Compound F798-0523 and application thereof in preparation of SARS-CoV-2 antiviral inhibitor

Abstract

The invention discloses a compound F798-0523 and an application of the compound F798-0523 in preparation of an SARS-CoV-2 antiviral inhibitor. The invention provides a compound which is named as a compound F798-0523 and is shown as a formula (I). The invention also provides an application of the compound F798-0523 or the pharmaceutically acceptable salt of the compound F798-0523 in preparation of a coronavirus inhibitor. The invention also provides an application of the compound F798-0523 or the pharmaceutically acceptable salt thereof in preparation of a medicine for treating and / or preventing coronavirus infection. The invention has popularization and application values for prevention and control of novel coronavirus. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to compound F798-0523 and its application in the preparation of SARS-CoV-2 antiviral inhibitors. Background Art

[0002] The coronavirus disease 2019, COVID-2019 is an acute respiratory infectious disease caused by the Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Variant viruses such as omicron and delta continue to emerge. Mutant viruses such as omicron contain a large number of mutation sites, showing super-spreadability, and weakening the protective effects of existing vaccines and neutralizing antibodies.

[0003] SARS-CoV-2 is a virus with an envelope structure and a linear single-stranded positive-sense RNA genome. The genome is about 29 kb. Approximately two-thirds of the 5′ end region encodes two open reading frames (ORFs), 1a and 1b, which are translated to form polyproteins pp1a and pp1ab after translation. These two polyproteins are cleaved by papain-like protease (PLpro) and main protease (Mpro) to produce nonstructural proteins (nsp) nsp1-nsp16. The latter one-third region encodes structural proteins, namely spike protein (S), envelope protein (E), membrane protein (M), nucleocapsid protein (N), and some accessory proteins.

[0004] The research and development of antiviral drugs is another important means to contain the epidemic and overcome virus mutations in addition to vaccination. In the past two years, a large number of scientific research results have revealed the key steps in the life cycle of SARS-CoV-2, clarified the functions of a number of important proteins involved in virus replication and infection, and provided potential new targets for drug design. More importantly, the spike protein at the virus invasion stage, the polymerase related to virus transcription and replication, and the main protease and papain protease for virus protein maturation and processing. In order to cope with the drug resistance mutations generated by drug targets in the future, it is necessary to explore new drug targets.

[0005] There is a non-conserved region in the nsp3 domain of coronaviruses. In 2003, when analyzing the first batch of SARS coronavirus genome sequences, it was considered a unique domain only present in the SARS coronavirus, so this region was called the "SARS unique domain" (SUD). The amino acid sequence homology between SARS-CoV-2 and the SUD of SARS-CoV is approximately 75%. SUD contains three subdomains, namely SUD-N, SUD-M, and SUD-C. Among them, SUD-N and SUD-M are collectively called SUDcore. SUD recognizes special RNA structures in the viral genome. The SUD-G4 interaction is a key factor in the regulation of the cytoplasmic transport of coronavirus RNA and sgRNA. In addition, SUD can also interact with host-related proteins, thereby improving the translation efficiency of viral proteins. Summary of the Invention

[0006] The object of the present invention is to provide compound F798-0523 and its application in the preparation of an antiviral inhibitor against SARS-CoV-2.

[0007] The present invention provides a compound named compound F798-0523, as shown in formula (Ⅰ);

[0008]

[0009] Compound F798-0523 has a molecular weight of 489.5 and its molecular formula is:

[0010] c1cc(sc1)Cn2c(=O)c3c(ccs3)n4c2nnc4SCC(=O)Nc5ccc(cc5F)F.

[0011] The target of compound F798-0523 is: SARS-CoV-2 SUD-core.

[0012] The present invention also provides the application of compound F798-0523 or a pharmaceutically acceptable salt thereof in the preparation of a coronavirus inhibitor.

[0013] The present invention also provides the application of compound F798-0523 or a pharmaceutically acceptable salt thereof in the preparation of a drug for the treatment and / or prevention of coronavirus infection.

[0014] The present invention also provides a coronavirus inhibitor, the active ingredient of which includes compound F798-0523 or a pharmaceutically acceptable salt thereof.

[0015] The present invention also provides a drug for the treatment and / or prevention of coronavirus infection, the active ingredient of which includes compound F798-0523 or a pharmaceutically acceptable salt thereof.

[0016] Specifically, the coronavirus described above is SARS-CoV-2.

[0017] The present invention also protects a method for preparing compound F798-0523.

[0018] The preparation method includes the following steps:

[0019] Compound M2 and compound M3 react to obtain compound F798-0523; compound M2 is shown as formula (Ⅱ); compound M3 is shown as formula (Ⅲ);

[0020]

[0021] As an example, compound M2 and compound M3 react according to the following method: Compound M2 (3.5 mmol) is dissolved in 30 ml of N,N-dimethylformamide, compound M3 (3.5 mmol) and K2CO3 (5.3 mmol) are added, and the mixture is stirred at room temperature for 5 h.

[0022] Compound M3 is obtained by reacting 2,4-difluoroaniline with chloroacetyl chloride.

[0023] As an example, 2,4-difluoroaniline and chloroacetyl chloride react according to the following method: 2,4-Difluoroaniline (3.5 mmol) is dissolved in dichloromethane to obtain a fluoroaniline solution; chloroacetyl chloride (3.85 mmol) is dissolved in dichloromethane to obtain a chloroacetyl chloride solution; the chloroacetyl chloride solution is added dropwise to the fluoroaniline solution under an ice bath, then triethylamine (7 mmol) is added, and then the reaction is carried out at room temperature for 5 hours.

[0024] As an example, the preparation method of compound M2 includes the following steps:

[0025] (1) Methyl 3-aminothiophene-2-carboxylate (1 eq, 15 mmol) is dissolved in 30 mL of dichloromethane, then the temperature is lowered to 0 °C, carbon disulfide (1.2 eq) is added dropwise and stirred for 10 min, then triethylamine (2 eq) is added dropwise, then the temperature is naturally raised to room temperature, and then the reaction is carried out for 18 h, and compound M1-a is obtained by purification;

[0026] (2) Compound M1-a is dissolved in 30 ml of tetrahydrofuran, then 2-thiophenemethylamine (1.2 eq) and triethylamine (2 eq) are added in sequence, the mixture is stirred at room temperature for 2 h, then the solvent is evaporated, the residue is dissolved in 30 ml of methanol, KOH (5.3 mmol) is added, and the mixture is stirred at room temperature for 30 min, and compound M1 is obtained by purification;

[0027] (3) Dissolve compound M1 (1 eq) in 30 ml of ethanol, then add anhydrous hydrazine (7.0 eq), and reflux at 80 °C for 18 h. Evaporate the solvent to dryness, dissolve the residue in 30 ml of ethanol, add potassium hydroxide (3 eq) and carbon disulfide (3 eq), and reflux at 80 °C for 18 h. Purify to obtain compound M2.

[0028] Compound M1-a is shown in formula (IV). Compound M1 is shown in formula (V).

[0029]

[0030] Specifically, the synthesis route of compound F798-0523 is shown in Figure 7 . This synthesis route has the advantages of easily available raw materials, environmental friendliness, and simple operation, and is suitable for large-scale industrial production.

[0031] In the said drug or the said inhibitor, in addition to the said compound as the active ingredient or the only active ingredient, a carrier may also be included.

[0032] Exemplarily, the carrier may be any one or any combination of a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, an adsorption carrier, a surfactant, or a lubricant.

[0033] Exemplarily, the carrier materials include but are not limited to water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly water-soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), and enteric-soluble carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.).

[0034] Exemplarily, the said drug or the said inhibitor can be made into various dosage forms, including but not limited to tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, freeze-dried powder injections, etc.

[0035] In the face of the impact of the novel coronavirus on the public health system, it is of great significance to discover new inhibitor targets and new small molecule inhibitors. Compound F798-0523 was obtained by the inventors of the present invention through primary screening (virtual screening) and antiviral effect screening (secondary screening), and is a new compound. The specific result of compound F798-0523 with SARS-CoV-2 SUD-core. Compound F798-0523 has the activity of inhibiting coronaviruses and can be used as a coronavirus inhibitor. The present invention also provides a preparation method of compound F798-0523. The present invention has the value of popularization and application for the prevention and control of the novel coronavirus. Description of the Drawings

[0036] Figure 1The binding mode of the drug to the N-terminal domain in the virtual screening of small molecule drugs targeting SARS-CoV-2 SUD.

[0037] Figure 2 Flowchart of virtual screening of small molecule drugs targeting SARS-CoV-2 SUD.

[0038] Figure 3 Cytotoxicity results of 11 small molecule inhibitors

[0039] Figure 4 Photo of the antiviral activity test of compound F798-0523.

[0040] Figure 5 Cytotoxicity results and antiviral activity results of compound F798-0523.

[0041] Figure 6 Schematic diagram of the affinity between compound F798-0523 and SUD-core protein.

[0042] Figure 7 Synthetic route of compound F798-0523.

[0043] Figure 8 1H NMR spectrum of compound F798-0523.

[0044] Figure 9 13C NMR spectrum of compound F798-0523. Figure 10 Mass spectrum of compound F798-0523. Specific implementation mode

[0045] The present invention will be further described in detail below in conjunction with specific implementation modes. The provided examples are only for clarifying the present invention and not for limiting the scope of the present invention. The following examples can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way. Animal virus: The public can obtain this biological material from the applicant in accordance with relevant national biosafety regulations. This biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0046] Unless otherwise specified, the experimental methods in the following examples are all conventional methods, which are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified. Unless otherwise specified, the quantitative tests in the following examples are all set up with three repeated experiments, and the results are averaged. The antiviral test was carried out in the State Key Laboratory of Emerging Infectious Diseases, Li Ka Shing Faculty of Medicine, The University of Hong Kong. The stock solution of the test compound was obtained by dissolving the test compound in DMSO. The culture medium containing the test compound was obtained by mixing 1 volume of the stock solution of the test compound and 100 volumes of DMEM culture medium containing 10% fetal bovine serum. The culture medium containing DMSO was obtained by mixing 1 volume of DMSO and 100 volumes of DMEM culture medium containing 10% fetal bovine serum.

[0047] The SARS-CoV-2 virus used in the examples was the wild-type SARS-CoV-2 virus HKU-001a strain, namely WTSARS-CoV-2 (HKU-001a strain). VeroE6-TMPRSS2 cells and WT SARS-CoV-2 (HKU-001a strain) are described in the following literature: Identification of the SARS-unique domain of SARS-CoV-2 as an antiviral target, Nature Communications|(2023)14:3999.

[0048] The MTT method was used to evaluate the cytotoxicity of the compounds in the examples. The MTT assay is a commonly used method for measuring the metabolism of living cells. Its principle is based on the ability of the mitochondrial respiratory chain in living cells to reduce the yellow MTT dye to form blue Formazan crystals. Since dead cells lack the essential succinate dehydrogenase, they cannot reduce MTT. These crystals can only be generated by living cells. The generated Formazan crystals can be dissolved in the MTT lysate, and then the OD value can be measured at 490 nm using an enzyme-linked immunosorbent assay (ELISA) reader to reflect the number of living cells.

[0049] The CPE test was used to evaluate the antiviral activity of the compounds in the examples. Cytopathic effect (CPE): refers to the fact that most viruses infect sensitive cells, proliferate in the cells and interact with them, which will cause the infected cells to aggregate, detach, fuse, form inclusion bodies, and even be damaged and die. These characteristic changes can be directly observed under a low-power microscope and are called cytopathic effects.

[0050] Example 1. Screening of Antiviral Compounds

[0051] I. Primary Screening

[0052] The inventors analyzed the high-resolution crystal structure of SUD-core through research and confirmed that SUD-core can be used as a new drug target. Based on the crystal structure obtained previously, the inventors carried out high-throughput virtual screening for druggable sites rich in polar amino acids and obtained 300 candidate small molecule inhibitors with relatively low binding free energies.

[0053] In the virtual screening of small molecule drugs targeting SARS-CoV-2 SUD, the binding mode of the drug to the N-terminal domain is shown in Figure 1 . Figure 1 In it: A: Overall diagram of small molecule binding; B: Local diagram of small molecule binding; C: Schematic diagram of 2D binding mode of small molecule binding (both blue and green arrows are hydrogen bonds, and the dotted lines of H-benzene rings show hydrogen-aromatic ring stacking interactions).

[0054] The flow chart of virtual screening of small molecule drugs targeting SARS-CoV-2 SUD is shown in Figure 2 . Figure 2 In it: A: Flow chart of virtual screening; B: Screening pocket of N-terminal domain; C: Screening pocket of C-terminal domain.

[0055] II. Secondary screening

[0056] The 300 small molecule inhibitors obtained from the primary screening were used as test compounds respectively. The antiviral activities of the compounds were evaluated by CPE. Further screening yielded 11 small molecule inhibitors.

[0057] The detailed parameters of the 11 small molecule inhibitors and the docking scores during the primary screening (the smaller the docking score value, the higher the binding activity) are shown in Table 1. Among the secondary screening of the 11 small molecule inhibitors, compound F798-0523 had the highest activity.

[0058] Table 1

[0059]

[0060]

[0061] Example 2. Cytotoxicity evaluation of 11 small molecule inhibitors

[0062] The test compounds were respectively: Compound 1 to Compound 11 in Table 1.

[0063] 1. Vero E6-TMPRSS2 cells were inoculated into 96-well plates (200 μL per well, containing 1000 - 10000 cells) and cultured in DMEM medium containing 10% fetal bovine serum for 3 days.

[0064] 2. After completing Step 1, aspirate and discard the supernatant. Add the culture medium containing the test compound (200 μL per well) to the test wells, and add the culture medium containing DMSO (200 μL per well) to the control wells. Incubate for 48 hours.

[0065] In the culture medium containing the test compound, the concentrations of the test compound are set to 100 μM, 25 μM, 6.25 μM, or 1.5625 μM respectively. Three replicate wells are set for each concentration of each test compound. Three replicate wells are set for the control wells.

[0066] 3. After completing Step 2, add 20 μL of 5 mg / ml MTT solution to each well and incubate for 4 hours.

[0067] 4. After completing Step 3, aspirate and discard the supernatant. Add 150 μL of DMSO to each well and shake for 10 min.

[0068] 5. Select a wavelength of 490 nm on the enzyme-linked immunosorbent assay monitor, measure the optical density value of each well, and record the results.

[0069] Take the average absorbance value of the control wells as 100% and calculate the relative values of the test wells.

[0070] The results are shown in Figure 3 (Numbers 1# to 14# correspond to Compounds 1 to 11 in Table 1 in sequence).

[0071] Example 3. CC of Compound F798-0523 50 value and EC 50 value

[0072] The test compound is: Compound F798-0523 prepared in Example 5.

[0073] I. Detecting the cytotoxicity of the test compound

[0074] 1. Inoculate VeroE6-TMPRSS2 cells into a 96-well plate (200 μL per well, containing 1000 - 10000 cells), and culture with DMEM culture medium containing 10% fetal bovine serum for 3 days.

[0075] 2. After completing Step 1, aspirate and discard the supernatant. Add the culture medium containing the test compound (200 μL per well) to the test wells, and add the culture medium containing DMSO (200 μL per well) to the control wells. Incubate for 48 hours.

[0076] In the culture medium containing the test compound, the test compound is set with gradient concentrations (1 - 100 μM). Three replicate wells are set for each concentration of the test compound. Three replicate wells are set for the control wells.

[0077] 3. After completing Step 2, add 20 μL of 5 mg / ml MTT solution to each well and incubate for 4 hours.

[0078] 4. After completing Step 3, aspirate the supernatant, add 150 μL of DMSO to each well, and shake for 10 min.

[0079] 5. Select a wavelength of 490 nm on the enzyme-linked immunosorbent assay monitor, measure the optical density value of each well, and record the results.

[0080] Take the average absorbance value of the control wells as 100%, calculate the relative values of each test well, which is the cell viability (%), and plot the cell viability curve at different gradient concentrations of the test compound.

[0081] The cell viability curve is shown in Figure 5 . CC 50 value (i.e., the compound concentration corresponding to 50% cell viability) is 68.34 μM.

[0082] II. Detection of the antiviral effect of the test compound

[0083] 1. Inoculate VeroE6-TMPRSS2 cells into a 96-well plate (200 μL per well, containing 1000 - 10000 cells), and culture with DMEM medium containing 10% fetal bovine serum for 3 days.

[0084] 2. After completing Step 1, aspirate the supernatant, add the culture medium containing the test compound to the test wells (200 μL per well), and add the culture medium containing DMSO to the control wells (200 μL per well), and incubate for 1 hour.

[0085] In the culture medium containing the test compound, the concentrations of the test compound are set to 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, or 1.5625 μM respectively. Each concentration of the test compound has 3 replicate wells. The control wells have 3 replicate wells.

[0086] 3. After completing Step 2, add SARS-CoV-2 virus (the virus infection dose is 0.01 MOI), and incubate for 2 hours.

[0087] 4. After completing Step 3, aspirate the supernatant, add the culture medium containing the test compound to the test wells (200 μL per well), and add the culture medium containing DMSO to the control wells (200 μL per well), culture for 36 hours, and then take pictures. The pictures are shown in Figure 4 . Figure 4 Among them, DMSO corresponds to the control wells.

[0088] In the culture medium containing the test compound, the concentration of the test compound is the same as that in Step 2.

[0089] 5. After completing step 4, the cytopathic effect (CPE) of the cells was identified under a microscope.

[0090] Taking the average CPE value of the control wells as 100%, the relative values of each test well were calculated, which were the relative values of infectious virus (%).

[0091] The curve of the relative value of infectious virus is shown in Figure 5 . EC 50 value (the compound concentration when the relative value of infectious virus is 50%) was 4.74 μM.

[0092] Experimental Example 4. Determination of the Binding Affinity between Compound F798-0523 and SUD-core Protein

[0093] Biolayer interferometry (BLI) is a label-free technique used to measure biomolecular interactions. This technique uses optical analysis methods and is achieved by analyzing the interference pattern formed by white light reflected from two surfaces. One surface is the surface layer where the protein is immobilized on the tip of the biosensor, and the other is the internal reference layer. When the ligand is immobilized on the surface of the biosensor tip and binds to the analyte in the solution, it causes an increase in the optical thickness of the biosensor tip, resulting in a wavelength shift. The wavelength shift can directly measure the thickness change of the biolayer. This technique can measure the intermolecular interactions in real time.

[0094] The BLI technique is widely used in the research and development of the interactions of small molecule compounds and antiviral drugs. In this experiment, the Octet RED96e analysis system (ForteBio) was used to study the binding affinity between SARS-CoV-2 SUD-core and small molecule inhibitors.

[0095] First, the inhibitor was dissolved in 100% DMSO, and the SARS-CoV-2 SUD-core protein (as shown in SEQ ID NO: 1) was biotinylated using a biotinylation reagent and immobilized on the SSA sensor. The biotinylation of the SARS-CoV-2 SUD-core protein was performed using a Biotin Quick Labeling Kit (Frdbbio).

[0096] 50 ng / μL of biotinylated SARS-CoV-2 SUD-core was immobilized on the SSA sensor for 1,800 seconds. The compound F798-0523 prepared in Example 5 was diluted to 100 μM using the reaction buffer (1×PBS, pH 7.4), and then incubated with the SARS-CoV-2 SUD-core that had been immobilized on the SSA sensor in the reaction buffer for 100 seconds. Finally, it was incubated in fresh reaction buffer for 120 seconds to dissociate the inhibitor and the protein.

[0097] Compound F798-0523 was serially diluted at different concentrations, and the binding kinetic parameters (Kd, Kon, Koff, and R 2 ) were recorded. R 2 is called the goodness of fit. When curve fitting the experimental data, the degree of agreement between the experimental data and the fitting function can be represented by the R 2 value. When R 2 is closer to 1, the degree of agreement is higher; when R 2 is closer to 0, the degree of agreement is lower. The Kd value reflects the affinity of the small molecule inhibitor binding to the SARS-CoV-2 SUD-core protein. The Kd value is an important indicator data for measuring the binding ability of SARS-CoV-2 SUD-core to the small molecule inhibitor. Koff, also known as the dissociation rate constant, is a constant used to evaluate the rate of reverse dissociation of the receptor-ligand complex to form the receptor and ligand. During the inhibitor screening process, the dissociation rate of the inhibitor from the target is a key factor affecting the drug efficacy. The slower the dissociation rate of the inhibitor from the target, the higher the possibility of exerting the drug efficacy.

[0098] The results are shown in Figure 6 . The corresponding KD value of compound F798-0523 is 11.97×10 -6 M, Kon = 44.4×10 1 1 / Ms, Koff = 5.31×10 -4 1 / s.

[0099] Example 5. Preparation and Characterization of Compound F798-0523

[0100] Methyl 3-aminothiophene-2-carboxylate, namely methyl 3-amino-2-thiophenecarboxylate, has the chemical formula C6H7NO2S and the CAS number 22288-78-4. Carbon disulfide dichloride, with the chemical formula CSCl2 and the CAS number 463-71-8. Triethylamine, with the chemical formula C6H 15 N and the CAS number 121-44-8. 2-Thiophenemethylamine, with the chemical formula C5H7N S and the CAS number 27757-85-3. Hydrazine anhydrous, with the chemical formula N2H4 and the CAS number 302-01-2. 2,4-Difluoroaniline, with the chemical formula C6H5F2N and the CAS number 367-25-9. Chloroacetyl chloride, with the chemical formula C2H2Cl2O and the CAS number 79-04-9.

[0101] I. Preparation of the Compound

[0102] The synthetic route of compound F798-0523 is shown in Figure 7 .

[0103] 1. In a 100 mL round-bottom flask, dissolve methyl 3-aminothiophene-2-carboxylate (1 eq, 15 mmol) in 30 mL of dichloromethane. Then cool the solution to 0 °C, and dropwise add carbon disulfide (1.2 eq) and stir for 10 min. Then dropwise add triethylamine (2 eq), and allow the temperature to rise to room temperature naturally. Then react for 18 hours. Then transfer the entire reaction system to 100 ml of water, and extract with dichloromethane three times (add 60 mL of dichloromethane each time). Combine the organic phases. Then treat the obtained organic phase successively as follows: wash with 70 ml of saturated NaCl aqueous solution, dry with anhydrous sodium sulfate, filter and collect the filtrate. Then concentrate the obtained filtrate under reduced pressure, and perform column chromatography (1000-mesh silica gel column), and elute with an elution solvent (the elution solvent is obtained by mixing 1 volume part of EA and 20 volume parts of PE, where EA refers to petroleum ether and PE refers to ethyl acetate). Collect the corresponding solution after column chromatography according to the molecular weight of the target product (199.26). Then evaporate the solvent of the obtained solution after column chromatography to obtain the product, which is compound M1-a.

[0104] 2. Dissolve all the products obtained in step 1 in 30 ml of tetrahydrofuran, then successively add 2-thiophenecarboxamide (1.2 eq) and triethylamine (2 eq), and stir at room temperature for 2 h. Then evaporate the solvent to obtain the product (the product contains compound M1-b).

[0105] 3. Dissolve all the products obtained in step 2 in 30 ml of methanol, add KOH (5.3 mmol), stir at room temperature for 30 min, then evaporate the solvent, dissolve the residue in water, then place it in an ice bath and add 1 M hydrochloric acid aqueous solution until pH = 4. Then filter and collect the filter residue, and recrystallize the filter residue with ethyl acetate to obtain the product, which is compound M1.

[0106] 4. Dissolve compound M1 (1 eq) in 30 ml of ethanol, then add anhydrous hydrazine (7.0 eq), reflux at 80 °C for 18 h, and then evaporate the solvent under nitrogen purge to obtain the product (the product contains compound M2-a).

[0107] 5. Dissolve all the products obtained in step 4 in 30 ml of ethanol, add potassium hydroxide (3 eq) and carbon disulfide (3 eq), reflux at 80 °C for 18 h, then evaporate the solvent, dissolve the residue in water, then place it in an ice bath and add 1 M hydrochloric acid aqueous solution until pH = 1. Then, extract with ethyl acetate three times (add 60 ml of ethyl acetate each time), and combine the organic phases. Then dry the obtained organic phase with anhydrous sodium sulfate, filter and collect the filtrate. Then concentrate the obtained filtrate under reduced pressure, and then perform column chromatography (1000-mesh silica gel column), and elute with an elution solvent (the elution solvent is obtained by mixing 1 volume part of EA and 20 volume parts of PE, EA refers to petroleum ether, and PE refers to ethyl acetate), and collect the corresponding post-column solution according to the molecular weight of the target product (320.4). Then evaporate the solvent from the obtained post-column solution to obtain the product, which is compound M2.

[0108] 6. Dissolve 2,4-difluoroaniline (3.5 mmol) in dichloromethane to obtain a fluoroaniline solution. Dissolve chloroacetyl chloride (3.85 mmol) in dichloromethane to obtain a chloroacetyl chloride solution. Drop the chloroacetyl chloride solution into the fluoroaniline solution under an ice bath, then add triethylamine (7 mmol), and then react at room temperature for 5 hours. Then transfer the entire reaction system to 100 ml of water, and then extract with ethyl acetate three times (add 50 mL of ethyl acetate each time), and combine the organic phases. Then perform the following treatments on the obtained organic phase in sequence: wash with 50 ml of saturated NaCl aqueous solution, dry with anhydrous sodium sulfate, filter and collect the filtrate. Then evaporate the solvent from the obtained filtrate, and then recrystallize with ethyl acetate to obtain the product, which is compound M3.

[0109] 7. Dissolve compound M2 (3.5 mmol) in 30 ml of N,N-dimethylformamide in a 100 mL round-bottom flask, add compound M3 (3.5 mmol) and K2CO3 (5.3 mmol), and stir at room temperature for 5 h. Then pour the entire reaction system into 100 ml of water, and then extract with ethyl acetate three times (add 50 ml of ethyl acetate each time), and combine the organic phases. Then perform the following treatments on the obtained organic phase in sequence: wash with 50 ml of saturated NaCl aqueous solution, dry with anhydrous sodium sulfate, filter and collect the filtrate. Then evaporate the solvent from the obtained filtrate, and recrystallize with ethyl acetate to obtain the product, named compound F798-0523.

[0110] II. Characterization

[0111] Characterize the compound F798-0523 obtained in step one.

[0112] Figure 8 It is the 1H NMR spectrum of compound F798-0523.

[0113] Figure 9 It is the carbon spectrum of compound F798-0523.

[0114] Figure 10 It is the mass spectrum of compound F798-0523.

[0115] According to Figure 8 、 Figure 9 and Figure 10 , the structural formula of compound F798-0523 can be confirmed as shown in formula (Ⅰ).

[0116]

[0117] The above has described the present invention in detail. For those skilled in the art, without departing from the gist and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modifications, uses or improvements of the present invention, including those that depart from the scope disclosed in this application and are made by conventional techniques known in the art. Some basic features can be applied according to the scope of the appended claims below.

Claims

1. A compound as shown in formula (I); 2. Use of the compound according to claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a coronavirus inhibitor.

3. The application according to claim 2, characterized in that: The coronavirus is SARS-CoV-2.

4. Use of the compound according to claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating and / or preventing coronavirus infection.

5. The application according to claim 4, characterized in that: The coronavirus is SARS-CoV-2.

6. A coronavirus inhibitor, the active ingredient of which comprises the compound according to claim 1 or a pharmaceutically acceptable salt thereof.

7. The coronavirus inhibitor according to claim 6, wherein: The coronavirus is SARS-CoV-2.

8. A drug for treating and / or preventing coronavirus infection, the active ingredient of which comprises the compound according to claim 1 or a pharmaceutically acceptable salt thereof.

9. The drug according to claim 8, characterized in that: The coronavirus is SARS-CoV-2.

10. A method for preparing the compound according to claim 1, comprising the following steps: Reacting compound M2 and compound M3 to obtain the compound according to claim 1; compound M2 is as shown in formula (II); compound M3 is as shown in formula (III);

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