Treatment or prevention of coronavirus infection

Abstract

A method for preventing or treating coronavirus infection. The method comprises: administering to a subject a therapeutically effective amount of a compound represented by formula (I) or a pharmaceutically acceptable salt, ester or prodrug thereof, wherein R1 is OH or H, and the coronavirus comprises at least one selected from 2019-nCov virus, HCov 229E virus, SARS virus, and MERS virus.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular, to the treatment or prevention of coronavirus infection. 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 April 27, 2020, there are nearly 3 million confirmed cases and more than 200,000 deaths around the world.

[0003] The Coronaviridae family (also known as “coronaviruses”) is an enveloped, positive-sense, single-stranded RNA virus. Coronaviruses can cause disease in mammals and birds. In humans, these viruses cause infections of the respiratory system, including the common cold, which is usually mild, but infections with rarer viruses like SARS (including the cold that causes COVID-19) and MERS can be fatal.

[0004] Therefore, it is urgent to research and develop a more effective method for treating or preventing coronavirus infection. Summary of the invention

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

[0006] The present invention is accomplished based on the following findings of the inventors:

[0007] Hydroxychloroquine (HCQ), also known as Plaquenil, is a drug used to prevent and treat malaria. Studies have shown that it can also be used as a treatment for COVID-19. Hydroxychloroquine (HCQ) has two enantiomers:

[0008] R-(-) Hydroxychloroquine;

[0009] S-(+) Hydroxychloroquine.

[0010] The inventors surprisingly found through experiments that S-(+)-hydroxychloroquine showed higher efficiency and activity in treating and / or preventing coronavirus infection.

[0011] In one aspect of the present invention, the present invention provides a method for treating or preventing coronavirus infection in a subject, comprising administering to the subject a therapeutically effective amount of a compound represented by formula (I) or a pharmaceutically acceptable salt, ester or prodrug thereof;

[0012]

[0013] Wherein, R1 is OH or H;

[0014] The coronavirus includes at least one selected from 2019-nCov virus, HCov 229E virus, SARS virus, and MERS virus.

[0015] In another aspect of the present invention, the present invention provides a pharmaceutical composition for treating or preventing coronavirus infection, the pharmaceutical composition comprising a compound represented by formula (I) or a pharmaceutically acceptable salt, ester or prodrug thereof;

[0016]

[0017] Wherein, R1 is OH or H;

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

[0019] In another aspect of the present invention, the present invention provides a compound represented by formula (I) or a pharmaceutically acceptable salt, ester or prodrug thereof for treating or preventing coronavirus infection in a subject;

[0020]

[0021] Wherein, R1 is OH or H;

[0022] The coronavirus includes at least one selected from 2019-nCov virus, HCov 229E virus, SARS virus, and MERS virus.

[0023] In another aspect of the present invention, the present invention provides a use of a compound represented by formula (I) or a pharmaceutically acceptable salt, ester or prodrug thereof in the preparation of a drug for treating or preventing coronavirus infection;

[0024]

[0025] Wherein, R1 is OH or H;

[0026] The coronavirus includes at least one selected from 2019-nCov virus, HCov 229E virus, SARS virus, and MERS virus.

[0027] In another aspect of the present invention, the present invention provides an air treatment agent, which comprises a compound represented by formula (I) or a pharmaceutically acceptable salt, ester or prodrug thereof;

[0028]

[0029] Wherein, R1 is OH or H.

[0030] In another aspect of the present invention, the present invention provides a device for treating air, comprising: a container containing the above-mentioned air treatment agent. Thus, viruses (especially coronaviruses) can be removed or killed by the device.

[0031] The above is merely a summary of certain aspects disclosed herein and is not limiting in nature. These and other aspects of the present invention and additional embodiments, features and advantages will be partially given in the following detailed description or learned through the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0033] Figure 1 The results of the activity of R-(-)-hydroxychloroquine and S-(+)-hydroxychloroquine against the new coronavirus;

[0034] Figure 2 R-(-)-hydroxychloroquine and S-(+)-hydroxychloroquine have an inhibitory effect on the activity of the new coronavirus;

[0035] Figure 3 The results of the anti-HcoV 229E activity of R-(-)-hydroxychloroquine and S-(+)-hydroxychloroquine are shown. DETAILED DESCRIPTION

[0036] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0037] definition

[0038] Unless otherwise specified, the following terms and phrases used in this invention shall have the following meanings:

[0039] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0040] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0041] As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0042] As used herein, the term "ester" refers to any ester of a compound, wherein any molecule with a -COOH functional group is generally referred to as a -C(O)OR having a substituent, or any molecule with an -OH functional group is generally referred to as an -OC(O)R functional group, wherein R can form a stable ester portion, which ester portion is any carbon-containing group, including but not limited to alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl, heterocycloalkyl and their substituted derivatives.

[0043] As used herein, the terms "pharmaceutically acceptable" and "pharmaceutically acceptable" refer to materials that are suitable for use in pharmaceutical formulations, are generally recognized as safe, are authorized for use by national regulatory agencies or state officials, or are listed in the pharmacopoeia or other recognized pharmacopeia for use in animals, and particularly in humans.

[0044] As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable diluent, adjuvant, excipient or carrier or other ingredient that can be used in conjunction with the compounds of the present invention.

[0045] As used herein, the term "pharmaceutically acceptable salt" refers to a desired pharmaceutically active salt, which may be enhanced. Pharmaceutically acceptable salts mixed with inorganic or organic acids include acid addition salts, metal salts and amine salts. Acid addition salts can be formed with inorganic and organic acids.

[0046] As used herein, the term "prodrug" refers to a biological system that, when administered, can produce any compound that is an active ingredient as a result of a spontaneous chemical reaction, an enzyme-catalyzed chemical reaction, or a photolysis and / or chemical process. Thus, a prodrug is a covalently modified analog or latent form of a therapeutically active compound. Non-limiting examples include esters, quaternary ammonium salt molecules, and ethylene glycol molecules, including prodrugs.

[0047] As used herein, the term "therapeutically effective amount" refers to an amount of a compound administered to an animal for treating a disease, which is sufficient to produce an effect for treating the disease.

[0048] A therapeutically effective amount of a compound for "treating" refers to or includes any of the following:

[0049] (1) To prevent the disease in animals that may be susceptible to it but do not yet experience or display the pathology or symptoms of the disease;

[0050] (2) inhibiting an animal that is experiencing or showing pathology or symptoms of the disease (i.e., preventing further development of the pathology and / or symptoms);

[0051] (3) Ameliorating pathology or symptoms of an animal experiencing or exhibiting a disease (i.e., reversing pathology and / or symptoms).

[0052] Compound

[0053] The present invention relates to a compound represented by formula (I) or a pharmaceutically acceptable salt, ester or prodrug thereof.

[0054]

[0055] Wherein, R1 is OH or H.

[0056] In particular, the present invention relates to a compound S-(+)-hydroxychloroquine represented by formula (III);

[0057]

[0058] Or the compound S-(+)-hydroxychloroquine represented by formula (IV).

[0059]

[0060] In some preferred embodiments of the present invention, the present invention proposes a compound S-(+)-hydroxychloroquine of formula (III). The inventors were surprised to find that S-(+)-hydroxychloroquine showed high efficiency and activity in treating or preventing coronavirus infection. The inventors proved through experiments that the biological activity of S-(+)-hydroxychloroquine is 3 times higher than that of R-(-)-hydroxychloroquine.

[0061] Certain salts, esters or prodrugs thereof can be used in treating or preventing subjects infected with coronavirus. In some examples, coronavirus includes at least one of 2019-nCov virus, HCov 229E virus, SARS virus, and MERS virus.

[0062] In some embodiments of the present invention, the compound represented by formula (I), (III) or (IV) can be used in the form of a pharmaceutically acceptable salt, and the pharmaceutically acceptable salt includes at least phosphate, hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, ethanesulfonate, toluenesulfonate, benzenesulfonate, acetate, trifluoroacetate, tartrate, maleate, succinate, citrate, benzoate, salicylate, ascorbate, adipate, alginate, arginine, aspartate, bisulfate, bisulfite, bromide, butyrate, camphorate, camphorsulfonate, octanoate, chloride, chlorobenzoate, cyclopentanepropionate, digluconate, dihydrogen phosphate, dinitrobenzoate, e), at least one of dodecylsulfate, fumarate, galacterate, galacturonate, glucoheptonate, gluconate, glutamate, glycerophosphate, hemisuccinate, hemisulfate, heptanoate, hexanoate, hippurate, 2-hydroxyethylsulfonate, hydrogen iodide, isethionate, isobutyrate, lactate, lactobionate, malate, malonate, mandelate, metaphosphate, methanesulfonate, methylbenzoate, hydrogen phosphate, 2-naphthalenesulfonate, nicotinate, oxalate, oleate, pamoate, pectinate, persulfate, phenylacetate, 3-phenylpropionate, phosphonate and phthalate.

[0063] Drug formulation and use

[0064] The compounds disclosed in the present invention can be formulated with conventional carriers and excipients, which meet the selected common practices. Tablets may contain excipients, adhesives, fillers, binders, etc. When used for oral administration, isotonic solutions are usually used, and aqueous dosage forms will be prepared in a sterile form.

[0065] The active ingredients can be administered alone, but are preferably formulated into a pharmaceutical formulation. The formulations of the present invention, whether for human or veterinary use, each formulation has at least one active ingredient, and one or more acceptable carriers and optionally other therapeutic ingredients.

[0066] The oral formulations of the present invention may be presented as discrete units, each unit comprising a predetermined amount of active ingredient, such as capsules, cachets or tablets, powders or granules, in aqueous or non-aqueous liquid solutions or suspensions, or water-in-oil emulsions or water-in-oil emulsions. The active ingredient may also be in the form of pills, cold drops or pastes.

[0067] The effective amount of the active ingredient is determined by the clinician using routine dose escalation studies taking into account at least the nature of the disease being treated, toxicity, whether the compound is used for prophylaxis (low doses) or for active viral infection, the drug formulation and method of administration.

[0068] In some preferred embodiments of the present invention, the present invention proposes a compound S-(+)-hydroxychloroquine shown in formula (III). The inventors were surprised to find that S-(+)-hydroxychloroquine showed high efficiency and activity in treating or preventing coronavirus infection. The inventors have proved through experiments that the biological activity of S-(+)-hydroxychloroquine is 3 times higher than that of R-(-)-hydroxychloroquine. The dosage of the compound shown in formula (I), (III) or (IV) is lower than the traditional dosage of chloroquine phosphate.

[0069] The compound or pharmaceutically acceptable salt, ester or prodrug thereof shown in formula (I) can be administered in the form of sol, fragrance, aerosol, partial inhalation, powder, granule, tablet, ointment, paste, emulsion, suspension, gas dispersion, solid dispersion, microparticle or pill. In some embodiments, the compound or pharmaceutically acceptable salt, ester or prodrug thereof shown in formula (I), (III) or (IV) can be administered in a daily dose (daily dosage) of less than 400 mg, for example 10 mg to 300 mg, and the compound shown in formula (I) is 10 to 150 mg.

[0070] In addition, it will be appreciated by those skilled in the art that the drugs of the present invention may also be used for combined drug therapy. The compound or pharmaceutically acceptable salt, ester or prodrug thereof represented by formula (I), (III) or (IV) may be combined with one or more active agents. Active agents suitable for combined use (or combined use) include, but are not limited to, corticosteroids, anti-inflammatory signal transduction regulators, β2 adrenergic receptor agonists bronchodilators, anticholinergics, mucolytics, hypertonic saline, agents for reducing cytokine storms, and other drugs for treating covid-19 viral infection, or mixtures thereof. In some preferred embodiments of the present invention, active agents suitable for combined use (or use in combination) include but are not limited to α-interferon, lopinavir, ritonavir, ribavirin, arbidol, remdesivir, oseltamivir, levofloxacin, moxifloxacin, azithromycin, linezolid, methylprednisolone, prednisone, hydrocortisone, drugs containing paracetamol, compound licorice, compound codeine, ambroxol, aminophylline, dihydroxypropyl theophylline, intravenous human immunoglobulin, vitamin C, Lianhua Qingwen, Qingkailing, Shuanghuanglian and Zhengchaihuyin.

[0071] In some embodiments, the present invention provides a method for treating or preventing COVID-19 virus infection, comprising: administering to a subject a therapeutically effective amount of a compound represented by formula (I) or a pharmaceutically acceptable salt, ester, hydrate or a prodrug thereof;

[0072]

[0073] Wherein, R1 is OH or H; the coronavirus includes at least one selected from 2019-nCov virus, HCov 229E virus, SARS virus, and MERS virus.

[0074] In some specific embodiments, the method of the present invention may further include administering a therapeutically effective amount of at least one other therapeutic agent or a combination thereof, including but not limited to corticosteroids, anti-inflammatory signaling regulators, β2 adrenergic receptor agonists bronchodilators, anticholinergics, mucolytics, hypertonic saline, agents that reduce cytokine storms, and other drugs for treating covid-19 viral infection, or a mixture thereof.

[0075] In some specific embodiments, the method of the present invention may further include administering a therapeutically effective amount of at least one of the following other therapeutic agents or a combination thereof, including but not limited to α-interferon, lopinavir, ritonavir, ribavirin, arbidol, remdesivir, oseltamivir, levofloxacin, moxifloxacin, azithromycin, linezolid, methylprednisolone, prednisone, hydrocortisone, drugs containing paracetamol, compound licorice, compound codeine, ambroxol, aminophylline, dihydroxypropyl theophylline, intravenous human immunoglobulin, vitamin C, Lianhua Qingwen, Qingkailing, Shuanghuanglian and Zhengchaihuyin.

[0076] In some specific embodiments, the compound represented by formula (I) or a pharmaceutically acceptable salt, ester, hydrate or a prodrug thereof can be administered in the form of a sol, a fragrance, an aerosol, a partial inhalant, a powder, a granule, a tablet, an ointment, a paste, an emulsion, a suspension, a gas dispersion, a solid dispersion, a microparticle or a pill.

[0077] In some specific embodiments, the daily dose of the compound of formula (I) or a pharmaceutically acceptable salt, ester, hydrate or prodrug thereof contains less than 400 mg of the compound of formula (I).

[0078] In some specific embodiments, the daily dose of the compound of formula (I) or the pharmaceutically acceptable salt, ester, hydrate or prodrug thereof contains 10 to 300 mg of the compound of formula (I).

[0079] In some specific embodiments, the daily dose of the compound of formula (I) or a pharmaceutically acceptable salt, ester, hydrate or prodrug thereof contains 10 to 150 mg of the compound of formula (I).

[0080] In other embodiments, the present invention provides a pharmaceutical composition for treating or preventing covid-19 virus infection, the pharmaceutical composition comprising a compound represented by formula (I) or a pharmaceutically acceptable salt, ester, hydrate or a prodrug thereof;

[0081]

[0082] Wherein, R1 is OH or H; the coronavirus includes at least one selected from 2019-nCov virus, HCov 229E virus, SARS virus, and MERS virus.

[0083] In some specific embodiments, the pharmaceutical composition of the present invention may further include, but is not limited to, corticosteroids, anti-inflammatory signal transduction regulators, β2 adrenergic receptor agonists bronchodilators, anticholinergics, mucolytics, hypertonic saline, agents for reducing cytokine storms, and other drugs for treating covid-19 viral infection, or mixtures thereof.

[0084] In some specific embodiments, the pharmaceutical composition of the present invention may further include, but is not limited to, α-interferon, lopinavir, ritonavir, ribavirin, arbidol, remdesivir, oseltamivir, levofloxacin, moxifloxacin, azithromycin, linezolid, methylprednisolone, prednisone, hydrocortisone, drugs containing paracetamol, compound licorice, compound codeine, ambroxol, aminophylline, dihydroxypropyl theophylline, intravenous human immunoglobulin, vitamin C, Lianhua Qingwen, Qingkailing, Shuanghuanglian and Zhengchaihuyin.

[0085] In some specific embodiments, the pharmaceutically acceptable salt comprises at least one selected from the following components: phosphate, hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, ethanesulfonate, toluenesulfonate, benzenesulfonate, acetate, trifluoroacetate, tartrate, maleate, succinate, citrate, benzoate, salicylate, ascorbate, adipate, alginate, arginine, aspartate, bisulfate, bisulfite, bromide, butyrate, camphorate, camphorsulfonate, caprylate, chloride, chlorobenzoate, cypionate, digluconate, dihydrogen phosphate, dinitrate , 2-hydroxyethylsulfonate, hydrogen iodide, isethionate, isobutyrate, lactate, lactobionate, malate, malonate, mandelate, metaphosphate, methanesulfonate, methylbenzoate, hydrogen phosphate, 2-naphthalenesulfonate, nicotinate, oxalate, oleate, pamoate, pectinate, persulfate, phenylacetate, 3-phenylpropionate, phosphonate, and phthalate.

[0086] In some specific embodiments, the compound represented by formula (I) or a pharmaceutically acceptable salt, ester or a prodrug thereof can be administered in the form of a sol, a fragrance, an aerosol, a partial inhalant, a powder, a granule, a tablet, an ointment, a paste, an emulsion, a suspension, a gas dispersion, a solid dispersion, microparticles or a pill.

[0087] In some specific embodiments, the dose of the compound represented by formula (I), (III) or (IV) or a pharmaceutically acceptable salt, ester or a prodrug thereof is less than 200 mg, less than 100 mg, less than 50 mg, less than 40 mg, less than 30 mg, less than 20 mg or less than 10 mg.

[0088] In other embodiments, the present invention provides a compound represented by formula (I) or a pharmaceutically acceptable salt, ester, hydrate or prodrug thereof for treating or preventing covid-19 virus infection;

[0089]

[0090] Wherein, R1 is OH or H; the coronavirus includes at least one selected from 2019-nCov virus, HCov 229E virus, SARS virus, and MERS virus.

[0091] In some specific embodiments, R1 is OH.

[0092] In some specific embodiments, the pharmaceutically acceptable salt comprises at least one selected from the following components: phosphate, hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, ethanesulfonate, toluenesulfonate, benzenesulfonate, acetate, trifluoroacetate, tartrate, maleate, succinate, citrate, benzoate, salicylate, ascorbate, adipate, alginate, arginine, aspartate, bisulfate, bisulfite, bromide, butyrate, camphorate, camphorsulfonate, caprylate, chloride, chlorobenzoate, cypionate, digluconate, dihydrogen phosphate, dinitrate , 2-hydroxyethylsulfonate, hydrogen iodide, isethionate, isobutyrate, lactate, lactobionate, malate, malonate, mandelate, metaphosphate, methanesulfonate, methylbenzoate, hydrogen phosphate, 2-naphthalenesulfonate, nicotinate, oxalate, oleate, pamoate, pectinate, persulfate, phenylacetate, 3-phenylpropionate, phosphonate, and phthalate.

[0093] In some specific embodiments, the compound represented by formula (I) or a pharmaceutically acceptable salt, ester or a prodrug thereof can be administered in the form of a sol, a fragrance, an aerosol, a partial inhalant, a powder, a granule, a tablet, an ointment, a paste, an emulsion, a suspension, a gas dispersion, a solid dispersion, microparticles or a pill.

[0094] In some specific embodiments, the daily dose of the compound of formula (I) or a pharmaceutically acceptable salt, ester, hydrate or prodrug thereof contains less than 400 mg of the compound of formula (I).

[0095] In some specific embodiments, the daily dose of the compound of formula (I) or the pharmaceutically acceptable salt, ester, hydrate or prodrug thereof contains 10 to 300 mg of the compound of formula (I).

[0096] In some specific embodiments, the daily dose of the compound of formula (I) or a pharmaceutically acceptable salt, ester, hydrate or prodrug thereof contains 10 to 150 mg of the compound of formula (I).

[0097] Air Treatment

[0098] Studies have shown that compounds represented by formula (I), (III) or (IV) can block the attachment or entry of viruses (such as coronaviruses, etc.), such as at least one of 2019-nCov virus, HCov 229E virus, SARS virus and MERS virus. Then, compounds represented by formula (I), (III) or (IV) can be used to treat air to protect people from viral infection.

[0099] In other embodiments, the present invention provides an air treatment agent, which includes a compound represented by formula (I) or a pharmaceutically acceptable salt, ester, hydrate or prodrug thereof;

[0100]

[0101] Wherein, R1 is OH or H.

[0102] In some specific embodiments, R1 is OH.

[0103] In some specific embodiments, the pharmaceutically acceptable salt includes at least one of the following components: phosphate, hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, ethanesulfonate, toluenesulfonate, benzenesulfonate, acetate, trifluoroacetate, tartrate, maleate, succinate, citrate, benzoate, salicylate, ascorbate, adipate, alginate, arginine, aspartate, bisulfate, bisulfite, bromide, butyrate, camphorate, camphorsulfonate, caprylate, chloride, chlorobenzoate, cypionate, digluconate, dihydrogen phosphate, dinitropropionate. Benzoate, dodecyl sulfate, fumarate, galactonate, galacturonate, gluconate, gluconate, glutamate, glycerophosphate, hemisuccinate, hemisulfate, heptanoate, hexanoate, hippurate, 2-hydroxyethyl sulfonate, hydrogen iodide, isethionate, isobutyrate, lactate, lactobionate, malate, malonate, mandelate, metaphosphate, methanesulfonate, methylbenzoate, hydrogen phosphate, 2-naphthalenesulfonate, nicotinate, oxalate, oleate, pamoate, pectinate, persulfate, phenylacetate, 3-phenylpropionate, phosphonate and phthalate.

[0104] In other embodiments, the present invention provides a device for treating air, the device comprising: a container containing the above-mentioned air treating agent. The device can be used in any form, such as a sprayer.

[0105] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0106] Example

[0107] Example 1: Preparation of the compound represented by formula (III) or (IV)

[0108]

[0109] 1. Preparation of (S)-(+)-2-(4-aminopentyl(ethyl)amino)ethanol:

[0110] Add a solution of 2-(4-aminopentyl(ethyl)amino)ethanol (100g, 0.57mol) in 2-propanol (200ml) to a solution of (+)-mandelic acid (42.6g, 0.28mol) in 2-propanol (200ml). Stir the solution overnight at room temperature. Filter to obtain a white solid. Suspend the solid in 35% sodium hydroxide aqueous solution (350ml) and extract with dichloromethane (3×100ml). Mix the extracts, dry (Na 2 SO 4 ), concentrated to obtain colorless oily (S)-(+)-2-(4-aminopentyl(ethyl)amino)ethanol (40.0 g, 40.0%).

[0111] Preparation of S-(+) Hydroxychloroquine:

[0112] A mixture of (S)-(+)-2-(4-aminopentyl(ethyl)amino)ethanol (38.0 g, 0.22 mol) and 4,7-dichloroquinoline (39.6 g, 0.2 mol) was heated at 125°C in a nitrogen atmosphere for 20 hours. After cooling, the mixture was transferred to a separatory funnel with 1M sodium hydroxide aqueous solution (300 ml) and dichloromethane (200 ml). The organic phase was separated and the aqueous phase was re-extracted with dichloromethane (2×100 ml). The organic phases were collected, mixed, and dried (using MgSO 4 ), concentrated to obtain crude S-(+)-hydroxychloroquine (60.5 g, 90.1%), then crystallized in 2-propanol (60 ml) and isopropyl acetate (80 ml), heated to 65°C to dissolve, slowly cooled to 10°C, filtered, the filter cake obtained was washed with ethyl acetate, and dried under reduced pressure at 70°C to obtain S-(+)-hydroxychloroquine (57.9, 86.2%).

[0113] Preparation of S-(+)-Hydroxychloroquine Sulfate:

[0114] 60% sulfuric acid solution (24g) was added dropwise to an anhydrous ethanol (250ml) solution containing S-(+)-hydroxychloroquine (50g, 0.15mol). The resulting solution was heated, stored and crystallized at 40-50°C for 3 hours, cooled at a temperature below 10°C, filtered and dried at 50°C to obtain white crystalline S-(+)-hydroxychloroquine sulfate (55.3g, yield 85%, purity 99.4%, chiral purity 98.7%).

[0115] 2. Preparation of (R)-(-)-2-(4-aminopentyl(ethyl)amino)ethanol:

[0116] A solution of 2-(4-aminopentyl(ethyl)amino)ethanol (100g, 0.57mol) in 2-propanol (200ml) was added to a solution of (-)-mandelic acid (42.6g, 0.28mol) in 2-propanol (200ml). The solution was stirred overnight at room temperature. A white solid was obtained by filtration. The solid was suspended in a 35% aqueous sodium hydroxide solution (350ml) and extracted with dichloromethane (3×100ml). The extract was mixed and dried (Na 2 SO 4 ), and concentrated to give (R)-(-)-2-(4-aminopentyl(ethyl)amino)ethanol (42.0 g, 42.0%) as a colorless oil.

[0117] Preparation of R-(-) Hydroxychloroquine:

[0118] A mixture of (R)-(-)-(4-aminopentyl(ethyl)amino)ethanol (40.0 g, 0.23 mol) and 4,7-dichloroquinoline (39.6 g, 0.2 mol) was heated at 125°C in a nitrogen atmosphere for 20 hours. After cooling, the mixture was transferred to a separatory funnel with 1M sodium hydroxide aqueous solution (300 ml) and dichloromethane (200 ml). The organic phase was separated and the aqueous phase was re-extracted with dichloromethane (2×100 ml). The organic phases were collected, mixed, and dried (MgSO 4 ), concentrated to obtain crude R-(-)-hydroxychloroquine (61.8 g, 92.0%), then crystallized in 2-propanol (60 ml) and isopropyl acetate (80 ml), heated to 65°C to dissolve, slowly cooled to 10°C, filtered, the filter cake obtained was washed with ethyl acetate, and dried under reduced pressure at 70°C to obtain R-(-)-hydroxychloroquine (57.4%, 85.4%).

[0119] Preparation of R-(-)-Hydroxychloroquine Sulfate:

[0120] 60% sulfuric acid solution (24g) was added dropwise to anhydrous ethanol (250ml) solution containing R-(-)-hydroxychloroquine (50g, 0.15mol). The resulting solution was heated, stored and crystallized at 40-50°C for 3 hours, cooled at a temperature below 10°C, filtered and dried at 50°C to obtain white crystalline R-(-)-hydroxychloroquine sulfate (56.7g, yield 87.1%, purity 99.4%, chiral purity 100%).

[0121] Example 2: Activity detection against the new coronavirus

[0122] The EC values ​​of R-(-)-hydroxychloroquine and S-(+)-hydroxychloroquine prepared in Example 1 were respectively detected. 50 Briefly, the 2019-nCoV virus was diluted to the corresponding concentration with 2% cell maintenance medium, and the virus was added to a 96-well plate so that each well contained 100 TCID50 of virus. After incubation at 37°C for 2 hours, the virus solution was removed, and the compound (R-(-)-hydroxychloroquine or S-(+)-hydroxychloroquine) diluted with 2% cell maintenance medium was tested, with 4 replicates for each concentration. Then, the culture medium was incubated at 37°C, 5% CO 2 The cells were cultured in an incubator for 48 hours. Only 2% of the cell maintenance solution was added to the cell control group, and no test compound was added. CPE (cytopathic effect) was observed every day. After 48 hours of culture, the viral RNA load in each well was detected, and the EC values ​​of R-(-)-hydroxychloroquine and S-(+)-hydroxychloroquine were calculated. 50 , the results are shown in Figure 1 and Table 1, where Figure 1 The vertical axis Infection% is the inhibition rate%.

[0123] Table 1:

[0124] name <![CDATA[EC 50 (μM)]]> R-(-)-Hydroxychloroquine (WS101-p1) 37.51 S-(+)-Hydroxychloroquine (WS202-p2) 8.36

[0125] The above results show that compared with R-(-)-hydroxychloroquine, the activity of S-(+)-hydroxychloroquine in inhibiting the replication of SARS-CoV-19 virus is more than 4 times higher, which indicates that the S-(+)-hydroxychloroquine enantiomer has strong chirality and is an excellent drug for the treatment of COVID-19.

[0126] Example 3: Activity test for preventing new coronavirus

[0127] Vero cells were purchased from ATCC Resource Center (USA). SARS-Cov-2 virus was isolated by the Institute of Military Medical Sciences in Beijing, collection code: 2019-nCoV BetaCoV / Beijing / AMMS01 / 2020.

[0128] Determination of EC of Drugs by Nucleic Acid Quantification 50The specific steps are as follows:

[0129] Vero cells were inoculated in a 96-well plate at a concentration of 10,000 / well one day in advance. The drug prepared in Example 1 was prepared into 400, 200, 100, 50, 25 and 12.5 μM using DMEM maintenance medium containing 2% fetal bovine serum (FBS), and added to the cell culture before infection. The cell culture supernatant was discarded and T705 (100 μL / well) with different drug concentrations was added. There were 4 wells for each drug, and 100 TCID50 virus solution was added to each drop. A positive drug control group, a virus control group (cells infected with the virus, not treated with compound drugs) and a normal cell control group (cells not infected with the virus, and not treated with compound drugs) were established and incubated at 37°C, 5% CO 2 The cells were cultured in an 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 50 For specific test results, see Figure 2 and Table 2, where Figure 2 The ordinate Infection% is the inhibition rate%.

[0130] Table 2:

[0131] name <![CDATA[EC 50 (μM)]]> R-(-)-Hydroxychloroquine (WS101-p1) 4.23 S-(+)-Hydroxychloroquine (WS202-p2) 1.91

[0132] The above results indicate that the antiviral activity of the hydroxychloroquine enantiomer in preventive treatment is increased by more than 4-7 times compared with the treatment after virus infection. Therefore, S-(+)-hydroxychloroquine can be used as a preventive treatment drug before or in the early stages of COVID-19 treatment.

[0133] Example 4: Anti-Hcov 229E Activity Test

[0134] (1) Test compounds and reference compounds

[0135] The test compound prepared in Example 1 was provided as a dry powder, and a stock solution of the test compound with a final concentration of 60 mM was prepared using 100% DMSO solution. The reference compound was provided by WuXi AppTec. The compound was set to 8 concentrations of 3-fold dilution, and then divided into two portions and subjected to 50% effective concentration (EC 50 ) and 50% cytotoxic concentration (CC 50 The highest concentrations tested are listed in Table 3. The final concentration of DMSO in the cell culture was 0.5%.

[0136] Table 3:

[0137]

[0138] (2) Cell lines and virus strains

[0139] HCoV 229E (ATCC VR-740) and MRC-5 cells (ATCC CCL-171) were obtained from ATCC. MRC-5 cells were maintained in the Minimum Essential Medium (MEM; Sigma) containing 10% FBS (Hyclone), 1% l-glutamine (Gibco), 1% NEAA (Gibco), and 1% penicillin-streptomycin (Hyclone). The assay medium was the Minimum Essential Medium supplemented with 5% FBS, 1% l-glutamine, 1% NEAA, and 1% penicillin-streptomycin.

[0140] (3) HCoV 229E CPE detection

[0141] MRC-5 cells were seeded at 20,000 cells per well in a 96-well plate and incubated at 37°C with 5% CO. 2 The next day, diluted compounds and viruses (200 TCID50 per well) were added to the culture medium continuously. The culture was incubated at 35°C and 5% CO 2 The cells were stored for 3 days under the same conditions until the virus control group (cells infected with the virus, not treated with the compound drug) showed significant CPE. Cell viability was detected using CellTiter Glo according to the manufacturer's instructions. The fluorescence signal was measured using Microplate Reader Synergy2 (Molecular Device). The antiviral activity of each compound was calculated based on the CPE inhibition effect at each concentration after normalization of the virus control group.

[0142] (4) Cytotoxicity test

[0143] The cytotoxicity of the compounds was evaluated under the same conditions, but without virus infection. Cell viability was measured using CellTiter Glo according to the manufacturer's instructions. The cytotoxicity at each concentration was then normalized to the medium control group (medium only) and CC was calculated. 50 value.

[0144] (5) Data analysis

[0145] The cytotoxicity and antiviral activity of the compounds were expressed as % inhibition rate and % cell viability, respectively, and calculated according to the following formula:

[0146] Inhibition rate (%) = (original data CPD-average VC) / (average CC-average VC) × 100;

[0147] Cell viability (%) = (raw data CPD-average MC) / (average CC-average MC) × 100;

[0148] Among them, the original data CPD represents the value of the compound-treated wells; the average VC, average CC and average MC represent the average values ​​of the virus control group, cell control group (cells not infected with virus or treated with compounds) and culture medium control group, respectively.

[0149] EC was calculated using GraphPad Prism software (version 5) and the equation log(inhibitor) vs. variable slope response. 50 and CC 50 Then calculate SI(CC 50 / EC 50 ). Results are shown in Figure 3 and Table 4, where Figure 3 The vertical axis Infection% is the inhibition rate% and viability% is the cell viability%.

[0150] Table 4:

[0151]

[0152] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0153] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. Use of the compound represented by formula (I) in the preparation of a medicament for treating or preventing coronavirus infection; The coronavirus is selected from at least one of 2019-nCov virus, HCov 229E virus, SARS virus, and MERS virus.

2. The use according to claim 1, characterized in that The compound represented by formula (I) is present in at least one form of sol, fragrance, aerosol, partial inhalant, powder, granule, tablet, paste, emulsion, suspension, gas dispersant, solid dispersant, microparticle and pill.

3. The use according to claim 1, characterized in that The dosage of the compound represented by formula (I) is less than 200 mg.

4. The use according to claim 1, characterized in that The dosage of the compound represented by formula (I) is less than 100 mg.

5. The use according to claim 1, characterized in that: The dosage of the compound represented by formula (I) is less than 50 mg.

6. The use according to claim 1, characterized in that: The dosage of the compound represented by formula (I) is less than 30 mg.

7. The use according to claim 1, characterized in that The dosage of the compound represented by formula (I) is less than 20 mg.

8. The use according to claim 1, characterized in that The dosage of the compound represented by formula (I) is less than 10 mg.

9. Use of a pharmaceutical composition in the preparation of a medicament for treating or preventing coronavirus infection; the pharmaceutical composition comprises a compound represented by formula (I), and optionally a pharmaceutically acceptable excipient or carrier; The coronavirus is selected from at least one of 2019-nCov virus, HCov 229E virus, SARS virus, and MERS virus.

Images