Treatment or prevention of coronavirus infection

Abstract

A method for treating or preventing a Coronaviridae infection in a subject, comprising administering a therapeutically effective amount of a compound of Formula I or a stereoisomer, tautomer, N-oxide, solvate, metabolite, pharmaceutically acceptable salt or prodrug thereof, and the Coronaviridae includes at least one selected from 2019-nCov virus, HCov 229E virus, SARS virus, MERS virus,

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to treating or preventing coronavirus infection in a subject. Background Art

[0002] The World Health Organization recently declared severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) a public health emergency of international concern. As of June 1, 2020, there are more than 6 million confirmed cases and more than 370,000 deaths worldwide.

[0003] The Coronaviridae (also called "coronaviruses") are a family of enveloped, positive-sense, single-stranded RNA viruses. The Coronaviridae cause disease in mammals and birds. In humans, the viruses cause respiratory infections, including the common cold, which are usually mild, although rare forms such as SARS (including the one that causes COVID-19) and MERS can be fatal.

[0004] Therefore, there is a growing need to research and develop more effective methods to treat or prevent Coronaviridae infections. Summary of the invention

[0005] The following is only an overview of some aspects of the present invention, but the present invention is not limited thereto. All references in this specification are incorporated herein by reference in their entirety. When the disclosure of this specification differs from the citation, the disclosure of this specification shall prevail.

[0006] During the research and development process, the inventors surprisingly found that WS-635 (compound of formula (I)) showed significantly high efficacy and activity for treating or preventing Coronaviridae infection in a variety of experimental tests.

[0007] In one aspect of the present disclosure, a method of treating or preventing a Coronaviridae infection in a subject is provided, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of Formula I or a stereoisomer, tautomer, N-oxide, solvate, metabolite, pharmaceutically acceptable salt or prodrug thereof,

[0008]

[0009] And the coronavirus family includes at least one selected from 2019-nCov virus, HCov 229E virus, SARS virus, and MERS virus.

[0010] In another aspect of the present disclosure, a pharmaceutical composition for treating or preventing a coronavirus infection is provided, comprising a compound of formula I or a stereoisomer, tautomer, N-oxide, solvate, metabolite, pharmaceutically acceptable salt or prodrug thereof,

[0011]

[0012] And the coronavirus family includes at least one selected from 2019-nCov virus, HCov 229E virus, SARS virus, and MERS virus.

[0013] In another aspect of the present disclosure, provided is a compound of Formula I or a stereoisomer, tautomer, N-oxide, solvate, metabolite, pharmaceutically acceptable salt or prodrug thereof for treating or preventing a Coronaviridae infection in a subject,

[0014]

[0015] And the coronavirus family includes at least one selected from 2019-nCov virus, HCov 229E virus, SARS virus, and MERS virus.

[0016] In another aspect of the present disclosure, there is provided a use of a compound of formula I or a stereoisomer, tautomer, N-oxide, solvate, metabolite, pharmaceutically acceptable salt or prodrug thereof in the preparation of a medicament for treating or preventing a Coronavirus infection,

[0017]

[0018] And the coronavirus family includes at least one selected from 2019-nCov virus, HCov 229E virus, SARS virus, and MERS virus.

[0019] The foregoing merely summarizes certain aspects disclosed herein and is not intended to be limiting in nature. These and other aspects of the invention as well as additional embodiments, features and advantages will become apparent through the following detailed description and through practice of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] These and other aspects and advantages of the embodiments of the present disclosure will become apparent and will be more readily understood from the following description with reference to the accompanying scheme and drawings, in which:

[0021] Figure 1 The results showed that WS-635 inhibits SARS-CoV-2 coronavirus at the cellular level;

[0022] Figure 2 showed the results of WS-635 anti-SARS-CoV-2 activity; and

[0023] Figure 3 The results of WS-635 activity against human coronavirus (HCoV) 229E are shown. DETAILED DESCRIPTION

[0024] definition

[0025] Unless otherwise stated, the following terms and phrases used herein shall have the following meanings:

[0026] "Ester" refers to any ester of a compound wherein any -COOH functional group of the molecule is a -C(O)OR functional group having a substituent, or wherein any -OH functional group of the molecule is a -OC(O)R functional group, wherein R can form a stable ester moiety, the ester moiety being any carbon-containing group including, but not limited to, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclyl, heterocycloalkyl, and substituted derivatives thereof.

[0027] "Pharmaceutically acceptable" means suitable for use in pharmaceutical preparations, generally recognized as safe, officially approved by a national or state regulatory agency or listed in the pharmacopoeia or other recognized pharmaceutical text for use in animals, and more particularly in humans.

[0028] "Pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient or vehicle or other component that is pharmaceutically acceptable and is administered in combination with the compounds of the present invention.

[0029] "Pharmaceutically acceptable salt" refers to a salt that can enhance its desired pharmacological activity. Its pharmaceutically acceptable salts with inorganic or organic acids include acid addition salts, metal salts and amine salts. Acid addition salts can be formed with inorganic acids and organic acids.

[0030] "Prodrug" refers to any compound that, upon administration, can produce its active ingredient drug by a biological system as a result of spontaneous chemical reactions, enzyme-catalyzed chemical reactions, photolysis, and / or metabolism by chemical processes. Thus, a prodrug is a covalently modified analog or latent form of a therapeutically active compound. Non-limiting examples include prodrug esters, quaternary ammonium molecules, diol molecules, and the like.

[0031] "Therapeutically effective amount" means that the compound is administered to an animal in an amount effective enough to treat a disease.

[0032] A therapeutically effective amount of a compound "treatment" means and includes any of the following uses:

[0033] (1) Prevention of diseases that may be predisposing animals to a disease but not yet experiencing or displaying the pathology or symptoms of the disease,

[0034] (2) inhibiting a disease is when an animal is experiencing or showing pathology or symptoms of a disease (i.e., preventing further development of the pathology and / or symptoms), or

[0035] (3) Amelioration of disease pathology or symptoms is the disease being experienced or manifested in the animal (i.e., reversal of pathology and / or symptoms).

[0036] Compound

[0037] The present disclosure relates to compounds of Formula I or stereoisomers, tautomers, N-oxides, solvates, metabolites, pharmaceutically acceptable salts or prodrugs thereof,

[0038]

[0039] When treating or preventing a Coronavirus infection in a subject in need thereof, a compound of Formula I or a stereoisomer, tautomer, N-oxide, solvate, metabolite, pharmaceutically acceptable salt or prodrug thereof is administered. And in some examples, the Coronavirus may include at least one selected from 2019-nCov virus, HCov 229E virus, SARS virus, MERS virus.

[0040] In some examples of the present disclosure, the compound of formula I can be used in the form of a pharmaceutically acceptable salt, and the pharmaceutically acceptable salt is at least one selected from the group consisting of phosphate, hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, ethanesulfonate, toluenesulfonate and benzenesulfonate, acetate, trifluoroacetate, tartrate, maleate, succinate, citrate, benzoate, salicylate and ascorbate, adipate, alginate, arginine, aspartate, bisulfate, bisulfite, bromide, butyrate, camphorate, camphorsulfonate, caprylate, chloride, chlorobenzoate, cyclopentanepropionate, digluconate, dihydrogen phosphate ... Nitrobenzoate, dodecylsulfate, fumarate, galactonate, galacturonate, glucoheptonate, gluconate, glutamate, glycerophosphate, hemisuccinate, hemisulfate, heptanoate, hexanoate, hippurate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, iodide, isethionate, isobutyrate, lactate, lactobionate, malate, malonate, mandelate, metaphosphate, methanesulfonate, methylbenzoate, monohydrogenphosphate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, oleate, pamoate, pectinate, persulfate, phenylacetate, 3-phenylpropionate, phosphate, phosphonate and phthalate.

[0041] Pharmaceutical preparations and uses

[0042] The compounds of the present disclosure can be formulated with conventional carriers and excipients, which meet the general practice selected. Tablets contain excipients, glidants, fillers, binders, etc. When intended for administration by oral administration, aqueous preparations will be prepared in sterile form, usually isotonic solutions.

[0043] Although it is possible for the active ingredient to be administered alone, it may be preferable to formulate it as a pharmaceutical formulation. The formulations of the present invention, whether for human or veterinary use, have at least one active ingredient together with one or more acceptable carriers and optionally other therapeutic ingredients.

[0044] The present invention suitable for oral formulation may be presented as a plurality of discrete units, each unit containing a predetermined amount of the active ingredient, such as capsules, cachets or tablets; powders or granules; in aqueous or non-aqueous liquid solutions or suspensions; or oil-in-water emulsions or water-in-oil emulsions. The active ingredient may also be in the form of a bolus, electuary or paste.

[0045] The effective amount of the active ingredient will depend at least on the nature of the disease being treated, toxicity, whether the compound is used for prophylaxis (low doses) or for active viral infection, the pharmaceutical formulation and method of administration, and is then determined by the clinician using routine dose escalation studies.

[0046] In some preferred embodiments of the present disclosure, the present disclosure relates to compounds of Formula I. The inventors surprisingly found that compounds of Formula I showed significantly high efficacy and activity for treating or preventing Coronaviridae infections in a variety of experimental tests.

[0047] Then, the compound of formula I or its pharmaceutically acceptable salt, ester or salt of prodrug or prodrug can be administered to the subject in the form of sol, perfumed water, aerosol, partial inhalation, powder, granule, tablet, ointment, paste, emulsion, suspension, gas dispersion, solid dispersion, microgranule, pill. And in some examples, the compound of formula I or its pharmaceutically acceptable salt, ester or prodrug is administered at a daily dose of less than 1000 mg, for example, 100 mg to 1000 mg, 100 to 500 mg, based on the compound of formula I.

[0048] In addition, those skilled in the art will appreciate that combination therapy may also be used. The compound of formula I or a pharmaceutically acceptable salt, ester or prodrug thereof may be combined with one or more active agents. In a non-limiting embodiment, suitable active agents for combination include one or more selected from the group comprising: corticosteroids, anti-inflammatory signal transduction regulators, β2-adrenergic receptor agonists bronchodilators, anticholinergics, mucolytics, hypertonic saline, agents for reducing cytokine storms, other drugs for treating covid-19 viral infection, or mixtures thereof. Then in some examples, a method for treating or preventing covid-19 viral infection in a subject is provided, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt, ester, hydrate or prodrug thereof,

[0049]

[0050] And in some specific examples, the provided methods may further include administering a therapeutically effective amount of at least one other therapeutic agent or a composition thereof, wherein the other therapeutic agent or a composition thereof is selected from the group consisting of: corticosteroids, anti-inflammatory signaling regulators, β2-adrenergic receptor agonist bronchodilators, anticholinergics, mucolytics, hypertonic saline, agents that reduce cytokine storms, other drugs used to treat covid-19 viral infection, or mixtures thereof.

[0051] In some specific examples, the compound of Formula I or a pharmaceutically acceptable salt, ester, hydrate or prodrug thereof is administered to a subject in the form of a sol, perfumed water, aerosol, partial inhalation agent, powder, granule, tablet, ointment, paste, emulsion, suspension, gas dispersion, solid dispersion, microgranule, or pill.

[0052] And in some specific examples, the compound of formula I or a pharmaceutically acceptable salt, ester, hydrate or prodrug thereof is administered at a daily dose of less than 1000 mg based on the compound of formula I.

[0053] And in some specific examples, the compound of formula I or a pharmaceutically acceptable salt, ester, hydrate or prodrug thereof is administered at a daily dose of 100 mg to 1000 mg based on the compound of formula I.

[0054] And in some specific examples, the compound of formula I or its pharmaceutically acceptable salt, ester, hydrate or prodrug is administered at a daily dose of 100 mg to 500 mg based on the compound of formula I.

[0055] In some other examples, a pharmaceutical composition for treating or preventing covid-19 virus infection in a subject in need thereof is provided, comprising a compound of formula I or a pharmaceutically acceptable salt, ester, hydrate or prodrug thereof,

[0056]

[0057] And in some specific examples, the composition further comprises at least one other therapeutic agent or a composition thereof, wherein the other therapeutic agent or a composition thereof is selected from the group comprising: corticosteroids, anti-inflammatory signal transduction regulators, β2-adrenergic receptor agonist bronchodilators, anticholinergics, mucolytics, hypertonic saline, agents for reducing cytokine storms, other drugs for treating covid-19 viral infection, or a mixture thereof.

[0058] And in some specific examples, the pharmaceutically acceptable salt is at least one selected from the group consisting of phosphate, hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, ethanesulfonate, toluenesulfonate and benzenesulfonate, acetate, trifluoroacetate, tartrate, maleate, succinate, citrate, benzoate, salicylate and ascorbate, adipate, alginate, arginine, aspartate, bisulfate, bisulfite, bromide, butyrate, camphorate, camphorsulfonate, octanoate, chloride, chlorobenzoate, cyclopentanepropionate, digluconate, dihydrogen phosphate, dinitrobenzoate, dodecylsulfide The salts described herein are bisphenol A, bisphenol A, bisphenol B, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A

[0059] And in some specific examples, the compound of formula I or its pharmaceutically acceptable salt, ester, hydrate or prodrug is in the form of sol, aromatic water, aerosol, partial inhalation, powder, granule, tablet, ointment, paste, emulsion, suspension, gas dispersion, solid dispersion, microgranule, pill.

[0060] And in some specific examples, the composition comprises a dose of less than 1000 mg, less than 800 mg, less than 600 mg, less than 500 mg, or less than 200 mg of a compound of Formula I or a pharmaceutically acceptable salt, ester, hydrate or prodrug thereof.

[0061] Then, the present disclosure provides a compound of formula I or a pharmaceutically acceptable salt, ester, hydrate or prodrug thereof for treating or preventing covid-19 virus infection in a subject,

[0062]

[0063] And in some specific examples, the pharmaceutically acceptable salt is at least one selected from the group consisting of phosphate, hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, ethanesulfonate, toluenesulfonate and benzenesulfonate, acetate, trifluoroacetate, tartrate, maleate, succinate, citrate, benzoate, salicylate and ascorbate, adipate, alginate, arginine, aspartate, bisulfate, bisulfite, bromide, butyrate, camphorate, camphorsulfonate, octanoate, chloride, chlorobenzoate, cyclopentanepropionate, digluconate, dihydrogen phosphate, dinitrobenzoate, dodecylsulfide The salts described herein are bisphenol A, bisphenol A, bisphenol B, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A, bisphenol A

[0064] And in some specific examples, the compound of formula I or its pharmaceutically acceptable salt, ester, hydrate or prodrug is in the form of sol, aromatic water, aerosol, partial inhalation, powder, granule, tablet, ointment, paste, emulsion, suspension, gas dispersion, solid dispersion, microgranule, pill.

[0065] And in some specific examples, the compound of formula I or a pharmaceutically acceptable salt, ester, hydrate or prodrug thereof is administered at a daily dose of less than 1000 mg based on the compound of formula I.

[0066] And in some specific examples, the compound of formula I or a pharmaceutically acceptable salt, ester, hydrate or prodrug thereof is administered at a daily dose of 100 mg to 1000 mg based on the compound of formula I.

[0067] And in some specific examples, the compound of formula I or its pharmaceutically acceptable salt, ester, hydrate or prodrug is administered at a daily dose of 100 mg to 500 mg based on the compound of formula I.

[0068] Example

[0069] Example 1. Evaluation of the inhibitory effect of WS-635 on SARS-CoV-2 coronavirus at the cellular level.

[0070] WS-635 was supplied by Waterstone Pharmaceuticals Inc. as a powder.

[0071] Viral cells are maintained by the Virology Laboratory of the Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences.

[0072] The SARS-CoV-2 coronavirus isolate (deposit code: 2019-nCoV BetaCoV / Beijing / AMMS01 / 2020) is deposited by the Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences.

[0073] Determination of EC of WS-635 by quantification of viral nucleic acid 50 :

[0074] Human Vero cells were purchased from ATCC (US), and the SARS-Cov-2 virus was isolated by the Beijing Institute of Military Medical Sciences with the accession code: 2019-nCoV BetaCoV / Beijing / AMMS01 / 2020.

[0075] 1). Determine the CC of the drug by MTS (Promega) method 50 . The details are as follows: Vero cells were seeded into 96-well cell culture plates at a concentration of 10,000 / well and cultured overnight at 37°C and 5% CO2. When the cells grew to a complete monolayer, a 2% FBS DMEM maintenance solution containing different concentrations of drugs (initial concentration of 400 μM) was added to 100 μL / well, and 4 replicate wells were measured at each concentration. Continue to culture and observe the cell status under a microscope every day. Until the 4th day of administration, 20 μL of MTS solution was added, incubated at 37°C for 1 hour, and then the OD490 value was measured.

[0076] 2) Determine the EC of the drug by nucleic acid quantification method 50 As follows: Vero cells were inoculated into a 96-well plate at a concentration of 10,000 / well one day in advance. The 2019-nCoV virus was diluted with 2% cell maintenance medium and added to the 96-well plate so that the virus content in each well was 100 TCID 50 WS-635 dissolved in DMSO was prepared at 400, 200, 100, 50, 25, and 12.5 μM using 2% FBS DMEM maintenance solution. The cell culture supernatant was discarded and different concentrations of T705 (100 μL / well) were added. Each drug was plated in 4 replicates and 100 TCID 50The virus solution was added to each drop. A positive drug control (10 μM remdesivir (RDV)), a virus control, and a normal cell control group were set up and cultured in a 37°C, 5% CO2 incubator. On the second day after infection, 50 μL of cell supernatant was taken from each well to extract nucleic acid. The viral load was detected by quantitative RT-PCR, and the EC was calculated by fitting the dose-response curve using graphpad prism 7. 50 .

[0077] 3) Select index SI = CC 50 / EC 50 .

[0078] The inhibitory effect of WS-635 on SARS-CoV-2 novel coronavirus was tested at the cellular level using a nucleic acid quantification method, and the EC value of WS-635 was calculated. 50 .

[0079] Figure 1 It was shown that WS-635 has an inhibitory effect on the 2019-nCoV coronavirus at the cellular level, and its EC 50 The CC 50 >100μM, SI>7.

[0080] Example 2. In vitro assay of WS-635 for SARS-CoV-2 replication

[0081] The experiment was conducted by the Chinese Institute of Virology in a P3 laboratory:

[0082] Human Vero cells (ATCC) were infected with the virus SARS-CoV-2 (2019-nCOV BetaCoV / Beijing / AMMS01 / 2020) to 100 TCID 50 The viral RNA load was measured by incubation at different compound concentrations.

[0083] Vero cells were seeded into 96-well plates at a concentration of 10,000 / well one day in advance. The 2019-nCoV virus was diluted with 2% cell maintenance medium and added to the 96-well plate so that the virus content in each well was 100 TCID 50 WS-635 dissolved in DMSO was prepared at 400, 200, 100, 50, 25, and 12.5 μM using 2% FBS DMEM maintenance solution. The cell culture supernatant was discarded and different concentrations of T705 (100 μL / well) were added. Each drug was plated in 4 replicates and 100 TCID 50The virus solution was added to each droplet. A positive drug control (10 μM remdesivir (RDV)), a virus control, and a normal cell control group were set up and cultured in a 37°C, 5% CO2 incubator. On the second day after infection, 50 μL of cell supernatant was taken from each well to extract nucleic acid. The viral load was detected by quantitative RT-PCR, and the EC was calculated by fitting the dose-response curve using graphpad prism7. 50 .

[0084] Inhibition and cytotoxicity curves are shown in Figure 2 As shown in Table 1. The figure shows that WS-635 inhibits SARS-CoV-2 (2019-nCOV BetaCoV / Beijing / AMMS01 / 2020) in a dose-dependent manner.

[0085] Table 1:

[0086] CPD ID <![CDATA[EC 50 (μM)]]> WS-635 5.5

[0087] Example 3. Evaluation of the antiviral activity of the test compound WS-635 against human coronavirus (HCoV) 229E in a cytopathic effect (CPE) assay.

[0088] 3.1. Test and reference compounds

[0089] The test compound WS-635 was provided by the sponsor as a dry powder and prepared as a 60 mM stock solution in 100% DMSO. The reference compound remdesivir was provided by WuXi AppTec. The compounds were tested in duplicate at 8 concentrations of 3-fold dilutions at 50% effective concentration (EC 50 ) and 50% cytotoxic concentration (CC 50 ). The highest concentrations tested are listed in Table 2. The final concentration of DMSO in the cell culture was 0.5%.

[0090] Table 2. Information of compounds

[0091]

[0092] 3.2. Cell lines and virus strains

[0093] HCoV 229E (ATCC VR-740) and MRC-5 cells (ATCC CCL-171) were obtained from ATCC. MRC-5 cells were maintained in minimal essential medium (Sigma) supplemented with 10% FBS (Hyclone), 1% L-glutamine (Gibco), 1% NEAA (Gibco) and 1% penicillin-streptomycin (Hyclone). Minimal essential medium supplemented with 5% FBS, 1% L-glutamine, 1% NEAA and 1% penicillin-streptomycin was used as assay medium.

[0094] 3.3. Reagents and instruments

[0095] The main reagent used in this assay is the luminescent cell viability assay kit CellTiter Glo (Promega). The main instrument used in this assay is the Microplate Reader Synergy2 (BioTek).

[0096] 3.4. Methods

[0097] The antiviral assays are summarized in Table 3.

[0098] Table 3. Antiviral assay methods

[0099] Virus cell Processing time (days) / end point Reference compounds Detection Reagents HCoV 229E MRC-5 3 / CPE Remdesivir CellTiter Glo

[0100] 3.4.1. HCoV 229E CPE determination

[0101] MRC-5 cells were seeded at 20,000 cells per well in a 96-well plate and incubated overnight at 37°C and 5% CO2. The next day, a plate containing serially diluted compounds and virus (200 TCID 50 ) in medium. The resulting culture was maintained at 35°C and 5% CO2 for another 3 days until viral infection in the virus control (cells infected with virus without compound treatment) showed significant CPE. Cell viability was measured using CellTiter Glo according to the manufacturer's manual. Luminescent signals were measured by Microplate Reader Synergy2 (Molecular Device). The antiviral activity of each compound was calculated based on the inhibition of CPE at each concentration normalized by the virus control.

[0102] Cytotoxicity assay

[0103] The cytotoxicity of the compounds was assessed simultaneously under the same conditions but without virus infection. Cell viability was measured using CellTiter Glo according to the manufacturer's manual. CC was then calculated based on the cytotoxicity at the test concentration normalized by the medium control (medium only). 50 value.

[0104] Data analysis

[0105] The antiviral activity and cytotoxicity of the compounds were expressed as % inhibition and % cell viability, respectively, and were calculated using the following formula:

[0106] Inhibition (%) = (original data CPD -average VC ) / (average CC -average VC )x 100

[0107] Cell viability (%) = (original data CPD -average MC ) / (average CC -average MC )x 100

[0108] The original data CPD Represents the values ​​of compound-treated wells; Average VC ,average CC and average MC The mean values ​​of virus control, cell control (cells not infected with virus or treated with compound) and medium control are shown respectively.

[0109] EC was calculated using GraphPad Prism software (version 5) using the equation log(inhibitor) vs. response with variable slope. 50 and CC 50 Then calculate SI(CC 50 / EC 50 ).

[0110] Results and discussion

[0111] The antiviral experiment was quality controlled using reference compounds. Remdesivir showed the expected antiviral activity and effect on cell viability, indicating the reliability of the experiment. Table 3 summarizes the antiviral and cytotoxicity results of the test compounds.

[0112] WS-635 showed inhibitory activity against HCoV 229E, EC 50 The value is 5.16 μM. It has obvious cytotoxicity to MRC-5 cells, CC 50 and SI values ​​were 46.79 μM and 9.07, respectively.

[0113] Inhibition and cytotoxicity curves are shown in Figure 3 As shown in Table 4. The figure shows that WS-635 has obvious cytotoxicity to MRC-5 cells infected with HCoV 229E and works by preventing the spread of the virus.

[0114] Table 4. EC of WS-635 50 and CC 50 value

[0115]

[0116] Throughout the specification, references to "embodiment", "some embodiments", "one embodiment", "another example", "example", "specific example" or "some examples" refer to the inclusion of a particular feature, structure, material or characteristic described in conjunction with the embodiment or example in at least one embodiment or example of the present disclosure. Thus, the appearance of words such as "in some embodiments", "in one embodiment", "in an embodiment", "in another example", "in an example", "in a specific example" or "in some examples" throughout the specification does not necessarily refer to the same embodiment or example of the present disclosure. Furthermore, particular features, structures, materials or characteristics may be combined in any suitable manner in one or more embodiments or examples.

[0117] Although exemplary embodiments have been shown and described, those skilled in the art will appreciate that the above embodiments should not be construed as limiting the present disclosure and that changes, substitutions and modifications may be made to the embodiments without departing from the spirit, principle and scope of the present disclosure.

Claims

1. Use of a compound of formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating or preventing coronavirus infection, (I)， And the coronavirus family is at least one selected from 2019-nCov virus and HCov 229E virus.

2. The use according to claim 1, wherein the pharmaceutically acceptable salt is at least one selected from the group consisting of phosphate, hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, ethanesulfonate, benzenesulfonate, acetate, tartrate, maleate, succinate, citrate, benzoate, salicylate, ascorbate, adipate, alginate, arginine, aspartate, bisulfite, bromide, butyrate, camphorate, camphorsulfonate, caprylate, chloride, chlorobenzoate, cyclopentane, benzoic acid ... Pentanepropionate, digluconate, dinitrobenzoate, fumarate, galactonate, galacturonate, glucoheptonate, gluconate, glutamate, hemisuccinate, heptanoate, hexanoate, hippurate, 2-hydroxyethanesulfonate, iodide, isethionate, lactate, lactobionate, malate, malonate, mandelate, methanesulfonate, methylbenzoate, 2-naphthalenesulfonate, nicotinate, oxalate, oleate, pamoate, pectinate, 3-phenylpropionate, phosphonate, and phthalate.

3. The use according to claim 1, wherein the pharmaceutically acceptable salt is at least one selected from the group consisting of toluenesulfonate, trifluoroacetate, hydrogen sulfate, dihydrogen phosphate, dodecyl sulfate, glycerophosphate, hemisulfate, isobutyrate, metaphosphate, monohydrogen phosphate, persulfate and phenylacetate.

4. The use according to claim 1, wherein the drug is in the form of a sol, aromatic water, aerosol, partial inhalation, powder, granule, tablet, ointment, paste, emulsion, suspension, gas dispersion, solid dispersion, microgranule, or pill.

5. The use according to claim 1, wherein the medicament comprises a dose of less than 1000 mg of the compound of formula I or a pharmaceutically acceptable salt thereof.

6. The use according to claim 1, wherein the medicament further comprises one or more pharmaceutically acceptable carriers, diluents or excipients.

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