing four-membered ring-linked pyrazolopyridine compound according to the present invention and its application, after in vitro atomization. MMAD: medium particle size, EF: emission fraction, FPF: fine particle fraction.

[0022] Figure 13 illustrates the synthesis of a class of pyrazolopyridine compounds A1-A12 with a nitrogen-containing four-membered ring linkage according to the present invention.

[0023] Figure 14 illustrates the synthesis of intermediates M5 to M11 of a type of aza-quaternary ring-linked pyrazolopyridine compounds according to the present invention.

[0024] Figure 15 illustrates the synthesis of a class of pyrazolopyridine compounds A13–A20 linked by a nitrogen-containing four-membered ring according to the present invention.

[0025] Figure 16 illustrates the synthesis of a class of pyrazolopyridine compounds B1–B6 linked by a nitrogen-containing four-membered ring according to the present invention.

[0026] Figure 17 illustrates the synthesis of a class of pyrazolopyridine compounds B7–B22 with a nitrogen-containing four-membered ring linkage according to the present invention.

[0027] Figure 18 illustrates the synthesis of a type of pyrazolopyridine compound B23, which is linked by a nitrogen-containing four-membered ring, according to the present invention.

[0028] Figure 19 illustrates the synthesis of a class of pyrazolopyridine compounds B24–B26, which are linked by a nitrogen-containing four-membered rings according to the present invention.

[0029] Figure 20 illustrates the synthesis of B27, a type of pyrazolidine compound linked by a nitrogen-containing four-membered ring, according to the present invention.

[0030] Figure 21 illustrates the synthesis of a class of pyrazolopyridine compounds C1-C2 linked by a nitrogen-containing four-membered ring according to the present invention.

[0031] Figure 22 illustrates the synthesis of a type of pyrazolopyridine compound C3 with a nitrogen-containing four-membered ring according to the present invention.

[0032] Figure 23 illustrates the synthesis of a class of nitrogen-containing four-membered ring-linked pyrazolopyridine compounds (C4-C8) according to the present invention.

[0033] Figure 24 illustrates the synthesis of a class of nitrogen-containing four-membered ring-linked pyrazolopyridine compounds (C9-C10) according to the present invention. Detailed Implementation

[0034] To enable those skilled in the art to understand the technical solution of the present invention more clearly, the present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0035] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0036] (1) Examples of representative compound structures

[0037]

[0038]

[0039]

[0040]

[0041] (2) Synthesis of representative compound structures

[0042] In this embodiment, the synthetic routes of compounds A1 to A12 are shown in Figure 13:

[0043] The synthesis of intermediate M1 was as follows: In a 250 mL round-bottom flask, 1-Boc-azacyclobutane-3-carboxylic acid (10 g, 49.7 mmol), cyclo(isopropyl)malonate (10.74 g, 74.5 mmol), and 4-dimethylaminopyridine (9.10 g, 1.5 mmol) were dissolved in 100 mL of dichloromethane. Under argon protection, a solution of N,N'-carbonyldiimidazole (9.67 g, 59.6 mmol) in 60 mL of dichloromethane was slowly added dropwise to the above system at 0 °C. The reaction mixture was allowed to rise naturally to room temperature and stirred overnight. The reaction was monitored by TLC. After the reaction was complete, a saturated citric acid aqueous solution was slowly added to the reaction system to adjust the pH to 5–6. Saturated brine (60 mL) was added, and the organic phase was separated. The aqueous phase was extracted twice with dichloromethane (30 mL). The organic phase was separated, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by vacuum distillation to obtain a yellow oily compound. The compound was dissolved in anhydrous ethanol (60 mL), heated to 80 °C, and stirred overnight. The reaction was monitored by TLC. After the reaction was complete, the reaction mixture was concentrated by vacuum distillation. The crude product was purified by silica gel column chromatography to give a pale yellow oily intermediate compound M1 (10.28 g, yield 76%). 1 H NMR (400MHz, CDCl3) δ4.20 (q, J = 7.2Hz, 2H), 4.13–4.01 (m, 4H), 3.67–3.56 (m, 1H), 3.47 (s, 2H), 1.43 (s, 9H), 1.29 (t, J = 7.2Hz, 3H).

[0044] The synthesis of intermediate M2a was as follows: In a 125 mL round-bottom flask, intermediate M1 (3.02 g, 11.13 mmol) was dissolved in acetonitrile (60 mL), and 2-aminopyridine (3.15 g, 33.39 mmol) and carbon tetrabromide (7.38 g, 22.26 mmol) were added. The reaction mixture was stirred overnight at 80 °C, and the reaction was monitored by TLC. After the reaction was completed, the crude product was concentrated by vacuum distillation. A saturated aqueous solution of citric acid (30 mL) was added, and the mixture was extracted twice with ethyl acetate (30 mL x 2). The organic phase was collected, washed with saturated brine, and purified by silica gel column chromatography after vacuum distillation to obtain a yellow oily intermediate compound M2a (2.58 g, yield 61%). 1 H NMR (400MHz, CDCl3) δ9.33(d,J=6.8Hz,1H),7.72(d,J=8.8Hz,1H),7.43(t,J=6.8Hz ,1H),7.03(t,J=6.8Hz,1H),4.50–4.27(m,7H),1.46(s,9H),1.44(t,J=6.8Hz,3H).

[0045] The synthesis of intermediate M3a was as follows: Intermediate M2a (2.58 g, 7.47 mmol) was dissolved in dichloromethane (40 mL) in a 125 mL round-bottom flask, and trifluoroacetic acid (4 mL) was added. The reaction mixture was stirred overnight at room temperature, and the reaction was monitored by TLC. After the reaction was complete, the mixture was concentrated by vacuum distillation to give a yellow oily intermediate compound M3a (1.76 g, 96% yield). 1 H NMR (400MHz, CDCl3) δ9.33(d,J=7.2Hz,1H),7.72(d,J=8.8Hz,1H),7.44(t,J=6.8Hz,1H ),7.01(t,J=6.8,1H),4.43–4.32(m,5H),4.46(q,J=7.2Hz,2H),1.47(t,J=7.2Hz,3H).

[0046] The synthesis of compound A1 was as follows: In a 50 mL round-bottom flask, intermediate M3a (100 mg, 0.41 mmol) and 2-chloroquinoline (80 mg, 0.49 mmol) were dissolved in anhydrous DMF (10 mL), and cesium carbonate (267 mg, 0.82 mmol) was added. The reaction mixture was heated to 110 °C overnight, and the reaction was monitored by TLC. After the reaction was complete, the cesium carbonate was filtered off, the filtrate was concentrated by vacuum distillation, and the crude product was purified by silica gel column chromatography to give a white solid compound A1 (72 mg, yield 62%). 1H NMR (500MHz, CDCl3) δ9.34(d,J=7.0Hz,1H),7.86(d,J=9.0Hz,1H),7.74(d,J=8.5Hz,1H),7. 68(d,J=9.0Hz,1H),7.58(d,J=8.0Hz,1H),7.52(t,J=7.5Hz,1H),7.39(t,J=7.5Hz,1H),7.20 (t,J=7.5Hz,1H),7.00(t,J=7.0Hz,1H),6.66(d,J=9.0Hz,1H),4.69(dd,J=14.5,7.0Hz,1H), 4.63(t,J=7.5Hz,4H),4.45(q,J=7.0Hz,2H),1.47(t,J=7.0Hz,3H),HRMS(ESI-TOF)m / z[M+H] + calcd for C 22 H 20 N4O2393.1659, found 373.1668.

[0047] In this embodiment, the synthetic route of intermediates M5 to M11 (see Figure 14) is as follows:

[0048] The synthesis of intermediate M5 was as follows: 2-quinolinone-4-carboxylic acid (9.45 g, 50.0 mmol) was added to a 125 mL round-bottom flask, followed by careful addition of phosphorus oxychloride (30 mL). The reaction mixture was heated to 110 °C and stirred overnight, with the reaction monitored by TLC. After the reaction was complete, phosphorus oxychloride was removed by vacuum distillation to give a yellow oily intermediate compound M5 (100% yield).

[0049] The synthesis of intermediate M6 was as follows: Dry methanol (40 mL) was slowly added dropwise to cooled intermediate M5 (50.0 mmol) at 0 °C. The reaction mixture was then stirred at room temperature for 2 hours, and the reaction was monitored by TLC. After the reaction was complete, saturated sodium bicarbonate solution was added to the reaction mixture at 0 °C to adjust the pH to 9–10. The aqueous phase was extracted twice with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate. The filtrate was concentrated by vacuum distillation, and the crude product was purified by silica gel column chromatography to give a white solid intermediate compound M6 (5.87 g, 53% yield in two steps). 1 HNMR (400MHz, CDCl3) δ8.73 (d, J = 8.8 Hz, 1H), 8.08 (d, J = 8.4 Hz, 1H), 7.91 (s, 1H), 7.79 (td, J = 6.8, 1.2 Hz, 1H), 7.66 (td, J = 6.8, 1.2 Hz, 1H), 4.05 (s, 3H).

[0050] The synthesis of intermediate 2-chloro-N-methylquinoline-4-carboxamide M7 was performed as follows: Cooled intermediate M5 (50.0 mmol) was dissolved in dry dichloromethane (40 mL), and methylamine hydrochloride (4.05 g, 60.0 mmol) and triethylamine (15.2 g, 150.0 mmol) were added. The reaction mixture was stirred at room temperature for 3 hours, and the reaction was monitored by TLC. After the reaction was complete, saturated sodium bicarbonate solution was added to the reaction mixture at 0 °C to adjust the pH to 9–10. The mixture was extracted twice with dichloromethane, and the organic phase was dried over anhydrous sodium sulfate. The filtrate was filtered and concentrated by vacuum distillation. The crude product was purified by silica gel column chromatography to give a white solid intermediate compound M7 (8.62 g, 78% yield in two steps). 1 HNMR(500MHz,DMSO-d6δ8.84(d,J=4.0Hz,1H),8.15(d,J=8.0Hz,1H),8.02(d,J=8.5Hz,1H), 7.88(td,J=7.0,1.0Hz,1H), 7.71(td,J=7.0,1.0Hz,1H), 7.65(s,1H), 2.87(d,J=4.5Hz,3H).

[0051] The synthesis of intermediate M8 was carried out as follows: using intermediate M5 (1.0 mmol) and 2,2-difluoroethylamine (97.2 mg, 1.2 mmol) as raw materials, and referring to the synthesis method of intermediate M7, a grayish-white solid intermediate compound M8 (60 mg, yield 48%) was obtained.

[0052] 1 H NMR (400MHz, CDCl3) δ8.14(d,J=8.4Hz,1H),8.05(d,J=8.4Hz,1H),7.79(t,J=7.2Hz,1H),7.63 (t,J=7.2Hz,1H),7.46(s,1H),6.41(brs,1H),6.07(tt,J=55.6,3.6Hz,1H),4.01–3.86(m,2H).

[0053] The synthesis of intermediate M9 was carried out as follows: using intermediate M5 (1.0 mmol) and N-methylpiperazine (120 mg, 1.2 mmol) as raw materials, and referring to the synthesis method of intermediate M7, a pale yellow solid intermediate compound M9 (60 mg, yield 48%) was obtained. 1HNMR(400MHz, CDCl3)δ8.06(d,J=8.4Hz,1H),7.83–7.75(m,2H),7.61(t,J=7.6Hz,1H),7.31(s ,1H),4.04–3.84(m,2H),3.77–3.66(m,1H),3.29–3.11(m,3H),2.64–2.51(m,2H),2.33(s,3H).

[0054] The synthesis of intermediate M10 was carried out as follows: using intermediate M5 (1.0 mmol) and tert-butyl 4,7-diazaspiro[2.5]octane-4-carboxylate (254 mg, 1.2 mmol) as raw materials, and referring to the synthesis method of intermediate M7, a pale yellow solid intermediate compound M10 (290 mg, yield 72%) was obtained. 1 H NMR(400MHz, CDCl3)δ8.06(d,J=8.0Hz,1H),7.82–7.73(m,2H),7.60(t,J=7.6Hz,1H),7.31(s,1H),4.17–4.06(m,0.5H),3.92 –3.70(m,2H),3.68–3.51(m,1H),3.43–3.17(m,2H),3.05–2.95(m,0.5H),1.49(s,9H),1.21–0.86(m,3H),0.70–0.36(m,1H).

[0055] The synthesis of intermediate M11 was carried out as follows: using intermediate M5 (1.0 mmol) and N,N-diethylethylenediamine (139 mg, 1.2 mmol) as raw materials, and following the synthesis method of intermediate M7, a pale yellow solid intermediate compound M11 (250 mg, yield 82%) was obtained. 1 HNMR (400MHz, CDCl3) δ8.24(d,J=8.4Hz,1H),8.05(d,J=8.0Hz,1H),7.77(td,J=7.2,1.2Hz,1H),7.61(td,J=7.2,1.2Hz,1H), 7.45(s,1H),6.90(brs,1H),3.60(dd,J=11.2,5.2Hz,2H),2.72(t,J=6.0Hz,2H),2.59(q,J=7.2Hz,4H),1.03(t,J=7.2Hz,6H).

[0056] The synthesis of compound A2 was carried out as follows: using intermediate M3a (100 mg, 0.41 mmol) and intermediate M6 (108 mg, 0.49 mmol) as raw materials, the synthesis method of compound A1 was followed to obtain white solid compound A2 (121 mg, yield 69%).1 HNMR(400MHz, CDCl3) δ9.34(d,J=6.8Hz,1H),8.45(d,J=8.0Hz,1H),7.77(d ,J=8.8Hz,1H),7.69(d,J=8.8Hz,1H),7.56(t,J=7.6Hz,1H),7.41(t,J=8.0H z,1H),7.32–7.24(m,2H),7.02(t,J=6.8Hz,1H),4.79–4.60(m,5H),4.47(q, J=7.2Hz,2H),4.01(s,3H),1.49(t,J=7.2Hz,3H).HRMS(ESI-TOF)m / z:[M+H] + calcd for C 24 H 22 N4O4431.1714, found 431.1715.

[0057] The synthesis of compound A3 was carried out as follows: using intermediate M3a (1.76 g, 7.18 mmol) and intermediate M7 (1.58 g, 7.18 mmol) as raw materials, the synthesis method of compound A1 was followed to obtain a pale yellow solid compound A3 (1.97 g, yield 64%). 1 HNMR(400MHz, CDCl3) δ9.34(d,J=6.8Hz,1H),7.88(d,J=8.4Hz,1H),7.75(brs,1 H),7.69(d,J=8.8Hz,1H),7.52(t,J=8.0Hz,1H),7.42(t,J=8.0Hz,1H),7.20(t,J =7.6Hz,1H),7.03(t,J=6.8Hz,1H),6.59(s,1H),4.70–4.50(m,5H),4.46(q,J=7. 2Hz,2H),3.11(d,J=5.2Hz,3H),1.49(t,J=7.2Hz,3H).HRMS(ESI-TOF)m / z:[M+H] + calcd for C 24 H 23 N5O3430.1874, found430.1888.

[0058] The synthesis of intermediate M4 was as follows: In a 125 mL round-bottom flask, intermediate A3a (1.97 g, 4.59 mmol) was dissolved in methanol / water (30 mL / 10 mL), and sodium hydroxide (550 mg, 13.76 mmol) was added. The reaction mixture was heated to 65 °C and stirred for 3 hours, and the reaction was monitored by TLC. After the reaction was complete, methanol was removed by vacuum distillation to obtain an aqueous solution of the crude product. The pH of the system was adjusted to 4–5 with 5 M hydrochloric acid, and a solid precipitated. The solid was filtered under reduced pressure, the filter cake was washed with water, and dried in air to obtain a white solid intermediate compound M4 (1.51 g, yield 82%). 1 H NMR (400MHz, CH3OD) δ9.33(d,J=6.8Hz,1H),7.88(d,J=8.0Hz,1H),7.75(s,1H),7.68(d,J=9.2Hz,1H),7.52(t,J=7.6Hz,1H),7.46– 7.39(m,1H),7.20(t,J=7.6Hz,1H),7.03(t,J=6.8Hz,1H),6.58(s,1H),4.63–4.61(m,1H),4.56–4.51(m,4H),3.13(d,J=4.8Hz,3H).

[0059] The synthesis of compound A4 was as follows: In a 50 mL round-bottom flask, intermediate M4 (100 mg, 0.25 mmol) was dissolved in anhydrous N,N-dimethylformamide (10 mL). Then, 2,2-difluoroethanol (41 mg, 0.50 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (58 mg, 0.30 mmol), and 4-dimethylaminopyridine (61 mg, 0.50 mmol) were added sequentially. The reaction mixture was stirred overnight at room temperature, and the reaction was monitored by TLC. After the reaction was complete, saturated brine was added, and the mixture was extracted with ethyl acetate. The organic phase was distilled under reduced pressure to obtain a crude product, which was purified by silica gel column chromatography to give a pale yellow solid compound A4 (87 mg, 75% yield). 1 H NMR (500MHz, DMSO-d6) δ9.23(d,J=6.5Hz,1H),8.64(d,J=2.0Hz,1H),7.89(d,J=7.5Hz,1H),7.80(d,J=9.0Hz,1H),7.68–7.59(m,2H),7.56(d,J=6.5 Hz,1H),7.33–7.19(m,2H),6.82(s,1H),6.53(t,J=54.5Hz,1H),4.74–4.6 2(m,3H),4.56(s,2H),4.48(s,2H),2.84(s,3H).HRMS(ESI-TOF)m / z:[M+H] +calcd for C 24 H 21 F2N5O3466.1685,found466.1690.

[0060] The synthesis of compound A5 was carried out as follows: using intermediate M4 (100 mg, 0.25 mmol) and isopropanol (30 mg, 0.5 mmol) as raw materials, and following the synthesis method of compound A4, a pale yellow compound A5 (66 mg, yield 60%) was obtained. 1 H NMR (500MHz, CDCl3) δ9.34(d,J=7.0Hz,1H),7.80(d,J=8.0Hz,1H),7.68(d,J=9.0Hz,1 H),7.63(d,J=8.0Hz,1H),7.48(t,J=7.5Hz,1H),7.41(t,J=7.5Hz,1H),7.13(t,J=7.5 Hz,1H),7.02(t,J=7.0Hz,1H),6.39(s,1H),5.41–5.30(m,1H),4.64–4.55(m,1H),4.5 3–4.40(m,4H),3.11(d,J=4.5Hz,3H),1.46(d,J=6.0Hz,6H).HRMS(ESI-TOF)m / z:[M+H] + calcd for C 25 H 25 N5O3444.2030,found 444.2032.

[0061] The synthesis of compound A6 was carried out as follows: using intermediate M4 (100 mg, 0.25 mmol) and oxetane-3-ol (37 mg, 0.5 mmol) as raw materials, and following the synthesis method of compound A4, a pale yellow compound A6 (77 mg, yield 68%) was obtained. 1HNMR(400MHz, CDCl3)δ9.28(d,J=6.8Hz,1H),7.84(d,J=8.0Hz,1H),7.72(d,J=9.2Hz,1H), 7.65(d,J=8.4Hz,1H),7.57–7.40(m,2H),7.17(t,J=7.6Hz,1H),7.06(t,J=6.8Hz,1H),6.78 (q,J=4.0Hz,1H),6.49(s,1H),5.81–5.69(m,1H),5.06(t,J=7.2Hz,2H),4.84(t,J=7.2Hz, 2H),4.72–4.61(m,1H),4.59–4.48(m,4H),3.10(d,J=4.8Hz,3H).HRMS(ESI-TOF)m / z:[M+H] + calcd for C 25 H 23 N5O4458.1823,found 458.1826.

[0062] The synthesis of compound A7 was carried out as follows: using intermediate M4 (100 mg, 0.25 mmol) and tetrahydropyran-4-ol (51 mg, 0.5 mmol) as raw materials, and following the synthesis method of compound A4, a pale yellow compound A7 (83 mg, yield 69%) was obtained.

[0063] 1 H NMR(500MHz, CDCl3)δ9.32(d,J=6.0Hz,1H),7.82(d,J=8.0Hz,1H),7.77–7.61(m,2H),7.50(t, J=7.5Hz,1H),7.44(t,J=7.5Hz,1H),7.15(t,J=6.5Hz,1H),7.06(t,J=6.0Hz,1H),6.44(s,1H) ,5.28(s,1H),4.66–4.58(m,1H),4.57–4.41(m,4H),4.13–4.01(m,2H),3.64(t,J=10.0Hz,2H) ,3.11(d,J=3.0Hz,3H),2.18–2.06(m,2H),1.98–1.81(m,2H).HRMS(ESI-TOF)m / z:[M+H]+calcd for C 27 H 27 N5O4486.2136,found 486.2135.

[0064] The synthesis of compound A8 was carried out as follows: using intermediate M3a (100 mg, 0.41 mmol) and intermediate M8 (133 mg, 0.49 mmol) as raw materials, the synthesis method of compound A1 was followed to obtain a pale yellow solid compound A8 (98 mg, yield 50%). 1 HNMR (400MHz, CDCl3) δ9.34(d,J=7.2Hz,1H),7.79(d,J=8.0Hz,1H),7.69(d,J=8.8Hz,1H),7.65(d,J =8.4Hz,1H),7.51(t,J=7.2Hz,1H),7.42(t,J=7.2Hz,1H),7.16(t,J=7.2Hz,1H),7.08(t,J=6.0Hz,1H ),7.03(td,J=6.8,0.8Hz,1H),6.49(s,1H),6.10(tt,J=56.0,4.0Hz,1H),4.70–4.60(m,1H),4.56–4. 50(m,4H),4.46(q,J=7.2Hz,2H),4.01–3.86(m,2H),1.48(t,J=7.2Hz,3H).HRMS(ESI-TOF)m / z:[M+H] + calcd for C 25 H 23 F2N5O3480.1842, found 480.1845.

[0065] The synthesis of compound A9 was carried out as follows: using intermediate M3a (100 mg, 0.41 mmol) and intermediate M9 (142 mg, 0.49 mmol) as raw materials, the synthesis method of compound A2 was followed to obtain a pale yellow solid compound A9 (134 mg, yield 66%). 1HNMR(500MHz, CDCl3)δ9.33(d,J=7.0Hz,1H),7.76(d,J=8.5Hz,1H),7.69(d,J=9.0Hz,1H),7.58–7.50(m,2 H),7.41(td,J=7.0,1.0Hz,1H),7.22(t,J=7.5Hz,1H),7.02(td,J=7.0,1.0Hz,1H),6.57(s,1H),4.74–4.6 2(m,3H),4.62–4.55(m,2H),4.45(q,J=7.0Hz,2H),4.04–3.97(m,1H),3.90–3.80(m,1H),3.22(t,J=5.0Hz ,2H),2.62–2.49(m,2H),2.31(s,3H),2.27–2.21(m,2H),1.47(t,J=7.0Hz,3H).HRMS(ESI-TOF)m / z:[M+H] + calcd for C 28 H 30 N6O3499.2452, found 499.2447.

[0066] The synthesis of compound A10 was carried out as follows: intermediate M3a (100 mg, 0.41 mmol) and intermediate M10 (196 mg, 0.49 mmol) were used as raw materials, and the synthesis method of compound A2 was followed. The protecting group was removed by trifluoroacetic acid to obtain a pale yellow solid compound A10 (135 mg, 65% yield in 2 steps). 1 H NMR (500MHz, CDCl3) δ9.35(d,J=7.0Hz,1H),7.75(dd,J=11.5,9.0Hz,1H),7.70(d,J=9.0Hz,1H),7.64–7.52(m ,2H),7.42(t,J=8.0Hz,1H),7.25(t,J=8.0Hz,1H),7.03(t,J=7.0Hz,1H),6.58(d,J=18.5Hz,1H),4.79–4.56(m ,5H),4.47(q,J=7.0Hz,2H),4.01–3.61(m,2H),3.23–3.14(m,1H),3.12–2.98(m,2H),2.80(t,J=4.5Hz,1H),1 .49(td,J=7.5,1.5Hz,3H),0.92–0.69(m,2H),0.60–0.48(m,1H),0.33–0.10(m,1H).HRMS(ESI-TOF)m / z:[M+H] + calcd for C 29 H30 N6O3511.2452, found 511.2477.

[0067] The synthesis of compound A11 was as follows: In a 50 mL round-bottom flask, intermediates M11 (305 mg, 1.0 mmol) and M3a (245 mg, 1.0 mmol) were dissolved in anhydrous DMF (20 mL), and cesium carbonate (652 mg, 2.0 mmol) was added. The reaction mixture was heated to 110 °C and reacted overnight, with the reaction monitored by TLC. After the reaction was complete, the cesium carbonate was filtered off, and the resulting filtrate was concentrated by vacuum distillation. The crude product was purified by silica gel column chromatography to give a yellow solid compound A11 (272 mg, yield 53%). 1 H NMR (400MHz, CDCl3) δ9.35(d,J=6.8Hz,1H),7.99(d,J=8.4Hz,1H),7.75(d,J=8.4Hz,1H),7.69(d,J=8.8H z,1H),7.54(td,J=7.2,1.2Hz,1H),7.42(td,J=7.2,1.2Hz,1H),7.23(t,J=8.0Hz,1H),7.03(td,J=6.8,1. 2Hz,1H),6.84–6.77(m,1H),6.76(s,1H),4.75–4.67(m,1H),4.65–4.59(m,4H),4.46(q,J=7.2Hz,2H),3.6 4–3.55(m,2H),2.71(t,J=6.0Hz,2H),2.58(q,J=7.2Hz,4H),1.48(t,J=7.2Hz,3H),1.03(t,J=7.2Hz,6H).

[0068] The synthesis of compound A12 was performed as follows: Following the synthetic method for compound A1, 2-chloroquinoline was reacted with intermediate M3b to yield a pale yellow solid compound A12 (142 mg, yield 43%). 1 HNMR (400MHz, CDCl3) δ9.22(d,J=6.8Hz,1H),7.90(d,J=8.8Hz,1H),7.77(d,J=8.4Hz,1H),7.62(d,J=7.6Hz,1H),7.55(t,J=7.2Hz,1H),7.23 (t,J=7.6Hz,2H),6.94(t,J=6.8Hz,1H),6.70(d,J=8.8Hz,1H),4.77–4.59(m,5H),4.47(q,J=7.2Hz,2H),2.65(s,3H),1.50(t,J=7.2Hz,3H).

[0069] In this embodiment, the synthetic routes for compounds A13 to A20 (see Figure 15) are as follows:

[0070] The synthesis of compound A13 was performed as follows: In a 50 mL round-bottom flask, intermediate M4 (100 mg, 0.25 mmol) was dissolved in anhydrous N,N-dimethylformamide (10 mL), followed by the sequential addition of methylamine hydrochloride (20 mg, 0.30 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (114 mg, 0.30 mmol), and diisopropylethylamine (97 mg, 0.75 mmol). The reaction mixture was stirred overnight at room temperature, and the reaction was monitored by TLC. After the reaction was complete, the mixture was concentrated by vacuum distillation, and the crude product was purified by silica gel column chromatography to give a white solid compound A13 (54 mg, yield 52%). 1 HNMR (500MHz, CD3OD) δ8.85(d,J=7.0Hz,1H),7.89(d,J=8.0Hz,1H),7.69(d,J=8. 0Hz,1H),7.59–7.52(m,2H),7.42(td,J=7.0,1.5Hz,1H),7.25(td,J=7.5,1.0Hz, 1H),7.01(td,J=7.0,1.0Hz,1H),6.77(s,1H),4.62(t,J=8.0Hz,2H),4.58–4.50( m,1H),4.43(t,J=8.0Hz,2H),3.00(s,3H),2.98(s,3H).HRMS(ESI-TOF)m / z:[M+H] + calcd for C 23 H 22 N6O2415.1877, found415.1875.

[0071] The synthesis of compound A14 was carried out as follows: using intermediate M4 (100 mg, 0.25 mmol) and 2-methoxyethylamine (23 mg, 0.30 mmol) as raw materials, and following the synthesis method of compound A13, a pale yellow solid compound A14 (54 mg, yield 47%) was obtained. 1H NMR(500MHz,CD3OD)δ8.83(d,J=7.0Hz,1H),7.89(dd,J=8.5,1.0Hz,1H),7.69(d,J=8 .5Hz,1H),7.60–7.52(m,2H),7.43(td,J=7.0,1.0Hz,1H),7.25(td,J=7.0,1.0Hz,1H) ,7.02(td,J=7.0,1.0Hz,1H),6.78(s,1H),4.67–4.61(m,2H),4.62–4.56(m,1H),4.49 –4.42(m,2H),3.65–3.63(m,4H),3.44(s,3H),2.98(s,3H).HRMS(ESI-TOF)m / z:[M+H] + calcd for C 25 H 26 N6O3459.2139,found 459.2139.

[0072] The synthesis of compound A15 was carried out as follows: using intermediate M4 (100 mg, 0.25 mmol) and 2,2-difluoroethylamine (24 mg, 0.30 mmol) as raw materials, and following the synthesis method of compound A13, a pale yellow solid compound A15 (66 mg, yield 57%) was obtained. 1 H NMR (500MHz, DMSO-d6) δ8.89(d,J=7.0Hz,1H),8.65(q,J=4.5Hz,1H),8.51(t,J=5.5Hz,1H),7.89(d, J=8.0Hz,1H),7.65(d,J=9.0Hz,1H),7.62(d,J=8.0Hz,1H),7.55(t,J=7.5Hz,1H),7.43(t,J=7.5Hz,1 H),7.24(t,J=7.5Hz,1H),7.07(t,J=7.0Hz,1H),6.81(s,1H),6.25(tt,J=56.0,3.5Hz,1H),4.63–4. 51(m,3H),4.38(t,J=6.5Hz,2H),3.84–3.73(m,2H),2.84(d,J=4.5Hz,3H).HRMS(ESI-TOF)m / z:[M+H] + calcd for C 24 H 22 F2N6O2465.1845, found 465.1842.

[0073] The synthesis of compound A16 was carried out as follows: using intermediate M4 (100 mg, 0.25 mmol) and cyclopropylamine (17 mg, 0.30 mmol) as raw materials, and following the synthesis method of compound A13, a pale yellow solid compound A16 (66 mg, yield 60%) was obtained. 1 H NMR (400MHz, DMSO-d6) δ8.86(d,J=6.8Hz,1H),8.62(d,J=4.4Hz,1H),8.27(d,J=3.6Hz,1H),7.89(d,J =7.6Hz,1H),7.66–7.59(m,2H),7.55(td,J=6.8,1.2Hz,1H),7.39(td,J=6.8,1.2Hz,1H),7.24(td,J= 7.2,0.8Hz,1H),7.03(td,J=6.8,0.8Hz,1H),6.81(s,1H),4.55–4.45(m,3H),4.40–4.30(m,2H),2.96 –2.87(m,1H),2.84(d,J=4.4Hz,3H),0.82–0.74(m,2H),0.71–0.63(m,2H).HRMS(ESI-TOF)m / z:[M+H] + calcd for C 25 H 24 N6O2441.2034, found 441.2036.

[0074] The synthesis of compound A17 was carried out as follows: using intermediate M4 (100 mg, 0.25 mmol) and N,N-diethylethylenediamine (35 mg, 0.30 mmol) as raw materials, and following the synthesis method of compound A13, a pale yellow solid compound A17 (82 mg, yield 66%) was obtained. 1 H NMR (500MHz, CD3OD) δ8.92(d,J=7.0Hz,1H),7.90(d,J=8.5Hz,1H),7.70(d,J=8.5Hz,1H),7.58(d ,J=9.0Hz,1H),7.55(d,J=7.5Hz,1H),7.45(t,J=7.5Hz,1H),7.26(t,J=7.5Hz,1H),7.04(t,J=7. 0Hz,1H),6.79(s,1H),4.68–4.59(m,3H),4.47(t,J=5.5Hz,2H),3.61(t,J=7.0Hz,2H),2.98(s,3 H),2.87(t,J=7.0Hz,2H),2.77(q,J=7.0Hz,4H),1.15(t,J=7.0Hz,6H).HRMS(ESI-TOF)m / z:[M+H]+ calcd for C 28 H 33 N7O2500.2768, found 500.2769.

[0075] The synthesis of compound A18 was carried out as follows: using intermediate M4 (100 mg, 0.25 mmol) and N,N-diethyl-N'-methylethylenediamine (39 mg, 0.30 mmol) as raw materials, and following the synthesis method of compound A13, a pale yellow solid compound A18 (83 mg, yield 65%) was obtained. 1 H NMR (500MHz, CDCl3) δ8.38(d,J=6.5Hz,1H),7.89(d,J=8.5Hz,1H),7.65(d,J=8.0Hz,1H),7.58(d,J=9.0H z,1H),7.50(t,J=7.5Hz,1H),7.25(t,J=8.5Hz,1H),7.19(t,J=7.5Hz,1H),6.95(q,J=4.0Hz,1H),6.86(t, J=6.5Hz,1H),6.56(s,1H),4.54–4.40(m,4H),4.24–4.14(m,1H),3.86–3.39(m,2H),3.09(s,3H),3.06(d ,J=4.5Hz,3H),2.77–2.57(m,2H),2.45(q,J=7.0Hz,4H),0.91(t,J=7.0Hz,6H).HRMS(ESI-TOF)m / z:[M+H] + calcd for C 29 H 35 N7O2514.2925, found 514.2922.

[0076] The synthesis of intermediate M13 was as follows: In a 125 mL round-bottom flask, A11 (272 mg, 0.53 mmol) was dissolved in methanol / water (20 mL / 5 mL), and sodium hydroxide (64 mg, 1.59 mmol) was added. The reaction mixture was heated to 65 °C and stirred for 3 hours, and the reaction was monitored by TLC. After the reaction was complete, methanol was removed by vacuum distillation to obtain an aqueous solution of the crude product. The pH of the system was adjusted to 4–5 with 5 M hydrochloric acid, and a solid precipitated. The solid was filtered under reduced pressure, the filter cake was washed with water, and dried in air to obtain a pale yellow solid M12 (236 mg, yield 92%).

[0077] The synthesis of compound A19 was carried out as follows: using intermediate M12 (100 mg, 0.21 mmol) and dimethylamine hydrochloride (20 mg, 0.25 mmol) as raw materials, and following the synthesis method of compound A13, a pale yellow solid compound A19 (98 mg, yield 77%) was obtained. 1 HNMR (500MHz, DMSO-d6) δ = 8.61 (t, J = 5.5Hz, 1H), 8.40 (d, J = 7.0Hz, 1H), 7.96 (d, J = 8.5Hz, 1H), 7.66–7 .59(m,2H),7.56(t,J=7.5Hz,1H),7.37(t,J=7.5Hz,1H),7.25(t,J=7.5Hz,1H),7.00(t,J=7.0Hz,1H), 6.78(s,1H),4.55(t,J=8.0Hz,2H),4.38(t,J=8.0Hz,2H),4.25-4.16(m,1H),3.40-3.34(m,2H),3.05( s,6H),2.61(t,J=6.5Hz,2H),2.54(q,J=7.0Hz,4H),0.99(t,J=7.0Hz,6H).HRMS(ESI-TOF)m / z::[M+H] + calcd for C 29 H 35 N7O2514.2925, found 514.2944.

[0078] The synthesis of compound A20 was carried out as follows: using intermediate M12 (100 mg, 0.21 mmol) and methoxymethylamine (18 mg, 0.30 mmol) as raw materials, and following the synthesis method of compound A13, a pale yellow solid compound A20 (95 mg, yield 72%) was obtained. 1HNMR (400MHz, CDCl3) δ8.63(d,J=6.8Hz,1H),8.00(d,J=8.4Hz,1H),7.74(d,J=8.4Hz,1H),7.64(d,J=9. 2Hz,1H),7.54(t,J=7.6Hz,1H),7.31(t,J=7.6Hz,1H),7.23(t,J=7.6Hz,1H),6.99–6.92(m,1H),6.90(t ,J=6.8Hz,1H),6.73(s,1H),4.66–4.48(m,4H),4.40–4.30(m,1H),3.62–3.55(m,2H),3.52(s,3H),3.45 (s,3H),2.70(t,J=6.0Hz,2H),2.57(q,J=7.2Hz,4H),1.03(t,J=7.2Hz,6H).HRMS(ESI-TOF)m / z::[M+Na] + calcd for C 29 H 35 N7O3552.2694, found 552.2693.

[0079] In this embodiment, the synthetic routes for compounds B1 to B6 (see Figure 16) are as follows:

[0080] The synthesis of intermediates M14a-f was as follows: Intermediate M1 (1.08 mg, 4.0 mmol) was dissolved in acetonitrile (20 mL) in a 125 mL round-bottom flask. Various R3- or R4-substituted 2-aminopyridine P-2-A derivatives (6.0 mmol) and carbon tetrabromide (1.99 g, 6.0 mmol) were added. The reaction mixture was stirred overnight at 80 °C, and the reaction was monitored by TLC. After the reaction was complete, the mixture was concentrated under reduced pressure by distillation. The crude product was purified by silica gel column chromatography to obtain the yellow oily intermediate compounds M14a-f.

[0081] Intermediate M14a, yield 67%. 1 H NMR (400MHz, CDCl3) δ9.33(d,J=6.8Hz,1H),7.72(d,J=8.8Hz,1H),7.43(d,J=8.0Hz ,1H),7.03(d,J=6.8Hz,1H),4.50–4.27(m,7H),1.46(s,9H),1.44(d,J=6.8Hz,3H).

[0082] Intermediate M14b, yield 50%. 1H NMR(500MHz, CDCl3)δ9.18(d,J=7.0Hz,1H),7.47(s,1H),6.86(dd,J=7.0,1.5Hz,1H),4.44–4.3 7(m,4H),4.36–4.28(m,2H),4.09–4.03(m,1H),2.46(s,3H),1.45(s,9H),1.43(t,J=7.5Hz,3H).

[0083] Intermediate M14c, yield 19%. 1 H NMR (500MHz, CDCl3) δ9.34 (dd, J=7.5, 5.5Hz, 1H), 7.34 (dd, J=9.0, 2.5Hz, 1H), 6.89 (td, J= 7.5, 2.5Hz, 1H), 4.46–4.37 (m, 4H), 4.36–4.28 (m, 3H), 1.46 (s, 9H), 1.44 (t, J = 7.5Hz, 3H).

[0084] Intermediate M14d, yield 41%. 1 H NMR(500MHz, CDCl3)δ9.15(s,1H),7.61(d,J=9.0Hz,1H),7.28(dd,J=9.0,1.5Hz,1H),4.45–4.3 7(m,4H),4.35–4.28(m,3H),4.09–4.02(m,1H),2.40(s,3H),1.45(s,9H),1.44(t,J=7.5Hz,3H).

[0085] Intermediate M14e, yield 22%. 1 H NMR(400MHz, CDCl3) δ9.32(dd,J=4.8,2.4Hz,1H),7.69(dd,J=10.0,5.2Hz,1H),7.35(td,J =7.6,2.4Hz,1H),4.49–4.37(m,4H),4.37–4.27(m,3H),1.46(s,9H),1.45(t,J=7.2Hz,3H).

[0086] Intermediate M14f, yield 35%. 1H NMR (400MHz, CDCl3) δ8.98 (d, J = 2.4Hz, 1H), 7.60 (d, J = 10.0Hz, 1H), 7.21 (dd, J = 9.6, 2.4Hz, 1H), 4.50–4.36 (m, 4H), 4.35–4.26 (m, 3H), 3.89 (s, 3H), 1.45 (s, 9H), 1.44 (t, J = 7.2Hz, 3H).

[0087] The synthesis of intermediates M15a-f was as follows: In a 50 mL round-bottom flask, intermediate 14a-f (1.0 mmol) was dissolved in methanol (10 mL) and water (2 mL), and sodium hydroxide (120 mg, 3.0 mmol) was added. The reaction mixture was stirred overnight at 60 °C, and the reaction was monitored by TLC. After the reaction was complete, the crude product was distilled under reduced pressure to obtain an aqueous solution. The pH was adjusted to 3-4 with saturated citric acid solution, and the mixture was extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate and concentrated to obtain white or pale yellow solid intermediates M15a-f (yield 76-88%), which did not require purification and were used directly in the next reaction.

[0088] The synthesis of intermediates M16a-f was as follows: In a 50 mL round-bottom flask, intermediate compound M15a-f (0.5 mmol) was dissolved in anhydrous N,N-dimethylformamide (10 mL). Dimethylamine hydrochloride (DMA·HCl) (1.0 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (228 mg, 0.6 mmol), and diisopropylethylamine (194 mg, 1.5 mmol) were added sequentially. The reaction mixture was stirred overnight at room temperature, and the reaction was monitored by TLC. After the reaction was complete, the crude product was concentrated by vacuum distillation. Saturated brine (30 mL) was added, and the mixture was extracted twice with ethyl acetate. The extract was dried over anhydrous sodium sulfate, filtered, and the crude product was concentrated by vacuum distillation. The crude product was purified by silica gel column chromatography to obtain pale yellow or white solid intermediate compounds M16a-f.

[0089] Intermediate M16a, white solid, yield 92%. 1 H NMR (400MHz, CDCl3) δ9.33(d,J=6.8Hz,1H),7.72(d,J=8.0Hz,1H),7.43(d,J=8.0Hz,1H) ,7.03(d,J=6.8Hz,1H),4.45–4.26(m,4H),3.97–3.84(m,1H),3.09(s,6H),1.46(s,9H).

[0090] Intermediate M16b, white solid, yield 84%. 1H NMR (400MHz, CDCl3) δ8.27 (d, J=6.8Hz, 1H), 7.40 (s, 1H), 6.71 (dd, J=6.8, 1.6Hz, 1H),4.40–4.21(m,4H),3.96–3.86(m,1H),3.09(s,6H),2.42(s,3H),1.45(s,9H).

[0091] Intermediate M16c, pale yellow solid, yield 89%. 1 H NMR (400MHz, CDCl3) δ8.42 (dd, J=7.6, 5.6Hz, 1H), 7.29 (dd, J=9.6, 2.4Hz, 1H), 6.76 (t d,J=7.2,2.4Hz,1H),4.39–4.19(m,4H),3.94–3.84(m,1H),3.10(s,6H),1.46(s,9H).

[0092] Intermediate M16d, white solid, yield 98%. 1 HNMR(400MHz, CDCl3)δ8.19(s,1H),7.55(d,J=9.2Hz,1H),7.14(dd,J=9.2,1.6Hz, 1H),4.38–4.20(m,4H),3.98–3.85(m,1H),3.10(s,6H),2.33(s,3H),1.45(s,9H).

[0093] Intermediate M16e, pale yellow solid, 91% yield. 1 H NMR (400MHz, CDCl3) δ8.38 (dd, J=4.4, 2.4Hz, 1H), 7.62 (dd, J=10.0, 5.2Hz, 1H), 7.23 ( td,J=8.0,2.4Hz,1H),4.37–4.21(m,4H),3.98–3.85(m,1H),3.10(s,6H),1.46(s,9H).

[0094] Intermediate M16f, white solid, yield 88%. 1 HNMR(400MHz, CDCl3) δ7.98(d,J=2.0Hz,1H),7.53(d,J=10.0Hz,1H),7.08(dd,J=9.6,2 .4Hz,1H),4.34–4.23(m,4H),3.95–3.84(m,1H),3.82(s,3H),3.11(s,6H),1.45(s,9H).

[0095] The synthesis of intermediates M17a-f was as follows: In a 50 mL round-bottom flask, compound M16a-f (0.5 mmol) was dissolved in methanol (5 mL), and concentrated hydrochloric acid (0.5 mL) was added. The reaction mixture was stirred overnight at room temperature, and the reaction was monitored by TLC. After the reaction was complete, the mixture was concentrated by vacuum distillation to obtain pale yellow solid intermediates M17a-f.

[0096] Intermediate M17a, yield 95%. 1 HNMR (400MHz, CD3OD) δ8.56(d,J=6.8Hz,1H),7.74(d,J=8.0Hz,1H),7.44(d,J=8.0 Hz, 1H), 7.07 (d, J = 6.8 Hz, 1H), 4.75–4.53 (m, 1H), 4.45–4.33 (m, 4H), 3.11 (s, 6H).

[0097] Intermediate M17b, yield 91%. 1 HNMR(400MHz,CD3OD)δ8.57(d,J=6.8Hz,1H),7.85(s,1H),7.46(dd,J=6.8, 1.2Hz,1H),4.74–4.64(m,1H),4.63–4.41(m,4H),3.14(s,6H),2.64(s,3H).

[0098] Intermediate M17c, yield 96%. 1 H NMR (400MHz, CD3OD) δ8.82 (dd, J=7.6, 5.2Hz, 1H), 7.94 (dd, J=8.0, 2.4Hz, 1H), 7.58(td,J=7.6,2.4Hz,1H),4.78–4.66(m,1H),4.65–4.45(m,4H),3.18(s,6H).

[0099] Intermediate M17d, yield 90%. 1 H NMR (500MHz, CD3OD) δ8.15(s,1H),7.57(d,J=9.0Hz,1H),7.36(dd,J=9.0,1.5Hz,1H),4.47–4.34(m,5H),3.09(s,6H),2.37(s,3H).

[0100] Intermediate M17e, yield 96%. 1H NMR (400MHz, CD3OD) δ8.78 (dd, J=7.6, 4.8Hz, 1H), 7.76 (dd, J=7.6, 2.4Hz, 1H), 7.58(td,J=7.6,2.4Hz,1H),4.72–4.63(m,1H),4.67–4.44(m,4H),3.15(s,6H).

[0101] Intermediate M17f, yield 94%. 1 HNMR (400MHz, CD3OD) δ8.19 (d, J=2.0Hz, 1H), 7.96 (d, J=10.0Hz, 1H), 7.84 (dd, J= 10.0,2.4Hz,1H),4.72–4.62(m,1H),4.61–4.41(m,4H),3.97(s,3H),3.16(s,6H).

[0102] In this embodiment, the synthesis of compounds B1–B6 was performed as follows: Intermediates M17a-f (0.25 mmol) and M7 (66 mg, 0.30 mmol) were dissolved in anhydrous DMF (10 mL) in a 50 mL flask, and cesium carbonate (244 mg, 0.75 mmol) was added. The reaction mixture was heated to 110 °C and reacted overnight, with the reaction monitored by TLC. After the reaction was complete, the cesium carbonate was removed by filtration, and the filtrate was distilled under reduced pressure to obtain crude products. These crude products were purified by silica gel column chromatography to yield pale yellow or white solid compounds B1–B6, respectively.

[0103] Compound B1, 75 mg, pale yellow solid, yield 77%. 1 H NMR (500MHz, CDCl3) δ8.37 (d, J = 6.0 Hz, 1H), 7.85 (d, J = 7.5 Hz, 1H), 7.61 (d, J = 8. 0Hz,1H),7.57(d,J=9.0Hz,1H),7.48(t,J=7.0Hz,1H),7.32(s,1H),7.26(t,J=8 .0Hz,1H),7.16(t,J=7.0Hz,1H),6.86(t,J=6.0Hz,1H),6.46(s,1H),4.49–4.33 (m,4H),4.10–4.01(m,1H),3.11(s,6H),3.05(s,3H).HRMS(ESI-TOF)m / z::[M+H] + calcd for C 24 H 24 N6O2429.2034, found 429.2033.

[0104] Compound B2, 38 mg, white solid, yield 69%.1 H NMR (500MHz, CDCl3) δ8.27(d,J=7.0Hz,1H),7.88(d,J=7.5Hz,1H),7.63(d,J=8.5Hz ,1H),7.50(td,J=7.0,1.0Hz,1H),7.33(s,1H),7.18(td,J=7.0,1.0Hz,1H),6.91(q, J=4.5Hz,1H),6.70(dd,J=7.0,1.5Hz,1H),6.53(s,1H),4.50–4.37(m,4H),4.13–4.0 4(m,1H),3.11(s,6H),3.07(d,J=5.0Hz,3H),2.39(s,3H).HRMS(ESI-TOF)m / z:[M+H] + calcd for C 25 H 26 N6O2443.2190, found 443.2189.

[0105] Compound B3, 50 mg, pale yellow solid, yield 72%. 1 H NMR (500MHz, CDCl3) δ8.41(dd,J=7.5,5.5Hz,1H),7.87(d,J=8.0Hz,1H),7.64(d,J=8.0Hz,1H),7.51(d,J=7.0Hz,1H),7.24–7.16(m,2H),6.87(q,J=5. 0Hz,1H),6.75(td,J=7.0,2.5Hz,1H),6.51(s,1H),4.50–4.35(m,4H),4.12 –3.97(m,1H),3.13(s,6H),3.07(d,J=5.0Hz,3H).HRMS(ESI-TOF)m / z:[M+H] + calcd for C 24 H 23 N6O2F447.1939, found 447.1933.

[0106] Compound B4, 49 mg, white solid, yield 75%. 1H NMR(500MHz, CDCl3)δ8.19(s,1H),7.87(d,J=8.0Hz,1H),7.63(d,J=8.0Hz,1H),7.53–7.45(m,2H),7.17(td,J=7.5,1.0Hz,1H),7.12(dd,J=9.5 ,1.5Hz,1H),6.98(q,J=4.5Hz,1H),6.51(s,1H),4.49–4.35(m,4H),3.12(s,6H),3.06(d,J=5.0Hz,3H),2.32(s,3H).HRMS(ESI-TOF)m / z:[M+H] + calcd for C 25 H 26 N6O2443.2190, found443.2193.

[0107] Compound B5, 52 mg, pale yellow solid, yield 66%. 1 H NMR (400MHz, DMSO) δ8.66 (q, J=4.4Hz, 1H), 8.51 (dd, J=4.4, 2.4Hz, 1H), 7.91 (d, J=8.0Hz, 1H ),7.70(dd,J=10.0,5.2Hz,1H),7.63(d,J=8.0Hz,1H),7.56(td,J=7.2,0.8Hz,1H),7.48(td ,J=8.0,2.4Hz,1H),7.25(t,J=7.2Hz,1H),6.82(s,1H),4.55(t,J=8.0Hz,2H),4.37(t,J=7. 2Hz,2H),4.26–4.15(m,1H),3.05(s,6H),2.85(d,J=4.4Hz,3H).HRMS(ESI-TOF)m / z:[M+Na] + calcd for C 24 H 23 N6O2F 469.1759,found469.1770.

[0108] Compound B6, 82 mg, white solid, yield 80%. 1H NMR (500MHz, CDCl3) δ7.99(d,J=2.0Hz,1H),7.90(d,J=8.0Hz,1H),7.68(d,J=8.0Hz ,1H),7.52(t,J=7.0Hz,1H),7.48(d,J=9.5Hz,1H),7.22(t,J=7.0Hz,1H),7.06(dd, J=9.5,2.5Hz,1H),6.60(s,1H),6.54(q,J=4.5Hz,1H),4.56–4.42(m,4H),4.16–4.0 6(m,1H),3.82(s,3H),3.15(s,6H),3.08(d,J=5.0Hz,3H).HRMS(ESI-TOF)m / z:[M+H] + calcd for C 25 H 26 N6O3459.2139, found 459.2142.

[0109] In this embodiment, the synthetic routes for compounds B7 to B22 (see Figure 17) are as follows:

[0110] The synthesis of intermediate M18 was as follows: In a 125 mL round-bottom flask, intermediates M17e (2.0 g, 7.62 mmol) and M6 (2.03 g, 9.15 mmol) were dissolved in anhydrous DMF (30 mL), and cesium carbonate (4.96 g, 15.24 mmol) was added. The reaction mixture was heated to 110 °C overnight, and the reaction was monitored by TLC. After the reaction was complete, the cesium carbonate was removed by filtration, and the filtrate was distilled under reduced pressure to obtain the crude product, which was purified by silica gel column chromatography to give a white solid compound M18 (1.64 g, yield 48%). 1 HNMR(500MHz, CDCl3)δ8.47(d,J=8.5Hz,1H),8.41(dd,J=4.0,2.5Hz,1H),7.78(d,J=8.5Hz,1H),7.62–7.55(m,2H),7.31(td,J=7.5,1.0Hz ,1H),7.21(td,J=7.5,2.0Hz,1H),7.17(s,1H),4.62(t,J=8.0Hz,2H),4.57(t,J=7.0Hz,2H),4.24–4.15(m,1H),4.01(s,3H),3.16(s,6H). 13C NMR(126MHz, CDCl3)δ166.89,162.69,157.81,153.40(d,J=237.8Hz),149.01,148.20(d,J=2.1Hz),144.08,136.40,129.88,126.94,1 25.49, 123.38, 119.92, 118.41 (d, J = 25.4Hz), 117.54 (d, J = 8.9Hz), 116.65 (d, J = 2.0Hz), 113.78, 113.45, 110.99, 56.26, 52.58, 28.55.

[0111] The synthesis of intermediate M19 was as follows: In a 125 mL round-bottom flask, intermediate M18 (1.64 g, 3.66 mmol) was dissolved in methanol (30 mL) and water (6 mL), and sodium hydroxide (440 mg, 11.0 mmol) was added. The reaction mixture was stirred overnight at 60 °C, and the reaction was monitored by TLC. After the reaction was complete, the crude product was distilled under reduced pressure to obtain an aqueous solution. The pH was adjusted to 3-4 with the addition of saturated citric acid solution, and a solid precipitated. The solid was filtered, the filter cake was washed with water, and dried in air to obtain a white solid compound M19 (1.46 g, 92% yield).

[0112] The synthesis of compounds B7, B8, B10, B11, and B15 was as follows: In a 50 mL flask, compound M19 (100 mg, 0.23 mmol) was dissolved in anhydrous N,N-dimethylformamide (10 mL). The corresponding amine compounds (0.28 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (105 mg, 0.28 mmol), and diisopropylethylamine (89 mg, 0.69 mmol) were added sequentially. The reaction mixture was stirred overnight at room temperature, and the reaction was monitored by TLC. After the reaction was complete, the crude product was concentrated by vacuum distillation. Saturated brine (30 mL) was added, and the mixture was extracted twice with ethyl acetate. The extract was dried over anhydrous sodium sulfate, filtered, and concentrated by vacuum distillation. The crude product was purified by silica gel column chromatography to obtain pale yellow or white solid compounds B7, B8, B10, B11, and B15, respectively.

[0113] Compound B7, 72 mg, pale yellow solid, yield 56%. 1H NMR (400MHz, DMSO) δ8.61(t,J=5.6Hz,1H),8.51(dd,J=4.4,2.4Hz,1H),7.97(d,J=8.0Hz,1H),7.70(dd,J=9.6 ,5.2Hz,1H),7.63(d,J=8.0Hz,1H),7.56(td,J=7.2,1.2Hz,1H),7.48(td,J=8.0,2.4Hz,1H),7.25(td,J=7.2,1 .2Hz,1H),6.78(s,1H),4.55(t,J=8.0Hz,2H),4.36(t,J=7.2Hz,2H),4.26–4.15(m,1H),3.48–3.38(m,2H),3.0 5(s,6H),2.61(t,J=6.8Hz,2H),2.54(dd,J=14.4,7.2Hz,4H),0.99(t,J=7.2Hz,6H).HRMS(ESI-TOF)m / z:[M+H] + calcd for C 29 H 34 N7O2F 532.2831,found532.2860.

[0114] Compound B8, 62 mg, pale yellow solid, yield 57%. 1 H NMR (400MHz, DMSO) δ8.65(t,J=5.6Hz,1H),8.50(dd,J=4.8,2.4Hz,1H),7.93(d,J=7.6Hz,1H),7.70( dd,J=10.0,5.2Hz,1H),7.63(d,J=8.0Hz,1H),7.56(td,J=6.8,1.2Hz,1H),7.48(td,J=8.0,2.0Hz,1H ),7.26(t,J=8.0,1.2Hz,1H),6.79(s,1H),4.55(t,J=8.0Hz,2H),4.37(t,J=8.0Hz,2H),4.26–4.14( m,1H),3.52–3.41(m,2H),3.05(s,6H),2.46(t,J=6.8Hz,2H),2.22(s,6H).HRMS(ESI-TOF)m / z:[M+H] + calcd for C 27 H 30 N7O2F 504.2518, found 504.2544.

[0115] Compound B10, 51 mg, pale yellow solid, yield 48%. 1HNMR(500MHz,DMSO-d6)δ9.79(brs,1H),9.28(brs,1H),9.11(t,J=6.0Hz,1H),8.51(d,J=2.0Hz ,1H),8.07(d,J=8.0Hz,1H),7.71(dd,J=10.0,5.0Hz,1H),7.65(d,J=7.5Hz,1H),7.59(t,J=6.5 Hz,1H),7.48(dt,J=8.0,2.0Hz,1H),7.28(t,J=7.0Hz,1H),7.09(s,1H),4.66–4.53(m,2H),4.4 7–4.35(m,2H),4.27–4.21(m,1H),4.20(d,J=6.0Hz,2H),3.05(s,6H).HRMS(ESI-TOF)m / z:[M+H] + calcd for C 25 H 24 N7O2FS 506.1769,found 506.1770.

[0116] Compound B11, 52 mg, pale yellow solid, yield 49%. 1 H NMR (400MHz, CDCl3) δ8.37(ddd,J=4.4,2.4,0.4Hz,1H),7.86–7.79(m,2H),7.59(d,J=8.0Hz,1H),7.55(ddd,J=9.6,5.2,0.4Hz,1H),7.50(td,J=6. 8,1.2Hz,1H),7.24–7.15(m,2H),6.59(s,1H),4.75(d,J=6.0Hz,2H),4.5 2–4.39(m,4H),4.13–4.04(m,1H),3.12(s,6H).HRMS(ESI-TOF)m / z:[M+H] + calcd for C 26 H 22 N7O3FS2564.1282, found 564.1307.

[0117] Compound B15 is a pale yellow solid with a yield of 56%. 1H NMR (400MHz, CDCl3) δ8.40(dd,J=4.0,2.4Hz,1H),7.89(d,J=8.4Hz,1H),7.74(d,J=8.4Hz,1H),7.62–7.50(m ,2H),7.30–7.17(m,2H),6.68(s,1H),6.41(d,J=7.2Hz,1H),4.58(t,J=8.0Hz,2H),4.52(t,J=8.0Hz,2H),4.3 1–4.21(m,1H),4.20–4.10(m,1H),3.47(dd,J=14.4,9.6Hz,1H),3.14(s,6H),2.93–2.77(m,4H),2.63(dd,J=1 4.4,4.0Hz,1H),2.13(dd,J=5.6,2.8Hz,1H),1.81–1.66(m,3H),1.62–1.49(m,1H).HRMS(ESI-TOF)m / z:[M+H] + calcd for C 30 H 32 N7O2F 542.2674, found 542.2680.

[0118] In a 50 mL round-bottom flask, compound M19 (100 mg, 0.23 mmol) was dissolved in anhydrous N,N-dimethylformamide (10 mL). The corresponding amine compounds (0.28 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (105 mg, 0.28 mmol), and diisopropylethylamine (89 mg, 0.69 mmol) were added sequentially. The reaction mixture was stirred overnight at room temperature, and the reaction was monitored by TLC. After the reaction was complete, the system was concentrated under reduced pressure to obtain a crude product. Saturated brine (30 mL) was added, and the mixture was extracted twice with ethyl acetate, dried over anhydrous sodium sulfate, and the filtrate was filtered. The crude product was concentrated under reduced pressure and purified by silica gel column chromatography to give a pale yellow or white solid compound. The obtained compound was dissolved in dichloromethane (20 mL), and trifluoroacetic acid (2 mL) was added. The reaction mixture was stirred overnight at room temperature, and the reaction was monitored by TLC. After the reaction was completed, the crude product was obtained by vacuum distillation. The pH was adjusted to 9-10 by adding saturated sodium bicarbonate solution. The product was extracted twice with ethyl acetate (10 mL). The organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and the filtrate was evaporated under vacuum. The crude product was purified by silica gel column chromatography to give pale yellow solids B9, B12-B14, B16 and B18.

[0119] Compound B9, 32 mg, white solid, yield 42%. 1H NMR (500MHz, CD3OD_SPE) δ8.44(dd,J=4.0,2.5Hz,1H),7.91(d,J=8.5Hz,1H),7.71(d,J=8 .5Hz,1H),7.64–7.54(m,2H),7.42(td,J=8.0,2.5Hz,1H),7.27(t,J=7.5Hz,1H),6.88(s, 1H),4.64(t,J=8.0Hz,2H),4.46(t,J=8.0Hz,2H),4.32–4.23(m,1H),3.49–3.37(m,2H),3 .22(dd,J=12.5,6.5Hz,1H),3.14(s,6H),1.20(d,J=6.5Hz,3H).HRMS(ESI-TOF)m / z:[M+H] + calcd for C 26 H 28 N7O2F 490.2361,found490.2356.

[0120] Compound B12, 57 mg, pale yellow solid, yield 38%. 1 H NMR (500MHz, CD3OD) δ8.47–8.45(m,1H),7.96(d,J=7.5Hz,1H),7.75(d,J=8.5Hz,1H),7.66(t,J=7.5Hz,1H ),7.63(dd,J=10.0,5.0Hz,1H),7.45(dt,J=8.0,2.5Hz,1H),7.36(t,J=7.5Hz,1H),6.96(s,1H),4.75(t,J= 8.5Hz,2H),4.72–4.66(m,1H),4.58–4.52(m,2H),4.37–4.29(m,1H),3.68(dd,J=12.0,7.0Hz,1H),3.57–3. 50(m,1H),3.49–3.39(m,2H),3.14(s,6H),2.51–2.41(m,1H),2.25–2.16(m,1H).HRMS(ESI-TOF)m / z:[M+H] + calcd for C 27 H 28 N7O2F502.2361, found 502.2358.

[0121] Compound B13, 52 mg, pale yellow solid, yield 38%. 11H NMR (500 MHz, DMSO) δ 8.79 (d, J = 7.5 Hz, 1H), 8.51 (dd, J = 4.5, 2.5 Hz, 1H), 7.84 (d, J = 8.0 Hz, 1H), 7.69 (dd, J = 10.0, 5.5 Hz, 1H), 7.63 (d, J = 8.0 Hz, 1H), 7.57 (td, J = 7.0, 1.0 Hz, 1H), 7.48 (td, J = 7.0, 2.5 Hz, 1H), 7.26 (td, J = 8.0, 1.0 Hz, 1H), 6.78 (s, 1H), 4.56 (t, J = 8.0 Hz, 2H), 4.37 (t, J = 8.0 Hz, 2H), 4.24–4.17 (m, 1H), 4.11–3.99 (m, 1H), 3.25–3.14 (m, 2H), 3.05 (s, 6H), 2.93–2.82 (m, 2H), 2.04–1.93 (m, 2H), 1.66–1.54 (m, 2H). HRMS (ESI-TOF) m / z: [M+H] + calcd for C 28 H 30 N7O2F 516.2518, found 516.2495。

[0122] Compound B14, 54 mg, pale yellow solid, yield 44%. 1 1H NMR (500 MHz, CD3OD) δ 8.48 (dd, J = 4.0, 2.0 Hz, 1H), 8.01 (d, J = 8.0 Hz, 1H), 7.87–7.80 (m, 2H), 7.66 (dd, J = 9.5, 5.0 Hz, 1H), 7.54 (dt, J = 6.0, 2.5 Hz, 1H), 7.49 (dt, J = 8.0, 2.5 Hz, 1H), 7.15 (s, 1H), 4.97 (t, J = 9.5 Hz, 2H), 4.73 (dd, J = 9.5, 6.0 Hz, 2H), 4.47–4.39 (m, 1H), 4.39–4.31 (m, 1H), 3.65 (dd, J = 12.5, 4.0 Hz, 1H), 3.35 (dt, J = 12.5, 4.0 Hz, 1H), 3.13 (s, 6H), 3.07–2.98 (m, 2H), 2.23–2.15 (m, 1H), 2.13–2.05 (m, 1H), 1.94–1.83 (m, 1H), 1.79–1.69 (m, 1H). HRMS (ESI-TOF) m / z: [M+H] + calcd for C 28 H 30 N7O2F 516.2518, found 516.2514。

[0123] Compound B16, 50 mg, pale yellow solid, yield 53%. 1 H NMR (400MHz, CDCl3) δ8.43–8.38(m,1H),7.75(t,J=9.2Hz,1H),7.58(m,3H),7.31–7.18(m,3H ),6.56(d,J=14.0Hz,1H),4.70–4.59(m,1H),4.59–4.49(m,3H),4.22–4.11(m,1H),4.04–3.59 (m,2H),3.19(dd,J=9.6,4.8Hz,1H),3.15(d,J=1.6Hz,6H),3.11–2.98(m,2H),2.84–2.77(m, 1H),0.85–0.67(m,2H),0.54(t,J=6.9Hz,1H),0.35–0.08(m,1H).HRMS(ESI-TOF)m / z::[M+Na] + calcd for C 29 H 30 N7O2F 550.2337, found 550.2358.

[0124] Compound B18, 45 mg, pale yellow solid, yield 47%. 1 H NMR(500MHz,CD3OD)δ8.47–8.41(m,1H),7.74(d,J=8.5Hz,1H),7.69–7.51(m,3H),7.43(td,J =8.0,2.5Hz,1H),7.31(q,J=8.0Hz,1H),6.87–6.73(m,1H),4.79–4.70(t,1H),4.69–4.58(m, 2H),4.46(t,J=7.0Hz,2H),4.33–4.23(m,1H),3.47(t,J=14.0Hz,1H),3.40–3.31(m,1H),3.2 8–3.16(m,2H),3.14(s,6H),3.10–2.77(m,2H),1.38–0.98(m,3H).HRMS(ESI-TOF)m / z:[M+H] + calcd for C 28 H 30 N7O2F 516.2518, found 516.2505.

[0125] The synthesis of compound B17 was as follows: In a 50 mL round-bottom flask, compound B16 (100 mg, 0.19 mmol) was dissolved in methanol (6 mL), followed by the addition of formaldehyde aqueous solution (0.2 mL, 37%) and sodium cyanoborohydride (36 mg, 0.57 mmol). The reaction mixture was stirred overnight at room temperature, and the reaction was monitored by TLC. After the reaction was complete, the crude product was concentrated by vacuum distillation. Saturated brine (10 mL) was added, and the mixture was extracted twice with ethyl acetate. The extract was dried over anhydrous sodium sulfate, and the filtrate was concentrated by vacuum distillation. The crude product was purified by silica gel column chromatography to give a pale yellow solid compound B17 (82 mg, 80% yield). 1 H NMR(500MHz,CD3OD)δ8.47–8.42(m,1H),7.73(dd,J=11.5,8.5Hz,1H),7.66–7.57(m,3H),7.43(td,J=8.0,2.0H z,1H),7.31(dd,J=14.0,6.5Hz,1H),6.73(d,J=8.5Hz,1H),4.72–4.57(m,2H),4.50–4.41(m,2H),4.34–4.23(m, 1H),4.11–3.83(m,1H),3.82–3.67(m,1H),3.47–3.37(m,1H),3.14(s,6H),3.10–3.01(m,1H),2.80(t,J=5.0Hz ,1H),2.46(d,J=3.5Hz,3H),0.84–0.71(m,2H),0.68–0.57(m,1H),0.41–0.08(m,1H).HRMS(ESI-TOF)m / z:[M+H] + calcd for C 30 H 32 N7O2F 542.2674,found 542.2669.

[0126] The synthesis of compound B19 was as follows: using compound B12 (80 mg, 0.16 mmol) as the starting material, and following the synthesis method of compound B17, a pale yellow solid compound B19 (48 mg, yield 59%) was obtained. 1H NMR (400MHz, CDCl3) δ8.40(s,1H),7.96(d,J=8.4Hz,1H),7.73(d,J=8.4Hz,1H),7.62–7.50(m,2 H),7.27–7.16(m,2H),7.07(d,J=6.8Hz,1H),6.71(s,1H),4.82–4.70(m,1H),4.57(t,J=8.0Hz, 2H),4.51(t,J=6.8Hz,2H),4.20–4.08(m,1H),3.14(s,6H),3.05–2.97(m,1H),2.87(d,J=10.0H z,1H),2.67–2.63(m,1H),2.50–2.44(m,1H),2.40(s,3H),2.33–2.27(m,1H),1.96–1.77(m,1H).

[0127] The synthesis of compound B20 was as follows: using compound B18 (100 mg, 0.19 mmol) as the starting material, and following the synthesis method of compound B17, a pale yellow solid compound B20 (74 mg, yield 72%) was obtained. 1 H NMR(400MHz, CDCl3)δ8.41(s,1H),7.81–7.72(m,1H),7.64–7.47(m,3H),7.28–7.18(m,2H),6.63–6.51(m,1H),4.72–4.50(m, 5H),4.21–4.10(m,1H),3.32–3.17(m,2H),3.15(s,6H),2.96–2.60(m,2H),2.30(s,3H),2.21–1.92(m,2H),1.20–0.84(m,3H).

[0128] The synthesis of compound B21 was as follows: using compound B13 (120 mg, 0.23 mmol) as the starting material, and following the synthesis method of compound B17, a pale yellow solid compound B21 (92 mg, yield 75%) was obtained. 1H NMR (400MHz, CDCl3) δ8.38 (dd, J=4.0, 2.4Hz, 1H), 7.86 (d, J=8.0Hz, 1H), 7.68 (d,J=8.4Hz,1H),7.60–7.47(m,2H),7.25–7.15(m,2H),6.66(d,J=8.0Hz,1H) ,6.58(s,1H),4.59–4.40(m,4H),4.15–3.99(m,2H),3.12(s,6H),2.91–2.78( m,2H),2.64–2.57(m,2H),2.29(s,3H),2.17–2.11(m,2H),1.75–1.59(m,2H).

[0129] The synthesis of compound B22 was carried out as follows: using compound B14 (120 mg, 0.23 mmol) as the starting material, and following the synthesis method of compound B17, a pale yellow solid compound B22 (96 mg, yield 78%) was obtained. 1 H NMR (400MHz, CDCl3) δ8.40 (dd, J=4.4, 2.4Hz, 1H), 7.95 (d, J=7.6Hz, 1H), 7.74 (d ,J=8.4Hz,1H),7.62–7.52(m,2H),7.28–7.17(m,2H),6.94(s,1H),6.71(s,1H), 4.58(t,J=8.0Hz,2H),4.52(t,J=6.8Hz,2H),4.41–4.33(m,1H),4.20–4.09(m,1 H),3.14(s,6H),2.52(m,4H),2.24(s,3H),1.88–1.71(m,2H),1.72–1.59(m,2H).

[0130] In this embodiment, the synthetic route of compound B23 (see Figure 18) is as follows:

[0131] The synthesis of intermediate M20 was as follows: In a 125 mL round-bottom flask, 2-(1-tert-butoxycarbonyl)azacyclobutane-3-yl)-6-fluoroimidazolo[1,2-a]pyridine-3-carboxylic acid (3.35 g, 10.0 mmol), N,O-dimethylhydroxylamine (1.46 g, 15.0 mmol), N,N-diisopropylethylamine (3.88 g, 30.0 mmol), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (4.18 g, 11.0 mmol) were dissolved in N,N-dimethylformamide (30 mL). The reaction mixture was stirred overnight at room temperature, and the reaction was monitored by TLC. After the reaction was complete, the filtrate was concentrated by vacuum distillation, and the crude product was purified by silica gel column chromatography to give a yellow solid intermediate compound M20 (2.27 g, yield 60%). 1 H NMR(400MHz, CDCl3) δ8.61(dd,J=4.6,2.4Hz,1H),7.64(dd,J=9.8,5.0Hz,1H),7.25(ddd,J=10.0,7 .4, 2.0Hz, 1H), 4.28 (d, J = 8.4Hz, 4H), 4.09 (q, J = 7.6Hz, 1H), 3.47 (s, 3H), 3.42 (s, 3H), 1.45 (s, 9H).

[0132] The synthesis of intermediate M21 was as follows: M20 (1.82 g, 4.82 mmol) and methylmagnesium bromide (5 mL, 14.44 mmol) were dissolved in tetrahydrofuran (40 mL) in a 125 mL round-bottom flask at 0 °C. The reaction mixture was then stirred overnight at room temperature, and the reaction was monitored by TLC. After the reaction was complete, a saturated ammonium chloride solution was added to the reaction mixture at 0 °C. The aqueous phase was extracted twice with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate. The filtrate was concentrated by vacuum distillation, and the crude product was purified by silica gel column chromatography to give a white solid intermediate compound M21 (1.01 g, yield 63%). 1 HNMR (400MHz, CDCl3) δ9.76 (dd, J=4.8, 2.0Hz, 1H), 7.71 (dd, J=9.6, 5.2Hz, 1H), 7.42(ddd,J=9.8,7.6,2.4Hz,1H),4.56–4.30(m,5H),2.55(s,3H),1.47(s,9H).

[0133] The synthesis of intermediate M22 was as follows: In a 50 mL round-bottom flask, compound M21 (200 mg, 0.6 mmol) was dissolved in methanol (10 mL), and concentrated hydrochloric acid (1.0 mL) was added. The reaction mixture was stirred overnight at room temperature, and the reaction was monitored by TLC. After the reaction was completed, the mixture was concentrated by vacuum distillation to give a white solid intermediate compound M22 (140 mg, 100% yield). 1 HNMR (400MHz, DMSO-d6) δ9.63 (dd, J=5.4, 2.4Hz, 1H), 7.88 (dd, J=9.8, 5.4Hz, 1H), 7.77–7. 69(m,1H),4.52(p,J=8.0Hz,1H),4.03(t,J=7.4Hz,2H),3.77(t,J=7.8Hz,2H),2.52(s,3H).

[0134] The synthesis of compound B23 was as follows: In a 50 mL round-bottom flask, intermediates M22 (100 mg, 0.43 mmol) and M7 (95 mg, 0.43 mmol) were dissolved in anhydrous DMF (10 mL), and cesium carbonate (420 mg, 1.29 mmol) was added. The reaction mixture was heated to 110 °C and reacted overnight, with the reaction monitored by TLC. After the reaction was complete, the cesium carbonate was removed by filtration, and the filtrate was distilled under reduced pressure to obtain the crude product, which was purified by silica gel column chromatography to give a white solid compound B23 (86 mg, yield 48%). 1 HNMR (400MHz, CDCl3) δ9.78(dd,J=5.2,2.4Hz,1H),7.91(d,J=8.4Hz,1H),7.71(d,J=8.0Hz,1H),7.67(dd,J=10.0,5.2Hz,1H),7.58–7.51( m,1H),7.43–7.38(m,1H),7.23(t,J=7.6Hz,1H),6.64(s,1H),6.35(q,J=4.8Hz,1H),4.68–4.55(m,5H),3.10(d,J=4.8Hz,3H),2.62(s,3H).

[0135] In this embodiment, the synthetic routes for compounds B24 to B26 (see Figure 19) are as follows:

[0136] The synthesis of intermediate M23 was as follows: In a 100 mL round-bottom flask, ethyl 2-(azacyclobutan-3-yl)-6-fluoroimidazolo[1,2-a]pyridine-3-carboxylate M14e (400 mg, 1.52 mmol) and 2-chloro-4-methylquinoline were dissolved in toluene (30 mL), followed by the addition of sodium tert-butoxide (438 mg, 4.56 mmol), x-Phos (145 mg, 0.30 mmol), and Pd2(dba)3 (139 mg, 0.15 mmol). The reaction mixture was heated at 100 °C overnight under argon protection. After the reaction was complete, the mixture was distilled under reduced pressure to obtain a crude product, which was purified by silica gel column chromatography to give a pale yellow solid intermediate compound M23 (128 mg, yield 54%). 1 HNMR(400MHz, CDCl3)δ9.33(dd,J=4.4,2.0Hz,1H),7.81–7.72(m,2H),7.65(dd,J=9.6,4.8Hz,1H),7.52(dt,J=6.8,1.2Hz,1H), 7.35–7.29(m,1H),7.26–7.20(m,1H),6.52(s,1H),4.71–4.55(m,5H),4.47(q,J=7.2Hz,2H),2.59(s,3H),1.49(t,J=7.2Hz,3H).

[0137] The synthesis of intermediate M24 was as follows: In a 50 mL round-bottom flask, intermediate M23 (295 mg, 0.73 mmol) was dissolved in methanol (10 mL) and water (3 mL), and sodium hydroxide (88 mg, 2.19 mmol) was added. The reaction mixture was stirred overnight at 60 °C, and the reaction was monitored by TLC. After the reaction was complete, the crude product was distilled under reduced pressure to obtain an aqueous solution. The pH was adjusted to 3-4 with 5 M hydrochloric acid solution, and a solid precipitated. The solid was filtered, the filter cake was washed with water, and dried in air to obtain a pale yellow solid intermediate compound M24 (240 mg, yield 88%).

[0138] The synthesis of intermediate M25 was as follows: In a 125 mL round-bottom flask, intermediate M24 (200 mg, 0.53 mmol), 3,6-diazabicyclo[3.1.1]heptane-6-carboxylic acid tert-butyl ester (126 mg, 0.64 mmol), N,N-diisopropylethylamine (205 mg, 1.59 mmol), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (242 mg, 0.64 mmol) were dissolved in N,N-dimethylformamide (10 mL). The reaction mixture was stirred overnight at room temperature, and the reaction was monitored by TLC. After the reaction was complete, the filtrate was concentrated by vacuum distillation, and the crude product was purified by silica gel column chromatography to give a white solid intermediate compound M25 (280 mg, yield 94.6%).1 H NMR (500MHz, CDCl3) δ8.17(s,1H),7.76(dd,J=8.0,4.0Hz,2H),7.62–7.55(m,2H),7.31(t,J=7.5Hz,1H),7.21(ddd,J=10.0,8.0,2. 5Hz,1H),6.46(s,1H),4.71(s,2H),4.61–4.53(m,2H),4.21(s,4H),3.93–3.43(m,3H),2.72–2.66(m,1H),2.56(s,3H),1.49(s,9H).

[0139] The synthesis of compound B24 was as follows: In a 50 mL round-bottom flask, intermediate M25 (280 mg, 0.5 mmol) was dissolved in dichloromethane (10 mL), and trifluoroacetic acid (1 mL) was carefully added. The reaction mixture was stirred overnight at room temperature, and the reaction was monitored by TLC. After the reaction was complete, the mixture was distilled under reduced pressure to obtain a crude product, which was purified by silica gel column chromatography to give a white solid compound B24 (195 mg, yield 85%). 1 H NMR (400MHz, CDCl3) δ8.27 (dd, J=3.6, 2.4Hz, 1H), 7.79 (dd, J=8.2, 1.2Hz, 1H), 7.75 (d, J=8.0H z,1H),7.59(dd,J=9.8,5.2Hz,1H),7.56–7.51(m,1H),7.25(dd,J=8.2,1.2Hz,1H),7.20(ddd, J=10.0,7.8,2.4Hz,1H),6.50(s,1H),4.60(t,J=8.1Hz,2H),4.56–4.48(m,2H),4.23–4.15(m, 1H), 3.94 (s, 2H), 3.81 (s, 4H), 2.81 (d, J = 9.0Hz, 1H), 2.63–2.51 (m, 3H), 1.63 (d, J = 9.4Hz, 1H).

[0140] The synthesis of compound B25 was as follows: In a 50 mL round-bottom flask, compound B24 (143 mg, 0.31 mmol) was dissolved in methanol (10 mL), followed by the addition of formaldehyde aqueous solution (1 mL, 37%) and sodium cyanoborohydride (59 mg, 0.94 mmol). The reaction mixture was stirred overnight at room temperature, and the reaction was monitored by TLC. After the reaction was complete, the crude product was concentrated by vacuum distillation. Saturated brine (10 mL) was added, and the mixture was extracted twice with ethyl acetate. The extract was dried over anhydrous sodium sulfate, and the filtrate was concentrated by vacuum distillation. The crude product was purified by silica gel column chromatography to give compound B25 (66 mg, 45% yield) as a white solid. 1H NMR(400MHz, CDCl3)δ8.22(dd,J=4.2,2.2Hz,1H),7.85–7.71(m,2H),7.59(dd, J=9.8,5.2Hz,1H),7.52(ddd,J=8.4,7.0,1.6Hz,1H),7.27–7.12(m,2H),6.50( d,J=1.2Hz,1H),4.60(t,J=8.2Hz,2H),4.56–4.48(m,2H),4.17(ddd,J=14.8,8 .4,6.4Hz,1H),3.92–3.53(m,5H),2.96–2.63(m,3H),2.59(s,3H),2.30(s,3H).

[0141] The synthesis of compound B26 was as follows: In a 50 mL flask, intermediate M24 (200 mg, 0.53 mmol) was dissolved in anhydrous N,N-dimethylformamide (10 mL). N-methylpiperazine (106 mg, 1.06 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (242 mg, 0.64 mmol), and diisopropylethylamine (206 mg, 1.59 mmol) were added sequentially. The reaction mixture was stirred overnight at room temperature, and the reaction was monitored by TLC. After the reaction was complete, the crude product was concentrated by vacuum distillation. Saturated brine (30 mL) was added, and the mixture was extracted twice with ethyl acetate. The extract was dried over anhydrous sodium sulfate, filtered, and concentrated by vacuum distillation. The crude product was purified by silica gel column chromatography to give compound B26 (76 mg, 31% yield) as a pale yellow or white solid. 1 HNMR (400MHz, CDCl3) δ8.36 (dd, J=4.2, 2.4Hz, 1H), 7.79–7.68 (m, 2H), 7.56–7.43 (m, 2H), 7.20–7.11 (m, 2H), 6. 44(s,1H),4.50(dt,J=20.0,7.8Hz,4H),4.18–4.06(m,1H),3.61(s,4H),2.53(s,3H),2.43(s,4H),2.28(s,3H).

[0142] In this embodiment, the synthetic route of compound B27 (see Figure 20) is as follows:

[0143] The synthesis of intermediate M26 was as follows: 2,4-Dibromoquinoline (1.0 g, 3.48 mmol) was dissolved in anhydrous 1,4-dioxane (20 mL) in a 50 mL round-bottom flask. The mixture was heated to 90 °C, and 40% HBr (4 mL) was added dropwise. The reaction mixture was heated at 90 °C overnight. After the reaction was complete, the crude product was concentrated by vacuum distillation and purified by silica gel column chromatography to give a grayish-white solid intermediate compound M26 (614 mg, yield 79%). 1 H NMR (400MHz, DMSO-d6) δ12.04(s,1H),7.82(dd,J=8.0,1.2Hz,1H),7.61(dt,J=7.2 ,1.2Hz,1H),7.36(dd,J=8.0,0.8Hz,1H),7.30(dt,J=7.2,1.2Hz,1H),7.03(s,1H).

[0144] The synthesis of intermediate M27 was as follows: In a 100 mL round-bottom flask, intermediate M26 (224 mg, 1.0 mmol) and 1-methyl-1,2,3,6-tetrahydropyridine-4-boronic acid pinacol ester (223 mg, 1.0 mmol) were dissolved in 1,4-dioxane (20 mL) and H2O (5 mL). K2CO3 (977 mg, 3.0 mmol) and [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride Pd(dppf)Cl2 (73 mg, 0.10 mmol) were then added. The reaction mixture was heated to 90 °C overnight under argon protection. After the reaction was complete, the mixture was distilled under reduced pressure to obtain a crude product, which was purified by silica gel column chromatography to give a pale yellow solid intermediate compound M27 (192 mg, yield 80%). 1 HNMR(400MHz, CDCl3)δ12.37(s,1H),7.72(d,J=7.6Hz,1H),7.53–7.42(m,2H),7.19(dt,J=8.0,0.8Hz,1 H), 6.55 (s, 1H), 5.84 (s, 1H), 3.25–3.15 (m, 2H), 2.75 (t, J = 5.6Hz, 2H), 2.58–2.51 (m, 2H), 2.48 (s, 3H).

[0145] The synthesis of intermediate M28 was as follows: In a 100 mL round-bottom flask, intermediate M27 (575 mg, 2.39 mmol) was dissolved in toluene (20 mL), followed by the addition of phosphorus oxychloride (2 mL). The reaction mixture was heated to 100 °C for 4 hours. After the reaction was complete, the mixture was distilled under reduced pressure to obtain a crude product, which was purified by silica gel column chromatography to give a pale yellow solid intermediate compound M28 (570 mg, yield 92%). 1HNMR (400MHz, CDCl3) δ7.96(s,1H),7.94(s,1H),7.64(dt,J=7.2,1.2Hz,1H),7.45(dt,J=7.2,1.2Hz,1H),7.14 (s,1H),5.85–5.76(m,1H),3.15(dd,J=6.0,2.8Hz,2H),2.70(t,J=5.6Hz,2H),2.57–2.48(m,2H),2.42(s,3H).

[0146] The synthesis of compound B27 was as follows: In a 100 mL round-bottom flask, intermediates M28 (570 mg, 2.20 mmol) and M17e (578 mg, 2.20 mmol) were dissolved in toluene (30 mL), followed by the addition of sodium tert-butoxide (634 mg, 6.60 mmol), x-Phos (210 mg, 0.44 mmol), and Pd2(dba)3 (201 mg, 0.22 mmol). The reaction mixture was heated at 100 °C overnight under argon protection. After the reaction was complete, the mixture was distilled under reduced pressure to obtain a crude product, which was purified by silica gel column chromatography to give a pale yellow solid compound B27 (156 mg, yield 15%). 1 H NMR(400MHz, CDCl3)δ8.36(dd,J=4.0,2.4Hz,1H),7.74(d,J=0.8Hz,1H),7.72(d,J=0.8Hz,1H),7.56–7.43(m,2H),7.23–7.1 0(m,2H),6.44(s,1H),4.60–4.40(m,4H),4.18–4.06(m,1H),3.76–3.50(m,4H),2.53(s,3H),2.51–2.33(m,4H),2.28(s,3H).

[0147] In this embodiment, the synthetic routes for compounds C1 to C2 (see Figure 21) are as follows:

[0148] The synthesis of intermediate M29 was as follows: Methyl 2-amino-5-methylbenzoate (1.65 g, 10.0 mmol) was dissolved in acetic acid (10 mL) in a 125 mL round-bottom flask. An aqueous solution of potassium cyanate (973 mg, 12.0 mmol) (10 mL) was slowly added dropwise. The reaction mixture was heated to 60 °C overnight, and the reaction was monitored by TLC. After the reaction was complete, the mixture was cooled to room temperature, and water (40 mL) was added to the reaction mixture. The mixture was stirred for 10 minutes, and the precipitated solid was filtered off. The filter cake was washed with water and dried in air to obtain a white solid intermediate compound M29 (1.68 g, yield 81%). 1H NMR (400MHz, DMSO-d6) δ9.57 (s, 1H), 8.26 (d, J = 8.4Hz, 1H), 7.69 (d, J = 1.6Hz, 1H), 7.33 (dd, J = 8.8, 2.0Hz, 1H), 6.54 (brs, 2H), 3.86 (s, 3H), 2.26 (s, 3H).

[0149] The synthesis of intermediate M30 was as follows: In a 125 mL round-bottom flask, intermediate M29 (1.68 g, 8.1 mmol) was dissolved in anhydrous ethanol (30 mL), and sodium hydroxide (645 mg, 16.2 mmol) was added. The reaction mixture was heated to 80 °C and refluxed overnight, and the reaction was monitored by TLC. After the reaction was complete, the mixture was cooled to room temperature, and a solid precipitated out. The solid was filtered, the filter cake was washed with water, and allowed to dry naturally in air to give a white solid intermediate compound M30 (1.42 g, 100% yield). 1 H NMR (400MHz, DMSO-d6) δ9.40 (brs, 1H), 7.47 (s, 1H), 7.09 (dd, J = 8.4, 2.0Hz, 1H), 6.79 (d, J = 8.4Hz, 1H), 2.22 (s, 3H).

[0150] The synthesis of intermediate M31 was as follows: In a 125 mL round-bottom flask, intermediate M30 (1.42 g, 8.1 mmol) and phosphorus oxychloride (20 mL) were mixed. The reaction mixture was heated to reflux at 110 °C overnight, and the reaction was monitored by TLC. After the reaction was complete, the mixture was cooled to room temperature, and phosphorus oxychloride was removed by vacuum distillation to obtain the crude product. Dichloromethane and triethylamine were slowly added to the cooled crude product until the pH of the system was 8–9. The mixture was concentrated under reduced pressure and purified by silica gel column chromatography to obtain a white solid intermediate compound M31 (1.33 g, yield 77%). 1 H NMR (400MHz, CDCl3) δ8.03–8.00(m,1H),7.90(d,J=8.8Hz,1H),7.82(dd,J=8.8,2.0Hz,1H),2.61(s,3H).

[0151] The synthesis of intermediate M32a was as follows: In a 125 mL round-bottom flask, intermediate M31 (891 mg, 4.18 mmol) was dissolved in anhydrous acetonitrile (40 mL), and 2,4-dimethoxybenzylamine (699 mg, 4.18 mmol) and diisopropylethylamine (1.62 g, 12.54 mmol) were added sequentially. The reaction mixture was heated at 50 °C overnight, and the reaction was monitored by TLC. After the reaction was complete, the mixture was cooled to room temperature, and a solid precipitated. The precipitated solid was filtered, the filter cake was washed with water, and dried in air to give a white solid intermediate compound M32a (1.32 g, 92% yield).1 HNMR(400MHz, CDCl3) δ7.63(d,J=8.4Hz,1H),7.51(dd,J=8.4,1.2Hz,1H),7.35(s,1H),7.33(d,J=8.4Hz,1 H), 6.54–6.45 (m, 2H), 6.25 (t, J = 4.4Hz, 1H), 4.76 (d, J = 5.2Hz, 2H), 3.88 (s, 3H), 3.81 (s, 3H), 2.46 (s, 3H).

[0152] The synthesis of intermediate M32b was as follows: using intermediate M31 (426 mg, 2.0 mmol) and 3,8-diazabicyclo[3.2.1]octane-3-carboxylic acid tert-butyl ester (424 mg, 2.0 mmol) as raw materials, and referring to the synthesis method of compound M32a, a white solid intermediate compound M32b (748 mg, yield 97%) was obtained. 1 HNMR(500MHz, CDCl3)δ7.70(d,J=8.5Hz,1H),7.59(s,1H),7.54(dd,J=8.5,1.5Hz,1H),4.57–4 .21(m,4H),3.77–3.42(m,2H),2.49(s,3H),2.00–1.89(m,2H),1.89–1.72(m,2H),1.52(s,9H).

[0153] The synthesis of intermediate M33a was as follows: In a 50 mL round-bottom flask, intermediates M32a (343 mg, 1.0 mmol) and M7 (262 mg, 1.0 mmol) were dissolved in anhydrous DMF (10 mL), and cesium carbonate (1.62 g, 3.0 mmol) was added. The reaction mixture was heated to 110 °C and reacted overnight, with the reaction monitored by TLC. After the reaction was complete, the cesium carbonate was removed by filtration, and the filtrate was distilled under reduced pressure to obtain the crude product, which was purified by silica gel column chromatography to give the pale yellow solid intermediate compound M33a (158 mg, yield 28%). 1HNMR (500MHz, CDCl3) δ8.42 (dd, J=4.0, 2.5Hz, 1H), 7.58 (dd, J=10.0, 5.0Hz, 1H), 7.46 (d, J=8. 0Hz,1H),7.34(dd,J=8.5,1.5Hz,1H),7.29(d,J=8.0Hz,1H),7.26(s,1H),7.19(td,J=7.5,2.5 Hz,1H),6.47(d,J=2.5Hz,1H),6.43(dd,J=8.0,2.5Hz,1H),6.06(brs,1H),4.70(d,J=5.5Hz,2 H),4.65–4.52(m,4H),4.13–4.05(m,1H),3.86(s,3H),3.79(s,3H),3.15(s,6H),2.38(s,3H).

[0154] The synthesis of intermediate M33b was as follows: using intermediates M32b (200 mg, 0.51 mmol) and M7 (135 mg, 0.51 mmol) as raw materials, and referring to the synthesis method of compound M33a, a pale yellow solid intermediate compound M33b (134 mg, yield 42%) was obtained. 1 H NMR (400MHz, CDCl3) δ8.44–8.39(m,1H),7.59(dd,J=10.0,4.8Hz,1H),7.49(d,J=8.4Hz,1H),7.45(s,1H),7.36(d,J=8.8Hz,1H),7.23–7.16( m,1H),4.60–4.45(m,4H),4.40–4.15(m,4H),4.13–4.01(m,1H),3.62– 3.31(m,2H),3.15(s,6H),2.40(s,3H),1.99–1.85(m,4H),1.51(s,9H).

[0155] The synthesis of compound C1 was as follows: In a 50 mL round-bottom flask, intermediate M33a (158 mg, 0.28 mmol) was dissolved in methanol (10 mL), and concentrated hydrochloric acid (1 mL) was carefully added. The reaction mixture was heated to 65 °C for 24 hours, and the reaction was monitored by TLC. After the reaction was complete, the mixture was distilled under reduced pressure to obtain a crude product, which was purified by silica gel column chromatography to give a pale yellow solid compound C1 (91 mg, yield 78%). 1H NMR (500MHz, DMSO-d6) δ8.49(dd,J=5.0,2.0Hz,1H),7.86(s,1H),7.71(dd,J=10.0,5.5Hz,1H),7.58(brs,2H),7.47(t,J=8.0Hz,1H),7.41(d,J=8.5 Hz,1H),7.27(d,J=8.5Hz,1H),4.43(t,J=8.0Hz,2H),4.27(t,J=8.0Hz,2H ),4.11–4.02(m,1H),3.03(s,6H),2.36(s,3H).HRMS(ESI-TOF)m / z:[M+H] + calcd for C 22 H 22 N7OF 420.1943, found 420.1946.

[0156] The synthesis of compound C2 was as follows: In a 50 mL round-bottom flask, intermediate M33b (134 mg, 0.22 mmol) was dissolved in dichloromethane (10 mL), and trifluoroacetic acid (1 mL) was carefully added. The reaction mixture was stirred overnight at room temperature, and the reaction was monitored by TLC. After the reaction was complete, the mixture was distilled under reduced pressure to obtain a crude product, which was purified by silica gel column chromatography to give a pale yellow solid compound C2 (88 mg, yield 79%). 1 H NMR(400MHz, CDCl3)δ8.42(s,1H),7.59(dd,J=9.6,5.2Hz,1H),7.48(d,J=8.8Hz,1H), 7.47(s,1H),7.35(d,J=8.8Hz,1H),7.20(t,J=7.6Hz,1H),4.66–4.44(m,4H),4.30–4.1 6(m,2H),4.14–4.02(m,1H),3.64–3.52(m,2H),3.46–3.33(m,2H),3.15(s,6H),2.48( brs,1H),2.40(s,3H),2.22–1.88(m,2H),1.85–1.72(m,2H).HRMS(ESI-TOF)m / z:[M+H] + calcd for C 28 H 31 N8OF 515.2678, found 515.2682.

[0157] In this embodiment, the synthetic route of compound C3 (see Figure 22) is as follows:

[0158] The synthesis of intermediate M34 was carried out as follows: using intermediate 2,4-dichloroquinazoline (1.0 g, 5.0 mmol) and tert-butyl 4,7-diazaspiro[2.5]octane-4-carboxylate (1.06 g, 5.0 mmol) as raw materials, and referring to the synthesis method of compound M32a, a pale yellow solid intermediate compound M34 (1.37 g, yield 73%) was obtained. 1 HNMR(500MHz, CDCl3)δ7.84–7.78(m,2H),7.73(td,J=7.0,1.0Hz,1H),7.42(td,J=7.0,1.0Hz,1H) ,3.94–3.89(m,2H),3.79–3.73(m,4H),1.50(s,9H),1.04(t,J=6.5Hz,2H),0.84(t,J=6.5Hz,2H).

[0159] The synthesis of intermediate M35 was carried out as follows: using intermediate M34 (187 mg, 0.50 mmol) and M17e (131 mg, 0.50 mmol) as raw materials, and referring to the synthesis method of compound M33a, a pale yellow solid intermediate compound M35 (71 mg, yield 57%) was obtained. 1 HNMR (400MHz, CDCl3) δ8.41 (dd, J=4.4, 2.4Hz, 1H), 7.65 (d, J=8.0Hz, 1H), 7.61–7.5 5(m,2H),7.54–7.48(m,1H),7.19(td,J=7.6,2.4Hz,1H),7.07(td,J=8.0,1.2Hz,1H) ,4.57(t,J=8.0Hz,2H),4.52(t,J=8.0Hz,2H),4.15–4.04(m,1H),3.78–3.69(m,4H), 3.58(s,2H),3.15(s,6H),1.49(s,9H),1.01(t,J=6.0Hz,2H),0.79(t,J=6.0Hz,2H).

[0160] The synthesis of compound C3 was as follows: using intermediate M32 (130 mg, 0.22 mmol) as the starting material, and following the synthesis method of compound C2, a pale yellow solid compound C3 (93 mg, yield 86%) was obtained. 1HNMR(500MHz, CDCl3)δ8.42(dd,J=4.5,2.5Hz,1H),7.69(d,J=8.0Hz,1H),7.62–7.55(m,2H) ,7.52(t,J=7.5Hz,1H),7.20(td,J=7.5,2.5Hz,1H),7.07(t,J=7.5Hz,1H),4.57(t,J=8.0Hz ,2H),4.52(t,J=8.0Hz,2H),4.15–4.04(m,1H),3.72–3.64(m,2H),3.54(s,2H),3.15(s,6H) ,3.14–3.11(m,2H),0.66(t,J=5.5Hz,2H),0.59(t,J=5.5Hz,2H).HRMS(ESI-TOF)m / z:[M+H] + calcd for C 27 H 29 N8OF 501.2521, found 501.2518.

[0161] In this embodiment, the synthetic routes for compounds C4 to C8 (see Figure 23) are as follows:

[0162] The synthesis of intermediate M36 was as follows: using compound M14e (825 mg, 2.27 mmol) as the starting material, and following the synthesis method of compound C1, a yellow solid compound M36 (526 mg, yield 88%) was obtained. 1 HNMR(400MHz,DMSO)δ9.22(dd,J=4.8,2.4Hz,1H),7.88(dd,J=9.6,5.2Hz,1H),7.70(td,J=8.0,2.4Hz,1H),4 .58–4.47(m,1H),4.36(q,J=7.2Hz,2H),4.08(t,J=8.0Hz,2H),3.92(t,J=8.8Hz,2H),1.37(t,J=7.2Hz,3H).

[0163] The synthesis of compound C4 was carried out as follows: using 2-chloro-4-methylquinazoline (357 mg, 2.0 mmol) and M36 (526 mg, 2.0 mmol) as raw materials, and referring to the synthesis method of intermediate M33a, a pale yellow solid compound C4 (559 mg, yield 69%) was obtained. 1HNMR(400MHz, CDCl3)δ9.33(dd,J=4.8,2.4Hz,1H),7.85(d,J=8.4Hz,1H),7.71–7.56(m,3H),7.32(td,J=7.6,2.4Hz,1H), 7.24–7.15(m,1H),4.75–4.60(m,5H),4.46(q,J=7.2Hz,2H),2.79(s,3H),1.49(t,J=7.2Hz,3H).HRMS(ESI-TOF)m / z:[M+H] + calcd for C 22 H 20 N5O2F 406.1674, found 406.1675.

[0164] The synthesis of intermediate M37 was as follows: Compound C4 (520 mg, 1.28 mmol) was dissolved in methanol (20 mL) and water (5 mL) in a 125 mL round-bottom flask, and sodium hydroxide (154 mg, 3.84 mmol) was added. The reaction mixture was stirred overnight at 60 °C, and the reaction was monitored by TLC. After the reaction was complete, the crude product was distilled under reduced pressure to obtain an aqueous solution. The pH was adjusted to 3-4 with the addition of saturated citric acid solution, and a solid precipitated. The solid was filtered, the filter cake was washed with water, and dried in air to obtain a white solid intermediate compound M37 (460 mg, 95% yield). 1 HNMR (500MHz, DMSO) δ9.82(dd,J=5.5,2.0Hz,1H),7.99(d,J=8.0Hz,1H),7.68(t,J=7.5Hz,1H),7.55(dd,J=10.0,5.5Hz,1H),7.51(d,J =8.5Hz,1H),7.31(t,J=7.5Hz,1H),7.24(t,J=7.5Hz,1H),5.12–5.00(m,1H),4.45(t,J=7.0Hz,2H),4.34(t,J=7.0Hz,2H),2.75(s,3H).

[0165] The synthesis of compound C5 was as follows: In a 50 mL flask, intermediate M37 (100 mg, 0.26 mmol) was dissolved in anhydrous N,N-dimethylformamide (8 mL). Compound methylamine hydrochloride (24 mg, 0.29 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (121 mg, 0.32 mmol), and diisopropylethylamine (103 mg, 0.78 mmol) were added sequentially. The reaction mixture was stirred overnight at room temperature, and the reaction was monitored by TLC. After the reaction was complete, the crude product was obtained by vacuum distillation and purified by silica gel column chromatography to give a pale yellow solid C5 (81 mg, 76% yield). 1 H NMR (400MHz, CDCl3) δ8.43(dd,J=4.0,2.4Hz,1H),7.86(d,J=8.4Hz,1H),7.68–7.61(m,2H),7.58(dd,J=10.0,5.2Hz ,1H),7.25–7.14(m,2H),4.72–4.53(m,4H),4.22–4.06(m,1H),3.15(s,6H),2.79(s,3H).HRMS(ESI-TOF)m / z:[M+H] + calcd for C 22 H 21 N6OF 405.1834,found405.1846.

[0166] The synthesis of intermediates M38a-c was as follows: In a 50 mL round-bottom flask, intermediate M37 (100 mg, 0.26 mmol) was dissolved in anhydrous N,N-dimethylformamide (8 mL). Various amines (0.29 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (121 mg, 0.32 mmol), and diisopropylethylamine (103 mg, 0.78 mmol) were added sequentially. The reaction mixture was stirred overnight at room temperature, and the reaction was monitored by TLC. After the reaction was complete, the crude product was obtained by vacuum distillation, and purified by silica gel column chromatography to give the yellow solid intermediate compounds M38a-c.

[0167] Intermediate M38a, 126 mg, yield 87%. 1H NMR(400MHz, CDCl3)δ9.32(s,1H),8.02(s,1H),7.88(d,J=8.4Hz,1H),7.70–7.62(m,2H),7.59(dd,J=9.6,5.2Hz,1H),7.31–7.27(m,2H),7.26–7.22 (m,1H),4.83–4.59(m,5H),4.41–4.30(m,1H),3.83–3.75(m,1H),3.58–3. 50(m,2H),2.81(s,3H),2.38–2.27(m,1H),2.10–1.90(m,2H),1.46(s,9H).

[0168] Intermediate M38b, 135 mg, yield 91%. 1 H NMR(500MHz, CDCl3)δ8.95(s,1H),7.92–7.83(m,2H),7.74(t,J=7.5Hz,1H),7.70(dd,J=9.5,5.0Hz,1H),7.35(t,J=7.5Hz,1H),7.29–7.14(m,2 H),4.99–4.83(m,2H),4.79–4.62(m,3H),4.22–4.08(m,3H),3.01–2.88 (m,2H),2.85(s,3H),2.07–1.98(m,2H),1.70–1.57(m,2H),1.46(s,9H).

[0169] Intermediate M38c, 230mg, 95%. 1 H NMR(500MHz, CDCl3)δ8.43(s,1H),7.87(d,J=8.5Hz,1H),7.68–7.62(m,2H),7.60(dd,J=10.0,5.0Hz,1H),7.25–7.19(m,2H),4.63(t,J=8.5Hz, 2H),4.57(t,J=7.5Hz,2H),4.17–4.08(m,1H),4.07–3.70(m,2H),3.48( m,4H),2.80(s,3H),1.50(s,9H),1.40–1.13(m,2H),1.04–0.48(m,2H).

[0170] Compound C6, 45 mg, yield 82%. 1H NMR (500MHz, CDCl3) δ9.25 (dd, J=4.5, 2.0Hz, 1H), 7.84 (d, J=8.5Hz, 1H), 7.67–7.58 (m, 2H), 7. 56(dd,J=10.0,5.0Hz,1H),7.26–7.17(m,2H),6.61(d,J=7.0Hz,1H),4.77–4.58(m,5H),4.50– 4.39(m,1H),3.26(dd,J=11.5,6.0Hz,1H),3.23–3.15(m,1H),3.08(dd,J=11.5,2.5Hz,1H),3. 04–2.96(m,1H),2.78(s,3H),2.37–2.24(m,1H),1.93–1.80(m,1H).HRMS(ESI-TOF)m / z:[M+H] + calcd for C 24 H 24 N7OF 446.2099, found 446.2098.

[0171] Compound C7, 35 mg, yield 79%. 1 H NMR (400MHz, CDCl3) δ9.24 (dd, J=4.8, 2.4Hz, 1H), 7.85 (d, J=8.0Hz, 1H), 7.68–7.58 (m,2H),7.56(dd,J=10.0,5.2Hz,1H),7.26–7.17(m,2H),6.04(d,J=8.0Hz,1H),4.77 –4.59(m,4H),4.48–4.35(m,1H),4.24–4.10(m,1H),3.30–3.17(m,2H),2.91–2.80( m,2H),2.78(s,3H),2.20–2.08(m,2H),1.72–1.55(m,2H).HRMS(ESI-TOF)m / z:[M+H] + calcd for C 25 H 26 N7OF 446.2099,found446.2098.

[0172] Compound C8, 48 mg, yield 74%. 1H NMR (400MHz, CDCl3) δ8.42(dd,J=3.6,2.4Hz,1H),7.87(d,J=8.0Hz,1H),7.69–7.61(m,2H),7.58(dd,J=9.6,5.2Hz,1H),7.25–7.16(m ,2H),4.67–4.53(m,4H),4.20–4.08(m,1H),4.04–3.27(m,4H),3.03(s,2H),2.79(s,3H),0.77–0.41(m,4H).HRMS(ESI-TOF)m / z:[M+H] + calcd for C 26 H 26 N7OF 472.2256,found472.2251.

[0173] In this embodiment, the synthetic route for compounds C9 to C10 (see Figure 24) is as follows:

[0174] The synthesis of compound C9 was as follows: 2-Chloroquinoxaline (164 mg, 1.0 mmol) and intermediate M3a (245 mg, 1.0 mmol) were dissolved in anhydrous DMF (10 mL) in a 125 mL flask, and cesium carbonate (977 mg, 3.0 mmol) was added. The reaction mixture was heated to 110 °C for 6 h, and the reaction was monitored by TLC. After the reaction was complete, the cesium carbonate was removed by filtration, and the filtrate was distilled under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography to give a pale yellow solid compound C9 (186 mg, 50% yield). 1 H NMR (400MHz, CDCl3) δ9.35(d,J=6.8Hz,1H),8.27(s,1H),7.89(dd,J=8.0,1.2Hz,1H),7.75–7.68(m,2H),7.57(dt,J=7.2,1.2Hz,1H),7.43(dt ,J=7.2,1.2Hz,1H),7.38(dt,J=7.2,1.2Hz,1H),7.04(td,J=6.8,1.2Hz,1H),4.82–4.67(m,5H),4.47(q,J=7.2Hz,2H),1.49(t,J=7.2Hz,3H).

[0175] The synthesis of compound C10 was carried out as follows: using 2-chloroquinoxaline (100 mg, 0.61 mmol) and intermediate M17e (159 mg, 0.61 mmol) as raw materials, and following the synthesis method of compound C9, a pale yellow solid compound C10 (128 mg, yield 54%) was obtained. 1H NMR (400MHz, DMSO-d6) δ8.51(dd,J=4.8,2.4Hz,1H),8.41(s,1H),7.86(d,J=8.8Hz,1H),7.72(dd,J=10.0,5.2Hz,1H),7.66–7.58(m,2H) ,7.48(dt,J=6.4,2.0Hz,1H),7.41(dt,J=6.4,2.0Hz,1H),4.65(t,J=8.4Hz,2H),4.44(t,J=8.4Hz,2H),4.30–4.20(m,1H),3.04(s,6H).

[0176] Example: Preparation method of compound B16 hydrochloride or trifluoroacetate.

[0177] Specifically, the process involves dissolving compound B16 in methanol in a 50 mL flask, slowly adding a 1.0 mol / L hydrochloric acid aqueous solution with stirring, and stirring the reaction mixture at room temperature for 1 hour.

[0178] After the reaction was completed, the system was concentrated by vacuum distillation to obtain a pale yellow solid hydrochloride B16·3HCl;

[0179] In a 50 mL flask, compound B16 was dissolved in dry dichloromethane, and 0.5 mL of trifluoroacetic acid was added with slow stirring.

[0180] The reaction mixture was stirred at room temperature for 4 hours.

[0181] After the reaction was completed, the system was concentrated by vacuum distillation to obtain a pale yellow oily trifluoroacetate B16·CF3COOH.

[0182] Similar organic acid salt products are obtained by reacting pyrazolopyridine compounds with a nitrogen-containing four-membered ring with other organic acids, including but not limited to hydrochloric acid, hydrobromic acid, hydrofluoric acid, phosphoric acid, acetic acid, oxalic acid, sulfuric acid, methanesulfonic acid, salicylic acid, trifluoroacetic acid, trifluoromethanesulfonic acid, naphthalenesulfonic acid, maleic acid, fumaric acid, citric acid, tartaric acid, succinic acid, malic acid, and glutamic acid.

[0183] (3) Performance testing of representative compounds

[0184] The following tests were performed on the pyrazolopyridine compounds linked to the aza-four-membered ring in each embodiment:

[0185] (3.1) Inhibition test of a class of aza-4-membered ring-linked pyrazolopyridine compounds on PDE10A (phosphodiesterase type 10A) activity

[0186] At room temperature, the pyrazolopyridine compound to be tested, linked to a four-membered ring, was reacted with 20,000–30,000 cpm of a solution containing 1.0 μg / mL recombinant clone PDE10A protein, 20 mmol / L Tris-HCl (pH 7.5), 4 mmol / L dithiothreitol, and 10 mmol / L MgCl2. 3 The H-cAMP mixture was incubated for 15 minutes, and then the reaction was stopped with 0.2 mol / L ZnSO4 and 0.2 mol / L Ba(OH)2, respectively. The unreacted ions in the supernatant were then measured using a PerkinElmer 2910 counter. 3 H-cAMP was used, and the experiment was repeated at least three times. The IC50 value for the inhibitory effect of aza-4-membered ring-linked pyrazolopyridine compounds on the activity of recombinant clone PDE10A protein was obtained through concentration testing and nonlinear regression calculations. 50 value.

[0187] The results of the activity inhibition tests of the aza-4-membered ring-linked pyrazolopyridine compounds against the recombinant clone PDE10A protein in each embodiment are shown in Table 1. Under the same conditions, the activity inhibition test results of the positive control (papaverine) against the recombinant clone PDE10A protein should be controlled within IC50. 50 = within the range of 50 to 100 nM, to ensure the IC50 of the measured compound. 50 The value data has a unified reference standard:

[0188] Table 1. Inhibitory activity of PDE10A protein by pyrazolidine compounds linked by aza-four-membered rings.

[0189]

[0190] (3.2) Metabolic stability test of a class of pyrazolopyridine compounds linked by a nitrogen-containing four-membered ring.

[0191] A 1 μmol / L concentration of the target aza-4-membered ring-linked pyrazolopyridine compound was incubated with 0.5 mg / mL rat liver microsomes (RLM) and 1.0 mmol / L NADPH in a 0.1 mol / L PBS buffer (pH 7.4) at 37 °C. The reaction was timed, and 30 μL of the incubation system was taken at 0, 5, 15, 30, and 60 min, respectively, and 200 μL of pre-cooled methanol (containing 20 μL of 1 μg / mL tolbutamide) was added to terminate the reaction. The remaining concentration of the target aza-4-membered ring-linked pyrazolopyridine compound in the system was measured using a Thermo ultra-high performance liquid chromatography-tandem mass spectrometry system, and the measurement was repeated three times. The half-life of the aza-4-membered ring-linked pyrazolopyridine compound metabolized by rat liver microsomes (RLM) was calculated by logarithmic concentration testing and linear regression. The larger the half-life of pyrazolopyridine compounds linked by a nitrogen-containing four-membered rings in rat liver microsomes (RLM), the slower the CYP metabolic enzymes metabolize the compound pair in vitro, and the more stable the compound may be in vivo.

[0192] The metabolic stability test results of representative pyrazolopyridine compounds linked by a nitrogen-containing four-membered ring are shown in Table 2. Among them, under the same conditions, the metabolic stability half-life T of the positive control (phenacetin) is... 1 / 2 Within the 30–50 min range, to ensure the half-life T of the compound being measured. 1 / 2 The data adheres to a unified reference standard:

[0193] Table 2. Results of metabolic stability tests for pyrazolidine compounds linked by a nitrogen-containing four-membered rings.

[0194]

[0195] (3.3) Selectivity test of pyrazolopyridine compounds linked by aza-four-membered rings to the PDEs superfamily

[0196] Taking the pyrazolopyridine compounds A11, B11 and B16 linked by a tert-atom ring as examples, the selectivity of the inhibitory effect of the pyrazolopyridine compounds linked by a tert-atom ring on the PDE family was tested; for the specific experimental procedure, refer to (1) the test method for the activity inhibition of PDE10A (phosphodiesterase type 10A) by the pyrazolopyridine compounds linked by a tert-atom ring.

[0197] The selectivity of pyrazolopyridine compounds A11, B11, and B16, linked by a nitrogen-containing four-membered ring, to PDE family enzymes is shown in Table 3.

[0198] Table 3. Selectivity test results of compounds A11, B11, and B16 for the PDEs superfamily.

[0199]

[0200]

[0201] Selectivity factor = IC 50 (PDEs) / IC 50 (PDE10A)

[0202] (3.4) Prediction of blood-brain barrier (BBB) ​​permeability of pyrazolopyridine compounds linked by aza-4-membered rings

[0203] Prepare a 5 mg / mL stock solution of the pyrazolopyridine compound to be tested with a four-membered ring using DMSO; take 20 μL of the 5 mg / mL stock solution of the pyrazolopyridine compound to be tested with a four-membered ring and add it to a mixture of KH2PO4-K2HPO4 buffer (pH 7.4, 50 mmol / L) and ethanol in a volume ratio of 7:3, and dilute the mixture to 100 μg / mL to prepare a secondary stock solution.

[0204] Porcine brain tissue extract (PBL) was dissolved in dodecane. The sample was coated onto a hydrophobic membrane (0.45 μm Hydrohobic High Protein Binding Immobilon-P Membrane). Then, 200 μL of a secondary stock solution of the 100 μg / mL aza-4-membered ring-linked pyrazolopyridine compound to be tested was added to the top of the hydrophobic membrane as a delivery pool, and 300 μL of the solution was added to the other side of the membrane as a receiving pool, ensuring full contact between the receiving solution and the hydrophobic membrane. After standing at room temperature for 12 hours, 200 μL of the secondary stock solution of the 100 μg / mL aza-4-membered ring-linked pyrazolopyridine compound to be tested was added to 300 μL of a mixture (pH 7.4, 50 mmol / L KH2PO4-K2HPO4 buffer / ethanol = 7:3, V / V), and mixed thoroughly to obtain the theoretical equilibrium concentration solution. The drug concentrations in the acceptor solution, theoretical equilibrium concentration solution, and blank well KH₂PO₄-K₂HPO₄ buffer were determined using a UV plate reader, and the effective permeability (Pe) of the compounds was calculated using Microsoft Office Excel software. Taking representative pyrazolopyridine compounds with aza-four-membered rings as examples in Table 4, their effective permeability Pe was tested. A higher Pe value indicates higher blood-brain barrier permeability. MP-10, a selective inhibitor of PDE10A and a candidate drug for treating schizophrenia, was used as a comparison. The test results are shown in Table 4.

[0205] Table 4. Blood-brain barrier permeability tests of pyrazolopyridine compounds linked by aza-four-membered rings.

[0206]

[0207]

[0208] Table 4 shows that MP-10 has the highest effective permeability Pe value, while when Pe ≥ 2.0 × 10⁻⁶... -6 cm·s -1 Compounds are typically predicted to have good blood-brain barrier permeability. Compared to MP-10, the pyrazolopyridine compounds linked by the aza-four-membered ring of this invention have lower blood-brain barrier permeability, indicating a smaller potential inhibitory effect on PDE10A in the central nervous system. This suggests a significant reduction in the potential side effects of central nervous system inhibition. Therefore, when the pyrazolopyridine compounds linked by the aza-four-membered ring of this invention are used to treat and / or prevent related diseases, including myocardial hypertrophy, myocardial remodeling, myocardial fibrosis, and myocardial injury, they will not produce a significant inhibitory effect on PDE10A in the central nervous system.

[0209] (3.5) Confirmatory experiments on the treatment of myocardial hypertrophy with pyrazolopyridine compounds linked by aza-four-membered rings.

[0210] Thirty-six C57 mice were randomly divided into six groups: Group 1 was the normal control group (control group); Group 2 was the model group; Group 3 was the low-dose group (2.5 mg / kg compound B16); Group 4 was the medium-dose group (5.0 mg / kg compound B16); Group 5 was the high-dose group (10.0 mg / kg compound B16); and Group 6 was the positive control group (propranolol group) (10 mg / kg compound B16).

[0211] Except for the normal control group (control group), which received physiological saline, the model group and other groups were subcutaneously injected with isoproterenol (ISO) 5.0 mg / kg / day for 14 consecutive days to induce myocardial hypertrophy and myocardial injury in C57 mice. Simultaneously, from the first day of modeling, the high-dose group was administered compound B16 at 10.0 mg / kg once daily by gavage, the medium-dose group was administered compound B16 at 5.0 mg / kg once daily by gavage, the low-dose group was administered compound B16 at 2.5 mg / kg once daily by gavage, the positive control group (propranolol group) was administered propranolol at 10 mg / kg once daily by gavage, while the normal control group and model group were administered an equal volume of solvent [1.6% DMSO + 98.4% CMC-Na (0.5%)] once daily by gavage for 14 consecutive days.

[0212] Twenty-four hours after the last administration, echocardiography was performed using a Technos MPX ultrasound system (ESAOTE, Italy) coupled with an 8.5-MHz image converter. Specifically, C57 mice were anesthetized with isoflurane, their chest area was shaved, and then echocardiography was performed at the level of the papillary muscle of the heart using a two-dimensional ultrasound-guided M-mode curve (probe frequency 8.5MHz). The cardiac function indicators detected included left ventricular ejection fraction (EF%), left ventricular fraction shortening (FS%), left ventricular end-diastolic volume, and left ventricular end-systolic volume. At the same time, biological samples were collected, and cardiac indices and the levels of biomarkers in myocardial tissue (expression levels of atrial natriuretic peptide (ANP) mRNA and β-myosin heavy chain (β-MHC) mRNA) were detected. The results are shown in Figures 1-5.

[0213] As shown in Figures 1-5, compared to the model group, the aza-4-membered ring-linked pyrazolopyridine compound B16 of the present invention can improve the heart-to-body weight ratio and heart-to-tibia ratio in C57 mice with ISO-induced myocardial hypertrophy and myocardial injury, improve the left ventricular end-systolic diameter and left ventricular end-diastolic diameter, increase the left ventricular ejection fraction and left ventricular shortening fraction, and reduce the mRNA content of myocardial tissue biomarkers ANP and β-MHC. At the same time, tissue section staining shows that the aza-4-membered ring-linked pyrazolopyridine compound B16 mentioned in the present invention can significantly improve myocardial hypertrophy and myocardial fibrosis induced by isoproterenol (ISO) and has a therapeutic effect of protecting the myocardium.

[0214] (3.6) Confirmatory experiments on the treatment of pulmonary fibrosis with pyrazolopyridine compounds linked by aza-four-membered rings

[0215] The efficacy verification experiment of compound B11 in treating pulmonary fibrosis was divided into 6 groups: low-dose group (5.0 mg / kg), high-dose group (10.0 mg / kg), dry powder inhaler intrapulmonary bolus administration group (B11 net content 0.5 mg / kg), positive control pirfenidone (PFD) gavage administration group (300 mg / kg), normal control group, and model group. Each group included 13 male SPF grade C57 mice, aged 6-8 weeks and weighing between 18-22 g. Except for the normal control group which used an equal volume of physiological saline, all other groups were modeled by intratracheal injection of 3 mg / kg bleomycin, with an administration volume of 5 mL / kg. After modeling, the mice were rotated to ensure uniform distribution of the drug solution and induce pulmonary fibrosis. Starting from the second day, [2.0% DMSO, 3.0% Solutol ( Mice were treated with B11 dissolved in HS-15 and 95% saline (V / V). The mice were then administered B11 via intraperitoneal injection (high dose H, 10.0 mg / kg) and B11 low dose L, 5.0 mg / kg; lung bolus administration of dry powder inhaler (0.5 mg / kg net B11); and gavage administration of pirfenidone mixed with CMC-Na (0.5%) (300 mg / kg) as a positive control for three consecutive weeks, with weekly body weight measurements. Lung administration of B11 was performed in anesthetized mice with endotracheal intubation equipment. Normal controls and model groups received an equal volume of saline. To evaluate the efficacy of B11 in treating pulmonary fibrosis, respiratory function in mice was monitored using dynamic whole-body phlometry (WBP) while awake. After drug treatment, all mice were kept awake and their respiratory function was tested using an animal respiratory system monitoring instrument. The test was performed in parallel in each group. The respiratory parameters included: airway narrowing index (Penh), end-expiratory apnea (EEP), and relaxation time (RT) reduction. Representative results are shown in Figure 6.

[0216] Subsequently, pulmonary function monitoring was performed in mice under anesthesia (PFT pulmonary function testing system): Animals were anesthetized with 0.45% sodium pentobarbital, their tracheas were cut open, and endotracheal tubes were inserted. The instrument monitored airway resistance (RI), dynamic compliance (Cdyn), total lung capacity (TLC), inspiratory capacity (IC), forced vital capacity (FVC), and forced expiratory volume in one second (FEV100). In the PFT method, vitamin B11 could improve bleomycin-induced abnormalities in airway resistance (RI), dynamic compliance (Cdyn), total lung capacity (TLC), inspiratory capacity (IC), forced vital capacity (FVC), and forced expiratory volume in one second (FEV100), as shown in Figure 7.

[0217] The above results indicate that B11 can improve the decline in lung function in mice with pulmonary fibrosis, regardless of whether it is the WBP or PFT method. The B11 dry powder inhaler group (net B11 content 0.5 mg / kg) showed the best effect, followed by the high-dose intraperitoneal injection group (10 mg / kg), and both were superior to the positive control drug PFD group. This shows that the B11 inhalation formulation can be effective at a lower dosage, which greatly reduces its potential toxic side effects.

[0218] Lung tissue samples from the same location were fixed and preserved at room temperature in 4% paraformaldehyde. Approximately 10 mg of lung tissue from the same location was preserved in RNAlater preservation solution at -80°C. The remaining lung tissue was flash-frozen in liquid nitrogen in cryovials and then preserved at -80°C. The obtained lung tissue sections were processed using routine H&E and Masson staining by a professional biotechnology company, and semi-quantitative scoring was performed randomly by relevant professionals. As shown in Figure 8, after B11 administration intervention, H&E staining showed improvement in alveolitis and pulmonary fibrosis, while Masson staining indicated a reduction in collagen fiber proliferation. Semi-quantitative scoring showed that the B11 lung administration group (net B11 content 0.5 mg / kg) had the best effect, followed by the high-dose intraperitoneal injection group (10 mg / kg), both of which were superior to the pirfenidone (PFD) gavage group. These results indicate that B11 can improve pulmonary fibrosis lesions in bleomycin-induced pulmonary fibrosis mice.

[0219] (4) Representative example: Preparation of dry powder inhalers (Figure 9)

[0220] (4.1) In this embodiment, the preparation method of compound B11 nano-suspension is as follows:

[0221] Compound B11 exhibits poor solubility in aqueous solutions containing lyophilization protectants, failing to achieve the desired drug loading. This issue is addressed by preparing a nano-suspension, as shown in Figure 9 (left). One or more of the following are selected as stabilizers for the nano-suspension: poloxamer F68, poloxamer F127, sodium dodecyl sulfate, phospholipids, polyvinyl alcohol, sodium cholate, sodium deoxycholate, polyethylene glycol succinate (TPGS), Tween 80, and Soluplus, and a 0.1-5% aqueous solution is prepared, denoted as solution A. Compound B11 is prepared into a saturated solution using solvents such as DMSO, acetone, methanol, or ethanol. A precision syringe pump is used to inject 5-20 times the volume of liquid A. The PTFE tube has an inner diameter of 0.3-1.0 mm, the injection rate is 0.1-1.0 mL / min, and the collection container is a 10 mL vial. The liquid in the collection vial is stirred at 1000-1500 rpm. The nano-suspension was dispersed by ultrasonication under ice bath conditions with a probe power of 20-60W for a total working time of 15-30 minutes, with an ultrasonic cycle of 2 seconds on and 1 second off. The B11 nano-suspension was then purified by centrifugation and ultrafiltration.

[0222] (4.2) Preparation method of compound B11 dry powder inhaler:

[0223] This study used ultrasonic spray freeze-drying technology to prepare dry powder inhalers. One or more of mannitol, sucrose, trehalose, glucose, and L-leucine were selected as freeze-drying protectants. The final type of freeze-drying protectant was determined based on the appearance of the dry powder, particle sphericity, geometric particle size, and drug content. The B11 nano-dispersion containing the freeze-drying protectant was subjected to ultrasonic spray freeze-drying, as shown in Figure 9 (right). The B11 nano-suspension containing the freeze-drying protectant was connected to a precision syringe pump via a syringe. A 50 mL beaker was used as the receiving container, with a stir bar added to the beaker to agitate the liquid nitrogen and prevent boiling over and particle agglomeration. The beaker was placed on an insulating sponge plate, and a small amount of liquid nitrogen was poured in for pre-cooling for several tens of seconds. After the boiling sound of the liquid nitrogen gradually weakened, about 30 mL of liquid nitrogen was poured in. The entire process was monitored, and the liquid nitrogen supply was ensured to be sufficient.

[0224] The ultrasonic spray freeze-drying process parameters are as follows: liquid solid content of 5-30%, liquid supply rate of 0.1-5 mL / min, PTFE tube inner diameter of 0.3-1.0 mm, frequency of frequency generator of 40-200 kHz, and amplitude of 20-80%. The collection container contains liquid nitrogen for instantaneous freezing. After collection, the mouth of the beaker is covered with perforated plastic wrap, and the liquid nitrogen is slowly evaporated until a small amount remains before being placed in a freeze dryer and dried at -50℃ and 0.05 mbar for 24 hours. The collected freeze-dried powder is stored in an environment with a relative humidity (RH) < 10%.

[0225] (4.3) Characterization of compound B11 nanosuspension (Figure 10)

[0226] B11 nanoparticle suspension particle size determination: The nanoparticle suspension was diluted with secondary water to a suitable concentration (slightly milky white), and measured using a nanoparticle size and Zeta potential analyzer (Zetasizer nano 2S). The material refractive index (RI) was set to 1.469 and the dispersion medium refractive index (RI) was set to 1.330. Each sample was measured three times. The results were analyzed using Origin 2021 software, and the results are shown in Figure 10A.

[0227] Morphology of B11 nanoparticles observed by transmission electron microscopy: A copper mesh (carbon film carrier) was placed face up on a metal support. After hydrophilic treatment of the film surface, the sample was prepared using the flotation method. The film-coated side of the carrier was floated on a droplet of nano-suspension (diluted with TPGS solution to an appropriate concentration). After 1 minute, the carrier was lifted with tweezers, and excess solution was absorbed with filter paper. The film-coated side of the carrier was then floated on phosphotungstic acid staining solution for 1 minute, and excess staining solution was absorbed with filter paper. The sample was located and photographed using a transmission electron microscope (JEM1400) at 120 kV. The results are shown in Figure 10B.

[0228] (4.4) Characterization of compound B11 dry powder inhaler

[0229] Drug loading measurement: Accurately weigh 5 mg of dry powder, dissolve the dry powder in 100 μL LDMSO, then dilute with 900 μL methanol, and determine according to chromatographic conditions. Record the peak area, calculate the concentration of compound B11 using the B11 standard curve, and calculate the drug loading data of the dry powder. The experiment was repeated three times in parallel.

[0230] Scanning electron microscopy observation of dry powder morphology: Non-conductive adhesive was attached to a metal plate for sample preparation. A small amount of dry powder sample was blown evenly onto the adhesive surface using a rubber bulb. The sample preparation surface should not be touched after preparation to prevent changes in sample morphology. Gold was sputtered for 150s using an ion sputtering instrument (J20 Ion Sputter Coater) at 5W and 6Pa to avoid obvious electrostatic phenomena in the porous structure. After preparation, the sample was placed in an EVO tungsten filament scanning electron microscope (EVOMA10) and photographed at 10.00kV and appropriate magnification. The results are shown in Figure 11A, and the particle size distribution is shown in Figure 11B. (4.5) In vitro atomization performance test of compound B11 dry powder inhaler

[0231] According to pharmacopoeia regulations, the aerodynamic properties of fine particles in inhaled formulations can be determined using a Next-Generation Impactor (NGI). An NGI is a cascaded impactor with seven stages and one microporous collector (MOC). The device includes detachable collection cups located on the same horizontal plane. Dry powder in the adapter is carried by the airflow through each stage of nozzles. The smaller the aerodynamic particle size, the closer the collected particles are to the airflow outlet. Except for the first stage, all other stages employ a porous design. The aerodynamic particle size of the particles trapped in each stage is primarily determined by the airflow velocity and the pore size of each stage.

[0232] The prepared drug-loaded dry powder was loaded into HPMC capsules No. 3 (stored in a drying cabinet), with an average of 9 mg of dry powder per capsule. The amount of dry powder loaded was accurately weighed. Silicone oil was sprayed onto the metal collection trays (S1 to S7 and MOC), and 15 mL of solvent was added to the central collection cup of the pre-separator. The device was then connected. The P1 line and output line on the flow control valve were connected to the DUSA line. The adapter and the generator with the capsule (without puncturing) were connected to the DUSA line. The pressure pump was turned on, and P1 was adjusted to 4.0 kPa. The DUSA line was removed, and the entire device was fully connected without connecting the adapter. The flow sensor was connected to the artificial throat, and the airflow velocity Q and other parameters (P2, P3, PA) were tested. The working parameters were set as follows: the number of working times was 10, and the working time was calculated according to formula [1]. The capsule was loaded into the generator. After confirming that it was punctured, the generator was loaded into the adapter. The working button was clicked to start working. After 10 capsules were filled, one determination was completed. The device was disassembled, and the deposited dry powder in the generator, adapter, artificial throat, pre-separator, and all collection trays was collected separately using small volumes of solvent. The collected liquid was diluted to volume with methanol, simultaneously dissolving and releasing the drug from the silicone oil. The drug content was determined using high-performance liquid chromatography (HPLC), and the results were used... DataAnalysisSoftware and Origin2021 were used for calculation and analysis, and the results are shown in Figure 12.

[0233]

[0234] The above description is merely a further embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope disclosed in the present invention, based on the technical solution and concept of the present invention, shall fall within the scope of protection of the present invention.

Claims

1. A class of pyrazolopyridine compounds or pharmaceutically acceptable salts thereof linked by a nitrogen-containing four-membered ring, characterized in that: Wherein: the substituents R1, R2, and R3 are independently H, halogen, and C, respectively. 1-3 Alkyl, C 1-3 One of the alkoxy groups; For quinoxaline (A): R6 and R7 substituted quinolines (B) Or R6, R 10 Substituted quinazoline (C): R4 is: One of them; R5 represents H and C. 1-5 One of the alkyl groups; R6 is an independent F that is either monosubstituted or disubstituted. 18 One of F, Cl or methyl; R7 can be methyl, -C(=O)-R, or R8, where R8 is: One of them; R9 is: One of them; R 10 Independently, it is one of methyl, ethyl, isopropyl, or R9; R is 2. The class of aza-tetra-membered ring-linked pyrazolopyridine compounds or pharmaceutically acceptable salts thereof according to claim 1, characterized in that, The pharmaceutically acceptable salt is a product obtained by reacting an acid with a pyrazolopyridine compound having a four-membered ring linked by an aza-type ring as shown in Formula (I); the acid is selected from one or more of hydrochloric acid, hydrobromic acid, hydrofluoric acid, phosphoric acid, oxalic acid, sulfuric acid, methanesulfonic acid, salicylic acid, trifluoroacetic acid, trifluoromethanesulfonic acid, naphthalenesulfonic acid, maleic acid, fumaric acid, citric acid, acetic acid, tartaric acid, succinic acid, malic acid, and glutamic acid.

3. A class of pyrazolopyridine compounds linked by a nitrogen-containing four-membered ring, characterized in that, The compound has the following structure:

4. The use of a class of aza-quaternary ring-linked pyrazolopyridine compounds according to any one of claims 1 to 3 in the preparation of cardioprotective drugs.

5. The use of a class of pyrazolidine compounds with a nitrogen-containing four-membered ring as described in any one of claims 1 to 3 in the preparation of drugs for treating pulmonary fibrosis.

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