CN112245428B

CN112245428B - Application of compound capable of inhibiting interaction of coronavirus Spike protein and ACE2

Info

Publication number: CN112245428B
Application number: CN202011532900.2A
Authority: CN
Inventors: 余伯阳; 刘秀峰; 梅杰; 周娅彤
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2020-12-23
Filing date: 2020-12-23
Publication date: 2021-03-19
Anticipated expiration: 2040-12-23
Also published as: WO2022134733A1; CN112245428A

Abstract

The invention discloses application of a compound capable of inhibiting interaction of coronavirus Spike protein and ACE 2. The structure is as follows, the application of the compound in preparing the medicine for treating and/or preventing SARS-CoV-2 novel coronavirus infection. Meanwhile, the compound can inhibit the interaction between the coronavirus Spike protein and the ACE2 protein, IC50<1 μ M, while being able to promote dissociation of the Spike-ACE2 complex. Can effectively inhibit the invasion of a novel coronavirus SARS-CoV-2 pseudovirus at a cellular level, IC50<2 μ M. The compound can be specifically combined in the RBD region, KD of the Spike protein<6 mu M, which shows that the compounds have very positive effect on preparing the medicaments for treating and/or preventing the coronavirus infection.

Description

Application of compound capable of inhibiting interaction of coronavirus Spike protein and ACE2

Technical Field

The invention relates to a pharmaceutical application, in particular to a compound capable of inhibiting interaction of coronavirus Spike protein and ACE 2.

Background

One virus SARS-CoV-2 is a new type of coronavirus, and the new type of coronavirus pneumonia caused by SARS-CoV-2 is named as COVID-2019 by WHO. To date, there is no effective method for treating this infectious disease. Therefore, there is a need to produce vaccines or antibody drugs to combat this disease or to screen for new drugs for the treatment of coronavirus diseases.

The Spike (Spike) protein of coronaviruses facilitates virus recognition and entry into target cells. Many research groups have shown that SARS-CoV-2 mediates cell infection by binding of the homotrimeric spike (S) glycoprotein to angiotensin converting enzyme 2 (ACE 2) through its Receptor Binding Domain (RBD), which is further proteolytically activated by human proteases. Targeting the interaction between the SARS-CoV-2 spur protein and the human ACE2 receptor is currently considered to be a key therapeutic strategy for coronavirus infection. The existing research shows that the neutralizing antibody aiming at SARS-CoV-2 or the soluble recombinant human ACE2 receptor can effectively prevent the protein-protein interaction of Spike-ACE2 and reduce the invasion of virus to human lung epithelial cells, thereby achieving the purpose of preventing or treating COVID-2019. However, similar to other protein therapies, there are many problems in the application of antibody or protein drugs, such as immunogenicity, poor oral bioavailability, poor product uniformity, instability during production and storage, and the use of protein drugs is still limited. The development of small molecule protein-protein interaction inhibitors is more challenging, but at the same time, many characteristics of the inhibitors are beneficial to drug development, such as the advantages of excellent tissue permeability, low immunogenicity, high stability, easiness in chemical synthesis production and the like. Therefore, the finding of the blocking agent of the interaction between the Spike protein and the ACE2 protein is of great significance for preparing the medicine for treating and/or preventing coronavirus infection.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to provide application of a compound capable of inhibiting interaction of coronavirus Spike protein and ACE 2.

The technical scheme is as follows: the invention provides an application of a compound capable of inhibiting interaction between a coronavirus Spike protein and ACE2 in preparation of a medicine for treating and/or preventing SARS-CoV-2 novel coronavirus infection, wherein the structure of the compound is as shown in formula I:

formula I

Wherein, R1 or R2 groups are respectively selected from H, F, Cl, Br, I and CF₃、CF₃O、CN、CH₃O、CH₃CH₂O、CH₃CH₂CH₂O、NH₂、CH₂NH₂、CH₃、CH₃CH₂、CH₃CH₂CH₂Or OH.

Further, the compounds are as follows:

the median inhibitory concentrations of compound 1, compound 2, and compound 3 were 0.96. mu.M, 0.10. mu.M, and 0.16. mu.M, respectively.

Further, the compound can specifically bind to the RBD region of the Spike protein.

Further, the compounds can inhibit the binding of coronavirus Spike protein and ACE2 protein, and can also promote the dissociation of Spike-ACE2 complex.

Further, the drug dosage form is an oral administration dosage form or a non-oral administration dosage form.

Further, the oral administration dosage form is tablet, powder, granule, capsule, emulsion or syrup.

Further, the non-oral administration dosage form is injection.

The preparation method of the compound capable of inhibiting the interaction between the coronavirus Spike protein and ACE2 comprises the following steps:

(1) drying ephedra at 40-50 ℃, crushing, sieving with a 24-100-mesh sieve, and taking the sieved powder for later use;

(2) adding ethanol with the weight percentage concentration of 70% -100% and the weight of 10-100 times of the weight of the ephedra powder into the ephedra powder for ultrasonic-assisted extraction, wherein the extraction temperature is 25-60 ℃, the extraction time is 0.5-2 h, and the ultrasonic frequency is 30-40 KHz, filtering, and removing a solvent to obtain an extract for later use;

(3) and (3) dissolving the product obtained in the step (2) in distilled water, performing ultrafiltration and centrifugation for 0.5-2 h, taking the lower-layer solution, concentrating under reduced pressure, recovering the solvent, and performing high performance liquid chromatography separation and fractional collection to obtain the

compounds

1, 2 and 3 respectively.

The high performance liquid phase separation conditions of step (3) include: the specification of the chromatographic column is C18,5 mu m,4.6 multiplied by 250 mm Grace vision HT; gradient elution is carried out by 0.1 percent aqueous formic acid solution-acetonitrile with the volume ratio of 95:5 to 65:35, the detection wavelength is 240-260nm, and the flow rate is 1-2 mL/min.

The coronavirus spike protein is a key protein for virus invasion, because it helps virus recognition and entry into target cells. Therefore, targeting the interaction between the coronavirus Spike protein and the human ACE2 receptor protein is a key treatment strategy for coronavirus infection, and the small molecule protein-protein interaction inhibitor between Spike and ACE2 can be applied to prevention and/or treatment of coronavirus infection mediated by Spike. The inventor adopts an activity-oriented separation strategy to separate a class of compounds taking kynurenic acid as a mother nucleus from natural products, and can inhibit the interaction between Spike protein and ACE2 protein, IC₅₀<1 μ M, while being able to promote dissociation of the Spike-ACE2 complex. Can effectively inhibit the invasion and IC of novel coronavirus SARS-CoV-2 pseudovirus at cellular level₅₀<2 μ M. The compound can be specifically combined in the RBD region, K, of the Spike protein_D<6 mu M, and the compounds can be found to be effective coronavirus invasion inhibitors.

The invention comprises a pharmaceutical composition, which contains a compound shown as a formula I or pharmaceutically acceptable salt thereof, or a corresponding isomer, a non-corresponding isomer or a racemate thereof as an active ingredient, and pharmaceutically acceptable excipients. The pharmaceutically acceptable excipient refers to any diluent, adjuvant or carrier that can be used in the pharmaceutical field. The compounds of the invention may be used in combination with other active ingredients, provided that they do not produce other adverse effects.

Has the advantages that: the compound of the invention can be combined with an RBD region combined with an ACE2 receptor on a Spike protein of SARS-CoV-2, efficiently inhibits the interaction between the RBD region of SARS-CoV-2 and ACE2, prevents viruses from entering cells, can promote the dissociation of a Spike-ACE2 compound, and can be used for preparing medicaments for treating and/or preventing SARS-CoV-2 novel coronavirus infection.

Drawings

FIG. 1 shows the results of dose-dependent blockade of the interaction between SARS-CoV-2 RBD and ACE2 protein by Compound 1 and determination of the semi-inhibitory concentration;

FIG. 2 shows the results of dose-dependent blockade of the interaction between SARS-CoV-2 RBD and ACE2 protein by Compound 2 and determination of the semi-inhibitory concentration;

FIG. 3 shows the results of dose-dependent blockade of the interaction between SARS-CoV-2 RBD and ACE2 protein by Compound 3 and determination of the semi-inhibitory concentration;

FIG. 4 shows the results of the activity of the compounds of the present invention in promoting the dissociation of the Spike and ACE2 complexesP＜0.01, ***P< 0.001 vs. solvent control);

FIG. 5 is the result of an affinity assay for binding of Compound 1 to SARS-CoV-2 RBD;

FIG. 6 is the result of an affinity assay for binding of Compound 2 to SARS-CoV-2 RBD;

FIG. 7 is the result of an affinity assay for binding of Compound 3 to SARS-CoV-2 RBD;

FIG. 8 shows the toxicity assay results of recombinant human ACE2 protein on 293T-ACE2 cells;

FIG. 9 shows the results of toxicity assay of Compound 1 on 293T (293T-ACE 2) cells overexpressing the ACE2 receptor;

FIG. 10 shows the toxicity assay of compound 2 against 293T-ACE2 cells;

FIG. 11 shows the results of toxicity assay of compound 3 against 293T-ACE2 cells;

FIG. 12 is a fluorescence microscopic image showing that

compounds

1, 2 and 3 inhibit the invasion of SARS-CoV-2 novel coronavirus pseudovirus into 293T-ACE2 cells;

FIG. 13 shows the results of measurement of the dose-dependent inhibition of SARS-CoV-2 coronavirus pseudovirus invasion into 293T-ACE2 cells by recombinant human ACE2 protein and the semi-inhibitory concentration;

FIG. 14 shows the measurement results of the dose-dependent inhibition of SARS-CoV-2 novel coronavirus pseudovirus invasion into 293T-ACE2 cells by Compound 1 and the semi-inhibitory concentration;

FIG. 15 shows the measurement results of the dose-dependent inhibition of SARS-CoV-2 novel coronavirus pseudovirus invasion into 293T-ACE2 cells and the semi-inhibitory concentration of compound 2;

FIG. 16 shows the measurement results of the dose-dependent inhibition of SARS-CoV-2 novel coronavirus pseudovirus invasion 293T-ACE2 cell by compound 3 and the semi-inhibitory concentration;

FIG. 17 is a model control group to which only the novel coronavirus S protein pseudovirus was added;

FIG. 18 is a positive control group to which a novel coronavirus S protein pseudovirus was added in admixture with 180 nM ACE2 recombinant protein;

FIG. 19 shows an experimental group to which a mixture of the novel coronavirus S protein pseudovirus and 5. mu.M Compound 2 was added;

FIG. 20 shows the measurement results of the dose-dependent inhibition of SARS-CoV-2 novel coronavirus pseudovirus invasion into Calu-3 cells and the semi-inhibitory concentration of Compound 2 of the present invention.

Detailed Description

Example 1

The preparation and structure identification of the compound of the invention.

Three compounds will be exemplified below (structures shown below):

the experimental method comprises the following steps:

the preparation method of the compound 1 comprises the following steps:

(1) drying dry grass stems of ephedra herb at 50 ℃, crushing, sieving with a 65-mesh sieve, and taking ephedra herb powder below the sieve for later use;

(2) adding ethanol with weight percentage concentration of 70% 50 times of the weight of the ephedra powder into the ephedra powder for ultrasonic wave auxiliary extraction, wherein the extraction temperature is 25 ℃, the extraction time is 0.5 h, and the ultrasonic frequency is 40 KHz, filtering, and removing a solvent to obtain an extract for later use;

(3) and (3) dissolving the product obtained in the step (2) in distilled water, performing ultrafiltration and centrifugation for 0.5 h, taking the lower-layer solution, performing reduced pressure concentration, recovering the solvent, and performing high performance liquid chromatography to obtain the compound 1.

Wherein the high performance liquid phase separation conditions of the step (3) comprise: the specification of the chromatographic column is C18,5 mu m,4.6 multiplied by 250 mm Grace vision HT; the mobile phase is 0.1% formic acid water solution (A) and acetonitrile (B), the gradient elution procedure is 0-10 min, 5% A; 10-50 min, 5% -20% A; 50-60 min, 20% -25% A; 60-75 min, 25-35% A; 75-76 min, 35% -5% A; 76-85 min, 5% A. The flow rate was 1.0 ml/min, the detection wavelength was 254 nm, the column temperature was kept at 35 ℃ and the retention time on the HPLC phase was about 8.4 min.

The preparation method of the compound 2 comprises the following steps:

(3) and (3) dissolving the product obtained in the step (2) in distilled water, performing ultrafiltration and centrifugation for 0.5 h, taking the lower-layer solution, performing reduced pressure concentration, recovering the solvent, and performing high performance liquid chromatography to obtain a compound 2.

Wherein the high performance liquid phase separation conditions of the step (3) comprise: the specification of the chromatographic column is C18,5 mu m,4.6 multiplied by 250 mm Grace vision HT; the mobile phase is 0.1% formic acid water solution (A) and acetonitrile (B), the gradient elution procedure is 0-10 min, 5% A; 10-50 min, 5% -20% A; 50-60 min, 20% -25% A; 60-75 min, 25-35% A; 75-76 min, 35% -5% A; 76-85 min, 5% A. The flow rate was 1.0 ml/min, the detection wavelength was 254 nm, the column temperature was kept at 35 ℃ and the retention time on the HPLC phase was about 13.9 min.

(III) A method for producing Compound 3, which comprises:

(3) and (3) dissolving the product obtained in the step (2) in distilled water, performing ultrafiltration and centrifugation for 0.5 h, taking the lower-layer solution, performing reduced pressure concentration, recovering the solvent, and performing high performance liquid chromatography to obtain a compound 3.

Wherein the high performance liquid phase separation conditions of the step (3) comprise: the specification of the chromatographic column is C18,5 mu m,4.6 multiplied by 250 mm Grace vision HT; the mobile phase is 0.1% formic acid water solution (A) and acetonitrile (B), the gradient elution procedure is 0-10 min, 5% A; 10-50 min, 5% -20% A; 50-60 min, 20% -25% A; 60-75 min, 25-35% A; 75-76 min, 35% -5% A; 76-85 min, 5% A. The flow rate was 1.0 ml/min, the detection wavelength was 254 nm, the column temperature was kept at 35 ℃ and the retention time on the HPLC phase was about 24.7 min.

The experimental results are as follows: compound 1, HR-ESI-MS (positive) gave M/z 206.0437 [ M + H [ ]]Determining the molecular formula as C₁₀H₇NO₄The 1H-NMR (500MHz, in DMSO) spectrum of the compound showed 7.86 (d, J = 9.0 Hz, 1H), 7.39 (d, J = 2.5 Hz, 1H), 7.24 (dd, J = 8.9, 2.5 Hz, 1H), 6.61 (s, 1H). Compound 1 was identified as 6-hydroxykynurenic acid by comparison with standard substances. Compound 2, HR-ESI-MS (positive) gave M/z 190.0487 [ M + H [ ]]Determining the molecular formula as C₁₀H₇NO₃The 1H-NMR (500MHz, in DMSO) spectrum of the compound showed 8.09 (d, J = 7.5 Hz, 1H), 7.96 (d, J = 8.4 Hz, 1H), 7.70 (t, J = 8.2 Hz, 1H), 7.37 (t, J = 7.5 Hz, 1H), 6.65 (s, 1H). Compound 2 was identified as kynurenic acid by comparison to the standard. Compound 3, HR-ESI-MS (positive) gave M/z 220.0592 [ M + H [ ]]Determining the molecular formula as C₁₁H₉NO₄1H-NMR (500MHz, in DMSO) spectra of the compounds showed 7.92 (d, J = 9.1 Hz, 1H), 7.47 (d, J = 2.9 Hz, 1H), 7.34 (dd, J = 9.3, 2.8)Hz, 1H), 6.60 (s, 1H), 3.85 (s, 3H). Compound 3 was identified as 6-methoxykynurenic acid by comparison with the standard.

Example 2

The compound of the invention inhibits the interaction between the RBD region of the novel coronavirus Spike protein of SARS-CoV-2 and the ACE2 receptor.

The experimental method comprises the following steps: competitive binding inhibition assay. To a 96-well microplate, 100. mu.l of 0.5. mu.g/ml SARS-Cov-2 RBD recombinant protein dissolved in a coating solution (50 mM carbonate buffer, pH 9.6) was added, and the mixture was left at 4 ℃ overnight. The next day, the uncoated proteins were removed by rinsing three times with 300 μ l of washing solution (phosphate buffer containing 0.05% Tween 20, pH 7.4). Then 300. mu.l of blocking solution (2% bovine serum albumin in wash, pH 7.4) was added and blocked at 37 ℃ for 1 h. After three rinses, 50. mu.l of the different concentrations of the test compound were mixed with 50. mu.l of 0.12. mu.g/ml biotinylated ACE2 and added to a 96-well plate and incubated for 1h at 37 ℃. After three rinses, 100 μ l of streptavidin-labeled horseradish peroxidase was added to each well and incubated for 1h at 37 ℃. After rinsing three times, 200. mu.l of a developing solution (sodium hydrogen phosphate-citric acid buffer containing 0.1 mg/ml TMB and 0.004% hydrogen peroxide, pH 5.5) was added to each well, and incubated at 37 ℃ for 20 minutes in the absence of light. After adding 50. mu.l of a reaction stop solution (1M sulfuric acid), the absorbance was read at 450 nm using a microplate reader. Raw data were analyzed using GraphPad Prism 6.0 software and relative inhibition was calculated. The measurement values are expressed as mean values of three independent experiments. + -. standard error of the mean value.

The experimental results are as follows: as shown in FIGS. 1-3, compounds 1-3 all dose-dependently blocked the interaction of SARS-CoV-2 RBD and ACE2 recombinant proteins at semi-inhibitory concentrations of 0.96. mu.M, 0.10. mu.M and 0.16. mu.M, respectively. The compound of the invention can effectively inhibit the interaction between the novel coronavirus Spike protein of SARS-CoV-2 and ACE2 receptor.

Example 3

The compound of the invention promotes the dissociation of the RBD region of the novel SARS-CoV-2 coronavirus Spike protein and the ACE2 receptor complex.

The experimental method comprises the following steps: competitionSexual binding inhibition assay. To a 96-well microplate, 100. mu.l of 0.5. mu.g/ml SARS-CoV-2 RBD recombinant protein dissolved in a coating solution (50 mM carbonate buffer, pH 9.6) was added, and the mixture was left at 4 ℃ overnight. The next day, the uncoated proteins were removed by rinsing three times with 300 μ l of washing solution (phosphate buffer containing 0.05% Tween 20, pH 7.4). Then 300. mu.l of blocking solution (2% bovine serum albumin in wash, pH 7.4) was added and blocked at 37 ℃ for 1 h. After three rinses, 50. mu.l of 0.12. mu.g/ml biotinylated ACE2 was added to 96-well plates and incubated for 1h at 37 ℃. After three rinses, 50. mu.l of the test compound at a concentration of 25. mu.M were added to the 96-well plate and incubated for 1h at 37 ℃. After three rinses, 100 μ l of streptavidin-labeled horseradish peroxidase was added to each well and incubated for 1h at 37 ℃. After rinsing three times, 200. mu.l of a developing solution (sodium hydrogen phosphate-citric acid buffer containing 0.1 mg/ml TMB and 0.004% hydrogen peroxide, pH 5.5) was added to each well, and incubated at 37 ℃ for 20 minutes in the absence of light. After adding 50. mu.l of a reaction stop solution (1M sulfuric acid), the absorbance was read at 450 nm using a microplate reader. Raw data were analyzed using GraphPad Prism 6.0 software. The measurement values are expressed as mean values of three independent experiments. + -. standard error of the mean value. Analysis between groups was performed using one-way analysis of variance,p<0.05 was considered statistically significant.

The experimental results are as follows: as shown in FIG. 4, the amount of ACE2 bound to SARS-CoV-2 RBD was significantly reduced compared to the solvent control group after addition of compounds 1-3, respectively. The compound of the invention promotes the dissociation of the RBD region of the novel SARS-CoV-2 coronavirus Spike protein and the ACE2 receptor complex.

Example 4

The compounds of the invention interact with the RBD region of the novel coronavirus Spike protein of SARS-CoV-2.

The experimental method comprises the following steps: surface Plasmon Resonance (SPR) experiments.

The SPR sensing technology, which uses the principle of surface plasmon resonance, absorbs a portion of light energy due to surface plasmon resonance occurring when light is coupled to a sensing chip, so that the light reflected from the sensing chip forms an SPR resonance signal. The SPR resonance signals are very sensitive to the change of the refractive index of the sample substance on the surface of the sensing chip, so that the substance information of the sample can be obtained by analyzing the SPR resonance images. Has extremely important application in the biosensing field of detecting protein-protein interaction and the like.

The experiment was tested using a BIAcore T200 instrument at 25 ℃ with a phosphate buffer (pH 7.4) as the mobile phase. Diluting SARS-CoV-2 RBD recombinant protein with 10 mmol/L sodium acetate buffer solution (pH 5.5, 5.0, 4.5 and 4.0, respectively) to a final concentration of 20 μ g/ml, performing pH screening by manual sample injection, and selecting the pH with the maximum coupling value for subsequent experiments. The SARS-CoV-2 RBD recombinant protein is coupled to a CM5 chip by adopting an amino coupling method, phosphate buffer is used as working buffer, and the SARS-CoV-2 RBD recombinant protein is diluted by 10 mmol/L sodium acetate buffer with the optimal pH value obtained by the screening to the final concentration of 20 mu g/ml. The chip surface was activated with a mixture of 0.2 mol/L EDC and 50 mmol/L NHS at a ratio of 1:1, injected at a flow rate of 10. mu.l/min, injected continuously for 7min, then injected with SARS-CoV-2 RBD recombinant protein solution, injected with 1 mol/L ethanolamine hydrochloric acid (pH 8.5) blocking solution for 7min, the activated chip surface was blocked, and the blocked blank channel was used as a negative control for the assay. Experiment the compound of the present invention with the highest concentration of 25 μ M is selected as ligand, and the ligand is injected into the SARS-CoV-2 RBD recombinant protein immobilized biosensor chip in twice serial diluent. 1:1 binding model for the assessment of binding kinetics. K was calculated by BIAcore T200 analysis software using kinetic model_DThe value is obtained.

The experimental results are as follows: as shown in FIGS. 5, 6 and 7, the affinity of Compound 1 to SARS-CoV-2 RBD recombinant protein was 0.60. mu.M, the affinity of Compound 2 to SARS-CoV-2 RBD recombinant protein was 5.28. mu.M, and the affinity of Compound 3 to SARS-CoV-2 RBD recombinant protein was 5.37. mu.M. Kinetic analysis data showed that compound 1 has a slightly stronger affinity for the SARS-CoV-2 RBD recombinant protein than

compound

2 or 3, but is an order of magnitude, suggesting that the compound backbone of our invention is the key structure for its function, R₁Or the change in the R2 substituent has no significant effect on the activity of the compound. The compounds of formula I may all haveAnd (4) activity. The compounds of the invention interact with the RBD region of the novel coronavirus Spike protein of SARS-CoV-2.

Example 5

The compound inhibits SARS-CoV-2 novel coronavirus S protein pseudovirus from invading 293T cells (293T-ACE 2) over expressing ACE2 receptor.

The experimental method comprises the following steps: experiment of invasion cell of new coronavirus S protein pseudovirus. SARS-CoV-2 new coronavirus S protein pseudovirus contains green fluorescent protein reporter gene, when virus invades cell, green fluorescent protein expression can be observed under fluorescent microscope, reflecting the number of virus invaded cell. Binding of the novel coronavirus S protein to the ACE2 receptor is a key step in viral entry into cells, and 293T cells overexpressing the ACE2 receptor (293T-ACE 2) were therefore selected to mimic viral entry. First, the MTT method was used to determine whether each compound had cytotoxicity or not, depending on the effect on the viability of the cells. 293T-ACE2 cells were plated into well plates and cultured in DMEM medium containing 10% fetal bovine serum at 37 ℃ for 24 hours, and after incubation of each well with 5% CO2, each compound was added and incubated for 24 hours. 293T-ACE2 cells were then placed in 100. mu.l 10% FBS-DMEM containing 500. mu.g/ml MTT. After 4 hours of incubation at 37 ℃, 150 μ l of dimethyl sulfoxide was added to each well to dissolve the crystals. The absorbance values were measured at 570 nm using a microplate reader. The virus invasion assay was then performed by spreading 293T-ACE2 cells evenly onto well plates and culturing the cells in DMEM medium containing 10% fetal bovine serum at 37 deg.C and 5% CO₂The experiment was started when the cells grew to 30% by culturing under the conditions. After mixing the same concentration of SARS-CoV-2 novel coronavirus S protein pseudovirus with different concentrations of test compound, it was incubated at 37 ℃ for 1h and then added to each well. After 6h incubation, the medium was replaced with fresh medium. After normal culture for 48h, the expression of green fluorescent protein in different cells is observed by photographing with a fluorescence microscope (20X), the expression of green fluorescent protein is quantitatively detected by a fluorescence microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 520 nm, and the number of virus invading 293T-ACE2 cells is analyzed. Raw data were analyzed using GraphPad Prism 6.0 software and phases were calculatedFor the inhibition rate. The measurement values are expressed as mean values of three independent experiments. + -. standard error of the mean value.

The experimental results are as follows: the compounds 1-3 can prevent the novel coronavirus S protein pseudovirus from entering 293T-ACE2 cells. As shown in FIGS. 8, 9, 10 and 11, the compounds 1-3 and the positive control hACE2 recombinant protein have no influence on the activity of 293T-ACE2 cells within a certain concentration range, which indicates that the compounds are good in safety. As shown in fig. 12, it can be clearly observed under a fluorescence microscope that model group 293T-ACE2 cells express a large amount of green fluorescent protein compared to 293T cells not expressing ACE2 receptor, indicating that ACE2 is a key receptor for helping the novel coronavirus S protein pseudovirus invade cells. Meanwhile, the positive control ACE2 recombinant protein and the compounds 1-3 are added to the model group, so that the green fluorescent protein expression is obviously reduced, and the virus invasion cells can be inhibited, and the cell protection effect is achieved. FIGS. 13, 14, 15, 16 show that both the ACE2 recombinant protein and compounds 1-3 can prevent the novel coronavirus S protein pseudovirus from entering 293T-ACE2 cells with dose-dependent inhibition at half-inhibitory concentrations of 69.62 nM, 1.09. mu.M, 0.44. mu.M and 0.75. mu.M, respectively. These results all indicate that the compounds of the present invention inhibit the invasion of SARS-CoV-2 novel coronavirus S protein pseudovirus into 293T cells overexpressing the ACE2 receptor.

Example 6

The compound of the invention inhibits the invasion of SARS-CoV-2 novel coronavirus S protein pseudovirus into human lung gland epithelial cells (Calu-3).

The experimental method comprises the following steps: experiment of invasion cell of new coronavirus S protein pseudovirus. SARS-CoV-2 new coronavirus S protein pseudovirus contains green fluorescent protein reporter gene, when virus invades cell, green fluorescent protein expression can be observed under fluorescent microscope, reflecting the number of virus invaded cell. Human lung glandular epithelial cells (Calu-3) were uniformly plated on a well plate, and the cells were cultured in MEM medium containing 10% fetal bovine serum at 37 ℃ under 5% CO₂The experiment was started when the cells grew to 50% by culturing under the conditions. The SARS-CoV-2 new coronavirus S protein pseudovirus and adenovirus are mixed at the same concentrationAfter mixing with the test compound at the same concentration, incubation was carried out at 37 ℃ for 1h, followed by addition to each well. After normal culture for 24h, the expression of green fluorescent protein in different cells is observed by photographing with a fluorescence microscope (20X), the expression level of the green fluorescent protein is quantitatively detected by a fluorescence microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 520 nm, and the number of virus invading Calu-3 cells is analyzed. Raw data were analyzed using GraphPad Prism 6.0 software and relative inhibition was calculated. The measurement values are expressed as mean values of three independent experiments. + -. standard error of the mean value.

The experimental results are as follows: as shown in FIGS. 17, 18 and 19, which are fluorescence microscope observation graphs of the compound 2 for inhibiting the SARS-CoV-2 novel coronavirus pseudovirus from invading human lung glandular epithelial cells (Calu-3), as shown in the figure, the compound 2 can prevent the novel coronavirus S protein pseudovirus from entering the Calu-3 cells. Among them, FIG. 17 is a model control group to which only the novel coronavirus S protein pseudovirus was added. FIG. 18 is a positive control group with the addition of a novel coronavirus S protein pseudovirus mixed with 180 nM ACE2 recombinant protein. FIG. 19 shows an experimental group to which a mixture of the novel coronavirus S protein pseudovirus and 5. mu.M Compound 2 was added. It can be observed under a fluorescence microscope that, compared with a model control group, after the positive control ACE2 recombinant protein and the compound 2 are added, the expression of the green fluorescent protein is obviously weakened, and both the positive control ACE2 recombinant protein and the compound can inhibit virus from invading cells to play a role in protection. FIG. 20 shows that compound 2 can prevent the novel coronavirus S protein pseudovirus from entering Calu-3 cells by dose-dependent inhibition at a semi-inhibitory concentration of 322.6 nM. These results all indicate that the compounds of the present invention inhibit the invasion of SARS-CoV-2 novel coronavirus S protein pseudovirus into human lung glandular epithelial cells.

Claims

1. The application of a compound capable of inhibiting interaction between a coronavirus Spike protein and ACE2 in the preparation of a medicine for treating and/or preventing SARS-CoV-2 novel coronavirus infection is disclosed, wherein the compounds are as follows:

。

2. use according to claim 1, characterized in that: the median inhibitory concentrations of compound 1, compound 2, and compound 3 were 0.96. mu.M, 0.10. mu.M, and 0.16. mu.M, respectively.

3. Use according to claim 1, characterized in that: the compound can be specifically combined in the RBD region of the Spike protein.

4. Use according to claim 1, characterized in that: the compounds may inhibit binding of coronavirus Spike protein to ACE2 protein or promote dissociation of Spike-ACE2 complex.

5. The use according to claim 1, wherein the pharmaceutical dosage form is an oral dosage form or a non-oral dosage form.

6. The use according to claim 5, wherein the oral administration is in the form of tablets, powders, granules, capsules, emulsions or syrups.

7. The use according to claim 5, wherein said non-oral dosage form is an injection.