Application of compound IMB-9C in preparation of medicine for resisting new coronavirus

Abstract

The invention provides application of a compound IMB-9C in preparation of a medicine for resisting new coronavirus, and belongs to the technical field of biological pharmacy. The chemical name of the compound IMB-9C is 2-(2-(3, 5-dimethyl-1H-pyrazol-1-yl)-6-oxo-4-phenylpyrimidine-1 (6H)-yl)-N-(3-nitrophenyl) acetamide, and the small molecule compound can block the combination of a HR1 structural domain and an HR2 structural domain of a spike protein S2 subunit of the new coronavirus in vitro and has the activity of inhibiting the new coronavirus and an Omemitron mutant strain. Toxicity detection shows that the compound IMB-9C has no growth inhibition activity on Vero, Huh-7 and HEK-293T cells at the concentration of 50 mu g / mL, and can be used for preparing drugs and / or preparations for resisting new coronavirus.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceuticals, and particularly to the application of compound IMB-9C in the preparation of drugs against the novel coronavirus. Background Art

[0002] SARS-CoV-2 belongs to the same β-coronavirus as the severe acute respiratory syndrome coronavirus and the Middle East respiratory syndrome virus, and is the seventh member of the human coronavirus family. The SARS-CoV-2 virus particles are oval or round in shape, with a diameter of 60-140 nm. Its genome is linear, single-stranded positive-strand RNA, with a total length of about 30 kb, mainly encoding three types of viral proteins: non-structural proteins, structural proteins, and accessory proteins. In just a few years, coronaviruses (CoVs) have caused three major infectious disease pandemics. Although there are now various types of vaccines and drugs approved for marketing, the frequent mutation of the virus makes the research and development of new drugs against SARS-CoV-2, especially the search for broad-spectrum small molecule new drugs that are highly effective, easy to absorb, not prone to drug resistance, and can resist a large class of viruses, more advantageous in terms of effectiveness and safety.

[0003] The life cycle of SARS-CoV-2 can be divided into basic steps such as entry, replication, protein synthesis and assembly, and exocytosis and release. During the virus entry process, it mainly relies on the binding of its surface Spike protein to the host cell surface receptor ACE2, enters the cell through membrane fusion or endocytosis, releases the genetic material of the viral genome, synthesizes its own nucleic acid and protein using the substances in the host cell, integrates into new virus particles, and finally is excreted by budding or exocytosis. Previous studies have shown that almost all proteins in the life cycle of SARS-CoV-2 can be used as drug design targets, including multiple domains of the Spike protein that play important roles in virus-receptor binding and membrane fusion, such as the Spike protein receptor binding domain (RBD), HR, etc.; enzymes related to viral RNA synthesis and replication, such as RNA-dependent RNA polymerase (RdRp), main protease (Mpro), papain-like protease (PLpro); specific receptors or enzymes, such as ACE2, transmembrane protease serine 2 (TMPRSS2), furin, etc.

[0004] Virus invasion of host cells is a crucial step in the infection process and an important period in the viral replication cycle. Inhibiting viral entry in the early stage of viral infection can reduce the chance of viral infection and also reduce the occurrence of drug resistance. Therefore, the viral membrane fusion protein is an ideal drug target. Inhibiting the viral membrane fusion function can lead to the termination of the replication cycle. Unlike the high variability of CoVs RBD, different CoVs have great differences in binding receptors. Even if the same receptor is used, different binding sites are often used. The HR domain of the S2 subunit is highly conserved in various CoVs. For example, the sequence similarities of the HR1 and HR2 domains of SARS-CoV-2 and SARS-CoV S2 subunits are 92.6% and 100%, respectively. They play a key role in CoVs infection by forming a six-helix bundle (6-HB), and the interaction mode of HR1 and HR2 is also conserved in CoVs and is shared by CoVs.

[0005] Therefore, it is of great significance to develop new broad-spectrum anti-coronavirus drugs by developing small molecule inhibitors that have both preventive and therapeutic effects, are not prone to drug resistance, have broad-spectrum anti-CoVs activity, and target and block the SARS-CoV-2HR1 / HR interaction. Summary of the invention

[0006] In view of this, the present invention provides the use of compound IMB-9C in the preparation of drugs against the new coronavirus. In the present invention, compound IMB-9C inhibits the new coronavirus by blocking the binding between the HR1 domain and the HR2 domain of the S2 subunit of the new coronavirus Spike protein, thereby exerting an effect of resisting the new coronavirus.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides the use of the compound 2-[2-(3,5-dimethyl-1H-pyrazol-1-yl)-6-oxo-4-phenylpyrimidin-1(6H)-yl]-N-(3-nitrophenyl)acetamide in the preparation of drugs and / or preparations against the new coronavirus, and the structural formula of the compound is shown in Formula I:

[0009]

[0010] Preferably, the coronavirus includes SARS-CoV-2 and its mutants.

[0011] The present invention also provides the use of the compound 2-[2-(3,5-dimethyl-1H-pyrazol-1-yl)-6-oxo-4-phenylpyrimidin-1(6H)-yl]-N-(3-nitrophenyl)acetamide in the preparation of a drug and / or preparation for blocking the binding of the HR1 domain and the HR2 domain of the SARS-CoV-2 Spike protein on a yeast two-hybrid model.

[0012] The present invention also provides the use of the compound 2-[2-(3,5-dimethyl-1H-pyrazol-1-yl)-6-oxo-4-phenylpyrimidin-1(6H)-yl]-N-(3-nitrophenyl)acetamide in the preparation of a drug and / or preparation for blocking the binding of the HR1 domain and the HR2 domain of the SARS-CoV-2 Spike protein.

[0013] The present invention also provides the use of the compound 2-[2-(3,5-dimethyl-1H-pyrazol-1-yl)-6-oxo-4-phenylpyrimidin-1(6H)-yl]-N-(3-nitrophenyl)acetamide in the preparation of a drug and / or preparation with pseudovirus activity; the pseudovirus has the same envelope protein as SARS-CoV-2 or its mutant strains.

[0014] The present invention also provides the use of the compound 2-[2-(3,5-dimethyl-1H-pyrazol-1-yl)-6-oxo-4-phenylpyrimidin-1(6H)-yl]-N-(3-nitrophenyl)acetamide in the preparation of a drug and / or preparation for inhibiting SARS-CoV-2 Spike protein-mediated cell-cell fusion.

[0015] The present invention also provides the use of the compound 2-[2-(3,5-dimethyl-1H-pyrazol-1-yl)-6-oxo-4-phenylpyrimidin-1(6H)-yl]-N-(3-nitrophenyl)acetamide in the preparation of a drug and / or preparation for binding to the HR1 domain or the HR2 domain of the in vitro purified SARS-CoV-2 Spike protein.

[0016] Preferably, the compound 2-[2-(3,5-dimethyl-1H-pyrazol-1-yl)-6-oxo-4-phenylpyrimidin-1(6H)-yl]-N-(3-nitrophenyl)acetamide binds to the HR1 domain or the HR2 domain of the in vitro purified SARS-CoV-2 Spike protein in a non-covalent manner.

[0017] The present invention also provides the use of the compound 2-[2-(3,5-dimethyl-1H-pyrazol-1-yl)-6-oxo-4-phenylpyrimidin-1(6H)-yl]-N-(3-nitrophenyl)acetamide in the preparation of a drug and / or preparation for altering the secondary structure of the interaction between the HR1 domain and the HR2 domain of the SARS-CoV-2 Spike protein.

[0018] Preferably, the drug and / or preparation comprises the compound 2-[2-(3,5-dimethyl-1H-pyrazol-1-yl)-6-oxo-4-phenylpyrimidin-1(6H)-yl]-N-(3-nitrophenyl)acetamide and pharmaceutical excipients.

[0019] By adopting the above technical solutions, the present invention has the following beneficial effects: The chemical name of the compound IMB-9C is 2-(2-(3,5-dimethyl-1H-pyrazol-1-yl)-6-oxo-4-phenylpyrimidin-1(6H)-yl)-N-(3-nitrophenyl)acetamide. This small molecule compound can block the binding of the HR1 domain and the HR2 domain of the SARS-CoV-2 Spike protein S2 subunit in vitro and has inhibitory activity against SARS-CoV-2 and Omicron mutant strains. Toxicity detection shows that the compound IMB-9C does not show growth inhibitory activity against Vero, Huh-7, and HEK-293T cells at a concentration of 50 μg / mL and can be used to prepare drugs and / or preparations against SARS-CoV-2. Description of the Drawings

[0020] Figure 1 Schematic diagram for the construction of the SARS-CoV-2 HR1-HR2 interaction model based on the yeast two-hybrid technology.

[0021] Figure 2 Enzyme digestion verification result diagram of the recombinant plasmids pAD-HR1 and pBD-HR2, where lane 1 is the original plasmid, lane 2 is the double enzyme digestion product of the recombinant plasmid, and lane 3 is the DNAmaker.

[0022] Figure 3 Growth of different strains in SD / -Leu-Trp-His-Ade (yeast defective medium).

[0023] Figure 4 Qualitative detection result diagram of the β-galactosidase activity of different strains.

[0024] Figure 5 Quantitative detection result diagram of the β-galactosidase activity of different strains.

[0025] Figure 6 Verification result diagram of the expression of HR1 and HR2 proteins.

[0026] Figure 7 Verification result diagram of the inhibitory activity of the HR1-HR2 interaction inhibitor Itraconazole on the yeast two-hybrid model.

[0027] Figure 8 Verification result diagram of the inhibitory activity of the compound IMB-9C on the HR1-HR2 interaction in the model.

[0028] Figure 9 Results diagram of the inhibitory activity of IMB-9C on the HR1-HR2 interaction in vitro detected by Native-PAGE technology.

[0029] Figure 10 Results diagram of the inhibitory activity of IMB-9C on pseudovirus.

[0030] Figure 11 Results diagram of the inhibitory activity of IMB-9C on SARS-CoV-2 and Omicron mutants.

[0031] Figure 12 Results diagram of the inhibitory activity of IMB-9C on cell-cell fusion.

[0032] Figure 13 Results diagram of the binding activity of IMB-9C with HR1 and HR2 respectively detected by SPR technology.

[0033] Figure 14 Results diagram of the binding mode of IMB-9C with HR1 and HR2 respectively detected by HPLC-Q-TOF-MS technology.

[0034] Figure 15 Results diagram of the effect of IMB-9C on the secondary structure of HR1+HR2 detected by circular dichroism spectroscopy.

[0035] Figure 16 Results diagram of the important amino acids predicted by molecular docking technology for the binding of IMB-9C with HR1.

[0036] Figure 17 Results diagram of the binding activity of IMB-9C with mutant HR1 detected by SPR technology.

[0037] Figure 18 Results diagram of the ADME prediction of compound IMB-9C.

[0038] Figure 19 Structural formula of compound IMB-9C.

[0039] Figure 20 Flow chart for obtaining the small molecule inhibitor IMB-9C of SARS-CoV-2 HR1-HR2 interaction. Specific implementation mode

[0040] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they cannot be construed as limiting the protection scope of the present invention.

[0041] Example 1. Synthesis of compound IMB-9C

[0042] (1) Dissolve 1.5 equivalents of KOH and 1.2 equivalents of thiourea in ethanol (1 g / 10 mL), and react at 80 °C under microwave irradiation for 9 h. Post-treatment: Concentrate the ethanol, dissolve the solid in water, adjust the pH to no more solid is produced with dilute hydrochloric acid solution, and filter the solid to obtain the crude intermediate 1, with a yield of about 30 - 40%;

[0043] (2) Dissolve the above intermediate 1 in ethanol, add 2 equivalents of sodium hydroxide solution, then add 2 equivalents of iodomethane, and react at 80 °C for 4 h. After TLC detection until the reaction is completed, post-treatment: Concentrate, then add dilute hydrochloric acid to adjust the pH to acidic, and filter to obtain intermediate 2;

[0044] (3) Add an excess of hydrazine hydrate to the above intermediate 2 and react at 100 °C. After TLC detection, the reaction is basically completed. Post-treatment: Cool the reaction solution to room temperature, pour it into ice water and stir to precipitate a solid. Dry it thoroughly to remove water to obtain intermediate 3;

[0045] (4) Dissolve the above intermediate 3 in an ethanol / acetic acid (volume ratio of ethanol / acetic acid is 1:3) solution, add 1.2 equivalents of acetylacetone, and react at 100 °C. After TLC monitoring until the reaction is completed, pour the reaction solution into ice water and stir well for 30 min, and filter the solid to obtain intermediate 4;

[0046] (5) Dissolve the above thoroughly dried compound 4 in a mixed solution of MeCN / DMF (volume ratio 1:1), add 1.2 equivalents of ethyl bromoacetate and 1.2 equivalents of potassium carbonate, and react at 60 °C for 4 h. Post-treatment: Add water, extract with dichloromethane to obtain the crude product;

[0047] (6) Dissolve the above crude product in methanol, then add 3 equivalents of 1 M aqueous sodium hydroxide solution and stir at room temperature for 1 h. Post-treatment: Adjust to acidic with dilute hydrochloric acid, filter the solid, and dry it;

[0048] (7) Dissolve the above solid in dichloromethane, then add 1.2 equivalents of triethylamine and 1.2 equivalents of HATU, stir at room temperature for 10 minutes, then add 1.1 equivalents of m-nitroaniline, and stir at room temperature for 3 h. Post-treatment: Wash with water, extract with dichloromethane. Purify by column chromatography (DCM / MeOH = 100 / 1) to obtain a white solid, which is compound IMB-9C.

[0049] Example 2. Construction of the yeast two-hybrid model for HR1-HR2 interaction

[0050] (1) Model construction strategy

[0051] The construction strategy is as Figure 1As shown, after the target gene was synthesized by total gene synthesis, it was ligated to the yeast two-hybrid plasmids pGADT7 and pGBKT7 via SmaI and PstI, and fused with the GAL4 promoter AD and BD domains located on the two plasmids for expression. The co-transformation of the two plasmids into HR1-HR2 interaction brought the AD and BD domains close to complete the GAL4 promoter activity, thus activating the expression of the reporter gene.

[0052] (2) Construction of plasmids

[0053] The DNA fragments encoding the HR1 (amino acids 918 - 983) and HR2 (amino acids 1162 - 1203) of the novel coronavirus synthesized by GenScript Biotech Corporation were cloned into the yeast two-hybrid vectors pGADT7 and pGBKT7 via NdeI, NotI and NdeI, EcoRV respectively, and the recombinant plasmids pAD-HR1 (pGADT7-HR1) and pBD-HR2 (pGBKT7-HR2) were successfully constructed. The plasmids were identified by restriction digestion. pAD-HR1 was verified by double digestion with SmaI and NotI, and pBD-HR2 was verified by double digestion with SmaI and PstI. The results of restriction digestion verification are as Figure 2 shown, confirming the correct construction of the recombinant plasmids.

[0054] (3) Co-transformation of the vectors into the AH109 host strain

[0055] 6 mL of the fresh cultured logarithmic-phase AH109 yeast cell culture was centrifuged at 5,000 g for 5 min at room temperature. The supernatant was discarded, and the cells were resuspended and washed with 1.5 mL of ultrapure water, then centrifuged at 6,000 g for 5 min at room temperature. The supernatant was discarded, and the pellet was resuspended with the buffer (20 μL of 10×TE, 20 μL of 1×LiAc and 160 μL of ultrapure water) to obtain the yeast competent cells.

[0056] 0.1 μg of pAD-HR1 and pBD-HR2 and 100 μg of salmon sperm DNA (salmon sperm DNA was boiled in boiling water for 20 min and then quickly placed in an ice bath before use) were added to a 1.5 mL centrifuge tube, mixed well, 0.1 mL of yeast competent cells were added and mixed well, and then 0.6 mL of sterilized PEG / LiAc solution (volume ratio 10×TE:10×LiAc:50% PEG4000 = 1:1:8) was added. The mixture was vortexed at high speed for 10 s to mix well. Then, it was cultured at 30 °C and 200 rpm for 30 min, heat shocked at 42 °C for 15 min, cooled on ice for 1 - 2 min, centrifuged at 14,000 rpm for 5 s at room temperature, and the supernatant was removed. The pellet was resuspended with 200 μL of sterilized water and spread on the SD / -Leu-Trp plate, and cultured inverted at 30 °C. The clones growing on the plate were inoculated on the SD / -Leu-Trp-His-Ade plate to observe the growth of the bacteria.

[0057] Protein T and protein 53 have a strong interaction, while there is no interaction between protein T and protein lam. The plasmids pAD-T, pBD-53 and pAD-T, pBD-lam(λ) were co-transformed in the same way to construct the positive control strain AH109(pAD-T + pBD-53) and the negative control strain AH109(pAD-T + pBD-λ); the plasmid pAD + pBD-HR2 was co-transformed into the AH109 strain, and the plasmids pAD-HR1 and pBD were co-transformed into the AH109 strain to detect the self-activation activity of the protein.

[0058] As Figure 3 shown, both AH109(pAD-HR1 + pBD-HR2) and the positive control strain AH109(pAD-T + pBD-53) can grow on the SD / -Leu-Trp-His-Ade (yeast defective medium) plate, while the HR2 self-activation strain, the HR1 self-activation strain and AH109 cannot grow on the quadruple-deficient plate, the same as the negative control strain AH109(pAD-T + pBD-λ), indicating that neither HR1 nor HR2 has self-activation, preliminarily proving the interaction between HR1 and HR2, and successfully constructing the HR1-HR2 interaction two-hybrid model.

[0059] (4) β-galactosidase activity detection

[0060] ① Qualitative detection of β-galactosidase activity

[0061] The yeast colonies growing on the SD / -Leu-Trp-His-Ade (yeast defective medium) were picked with a sterile toothpick onto a clean filter paper, with the colony side facing up, placed in liquid nitrogen for 10 s, thawed at room temperature, and then the filter paper was placed on another clean filter paper pre-soaked in the Zbuffer / X-gal solution, incubated at 30 °C, and observed whether the colonies turned blue. Those turning blue within 8 h were positive, and those without color change were negative. The results are as Figure 4 shown, like the positive control strain AH109(pAD-T + pBD-53), the colony spots of AH109(pAD-HR1 + pBD-HR2) all showed blue, indicating β-galactosidase hydrolysis activity; AH109(pAD + pBD-HR2), AH109(pAD-HR1 + pBD), AH109 yeast and the negative control strain AH109(pAD-T + pBD-λ) did not show blue.

[0062] ② Quantitative detection of β-galactosidase activity

[0063] Collect the yeast growing in the liquid medium SD / -Leu-Trp-His-Ade (yeast defective medium), vortex and mix to measure OD 600Value. Transfer each tube of bacterial liquid to three 1.5 mL EP tubes, and centrifuge at 14,000 rpm for 30 s. Discard the supernatant, add 1.5 mL of Z buffer (0.1 M Na 2 HPO 4 , 35 mM NaH 2 PO 4 , 10 mM KCl, and 1 mM MgSO 4 , pH 7.0) to each tube to resuspend the cells. Centrifuge again at 14,000 rpm for 30 s and discard the supernatant. Resuspend the cells with 300 μL of Z buffer. Transfer 0.1 mL of the cell suspension to another clean EP tube. Place this EP tube in liquid nitrogen for 0.5 - 1 min, and then in a 37 °C water bath for 0.5 - 1 min. Freeze-thaw more than 2 times to ensure complete cell lysis. Set up a blank control with 100 μL of Z buffer.

[0064] Add 0.7 mL of Z buffer (containing 0.27% β-mercaptoethanol) to each EP tube (including the blank control). Quickly add 160 μL of ONPG (dissolved in Z buffer, 4 mg / mL) to each EP tube, and place the EP tubes in 30 °C. Start timing. When yellow appears in the EP tubes, add 0.4 mL of 1 M Na 2 CO 3 to terminate the reaction. Record the time t used. Centrifuge the liquid in the EP tubes at 14,000 rpm for 10 min. Then, transfer the supernatant to a clean cuvette (do not aspirate the precipitate to avoid affecting colorimetry), and measure OD 420 (compared with the blank control tube). OD 420 should be between 0.02 - 1.0. Calculate the β-galactosidase activity.

[0065] β-gal units = 1000 × OD 420 / (t × V × OD 600 ) V = 0.1 mL × 5

[0066] The results are as Figure 5 shown. The enzyme activity of the positive control bacterium AH109 (pAD-T + pBD-53) is 2.82, proving the reliability of the experiment; the model bacterium AH109 (pAD-HR1 + pBD-HR2) shows considerable β-galactosidase activity, with an enzyme activity value of 2.33, while the negative control bacterium AH109 (pAD-T + pBD-λ) has weak activity, with an enzyme activity value of only 0.31. These results indicate that HR1 and HR2 show interactions in the yeast two-hybrid system, and the model construction is successful.

[0067] (5) Verification of the expression of HR1 and HR2 proteins

[0068] ① Extraction of total yeast protein

[0069] Inoculate AH109 (pAD-HR1 + pBD-HR2) into 4 mL of SD–Ade / –His / –Leu / –Trp (yeast defective medium), and inoculate the AH109 strain into 4 mL of YPD medium. Incubate at 30 °C and 220 rpm for 48 h; centrifuge at 1000 g at room temperature for 5 min to collect the cells, and resuspend the cells with 200 μL of PBS buffer. Break the suspension with a cell disruptor, collect the lysate, centrifuge at 12000 rpm, and collect the supernatant; add 5× protein loading buffer to the supernatant sample and incubate in a metal bath at 100 °C for 10 min. Analyze the protein expression by western-blot. The pGADT7 (AD) plasmid contains the hemagglutinin HA antigen, so the HR1 protein carries an HA tag, and the pGBKT7 (BD) contains the c-myc antigen, and HR2 carries a c-Myc tag. The corresponding tag antibodies can be used to detect the protein expression. The results are as Figure 6 shown. There is no obvious band in AH109 (band 1), and the protein expression of HR1 and HR2 with the corresponding tags in yeast can be detected in the model bacteria (band 2).

[0070] (6) Verification of the inhibitory activity of the HR1-HR2 interaction inhibitor Itraconazole on the yeast two-hybrid model

[0071] Itraconazole is a known inhibitor of the HR1-HR2 interaction. Inoculate the model yeast AH109 (pAD-HR1 + pBD-HR2) and the positive control strain AH109 (pAD-T + pBD-53) into the SD / -Trp-Leu-His-Ade quadruple-deficient liquid medium (yeast defective medium), and incubate AH109 in YPD medium at 220 rpm and 30 °C until the OD 600 reaches 0.6 to 0.8. Dilute the bacterial solution with SD / -Trp-Leu-His-Ade quadruple-deficient liquid medium and YPD at a ratio of 1:1000, add it to a 96-well plate, and add serially diluted Itraconazole to make its final concentrations 100, 50, 25, 12.5, 6.25, 3.125, 1.56 μg / mL respectively. Incubate statically at 30 °C for 48 h, observe the growth inhibition of Itraconazole on the three yeast strains, and the concentration at which the growth of the yeast is completely inhibited is judged as the MIC.

[0072] As Figure 7As shown, the MIC of Itraconazole on AH109 (pAD-HR1 + pBD-HR2) was 3.125 μg / mL, and the MIC on AH109 (pAD-T + pBD-53) was 12.5 μg / mL, indicating a specific inhibitory effect on the existence of HR1 / HR2 interaction and demonstrating that this yeast two-hybrid system can be used for the activity measurement of HR1-HR2 interaction inhibitors.

[0073] Example 3. Verification of the inhibitory activity of compound IMB-9C on the yeast two-hybrid model

[0074] (1) Inhibitory activity of compound IMB-9C on the growth of the model

[0075] The model yeast strain AH109 (pAD-HR1 + pBD-HR2) and the positive control strain AH109 (pAD-T + pBD-53) were respectively inoculated into SD / -Trp-Leu-His-Ade quadruple-deficient liquid medium and cultured at 220 rpm and 30 °C until the OD 600 reached 0.6 to 0.8. The bacterial solution was diluted 1:1000 with SD / -Trp-Leu-His-Ade quadruple-deficient liquid medium, and 196 μL / well was added to a 96-well plate. 4 μL of the serially diluted compound IMB-9C was added to make the final concentrations 100, 50, 25, 12.5, 6.25, 3.125, and 1.56 μg / mL respectively. It was statically cultured at 30 °C for 48 h, and the growth inhibition of the compound on the two yeast strains was observed, and the MIC was measured.

[0076] As Figure 8 shown in A of, the MIC of compound IMB-9C on the model strain was 3.125 μg / mL, and the MIC on the positive control strain was 25 μg / mL. IMB-9C had a relatively specific inhibitory activity on the model strain.

[0077] (2) β-galactosidase activity detection

[0078] The model AH109 (pAD-HR1 + pBD-HR2) was cultured overnight at 30 °C in the defective medium SD / -Trp-Leu-His-Ade until the logarithmic growth phase. 50 μL of the overnight cultured cells were transferred to 5 mL of the defective medium SD / -Leu / -Trp at a ratio of 1:100. At the same time, the corresponding concentration of the compound (final concentrations of 3.125, 1.56, 0.78, 0.39, 0.18 μg / mL) was added, and the culture was continued, and a drug-free control was also set up. After 24 h, the cells were collected, and β-galactosidase activity quantitative detection was performed according to the method described in Example 1. After obtaining the β-gal units, the percentage of the drug-added group relative to the drug-free control was calculated.

[0079] The results are as Figure 8 shown in B. The β-galactosidase activity of the drug-free control bacteria was 100%, while the activity of the bacteria treated with 0.18 μg / mL of IMB-9C was 43.17% of that of the drug-free control bacteria. As the concentration of IMB-9C increased, the activity value showed a dose-dependent decrease. At 3.125 μg / mL, the activity was only 0.39% of that of the drug-free control bacteria.

[0080] Example 4. Cytotoxicity Detection of Compound IMB-9C

[0081] The cytotoxicity of IMB-9C was detected using a CCK-8 kit (Cell Counting Kit-8). HEK-293T cells were taken out from liquid nitrogen, resuscitated in a 37 °C water bath, centrifuged at 800 rpm for 5 min, the supernatant was discarded, the cells were resuspended with 1 mL of 1640 medium containing 10% fetal bovine serum, transferred into a culture dish, filled up with 9 mL of medium, and cultured at 37 °C, 5% CO 2 conditions; when the cells grew to cover more than 80% of the culture dish area, they were digested with trypsin and passaged; the cells in the logarithmic phase were plated at a density of 8000 cells / well, cultured until they adhered, and the outermost circle was sealed with sterile water; the active compounds obtained by screening were added to each well to make the final concentration 50 μg / mL, and the cells were further cultured for 48 h; 10 μL of CCK-8 detection reagent was added to each well, and after culturing in the dark for 2 - 4 h, the absorbance value at 450 nm was read with an enzyme-linked immunosorbent assay (ELISA) reader, and compared with the cell control group without the compound to calculate the relative cell survival rate. The toxicity of compound IMB-9C to Vero cells and Huh-7 cells was detected by the same method.

[0082] The results showed (Table 1) that compound IMB-9C did not show growth inhibitory activity against Vero, Huh-7, and HEK-293T cells at a concentration of 50 μg / mL.

[0083] Table 1 Relative Survival Rate of IMB-9C

[0084] Vero Huh-7 HEK-293T IMB-9C (50 μg / mL) 131％ 113％ 71％

[0085] Example 5 Native-PAGE Detection of the Inhibitory Activity of Compound IMB-9C on the Interaction between HR1 and HR2

[0086] HR1 was incubated with IMB-9C (final concentrations of 50, 25, 12.5, 6.25, 3.125, 1.56 μM) at 37 °C for 1 h, then HR2 was added and incubated at 37 °C for 30 min. Control groups of HR1 group, HR2 group and HR1+HR2 mixed group were set up. HR1 and HR2 were diluted with PBS. The final concentration of HR1 in the HR1 group was 40 μM, the final concentration of HR2 in the HR2 group was 40 μM, and the final concentrations of both HR1 and HR2 in the HR1+HR2 mixed group system were 40 μM. 4× non-denaturing loading buffer was added to the protein samples to make the final concentration of Loading buffer 1×. The precast gel was fixed in the electrophoresis tank, Tris-glycine-Native electrophoresis buffer was added, 20 μL of protein sample was loaded into each well, electrophoresis was carried out at 4 °C, the stacking gel was electrophoresed at a constant voltage of 80 V for 30 min. After the protein bands completely entered the separating gel, the voltage was adjusted to 120 V and electrophoresed for about 1 h. Electrophoresis was stopped when the bromophenol blue ran to the edge but not completely out of the gel. After electrophoresis, the separating gel part was placed in Coomassie Brilliant Blue staining for 30 min and photographed with a gel imaging system (Bio-rad).

[0087] It can be seen from Figure 9 that after adding different concentrations of IMB-9C, the formation of HR1+HR2 was inhibited and showed concentration dependence, indicating that IMB-9C blocked the interaction between HR1 and HR2.

[0088] Example 6 Detection of the pseudovirus inhibitory activity of compound IMB-9C

[0089] VSV-G pseudotyped ΔG-luciferase VSV (Kerafast, America) was inoculated into BHK-21 cells (Kerafast) overexpressing the S proteins of SARS-CoV-2 wild type and different variants (wild type, α, β, δ, γ, Mu, BA.2, etc.). After 2 h at 37 °C, the inoculum was removed and the cells were re-treated with DMEM medium containing 5% FBS and VSV-G antibody (I1, mouse hybridoma supernatant from CRL-2700; ATCC; 1:100). After 24 h, the pseudovirus particles were collected, centrifuged at 1320 g to remove cell debris, and stored at -80 °C until use. To determine the effect of the compound on virus entry, different concentrations of IMB-9C (0.8 μM to 100 μM) diluted in DMSO were mixed with the pseudovirus. HEK-293T-ACE2 cells were treated with the IMB-9C and pseudovirus mixture for 24 h. The activity of firefly luciferase was quantitatively determined by the Glo luciferase assay (E2620, Promega).

[0090] The luciferase luminescence value was detected, and the inhibition rate was calculated according to the following formula:

[0091] Inhibition rate (%) = [1 - (RLU of sample - RLU of negative control) / (RLU of positive control - RLU of negative control)] * 100%

[0092] The quantitative results of the neutralizing activity of compound IMB-9C against pseudovirus are as Figure 10 shown in Table 2. The inhibition rates of compound IMB-9C are 68.1% and 42.46% respectively.

[0093] Table 2: IC 50 s (μM) of IMB-9C against pseudovirus

[0094] WT Alpha Beta Mu Gamma Delta BA.1 BA.2 BA.4 BA.5 <![CDATA[IC 50 > 32.15 34.98 23.86 17.29 17.13 23.14 NA 16.42 NA NA

[0095] Example 7. Detection of the inhibitory activity of compound IMB-9C against SARS-CoV-2

[0096] The SARS-CoV-2 HKU-001a strain (GenBank accession number: MT230904) is a wild-type strain (WT) isolated from a nasopharyngeal aspirate specimen of a patient diagnosed with COVID-19 infection. All other variants, including Beta, Delta, BA.1, BA.2, BA.4, and BA.5, were also isolated. The antiviral effect (MOI = 0.001) was detected by the cytopathic effect (CPE) experiment for 72 - 96 h. The compound concentration that did not show CPE was recorded as the effective concentration (EC).

[0097] Cell supernatant samples collected at 24 h p.i. were used for qRT-PCR analysis of virus replication (MOI = 0.1). Total RNA was extracted from the virus supernatant lysed with RLT buffer using the QIAamp Viral RNA Mini Kit (Qiagen), and the SARS-CoV-2 virus load was quantified using the QuantiNova Probe RT-PCR Kit (Qiagen) and the LightCycler 480 real-time PCR system (Roche).

[0098] The results showed that IMB-9C could inhibit the CPE of live virus at a relatively low concentration. The effective concentrations of IMB-9C against SARS-CoV-2 and different mutant strains are shown in Table 3. Further verification of the CPE results using qRT-PCR, as Figure 11 shown, the IC 50 of IMB-9C on the SARS-CoV-2 wild strain is 1.948 μM.

[0099] Table 3: IC 50 (μM) and CC 50 (μM) of IMB-9C against SARS-CoV-2 and mutant strains

[0100] WT Alpha Beta Mu Gamma Delta BA.1 BA.2 BA.4 BA.5 <![CDATA[IC 50 > 3.1-1.6 NA 1.6-0.8 NA NA 1.6-0.8 0.8-0.4 0.8-0.4 0.4 0.8-0.4 <![CDATA[CC 50 > >112.5 >112.5 >112.5 >112.5 >112.5 >112.5 >112.5 >112.5 >112.5 >112.5 SI* >70.3 NA >140.6 NA NA >140.6 >281.3 >281.3 >281.3 >281.3

[0101] SI* = CC 50 / IC 50

[0102] Example 8. Blocking of cell fusion by compound IMB-9C

[0103] Using HEK-293T cells expressing the SARS-CoV-2 Spike protein as effector cells and Vero cells expressing ACE2 on the cell membrane surface as target cells, when the two types of cells are co-cultured, due to the binding of the Spike protein to ACE2, membrane fusion occurs between the two types of cells and the cells swell. EGFP protein is simultaneously expressed in HEK-293T cells expressing the Spike protein, making the HEK-293T cells have strong fluorescence, while the fluorescence of the fused cells weakens. The cell fusion experiment can simulate the process of virus infecting the host. Interaction blockers can block the formation of 6-HB by the interaction of Spike HR1 and HR2 to inhibit the occurrence of membrane fusion, thereby reducing the number of fused cells, manifested as the recovery of fluorescence intensity.

[0104] Plate HEK-293T cells in a 6-well plate at a density of 500,000 cells per well and incubate overnight until they adhere; add 6 μL of Lipofectamine TM liposome diluted with 250 μL of serum-free Opti-MEM medium to each well 2000 , and incubate at room temperature for 5 min; dilute 4 μg of pAAV-IRES-EGFP or pAAV-IRES-EGFP-SARS-CoV-2-S plasmid with 250 μL of serum-free Opti-MEM medium in each well, mix it with the liposome dilution in equal volume, and place it at room temperature for 20 min; add 1.5 mL of complete medium to the mixed solution of the plasmid and the liposome, pipette and mix well; aspirate the original culture medium in the 6-well plate, and carefully add the above transfection medium to the adherent cells; after incubating at 37 °C for 6 h, aspirate the transfection medium, wash the cell surface with PBS, add complete medium, and culture at 37 °C for 48 h; digest the Vero cells and mix them with the transfected cells, then plate them in a 96-well plate. The number of Vero cells is about 1 / 5 of the number of HEK-293T cells. At the same time, add different concentrations of compound IMB-9C (12.5, 25, and 50 μM), set 3 replicates, and take pictures and images with a high-content system after culturing at 37 °C for 12 h.

[0105] The results are as Figure 12, HEK-293T cells expressing only EGFP were co-cultured with Vero for 24 h. Since there was no cell fusion phenomenon, the fluorescence signal was strong and concentrated. After co-culturing HEK-293T cells transfected with SARS-CoV-2-S / EGFP with Vero, it was observed that the fluorescence intensity became weaker and the fluorescence area became larger, indicating that cell fusion had occurred. With the addition of compound IMB-9C, the number of fused cells decreased and the fluorescence intensity recovered to a certain extent, indicating that cell fusion was inhibited and showed concentration dependence.

[0106] Example 9. Detection of the affinity between compound IMB-9C and HR1, HR2 by SPR method

[0107] Filter the experimental buffers PBST and PBST containing 5% DMSO through a 0.22 μM filter membrane, degas them under vacuum, and wash the pipeline with the degassed buffer. Dissolve 153 mg of EDC and 23 mg of NHS in 2 mL of deionized water respectively, and filter through a 0.22 μM filter membrane. Mix EDC and NHS at a ratio of 1:1 and inject them. Set the flow rate to 10 μL / min and inject for 7 min to complete the chip activation. Dilute the protein to 80 μg / mL with a sodium acetate solution of appropriate pH value, fix the protein in the left channel, set the flow rate to 10 μL / min and inject for 7 min, and use the right channel as a blank control. When the protein is fixed to an ideal signal, inject 1 M ethanolamine (pH 8.5) for 8 min to block the sites on the chip surface that are not bound to the protein. Dilute the compound with PBST buffer containing 5% DMSO, and the final concentrations are 200, 100, and 50 μM respectively. The binding process lasts for 2.5 min and the natural dissociation lasts for 5.5 min, and set the flow rate to 25 μL / min. Observe the binding of the compound to the protein fixed on the chip at different concentrations. Use Trace Drawer software to calculate the equilibrium dissociation constant (KD) of the compound and the protein.

[0108] As Figure 13 , as shown in Table 4, compound IMB-9C can bind to both HR1 and HR2, and the KD values are 5.67×10 -7 M and 3.68×10 -5 M.

[0109] Table 4: Detection results of the affinity between IMB-9C and HR1, HR2

[0110]

[0111]

[0112] Example 10. Detection of the binding mode between compound IMB-9C and HR1, HR2 by HPLC-Q-TOF-MS method

[0113] Purified SARS-CoV-2 HR1 (5 μM) or HR2 (5 μM) was incubated with IMB-9C (500 μM) in Tris-buffered saline (TBS, containing 10 mM Tris, 50 mM NaCl, pH 8.0) at room temperature for 30 min (the experimental group and the control group were set up in parallel), and then subjected to HPLC-Q-TOF-MS injection detection.

[0114] As Figure 14 shown, there was no change in the molecular weight of the polypeptide detected by HPLC-Q-TOF-MS before and after the incubation of IMB-9C with HR1 or HR2, indicating that its binding to HR1 and HR2 was non-covalent.

[0115] Example 11. CD detection of the effect of compound IMB-9C on the secondary structure of HR1+HR2

[0116] After co-incubating HR1 (10 μM) + HR2 (10 μM) and HR1 (10 μM) + HR2 (10 μM) + IMB-9C (10 μM) at ambient temperature for 30 min, CD spectra in the far-ultraviolet region (200 - 260 nm) were collected on a Chirascan plus circular dichroism spectrometer (UK Applied Photophysics Ltd). The scanning speed was 120 nm / min, the time constant was 1 s, the bandwidth was 1 nm, and the scans were collected three times. The average molecular weight of the residues was set to 110 for calculating the mean residue ellipticity. According to the manufacturer's instructions, the data were processed by smoothing and subtracting the original buffer spectrum. Three independent experiments were performed for each sample. CDNN was used to perform spectral deconvolution on the CD data to determine the relative amounts of α-helix, β-turn, β-sheet, and random coil.

[0117] As Figure 15 shown in Table 5, after incubation of HR1+HR2 with compound IMB-9C, the secondary structure of the protein changed significantly, with the α-helix content increasing by 5.64% and the β-turn content increasing by 7.6%, indicating that the co-incubation with compound IMB-9C changed the protein secondary structure of HR1+HR2.

[0118] Table 5: Effect of IMB-9C on the secondary structure of HR1+HR2

[0119]

[0120] Example 12. Molecular docking to predict the important amino acid sites for the binding of compound IMB-9C to HR1

[0121] Since the affinity of IMB-9C for HR1 is higher than that for HR2, molecular docking of IMB-9C with HR1 was performed to determine its possible binding mode with HR1. The crystal structure of the core of the S2 subunit after membrane fusion of the new coronavirus with the receptor was obtained from the Protein Data Bank (PDB) (PDB ID: 6LXT; https: / / www.rcsb.org / structure / 6lxt). In the docking, the pocket of HR1 used the entire HR1 protein. First, water molecules were extracted from the PDB file, and polar hydrogens were added to the same PDB file. The PDB structure of the ligand molecule was created using ACD chemsketch software (https: / / www.acdlabs.com / resources / freeware / chemsketch / ), and then converted to the pdbqt format using AutoDock Vina.

[0122] The docking program Autodockvina 33 was used to calculate the interactions between 9 flexible structures of IMB-9C and the rigid binding site. After docking, the conformation of the compound was analyzed, and (H-bond and hydrophobic) interactions and three-dimensional models were generated using UCSF Chimera 34, LIGPLOT 35, or PyMol (Schrodinger LLC).

[0123] The results are as Figure 16 shown. IMB-9C can enter the pocket of the α-helical structure formed between HR1 and HR2. It forms hydrogen bonds with the K933 residue and hydrophobic interactions with the A930, I931, K933, I934, Q935, S937, L938, S939, S940, T941, T942, and L945 residues, indicating that these 12 amino acids may be the sites that play important roles in the interaction between the compound IMB-9C and HR1.

[0124] Example 13 Construction of a Mutant Yeast Two-Hybrid Model to Detect the Compound IMB-9C

[0125] The pAD-HR1 plasmid with mutations at these 12 sites (A930, I931, K933, I934, Q935, S937, L938, S939, S940, T941, T942, and L945) was synthesized by total gene synthesis, and the MIC of IMB-9C was detected on the mutant yeast two-hybrid model.

[0126] pAD-HR1 with mutations in I934, Q935, S937, L938, S939, S940, T942 mutantThe plasmid and pBD-HR2 cannot grow on the quadruple dropout medium, indicating that these 7 amino acids may play a decisive role in the interaction between HR1 and HR2, and mutation leads to the loss of interaction. The pAD-HR1 with mutations in the remaining 5 amino acids (A930, I931, K933, T941, L945) mutant The plasmid and pBD-HR2 can grow on the quadruple dropout medium. Further, the MIC of compound IMB-9C against these 5 groups of yeast two-hybrids was detected. As shown in Table 6, compared with the original yeast two-hybrid system, the MIC increased by 5-8 times.

[0127] Table 6: MIC of IMB-9C against the mutant yeast two-hybrid model (μg / mL)

[0128] A930 I931 K933 T941 L945 wild MIC 50 25 25 12.5 12.5 3.125

[0129] Example 14. SPR detection of the affinity between compound IMB-9C and mutant HR1

[0130] An HR1 polypeptide fragment with mutations in 5 amino acid sites (A930, I931, K933, T941, L945) was synthesized, and SPR was used to study the change in the affinity between IMB-9C and mutant HR1.

[0131] As Figure 17 , shown in Table 7, compared with the original HR1 polypeptide, the affinity between the mutant HR1 and compound IMB-9C decreased by 2-3 orders of magnitude.

[0132] Table 7: Detection of the affinity between IMB-9C and mutant HR1

[0133] Kon (1 / Ms) Koff (1 / S) KD (M) HR1-A930G <![CDATA[7.80×10 3 > <![CDATA[9.86×10 -2 > <![CDATA[1.27×10 -5 > HR1-I931G <![CDATA[3.74×10 2 > <![CDATA[1.01×10 -1 > <![CDATA[2.69×10 -4 > HR1-K933G <![CDATA[2.62×10 4 > <![CDATA[3.76×10 -1 > <![CDATA[1.44×10 -5 > HR1-T941G <![CDATA[8.16×10 3 > <![CDATA[4.37×10 -1 > <![CDATA[5.36×10 -5 > HR1-L945G <![CDATA[1.67×10 3 > <![CDATA[5.38×10 -1 > <![CDATA[3.21×10 -4 >

[0134] Example 15. ADME prediction of compound IMB-9C

[0135] The online website http: / / www.swissadme.ch / was used to predict the ADME pharmacokinetic properties of compound IMB-9C. The operation process is as follows: The structure of the active compound was converted into the SMILES format using Chemdraw software and input into the search box, and then click run to predict the in vivo pharmacokinetic properties of the compound.

[0136] The ADMET prediction results are as Figure 18 , shown in Table 8. The results show that compound IMB-9C follows the five-fold rule, has good intestinal absorption, cannot cross the blood-brain barrier, is slightly soluble in water, does not inhibit CYP2D6, and has inhibitory activity against CYP3A4, CYP2C9, and CYP2C19.

[0137] Table 8: ADMET Prediction of IMB-9C

[0138]

[0139] As can be seen from the above examples, the present invention provides the use of the compound IMB-9C in the preparation of drugs against the novel coronavirus. The compound IMB-9C of the present invention has an inhibitory effect on the novel coronavirus and its mutant strains, and can be used to prepare drugs and / or preparations against the novel coronavirus.

[0140] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. Use of the compound 2-[2-(3,5-dimethyl-1H-pyrazol-1-yl)-6-oxo-4-phenylpyrimidin-1(6H)-yl]-N-(3-nitrophenyl)acetamide in the preparation of drugs and / or preparations against the new coronavirus, the structural formula of the compound is shown in Formula I:

2. The use according to claim 1, characterized in that: The coronaviruses include SARS-CoV-2 and its mutant strains.

3. Use of the compound 2-[2-(3,5-dimethyl-1H-pyrazol-1-yl)-6-oxo-4-phenylpyrimidin-1(6H)-yl]-N-(3-nitrophenyl)acetamide in the preparation of drugs and / or preparations that block the binding between the HR1 domain and the HR2 domain of the S2 subunit of the SARS-CoV-2 Spike protein in a yeast two-hybrid model.

4. Use of the compound 2-[2-(3,5-dimethyl-1H-pyrazol-1-yl)-6-oxo-4-phenylpyrimidin-1(6H)-yl]-N-(3-nitrophenyl)acetamide in the preparation of drugs and / or preparations that block the binding between the HR1 domain and the HR2 domain of the S2 subunit of the SARS-CoV-2 Spike protein.

5. Use of the compound 2-[2-(3,5-dimethyl-1H-pyrazol-1-yl)-6-oxo-4-phenylpyrimidin-1(6H)-yl]-N-(3-nitrophenyl)acetamide in the preparation of drugs and / or preparations with anti-pseudovirus activity; the pseudovirus has the same envelope protein as SARS-CoV-2 or its mutant strains.

6. Use of the compound 2-[2-(3,5-dimethyl-1H-pyrazol-1-yl)-6-oxo-4-phenylpyrimidin-1(6H)-yl]-N-(3-nitrophenyl)acetamide in the preparation of drugs and / or preparations for inhibiting cell-cell fusion mediated by the SARS-CoV-2 Spike protein.

7. Use of the compound 2-[2-(3,5-dimethyl-1H-pyrazol-1-yl)-6-oxo-4-phenylpyrimidin-1(6H)-yl]-N-(3-nitrophenyl)acetamide in the preparation of drugs and / or preparations that bind to the HR1 domain or HR2 domain of the in vitro purified novel coronavirus Spike protein.

8. The use according to claim 7, characterized in that: The compound 2-[2-(3,5-dimethyl-1H-pyrazol-1-yl)-6-oxo-4-phenylpyrimidin-1(6H)-yl]-N-(3-nitrophenyl)acetamide binds to the HR1 domain or HR2 domain of the in vitro purified SARS-CoV-2 Spike protein in a non-covalent manner.

9. Use of the compound 2-[2-(3,5-dimethyl-1H-pyrazol-1-yl)-6-oxo-4-phenylpyrimidin-1(6H)-yl]-N-(3-nitrophenyl)acetamide in the preparation of drugs and / or preparations that change the secondary structure of the interaction between the HR1 domain and the HR2 domain of the SARS-CoV-2 Spike protein.

10. The use according to any one of claims 1 to 9, characterized in that: The medicine and / or preparation comprises the compound 2-[2-(3,5-dimethyl-1H-pyrazol-1-yl)-6-oxo-4-phenylpyrimidin-1(6H)-yl]-N-(3-nitrophenyl)acetamide and pharmaceutical excipients.

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