Cinnamamide 3CL protease covalent inhibitor, preparation method thereof and application of cinnamamide 3CL protease covalent inhibitor in preparation of anti-coronavirus drugs

Abstract

The invention discloses a cinnamamide 3CL protease covalent inhibitor, a preparation method thereof and application of the cinnamamide 3CL protease covalent inhibitor in preparation of anti-coronavirus drugs, and belongs to the field of medicinal chemistry and medicine. In the structure of the cinnamamide 3CL protease covalent inhibitor, an alpha, beta-unsaturated amido bond can be used as an electrophilic active group to be subjected to an addition reaction with a thiol group of 3CL protease catalytic cysteine Cys145 to form a covalent thioether bond, and meanwhile, a covalent thioether bond is formed through hydrogen-bond interaction between the amido group and key residues such as His41 or Glu166 and the like, so that the covalent thioether bond is formed. The compound can stabilize a covalent bond formed by an active site of 3CLpro, and has the potential of effectively inhibiting the replication and activity of coronavirus by inhibiting the activity of 3CLpro, especially diseases caused by coronavirus such as MERS-CoV, SARS-CoV or SARS-CoV-2. The invention discloses the high therapeutic index and low half effective concentration of the compound in the antiviral field for the first time, and shows excellent pharmacokinetic properties and virus inhibition effect.

Description

Technical Field

[0001] The present invention belongs to the fields of medicinal chemistry and pharmaceutical technology, and particularly relates to covalent inhibitors of 3CL protease of cinnamamide type, their preparation methods and applications in the preparation of anti-coronavirus drugs. Background Art

[0002] 3CL protease, also known as 3CL pro or M pro or nsp5, is a key enzyme in coronaviruses and is crucial for the virus life cycle. This enzyme plays a central role in the replication and transcription processes of coronaviruses, and thus has become an important target in the research and development of antiviral drugs. 3CL protease is a cysteine protease with specific substrate recognition characteristics and active site structures. It usually consists of two similar domains, which form an active cleft similar to scissors in the spatial structure, specifically for cleaving polyprotein precursors. The characteristic of this structure is that it contains a catalytic cysteine residue and a histidine residue that composes the substrate binding site at its active site. The main function of 3CL protease is to process the polyprotein precursors pp1a and pp1ab of coronaviruses, and release functional and diverse non-structural proteins (NSPs) by recognizing and cleaving specific cleavage sites. Due to the key role of 3CL protease in the virus life cycle and the absence of a direct homologous protein in human host cells, it has become an ideal antiviral target. Inhibiting the activity of this enzyme can effectively block virus replication and slow down or prevent the progression of virus infection.

[0003] Currently, more than 1750 3CL pro inhibitors are at different stages of research and development globally, among which 5 have entered clinical phase III trials, and 4 have obtained market approval. For example, Paxlovid (nirmatrelvir / ritonavir) has been successfully marketed and widely used, becoming an effective drug in the fight against the SARS-CoV-2 epidemic. However, the oral bioavailability of Paxlovid is relatively low, and it is easily affected by P-glycoprotein resulting in drug efflux. At the same time, potential drug-drug interactions when used in combination are also a major limitation in its application. Among the existing 3CL pro inhibitors, although peptidomimetic irreversible inhibitors show good activity, these inhibitors usually have problems of low metabolic stability and low cell membrane permeability, and their pharmacokinetic properties are not ideal. In addition, potential off-target effects and toxicity problems are also the main factors hindering their further development and wide application. Therefore, although 3CL proIt has been proven to be a well-established target in the antiviral treatment of coronaviruses, and the research and development of related inhibitors still have important scientific value and practical significance. Future research needs to focus on optimizing the efficacy and safety of these inhibitors, improving their bioavailability, reducing drug interactions, and minimizing off-target effects and toxicity, so as to develop a safer and more effective second-generation 3CL pro inhibitors.

[0004] The coronavirus SARS-CoV-2, belonging to the β-coronavirus family, is the seventh known member of this family and is closely related to the severe acute respiratory syndrome coronavirus (SARS-CoV) and the Middle East respiratory syndrome coronavirus (MERS-CoV). Although SARS-CoV-2 is similar to SARS-CoV in many biological characteristics, it has a lower fatality rate but significantly higher infectivity than SARS-CoV. The viral particles of SARS-CoV-2 are usually round or oval, with a diameter of approximately 60-140 nm. Its genome is a linear single-stranded positive-sense RNA, about 30 kb in length, encoding a variety of proteins including non-structural proteins, structural proteins, and some accessory proteins. Among the proteins encoded by SARS-CoV-2, 3-chymotrypsin-like protease (3CL pro ) and papain-like protease (PL pro ) are crucial for the viral replication and transcription processes. 3CL pro is particularly critical because it is responsible for cleaving the viral polyprotein precursor to generate active proteins, thus driving the viral replication process. Notably, 3CL pro shows a high degree of structural conservation in various coronaviruses including MERS-CoV and SARS-CoV ( Figure 1 ), and this structural similarity makes 3CL pro an ideal drug target suitable for developing inhibitors that can block the replication and transcription of multiple coronaviruses. Therefore, it not only has important value in scientific research but also shows broad application prospects in new drug development. Summary of the Invention

[0005] In order to overcome the above-mentioned disadvantages of the prior art, the purpose of the present invention is to provide covalent inhibitors of cinnamamide 3CL protease, their preparation methods, and their applications in the preparation of anti-coronavirus drugs.

[0006] To achieve the above purpose, the present invention adopts the following technical solutions:

[0007] In the first aspect of the present invention, cinnamamide-based 3CL protease covalent inhibitors are disclosed, which are compounds of formula I or solvate compounds, enantiomers, diastereomers, tautomers or mixtures thereof in any proportion of pharmaceutically acceptable salts thereof;

[0008] The structural formula of the compound of formula I is:

[0009]

[0010] Wherein, R 1 、R 2 and R 3 are each independently selected from -H, -CH 3 , -F, -Cl, -Br, -CN, -NO 2 , i-Pr, t-Bu

[0011] Preferably, the compound of formula I is combined with a salt formed with one or more acids, and the types of the acids are hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, fumaric acid, maleic acid, oxalic acid, malonic acid, succinic acid, citric acid, malic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, glutamic acid or aspartic acid.

[0012] In the second aspect of the present invention, a preparation method of cinnamamide-based 3CL protease covalent inhibitors is disclosed. A cinnamoyl chloride compound is mixed and reacted with an intermediate to obtain a cinnamamide-based 3CL protease covalent inhibitor;

[0013] Wherein, the cinnamoyl chloride compound is R 1 、R 2 and R 3 are each independently selected from -H, -CH 3 , -F, -Cl, -Br, -CN, -NO 2 , i-Pr, t-Bu The intermediate is

[0014] Preferably, the molar ratio of the cinnamoyl chloride compound to the intermediate is 1.2:1.

[0015] Preferably, the preparation method of the cinnamoyl chloride compound is: a cinnamic acid compound is mixed and reacted with thionyl chloride to obtain a cinnamoyl chloride compound;

[0016] Wherein, the cinnamic acid compound is R 1 、R 2 and R 3 are each independently selected from -H, -CH 3 , -F, -Cl, -Br, -CN, -NO2 i-Pr t-Bu The molar ratio of the cinnamic acid compound to thionyl chloride is 1:1.3.

[0017] Preferably, the preparation method of the intermediate is as follows: 3-tert-butyl-6-(ethylthio)-1,3,5-triazine-2,4(1H,3H)-dione undergoes an alkylation reaction with 2,4,5-trifluorobenzyl bromide to obtain compound a; compound a is de-tert-butylated to obtain compound b; compound b is mixed and reacted with propargyl bromide to obtain compound c; compound c is mixed and reacted with 6-chloro-2-methyl-2H-indazol-5-amine to obtain compound d; compound d, azidotrimethylsilane, and sodium L-ascorbate are dissolved in N,N-dimethylacetamide solution for reaction to obtain the intermediate;

[0018] Among them, compound a is Compound b is Compound c is Compound d is The molar ratio of 3-tert-butyl-6-(ethylthio)-1,3,5-triazine-2,4(1H,3H)-dione to 2,4,5-trifluorobenzyl bromide is 1:1.2, the molar ratio of compound b to propargyl bromide is 1:2.2, the molar ratio of compound c to 6-chloro-2-methyl-2H-indazol-5-amine is 1:1.4, the molar ratio of compound d to azidotrimethylsilane is 1:1.8, and the molar ratio of compound d to sodium L-ascorbate is 1:1.2.

[0019] In the third aspect of the present invention, a pharmaceutical composition against coronavirus is disclosed, and the pharmaceutical composition includes the above-mentioned cinnamide 3CL protease covalent inhibitor and other drugs with anti-coronavirus activity.

[0020] In the fourth aspect of the present invention, the application of the above-mentioned cinnamide 3CL protease covalent inhibitor or the above-mentioned anti-coronavirus pharmaceutical composition in the preparation of drugs for preventing and / or treating coronavirus is disclosed.

[0021] Preferably, the drug contains one or more pharmaceutically acceptable carriers or excipients.

[0022] Preferably, the coronavirus protease is SARS-CoV-2 3CL pro .

[0023] Preferably, the concentration of the cinnamide 3CL protease covalent inhibitor is 0.1 - 0.5 μM.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The covalent inhibitor of 3CL protease of cinnamamide provided by the present invention, the α,β-unsaturated amide bond in its structure can serve as an electrophilic active group, react with the thiol group (-SH) of the catalytic cysteine Cys145 of 3CL protease to form a covalent thioether bond. At the same time, through the amide group forming hydrogen bond interactions with key residues such as His41 or Glu166, it can stabilize the covalent bond formed by the active site of 3CL pro Thereby, the covalent inhibitor of 3CL protease of cinnamamide has the potential to effectively inhibit the replication and activity of coronaviruses by inhibiting the activity of 3CL pro , especially diseases caused by coronaviruses such as MERS-CoV, SARS-CoV or SARS-CoV-2. The present invention for the first time reveals the high therapeutic index and low half-maximal effective concentration (EC 50 ) of this type of compound in the field of antiviral, demonstrating excellent pharmacokinetic properties and virus inhibition effects. This discovery not only overcomes the common problems of insufficient efficacy and short action time in existing antiviral drugs, but also significantly enhances the clinical application potential of 3CL pro as a drug target. This new inhibition mechanism can provide a safer and more effective treatment option for the market, and has important clinical significance for current and future potential coronavirus infection epidemics. Especially in public health crises such as the COVID-19 pandemic, the covalent inhibitor of 3CL protease of cinnamamide can be used as an effective treatment means to help reduce the spread of the virus and the severity of the disease, significantly promoting the progress in the field of antiviral drug research and development. In summary, the covalent inhibitor of 3CL protease of cinnamamide has important research value and application prospects. Developing this inhibitor can fill the gap in anti-coronavirus drugs, and can not only be used to treat existing coronavirus pneumonia diseases, but also show superior potential in preventing virus infections.

[0026] Furthermore, the provided representative compounds have excellent structure-activity characteristics, meet the requirements for inhibiting coronavirus 3CL pro , and have strong virus inhibition ability. Through precise chemical design, these compounds can specifically bind to the active site of 3CL pro , showing a high inhibition efficiency, and then effectively blocking the virus life cycle. They can be developed into covalent inhibitors of 3CL pro against coronaviruses, especially the 3CL pro protease of SARS-CoV-2.

[0027] Furthermore, the salification reaction of covalent inhibitors of 3CL protease with cinnamides not only maintains the original inhibitory activity but also improves the bioavailability, solubility, and stability of the inhibitor, making it more suitable for absorption and distribution in pharmaceutical applications. The introduction of these salt compounds can provide more options and optimization space for the clinical development and application of this series of inhibitors, enhancing their potential as antiviral drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 For MERS-CoV 3CL pro (PDB ID 4RSP, green), SARS-CoV 3CL pro (PDB ID 1Q2W, red), SARS-CoV-2 3CL pro (PDB ID 6LU7, blue) superimposed simulation diagram;

[0029] Figure 2 1H NMR spectrum of compound 1 synthesized in Example 1 of the present invention;

[0030] Figure 3 13C NMR spectrum of compound 1 synthesized in Example 1 of the present invention;

[0031] Figure 4 1H NMR spectrum of compound 3 synthesized in Example 3 of the present invention;

[0032] Figure 5 13C NMR spectrum of compound 3 synthesized in Example 3 of the present invention;

[0033] Figure 6 Inhibitory activity test result diagram of compound 1 of the present invention against 3CL pro ;

[0034] Figure 7 Inhibitory activity test result diagram of compound 3 of the present invention against 3CL pro ;

[0035] Figure 8 Inhibitory activity test result diagram of compound 17 of the present invention against 3CL pro ;

[0036] Figure 9 Inhibitory efficiency diagram of the test drug of the present invention against A549 cells;

[0037] Figure 10 Inhibitory efficiency diagram of the test drug of the present invention against HEK293 cells;

[0038] Figure 11 Inhibitory efficiency diagram of the test drug of the present invention against HepG2 cells. Detailed implementation mode

[0039] To enable those skilled in the art to understand the features and effects of the present invention, the following provides only a general description and definition of the terms and phrases mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used herein shall have the ordinary meaning understood by those skilled in the art with respect to the present invention. In case of conflict, the definition in this specification shall prevail.

[0040] The theories or mechanisms described and disclosed herein, whether correct or incorrect, shall not in any way limit the scope of the present invention, that is, the content of the present invention can be implemented without being limited by any specific theory or mechanism.

[0041] In this document, for the sake of brevity of description, all possible combinations of all technical features in each embodiment or example are not described. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each embodiment or example can be combined arbitrarily, and all possible combinations should be considered as the scope described in this specification.

[0042] The present invention provides covalent inhibitors of cinnamamide 3CL protease, which are various derivative forms of the compound of formula I or its pharmaceutically acceptable salts, including but not limited to its solvate compounds, enantiomers, diastereoisomers, tautomers, and any proportion mixtures of these forms, including racemic mixtures;

[0043] The structural formula of the compound of formula I is as follows:

[0044]

[0045] Wherein, R 1 , R 2 and R 3 are each independently selected from -H, -CH 3 , -F, -Cl, -Br, -CN, -NO 2 , i-Pr, t-Bu

[0046] The pharmaceutically acceptable salt is a covalent inhibitor of cinnamamide 3CL protease that inhibits the activity of coronavirus 3CL protease, and this inhibitor can form a salt complex with one or more acids. The specific types of acids include but are not limited to hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, fumaric acid, maleic acid, oxalic acid, malonic acid, succinic acid, citric acid, malic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, glutamic acid or aspartic acid.

[0047] The present invention provides a method for preparing the above-mentioned covalent inhibitors of 3CL protease of cinnamamide class, comprising the following steps:

[0048] (1) Synthesize compound a: 3-(tert-butyl)-6-(ethylthio)-1-(2,4,5-trifluorobenzyl)-1,3,5-triazine-2,4(1H,3H)-dione

[0049]

[0050] Using 3-(tert-butyl)-6-(ethylthio)-1,3,5-triazine-2,4(1H,3H)-dione as the starting material, the target compound a is prepared by alkylation reaction with 2,4,5-trifluorobenzyl bromide. This alkylation reaction is carried out in the medium of acetonitrile, and potassium carbonate is used as the basic catalyst to improve the reaction efficiency. The reaction system is stirred and refluxed at 85 °C for 14 hours to ensure sufficient reaction. After the reaction is completed, the reaction mixture is concentrated by removing the solvent through the method of vacuum distillation. Then, the reaction product is purified by column chromatography technology, and finally compound a is obtained;

[0051] Among them, the molar ratio of 3-(tert-butyl)-6-(ethylthio)-1,3,5-triazine-2,4(1H,3H)-dione to 2,4,5-trifluorobenzyl bromide is 1:1.2.

[0052] (2) Synthesize compound b: 6-(ethylthio)-1-(2,4,5-trifluorobenzyl)-1,3,5-triazine-2,4(1H,3H)-dione

[0053]

[0054] Dissolve the compound a synthesized in step (1) in a 50% trifluoroacetic acid dichloromethane solution, and stir and react at room temperature for 5 hours to remove the tert-butyl group. The reaction mixture is concentrated by vacuum distillation technology to remove the solvent, and finally the target compound b is obtained;

[0055] Among them, the molar ratio of compound a to the 50% trifluoroacetic acid dichloromethane solution is 1:6; the solvents used are trifluoroacetic acid and dichloromethane.

[0056] (3) Synthesize compound c: 6-(ethylthio)-3-(prop-2-yn-1-yl)-1-(2,4,5-trifluorobenzyl)-1,3,5-triazine-2,4(1H,3H)-dione

[0057]

[0058] Dissolve the compound b prepared in step (2) and propargyl bromide in acetonitrile, add potassium carbonate as a catalyst, and reflux and stir the reaction at 85 °C for 7 hours. Concentrate the reaction liquid under reduced pressure to remove the solvent to obtain compound c;

[0059] Among them, the molar ratio of compound b to propargyl bromide is 1:2.2; the solvent used is acetonitrile.

[0060] (4) Synthesis of compound d: (E)-6-((6-chloro-2-methyl-2H-indazol-5-yl)imino)-3-(prop-2-yn-1-yl)-1-(2,4,5-trifluorobenzyl)-1,3,5-triazine-2,4-dione

[0061]

[0062] Dissolve the compound c prepared in step (3) and 6-chloro-2-methyl-2H-indazol-5-amine in tert-butanol, add acetic acid to adjust the pH value to acidic, reflux and stir the reaction at 85 °C for 7 hours, neutralize the acetic acid in the system with saturated sodium bicarbonate solution, concentrate the reaction liquid under reduced pressure to remove the solvent, and then separate by column chromatography to obtain compound d;

[0063] Among them, the molar ratio of compound c to 6-chloro-2-methyl-2H-indazol-5-amine is 1:1.4; the solvent used is tert-butanol.

[0064] (5) Synthesis of intermediate: (E)-3-((1H-1,2,3-triazol-5-yl)methyl)-6-((6-chloro-2-methyl-2H-indazol-5-yl)imino)-1-(2,4,5-trifluorobenzyl)-1,3,5-triazine-2,4-dione

[0065]

[0066] Dissolve the compound d prepared in step (4), trimethylsilyl azide (TMSN 3 ) and sodium L-ascorbate in a mixed solvent of N,N-dimethylacetamide and water, add copper sulfate pentahydrate as a catalyst, and reflux and stir the reaction at 100 °C under argon protection for 18 hours. After concentrating the reaction liquid under reduced pressure to remove the solvent, separate by column chromatography to obtain the required intermediate;

[0067] Among them, the molar ratio of compound d to trimethylsilyl azide is 1:1.8, the molar ratio of compound d to sodium L-ascorbate is 1:1.2; the molar ratio of compound d to copper sulfate pentahydrate is 1:0.2. In the mixed solvent of N,N-dimethylacetamide and water, the volume ratio of N,N-dimethylacetamide to water is 9:4.

[0068] (6) Synthesis of cinnamoyl chloride compounds

[0069]

[0070] The cinnamic acid compounds are stirred and reacted with thionyl chloride at room temperature for 3 hours to obtain cinnamoyl chloride compounds;

[0071] Among them, the structures of the cinnamic acid compounds are shown in Table 1, and the molar ratio of cinnamic acid to thionyl chloride is 1:1.3; the solvent used is thionyl chloride.

[0072] Table 1 Cinnamic acid compounds

[0073]

[0074]

[0075]

[0076] (7) Synthesis of cinnamide 3CL protease covalent inhibitors

[0077]

[0078] The cinnamoyl chloride compounds prepared in step (6) and the intermediate prepared in step (5) are dissolved in a mixed solvent, stirred and reacted at room temperature for 4 hours. The reaction liquid is concentrated under reduced pressure to remove the solvent, and then separated by column chromatography to obtain cinnamide 3CL protease covalent inhibitors;

[0079] Among them, the molar ratio of cinnamoyl chloride compounds to the intermediate is 1.2:1; the mixed solvent used is obtained by mixing N,N-dimethylformamide:triethylamine:tetrahydrofuran = 1:3:12.

[0080] In this article, the cinnamide 3CL protease covalent inhibitors can be used alone or in combination with other reagents having preventive and / or therapeutic activities against coronaviruses to prepare combination drugs for preventing and / or treating coronavirus infections. In addition, the inhibitor can also be mixed with one or more pharmaceutical excipients such as excipients and diluents to form oral drugs in various dosage forms, including tablets, capsules, granules, syrups, premixes or pellets; or further processed into liniments or injections suitable for non-oral administration methods.

[0081] In this text, the covalent inhibitors of cinnamide 3CL protease can be used alone or in combination with other drugs having anti-coronavirus activity to prepare a pharmaceutical composition for preventing or treating coronavirus infection. In addition, the inhibitor can also be mixed with various excipients, diluents and other pharmaceutical auxiliary materials to prepare various dosage forms for administration, such as oral tablets, capsules, granules, syrups, premixes or pellets, as well as non-oral liniments or injections, etc. These different preparation forms increase the applicability of the drug and the compliance of patients, providing diverse treatment options to meet different treatment needs and patient conditions. In this way, it aims to provide a comprehensive solution to combat the severe coronavirus epidemic and reduce the public health burden.

[0082] In this text, the coronaviruses mentioned include but are not limited to MERS-CoV, SARS-CoV and SARS-CoV-2, and the diseases caused by the involved coronaviruses include various infectious diseases and their complications caused by SARS-CoV, MERS-CoV, and SARS-CoV-2. These viruses are all severe respiratory pathogens, and the diseases they cause can range from mild dyspnea to severe acute respiratory syndrome.

[0083] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

[0084] Conventional instrument equipment in the art is used in the following embodiments. For the experimental methods without specific conditions indicated in the following embodiments, they are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. Various raw materials are used in the following embodiments, and unless otherwise stated, commercially available products with conventional specifications in the art are used.

[0085] I. Specific embodiments for synthesizing Compounds 1-20

[0086] The structural formula of the representative compound of the compound of Formula I is shown as follows:

[0087]

[0088]

[0089] The following gives specific embodiments for synthesizing the above representative compounds.

[0090] Example 1

[0091] Preparation of Compound 1: (E)-6-((6-chloro-2-methyl-2H-indazol-5-yl)imino)-3-((1-cinnamoyl-1H-1,2,3-triazol-5-yl)methyl)-1-(2,4,5-trifluorobenzyl)-1,3,5-triazine-2,4-dione

[0092] (1) Preparation of Compound a

[0093]

[0094] 3-tert-Butyl-6-(ethylthio)-1,3,5-triazine-2,4(1H,3H)-dione (687.90 mg, 3.0 mmol), 2,4,5-trifluorobenzyl bromide (810.04 mg, 3.6 mmol), and potassium carbonate (829.20 mg, 6.0 mmol) were placed in a reactor for alkylation reaction. They were dissolved in 15 mL of acetonitrile, heated under reflux, and stirred for 14 hours with TLC monitoring. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent. The resulting solid residue was washed with saturated sodium chloride aqueous solution, extracted with ethyl acetate, the organic phase was collected, and purified by column chromatography (using petroleum ether:ethyl acetate (V:V) = 4:1 as the mobile phase), and dried to obtain 942.74 mg of Compound a with a yield of 84.16%.

[0095] (2) Preparation of Compound b

[0096]

[0097] Compound a (1120.17 mg, 3.0 mmol) prepared in step (1) was placed in a reactor, and 18 ml of 50% trifluoroacetic acid dichloromethane solution was added. The reaction was stirred at room temperature for 5 hours to remove the tert-butyl group. After the reaction was completed, the solvent was removed by concentration under reduced pressure. The residue was washed three times with 150 mL of saturated sodium bicarbonate aqueous solution and dried to obtain 881.34 mg of Compound b with a yield of 92.59%.

[0098] (3) Preparation of Compound c

[0099]

[0100] Compound b (951.87 mg, 3.0 mmol), propargyl bromide (568.94 μL, 6.6 mmol), and potassium carbonate (829.20 mg, 6.0 mmol) were dissolved in 6 mL of acetonitrile, and the reaction was refluxed and stirred at 85 °C for 7 hours with TLC monitoring of the reaction progress. After the reaction was completed, the solvent was removed by concentration under reduced pressure. The resulting solid residue was washed three times with 120 mL of water once and dried to obtain 960.59 mg of Compound c with a yield of 90.11%.

[0101] (4) Preparation of Compound d

[0102]

[0103] Dissolve compound c (1066.02 mg, 3.0 mmol) and 6-chloro-2-methyl-2H-indazol-5-amine (762.80 mg, 4.2 mmol) in 8 mL of tert-butanol, add acetic acid (1143.80 μL, 20 mmol) to provide an acidic environment, and reflux and stir the reaction at 85 °C for 7 hours. Monitor the reaction progress by TLC, concentrate the solvent under reduced pressure, wash the residue with 150 mL of water, and then separate it by column chromatography (the eluent is dichloromethane:methanol = 24:1), and recrystallize with methanol to obtain 971.93 mg of compound d, with a yield of 68.23%.

[0104] (5) Preparation of Intermediate

[0105]

[0106] Dissolve compound d (1424.49 mg, 3.0 mmol), trimethylsilyl azide (TMSN 3 )(710.28 μL, 5.4 mmol) and sodium L-ascorbate (237.73 mg, 1.2 mmol) in 60 mL of N,N-dimethylacetamide:water = 9:4 solvent, and reflux and stir the reaction at 100 °C under argon protection for 18 hours. After the reaction is completed, concentrate the solvent under reduced pressure, wash the residue with 100 mL of water, and then separate it by column chromatography (the eluent is dichloromethane:methanol = 15:1), and recrystallize with acetone-water to obtain 824.33 mg of the intermediate, with a yield of 53.06%.

[0107] (6) Preparation of Cinnamoyl Chloride

[0108]

[0109] React cinnamic acid (148.16 mg, 1.0 mmol) with thionyl chloride (94.30 μL, 1.3 mmol) at room temperature with stirring for 3 hours to obtain 85.35 mg of cinnamoyl chloride, with a yield of 51.23%.

[0110] (7) Preparation of (E)-6-((6-chloro-2-methyl-2H-indazol-5-yl)imino)-3-((1-cinnamoyl-1H-1,2,3-triazol-5-yl)methyl)-1-(2,4,5-trifluorobenzyl)-1,3,5-triazine-2,4-dione

[0111]

[0112] Cinnamoyl chloride (199.92 mg, 1.2 mmol) and 3-((1H-1,2,3-triazol-5-yl)methyl)-6-((6-chloro-2-methyl-2H-indazol-5-yl)imino)-1-(2,4,5-trifluorobenzyl)-1,3,5-triazinan-2,4-dione (510.36 mg, 1.0 mmol) were dissolved in 26 mL of DMF:DEA:THF (1:2:10), and the mixture was stirred at room temperature for 4 hours to obtain 1485.29 mg of the compound, with a yield of 74.89%. The results of 1H NMR and 13C NMR are as Figure 2 and Figure 3 shown.

[0113] 1 H NMR (600 MHz, DMSO-d6) δ 10.76 (s, 1H), 7.78 - 7.75 (m, 4H), 7.59 (d, J = 16.1 Hz, 2H), 7.25 (t, J = 8.8 Hz, 4H), 6.50 (d, J = 16.0 Hz, 2H), 5.54 (s, 1H), 5.21 (s, 2H), 4.97 (d, J = 6.7 Hz, 2H), 4.14 (s, 3H).

[0114] 13 C NMR (151 MHz, DMSO-d6) δ 165.52, 161.94, 160.30, 154.12, 151.81, 150.84, 150.17, 144.57, 140.65, 139.58, 135.29, 133.38, 131.75, 128.91, 128.88, 128.51, 128.46, 126.93, 124.57, 117.15, 117.13, 116.38, 114.70, 113.93, 106.68, 45.39, 40.02, 34.64.

[0115] Example 2

[0116] Compound 2: Preparation of (E)-6-((6-chloro-2-methyl-2H-indazol-5-yl)imino)-3-((1-((E)-3-(p-tolyl)acryloyl)-1H-1,2,3-triazol-5-yl)methyl)-1-(2,4,5-trifluorobenzyl)-1,3,5-triazine-2,4-dione.

[0117]

[0118] The preparation method was referred to Example 1, and only the corresponding raw materials needed to be replaced. The yield of the obtained compound 2 was 70.72%.

[0119] 11H NMR (600 MHz, DMSO-d6) δ 10.71 (s, 1H), 8.00 - 7.98 (m, 2H), 7.57 (d, J = 12.3 Hz, 5H), 7.38 (m, 2H), 6.95 (m, 1H), 6.67 (d, J = 18.1 Hz, 2H), 4.49 - 4.40 (m, 4H), 3.95 (s, 3H), 2.42 (s, 3H).

[0120] 13 13C NMR (151 MHz, DMSO-d6) δ 165.86, 162.11, 156.65, 153.98, 152.75, 148.72, 147.04, 144.92, 141.77, 139.20, 137.65, 133.71, 132.23, 130.92, 128.94, 128.76, 128.04, 127.18, 124.60, 118.86, 118.09, 117.13, 114.44, 111.76, 106.12, 44.81, 41.01, 37.87, 21.35.

[0121] Example 3

[0122] Compound 3: Preparation of (E)-6-((6-chloro-2-methyl-2H-indazol-5-yl)imino)-3-((1-((E)-3-(4-fluorophenyl)acryloyl)-1H-1,2,3-triazol-5-yl)methyl)-1-(2,4,5-trifluorobenzyl)-1,3,5-triazine-2,4-dione.

[0123]

[0124] The preparation method refers to Example 1, and only the corresponding raw materials need to be replaced. The yield of the obtained Compound 3: 72.88%. The results of 1H NMR and 13C NMR are as Figure 4 and Figure 5 shown.

[0125] 1 1H NMR (600 MHz, DMSO-d6) δ 11.04 (s, 1H), 8.23 (s, 1H), 7.76 - 7.73 (m, 4H), 7.43 (td, J = 6.9, 3.3 Hz, 4H), 6.70 (d, J = 16.1 Hz, 1H), 6.41 (s, 2H), 5.23 (d, J = 54.2 Hz, 2H), 5.00 (s, 2H), 4.13 (s, 3H).

[0126] 1313C NMR (151 MHz, DMSO-d6) δ 165.55, 164.65, 160.29, 156.26, 155.81, 152.18, 151.33, 149.63, 142.53, 141.87, 138.73, 132.21, 131.97, 128.48, 128.19, 126.88, 126.34, 126.16, 118.74, 118.41, 117.24, 115.80, 114.78, 111.51, 106.88, 52.89, 49.45, 33.76.

[0127] Example 4

[0128] Compound 4: Preparation of (E)-6-((6-chloro-2-methyl-2H-indazol-5-yl)imino)-3-((1-((E)-3-(4-chlorophenyl)acryloyl)-1H-1,2,3-triazol-5-yl)methyl)-1-(2,4,5-trifluorobenzyl)-1,3,5-triazine-2,4-dione.

[0129]

[0130] The preparation method refers to Example 1, and only the corresponding raw materials need to be replaced. The yield of the obtained Compound 4: 75.83%.

[0131] 1 1H NMR (600 MHz, DMSO-d6) δ 10.74 (s, 1H), 8.01 - 7.99 (m, 2H), 7.62 (d, J = 6.8 Hz, 7H), 6.90 (m, 1H), 6.70 (d, J = 12.5 Hz, 2H), 4.47 - 4.39 (m, 4H), 3.94 (s, 3H).

[0132] 13 13C NMR (151 MHz, DMSO-d6) δ 164.84, 162.16, 156.64, 153.33, 152.77, 148.13, 147.03, 144.98, 141.79, 139.55, 134.61, 133.89, 133.04, 130.49, 129.25, 128.97, 128.13, 127.81, 124.69, 118.85, 118.36, 117.19, 114.45, 111.76, 106.16, 44.85, 41.05, 37.95.

[0133] Example 5

[0134] Preparation of Compound 5: (E)-3-((1-((E)-3-(4-bromophenyl)acryloyl)-1H-1,2,3-triazol-5-yl)methyl)-6-((6-chloro-2-methyl-2H-indazol-5-yl)imino)-1-(2,4,5-trifluorobenzyl)-1,3,5-triazine-2,4-dione

[0135]

[0136] The preparation method refers to Example 1, and only the corresponding raw materials need to be replaced. The yield of the obtained Compound 5: 77.93%.

[0137] 1 H NMR(600MHz, DMSO-d6) δ10.71(s, 1H), 8.00 - 7.96(m, 2H), 7.72(d, J = 12.2Hz, 7H), 6.91(m, 1H), 6.65(d, J = 6.5Hz, 2H), 4.49 - 4.42(m, 4H), 3.96(s, 3H).

[0138] 13 C NMR(151MHz, DMSO-d6) δ165.02, 162.22, 156.53, 153.34, 152.86, 148.32, 147.85, 144.92, 141.76, 139.24, 134.22, 133.39, 131.54, 130.98, 128.73, 128.68, 128.41, 124.65, 122.36, 118.89, 118.25, 117.14, 114.44, 111.72, 106.18, 44.82, 41.04, 37.98.

[0139] Example 6

[0140] Preparation of Compound 6: 4-((E)-3-(5-(((E)-4-((6-chloro-2-methyl-2H-indazol-5-yl)imino)-2,6-dioxo-3-(2,4,5-trifluorobenzyl)-1,3,5-triazin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)-3-oxoprop-1-en-1-yl)benzonitrile

[0141]

[0142] The preparation method refers to Example 1, and only the corresponding raw materials need to be replaced. The yield of the obtained Compound 6: 76.49%.

[0143] 11H NMR (600 MHz, DMSO-d6) δ 10.75 (s, 1H), 8.03 - 7.98 (m, 2H), 7.86 - 7.82 (m, 2H), 7.73 (d, J = 5.9 Hz, 2H), 7.62 (s, 1H), 7.58 - 7.56 (m, 2H), 6.94 (s, 1H), 6.67 (d, J = 6.1 Hz, 2H), 4.51 - 4.46 (m, 4H), 3.94 (s, 3H).

[0144] 13 13C NMR (151 MHz, DMSO-d6) δ 165.82, 162.14, 161.28, 156.69, 153.34, 152.74, 147.03, 144.91, 141.71, 139.57, 139.13, 133.25, 132.13, 130.97, 128.88, 128.65, 128.14, 124.63, 118.67, 118.45, 118.06, 117.13, 114.48, 111.80, 111.02, 106.15, 44.83, 41.22, 37.91.

[0145] Example 7

[0146] Compound 7: Preparation of (E)-6-((6-chloro-2-methyl-2H-indazol-5-yl)imino)-3-((1-((E)-3-(4-nitrophenyl)acryloyl)-1H-1,2,3-triazol-5-yl)methyl)-1-(2,4,5-trifluorobenzyl)-1,3,5-triazine-2,4-dione.

[0147]

[0148] The preparation method refers to Example 1, and only the corresponding raw materials need to be replaced. The yield of the obtained Compound 7: 75.01%.

[0149] 1 1H NMR (600 MHz, DMSO-d6) δ 10.72 (s, 1H), 8.38 (s, 2H), 8.03 (d, J = 8.3 Hz, 4H), 7.83 (s, 1H), 7.68 - 7.67 (m, 1H), 7.60 (s, 1H), 6.94 (d, J = 5.7 Hz, 2H), 6.55 - 6.53 (m, 1H), 4.49 - 4.43 (m, 4H), 3.93 (s, 3H).

[0150] 1313C NMR (151 MHz, DMSO-d6) δ 165.91, 162.15, 156.84, 153.53, 152.82, 148.23, 147.25, 147.06, 144.78, 141.79, 141.01, 139.25, 133.97, 130.53, 129.06, 128.73, 128.31, 124.26, 123.86, 118.75, 118.05, 117.41, 114.14, 111.75, 106.71, 44.28, 41.10, 37.28.

[0151] Example 8

[0152] Compound 8: Preparation of (E)-6-((6-chloro-2-methyl-2H-indazol-5-yl)imino)-3-((1-((E)-3-(4-isopropylphenyl)acryloyl)-1H-1,2,3-triazol-5-yl)methyl)-1-(2,4,5-trifluorobenzyl)-1,3,5-triazine-2,4-dione.

[0153]

[0154] The preparation method refers to Example 1, and only the corresponding raw materials need to be replaced. The yield of the obtained Compound 8: 73.88%.

[0155] 1 1H NMR (600 MHz, DMSO-d6) δ 10.70 (s, 1H), 7.99 - 7.98 (m, 2H), 7.65 (d, J = 11.3 Hz, 5H), 7.28 (s, 2H), 6.97 - 6.96 (m, 1H), 6.64 (d, J = 4.1 Hz, 2H), 5.02 (s, 1H), 4.48 - 4.41 (m, 4H), 3.95 (s, 3H), 1.19 (s, 6H).

[0156] 13 13C NMR (151 MHz, DMSO-d6) δ 164.16, 162.21, 156.36, 155.73, 153.27, 147.77, 147.53, 147.02, 144.94, 141.73, 139.21, 133.81, 132.47, 130.93, 128.72, 128.69, 128.33, 126.01, 124.56, 118.38, 118.14, 117.15, 114.46, 111.73, 106.12, 44.82, 41.01, 37.86, 33.23, 23.35.

[0157] Example 9

[0158] Preparation of Compound 9: (E)-3-((1-((E)-3-(4-(tert-Butyl)phenyl)acryloyl)-1H-1,2,3-triazol-5-yl)methyl)-6-((6-chloro-2-methyl-2H-indazol-5-yl)imino)-1-(2,4,5-trifluorobenzyl)-1,3,5-triazine-2,4-dione.

[0159]

[0160] The preparation method refers to Example 1, and only the corresponding raw materials need to be replaced. The yield of the obtained Compound 9: 71.17%.

[0161] 1 H NMR (600 MHz, DMSO-d6) δ 10.69 (s, 1H), 8.05 - 8.03 (m, 2H), 7.75 (d, J = 12.9 Hz, 5H), 7.27 (s, 2H), 6.94 - 6.92 (m, 1H), 6.65 (d, J = 4.9 Hz, 2H), 4.47 - 4.40 (m, 4H), 3.96 (s, 3H), 1.34 (s, 9H).

[0162] 13 C NMR (151 MHz, DMSO-d6) δ 165.03, 162.51, 156.37, 153.32, 152.71, 150.53, 148.15, 147.82, 144.98, 141.06, 139.23, 133.92, 132.12, 130.91, 128.76, 128.53, 128.25, 124.92, 124.66, 118.87, 118.31, 117.15, 114.43, 111.79, 106.14, 44.83, 41.02, 37.83, 34.15, 31.36.

[0163] Example 10

[0164] Compound 10: (E)-6-((6-chloro-2-methyl-2H-indazol-5-yl)imino)-3-((1-((E)-3-(4-methoxyphenyl)acryloyl)-1H-1,2,3-triazol-5-yl)methyl)-1-(2,4,5-trifluorobenzyl)-1,3,5-triazine-2,4-dione.

[0165]

[0166] The preparation method refers to Example 1, and only the corresponding raw materials need to be replaced. The yield of the obtained Compound 10: 70.23%.

[0167] 1 1H NMR (600 MHz, DMSO-d6) δ 10.77 (s, 1H), 8.02 - 8.00 (m, 2H), 7.69 (d, J = 10.1 Hz, 5H), 7.04 (s, 2H), 6.93 - 6.91 (m, 1H), 6.66 (d, J = 6.1 Hz, 2H), 4.53 - 4.44 (m, 4H), 3.89 (s, 3H), 3.79 (s, 3H).

[0168] 13 13C NMR (151 MHz, DMSO-d6) δ 165.12, 162.93, 159.98, 156.69, 155.21, 152.37, 148.89, 147.01, 144.97, 141.73, 139.04, 133.11, 130.93, 130.21, 128.79, 128.56, 127.59, 124.62, 118.80, 118.03, 117.12, 114.49, 114.21, 111.72, 106.12, 55.82, 44.89, 41.02, 37.89.

[0169] Example 11

[0170] Compound 11: Preparation of (E)-6-((6-chloro-2-methyl-2H-indazol-5-yl)imino)-3-((1-((E)-3-(m-tolyl)acryloyl)-1H-1,2,3-triazol-5-yl)methyl)-1-(2,4,5-trifluorobenzyl)-1,3,5-triazine-2,4-dione.

[0171]

[0172] The preparation method refers to Example 1, and only the corresponding raw materials need to be replaced. The yield of the obtained Compound 11: 69.97%.

[0173] 1 1H NMR (600 MHz, DMSO-d6) δ 10.70 (s, 1H), 8.00 - 7.97 (m, 2H), 7.71 (d, J = 4.3 Hz, 2H), 7.62 (s, 1H), 7.55 - 7.52 (m, 1H), 7.17 - 7.15 (m, 2H), 6.95 - 6.93 (m, 2H), 6.73 (d, J = 5.2 Hz, 2H), 4.49 - 4.41 (m, 4H), 3.96 (s, 3H), 2.43 (s, 3H).

[0174] 1313C NMR (151 MHz, DMSO-d6) δ 164.75, 162.41, 156.97, 153.36, 152.65, 148.11, 147.15, 144.96, 141.28, 139.16, 138.01, 135.47, 133.42, 130.32, 128.76, 128.57, 128.15, 128.02, 126.76, 125.50, 124.05, 118.94, 118.41, 117.58, 114.25, 111.37, 106.49, 44.91, 41.35, 37.16, 21.73.

[0175] Example 12

[0176] Compound 12: Preparation of (E)-6-((6-chloro-2-methyl-2H-indazol-5-yl)imino)-3-((1-((E)-3-(3-fluorophenyl)acryloyl)-1H-1,2,3-triazol-5-yl)methyl)-1-(2,4,5-trifluorobenzyl)-1,3,5-triazine-2,4-dione.

[0177]

[0178] The preparation method refers to Example 1, and only the corresponding raw materials need to be replaced. The yield of the obtained Compound 12: 68.19%.

[0179] 1 1H NMR (600 MHz, DMSO-d6) δ 10.68 (s, 1H), 8.03 - 7.96 (m, 2H), 7.69 (d, J = 4.1 Hz, 2H), 7.63 (s, 1H), 7.41 (s, 1H), 7.27 - 7.25 (m, 1H), 6.63 (d, J = 8.9 Hz, 3H), 6.61 (d, J = 6.7 Hz, 2H), 4.51 - 4.46 (m, 4H), 3.99 (s, 3H).

[0180] 13 13C NMR (151 MHz, DMSO-d6) δ 165.98, 162.81, 162.19, 156.99, 153.65, 152.68, 148.05, 147.83, 144.54, 141.25, 139.67, 136.97, 133.78, 130.86, 130.15, 128.29, 128.06, 124.36, 124.13, 118.91, 118.07, 117.50, 114.72, 114.30, 113.83, 111.67, 106.25, 44.17, 41.15, 37.92.

[0181] Example 13

[0182] Preparation of Compound 13: (E)-6-((6-chloro-2-methyl-2H-indazol-5-yl)imino)-3-((1-((E)-3-(3-chlorophenyl)acryloyl)-1H-1,2,3-triazol-5-yl)methyl)-1-(2,4,5-trifluorobenzyl)-1,3,5-triazine-2,4-dione.

[0183]

[0184] The preparation method refers to Example 1, and only the corresponding raw materials need to be replaced. The yield of the obtained Compound 13: 73.76%.

[0185] 1 H NMR (600 MHz, DMSO-d6) δ 10.72 (s, 1H), 8.01 - 7.94 (m, 2H), 7.69 (d, J = 5.8 Hz, 2H), 7.59 (s, 1H), 7.48 - 7.45 (m, 1H), 7.29 (d, J = 10.3 Hz, 3H), 6.95 (m, 1H), 6.24 (d, J = 4.3 Hz, 2H), 4.47 - 4.40 (m, 4H), 3.96 (s, 3H).

[0186] 13 C NMR (151 MHz, DMSO-d6) δ 165.91, 162.56, 156.80, 153.17, 152.41, 148.43, 147.44, 144.82, 141.69, 139.02, 136.91, 134.45, 133.47, 130.93, 130.17, 128.72, 128.25, 128.03, 126.87, 126.66, 124.59, 118.96, 118.01, 117.12, 114.74, 111.83, 106.11, 44.38, 41.95, 37.27.

[0187] Example 14

[0188] Preparation of Compound 14: (E)-3-((1-((E)-3-(3-bromophenyl)acryloyl)-1H-1,2,3-triazol-5-yl)methyl)-6-((6-chloro-2-methyl-2H-indazol-5-yl)imino)-1-(2,4,5-trifluorobenzyl)-1,3,5-triazine-2,4-dione.

[0189]

[0190] The preparation method refers to Example 1, and only the corresponding raw materials need to be replaced. The yield of the obtained compound 14: 70.62%.

[0191] 1 H NMR(600MHz,DMSO-d6)δ10.78(s,1H),8.00-7.95(m,2H),7.70(d,J=7.2Hz,2H),7.62(d,J=8.8Hz,3H),7.41-7.39(m,1H),7.28(s,1H),6.97(s,1H),6.70-6.67(d,J=4.4Hz,2H),4.50-4.44(m,4H),3.93(s,3H).

[0192] 13 C NMR(151MHz,DMSO-d6)δ165.25,162.38,156.63,153.48,152.80,148.77,147.06,144.73,141.12,139.44,137.02,133.59,133.26,130.75,130.40,129.26,128.81,128.13,127.25,124.14,123.21,118.78,118.14,117.58,114.79,111.16,106.39,44.97,41.04,37.33.

[0193] Example 15

[0194] Compound 15: Preparation of (E)-6-((6-chloro-2-methyl-2H-indazol-5-yl)imino)-3-((1-((E)-3-(2-fluorophenyl)acryloyl)-1H-1,2,3-triazol-5-yl)methyl)-1-(2,4,5-trifluorobenzyl)-1,3,5-triazine-2,4-dione.

[0195]

[0196] The preparation method refers to Example 1, and only the corresponding raw materials need to be replaced. The yield of the obtained compound 15: 63.37%.

[0197] 11H NMR (600 MHz, DMSO-d6) δ 10.71 (s, 1H), 8.05 (d, J = 7.5 Hz, 3H), 7.72 (s, 1H), 7.63 (s, 1H), 7.33 - 7.30 (m, 1H), 7.18 (d, J = 5.7 Hz, 3H), 6.95 - 6.93 (m, 1H), 6.69 - 6.66 (m, 1H), 6.41 (s, 1H), 4.48 - 4.40 (m, 4H), 3.91 (s, 3H).

[0198] 13 13C NMR (151 MHz, DMSO-d6) δ 165.02, 162.44, 161.87, 156.51, 153.29, 152.54, 152.15, 148.55, 144.63, 141.96, 139.14, 133.81, 130.54, 129.61, 128.34, 128.09, 128.01, 124.34, 124.16, 123.44, 118.85, 118.35, 117.87, 115.47, 114.03, 111.76, 106.11, 44.99, 41.26, 37.72.

[0199] Example 16

[0200] Compound 16: Preparation of (E)-6-((6-chloro-2-methyl-2H-indazol-5-yl)imino)-3-((1-((E)-3-(3,4-dimethylphenyl)acryloyl)-1H-1,2,3-triazol-5-yl)methyl)-1-(2,4,5-trifluorobenzyl)-1,3,5-triazine-2,4-dione.

[0201]

[0202] The preparation method refers to Example 1, and only the corresponding raw materials need to be replaced. The yield of the obtained compound 16: 67.92%.

[0203] 1 1H NMR (600 MHz, DMSO-d6) δ 10.77 (s, 1H), 8.01 - 7.98 (m, 2H), 7.75 (d, J = 7.4 Hz, 2H), 7.62 (s, 1H), 7.50 - 7.48 (m, 1H), 7.19 (d, J = 8.2 Hz, 2H), 6.94 - 6.92 (m, 1H), 6.71 - 6.65 (d, J = 7.6 Hz, 2H), 4.51 - 4.44 (m, 4H), 3.94 (s, 3H), 2.35 - 2.31 (m, 6H).

[0204] 13 13C NMR (151 MHz, DMSO-d6) δ 165.50, 162.95, 156.58, 153.29, 152.38, 148.47, 147.02, 144.21, 141.70, 139.36, 136.79, 136.03, 133.29, 132.95, 132.04, 131.84, 130.72, 128.22, 128.05, 125.28, 124.51, 118.87, 118.58, 117.83, 114.70, 111.88, 106.19, 44.40, 41.12, 37.11, 19.63, 18.55.

[0205] Example 17

[0206] Compound 17: Preparation of (E)-6-((6-chloro-2-methyl-2H-indazol-5-yl)imino)-3-((1-((E)-3-(3,4-difluorophenyl)acryloyl)-1H-1,2,3-triazol-5-yl)methyl)-1-(2,4,5-trifluorobenzyl)-1,3,5-triazine-2,4-dione.

[0207]

[0208] The preparation method refers to Example 1, and only the corresponding raw materials need to be replaced. The yield of the obtained compound 17: 66.03%.

[0209] 1 1H NMR (600 MHz, DMSO-d6) δ 10.70 (s, 1H), 8.00 - 7.96 (m, 2H), 7.72 (d, J = 6.8 Hz, 2H), 7.60 (s, 1H), 7.48 - 7.45 (m, 1H), 7.10 (s, 1H), 6.94 - 6.91 (m, 2H), 6.77 (d, J = 6.6 Hz, 2H), 4.49 - 4.43 (m, 4H), 3.94 (s, 3H).

[0210] 1313C NMR (151 MHz, DMSO-d6) δ 165.14, 162.32, 156.45, 153.78, 152.45, 149.95, 148.52, 147.97, 147.17, 144.73, 141.89, 139.23, 133.63, 132.11, 130.70, 128.58, 128.10, 126.84, 125.45, 124.52, 118.91, 118.08, 117.27, 114.75, 112.15, 111.07, 106.88, 44.79, 41.94, 37.96.

[0211] Example 18

[0212] Compound 18: Preparation of (E)-6-((6-chloro-2-methyl-2H-indazol-5-yl)imino)-3-((1-((E)-3-(3,4-dichlorophenyl)acryloyl)-1H-1,2,3-triazol-5-yl)methyl)-1-(2,4,5-trifluorobenzyl)-1,3,5-triazine-2,4-dione.

[0213]

[0214] The preparation method refers to Example 1, and only the corresponding raw materials need to be replaced. The yield of the obtained compound 18: 67.54%.

[0215] 1 1H NMR (600 MHz, DMSO-d6) δ 10.72 (s, 1H), 8.04 - 7.96 (m, 2H), 7.75 (d, J = 6.6 Hz, 2H), 7.63 (d, J = 8.1 Hz, 2H), 7.49 - 7.47 (m, 1H), 7.31 (s, 1H), 6.91 - 6.90 (m, 1H), 6.73 (d, J = 7.3 Hz, 2H), 4.48 - 4.41 (m, 4H), 3.95 (s, 3H).

[0216] 13 13C NMR (151 MHz, DMSO-d6) δ 165.91, 162.45, 156.32, 153.05, 152.72, 148.35, 147.62, 144.36, 141.74, 139.16, 134.34, 133.74, 133.46, 132.38, 130.80, 130.13, 128.35, 128.11, 127.61, 127.26, 124.33, 118.99, 118.07, 117.61, 114.77, 111.62, 106.49, 44.87, 41.06, 37.28.

[0217] Example 19

[0218] Preparation of Compound 19: (E)-6-((6-chloro-2-methyl-2H-indazol-5-yl)imino)-3-((1-((E)-3-(3,4-dibromophenyl)acryloyl)-1H-1,2,3-triazol-5-yl)methyl)-1-(2,4,5-trifluorobenzyl)-1,3,5-triazine-2,4-dione.

[0219]

[0220] The preparation method refers to Example 1, and only the corresponding raw materials need to be replaced. The yield of the obtained Compound 19: 70.03%.

[0221] 1 H NMR (600 MHz, DMSO-d6) δ 10.76 (s, 1H), 8.01 - 7.99 (m, 2H), 7.72 (d, J = 6.9 Hz, 2H), 7.61 (d, J = 7.6 Hz, 2H), 7.44 - 7.42 (m, 2H), 6.93 (s, 1H), 6.70 (d, J = 6.6 Hz, 2H) 4.49 - 4.41 (m, 4H), 3.93 (s, 3H).

[0222] 13 C NMR (151 MHz, DMSO-d6) δ 165.31, 162.61, 156.90, 153.24, 152.65, 148.31, 147.96, 144.91, 141.58, 139.40, 136.22, 133.95, 133.21, 132.05, 130.98, 128.87, 128.34, 127.08, 125.81, 124.63, 123.22, 118.62, 118.25, 117.95, 114.08, 111.84, 106.46, 44.23, 41.21, 37.17.

[0223] Example 20

[0224] Preparation of Compound 20: 4-((E)-3-(5-(((E)-4-((6-chloro-2-methyl-2H-indazol-5-yl)imino)-2,6-dioxo-3-(2,4,5-trifluorobenzyl)-1,3,5-triazin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)-3-oxoprop-1-en-1-yl)phthalonitrile.

[0225]

[0226] The preparation method refers to Example 1, and only the corresponding raw materials need to be replaced. The yield of the obtained compound 20: 64.92%.

[0227] 1 H NMR(600MHz,DMSO-d6)δ10.80(s,1H),8.02(d,J=5.9Hz,3H),7.75(d,J=12.1Hz,4H),7.63(s,1H),6.93-6.90(m,1H),6.71(d,J=6.8Hz,2H),4.56-4.45(m,4H),3.91(s,3H).

[0228] 13 C NMR(151MHz,DMSO-d6)δ165.70,162.62,156.28,153.95,152.29,148.11,147.36,145.03,141.34,140.28,139.49,133.92,133.45,133.02,131.07,130.82,128.68,128.21,124.66,118.83,118.52,117.41,115.92,115.89,115.81,115.08,114.62,111.82,105.97,44.48,41.16,37.93.

[0229] Example 21

[0230] Compound 21: Preparation of (E)-6-((6-chloro-2-methyl-2H-indazol-5-yl)imino)-3-((1-((E)-3-(3,4-dinitrophenyl)acryloyl)-1H-1,2,3-triazol-5-yl)methyl)-1-(2,4,5-trifluorobenzyl)-1,3,5-triazine-2,4-dione.

[0231]

[0232] The preparation method refers to Example 1, and only the corresponding raw materials need to be replaced. The yield of the obtained compound 21: 67.90%.

[0233] 11H NMR (600 MHz, DMSO-d6) δ 10.68 (s, 1H), 8.45 (d, J = 7.3 Hz, 2H), 8.14 (s, 1H), 8.03 - 7.97 (m, 2H), 7.83 (s, 1H), 7.66 - 7.64 (m, 1H), 7.59 (s, 1H), 6.97 (d, J = 6.5 Hz, 2H), 6.65 - 6.63 (m, 1H), 4.50 - 4.42 (m, 4H), 3.93 (s, 3H).

[0234] 13 13C NMR (151 MHz, DMSO-d6) δ 165.38, 162.77, 156.39, 153.70, 152.01, 148.03, 147.09, 144.62, 144.36, 144.10, 143.97, 141.54, 139.23, 135.32, 133.12, 130.66, 128.86, 128.52, 124.85, 124.51, 120.29, 118.93, 118.23, 117.07, 116.65, 111.96, 106.39, 44.66, 41.39, 37.92.

[0235] II. Biological Activity Assay

[0236] 1. 3CL pro Inhibitory Activity Test

[0237] In this study, the fluorescence resonance energy transfer (FRET) technique was used to systematically evaluate the inhibitory effect of the studied compounds on SARS-CoV-2 3CL pro . The specific method is as follows:

[0238] (a) Compound Preparation

[0239] After accurately weighing the target compound, it was formulated with dimethyl sulfoxide (DMSO) to a predetermined concentration gradient, 5 μL for each gradient, and prepared for subsequent experiments.

[0240] (b) Experimental Setup

[0241] The above compound solution and 91 μL of the detection reagent (Assay Reagent, which is a mixture of Assay Buffer and 2019-nCoV Mpro / 3CL pro in a ratio of 90:1, source: Shanghai Beyotime Biotechnology Co., Ltd.) were successively added to a black 96-well plate and mixed well.

[0242] (c) Incubation and Substrate Addition

[0243] Incubate for 10 minutes at 37 °C in the dark. Subsequently, quickly add 4 μL of Substrate (100 μM Dabcyl-KTSAVLQSGFRKME-Edans, source: Shanghai Beyotime Biotechnology Co., Ltd.) to each reaction well and mix again.

[0244] (d) Fluorescence measurement

[0245] After continuing to incubate for 5 minutes at 37 °C in the dark, use the Varioskan Flash multimode microplate reader from Thermo Fisher Scientific to measure the fluorescence signal within 5 to 30 minutes, setting the excitation wavelength at 340 nm and the emission wavelength at 490 nm.

[0246] (e) Control and data analysis

[0247] In the experiment, the Assay Reagent without the compound was set as the 100% enzyme activity control, and the Assay Buffer without SARS-CoV-2 Mpro / 3CL pro was set as the blank control. At the same time, S-216722 (Shandong Xuanshuo Pharmaceutical Technology Co., Ltd.) and PF-07321332 (Jinan Jianfeng Chemical Co., Ltd.) were used as positive controls for comparison. All data were analyzed by nonlinear regression using GraphPad Prism software to calculate the IC 50 value of each sample.

[0248]

[0249] The experimental results are shown in Tables 2 and 3 (in Table 2, the column where the IC 50 is located, A: IC 50 < 200 nM, B: IC 50 = 200 - 500 nM, C: IC 50 = 500 - 1000 nM, D: IC 50 > 1000 nM). The compounds in the examples all have inhibitory activity against 3CL pro . Among them, Compounds 1, 3, and 17 have stronger inhibitory effects on 3CL pro , and their IC 50 values are all below 200 nM.

[0250] Table 2 Inhibitory activity of Compounds 1 - 21 against 3CL pro

[0251] Compound number <![CDATA[IC 50 (nM)]]> 1 A 2 C 3 A 4 B 5 C 6 C 7 B 8 C 9 B 10 B 11 C 12 B 13 C 14 B 15 D 16 C 17 A 18 B 19 B 20 C 21 D

[0252] Table 3 Inhibitory activity of Compounds 1, 3, 17, S-217622, and PF-07321332 against 3CL pro ​

[0253]

[0254] The results in Table 2 and Table 3 show that compounds 1-21 have inhibitory effects on 3CL pro to varying degrees. Specifically, the half-maximal inhibitory concentration (IC 50 ) values of compounds 1, 3, and 17 are all lower than 200 nanomoles (nM), which emphasizes the effective inhibitory activity of the covalent inhibitors of 3CL protease of cinnamamides in the present invention against the main protease of SARS-CoV-2 (3CL pro ). Further quantitative analysis reveals that, as Figures 6 to 8 shown, compounds 1, 3, and 17 exhibit the highest inhibitory activity among all the tested compounds. Among them, compound 1 shows a more superior inhibitory effect compared to compounds 3 and 17, and its data performance supports the further development and application of compound 1 as a potential anti-coronavirus drug. These findings not only demonstrate the potential of these compounds in virus inhibition but also provide strong experimental support for further drug development research. By carefully analyzing the IC 50 values, the antiviral activities of these compounds can be more accurately evaluated and optimized, providing effective treatment options for dealing with SARS-CoV-2 and its variants.

[0255] 2, 3CL pro Cytotoxicity test

[0256] (a) Cell culture and seeding

[0257] In this study, the cytotoxic effects of compounds 1, 3, and 17 on cells in vitro were preliminarily evaluated by the MTT method. HepG2, HEK293, and A549 cells in the logarithmic growth phase were used in the experiment. After being treated with trypsin, the cells were converted into a single-cell suspension, and the cell concentration was adjusted to 5×10 4 cells / mL. Under sterile conditions, 180 μL of the cell suspension was seeded into each well of a 96-well plate, and then the plate was placed in an incubator at 37°C and 5% CO 2 for 24 hours to form a single-layer cell membrane at the bottom of the well plate.

[0258] (b) Drug treatment and secondary incubation

[0259] After incubation, 20 μL of the test drug solution with different concentrations was added to each well, and 6 replicates were set up simultaneously to enhance the repeatability of the experiment. S-216722 (Shandong Xuanshuo Pharmaceutical Technology Co., Ltd.) and PF-07321332 (Jinan Jianfeng Chemical Co., Ltd.) were used as positive controls for comparison in the experiment. After treatment, the culture plate was placed back in an incubator at 37°C and 5% CO 2Continue to incubate for 24 hours in the environment of

[0260] (c) MTT activity detection

[0261] After the cells are incubated again, add 20 μL of 5 g / L MTT solution to each well and continue to culture for 4 hours under the same incubation conditions. Thereafter, gently remove the supernatant in the wells and add 100 μL of DMSO to each well. Place the culture plate on a shaker and shake it at a low speed for 10 minutes to ensure that the formazan crystals are completely dissolved. Use an enzyme-linked immunosorbent assay reader to measure the absorbance value of each well at a wavelength of 490 nm, and calculate the cell survival rate of each well accordingly.

[0262] (d) Data analysis and results

[0263] The relevant cell survival data and graphic analysis results are shown in Tables 4 to 6 and Figures 9 to 11 . These detailed results provide a scientific basis for in-depth evaluation of the safety of the tested compounds, facilitating subsequent research and the drug development process.

[0264]

[0265] Table 4 Tested drugs on A549 cells

[0266]

[0267] Table 5 Tested drugs on HEK293 cells

[0268]

[0269] Table 6 Tested drugs on HepG2 cells

[0270]

[0271] Tables 4 to 6 summarize the inhibition efficiency data of PF-07321332, S-217622, and Compounds 1, 3, and 17 on A549, HEK293, and HepG2 cells, in combination with Figures 9 to 11A detailed analysis was carried out. From these data, it can be observed that at all tested concentrations, Compound 1 showed lower toxicity than PF-07321332 and S-217622 in inhibiting HepG2 and HEK293 cells. When treating A549 cells, Compound 1 showed relatively low toxicity at low concentrations. Additionally, Compounds 3 and 17 showed slightly higher toxicity to these three types of cells than PF-07321332 and S-217622 in high-concentration treatments of 400 nM and 200 nM. However, when the concentration was reduced to 12.5 nM or lower, the survival rates of these two compounds in HEK293 and HepG2 cells were higher than those of PF-07321332 and S-217622, but still lower than that of Compound 1. These analyses clarified that Compound 1 not only showed high inhibitory activity against 3CL pro but also had low cytotoxicity, indicating its research value as a potential antiviral drug, and it is recommended to conduct in-depth research on it.

[0272] The above content is only to illustrate the technical idea of the present invention and cannot be used to limit the protection scope of the present invention. Any modifications made on the basis of the technical solution according to the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A cinnamamide 3CL protease covalent inhibitor, characterized in that: A solvate, enantiomer, diastereomer, tautomer or a mixture thereof in any ratio of a compound of formula I or a pharmaceutically acceptable salt thereof; The structural formula of the compound of formula I is: Wherein, R1, R2 and R3 are each independently selected from -H -CH3 -F -Cl -Br -CN -NO2 i-Pr t-Bu 2. The cinnamamide 3CL protease covalent inhibitor according to claim 1, characterized in that: Representative compounds are as follows:

3. The cinnamamide 3CL protease covalent inhibitor according to claim 1 or 2, characterized in that: The compound of formula I is complexed with a salt formed with one or more acids, wherein the acid is hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, fumaric acid, maleic acid, oxalic acid, malonic acid, succinic acid, citric acid, malic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, glutamic acid or aspartic acid.

4. The method for preparing the cinnamamide 3CL protease covalent inhibitor according to any one of claims 1 to 3, characterized in that: The cinnamoyl chloride compound is mixed with the intermediate to react to obtain a cinnamoyl amide 3CL protease covalent inhibitor; Among them, cinnamoyl chloride compounds are R1, R2 and R3 are each independently selected from -H -CH3 -F -Cl -Br -CN -NO2 i-Pr t-Bu The intermediate is 5. The method for preparing the cinnamamide 3CL protease covalent inhibitor according to claim 4, characterized in that: The molar ratio of the cinnamoyl chloride compound to the intermediate is 1.2:

1.

6. A pharmaceutical composition for resisting coronavirus, characterized in that: The pharmaceutical composition comprises the cinnamamide 3CL protease covalent inhibitor according to any one of claims 1 to 3 and other drugs with anti-coronavirus activity.

7. Use of the cinnamamide 3CL protease covalent inhibitor according to any one of claims 1 to 3 or the anti-coronavirus pharmaceutical composition according to claim 6 in the preparation of a drug for preventing and / or treating coronavirus.

8. The use according to claim 7, characterized in that: The medicament contains one or more pharmaceutically acceptable carriers or excipients.

9. The use according to claim 7, characterized in that: The coronavirus protease is SARS-CoV-23CL pro .

10. The use according to claim 7, characterized in that: The concentration of the cinnamamide 3CL protease covalent inhibitor is 0.1-0.5 μM.

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