WO2021217574A1

WO2021217574A1 - Treatment or prevention of coronaviridae infection

Info

Publication number: WO2021217574A1
Application number: PCT/CN2020/088241
Authority: WO
Inventors: Jian Cui; Minglong HU; Ying Liang; Yao Yu; Yuan MENG; Tongtong Li; Along ZHAO; Faming Zhang
Original assignee: Waterstone Pharmaceuticals (Wuhan) Co., Ltd.
Priority date: 2020-04-30
Filing date: 2020-04-30
Publication date: 2021-11-04
Also published as: CN115515935A

Abstract

A method of treating or preventing a Coronaviridae infection in a subject comprising administrating a therapeutically effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt, ester or prodrug thereof to the subject in need thereof, wherein R1 is OH or H, and the Coronaviridae comprises at least one selected from 2019-nCov virus, HCov 229E virus, SARS virus, MERS virus.

Description

Treatment or Prevention of Coronaviridae Infection

FIELD OF THE INVENTION

The present invention relates to the field of medicinal technology, in particular, to the treatment or prevention of Coronaviridae infection in a subject.

BACKGROUND OF THE INVENTION

The World Health Organization has recently declared the severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) a public health emergency of international concern. As of April 27, 2020, there are almost 3,000,000 confirmed cases and more than 200,000 death cases all around the world.

Coronaviridae (also may be known as “Coronaviruses” ) is a family of enveloped, positive-sense, single-stranded RNA viruses. Coronaviruses may cause diseases in mammals and birds. In humans, the viruses cause respiratory infections, including the common cold, which are typically mild, though rarer forms such as SARS (including the one causing COVID-19) and MERS can be lethal.

Therefore there is an increasing demand for the research and development of more effective method for treating or preventing a Coronaviridae infection.

SUMMARY OF THE INVENTION

The following is only an overview of some aspects of the present invention, but is not limited thereto. All references of this specification are incorporated herein by reference in their entirety. When the disclosure of this specification is different with citations, the disclosure of this specification shall prevail.

The present invention is based on the following findings: Hydroxychloroquine (HCQ) , sold under the brand name Plaquenil, is a medication used to prevent and treat malaria, it is also being studied as a treatment for coronavirus disease 2019 (COVID-19) . Hydroxychloroquine (HCQ) has two enantiomers as follows:

Formula II R- (-) -Hydroxychloroquine

Formula III S- (+) -Hydroxychloroquine

It is surprisingly found by the inventors that an S- (+) -Hydroxychloroquine showed significantly higher efficiency and activity for treating or preventing a Coronaviridae infection in several experimental tests.

In one aspect of present disclosure, there is provided a method of treating or preventing a Coronaviridae infection in a subject comprising administrating a therapeutically effective amount of a compound of Formula I or a pharmaceutically acceptable salt, ester or prodrug thereof to the subject in need thereof

wherein R1 is OH or H,

and the Coronaviridae comprises at least one selected from 2019-nCov virus, HCov 229E virus, SARS virus, MERS virus.

In another aspect of present disclosure, there is provided a pharmaceutical composition for treating or preventing Coronaviridae virus infection comprising a compound of Formula I or a pharmaceutically acceptable salt, ester or prodrug thereof to the subject in need thereof

wherein R1 is OH or H,

In another aspect of present disclosure, there is provided compound of Formula I or a pharmaceutically acceptable salt, ester or prodrug thereof, for use in treating or preventing Coronaviridae infection in a subject,

wherein R1 is OH or H,

In another aspect of present disclosure, there is provided a use of a compound of Formula I or a pharmaceutically acceptable salt, ester or prodrug thereof in the manufacture of a medicament for treating or preventing Coronaviridae infection.

wherein R1 is OH or H,

In another aspect of present disclosure, there is provided an agent for treating air comprising a compound of Formula I or a pharmaceutically acceptable salt, ester or prodrug thereof,

wherein R1 is OH or H.

In another aspect of present disclosure, there is provided an apparatus for treating air comprising: a container containing the agent described above. Then the virus especially coronavirus may be removed or killed by this apparatus.

The foregoing merely summarizes certain aspects disclosed herein and is not intended to be limiting in nature. These aspects and other aspects and additional embodiments, features, and advantages of the invention will be apparent from the following detailed description and through practice of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other aspects and advantages of embodiments of the present disclosure will become apparent and more readily appreciated from the following descriptions made with reference the accompanying schemes and drawings, in which:

Fig. 1 shows R- (-) -Hydroxychloroquine and S- (+) -Hydroxychloroquine anti-2019-nCov virus activity results;

Fig. 2 shows R- (-) -Hydroxychloroquine and S- (+) -Hydroxychloroquine preventing 2019-nCov virus activity results; and

Fig. 3 shows R- (-) -Hydroxychloroquine and S- (+) -Hydroxychloroquine anti HcoV 229E activity results.

EMOBODIMENT

Definition

Unless otherwise indicated, otherwise the following terms and phrases as used herein will have the following meanings:

"Esters" refers to any ester of the compound, wherein the molecule of any-COOH functional group is-C (O) OR function with a substituent, or wherein any of the molecule-OH functional group is-OC (O) R functional groups, wherein R may be formed a stable ester moiety of the ester moiety is any carbon-containing groups, including but not limited to alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclyl, heterocycloalkyl and substituted derivatives thereof.

"Pharmaceutically acceptable" refers to a suitable for use in a pharmaceutical formulation, generally regarded as safe, a regulatory agency of the country or a state government official authorized to be used by, or column in the Pharmacopeia or other generally recognized pharmacopeia for use in animals and particularly for use in humans.

"Pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable diluent, adjuvant, excipient or carrier or other component, and is administered in combination with a compound of the present invention.

"Pharmaceutically acceptable salt" refers to the desired pharmacologically active salt thereof can be enhanced. Pharmaceutically acceptable salts thereof with inorganic or organic acids include the acid addition salts, metal salts and amine salts. Acid addition salts may be formed with inorganic acids and organic acids.

"Prodrug" refers to a biological system at the time of administration, as a spontaneous chemical reaction, an enzyme-catalyzed chemical reactions, photolysis and/or metabolic result of chemical processes, can generate any compound of which the active ingredient drug substance. Thus, the prodrug is a therapeutically active compound covalently modified analogs or latent form. A non-limiting example includes a pro-drug ester, quaternary ammonium molecules, glycol molecules and the like.

"a therapeutically effective amount" means the compound was administered to an animal for the treatment of diseases, the treatment of the disease in an amount sufficient to effect.

A therapeutically effective amount of a compound "treating" means and includes any of the application:

(1) prevention may be predisposed to the disease but still does not yet experience or display the pathology or symptomatology of the disease in animals that develop the disease,

(2) inhibiting the disease is experiencing or displaying the pathology or symptomatology of the disease in an animal of the (i.e., arresting further development of the pathology and/or symptomatology) , or

(3) improve the pathology or symptomatology of the disease is experiencing or displaying the disease in an animal (i.e., reversing the pathology and/or symptomatology) .

COMPOUND

The present disclosure relates to a compound of Formula I or a pharmaceutically acceptable salt, ester or prodrug thereof

wherein R1 is OH or H.

Especially, the present disclosure relates to a compound S- (+) -Hydroxychloroquine of Formula III

or a compound S- (+) -Chloroquine of Formula IV.

In some preferred embodiments of present disclosure, the present disclosure relates to a compound S- (+) -Hydroxychloroquine of Formula III. It is surprisingly found by the inventors that an S- (+) -Hydroxychloroquine showed significantly higher efficiency and activity for treating or preventing a Coronaviridae infection in several experimental tests. It was demonstrated in some experimental tests by the inventors that S- (+) -Hydroxychloroquine showed a higher level bio-activity at a fold of 3 than R- (-) -Hydroxychloroquine.

In some salt, ester or prodrug thereof when treating or preventing a Coronaviridae infection in a subject in need thereof. And in some examples, the Coronaviridae may comprise at least one selected from 2019-nCov virus, HCov 229E virus, SARS virus, MERS virus.

In some examples of present disclosure, the compound of Formula I, III or IV may be used in a form a pharmaceutically acceptable salt, and the pharmaceutically acceptable salt is at least one selected from a group comprising phosphate, hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, ethanesulfonate, toluenesulfonate and benzenesulfonate, acetate, trifluoroacetate, tartrate, maleate, succinate, citrate, benzoate, salicylate and ascorbate, adipate, alginate, arginate, aspartate, bisulfate, bisulfite, bromide, butyrate, camphorate, camphorsulfonate, caprylate, chloride, chlorobenzoate, cyclopentanepropionate, digluconate, dihydrogenphosphate, dinitrobenzoate, dodecylsulfate, fumarate, galacterate, galacturonate, glucoheptaoate, gluconate, glutamate, glycerophosphate, hemisuccinate, hemisulfate, heptanoate, hexanoate, hippurate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, iodide, isethionate, iso-butyrate, 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.

Pharmaceutical Formulation and Use

The compound of present disclosure may be formulated with conventional carriers and excipients, carriers and excipients which will meet the selected common practice. Tablets containing excipients, glidants, fillers, binders and the like. An aqueous formulation will be made in the form of a sterile, when it is intended for administration of the administered orally, typically isotonic solution.

While the active ingredient can be administered alone, but may preferably be formulated into pharmaceutical formulation. The formulations of the present invention, whether for human or veterinary use in formulations, each having at least one active ingredient, and one or more acceptable carriers and optionally other therapeutic ingredients.

The present invention suitable for oral administration of the formulations may be presented as discrete units, each containing a predetermined quantity of active ingredient such as capsules, cachets or tablets; a powder or granules; in an aqueous or non-aqueous liquid solutions or suspensions; or oil in water emulsion or a water in oil emulsion. The active ingredients may also be a bolus, electuary or paste form.

An effective amount of an active ingredient at least depending upon the nature of the disease being treated, toxicity, the compound is used for prophylaxis (low dose) or for the active viral infection, pharmaceutical formulation and administration method, and then using a conventional dose escalation studies by the clinician to make a decision.

In some preferred embodiments of present disclosure, the present disclosure relates to a compound S- (+) -Hydroxychloroquine of Formula III. It is surprisingly found by the inventors that an S- (+) -Hydroxychloroquine showed significantly higher efficiency and activity for treating or preventing a Coronaviridae infection in several experimental tests. It was demonstrated in some experimental tests by the inventors that S- (+) -Hydroxychloroquine showed a higher level bio-activity at a fold of 3 than R- (-) -Hydroxychloroquine. Then the compound of Formula I, III or IV may be used at a dose lower than the traditional dose of chloroquine phosphate.

Then the compound of Formula I or the pharmaceutically acceptable salt, ester or prodrug thereof may be administrated to the subject in a form of sol, aromatic water, aerosol, partial inhalant, powder, granule, tablets, ointment, paste, emulsion, suspension, gas dispersion, solid dispersion, microparticle, pill. And in some examples, the compound of Formula I, III or IV or a pharmaceutically acceptable salt, ester or prodrug thereof may be administrated at a daily dose of lower than 400 mg, for example 10 mg to 300 mg, 10～150 mg in term of the compound of Formula I.

Additionally the person skilled in the art may understand that combination therapy may also be used. The compounds of the compound of Formula I, III or IV or a pharmaceutically acceptable salt, ester or prodrug thereof may bein combination with one or more active agents. Suitable active agents for use in combination a non-limiting embodiments include one or more selected from the group comprising a corticosteroid, an anti-inflammatory signal transduction modulator, a β2-adrenoreceptor agonist bronchodilator, an anticholinergic, a mucolytic agent, hypertonic saline, agent for reducing cytokine storm, other drugs for treating a covid-19 virus infection, or mixtures thereof. In some preferred embodiments of present disclosure, the at least one other therapeutic agent or composition thereof is selected from the group comprising Alpha-interferon, Lopinavir, Ritonavir, Ribavirin, Arbidol, Remdesivir, Oseltamivir, Levofloxacin, moxifloxacin, azithromycin, linezolid, methylprednisolone, prednisone, hydrocortisone, drugs containing paracetamol, compound liquorice, compound codeine, ambroxol, aminophylline, dihydroxypropyltheophylline, intravenous injection of human immunoglobulin, vitamin C, Lianhua Qingwen, Qingkailing, Shuanghuanglian, Zhengchaihuyin.

Then in some examples, it is provided a method of treating or preventing covid-19 virus infection in a subject comprising administrating a therapeutically effective amount of a compound of Formula I or a pharmaceutically acceptable salt, ester, hydrate or prodrug thereof to the subject in need thereof

wherein R1 is OH or H.

And in some specific examples, it is provided that the method may further comprise administering a therapeutically effective amount of at least one other therapeutic agent or composition thereof selected from the group comprising a corticosteroid, an anti-inflammatory signal transduction modulator, a β2-adrenoreceptor agonist bronchodilator, an anticholinergic, a mucolytic agent, hypertonic saline, agent for reducing cytokine storm, other drugs for treating a covid-19 virus infection, or mixtures thereof.

And in some specific examples, it is provided that the method may further comprise administering a therapeutically effective amount of at least one other therapeutic agent or composition thereof selected from the group comprising Alpha-interferon, Lopinavir, Ritonavir, Ribavirin, Arbidol, Remdesivir, Oseltamivir, Levofloxacin, moxifloxacin, azithromycin, linezolid, methylprednisolone, prednisone, hydrocortisone, drugs containing paracetamol, compound liquorice, compound codeine, ambroxol, aminophylline, dihydroxypropyltheophylline, intravenous injection of human immunoglobulin, vitamin C, Lianhua Qingwen, Qingkailing, Shuanghuanglian, Zhengchaihuyin.

And in some specific examples, the compound of Formula I or the pharmaceutically acceptable salt, ester, hydrate or prodrug thereof is administrated to the subject in a form of sol, aromatic water, aerosol, partial inhalant, powder, granule, tablets, ointment, paste, emulsion, suspension, gas dispersion, solid dispersion, microparticle, pill.

And in some specific examples, compound of Formula I or a pharmaceutically acceptable salt, ester, hydrate or prodrug thereof is administrated at a daily dose of lower than 400 mg in term of the compound of Formula I.

And in some specific examples, the compound of Formula I or a pharmaceutically acceptable salt, ester, hydrate or prodrug thereof is administrated at a daily dose of 10 mg to 300 mg in term of the compound of Formula I.

And in some specific examples, the compound of Formula I or a pharmaceutically acceptable salt, ester, hydrate or prodrug thereof is administrated at a daily dose of 10 mg to 150 mg in term of the compound of Formula I.

In some other examples, it is provided a pharmaceutical composition to treat or prevent covid-19 virus infection comprising a compound of Formula I or a pharmaceutically acceptable salt, ester, hydrate or prodrug thereof to the subject in need thereof

wherein R1 is OH or H.

And in some specific examples, R1 is OH.

And in some specific examples, the composition further comprises at least one other therapeutic agent or composition thereof selected from the group comprising a corticosteroid, an anti-inflammatory signal transduction modulator, a β2-adrenoreceptor agonist bronchodilator, an anticholinergic, a mucolytic agent, hypertonic saline, agent for reducing cytokine storm, other drugs for treating a covid-19 virus infection, or mixtures thereof.

And in some specific examples, the composition further comprises at least one other therapeutic agent or composition thereof selected from the group comprising Alpha-interferon, Lopinavir, Ritonavir, Ribavirin, Arbidol, Remdesivir, Oseltamivir , levofloxacin, moxifloxacin, azithromycin, linezolid, methylprednisolone, prednisone, hydrocortisone, drugs containing paracetamol, compound liquorice, compound codeine, ambroxol, aminophylline, dihydroxypropyltheophylline, intravenous injection of human immunoglobulin, vitamin C, Lianhua Qingwen, Qingkailing, Shuanghuanglian, Zhengchaihuyin.

And in some specific examples, the pharmaceutically acceptable salt is at least one selected from a group comprising phosphate, hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, ethanesulfonate, toluenesulfonate and benzenesulfonate, acetate, trifluoroacetate, tartrate, maleate, succinate, citrate, benzoate, salicylate and ascorbate, adipate, alginate, arginate, aspartate, bisulfate, bisulfite, bromide, butyrate, camphorate, camphorsulfonate, caprylate, chloride, chlorobenzoate, cyclopentanepropionate, digluconate, dihydrogenphosphate, dinitrobenzoate, dodecylsulfate, fumarate, galacterate, galacturonate, glucoheptaoate, gluconate, glutamate, glycerophosphate, hemisuccinate, hemisulfate, heptanoate, hexanoate, hippurate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, iodide, isethionate, iso-butyrate, 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.

And in some specific examples, the compound of Formula I or the pharmaceutically acceptable salt, ester, hydrate or prodrug thereof is in a form of sol, aromatic water, aerosol, partial inhalant, powder, granule, tablets, ointment, paste, emulsion, suspension, gas dispersion, solid dispersion, microparticle, pill.

And in some specific examples, the composition comprises the compound of Formula I or the pharmaceutically acceptable salt, ester, hydrate or prodrug thereof at a dose of lower than 200 mg, lower than 100 mg, lower than 50 mg, lower than 40 mg, 30 mg, 20 mg or 10 mg.

Then it is provided in the present disclosure compound of Formula I or a pharmaceutically acceptable salt, ester, hydrate or prodrug thereof, for use in treating or preventing covid-19 virus infection in a subject,

wherein R1 is OH or H.

And in some specific examples, R1 is OH.

And in some specific examples, the compound of Formula I or a pharmaceutically acceptable salt, ester, hydrate or prodrug thereof is administrated at a daily dose of lower than 400 mg in term of the compound of Formula I.

And in some specific examples, the compound of Formula I or a pharmaceutically acceptable salt, ester, hydrate or prodrug thereof is administrated at a daily dose of 10mg to 300 mg in term of the compound of Formula I.

And in some specific examples, the compound of Formula I or a pharmaceutically acceptable salt, ester, hydrate or prodrug thereof is administrated at a daily dose of 10mg to 150 mg in term of the compound of Formula I.

Treating Air

It is demonstrated that the compound of Formula I, III or IV may block the attachment or entry of the virus such as Coronaviridae, for example at least one selected from 2019-nCov virus, HCov 229E virus, SARS virus, MERS virus. Then the compound of Formula I, III or IV may be used to treat air to provide an atmosphere protecting the person from virus infection

Then an agent for treating air may be provided and the agent comprises a compound of Formula I or a pharmaceutically acceptable salt, ester, hydrate or prodrug thereof

wherein R1 is OH or H.

And in some specific examples, R1 is OH.

And in some specific examples, the pharmaceutically acceptable salt is at least one selected from a group comprising phosphate, hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, , ethanesulfonate, toluenesulfonate and benzenesulfonate, acetate, trifluoroacetate, tartrate, maleate, succinate, citrate, benzoate, salicylate and ascorbate, adipate, alginate, arginate, aspartate, bisulfate, bisulfite, bromide, butyrate, camphorate, camphorsulfonate, caprylate, chloride, chlorobenzoate, cyclopentanepropionate, digluconate, dihydrogenphosphate, dinitrobenzoate, dodecylsulfate, fumarate, galacterate, galacturonate, glucoheptaoate, gluconate, glutamate, glycerophosphate, hemisuccinate, hemisulfate, heptanoate, hexanoate, hippurate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, iodide, isethionate, iso-butyrate, 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.

And then it is provided an apparatus for treating air comprising: a container containing the agent described above. The apparatus may be used in any form, such as sprayer.

Examples

Example 1. Preparation of compound of Formula III and IV

Preparation of (S) - (+) -2- (4-Aminopentyl (ethyl) amino) ethanol

A solution of 2- (4-Aminopentyl (ethyl) amino) ethanol (100g, 0.57mol) in 2-propanol (200 ml) was added to a solution of (+) -mandelic acid (42.6g, 0.28mol) in 2-propanol (200ml) . The solution was stirred overnight at room temperature. Filtration gave white solid. The solid was suspended in 35%aqueous sodium hydroxide (350 ml) and extracted with dichloromethane (3x 100ml) . The extracts were combined, dried (using Na ₂SO ₄) and concentrated to give (S) - (+) -2-(4-Aminopentyl (ethyl) amino) ethanol (40.0g, 40.0%) as a colourless oil.

Preparation of S- (+) -Hydroxychloroquine

A mixture of (S) - (+) -2- (4-Aminopentyl (ethyl) amino) ethanol (38.0g, 0.22mol) and 4, 7-dichloroquinoline (39.6g, 0.2mol) was heated at 125℃ in a nitrogen atmosphere for 20 hours. After cooling, the mixture was transferred into a separating funnel using 1M aqueous sodium hydroxide (300ml) and dichloromethane (200 ml) . The organic phase was separated and the aqueous phase was re-extracted with dichloromethane (2 x 100 ml) . The organic phases were combined, dried (using MgSO ₄) and concentrated to give a S- (+) -Hydroxychloroquine crude (60.5g, 90.1%) which were crystallized from 2-propanol (60 ml) and Isopropyl acetate (80 ml) , heated at 65℃ to dissolve the solution, slowly cooled to 10℃, filtered, and the resulting filter cake was washed with ethyl acetate, and decompression dried at 70℃ to get S- (+) -hydroxychloroquine (57.9, 86.2%) .

Preparation of S- (+) -Hydroxychloroquine sulfate

A solution of 60%sulfuric acid solution (24g) was added dropwise to a solution of S- (+) -Hydroxychloroquine (50g, 0.15mol) in anhydrous ethanol (250ml) . The resulting solution were heated at 40-50℃, preserved and crystallized for 3 hours, after cooling at a temperature below 10℃, filtered and dried at 50℃, then gave white crystals S- (+) -hydroxychloroquine sulfate (55.3g, yield: 85%, purity: 99.4%; Chiral purity: 98.7%) .

Preparation of (R) - (-) -2- (4-Aminopentyl (ethyl) amino) ethanol

A solution of 2- (4-Aminopentyl (ethyl) amino) ethanol (100g, 0.57mol) in 2-propanol (200 ml) was added to a solution of (-) -mandelic acid (42.6g, 0.28mol) in 2-propanol (200ml) . The solution was stirred overnight at room temperature. Filtration gave white solid. The solid was suspended in 35%aqueous sodium hydroxide (350 ml) and extracted with dichloromethane (3x 100ml) . The extracts were combined and dried (using Na ₂SO ₄) and concentrated to give (R) - (-) -2- (4-Aminopentyl (ethyl) amino) ethanol (42.0g, 42.0%) as a colourless oil.

Preparation of R- (-) -Hydroxychloroquine

A mixture of (R) - (-) -2- (4-Aminopentyl (ethyl) amino) ethanol (40.0g, 0.23mol) , 4, 7-dichloroquinoline (39.6g, 0.2mol) was heated at 125℃ in a nitrogen atmosphere for 20 hours. After cooling, the mixture was transferred into a separating funnel using 1M aqueous sodium hydroxide (300ml) and dichloromethane (200 ml) . The organic phase was separated and the aqueous phase was re-extracted with dichloromethane (2 x 100 ml) . The organic phases were combined, dried (MgSO ₄) and concentrated to give R- (-) -Hydroxychloroquine crude (61.8g, 92.0%) which were crystallized from 2-propanol (60 ml) and isopropyl acetate (80 ml) , heated at 65℃ to dissolve the solution, slowly cooled to 10℃, filtered, and the resulting filter cake was washed with ethyl acetate, and decompression dried at 70℃ to get R- (-) -Hydroxychloroquine (57.4, 85.4%) .

Preparation of R- (-) -Hydroxychloroquine sulfate

A solution of 60%sulfuric acid solution (24g) was added dropwise to a solution of R- (-) - Hydroxychloroquine (50g, 0.15mol) in anhydrous ethanol (250ml) . The mixture was heated at 40-50℃, preserved and crystallized for 3 hours, cooled at a temperature below 10℃, filtered and dried at 50℃, to give white crystals R- (-) -hydroxychloroquine sulfate (56.7g, yield: 87.1%, purity: 99.4%; Chiral purity: 100%) .

Example 2. Activity test of anti-2019-nCov virus

EC ₅₀ of R- (-) -Hydroxychloroquine and S- (+) -Hydroxychloroquine were tested respectively. Briefly, 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 contains 100TCID50 of virus. The plated was incubated at 37 ℃ for 2 hours, the virus solution was removed and testing compound (R- (-) -Hydroxychloroquine or S- (+) -Hydroxychloroquine) was added at a series of concentration diluted with 2%cell maintenance medium, 4 replicates for each concentration. Then continue incubating at 37 ℃ in 5%CO ₂ incubator for 48 hours. The cell control group was only added with 2%cell maintenance medium without any testing compound. Observe the CPE every day. After 48 hours of culture, the viral RNA load of each well was detected, and the EC ₅₀ of R- (-) -Hydroxychloroquine and S- (+) -Hydroxychloroquine were calculated and summarized as shown in Figure 1 and Table 1.

Table1:

Name	EC ₅₀ (μM)
R- (-) -Hydroxychloroquine (WS101-p1)	37.51
S- (+) -Hydroxychloroquine (WS202-p2)	8.36

The results above show that S- (+) -Hydroxychloroquine shows greater than 4 fold activity compare with R- (-) -Hydroxychloroquine in inhibiting SARS-CoV-19 viral replication, which strongly indicates chiral selectivity and the S- (+) -Hydroxychloroquine enantiomer as a superior therapeutics for the treatment of COVID-19.

Example 3. Activity test of preventing 2019-nCov virus

Vero cell was purchased from ATCC (US) , The SARS-Cov-2 virus was isolated by Beijing Military Medical Research Institute with the collection code: 2019-nCoV BetaCoV/Beijing/AMMS01/2020.

EC ₅₀ of the drug was determined by nucleic acid quantitative method. As follows: Vero cells were inoculated into 96 well plates at a concentration of 10000 /well one day in advance. The drug was prepared into 400, 200, 100, 50, 25 and 12.5 μM with DMEM maintenance solution of 2%FBS and added into the cell culture before infection. The cell culture supernatant was discarded, and T705 (100 μL /well) with different drug concentrations was added. Each drug had 4 multiple holes, and then 100 TCID50 virus solution was added to each drop. The positive drug control, virus control and normal cell control groups were set up and cultured in 37 ℃, 5%CO ₂ incubator. On the second day after infection, 50 μL cell supernatant was taken from each pore to extract nucleic acid. The viral load was detected by quantitative RT-PCR and EC ₅₀ was calculated as shown in Figure 2 and Table 2.

Table 2:

Name	EC ₅₀ (μM)
R- (-) -Hydroxychloroquine (WS101-p1)	4.23
S- (+) -Hydroxychloroquine (WS202-p2)	1.91

The results above show that prophylatic treatment of Hydroxychloroquine enantiomers showed an over 4-7 folds more anti-viral activity than treatment after viral infection, so S- (+) -Hydroxychloroquine can be used as a prophylactic therapeutics before or in early stage of the COVID-19 treatment.

Example 4. Activity test of anti HcoV 229E

(1) Test and reference compounds

Test compounds were provided in dry powders and prepared as 60 mM stock solutions in 100%DMSO solution. Reference compoundwas provided by WuXi AppTec. Compounds were tested at 8 concentrations, 3-fold dilutions, in duplicate for 50%effective concentration (EC ₅₀) and 50%cytotoxicity concentration (CC ₅₀) determinations. The highest concentrations tested were listed in Table 3. The final concentration of DMSO in cell culture was 0.5%.

Table 3:

(2) Cell line and Virus strain

HCoV 229E (ATCC VR-740) and MRC-5 cells (ATCC CCL-171) were acquired from the ATCC. MRC-5 cells are maintained in the Minimum Essential Medium (Sigma) supplemented with 10%FBS (Hyclone) , 1%L-glutamine (Gibco) , 1%NEAA (Gibco) and 1%penicillin-streptomycin (Hyclone) . Minimum Essential Medium supplemented with 5%FBS, 1%L-glutamine, 1%NEAA and 1%penicillin-streptomycin was used as the assay medium.

(3) HCoV 229E CPE assay

In 96-well plates, MRC-5 cells were seeded at 20,000 cells per well and cultured at 37℃and 5%CO2 overnight. Next day, the medium containing serially diluted compounds and virus (200 TCID50 per well) was added. The resulting cultures were kept at 35℃ and 5%CO2 for additional 3 days until that virus infection in the virus control (cells infected with virus, without compound treatment) displays significant CPE. Cell viability was measured with CellTiter Glo according to the manufacturer’s manual. The luminescent signal was 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.

(4) Cytotoxicity assay

Cytotoxicity of compounds was assessed under the same conditions but without virus infection, in parallel. Cell viability was measured with CellTiter Glo according to the manufacturer’s manual. CC ₅₀ values were then calculated based on cytotoxicity at the test concentrations normalized by the medium control (medium only) .

(5) Data analysis

Antiviral activity and cytotoxicity of compounds were expressed as %inhibition and %cell viability, respectively, and calculated with the formulas below:

Inhibition (%) = (Raw data CPD –Average VC) / (Average CC –Average VC) x 100

Cell Viability (%) = (Raw data CPD –Average MC) / (Average CC –Average MC) x 100

Where the Raw data CPD indicates the values of the compound-treatment wells; Average VC, Average CC and Average MC indicate the average values of the virus control, cell control (cells without virus infection or compound treatment) and medium control, respectively.

EC ₅₀ and CC ₅₀ values were calculated using GraphPad Prism software (Version 5) and using the equation log (inhibitor) vs. response with variable slope. SI (CC ₅₀/EC ₅₀) was then calculated. The results were summarized as shown in Figure 3 and Table 4.

Table 4:

Reference throughout this specification to "an embodiment" , "some embodiments" , "one embodiment" , "another example" , "an example" , "a specific example" or "some examples" means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present disclosure. Thus, the appearances of the phrases 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, " in various places throughout this specification are not necessarily referring to the same embodiment or example of the present disclosure. Furthermore, the particular features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments or examples.

Although explanatory embodiments have been shown and described, it would be appreciated by those skilled in the art that the above embodiments cannot be construed to limit the present disclosure, and changes, alternatives, and modifications can be made in the embodiments without departing from spirit, principles and scope of the present disclosure.

Claims

A method of treating or preventing a Coronaviridae infection in a subject comprising administrating a therapeutically effective amount of a compound of Formula I or a pharmaceutically acceptable salt, ester or prodrug thereof to the subject in need thereof

wherein R1 is OH or H,

and the Coronaviridae comprises at least one selected from 2019-nCov virus, HCov 229E virus, SARS virus, MERS virus.
The method of claim 1, wherein R1 is OH.
The method of claim 1, further comprising administering a therapeutically effective amount of at least one other therapeutic agent or composition thereof selected from the group comprising a corticosteroid, an anti-inflammatory signal transduction modulator, a β2-adrenoreceptor agonist bronchodilator, an anticholinergic, a mucolytic agent, hypertonic saline, agent for reducing cytokine storm, other drugs for treating a covid-19 virus infection, or mixtures thereof.
The method of claim 1, further comprising administering a therapeutically effective amount of at least one other therapeutic agent or composition thereof selected from the group comprising Alpha-interferon, Lopinavir, Ritonavir, Ribavirin, Arbidol, Remdesivir, Oseltamivir , levofloxacin, moxifloxacin, azithromycin, linezolid, methylprednisolone, prednisone, hydrocortisone, drugs containing paracetamol, compound liquorice, compound codeine, ambroxol, aminophylline, dihydroxypropyltheophylline, intravenous injection of human immunoglobulin, vitamin C, Lianhua Qingwen, Qingkailing, Shuanghuanglian, Zhengchaihuyin.
The method of claim 1, wherein the pharmaceutically acceptable salt is at least one selected from a group comprising phosphate, hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, ethanesulfonate, toluenesulfonate and benzenesulfonate, acetate, trifluoroacetate, tartrate, maleate, succinate, citrate, benzoate, salicylate and ascorbate, adipate, alginate, arginate, aspartate, bisulfate, bisulfite, bromide, butyrate, camphorate, camphorsulfonate, caprylate, chloride, chlorobenzoate, cyclopentanepropionate, digluconate, dihydrogenphosphate, dinitrobenzoate, dodecylsulfate, fumarate, galacterate, galacturonate, glucoheptaoate, gluconate, glutamate, glycerophosphate, hemisuccinate, hemisulfate, heptanoate, hexanoate, hippurate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, iodide, isethionate, iso-butyrate, 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.
The method of claim 1, wherein the compound of Formula I or the pharmaceutically acceptable salt, ester or prodrug thereof is administrated to the subject in a form of sol, aromatic water, aerosol, partial inhalant, powder, granule, tablets, ointment, paste, emulsion, suspension, gas dispersion, solid dispersion, microparticle, pill.
The method of claim 1, wherein the compound of Formula I or a pharmaceutically acceptable salt, ester or prodrug thereof is administrated at a daily dose of lower than 400 mg in term of the compound of Formula I.
The method of claim 1, wherein the compound of Formula I or a pharmaceutically acceptable salt, ester or prodrug thereof is administrated at a daily dose of 10mg to 300 mg in term of the compound of Formula I.
The method of claim 1, wherein the compound of Formula I or a pharmaceutically acceptable salt, ester or prodrug thereof is administrated at a daily dose of 10mg to 150 mg in term of the compound of Formula I.
A pharmaceutical composition for treating or preventing Coronaviridae virus infection comprising a compound of Formula I or a pharmaceutically acceptable salt, ester or prodrug thereof to the subject in need thereof

wherein R1 is OH or H,

and the Coronaviridae comprises at least one selected from 2019-nCov virus, HCov 229E virus, SARS virus, MERS virus.
The pharmaceutical composition of claim 10, wherein R1 is OH.
The pharmaceutical composition of claim 10, further comprising at least one other therapeutic agent or composition thereof selected from the group comprising a corticosteroid, an anti-inflammatory signal transduction modulator, a β2-adrenoreceptor agonist bronchodilator, an anticholinergic, a mucolytic agent, hypertonic saline, agent for reducing cytokine storm, other drugs for treating a covid-19 virus infection, or mixtures thereof.
The pharmaceutical composition of claim 10, further comprising at least one other therapeutic agent or composition thereof selected from the group comprising Alpha-interferon, Lopinavir, Ritonavir, Ribavirin, Arbidol, Remdesivir, Oseltamivir, levofloxacin, moxifloxacin, azithromycin, linezolid, methylprednisolone, prednisone, hydrocortisone, drugs containing paracetamol, compound liquorice, compound codeine, ambroxol, aminophylline, dihydroxypropyltheophylline, intravenous injection of human immunoglobulin, vitamin C, Lianhua Qingwen, Qingkailing, Shuanghuanglian, Zhengchaihuyin.
The The pharmaceutical composition of claim 10, wherein the pharmaceutically acceptable salt is at least one selected from a group comprising phosphate, hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, ethanesulfonate, toluenesulfonate and benzenesulfonate, acetate, trifluoroacetate, tartrate, maleate, succinate, citrate, benzoate, salicylate and ascorbate, adipate, alginate, arginate, aspartate, bisulfate, bisulfite, bromide, butyrate, camphorate, camphorsulfonate, caprylate, chloride, chlorobenzoate, cyclopentanepropionate, digluconate, dihydrogenphosphate, dinitrobenzoate, dodecylsulfate, fumarate, galacterate, galacturonate, glucoheptaoate, gluconate, glutamate, glycerophosphate, hemisuccinate, hemisulfate, heptanoate, hexanoate, hippurate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, iodide, isethionate, iso-butyrate, 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.
The pharmaceutical composition of claim 10, wherein the compound of Formula I or the pharmaceutically acceptable salt, ester or prodrug thereof is in a form of sol, aromatic water, aerosol, partial inhalant, powder, granule, tablets, ointment, paste, emulsion, suspension, gas dispersion, solid dispersion, microparticle, pill.
The pharmaceutical composition of claim 10, comprising the compound of Formula I or the pharmaceutically acceptable salt, ester or prodrug thereof at a dose of lower than 200mg, lower than 100mg, lower than 50mg, lower than 30mg, 20mg or 10mg.
A compound of Formula I or a pharmaceutically acceptable salt, ester or prodrug thereof, for use in treating or preventing Coronaviridae infection in a subject,

wherein R1 is OH or H,

and the Coronaviridae comprises at least one selected from 2019-nCov virus, HCov 229E virus, SARS virus, MERS virus.
The compound of claim 17, wherein R1 is OH.
The compound of claim 17, wherein the pharmaceutically acceptable salt is at least one selected from a group comprising phosphate, hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, ethanesulfonate, toluenesulfonate and benzenesulfonate, acetate, trifluoroacetate, tartrate, maleate, succinate, citrate, benzoate, salicylate and ascorbate, adipate, alginate, arginate, aspartate, bisulfate, bisulfite, bromide, butyrate, camphorate, camphorsulfonate, caprylate, chloride, chlorobenzoate, cyclopentanepropionate, digluconate, dihydrogenphosphate, dinitrobenzoate, dodecylsulfate, fumarate, galacterate, galacturonate, glucoheptaoate, gluconate, glutamate, glycerophosphate, hemisuccinate, hemisulfate, heptanoate, hexanoate, hippurate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, iodide, isethionate, iso-butyrate, 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.
The compound of claim 17, wherein the compound of Formula I or the pharmaceutically acceptable salt, ester or prodrug thereof is administrated to the subject in a form of sol, aromatic water, aerosol, partial inhalant, powder, granule, tablets, ointment, paste, emulsion, suspension, gas dispersion, solid dispersion, microparticle, pill.
The compound of claim 17, wherein the compound of Formula I or a pharmaceutically acceptable salt, ester or prodrug thereof is administrated at a daily dose of lower than 400 mg in term of the compound of Formula I.
The compound of claim 17, wherein the compound of Formula I or a pharmaceutically acceptable salt, ester or prodrug thereof is administrated at a daily dose of 10 mg to 300 mg in term of the compound of Formula I.
The compound of claim 17, wherein the compound of Formula I or a pharmaceutically acceptable salt, ester or prodrug thereof is administrated at a daily dose of 10 mg to 150 mg in term of the compound of Formula I.
Use a compound of Formula I or a pharmaceutically acceptable salt, ester or prodrug thereof in the manufacture of a medicament for treating or preventing Coronaviridae infection

wherein R1 is OH or H,

and the Coronaviridae comprises at least one selected from 2019-nCov virus, HCov 229E virus, SARS virus, MERS virus.
The use of claim 24, wherein R1 is OH.
The use of claim 24, wherein the pharmaceutically acceptable salt is at least one selected from a group comprising phosphate, hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, ethanesulfonate, toluenesulfonate and benzenesulfonate, acetate, trifluoroacetate, tartrate, maleate, succinate, citrate, benzoate, salicylate and ascorbate, adipate, alginate, arginate, aspartate, bisulfate, bisulfite, bromide, butyrate, camphorate, camphorsulfonate, caprylate, chloride, chlorobenzoate, cyclopentanepropionate, digluconate, dihydrogenphosphate, dinitrobenzoate, dodecylsulfate, fumarate, galacterate, galacturonate, glucoheptaoate, gluconate, glutamate, glycerophosphate, hemisuccinate, hemisulfate, heptanoate, hexanoate, hippurate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, iodide, isethionate, iso- butyrate, 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.
The use of claim 24, wherein the compound of Formula I or the pharmaceutically acceptable salt, ester or prodrug thereof is in a form of sol, aromatic water, aerosol, partial inhalant, powder, granule, tablets, ointment, paste, emulsion, suspension, gas dispersion, solid dispersion, microparticle, pill.
The use of claim 24, wherein the medicament comprises the compound of Formula I or the pharmaceutically acceptable salt, ester or prodrug thereof at a dose of lower than 200 mg, lower than 100 mg, lower than 50 mg, lower than 30 mg, 20 mg or 10 mg.
An agent for treating air comprising a compound of Formula I or a pharmaceutically acceptable salt, ester or prodrug thereof,

wherein R1 is OH or H.
The agent of claim 29, wherein R1 is OH.
The agent of claim 29, wherein the pharmaceutically acceptable salt is at least one selected from a group comprising phosphate, hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, ethanesulfonate, toluenesulfonate and benzenesulfonate, acetate, trifluoroacetate, tartrate, maleate, succinate, citrate, benzoate, salicylate and ascorbate, adipate, alginate, arginate, aspartate, bisulfate, bisulfite, bromide, butyrate, camphorate, camphorsulfonate, caprylate, chloride, chlorobenzoate, cyclopentanepropionate, digluconate, dihydrogenphosphate, dinitrobenzoate, dodecylsulfate, fumarate, galacterate, galacturonate, glucoheptaoate, gluconate, glutamate, glycerophosphate, hemisuccinate, hemisulfate, heptanoate, hexanoate, hippurate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, iodide, isethionate, iso-butyrate, 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.
An apparatus for treating air comprising:

a container containing the agent of any one of claims 29 to 31.