A novel coronavirus nsp16 inhibitor and its preparation method, virtual screening method and application

Abstract

The present invention relates to the field of biological and pharmaceutical technology, and in particular to a novel coronavirus nsp16 inhibitor and its preparation method, virtual screening method and application. The novel coronavirus nsp16 inhibitor has a structure shown in formula (I), can effectively inhibit the activity of nsp16 in vitro, and has the potential to prevent and / or treat novel coronavirus infection. In addition, its preparation method is simple, and the virtual screening method is efficient and effective; #imgabs0#

Description

Technical Field

[0001] The present invention relates to the field of biology and pharmaceutical technology, and in particular to a novel coronavirus nsp16 inhibitor and a preparation method, a virtual screening method and application thereof. Background Art

[0002] One of the attractive drug targets among the many targets of the new coronavirus is nsp16 (non-structural protein 16). As a part of the methyltransferase, its function is mainly involved in the capping modification of the 5'-end of RNA, similar to eukaryotic organisms, to escape the immune attack of the host system. The specific mechanism of this target is: the initial cap core structure (cap-0) of the new coronavirus is formed at the 5'-end of RNA. First, RNA 5'-triphosphatase (RTPase / nsp13) hydrolyzes the newly generated RNA into ppRNA and removes the terminal γ-phosphate; then Gppp-RNA is formed under the catalysis of RNA guanosyltransferase (GTase / nsp12); then the N7 position of the substrate is methylated by the action of N7-methyltransferase (nsp14) to form cap-0; finally, the 2'O position of the first nucleotide ribose of the substrate is methylated under the action of 2-O'-methyltransferase (nsp10 / nsp16) to form cap-1. In the methylation reaction, the methyl donor is S-adenosyl-methionine (SAM / AdoMet).

[0003] The druggable site for targeting nsp16 is mainly the SAM pocket, which is close to the substrate m 7 GpppA-RNA and four highly conserved catalytic motifs (KDKE) are closely related to the active function of methyltransferases. Currently reported related active inhibitors include Sinefungin (SFG) and its analogs WZ16, SS148, etc. Although this type of molecule has good activity, reaching the micromolar or even nanomolar level, the SAM analogs have a common structure and poor specificity, and have off-target effects, and are prone to act on other methyltransferase proteins in the human body. For example, Sinefungin has been shown to occupy the SAM binding pocket of human N7 methyltransferase in the crystal structure (PDB ID: 3EPP). Therefore, it is necessary to develop more inhibitors with novel structures and stronger targeting for the treatment of new coronavirus infection.

[0004] Computer-aided Virtual Screening (CAVS) refers to the use of high-performance computers such as the Tianhe-2 supercomputer to perform efficient virtual screening of virtual compound libraries. With the rapid advancement of structural analysis technologies (such as X-ray, nuclear magnetic resonance, cryo-electron microscopy, etc.), the available structures in the Protein Data Bank (PDB) have increased in the past few decades, and structure-based virtual screening (SBVS) has become one of the most prominent types of virtual screening, accounting for an average of 57.6%. However, existing virtual screening technologies only rely on scoring functions to score and rank the docking conformations to be screened, which has a high probability of false positives, low reliability of results, and low screening efficiency. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes a novel coronavirus nsp16 inhibitor and its preparation method, virtual screening method and application, the inhibitor has a novel structure, can effectively inhibit the activity of nsp16 methyltransferase in vitro, has the potential to prevent and / or treat novel coronavirus infection, and the preparation method is simple, the virtual screening method is efficient and effective.

[0006] In a first aspect of the present invention, there is provided a compound represented by formula (I) or a pharmaceutically acceptable salt thereof,

[0007]

[0008] The chlorine atom, the nitrogen atom in the sulfonamide group and the nitrogen atom on the amide bond in the compound of formula (I) interact with the amino acid residues C6913, D6897 and G6869 of nsp16 respectively. In addition, the hydroxyl group in the compound of formula (I) can interact with the amino acid residues D6928 and K6968 of nsp16. The in vitro activity test data also showed that the compound of formula (I) showed good inhibitory activity against nsp16, IC 50 The value is 40.86 μM. Therefore, the compound of formula (I) of the present invention can provide a new structural idea for the development of nsp16 inhibitors.

[0009] The second aspect of the present invention provides a method for preparing a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, comprising the following steps:

[0010] (1) 4-methoxybenzenesulfonyl chloride is subjected to nitration reaction to obtain intermediate (II);

[0011] (2) intermediate (II) reacts with m-chloroaniline to obtain intermediate (III);

[0012] (3) Intermediate (III) is subjected to reduction reaction to obtain intermediate (IV);

[0013] (4) intermediate (IV) reacts with 3-furoyl chloride to obtain intermediate (V);

[0014] (5) Demethylating the intermediate (V) to obtain a compound represented by formula (I);

[0015] The chemical structural formulas of the intermediates (II) to (V) are as follows:

[0016]

[0017] According to some embodiments of the present invention, in step (1), the reagents used in the nitration reaction include concentrated sulfuric acid and concentrated nitric acid;

[0018] Furthermore, the nitrification reaction time is 6 to 10 hours, and the nitrification reaction temperature is 0°C to 4°C.

[0019] According to some embodiments of the present invention, in step (2), the reaction is carried out in the presence of a solvent, and the solvent is pyridine;

[0020] Furthermore, the reaction time is 5 to 30 minutes, and the reaction temperature is 0°C to 4°C.

[0021] According to some embodiments of the present invention, in step (3), the reagents used in the reduction reaction include iron powder and acetic acid;

[0022] Furthermore, the reduction reaction is carried out in the presence of a solvent, and the solvent is a methanol-water mixed solution;

[0023] Furthermore, the reduction reaction time is 1 to 2 hours, and the reduction reaction temperature is 60° C. to 100° C.

[0024] According to some embodiments of the present invention, in step (4), the reaction is carried out in the presence of a solvent, and the solvent is dichloromethane;

[0025] Furthermore, the reaction time is 0.5 to 2 hours, and the reaction temperature is 0°C to 4°C.

[0026] According to some embodiments of the present invention, in step (5), the reagent used for demethylation includes boron tribromide;

[0027] Further, the demethylation is carried out under the conditions of dichloromethane as solvent and nitrogen protection;

[0028] Furthermore, the reaction temperature of the demethylation is -30°C to -10°C, and the reaction time is 1 to 4 hours.

[0029] The third aspect of the present invention provides a virtual screening method for a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, comprising the following steps:

[0030] S1, the three-dimensional structure of nsp16 and the acquisition and processing of small molecule compound libraries;

[0031] S2, re-docking sinefungin with nsp16 obtained in step S1, adjusting the docking parameters according to the degree of overlap between the docked conformation of sinefungin and the original structural conformation, and constructing a virtual screening system;

[0032] S3, using the virtual screening system constructed in step S2 to screen the small molecule compound library obtained in step S1 to obtain candidate compounds;

[0033] S4. Perform in vitro activity screening on the candidate compounds obtained in step S3 to obtain the compound represented by formula (I).

[0034] According to some embodiments of the present invention, in step S1, the three-dimensional structure of nsp16 can be obtained from a database commonly used in the art, including but not limited to the RCSB protein crystal database;

[0035] Furthermore, the three-dimensional structure of nsp16 includes nsp16, its cofactor SAM and substrate m 7 The crystal structure of the complex of GpppA (PDB ID: 6WVN), and the crystal structure of the complex of nsp16 and its cofactor SAM (PDB ID: 6W4H).

[0036] The present invention uses both containing and not containing substrate m 7 Computer-assisted virtual screening of two nsp16 three-dimensional structures of GpppA, the 6W4H crystal structure does not contain substrate m 7 GpppA, while 6WVN is the substrate m 7 GpppA, whether the substrate occupies the RNA groove has a certain influence on the docking results, mainly by affecting the binding mode and scoring of the docking conformation, thereby affecting the docking results. Therefore, using two different states of nsp16 crystal structures is conducive to improving the screening success rate. According to the experimental results, two of the nine compounds with preliminary nsp16 inhibitory effects came from the results of 6W4H screening (ZINC55183218, ZINC71479437), which also verifies the use of both containing and not containing substrate m 7 The crystal structure of nsp16 of GpppA is helpful to improve the screening success rate.

[0037] According to some embodiments of the present invention, in step S1, after obtaining the three-dimensional structure of nsp16, the MOLPROBITY software is used to detect various stereochemical parameters of the three-dimensional structure.

[0038] According to some embodiments of the present invention, in step S1, the processing of the three-dimensional structure of nsp16 includes deleting co-crystal ligands and crystal water, assigning histidine proton states, adding disulfide bonds, hydrogen atoms and missing heavy atoms, and minimizing the structure using the OPLS_2005 force field.

[0039] According to some embodiments of the present invention, in step S1, the small molecule compound library can be obtained from a commonly used database in the art, including but not limited to the ZINC compound library, the FDA-approved compound library, the ChEMBL compound library, the Maybridge compound library, etc.; in the present invention, the ZINC compound library is preferred; wherein all compounds must satisfy Lipinski's five rules;

[0040] Furthermore, the compounds in the small molecule compound library of the present invention must satisfy Mwt=400 and logP=4.

[0041] Further limiting the molecular weight and lipid-water partition coefficient is helpful to reduce the screening cost and increase the screening success rate.

[0042] According to some embodiments of the present invention, in step S1, processing the small molecule compound library includes using the OPLS_2005 force field to perform energy optimization on each compound.

[0043] According to some embodiments of the present invention, in step S2, the docking parameters are:

[0044] When the three-dimensional structure of nsp16 does not contain the substrate m 7 In the case of GpppA, the OD2 atom of amino acid ASP6897 in nsp16 was used as the docking center, and the box size was Generate a grid file; or,

[0045] When the three-dimensional structure of nsp16 contains the substrate m 7 GpppA, with substrate m 7 The 2'O position of GpppA served as the docking center, and the box size was Generate a grid file.

[0046] According to some embodiments of the present invention, in step S3, the screening process includes the following steps:

[0047] The high-throughput virtual screening mode, standard precision mode, and extra precision mode of Glide in the Schrödinger software were used for screening in turn, and the top 10% of the scoring compounds were selected each time; then the MMGBSA module in the Schrödinger software was used for fine screening, and the compounds with higher binding energy than sinefungin were retained; finally, the binding energy size and manual visual inspection analysis were performed.

[0048] According to some embodiments of the present invention, in step S3, the items analyzed by manual visual inspection include at least one of hydrogen bonding, hydrophobic interaction, π-π interaction, and σ-π interaction between the compound and the SAM pocket of nsp16.

[0049] The fourth aspect of the present invention provides the use of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing and / or treating novel coronavirus infection.

[0050] According to some embodiments of the present invention, the drug is a nsp16 inhibitor.

[0051] According to some embodiments of the present invention, the novel coronavirus is SARS-CoV-2.

[0052] The fifth aspect of the present invention provides the use of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof in the preparation of an nsp16 inhibitor.

[0053] The novel coronavirus nsp16 inhibitor of the present invention can also be combined with one or more pharmaceutically acceptable carriers to form a pharmaceutical composition. The carriers include conventional diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants, etc. in the pharmaceutical field.

[0054] The novel coronavirus nsp16 inhibitor of the present invention can be prepared into various forms such as injection, tablets, powders, granules, capsules, oral liquids, ointments, creams, etc. The above-mentioned various dosage forms of drugs can be prepared according to conventional methods in the pharmaceutical field, and introduced into the body such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue by injection, spraying, nasal drops, eye drops, penetration, absorption, physical or chemical mediation methods; or introduced into the body after being mixed or wrapped with other substances.

[0055] definition:

[0056] “Interactions” refer to the van der Waals, hydrogen bonding, hydrophobic, and charge interactions of each compound with the amino acid residues in the binding pocket after docking.

[0057] "Lipinski's Five Rules", also known as Lipinski's Five Rules, mean that the molecular weight of the compound is less than 500 Daltons, the number of hydrogen bond donors in the compound structure does not exceed 5, the number of hydrogen bond acceptors in the compound does not exceed 10, the logarithm of the compound's lipid-water partition coefficient logP is between -2 and 5, and the number of rotatable bonds in the compound does not exceed 10; Lipinski's Five Rules are often used to guide the drugability screening of compounds.

[0058] "Pharmaceutically acceptable salts" refer to salts of compounds of the invention, prepared from compounds of the invention with relatively nontoxic acids or bases. When compounds of the invention contain relatively acidic functional groups, base addition salts may be obtained by contacting a neutral form of such compounds with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino or magnesium salts or similar salts. When compounds of the invention contain relatively basic functional groups, acid addition salts may be obtained by contacting a neutral form of such compounds with a sufficient amount of acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts, such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts, such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid and methanesulfonic acid and the like; also include salts of amino acids (such as arginine, etc.), and salts of organic acids such as glucuronic acid (see Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science 66: 1-19 (1977)). Certain specific compounds of the present invention contain basic and acidic functional groups, and thus can be converted into either base or acid addition salts.

[0059] Beneficial effects:

[0060] The present invention is based on the filtering of layer-by-layer virtual screening and combined with artificial visual detection and analysis, and the selected compounds are tested for in vitro activity; the results show that 2 of the 13 compounds screened have good nsp16 inhibitory activity (<50μM). It is proved that the virtual screening system constructed by the present invention is accurate and reliable, and can quickly and efficiently obtain potential inhibitors from a complex compound library, improve the screening efficiency, and save experimental costs and time; further through subsequent in vitro activity experiments, the virtual screening results are further verified for biological activity, which improves the accuracy of the screening results and reduces the false positive rate. In addition, the lead compound with nsp16 inhibitory activity obtained by the present invention has a simple preparation process and can be further developed as an anti-new coronavirus drug.

[0061] Other features and advantages of the present invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0063] Figure 1 This is the basic overview of the virtual screening of the present invention;

[0064] Figure 2 It is the three-dimensional structure of nsp16 (PDB ID: 6WVN) and the SAM pocket;

[0065] Figure 3 is the three-dimensional structure of nsp16 (PDB ID: 6W4H) and the SAM pocket;

[0066] Figure 4 The superposition of the docked conformation (pink) and the original structure (cyan) of sinefungin (SFG) (PDB ID: 6WVN);

[0067] Figure 5 The superposition of the docked conformation (pink) and the original structure (cyan) of sinefungin (SFG) (PDB ID: 6W4H);

[0068] Figure 6 is a virtual screening flow chart;

[0069] Figure 7 is the binding mode of the inhibitor molecule sinefungin (SFG);

[0070] Figure 8 It is the chemical structure diagram of 13 hit compounds from virtual screening;

[0071] Fig. 9is the IC of compounds 1 and 2 relative to nsp16 50 curve;

[0072] Fig.10 is the docking diagram of compound-1 (ZINC12778720) and nsp16 protein;

[0073] Fig.11 It is the docking diagram of compound-2 (ZINC95190922) and nsp16 protein. DETAILED DESCRIPTION

[0074] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0075] The design concept of the present invention is as follows: Figure 1 As shown in the figure, relying on the Tianhe-2 supercomputing platform, the molecular docking software Schrodinger was used to perform structure-based virtual screening of small molecule compounds in the ZINC molecular database. The docking conformations were scored and evaluated through a series of rigorous screening processes, and then potential molecules with high scores and good binding modes were selected through manual visual inspection and analysis to improve the hit rate of virtual screening. Finally, the compounds were selected for in vitro enzyme activity verification, and the lead compounds with nsp16 inhibitory activity were screened out.

[0076] Example 1: A virtual screening method for nsp16 inhibitors

[0077] The specific steps include:

[0078] (1) Acquisition and processing of the three-dimensional structure of nsp16 methyltransferase

[0079] The protein database of Protein Data Bank (https: / / www.rcsb.org / ) was used to obtain The high-resolution three-dimensional structure of nsp16 (PDB ID: 6WVN) shows nsp16 with its cofactor SAM and substrate m 7 The crystal structure of the complex of GpppA (SAM binding pocket with nsp16 as shown in Figure 2) Figure 2 In addition, no substrate m 7The nsp16 protein structure of GpppA (PDB ID: 6W4H) was also used for virtual screening to increase the success rate. This structure is the crystal structure of the complex between nsp16 and its cofactor SAM with a resolution of (The binding pocket of SAM and nsp16 is as follows Figure 3 shown).

[0080] In order to verify the reliability of the model, the Molprobity website (http: / / molprobity.biochem.duke.edu / index.php) was used to evaluate the model quality of 6WVN and 6W4H structures (Tables 1, 2).

[0081] Table 1 List of parameters used to evaluate the quality of the selected model (PDB ID: 6WVN)

[0082]

[0083] * 100 th Indicates the best quality among models with similar resolution range, 0 th Indicates the worst model.

[0084] Table 2 List of parameters used to evaluate the quality of the selected model (PDB ID: 6W4H)

[0085]

[0086]

[0087] * 100 th Indicates the best quality among models with similar resolution range, 0 th Indicates the worst model.

[0088] Using Schrodinger (Release 2019-2, LLC, New York, NY, 2019) software package, including: (1) deletion of cocrystal ligands and crystallographic water; (2) assignment of histidine proton states; (3) addition of disulfide bonds, hydrogen atoms, and missing heavy atoms; and (4) minimization of the structure using the OPLS_2005 force field.

[0089] (2) Acquisition and processing of small molecule compound libraries

[0090] 210,000 small molecule structures (Mwt = 400, logP = 4) were obtained from the ZINC15 database (https: / / zinc.docking.org). These molecules are all lead-like compounds and conform to Lipinski's five rules. The LigPrep module in the Schrödinger software package was used for ligand preparation, and the OPLS_2005 force field was also used to generate a low-energy conformation for each ligand molecule for subsequent virtual screening.

[0091] (3) Constructing a virtual screening system based on the structural information prepared in step (1)

[0092] The Receptor Grid Generation module in the Schrödinger software package was used to prepare the grid file. The 2'O position of the substrate GpppA was selected as the docking center in the 6WVN model, and the box size was The 6W4H model selects the OD2 atom of amino acid ASP6897 as the docking center, and the box size is The optimization of the parameters was determined by comparing the conformation of the inhibitor molecule sinefungin (SFG) after docking with the original structure ( Figure 4 , 5).

[0093] (4) Using the virtual screening system constructed in step (3), the small molecule compound library in step (2) is subjected to structure-based virtual screening, and the final candidate small molecules are obtained through manual visual inspection and analysis.

[0094] The Ligand Docking module was used for molecular docking, and the prepared ligand molecules (213010) were docked to the SAM pockets of the prepared target proteins (the flowchart is shown in Figure 6 shown).

[0095] First, the high-throughput virtual screening (HTVS) mode of Glide in Schrödinger software was used for the first round of preliminary screening, and the top 10% of the compounds (21,301) were selected for the second round of rescreening using the standard precision (SP) mode. Then, the top 10% of the compounds (2130) were selected for the third round of rescreening using the extra precision (XP) mode. Then, the top 10% of the compounds (213) were selected for the final round of fine screening using the MMGBSA module in Schrödinger software. The compounds with higher binding energy than the control SFG were retained (6WVN: 143, 6W4H: 82). Finally, the binding energy and manual visual inspection analysis (refer to Figure 7SFG forms key interactions, including hydrogen bonds with key amino acids such as D6912, C6913, L6898, D6897, D6928, G6979, and N6841, π-π hydrophobic interactions with F6947, and σ-π non-bonded interactions with L6898. 13 compounds with potential nsp16 inhibitory activity were selected (6WVN: 6, 6W4H: 7). The detailed information of the compounds is shown in Table 3 and Figure 8 .

[0096] (5) Conducting in vitro biochemical activity tests on the candidate small molecules obtained in step (4)

[0097] The compounds selected according to the virtual screening results were tested for in vitro biochemical activity, and the methyltransferase activity was determined in a 30uL system: 4× buffer (80mM Tris-HCl, pH 8.0; 200mM NaCl; 4mM EDTA; 12mM MgCl2; 0.4mg / mL BSA; 4mM DTT), 0.01mM SAM, 1μg of purified protein (the molar ratio of nsp10 to nsp16 was 6:1), 2μg of m 7 GpppA-RNA substrate, 0.5uCi of S-adenosyl[methyl-3H]methionine (67.3Ci / mmol, 0.5uCi / uL), 37℃ water bath for 1h, 3H labeled product separated in DEAE-Sephadex column, quantified by liquid scintillation, added 50uM of the sample to be tested, and observed the inhibition rate (results in Table 3). Select compounds with inhibition rate greater than 50% for the next test: add different concentrations of the test compound (0.1, 0.5, 1, 2, 5, 10, 20, 50μM), use DMSO and irrelevant drugs as negative controls, use Sinefugin as positive control, IC 50 The values ​​were calculated from the concentration-inhibition rate curves using GraphpadPrism 8.0.

[0098] Table 3 Inhibition rates and scores of 13 potential molecules on nsp16

[0099]

[0100] The method of the present invention obtained 13 compounds through four rounds of virtual screening and artificial visual detection analysis, of which 9 had preliminary potential nsp16 inhibition effects. After testing, compound 1 (ZINC12778720) had the best anti-nsp16 activity (IC 50 =40.86 μM), followed by compound 2 (ZINC95190922), whose IC 5048.15μM( Fig. 9 ), IC of compound ZINC2618640 50 The value was greater than 50 μM (the activity was relatively poor and no further study was conducted). The binding modes of compounds 1 and 2 with nsp16 protein are shown in Fig.10 , 11 .

[0101] Example 2, Synthesis route of compound 1 (ZINC12778720)

[0102]

[0103] The specific synthesis steps are as follows:

[0104] S1. Weigh 3.09 g of p-methoxybenzenesulfonyl chloride and add it to a 50 mL flask. At room temperature, use a rubber pipette to draw 20 mL of concentrated sulfuric acid, then place the flask in a 0°C ice bath, slowly add 1 mL of concentrated nitric acid with a rubber pipette while stirring. After reacting for 8 hours in a 0°C ice bath, pour the reaction system into ice water, extract the reaction solution three times with ether, dry the organic phase with anhydrous sodium sulfate, remove the solvent by vacuum rotary evaporation, and obtain intermediate (II) by rapid column chromatography (petroleum ether / ethyl acetate).

[0105] S2. Weigh 1.4 g of m-chloroaniline and add it to a 50 mL flask. After adding 7 mL of pyridine solvent, place the flask in a 0°C ice bath. Add 2.5 g of intermediate (II) slowly in batches with a spoon while stirring. After reacting in a 0°C ice bath for 15 min, remove the solvent by vacuum rotary evaporation, and obtain intermediate (III) by rapid column chromatography (petroleum ether / ethyl acetate).

[0106] S3. Weigh 3.42 g of intermediate (III) and 2.8 g of iron powder and add them to a 50 mL flask. Add 15 mL of acetic acid, 2 mL of methanol and 2 mL of water to the flask respectively, place the flask in an 80°C oil bath and react for 1.5 h. After the reaction is completed, filter the reaction system to remove the remaining iron powder in the reaction system, remove a large amount of acetic acid by vacuum rotary evaporation and adjust the pH value of the reaction system to neutral. Then, extract with dichloromethane and saturated brine three times, then dry the organic phase with anhydrous sodium sulfate, remove the solvent by vacuum rotary evaporation, and obtain intermediate (IV) by rapid column chromatography (petroleum ether / dichloromethane).

[0107] S4. Weigh 0.34 g of intermediate (Ⅳ) and add it to a 50 mL flask. Add 10 mL of anhydrous dichloromethane to the flask, then place the flask in a 0°C ice bath, dropwise add 0.13 g of 3-furoyl chloride while stirring, and continue to react in a 0°C ice bath for 1 h. After the reaction is completed, remove the solvent by vacuum rotary evaporation, and obtain intermediate (V) by rapid column chromatography (petroleum ether / ethyl acetate).

[0108] S5. Weigh 81.2 mg of intermediate (V) and add it to a 25 mL flask. Under nitrogen protection, add 6 mL of anhydrous dichloromethane to the flask, then place the flask at -20°C, slowly add 600 μL of 1 M boron tribromide n-hexane solution while stirring, and react at -20°C for 3 h. After the reaction, add 5 mL of methanol to quench the remaining boron tribromide in the reaction system, remove the solvent by vacuum rotary evaporation, and obtain compound-1 (ZINC12778720) by rapid column chromatography (petroleum ether / ethyl acetate).

[0109] The characterization data of compound-1 (ZINC12778720) are as follows:

[0110] White solid, yield 62%, 1 H NMR(500MHz,DMSO)δ10.98(s,1H),10.46(s,1H),9.31(s,1H),8.43(s,1H),8.23(s,1H ),7.79(s,1H),7.46(dd,J=8.5Hz,1.9Hz,1H),7.25(t,J=8.1Hz,1H),7.05(m,5H).13C NMR (125MHz, DMSO) δ160.55,153.31,146.15,144.35,139.59,133.31,130.86,12 8.96,125.76,124.84,123.35,122.97,122.39,118.70,117.63,115.58,109.16.

[0111] In summary, the present invention relies on the Tianhe-2 supercomputing platform to perform structure-based virtual screening on the nsp16 target, and quickly and efficiently screens out 13 ligand molecules from about 210,000 small molecules (Mwt=400, logp=4) in the ZINC library. The in vitro activity test verifies that two molecules have preliminary enzymatic activity, namely compound 1 (ZINC12778720, IC 50 =40.86 μM) and compound 2 (ZINC95190922, IC 50 =48.15 μM), and the hit rate reached 2 / 13, proving that the screening method of the present invention is effective. In addition, the active molecules screened out have a more novel structure than the SAM analogs reported in the literature, and are expected to be developed into nsp16 inhibitors with high specificity, good drugability, and feasible synthesis through subsequent structural modification.

[0112] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A compound represented by formula (I) or a pharmaceutically acceptable salt thereof, 2. A method for preparing the compound represented by formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, It is characterized in that The following steps are involved: (1) 4-methoxybenzenesulfonyl chloride is subjected to nitration reaction to obtain intermediate (II); (2) intermediate (II) reacts with m-chloroaniline to obtain intermediate (III); (3) Intermediate (III) is subjected to reduction reaction to obtain intermediate (IV); (4) intermediate (IV) reacts with 3-furoyl chloride to obtain intermediate (V); (5) Demethylating the intermediate (V) to obtain a compound represented by formula (I); The chemical structural formulas of the intermediates (II) to (V) are as follows:

3. A virtual screening method for a compound represented by formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, It is characterized in that The following steps are involved: S1, the three-dimensional structure of nsp16 and the acquisition and processing of small molecule compound libraries; S2, re-docking sinefungin with nsp16 obtained in step S1, adjusting the docking parameters according to the degree of overlap between the docked conformation of sinefungin and the original structural conformation, and constructing a virtual screening system; S3, using the virtual screening system constructed in step S2 to screen the small molecule compound library obtained in step S1 to obtain candidate compounds; S4. Perform in vitro activity screening on the candidate compounds obtained in step S3 to obtain the compound represented by formula (I).

4. The virtual screening method according to claim 3, It is characterized in that In the step S1, the processing of the three-dimensional structure of nsp16 includes the deletion of co-crystal ligands and crystal water, the identification of the proton state of histidine, the addition of disulfide bonds, hydrogen atoms and missing heavy atoms, and the minimization of the structure using the OPLS_2005 force field; And / or, the processing of the small molecule compound library includes energy optimization of each compound using the OPLS_2005 force field.

5. The virtual screening method according to claim 3, It is characterized in that In step S2, the docking parameters are: When the three-dimensional structure of nsp16 does not contain the substrate m 7 In the case of GpppA, the OD2 atom of amino acid ASP6897 in nsp16 was used as the docking center, and the box size was Generate grid file; or, When the three-dimensional structure of nsp16 contains the substrate m 7 GpppA, with substrate m 7 The 2'O position of GpppA served as the docking center, and the box size was Generate a grid file.

6. The virtual screening method according to claim 3, It is characterized in that In step S3, the screening process includes the following steps: The high-throughput virtual screening mode, standard precision mode, and extra precision mode of Glide in the Schrödinger software were used for screening in turn, and the top 10% of the scoring compounds were selected each time; then the MMGBSA module in the Schrödinger software was used for fine screening, and the compounds with higher binding energy than sinefungin were retained; finally, the binding energy size and manual visual inspection analysis were performed.

7. Use of the compound represented by formula (I) according to claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing and / or treating novel coronavirus infection.

8. The use according to claim 7, It is characterized in that The drug is a nsp16 inhibitor.

9. The use according to claim 7, It is characterized in that The new coronavirus is SARS-CoV-2.

10. Use of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of an nsp16 inhibitor.

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