A pyrazolo pyridine compound, and a preparation method and application thereof

Abstract

The application belongs to the technical field of medicine synthesis, and particularly relates to a pyrazolopyridine compound and a preparation method and application thereof. The pyrazolopyridine compound provided by the application has a structure as shown in formula I. The pyrazolopyridine compound provided by the application has obvious protection on damaged myocardial cells. Meanwhile, the compound can play an effective myocardial protection role by improving myocardial cell activity, reducing lactate dehydrogenase release, and reducing the expression of pro-inflammatory factors TNF-alpha, IL-18, IL-1beta and IL-6. Therefore, the application not only enriches the molecular bank of myocardial protection drugs, and provides a theoretical basis and a candidate skeleton for developing a new myocardial protection drug, and has a wide application prospect in the preparation of the pyrazolopyridine compound and the development of a myocardial protection drug with more excellent activity.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical synthesis technology, specifically relating to a pyrazolopyridine compound, its preparation method, and its application. Background Technology

[0002] Cardiovascular disease (CVD) is one of the leading causes of death worldwide. Early screening and timely intervention are crucial for improving the prognosis and long-term survival benefits of CVD patients. In recent years, with the continuous discovery and application of various biomarkers, the early warning capability of cardiovascular disease has been significantly improved. However, with the increasingly serious aging of the population, the incidence rate continues to rise, and the related medical burden remains relatively heavy.

[0003] Myocardial cell damage is the pathological basis for the development of many cardiovascular diseases, and is widely present in the processes of myocardial infarction, myocardial fibrosis, myocardial hypertrophy, and atherosclerosis. Myocardial infarction (MI) is primarily caused by coronary artery thrombosis, characterized by myocardial necrosis and compensatory ventricular remodeling following myocardial ischemia. If the thrombus is not cleared in time, it will lead to persistent coronary artery obstruction, prolonging myocardial ischemia time and causing irreversible myocardial damage. Furthermore, it can induce myocardial infarction and heart failure through oxidative stress. Currently, the most widely used treatment strategy for myocardial infarction is to restore blood supply to the ischemic myocardium. However, while reperfusion can save ischemic myocardium, it can also cause myocardial ischemia-reperfusion injury (MIRI), affecting the internal structure and function of the myocardium. In addition, MIRI often occurs during cardiac surgeries such as heart transplantation, arterial bypass surgery, and cardiopulmonary resuscitation, and is directly related to the high mortality rate and poor prognosis of cardiovascular diseases. Myocardial fibrosis is characterized by extracellular collagen accumulation without cardiomyocyte necrosis. However, over time, chronic stress overload leads to cardiomyocyte necrosis and alternating fibrosis, a process that is irreversible. Fibrosis restricts the heart's oxygen and nutrient supply, causing changes in electrical conduction and structure, making patients more susceptible to arrhythmias and heart failure. Myocardial hypertrophy is a compensatory response of the heart to long-term stress overload (such as hypertension and aortic stenosis). It is characterized by increased cardiomyocyte volume, increased protein synthesis, and reactivation of embryonic genetic programs. Persistent pathological hypertrophy eventually leads to heart failure, arrhythmias, and sudden death. Therefore, the dynamic homeostasis of cardiomyocytes plays a crucial role in maintaining the normal physiological activity of myocardial tissue.

[0004] As awareness of the severe consequences of myocardial injury continues to grow, research in the field of myocardial protection has also seen rapid development. This includes percutaneous coronary intervention (PCI), which restores myocardial blood flow by opening occluded blood vessels through balloon dilation or stent implantation; nanoparticles created by covalently binding adenosine with natural lipid squalene using nanomedicine delivery systems to reduce infarct size; and basic preventative drugs such as lipid-lowering drugs, antihypertensive drugs, antiplatelet drugs, and anticoagulants. While these treatment strategies have the ability to reduce myocardial injury, their clinical application is limited by various factors, resulting in less than ideal therapeutic effects. Furthermore, classic myocardial protective drugs such as beta-blockers, angiotensin-converting enzyme inhibitors / angiotensin receptor blockers (ACEIs / ARBs), and statins, although widely used, often require combination therapy and may be accompanied by numerous adverse reactions.

[0005] Therefore, there is an urgent clinical need to expand the types of candidate compounds and drugs with cardioprotective effects, thereby promoting the research and development of drugs for myocardial injury and guiding the implementation of combination drug regimens for multiple aspects of myocardial injury. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a pyrazolopyridine compound, its preparation method, and its applications. The pyrazolopyridine compound provided by this invention has a novel structure and exhibits good protective effects on cardiomyocytes, providing a theoretical basis and structural entity for the development of more cardiomyocyte protective drugs.

[0007] To achieve the above objectives, the first aspect of the present invention adopts the following technical solution: A pyrazolopyridine compound, the structure of which is shown in Formula I: ; In Equation I, R1 is One of them; R2 is One of them.

[0008] To enhance the cardioprotective effect of the compounds, as a preferred embodiment, the pyrazolopyridine compounds are selected from compounds with the following structures, denoted sequentially as compounds L-1 to L-20:

[0009] The second aspect of this invention is the following technical solution: A method for preparing the pyrazolopyridine compound as described above employs the following technical route:

[0010] When R2 is The method for preparing the pyrazolopyridine compound includes the following steps: (1) React raw material A and raw material B in a solvent to obtain intermediate C; (2) Intermediate C and raw material D are reacted in a solvent, and then hydrolyzed in the presence of an alkaline substance to obtain intermediate E; (3) Intermediate E is reacted with Boc hydrazine and a condensing agent in a solvent to obtain intermediate F; (4) Remove the protecting group from intermediate F in a solvent to obtain intermediate G; (5) React intermediate G with raw material H in a solvent to obtain intermediate I; (6) Intermediate I was refluxed in a solvent to obtain intermediate J; (7) React intermediate J with an alkaline substance in a solvent to obtain compound K, which is the pyrazolopyridine compound; When R2 is The method for preparing the pyrazolopyridine compound includes the following steps: (S1) React raw material A and raw material B in a solvent to obtain intermediate C; (S2) Intermediate C and raw material D are reacted in a solvent, and then hydrolyzed in the presence of an alkaline substance to obtain intermediate E; (S3) Intermediate E is reacted with Boc hydrazine and a condensing agent in a solvent to obtain intermediate F; (S4) Remove the protecting group from intermediate F in a solvent to obtain intermediate G; (S5) Intermediate G is reacted with raw material H in a solvent to obtain intermediate I; (S6) Intermediate I was refluxed in a solvent to obtain intermediate J; (S7) Intermediate J is reacted with an alkaline substance in a solvent to obtain compound K; (S8) Compound K was reacted with O-(tetrahydro-2H-pyran-2-yl)hydroxylamine and a condensing agent in a solvent to give intermediate L; (S9) The intermediate L is reacted with an acidic substance in a solvent to obtain compound M, which is the pyrazolopyridine compound. When R2 is The method for preparing the pyrazolopyridine compound includes the following steps: (i) React raw material A and raw material B in a solvent to obtain intermediate C; (ii) Intermediate C is reacted with raw material D in a solvent, and then hydrolyzed in the presence of an alkaline substance to obtain intermediate E; (iii) Intermediate E is reacted with Boc hydrazine and a condensing agent in a solvent to obtain intermediate F; (iv) Remove the protecting group from intermediate F in a solvent to obtain intermediate G; (v) Intermediate G is reacted with starting material H in a solvent to obtain intermediate I; (vi) Intermediate I is refluxed in a solvent to obtain intermediate J; (vii) Intermediate J is reacted with a basic substance in a solvent to obtain compound K; (viii) Compound K is reacted with Boc-hydrazine and a condensing agent in a solvent to give intermediate N; (ix) The intermediate N is reacted with an acidic substance in a solvent to obtain compound O, which is the pyrazolopyridine compound.

[0011] As a preferred embodiment, the raw material A is one of acetophenone, m-methylacetophenone, p-methylacetophenone, m-chloroacetophenone, p-chloroacetophenone, m-bromoacetophenone, p-bromoacetophenone, m-trifluoromethylacetophenone, p-trifluoromethylacetophenone, p-n-propylacetophenone, p-ethylacetophenone, p-methoxyacetophenone, and p-dimethylaminoacetophenone.

[0012] As a preferred embodiment, the alkaline substance is one or more of triethylamine, sodium hydroxide, and sodium bicarbonate.

[0013] As a preferred embodiment, the condensing agent is N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate or N,N-diisopropylethylamine.

[0014] As a preferred embodiment, the acidic substance is one or more of trifluoroacetic acid, hydrochloric acid, formic acid, and acetic acid.

[0015] As a preferred embodiment, the solvent is one or more of tetrahydrofuran, acetic acid, methanol, N,N-dimethylformamide, dichloromethane, water, and phosphorus oxychloride.

[0016] The third aspect of this invention is the following technical solution: The application of a pyrazolopyridine compound as described above in the preparation of a cardioprotective drug.

[0017] As a preferred embodiment, the mechanism of action of the cardioprotective drug includes one or more of the following: enhancing the activity of cardiomyocytes, reducing the release of lactate dehydrogenase from cardiomyocytes, and reducing the expression level of pro-inflammatory factors in cardiomyocytes.

[0018] The technical solution of the present invention has the following beneficial effects: The pyrazolopyridine compounds provided by this invention possess a novel pyrazolopyridine structural skeleton. Experimental results show that the pyrazolopyridine compounds of this invention have a significant protective effect against damaged cardiomyocytes. Furthermore, mechanistic investigation revealed that these compounds can enhance cardiomyocyte activity, reduce lactate dehydrogenase release, and decrease the expression of pro-inflammatory factors TNF-α, IL-18, IL-1β, and IL-6, indicating that they exert cardioprotective effects by inhibiting inflammatory responses and mitigating cell damage.

[0019] Furthermore, the method for preparing the compounds provided by this invention has mild synthesis conditions, is easy to implement, and the compounds synthesized by this method have the characteristics of high yield and high purity.

[0020] Therefore, this invention not only enriches the molecular library of cardioprotective drugs, providing a theoretical basis and candidate skeletons for the development of novel cardioprotective drugs, but also has broad application prospects in the preparation of pyrazolopyridine compounds and the development of cardioprotective drugs with better activity. Attached Figure Description

[0021] Figure 1 The results show the protective effect of compound L-9 at different concentrations on a cardiomyocyte injury model in this invention; where A represents cell survival rate and B represents cell morphology under different treatment conditions. Figure 2 The effect of compound L-9 in this invention on the release rate of lactate dehydrogenase (LDH); Figure 3 The effect of pretreatment with compound L-9 in this invention on the expression of inflammation-related gene mRNA in cardiomyocytes; wherein, A is the mRNA expression level of TNF-α; B is the mRNA expression level of IL-18; C is the mRNA expression level of IL-1β; and D is the mRNA expression level of IL-6. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described clearly and completely below in conjunction with specific embodiments. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0023] Regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The terms "comprising," "including," "having," "containing," etc., used in this invention are open-ended, meaning they include but are not limited to. Unless otherwise stated, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. Although only preferred methods and materials are described in this invention, any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this invention.

[0024] In the following embodiments of the present invention, the structure of the compound was determined by nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HR-MS).

[0025] Example 1

[0026] This embodiment provides a pyrazolopyridine compound, denoted as compound L-1, with the following structural formula: .

[0027] The specific preparation route and process of compound L-1 are as follows:

[0028] (1) Acetophenone (1.0 mmol) and diethyl oxalate (1.1 mmol) were placed in a 250 mL round-bottom flask and dissolved in 30 mL of tetrahydrofuran. Sodium hydride (3 mmol) was slowly added while stirring at room temperature (15~30℃). After stirring at room temperature for 30 min, the mixture was transferred to an oil bath at 55℃ and reacted for 5 h. After the reaction was complete by TLC (petroleum ether: ethyl acetate = 1:1, v / v), tetrahydrofuran was removed by vacuum distillation. 30 mL of water was added and mixed. Formic acid was added dropwise to adjust the pH to 5~6 while stirring. A solid precipitated out. The mixture was filtered to obtain intermediate a.

[0029] (2) Intermediate a (1 mmol) and 5-amino-1-ethylpyrazole (1 mmol) were placed in a 100 mL round-bottom flask, 20 mL of glacial acetic acid was added, and the mixture was dissolved by sonication. The mixture was then transferred to an oil bath at 100 °C and heated for 5 h. After the reaction was complete by TLC (petroleum ether: ethyl acetate = 1:1, v / v), the mixture was cooled to room temperature and the solvent glacial acetic acid was removed by vacuum distillation. Then, 30 mL of methanol, sodium hydroxide solution (10 mmol), and water were added in sequence and mixed. The mixture was then transferred to 70 °C and hydrolyzed for 2 h. After the hydrolysis was complete by TLC (petroleum ether: ethyl acetate = 1:1, v / v), most of the solvent was removed by vacuum distillation. 30 mL of water was added and mixed. Formic acid was added dropwise to adjust the pH of the solution to 5-6. The filter cake was collected by suction filtration to obtain intermediate b.

[0030] (3) Intermediate b (1 mmol), tert-butyl hydrazine formate (1.5 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (HATU, 1.5 mmol), and N,N-diisopropylethylamine (2 mmol) were placed in a 100 mL round-bottom flask and N,N-dimethylformamide (25 mL) was added as solvent. The reaction was carried out at room temperature for 6 h. The reaction was monitored by TLC (petroleum ether:ethyl acetate = 1:1, v / v) and the reaction was complete. The mixture was extracted three times with water and ethyl acetate. The upper organic phases were combined, washed with saturated brine, and the organic phase was removed by vacuum distillation. The mixture was purified by column chromatography (petroleum ether:ethyl acetate = 5:1, v / v) to obtain intermediate c.

[0031] (4) Dissolve intermediate c (1 mmol) in dichloromethane, add trifluoroacetic acid dropwise while monitoring continuously until the protecting group is removed. If the solid adheres to the wall, discard the dichloromethane solvent directly. If it does not adhere to the wall, remove the dichloromethane by vacuum distillation. Add water and mix, then add saturated sodium bicarbonate solution to adjust the pH to neutral. After filtration, dry the filter cake to obtain intermediate d.

[0032] (5) Dissolve intermediate d (1 mmol) in dichloromethane and adjust the pH to 8-9 by adding triethylamine dropwise. Separately, dissolve methyl 4-chloroformylbenzoate (1.2 mmol) in dichloromethane and add it dropwise to the reaction solution containing intermediate d under ice bath conditions. After reacting for 3 h, a white solid precipitates. Monitor the reaction by TLC (petroleum ether:ethyl acetate = 1:1, v / v). After the reaction is complete, filter directly and dry the filter cake to obtain intermediate e.

[0033] (6) Intermediate e (1 mmol) was directly dissolved in phosphorus oxychloride and refluxed at 90 °C for 8 h to carry out the cyclization reaction. After the reaction was completed, the reaction solution was added dropwise to ice water under ice bath conditions to quench the reaction. After the temperature dropped to room temperature, the product was filtered. The product obtained by filtration was dissolved in ethyl acetate and purified by column chromatography (petroleum ether: ethyl acetate = 8:1, v / v) to obtain intermediate f.

[0034] (7) Dissolve intermediate f (1 mmol) in methanol, add sodium hydroxide (10 mmol) and 30 mL of water, transfer to an oil bath at 70 °C and heat for 2 h to hydrolyze. TLC monitoring (petroleum ether: ethyl acetate = 1:1, v / v) showed complete hydrolysis. Remove the solvent by vacuum distillation, add water and mix, then add formic acid to adjust the pH of the solution to a weak acid. Filter the solution to obtain intermediate g.

[0035] (8) Dissolve intermediate g (1 mmol) in 15 mL of dichloromethane, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2 mmol) and N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (HATU, 0.2 mmol), stir at room temperature for 0.5 h, add O-(tetrahydro-2H-pyran-2-yl)hydroxylamine (1.5 mmol), and continue the reaction for 3 h. After monitoring the reaction by TLC, extract three times with dichloromethane and water, combine the lower organic phases, wash with saturated brine, and purify by column chromatography (eluting agent: ethyl acetate: petroleum ether = 5:1) to obtain intermediate h.

[0036] (9) The intermediate h (1 mmol) was dissolved in 30 mL of dichloromethane. While stirring, 3 mL of trifluoroacetic acid was slowly added dropwise to remove the protecting group. The precipitated solid was filtered, and the filter cake was ultrasonically dispersed in water. The solution was adjusted to a weakly acidic state with saturated sodium bicarbonate solution and filtered again to obtain the pyrazolopyridine compound of this example, namely compound L-1. The yield of compound L-1 was 72%, a white solid with a melting point of 214.8-215.7 °C. The NMR characterization results were as follows: 1 H NMR (400 MHz, DMSO- d 6) δ8.50 (s, 1H, Ar-H), 8.37 – 8.30 (m, 3H, Ar-H), 8.27 (d, J = 6.6 Hz, 2H, Ar-H), 8.01 (d, J = 8.2 Hz, 2H, Ar-H), 7.58 – 7.49 (m, 3H, Ar-H), 4.57 (q, J =7.2 Hz, 2H, CH2), 1.50 (t, J = 7.2 Hz, 3H, CH3). 13 C NMR (101 MHz, DMSO- d 6) δ164.26, 162.99, 162.53, 155.83, 150.17, 137.58, 136.07, 131.68, 130.11,128.96, 127.89, 127.40, 127.28, 125.09, 124.43, 111.58, 109.26, 41.77, 14.80.HR-MS (ESI), calcd. C 23 H 18 N6O3, [M + H] + m / z: 427.1518, found: 427.1504.

[0037] Example 2 This embodiment provides a pyrazolopyridine compound, denoted as compound L-2, with the following structural formula: .

[0038] The preparation method of compound L-2 differs from that of Example 1 in that acetophenone in step (1) is replaced with m-methylacetophenone, while the other steps are the same as in Example 1. Due to the change in raw materials, the structure of the product obtained in each step of the preparation process also changes accordingly. The yield of compound L-2 prepared in this example is 75%, it is a white solid with a melting point of 223.8-224.3℃. The NMR characterization results are as follows: 1 H NMR (400 MHz, DMSO- d 6) δ 8.57 (s, 1H, Ar-H), 8.43 – 8.34 (m, 3H, Ar-H), 8.11 (d, J = 8.0 Hz, 2H, Ar-H), 8.03 (d, J = 8.1 Hz, 2H, Ar-H), 7.47(t, J = 7.6 Hz, 1H, Ar-H), 7.35 (d, J = 7.5 Hz, 1H, Ar-H), 4.62 (q, J = 7.2Hz, 2H, CH2), 2.46 (s, 3H, CH3), 1.53 (t, J = 7.2 Hz, 3H, CH3). 13 C NMR (101MHz, DMSO- d 6) δ 164.28, 162.93, 162.55, 155.95, 150.17, 138.19, 137.55,136.18, 131.67, 130.76, 128.84, 127.85, 127.79, 127.26, 125.01, 124.64,124.38, 111.60, 109.21, 41.72, 21.17, 14.80. HR-MS (ESI), calcd. C 24 H 20 N6O3, [M+ H] + m / z : 441.1675, found: 441.1661.

[0039] Example 3 This embodiment provides a pyrazolopyridine compound, denoted as compound L-3, with the following structural formula: .

[0040] The preparation method of compound L-3 differs from that of Example 1 in that acetophenone in step (1) is replaced with p-methylacetophenone, while the other steps are the same as in Example 1. Due to the change in raw materials, the structure of the product obtained in each step of the preparation process also changes accordingly. The yield of compound L-3 prepared in this example is 70%, it is a white solid with a melting point of 224.6-227.8℃. The NMR characterization results are as follows: ¹H NMR (400 MHz, DMSO-d6) δ 8.54 (s, ¹H, Ar-H), 8.40 – 8.34 (m, 3H, Ar-H), 8.23 ​​– 8.20 (m, 2H, Ar-H), 8.03 (d, J = 8.1 Hz, 2H, Ar-H), 7.37 (d, J = 8.0 Hz, 2H, Ar-H), 4.60 (q, J = 7.2 Hz, 2H, CH2), 2.39 (s, 3H, CH3), 1.52 (t, J = 7.2 Hz, 3H, CH3). ¹³C NMR (101 MHz, DMSO-d6) δ 164.17, 162.51, 155.65, 150.10, 139.86, 136.02, 134.70, 131.61, 129.50, 127.85, 127.24,127.17, 126.83, 125.08, 124.26, 111.15, 109.02, 41.67, 20.91, 14.77. HR-MS(ESI), calcd. C 24 H 20 N6O3, [M + H]+ m / z: 441.1675, found: 441.1661.

[0041] Example 4 This embodiment provides a pyrazolopyridine compound, denoted as compound L-4, with the following structural formula: .

[0042] The preparation method of compound L-4 differs from that of Example 1 in that acetophenone in step (1) is replaced with m-chloroacetophenone, while the other steps are the same as in Example 1. Due to the change in raw materials, the structure of the product obtained in each step of the preparation process also changes accordingly. The yield of compound L-4 prepared in this example is 75%, a white solid with a melting point of 227.4-229.1℃. The NMR characterization results are as follows:1 H NMR (400 MHz, DMSO- d 6) δ 8.62 (s, 1H, Ar-H), 8.51 (s, 1H, Ar-H), 8.39 (d, J = 7.0 Hz, 3H, Ar-H), 8.32 (s, 1H, Ar-H), 8.03 (d, J = 8.0 Hz, 2H, Ar-H), 7.62 (s, 2H, Ar-H), 4.66 (q, J = 7.2 Hz, 2H, CH2), 1.54 (t, J = 7.2Hz, 3H, CH3). 13 C NMR (101 MHz, DMSO- d 6) δ 164.31, 162.68, 162.32, 154.04,149.93, 139.55, 136.48, 133.90, 130.74, 129.82, 127.78, 127.20, 124.76,124.55, 111.59, 109.59, 41.78, 14.75. HR-MS (ESI), calcd. C 23 H 17 ClN6O3, [M + H] + m / z : 461.1129, found: 461.1115.

[0043] Example 5 This embodiment provides a pyrazolopyridine compound, denoted as compound L-5, with the following structural formula: .

[0044] The preparation method of compound L-5 differs from that of Example 1 in that acetophenone in step (1) is replaced with p-chloroacetophenone, while the other steps are the same as in Example 1. Due to the change in raw materials, the structure of the product obtained in each step of the preparation process also changes accordingly. The yield of compound L-5 prepared in this example is 70%, it is a white solid with a melting point of 221.4-224.3℃. The NMR characterization results are as follows: 1 H NMR (400 MHz, DMSO- d 6) δ 8.61 (s, 1H, Ar-H), 8.48 (s, 1H, Ar-H), 8.38 (d, J = 8.1 Hz, 4H, Ar-H), 8.03 (d,J = 8.0 Hz, 2H, Ar-H), 7.65 (d, J =8.2 Hz, 2H, Ar-H), 4.64 (d, J = 7.8 Hz, 2H, CH2), 1.53 (t, J = 7.1 Hz, 3H,CH3). 13 C NMR (101 MHz, DMSO- d 6) δ 164.23, 162.38, 154.37, 150.00, 136.24,135.00, 128.95, 127.85, 127.21, 124.97,124.51, 109.37,41.76,14.80. HR-MS(ESI), calcd. C 23 H 17 ClN6O3, [M + H] + m / z : 461.1129, found: 461.1113.

[0045] Example 6 This embodiment provides a pyrazolopyridine compound, denoted as compound L-6, with the following structural formula: .

[0046] The preparation method of compound L-6 differs from that of Example 1 in that acetophenone in step (1) is replaced with m-bromoacetophenone, while the other steps are the same as in Example 1. Due to the change in raw materials, the structure of the product obtained in each step of the preparation process also changes accordingly. The yield of compound L-6 prepared in this example is 72%, a pale yellow solid with a melting point of 218.5-221.6℃. The NMR characterization results are as follows: 1 H NMR (400 MHz, DMSO- d 6) δ 8.54 (s, 1H, Ar-H), 8.45 (d, J = 1.9 Hz,1H, Ar-H), 8.40 (s, 1H, Ar-H), 8.35 (d, J = 8.0 Hz, 2H, Ar-H), 8.30 (d, J =7.5 Hz, 1H, Ar-H), 8.03 (d, J = 8.1 Hz, 2H, Ar-H), 7.72 (d, J = 7.9 Hz, 1H,Ar-H), 7.53 (t, J= 7.8 Hz, 1H, Ar-H), 4.61 (q, J = 7.2 Hz, 2H, CH2), 1.52(t, J = 7.2 Hz, 3H, CH3). 13 C NMR (101 MHz, DMSO- d 6) δ 164.27, 162.91, 162.36,153.97, 149.93, 139.76, 136.09, 132.75, 131.70, 130.99, 129.68, 127.87,127.27, 126.41, 125.00, 124.53, 122.49, 111.57, 109.59, 41.79, 14.75. HR-MS(ESI), calcd. C 23 H 17 BrN6O3, [M + H] + m / z : 505.0624, found: 505.0609. Example 7 This embodiment provides a pyrazolopyridine compound, denoted as compound L-7, with the following structural formula: .

[0047] The preparation method of compound L-7 differs from that of Example 1 in that acetophenone in step (1) is replaced with p-bromoacetophenone, while the other steps are the same as in Example 1. Due to the change in raw materials, the structure of the product obtained in each step of the preparation process also changes accordingly. The yield of compound L-7 prepared in this example is 72%, a white solid with a melting point of 227.3-229.9℃. The NMR characterization results are as follows: 1 H NMR (400 MHz, DMSO- d 6) δ 8.57 (s, 1H, Ar-H), 8.42 (s, 1H, Ar-H), 8.36 (d, J = 8.1 Hz, 2H, Ar-H), 8.28 (d, J = 8.5 Hz, 2H, Ar-H), 8.03 (d, J =8.3 Hz, 2H, Ar-H), 7.80 – 7.72 (m, 2H, Ar-H), 4.61 (q, J = 7.2 Hz, 2H, CH2), 1.52 (t, J = 7.2 Hz, 3H, CH3).13 C NMR (101 MHz, DMSO- d 6) δ 164.27, 162.94,162.42, 154.50, 150.04, 136.62, 136.07, 131.88, 131.74, 129.31, 127.89,127.27, 125.05, 124.56, 123.92, 111.32, 109.44, 41.80, 14.78. HR-MS (ESI),calcd. C 23 H 17 BrN6O3, [M + H] + m / z : 505.0624, found: 505.0610.

[0048] Example 8 This embodiment provides a pyrazolopyridine compound, denoted as compound L-8, with the following structural formula: .

[0049] The preparation method of compound L-8 differs from that of Example 1 in that acetophenone in step (1) is replaced with m-trifluoromethylacetophenone, while the other steps are the same as in Example 1. Due to the change in raw materials, the structure of the product obtained in each step of the preparation process also changes accordingly. The yield of compound L-8 prepared in this example is 72%, a pale yellow solid with a melting point of 219.6-224.4℃. The NMR characterization results are as follows: 1 H NMR (400 MHz, DMSO- d 6) δ 8.61 (s, 3H, Ar-H), 8.53 (s, 1H, Ar-H), 8.37 (d, J = 8.4 Hz, 2H, Ar-H), 8.03 (d, J = 8.2 Hz, 2H, Ar-H), 7.91(d, J = 7.8 Hz, 1H, Ar-H), 7.83 (t, J = 7.7 Hz, 1H, Ar-H), 4.64 (q, J = 7.2Hz, 2H, CH2), 1.54 (t, J = 7.2 Hz, 3H, CH3). 13 C NMR (101 MHz, DMSO- d6) δ164.33, 162.94, 162.37, 153.98, 149.99, 138.51, 136.12, 131.76, 131.40,130.10, 129.96, 129.64, 127.88, 127.28, 126.50, 124.99, 124.69, 123.58,122.81, 111.66, 109.73, 41.82, 14.70. HR-MS (ESI), calcd. C 24 H 17 F3N6O3, [M + H] + m / z : 495.1392, found: 495.1378.

[0050] Example 9 This embodiment provides a pyrazolopyridine compound, denoted as compound L-9, with the following structural formula: .

[0051] The preparation method of compound L-9 differs from that of Example 1 in that acetophenone in step (1) is replaced with p-trifluoromethylacetophenone, while the other steps are the same as in Example 1. Due to the change in raw materials, the structure of the product obtained in each step of the preparation process also changes accordingly. The yield of compound L-9 prepared in this example is 71%, a pale yellow solid with a melting point of 221.4-225.9℃. The NMR characterization results are as follows: 1 H NMR (400 MHz, DMSO- d 6 ) δ 8.61 (d, J = 12.8 Hz, 1H, Ar-H), 8.54 (d, J = 8.5 Hz, 3H, Ar-H), 8.37 (d, J = 7.9 Hz, 2H, Ar-H), 8.06–7.97 (m,2H, Ar-H), 7.94 (d, J = 7.6 Hz, 2H, Ar-H), 4.65 (d, J = 7.8 Hz, 2H, CH2), 1.54 (t, J = 7.2 Hz, 3H, CH3). 13 C NMR (101 MHz, DMSO- d6) δ 164.34, 162.38,154.08, 150.39, 150.06, 141.31, 131.80, 128.15, 128.07, 127.87, 127.61,127.26, 127.16, 125.80, 124.99, 124.72, 112.10, 109.79, 41.87, 14.76. HR-MS(ESI), calcd. C 24 H 17 F3N6O3, [M + H] + m / z : 495.1392, found: 495.1379.

[0052] Example 10 This embodiment provides a pyrazolopyridine compound, denoted as compound L-10, with the following structural formula: .

[0053] The preparation method of compound L-10 differs from that of Example 1 in that acetophenone in step (1) is replaced with p-n-propylacetophenone, while the other steps are the same as in Example 1. Due to the change in raw materials, the structures of the products obtained in each step of the preparation process also change accordingly. The yield of compound L-10 prepared in this example is 74%, a white solid with a melting point of 230.6-233.8℃. The NMR characterization results are as follows: 1 H NMR (400 MHz, DMSO- d 6) δ 8.55 (s, 1H, Ar-H), 8.41 – 8.33 (m, 3H, Ar-H), 8.22 (d, J = 8.1 Hz, 2H, Ar-H), 8.02 (d, J = 8.2 Hz, 2H, Ar-H), 7.38(d, J = 8.0 Hz, 2H, Ar-H), 4.60 (q, J = 7.3 Hz, 2H, CH2), 2.64 (t, J = 7.6Hz, 2H, CH2), 1.65 (q, J = 7.4 Hz, 2H, CH2), 1.51 (t, J = 7.2 Hz, 3H, CH3), 0.94 (t, J = 7.3 Hz, 3H, CH3). 13 C NMR (101 MHz, DMSO-d 6) δ 164.25, 162.94,162.56, 155.90, 150.19, 144.49, 136.13, 135.14, 131.66, 128.92, 127.87,127.32, 127.26, 125.06, 124.35, 111.41, 109.08, 41.72, 37.01, 23.94, 14.80,13.66. HR-MS (ESI), calcd. C 26 H 24 N6O3, [M + H] + m / z : 469.1988, found: 469.1975.

[0054] Example 11 This embodiment provides a pyrazolopyridine compound, denoted as compound L-11, with the following structural formula: .

[0055] The preparation method of compound L-11 differs from that of Example 1 in that acetophenone in step (1) is replaced with p-ethylacetophenone, while the other steps are the same as in Example 1. Due to the change in raw materials, the structure of the product obtained in each step of the preparation process also changes accordingly. The yield of compound L-11 prepared in this example is 75%, it is a white solid with a melting point of 227.2-230.4℃. The NMR characterization results are as follows: 1 H NMR (400 MHz, DMSO- d 6) δ 8.55 (s, 1H, Ar-H), 8.42 – 8.30 (m, 3H, Ar-H), 8.23 ​​(d, J = 8.1 Hz, 2H, Ar-H), 8.03 (d, J = 8.2 Hz, 2H, Ar-H), 7.41(d, J = 8.0 Hz, 2H, Ar-H), 4.61 (q, J = 7.2 Hz, 2H, CH2), 2.70 (q, J = 7.5Hz, 2H, CH2), 1.52 (t, J = 7.2 Hz, 3H, CH3), 1.25 (t, J = 7.6 Hz, 3H, CH3). 13 CNMR (101 MHz, DMSO- d6) δ 164.24, 162.54, 155.85, 150.17, 146.07, 135.09,131.65, 128.34, 127.85, 127.38, 127.24, 125.03, 124.34, 111.36, 109.07,41.71, 27.99, 15.39, 14.79. HR-MS (ESI), calcd. C 25 H 22 N6O3, [M + H] + m / z :455.1831, found: 455.1835.

[0056] Example 12 This embodiment provides a pyrazolopyridine compound, denoted as compound L-12, with the following structural formula: .

[0057] The preparation method of compound L-12 differs from that of Example 1 in that acetophenone in step (1) is replaced with p-methoxyacetophenone, while the other steps are the same as in Example 1. Due to the change in raw materials, the structure of the product obtained in each step of the preparation process also changes accordingly. The yield of compound L-12 prepared in this example is 70%, a yellow solid with a melting point of 210.3-215.4℃. The NMR characterization results are as follows: 1 H NMR (400 MHz, DMSO- d 6) δ 8.53 (d, J = 6.1 Hz, 1H, Ar-H), 8.37(d, J = 6.5 Hz, 2H, Ar-H), 8.28 (d, J = 8.2 Hz, 1H, Ar-H), 8.22 (d, J = 8.5Hz, 1H, Ar-H), 8.00 (q, J = 7.0 Hz, 3H, Ar-H), 7.91 – 7.85 (m, 1H, NH), 7.20– 7.02 (m, 2H, Ar-H), 4.59 (dd, J = 11.3, 4.8 Hz, 2H, CH2), 3.84 (s, 3H, CH3), 1.51 (t, J = 7.2 Hz, 3H, CH3). 13 C NMR (101 MHz, DMSO- d6) δ 164.29, 163.83,162.82, 162.66, 161.02, 155.68, 150.27, 136.26, 135.94, 135.84, 128.95,127.89, 127.57, 127.27, 127.08, 126.84, 124.42, 114.35, 108.81, 55.36, 41.72,14.84.

[0058] Example 13 This embodiment provides a pyrazolopyridine compound, denoted as compound L-13, with the following structural formula: .

[0059] The preparation method of compound L-13 differs from that of Example 1 in that acetophenone in step (1) is replaced with p-dimethylaminoacetophenone, while the other steps are the same as in Example 1. Due to the change in raw materials, the structure of the product obtained in each step of the preparation process also changes accordingly. The yield of compound L-13 prepared in this example is 74%, a yellow solid with a melting point of 215.7-218.8℃. The NMR characterization results are as follows: 1 H NMR (400 MHz, DMSO- d 6) δ 8.54 – 8.38 (m, 1H, Ar-H), 8.37 –8.22 (m, 3H, Ar-H), 8.15 (dt, J = 6.1, 2.1 Hz, 2H, Ar-H), 8.06 – 7.97 (m, 2H, Ar-H), 7.95 (dd, J = 8.6, 2.6 Hz, 1H, NH), 6.80 (d, J = 9.1 Hz, 2H, Ar-H), 4.54 (d, J = 7.3 Hz, 2H, CH2), 2.98 (d, J = 4.1 Hz, 6H, 2CH3), 1.47 (t, J =7.3 Hz, 3H, CH3). 13 C NMR (101 MHz, DMSO- d6) δ 165.69, 164.80, 164.34, 163.31,163.17, 156.19, 150.77, 136.46, 132.22, 130.54, 128.40, 128.08, 127.79,127.60, 127.36, 125.85, 125.55, 124.93, 114.86, 111.64, 109.32, 45.47, 42.23,15.35.

[0060] Example 14 This embodiment provides a pyrazolopyridine compound, denoted as compound L-14, with the following structural formula: .

[0061] The preparation method of compound L-14 differs from that of Example 6 in that steps (8) and (9) are omitted, while other steps are the same as in Example 6. Due to the change in process, the structure of the product obtained in each step of the preparation also changes accordingly. The yield of compound L-14 prepared in this example is 45%, a white solid with a melting point of 258.9-261.3℃. The NMR characterization results are as follows: 1 H NMR (400MHz, DMSO- d 6) δ 8.51 (s, 1H, Ar-H), 8.42 (t, J = 1.9 Hz, 1H, Ar-H), 8.37 (s,1H, Ar-H), 8.33 (d, J = 8.6 Hz, 2H, Ar-H), 8.28 (d, J = 8.1 Hz, 1H, Ar-H), 8.15 (d, J = 8.5 Hz, 2H, Ar-H), 7.70 (dd, J = 7.9, 1.1 Hz, 1H, Ar-H), 7.51(t, J = 7.9 Hz, 1H, Ar-H), 4.59 (q, J = 7.2 Hz, 2H, CH2), 1.52 (t, J = 7.3Hz, 3H, CH3). 13 C NMR (101 MHz, DMSO- d6) δ 166.80, 164.29, 162.43, 154.01,149.96, 139.80, 132.76, 131.71, 131.01, 130.09, 129.69, 127.22, 126.45,125.89, 124.56, 123.30, 122.49, 111.64, 109.63, 41.80, 14.76.

[0062] Example 15 This embodiment provides a pyrazolopyridine compound, denoted as compound L-15, with the following structural formula: .

[0063] The preparation method of compound L-15 differs from that of Example 7 in that steps (8) and (9) are omitted, while the other steps are the same as in Example 7. Due to the change in raw materials, the structure of the product obtained in each step of the preparation process also changes accordingly. The yield of compound L-15 prepared in this example is 78%, a pale yellow solid with a melting point of 257.1-260.5℃. The NMR characterization results are as follows: 1 H NMR (400MHz, DMSO- d 6) δ 8.64 – 8.45 (m, 1H, Ar-H), 8.43 – 8.34 (m, 3H, Ar-H), 8.27(d, J = 8.3 Hz, 2H, Ar-H), 8.23 ​​– 8.08 (m, 3H, Ar-H), 7.75 (d, J = 8.6 Hz, 1H, Ar-H), 4.61 (q, J = 7.5 Hz, 2H, CH2), 1.52 (t, J = 7.3 Hz, 3H, CH3). 13 CNMR (101 MHz, DMSO- d 6) δ 166.48, 164.10, 162.46, 154.39, 149.98, 136.56,133.97, 131.83, 131.66, 130.13, 129.24, 127.28, 126.34, 124.41, 123.90,111.24, 109.39, 41.77, 14.76.

[0064] Example 16 This embodiment provides a pyrazolopyridine compound, denoted as compound L-16, with the following structural formula: .

[0065] The preparation method of compound L-16 differs from that of Example 8 in that steps (8) and (9) are omitted, while other steps are the same as in Example 8. Due to the change in raw materials, the structure of the product obtained in each step of the preparation process also changes accordingly. The yield of compound L-16 prepared in this example is 72%, a white solid with a melting point of 265.2-268.7℃. The NMR characterization results are as follows: 1 H NMR (400MHz, DMSO- d 6) δ 8.54 (dd, J = 13.3, 9.1 Hz, 3H, Ar-H), 8.43 (dd, J = 7.5, 3.1Hz, 1H, Ar-H), 8.31 (d, J = 7.8 Hz, 2H, Ar-H), 8.14 (d, J = 7.9 Hz, 2H, Ar-H), 7.88 – 7.77 (m, 2H, Ar-H), 4.59 (q, J = 7.7 Hz, 2H, CH2), 1.56 – 1.47 (t,3H, CH3). 13 C NMR (101 MHz, DMSO- d 6) δ 166.62, 164.27, 162.43, 153.97, 149.98,138.50, 134.81, 131.73, 131.39, 130.11, 127.24, 126.51, 125.52, 124.64,123.57, 111.67, 109.72, 41.82, 14.69.

[0066] Example 17 This embodiment provides a pyrazolopyridine compound, denoted as compound L-17, with the following structural formula: .

[0067] The preparation method of compound L-17 differs from that of Example 11 in that steps (8) and (9) are omitted, while other steps are the same as in Example 11. Due to the change in raw materials, the structure of the product obtained in each step of the preparation process also changes accordingly. The yield of compound L-17 prepared in this example is 74%, a white solid with a melting point of 266.4-269.8℃. The NMR characterization results are as follows: 1 H NMR (400MHz, DMSO- d6) δ 13.42 (s, 1H, OH), 8.46 (s, 1H, Ar-H), 8.33 – 8.27 (m, 3H, Ar-H), 8.18 – 8.11 (m, 4H, Ar-H), 7.35 (d, J = 8.3 Hz, 2H, Ar-H), 4.55 (q, J = 7.1 Hz, 2H, CH2), 2.65 (q, J = 7.5 Hz, CH2), 1.50 (t, J = 7.2 Hz, 3H, CH3), 1.21 (t, J = 7.6 Hz, 3H, CH3). 13 C NMR (101 MHz, DMSO- d 6) δ 166.48, 164.06,162.59, 155.77, 150.13, 146.01, 135.04, 133.93, 131.57, 130.12, 128.28,127.32, 127.27, 126.40, 124.20, 111.31, 109.02, 41.68, 27.95, 15.33, 14.75.

[0068] Example 18 This embodiment provides a pyrazolopyridine compound, denoted as compound L-18, with the following structural formula: .

[0069] The preparation method of compound L-18 differs from that of Example 1 in that steps (8) and (9) are omitted, while other steps are the same as in Example 1. Due to the change in raw materials, the structure of the product obtained in each step of the preparation process also changes accordingly. The yield of compound L-18 prepared in this example is 42%, a white solid with a melting point of 259.3-263.4℃. The NMR characterization results are as follows: 1 H NMR (400MHz, DMSO- d 6) δ 8.52 (s, 1H, Ar-H), 8.37 – 8.32 (m, 3H, Ar-H), 8.30 – 8.26 (m, 2H, Ar-H), 8.15 (d, J = 8.4 Hz, 2H, Ar-H), 7.54 (dd, J = 11.7, 7.1 Hz,3H, Ar-H), 4.59 (q, J= 7.3 Hz, 2H, CH2), 1.51 (t, J = 7.2 Hz, 3H, CH3). 13 CNMR (101 MHz, DMSO- d 6) δ 166.65, 164.18, 162.60, 155.81, 150.17, 137.57,134.48, 131.65, 130.13, 130.08, 128.94, 127.38, 127.28, 126.26, 124.39,111.61, 109.26, 41.76, 14.78.

[0070] Example 19 This embodiment provides a pyrazolopyridine compound, denoted as compound L-19, with the following structural formula: .

[0071] The specific preparation route and method for compound L-19 are as follows:

[0072] (1) Place p-bromoacetophenone (1.0 mmol) and diethyl oxalate (1.1 mmol) in a 250 mL round-bottom flask, add 30 mL of tetrahydrofuran and mix. While stirring at room temperature, slowly add sodium hydride (3 mmol). After stirring at room temperature for 30 min, transfer to an oil bath at 55 °C and react for 5 h. After the reaction is complete by TLC (petroleum ether: ethyl acetate = 1:1, v / v), remove tetrahydrofuran by vacuum distillation, add 30 mL of water and mix. While stirring, add formic acid dropwise to adjust the pH to 5-6. A solid precipitates out. Filter to obtain intermediate a'.

[0073] (2) Intermediate a' (1 mmol) and 5-amino-1-ethylpyrazole (1 mmol) were placed in a 100 mL round-bottom flask, 20 mL of glacial acetic acid was added, and the mixture was dissolved by sonication. The mixture was then transferred to an oil bath at 100 °C and heated for 5 h. After the reaction was complete by TLC (petroleum ether: ethyl acetate = 1:1, v / v), the mixture was cooled to room temperature and the glacial acetic acid was removed by vacuum distillation. Then, 30 mL of methanol, 10 mmol of sodium hydroxide solution, and water were added in sequence and the mixture was transferred to 70 °C for hydrolysis for 2 h. After the hydrolysis was complete by TLC (petroleum ether: ethyl acetate = 1:1, v / v), the solvent was removed by vacuum distillation, water was added, and formic acid was added dropwise to adjust the pH of the solution to 5-6. The filter cake was collected by suction filtration to obtain intermediate b'.

[0074] (3) Intermediate b' (1 mmol), tert-butyl hydrazinoate (1.5 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (1.5 mmol), and N,N-diisopropylethylamine (2 mmol) were placed in a 100 mL round-bottom flask and N,N-dimethylformamide (25 mL) was added as solvent. The reaction was carried out at room temperature for 6 h. The reaction was monitored by TLC (petroleum ether: ethyl acetate = 1:1) and the reaction was complete. The mixture was extracted three times with water and ethyl acetate. The upper organic phases were combined, washed with saturated brine, and the organic phase was removed by vacuum distillation. The mixture was purified by column chromatography (petroleum ether: ethyl acetate = 5:1, v / v) to obtain intermediate c'.

[0075] (4) Dissolve intermediate c' (1 mmol) in dichloromethane solvent, add trifluoroacetic acid dropwise while monitoring continuously until the protecting group is removed. If the solid adheres to the wall, discard the dichloromethane solvent directly. If it does not adhere to the wall, remove the dichloromethane by vacuum distillation, add water, add saturated sodium bicarbonate solution to adjust the pH to neutral, filter and dry the filter cake to obtain intermediate d'.

[0076] (5) Dissolve intermediate d' (1 mmol) in dichloromethane and add triethylamine dropwise to adjust the pH to 8-9; dissolve methyl 4-chloroformylbenzoate (1.2 mmol) in dichloromethane and add it dropwise to the reaction solution containing intermediate d' under ice bath conditions. After reacting for 3 h, a white solid precipitates. Monitor the reaction by TLC (petroleum ether: ethyl acetate = 1:1, v / v). After the reaction is complete, filter directly and dry the filter cake to obtain intermediate e'.

[0077] (6) The intermediate e' (1 mmol) was directly dissolved in phosphorus oxychloride and refluxed at 90 °C for 8 h to carry out the ring-closure reaction. After the reaction was completed, the reaction solution was added dropwise to ice water under ice bath conditions to quench the reaction. After the temperature dropped to room temperature, the product was filtered. The product obtained by filtration was dissolved in ethyl acetate and purified by column chromatography (petroleum ether: ethyl acetate = 8:1, v / v) to obtain the intermediate f'.

[0078] (7) Dissolve intermediate f' (1 mmol) in methanol, add sodium hydroxide (10 mmol) and 30 mL of water, transfer to 70 °C oil bath for heating and hydrolysis for 2 h, monitor by TLC (petroleum ether: ethyl acetate = 1:1, v / v) hydrolysis is complete, remove solvent by vacuum distillation, add water, add formic acid to adjust the pH of the solution to 5~6, filter and take filter cake to obtain intermediate g'.

[0079] (8) Dissolve intermediate g' (1 mmol) in 15 mL of dichloromethane, add Boc-hydrazine (1.2 mmol), N,N-diisopropylethylamine (2.5 mmol), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (1.5 mmol) and react for 4 h. Monitor the reaction by TLC (petroleum ether: ethyl acetate = 3:1, v / v). After the reaction is complete, remove the organic phase by vortexing and perform column chromatography (eluent: petroleum ether: ethyl acetate = 5:1) to obtain intermediate h'.

[0080] (9) After ultrasonically dispersing intermediate h' with water, hydrochloric acid was added dropwise to remove the Boc group, followed by filtration to obtain the pyrazolopyridine compound of this example, namely compound L-19. Compound L-19 had a yield of 75%, was a pale yellow solid, and had a melting point of 260.5-263.7℃. NMR characterization results were as follows: 1 H NMR (400 MHz, DMSO- d 6) δ 8.51 (s, 1H, Ar-H), 8.38 – 8.29 (m, 3H, Ar-H), 8.25 – 8.21 (m, 2H, Ar-H), 8.11 (d, J = 8.6 Hz, 2H, Ar-H), 7.72 (d, J = 8.6 Hz, 2H, Ar-H), 4.57 (q, J = 7.3 Hz, 2H, CH2), 1.50 (t, J = 7.2 Hz, 3H, CH3). 13 C NMR (101 MHz, DMSO- d 6) δ 164.68, 164.22,154.48, 150.03, 136.61, 135.85, 131.87, 131.74, 129.31, 128.15, 127.24,125.26, 124.54, 123.91, 111.72, 111.33, 109.43, 41.78, 14.79.

[0081] Example 20 This embodiment provides a pyrazolopyridine compound, denoted as compound L-20, with the following structural formula: .

[0082] The preparation method of compound L-20 differs from that of Example 19 in that p-bromoacetophenone in step (1) is replaced with acetophenone, while the other steps are the same as in Example 19. Due to the change in raw materials, the structure of the product obtained in each step of the preparation process also changes accordingly. The yield of compound L-20 prepared in this example is 35%, it is a white solid with a melting point of 261.7-263.9℃. The NMR characterization results are as follows: 1 H NMR (400 MHz, DMSO- d 6) δ 8.49 (s, 1H, Ar-H), 8.37 – 8.34 (m, 3H, Ar-H), 8.27 – 8.23 ​​(m, 2H, Ar-H), 8.13 (d, J = 8.5 Hz, 2H, Ar-H), 7.56 – 7.48(m, 3H, Ar-H), 4.55 (q, J = 7.3 Hz, 2H, CH2), 1.47 (t, J = 7.2 Hz, 3H, CH3). 13 C NMR (101 MHz, DMSO- d 6) δ 164.82, 164.11, 162.62, 155.82, 150.17, 137.54,134.43, 131.67, 130.10, 128.95, 128.56, 127.39, 126.00, 124.40, 111.63,109.25, 41.76, 14.79.

[0083] The technical effects of the present invention will be explained below with reference to specific experimental examples.

[0084] Experimental Example 1: Test of Cardiomyocyte Protective Effect Frozen H9c2 cells (rat cardiomyocytes) were revived and then passaged at the required ratio to the logarithmic growth phase. After culture, the cells were digested with trypsin, resuspended in complete culture medium, and the cell density was adjusted to 5 × 10⁶ cells / year. 4H9c2 cell suspension was obtained by measuring cells / mL. 100 μL of H9c2 cell suspension (i.e., 5000 cells per well) was added to each well of a 96-well plate. The plate was gently shaken to ensure even cell distribution, and the plates were incubated at 37°C with 5% CO2 for 24 h. After incubation, cell adhesion and growth were observed using an inverted microscope to ensure cell confluence ≥80%. A 300 μM H2O2 solution was prepared using culture medium. The old culture medium in the 96-well plate was discarded, and 100 μL of 300 μM H2O2 solution was added to each well. The plates were incubated at 37°C with 5% CO2 for 1.5 h. Cell viability was then assessed using the MTT assay to establish a cell damage model. When the H9c2 cell confluence was ≥80%, H9c2 cells were incubated for 24 h with different concentrations of pyrazolopyridine compounds (5, 10, 20, 40, 80 μM) from Examples 1-20 of the present invention to evaluate the toxicity of pyrazolopyridine compounds to H9c2 cells. The optimal concentration of the compound was found to be 10 μM.

[0085] In the specific experiment, when the confluence of H9c2 cells was ≥80%, H9c2 cells were incubated for 24 h with the pyrazolopyridine compound (10 μM) of the present invention. After incubation, the culture medium was discarded, fresh culture medium was added, and cardiomyocyte injury was induced by treatment with 300 μM H2O2 solution for 1.5 h to evaluate the protective effect of the pyrazolopyridine compound on the cardiomyocyte injury model. Luteolin (LUT, 10 μM) was used as a positive control. All the above groups were in triplicate. After incubation, 20 μL of MTT solution was added to each well, gently mixed, and the 96-well plate was placed in a 37°C, 5% CO2 incubator in the dark for 4 h. Then the supernatant of the 96-well plate was discarded (care should be taken to avoid touching the formazan crystals at the bottom of the well), 150 μL of dimethyl sulfoxide (DMSO) was added to each well, and the plate was shaken at room temperature in the dark for 10 min to fully dissolve the formazan crystals. The absorbance of each well was measured at 490 nm and the data were recorded to calculate the cell viability. Experimental data are expressed as mean ± standard deviation (Mean ± SD) of three independent trials. A p-value < 0.05 was considered statistically significant. The evaluation results of the cardioprotective ability of pyrazolopyridine compounds are shown in Table 1. In Table 1, "PF" represents the ratio of the survival rate of injured cardiomyocytes with and without the compound pretreatment.

[0086] Table 1. Results of the determination of the protective ability of pyrazolopyridine compounds against cardiomyocytes.

[0087] The results in Table 1 show that, compared with the H2O2 group, the survival rate of cardiomyocytes was significantly improved under the action of the 10 μM concentration of the compound, indicating that the pyrazolopyridine compounds of the present invention have different degrees of protective effect on cardiomyocytes, and some compounds have a better protective effect than the positive control LUT.

[0088] Experimental Example 2: Investigation into the Mechanism of Cardioprotective Effect of Compounds Based on the results of the evaluation of the activity of the compound on cardiomyocytes in Experiment Example 1, this invention takes compound L-9 as an example to explore its mechanism of action in cardioprotection.

[0089] (i) Cardiac cell viability and morphological testing To systematically evaluate the cardioprotective activity of compound L-9, three dosage groups were set up with low, medium, and high concentrations of 0.625 μM, 2.5 μM, and 10 μM, respectively. H2c9 cardiomyocytes were pretreated with compound L-9 at the above concentrations for 24 h, followed by treatment with 150 μM H2O2 for 2 h to induce a myocardial injury model. Cell viability was determined by the MTT assay, and cell morphology images of each treatment group were recorded.

[0090] Figure 1 The results show the protective effect of compound L-9 at different concentrations on a cardiomyocyte injury model. Figure 1 In the figures, A represents cell survival rate; B represents cell morphology under different treatment conditions, with a scale bar of 200 μM. CON is the control group; H2O2 is the model group; H2O2+L, H2O2+M, and H2O2+H are the L-9 treatment groups at 0.625, 2.5, and 10 μM, respectively; H2O2+LUT is the 10 μM luteolin (LUT) treatment group. # P < 0.05, ## P < 0.01, ### P < 0.001 indicate comparison with the CON group; *P < 0.05, ** P < 0.01, ***P < 0.001 indicate comparison with the myocardial injury model group.

[0091] like Figure 1 As shown in Figure A, compared with the control group (CON), the cell survival rate of the model group (H2O2) was significantly reduced, indicating that the injury model was successfully constructed. The cell survival rates of all groups pretreated with compound L-9 were higher than those of the model group, and the cardiomyocyte survival rate increased in a dose-dependent manner with increasing drug concentration. At a dose of 10 μM, the protective effect of compound L-9 on cardiomyocytes was comparable to that of the positive control luteolin (LUT).

[0092] like Figure 1As shown in Figure B, the protective effect was further verified at the cellular morphology level. In the normal control group, cells exhibited regular morphology, a typical spindle shape, plump cell bodies, clear nuclear boundaries, and high cell density. In contrast, cells in the model group showed significant shrinkage, irregular morphology, and diffuse nuclear distribution, accompanied by substantial cell death. Pretreatment with different concentrations of compound L-9 showed a dose-dependent improvement in cell morphology, manifested as reduced cell shrinkage, and the overall cell condition was significantly better than the model group. These results, corroborated by the cell viability results, all indicate that compound L-9 has a clear cardioprotective effect.

[0093] (ii) Lactate dehydrogenase (LDH) test Lactic dehydrogenase (LDH) is a commonly used biochemical indicator for evaluating the degree of cell damage, and its release rate is directly proportional to the number of dead cells. Cells were grouped and treated with gradient concentrations of the test compound according to the grouping protocol, and co-cultured for 24 h. Then, hydrogen peroxide was added to the culture system, and treatment continued for another 2 h. Next, the cells were centrifuged at 4000 rpm for 5 min using a multi-well plate centrifuge. 250 μL of LDH releasing agent was injected into each well, and the mixture was gently agitated with a pipette. The culture plate was transferred to a 37°C incubator containing 5% carbon dioxide and incubated for 60 min. After incubation, the cells were centrifuged again for 5 min. 60 μL of LDH assay working solution was added to each well (this process requires protection from light), and the cells were incubated at room temperature in the dark for 30 min. The OD value was then measured at 490 nm using a microplate reader. The LDH release rate (mU / mL) in the test sample is calculated as follows: (OD value of sample well - OD value of sample control well) / (OD value of standard well - OD value of standard control well) × standard concentration (mU / mL). The effect of compound L-9 on lactate dehydrogenase (LDH) release rate is shown in the figure. Figure 2 As shown. Figure 2 In this study, CON served as the control group; H2O2 served as the model group; H2O2+L, H2O2+M, and H2O2+H were the L-9 treatment groups at 0.625, 2.5, and 10 μM, respectively; and H2O2+LUT was the 10 μM luteolin (LUT) treatment group. # P < 0.05, ## P < 0.01, ### P < 0.001 indicate comparisons with the CON group; * P < 0.05, ** P < 0.01, *** P < 0.001 indicate comparisons with the myocardial injury model group.

[0094] Depend on Figure 2It was found that the LDH release rate in the H2O2 model group was significantly higher than that in the control group (CON), indicating severe cell damage. Furthermore, after pretreatment with different concentrations of compound L-9, the release rate gradually decreased with increasing concentration, demonstrating a clear dose-dependent effect.

[0095] (iii) Expression test of inflammation-related gene mRNA in cardiomyocytes The occurrence and development of myocardial injury involves multiple signaling pathways. To explore the potential molecular mechanism by which compound L-9 exerts its cardioprotective effect, this invention employs polymerase chain reaction (PCR) technology to detect changes in the mRNA expression of genes related to inflammation, apoptosis, pyroptosis, and autophagy in a myocardial injury cell model. H9c2 cells were grouped, treated with drugs, and the model was constructed according to the experimental protocol described above. Total RNA was extracted from each tissue cell according to the instructions of the RNA extraction kit, and its concentration was determined using quantitative real-time immunoassay. The total RNA was reverse transcribed into cDNA. Finally, PCR was used to detect the mRNA expression of each gene group, and the data were processed using GraphPad Prism 8.0.

[0096] Figure 3 The effect of pretreatment with compound L-9 on the expression of inflammation-related genes mRNA in cardiomyocytes; where A is the mRNA expression level of TNF-α; B is the mRNA expression level of IL-18; C is the mRNA expression level of IL-1β; and D is the mRNA expression level of IL-6. Figure 3 In this study, CON served as the control group; H2O2 served as the model group; and H2O2+L, H2O2+M, and H2O2+H were the L-9 treatment groups at concentrations of 0.625, 2.5, and 10 μM, respectively. # P < 0.05, ## P < 0.01, ### P < 0.001 indicate comparisons with the CON group; * P < 0.05, ** P < 0.01, *** P < 0.001 indicate comparisons with the myocardial injury model group.

[0097] like Figure 3 As shown in Figures A through D, pretreatment with compound L-9 reduced the expression of some pro-inflammatory cytokines. Compared to the CON group, the H2O2 group showed significantly increased mRNA levels of pro-inflammatory cytokines TNF-α, IL-18, IL-1β, and IL-6. However, pretreatment with compound L-9, compared to the H2O2 group, significantly reduced the expression of IL-18 mRNA in a dose-dependent manner, and also significantly reduced the expression of IL-1β and IL-6 mRNA. These results indicate that compound L-9 can alleviate cardiomyocyte damage by reducing the mRNA expression of pro-inflammatory factors.

[0098] In summary, the results of this invention, through structural design and structure-activity relationship studies of pyrazolopyridine compounds and cardioprotective activity tests, confirm that the compounds of this invention possess superior protective capabilities against cardiomyocytes, and also explore the mechanism of their cardioprotective effect. Therefore, this invention not only enriches the skeletal types of this class of compounds in cardioprotective drugs, but also reveals that these compounds enhance cardioprotective activity through anti-inflammatory effects, providing a theoretical basis for exploring the mechanism of action of these drugs.

[0099] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A pyrazolopyridine compound, characterized in that, The structure is shown in Equation I: ； In Equation I, R1 is One of them; R2 is One of them.

2. The pyrazolopyridine compound according to claim 1, characterized in that, The pyrazolopyridine compounds are selected from compounds with the following structures, and are designated as compounds L-1 to L-20 in sequence:

3. A method for preparing a pyrazolopyridine compound as described in claim 1 or 2, characterized in that, The following technical approach is adopted: ； When R2 is The method for preparing the pyrazolopyridine compound includes the following steps: (1) React raw material A and raw material B in a solvent to obtain intermediate C; (2) Intermediate C and raw material D are reacted in a solvent, and then hydrolyzed in the presence of an alkaline substance to obtain intermediate E; (3) Intermediate E is reacted with Boc hydrazine and a condensing agent in a solvent to obtain intermediate F; (4) Remove the protecting group from intermediate F in a solvent to obtain intermediate G; (5) React intermediate G with raw material H in a solvent to obtain intermediate I; (6) Intermediate I was refluxed in a solvent to obtain intermediate J; (7) React intermediate J with an alkaline substance in a solvent to obtain compound K, which is the pyrazolopyridine compound; When R2 is The method for preparing the pyrazolopyridine compound includes the following steps: (S1) React raw material A and raw material B in a solvent to obtain intermediate C; (S2) Intermediate C and raw material D are reacted in a solvent, and then hydrolyzed in the presence of an alkaline substance to obtain intermediate E; (S3) Intermediate E is reacted with Boc hydrazine and a condensing agent in a solvent to obtain intermediate F; (S4) Remove the protecting group from intermediate F in a solvent to obtain intermediate G; (S5) Intermediate G is reacted with raw material H in a solvent to obtain intermediate I; (S6) Intermediate I was refluxed in a solvent to obtain intermediate J; (S7) Intermediate J is reacted with an alkaline substance in a solvent to obtain compound K; (S8) Compound K was reacted with O-(tetrahydro-2H-pyran-2-yl)hydroxylamine and a condensing agent in a solvent to give intermediate L; (S9) The intermediate L is reacted with an acidic substance in a solvent to obtain compound M, which is the pyrazolopyridine compound. When R2 is The method for preparing the pyrazolopyridine compound includes the following steps: (i) React raw material A and raw material B in a solvent to obtain intermediate C; (ii) Intermediate C is reacted with raw material D in a solvent, and then hydrolyzed in the presence of an alkaline substance to obtain intermediate E; (iii) Intermediate E is reacted with Boc hydrazine and a condensing agent in a solvent to obtain intermediate F; (iv) Remove the protecting group from intermediate F in a solvent to obtain intermediate G; (v) Intermediate G is reacted with starting material H in a solvent to obtain intermediate I; (vi) Intermediate I is refluxed in a solvent to obtain intermediate J; (vii) Intermediate J is reacted with a basic substance in a solvent to obtain compound K; (viii) Compound K is reacted with Boc-hydrazine and a condensing agent in a solvent to give intermediate N; (ix) The intermediate N is reacted with an acidic substance in a solvent to obtain compound O, which is the pyrazolopyridine compound.

4. The method for preparing pyrazolopyridine compounds according to claim 3, characterized in that, The raw material A is one of the following: acetophenone, m-methylacetophenone, p-methylacetophenone, m-chloroacetophenone, p-chloroacetophenone, m-bromoacetophenone, p-bromoacetophenone, m-trifluoromethylacetophenone, p-trifluoromethylacetophenone, p-n-propylacetophenone, p-ethylacetophenone, p-methoxyacetophenone, and p-dimethylaminoacetophenone.

5. The method for preparing pyrazolopyridine compounds according to claim 3, characterized in that, The alkaline substance is one or more of triethylamine, sodium hydroxide, and sodium bicarbonate.

6. The method for preparing pyrazolopyridine compounds according to claim 3, characterized in that, The condensing agent is N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate or N,N-diisopropylethylamine.

7. The method for preparing pyrazolopyridine compounds according to claim 3, characterized in that, The acidic substance is one or more of trifluoroacetic acid, hydrochloric acid, formic acid, and acetic acid.

8. The method for preparing pyrazolopyridine compounds according to claim 3, characterized in that, The solvent is one or more of tetrahydrofuran, acetic acid, methanol, N,N-dimethylformamide, dichloromethane, water, and phosphorus oxychloride.

9. The use of a pyrazolopyridine compound as described in claim 1 or 2 in the preparation of a cardioprotective drug.

10. The use of the pyrazolopyridine compound according to claim 9 in the preparation of a cardioprotective drug, characterized in that, The mechanisms of action of the cardioprotective drugs include one or more of the following: enhancing the activity of cardiomyocytes, reducing the release of lactate dehydrogenase from cardiomyocytes, and reducing the expression levels of pro-inflammatory factors in cardiomyocytes.

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