WO2022144779A1

WO2022144779A1 - Sulfonamide derivatives, compositions comprising same and uses thereof against rna viruses

Info

Publication number: WO2022144779A1
Application number: PCT/IB2021/062399
Authority: WO
Inventors: Kenkichi Masutomi; Mami YASUKAWA; Koichi Watashi; Teruki Honma; Shun-Ichi Sekine; Hiroo Koyama; Takehiro Fukami
Original assignee: National Cancer Center; Japan As Represented By Director General Of National Institute Of Infectious Diseases; Riken; Gf Mille Inc.; Solstar Pharma Inc.
Priority date: 2020-12-29
Filing date: 2021-12-28
Publication date: 2022-07-07
Also published as: JP2022104747A

Abstract

Described herein are sulfonamide derivatives of Formula (I) capable of inhibiting RNA dependent RNA polymerase. Also described are pharmaceutical compositions comprising these compounds and the uses thereof in the prophylaxis and/or treatment of viral infections.

Description

SULFONAMIDE DERIVATIVES, COMPOSITIONS COMPRISING SAME AND USES THEREOF AGAINST RNA VIRUSES

CROSS REFERENCE TO RELATED APPLICATION

[0001] The present application claims priority to Japanese patent application JP 2020- 219885 filed on December 29, 2020, the contents of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] The invention relates to the field of viral infections, and more particularly to sulfonamide derivatives capable of inhibiting RNA dependent RNA polymerase, and to the use thereof as antiviral agents.

BACKGROUND OF THE INVENTION

[0003] The COVID-19 pandemic, also known as the coronavirus pandemic, is an ongoing global pandemic of coronavirus disease 2019 (COVID-19), caused by a singlestranded RNA virus referred to as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). As of today, ritonavir (Paxlovid™, Pfizer) and molnupiravir (Lagevrio™, Merck) are the only two oral drugs that have been approved by the U.S. FDA for antiviral activity against SARS-CoV-2.

[0004] RNA viruses having a single-strand RNA as a genome includes coronaviruses (e.g., SARS-CoV-2), hepatitis C virus (HCV), influenza virus, Ebola virus, polio virus, dengue virus, West Nile fever virus, and the like. A common biological feature of these RNA viruses is the use of a RNA dependent RNA polymerase (RdRP) for genome replication (i.e., RNA replication). In other words, since RdRP is considered to be an essential enzyme for life cycle of RNA viruses, there is a need for antiviral agents that display therapeutic effects by inhibiting RdRP.

[0005] The drug remdesivir is being studied and has already been approved for the treatment of SARS-CoV-2. However, remdesivir in not clinically convenient since is must be given by infusion into the vein. Therefore, there is still a need for a highly effective antiviral agent that can be administered orally. [0006] WO 2001/00579 describes compounds that modulate the PPAR gamma receptor for the treatment lipid-related diseases and diabetes. WO 2001/05793 describes sulfonamide derivatives for the treatment of depression, diabetes, obesity and CNS disorders. The following particular benzenesulfonate compound has been described for many therapeutic uses:

[0007] For instance, that compound has been described in WO 2005/033074 for treating diabetes, it has been described in WO 2020/205025 and WO 2021/076616 for treating non-alcoholic fatty liver diseases (NAFLD), it has been described in WO 2020/243058 for treating cancer, and it has been described in WO 2018/035446 for the treatment of blood cancers. However, these international PCT patent publications generally describe this molecule as a PPAR gamma inhibitor and neither of them describe nor suggest using that compound as antiviral agents, let alone for inhibiting RdRP.

[0008] As such there is still an important medical need for new antiviral agents and more particularly for therapeutically effective inhibitors of RdRP. There is also a need for pharmaceutical compounds and pharmaceutical compositions that are effective against RNA viruses such as Covid-19 and that can be administered orally.

[0009] The present invention addresses these needs and other needs as it will be apparent from the review of the disclosure and description of the features of the invention hereinafter.

BRIEF SUMMARY OF THE INVENTION

[00010] According to one aspect, the invention relates to a pharmaceutical composition for the treatment of a virus infection, the composition comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof:

wherein

Ri and R₂ are each independently a halogen atom, and m and n are each independently an integer between 0 - 3; and a pharmaceutically acceptable carrier or excipient.

[00011] According to another aspect, the invention relates to the use of a compound of Formula (I) as defined herein, or a pharmaceutically acceptable salt thereof, for the treatment of a virus infection.

[00012] According to another aspect, the invention relates to a method for interfering with virus replication, comprising contacting said virus with a compound of Formula (I) as defined herein, or a pharmaceutically acceptable salt thereof, as defined herein.

[00013] According to another aspect, the invention relates to a method for the treatment of a virus infection, comprising administering to a subject in need thereof a compound of Formula (I) as defined herein, or a pharmaceutically acceptable salt thereof.

[00014] In embodiments, the compound of Formula (I) is represented by Formula (la) as defined herein, or it is represented by one of the following formulas (RK-X), (RK-Y) or (RK-Z) as defined herein.

[00015] Additional aspects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of preferred embodiments which are exemplary and should not be interpreted as limiting the scope of the invention. BRIEF DESCRIPTION OF THE FIGURES

[00016] For the invention to be readily understood, embodiments of the invention are illustrated by way of example in the accompanying figures.

[00017] Figure 1A is a diagram depicting a testing method for evaluating inhibitory activity of tested compounds by RNA quantification, in accordance with Example 1.

[00018] Figure 1 B is a panel of line graphs depicting results of virus RNA quantifications with compounds RK-X, RK-Y and RK-Z, in accordance with Example 1.

[00019] Figure 2 is a panel depicting inhibition of RNA-dependent RNA polymerase (RdRp) with increasing concentration of compound RK-X, in accordance with Example 2. Fig. 2A: Gel image. Fig. 2B: Bar graph.

[00020] Figure 3A are bar graphs showing clinical score data of infected K18 mice treated with compound RK-X, in accordance with Example 3. Left panel shows box and violin plot and right panel shows mean ± SD.

[00021] Figure 3B is line graph with survival curves depicting increased survival of infected mice treated with compound RK-X, in accordance with Example 3.

[00022] Further details of the invention and its advantages will be apparent from the detailed description included below.

DETAILED DESCRIPTION OF EMBODIMENTS

[00023] In the following description of the embodiments, references to the accompanying figures are illustrations of an example by which the invention may be practiced. It will be understood that other embodiments may be made without departing from the scope of the invention disclosed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. General overview

[00024] The invention provides compounds, pharmaceutical compositions and uses thereof in the prophylaxis and/or treatment of viral infections.

[00025] Particularly, the present inventors have identified sulfonamide derivatives with antiviral activity, i.e. “antiviral compound”. In embodiments, antiviral compounds and antiviral compositions in accordance with the invention are effective in at least one of: (i) impairing cellular replication of RNA viruses; (ii) inhibiting virus infection; and (iii) reducing cellular viral load. In preferred embodiments, the compounds and compositions are effective in inhibiting the virus RNA dependent RNA polymerase activity. Antiviral compounds, pharmaceutical compositions and therapeutic uses

[00026] One aspect of the invention concerns compounds, as well as pharmaceutical compositions and uses thereof, as antiviral agents.

[00027] In one embodiment the compound is a sulfonamide compound of

Formula (I), or a pharmaceutically acceptable salt thereof:

wherein

Ri and R₂ are each independently a halogen atom, and m and n are each independently an integer between 0-3. [00028] In embodiments the compound of Formula (I) is a compound represented by the Formula (la):

wherein Ria, Rib, and R_2a are each independently a halogen atom, and

R₂b is a hydrogen atom or a halogen atom.

[00029] In embodiments, the compound of Formula (I) is represented by one of the following Formulas (RK-X), (RK-Y) or (RK-Z):

[00030] Compounds in accordance with the present invention can be used in either a free form or in the form of a “pharmaceutically acceptable salt”. Accordingly, the present invention encompasses pharmaceutically acceptable salts of any of the sulfonamide derivatives defined herein, including pharmaceutically acceptable salts of compounds of Formula (I), Formula (la), Formula (RK-X), Formula (RK-Y) and Formula (RK-Z).

[00031] The compounds of the present invention and salts thereof can be produced by those skilled in the art using any suitable methods, including for instance the method described in international PCT publication WO 2001/00579 (e.g., Example 178, 180 etc.)

[00032] Pharmaceutically acceptable salts of the compounds of the present invention include those derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of pharmaceutically acceptable salt include salts with inorganic acids include, but are not limited to, besylic acid salt (besylate), tosylic acid salt (tosylate) and mesylic acid salt (mesylate), hydrochloric acid salt, hydrobromic acid salt, sulfuric acid salt, phosphoric acid salt, and the like, salts with organic acids such as acetic acid salt, fumaric acid salt, oxalic acid salt, citric acid salt, methanesulfonic acid salt, benzenesulfonic acid salt, tosylic acid salt, maleic acid salt, and the like; salts with amino acids such as glycine salt, lysine salt, arginine salt, ornithine salt, glutamic acid salt, aspartic acid salt and the like; and the like. Examples of suitable salts are described in international PCT publication WO 2001/00579.

[00033] It is also conceivable to use the compounds of the present invention the form of a “prodrug”. Design of prodrugs is within the ordinary skill in the art and has been described in the literature (e.g., “Development of Pharmaceuticals”, Vol. 7, Design of Molecules, p.163-198, Published by Hirokawa Shoten (1990)).

[00034] Antiviral compounds and compositions in accordance with the present invention may provide substantial therapeutic benefits to subjects, particularly human subjects suffering, or susceptible to suffer, from a viral infection or viral disease such as COVID-19. Therefore, delivering an antiviral compound in accordance with the present invention may have useful pharmaceutical applications in the prophylaxis and the treatment of viral infections and diseases in mammalian subjects. [00035] Accordingly, one additional aspect of the invention concerns the use of an antiviral compound as defined herein, in the treatment of a viral disease of a mammalian subject in need thereof. The antiviral compound of the invention may be useful in: (i) treating viral infections (e.g., coronavirus-related disease such as COVID-19) in subjects; (ii) preventing, interfering and/or reducing viral infections and/or propagation in subjects (e.g., a human subject), (iii) preventing, interfering and/or reducing virus transcription and/or viral genome RNA replication; and (iv) reducing the load of viruses infection (e.g., corona viruses) in a subject, particularly in a human subject know (or suspected) of being infected with the virus SARS-CoV-2 causing COVID-19. According to one particular aspect, treatment of a subject comprises reducing viruses replication, thereby reducing the viral load in an infected subject and/or preventing spreading of virus infection to uninfected neighboring cells.

[00036] The antiviral compounds and compositions of the present invention may potentially be used against various types of viruses, including those of Group I (doublestranded DNA viruses), Group II (single-stranded DNA viruses), Group III (doublestranded RNA viruses), Group IV (positive sense single-stranded RNA viruses), Group V (negative sense single-stranded RNA viruses), Group VI (single-stranded RNA viruses with a DNA intermediate in their life cycle) and Group VII (double-stranded DNA viruses with an RNA intermediate in their life cycle).

[00037] In embodiments, the virus is an RNA virus. In embodiments, the virus is a single-stranded RNA virus. In embodiments, the virus is a corona virus, hepatitis C virus (HCV), influenza virus, Ebola virus, polio virus, dengue virus, enterovirus, coxsackievirus, measles virus, and/or a West Nile fever virus. In particular embodiments, the virus is a corona virus including, but not limited to, SARS-CoV-2.

[00038] In embodiments, the virus infection is severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), COVID-19, hepatitis C, influenza, Ebola hemorrhagic fever, polio, dengue fever, hand foot and mouth disease, measles, West Nile fever and the like. [00039] Related aspects of the invention concern pharmaceutical compositions comprising an effective amount of an antiviral compound as defined herein. As used herein, the term “pharmaceutical composition” refers to the presence of at least one of compound (or salt) as defined herein, and at least one pharmaceutically acceptable carrier, diluent, vehicle or excipient.

[00040] In embodiments, the compound of the present invention and related composition is not for use as a PPAR gamma (PPARy) inhibitor, nor for the treatment of diseases associated with inhibition of PPAR gamma activity. In embodiments, the compound of the present invention and the related pharmaceutical composition are not for the treatment of depression, diabetes, obesity or CNS disorders. In embodiments, the compound of the present invention and the related pharmaceutical composition are not for treating non-alcoholic fatty liver diseases (NAFLD). In embodiments, the compound of the present invention and the related pharmaceutical composition are not for treating cancers, e.g., blood cancers.

[00041] The pharmaceutical composition of the invention may be formulated in accordance to desired parameters such as dosage, the patient population, route of administration, etc. Examples of the pharmaceutical composition include, but are not limited to, tablet (including sugar-coated tablet, film-coated tablet, sublingual tablet, orally disintegrating tablet, buccal tablet and the like), pill, powder, granule, capsule (including soft capsule, microcapsule), troche, syrup, liquid, emulsion, suspension, controlled- release preparation (e.g., immediate-release preparation, sustained-release preparation, sustained-release microcapsule), aerosol, film (e.g., orally disintegrable film, oral mucosaadhesive film), injection (e.g., subcutaneous injection, intravenous injection (e.g., bolus), intramuscular injection, intraperitoneal injection), intravenous drip infusion, transdermal absorption type preparation, ointment, lotion, patch, suppository (e.g., rectal suppository, vaginal suppository), pellet, nasal preparation, transpulmonary preparation (inhalant), eye drop and the like. Among these, the form of a pharmaceutical composition suitable for oral administration is preferred.

[00042] "Pharmaceutically acceptable carrier, vehicle or excipient" refers to a diluent, adjuvant, excipient, carrier or drug delivery substance(s) with which a compound is administered. The term "pharmaceutically acceptable" refers to drugs, medicaments, inert ingredients, etc., which are suitable for use in treatment of a viral infection in subjects (preferably mammals such as humans) without undue toxicity, incompatibility, instability, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio. It preferably refers to a compound or composition that is approved or approvable by a regulatory agency of the Federal or state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals and more particularly in humans.

[00043] Specific examples of pharmaceutically acceptable carriers include excipients (e.g., lactose, sucrose, D-mannitol, starch, cornstarch, crystalline cellulose, light anhydrous silicic acid etc.), lubricants (e.g., magnesium stearate, talc, colloid silica etc.), binders (e.g., crystalline cellulose, sucrose, D-mannitol, dextrin, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, starch, sucrose, gelatin, methylcellulose, carboxymethylcellulose sodium etc.), disintegrant (e.g., starch, carboxymethylcellulose, carboxymethylcellulose calcium, sodium carboxymethyl starch, L-hydroxypropylcellulose etc.), and the like which be used for solid preparations.

[00044] For liquid preparations, the pharmaceutically acceptable vehicle, carrier or excipient can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol and the like), suitable mixtures thereof, and vegetable oils. Additional examples of pharmaceutically acceptable vehicles include, but are not limited to: Water for Injection USP; aqueous vehicles such as, but not limited to, Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection; water- miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and polypropylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate. Additional examples of carriers, vehicle or excipients may include solubilizing agents (e.g., polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, triethanolamine, sodium carbonate, sodium citrate etc.), suspending agents (surfactants, for example, stearyl triethanolamine, sodium lauryl sulfate, lauryl aminopropionic acid, lecithin, benzalkonium chloride, glycerin monostearate and the like; hydrophilic polymers, for example, poly(vinyl alcohol), polyvinylpyrrolidone, carboxymethylcellulose sodium, methylcellulose, hydroxymethylcellulose, hydroxypropylcellulose and the like, etc.), isotonic agents (e.g., glucose, D-sorbitol, sodium chloride, glycerol, D-mannitol etc.), buffering agents (buffer solutions, for example, phosphate, citrate and the like, etc.), and soothing agents (e.g., benzyl alcohol etc.). Prevention of the action of microorganisms in the composition can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, isotonic agents are included, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin. Where necessary, preparation additives such as antiseptic (e.g., p-oxybenzoic acid esters, chlorobutanol, benzyl alcohol, sorbic acid etc.), antioxidant (e.g., sulfite, ascorbic acid, a-tocopherol etc.), colorant, sweetening agent, and the like may further be added.

[00045] One particular aspect concerns the use of a therapeutically effective amount of one or more antiviral compound as defined herein for prophylaxis and/or treatment of viral infections in subjects.

[00046] As used herein, the term "therapeutically effective amount" or “effective amount" means the amount of compound that, when administered to a subject for treating or preventing a particular disorder, disease or condition, is sufficient to effect such treatment or prevention of that disorder, disease orcondition. Dosages and therapeutically effective amounts may vary, for example, depending upon a variety of factors including the activity of the specific agent employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, and any drug combination, if applicable, the effect which the practitioner desires the compound to have upon the subject and the properties of the compounds (e.g. bioavailability, stability, potency, toxicity, etc.), and the particular disorder(s), disease(s) or condition(s) the subject is suffering from. In addition, the therapeutically effective amount may depend on the severity of the disease state, or underlying disease or complications. Such appropriate doses may be determined using any available assays. In embodiments a dose of the compound according to the invention is about 0.1 to about 6000 mg in the case of oral administration (e.g., a dose of about 0.1 mg, about 0.5 mg, or about 1 mg, or about 5 mg, or about 10 mg, or about 50 mg, or about 100 mg, or about 500 mg, or about 1000 mg, or about 2500 mg, or about 5000 mg, or about 6000 mg). In embodiments a dose of the compound according to the invention is about 0.1 to about 600 mg in the case of parenteral administration (e.g., a dose of about 0.1 mg, about 0.5 mg, or about 1 mg, or about 5 mg, or about 10 mg, or about 50 mg, or about 100 mg, or about 250 mg, or about 500 mg, or about 600 mg). A dose may be administered in 1 to 3 divided portions per day. It is also conceivable to have two modes of treatment, for instance a low dose for moderate infection (e.g., 5 mg or more/day) and a high dose for severe infection (e.g., 1000 mg/day). It may be possible to switch from one type of treatment to the other, as appropriate. In embodiments the dose is adjusted according to body weight, for instance about 10 mg/kg to about 200 mg/kg four times a day (QID), or about 25 mg/kg to about 100 mg/kg QID, or about 50 mg I kg QID. When administered in the form of a sustained- release preparation, a dose may be administered every other day or at longer intervals (e.g., every 2, 3, 4, 5, 6 or 7 days).

[00047] The active compound, i.e. antiviral compound, may be formulated prior to administration into pharmaceutical compositions using available techniques and procedures. Formulations of the antiviral compound may be prepared so as to provide a pharmaceutical composition in a form suitable for any route of administration such as oral administration, injections (e.g., subcutaneous, intramuscular, intrathecal, intravenous, intra-nasal), sublingual, buccal, rectal, vaginal, ocular, otic, nasal, inhalation (e.g., intratracheal, pulmonary), nebulization, cutaneous, and transdermal administration. In preferred embodiments the route of administration is inhalation and/or nebulization. It is conceivable that the antiviral compounds be formulated under the form of nanoparticles, liposomes or any other suitable controlled release system for a chronology delivery, an extended release, and/or a targeted delivery.

[00048] Pharmaceutical compositions or formulations may, where appropriate, be conveniently presented in discrete dosage units and may be prepared by any of the methods well-known in the art of pharmaceutical formulation. All methods include the step of bringing together the active pharmaceutical ingredient(s) with liquid carriers or finely divided solid carriers or both as the need dictates. When appropriate, the above-described formulations may be adapted so as to provide sustained release of the active pharmaceutical ingredient. Sustained release formulations well-known to the art include the use of a bolus injection, continuous infusion, biocompatible polymers or liposomes. In preferred embodiments, the compositions according to the invention are formulated for oral administration.

[00049] A pharmaceutical composition in accordance with the present invention may further comprise at least of an antiviral drug, a steroid, an anti-inflammatory agent, a proteolytic enzyme inhibitor, a drug for the treatment of severe cytokine storm syndrome and acute respiratory distress syndrome, and interferon. In embodiments, the antiviral drug is selected from remdesivir, nelfinavir, favipiravir, lopinavir, and chloroquine. In embodiments, the steroid is selected from dexamethasone and ciclesonide. In embodiments, the antiviral drug is selected from approved antiviral agents and antiviral compounds being tested in human subjects for treatment of viral infection(s) including, but not limited to, hydroxychloroquine, ribavirin (Virazole®), lopinavir, ritonavir, remdesivir (Veklury®), favipiravir, colchicine and ivermectine, etc. In embodiments, the antiviral drug is an anti-inflammatory agent such as hydroxychloroquine and the like. In embodiments, the antiviral drug is a proteolytic enzyme inhibitors such as nafamostat, camostat and the like. In embodiments, the anti-inflammatory agent is a drug for the treatment of severe cytokine storm syndrome and acute respiratory distress syndrome including, but not limited to, anti-l L-6R antibodies (e.g., tocilizumab, sarilumab and the like), JAK inhibitors (e.g., baricitinib, ruxolitinib and the like), BTK inhibitors (e.g., acalabrutinib and the like), anti-complement antibodies (e.g., ravulizumab and the like), and the like.

[00050] An additional related aspect concerns therapeutic methods (e.g. for treating a viral infection) comprising administering a therapeutically effective amount of one or more antiviral compound or composition as described herein to a subject. The invention also encompasses methods, compounds, and pharmaceutical compositions for the treatment of a virus infection in mammals including, but not limited to, corona viruses-related diseases and infections. In embodiments, the viral disease is a corona virus-related disease such as COVID-19. [00051] As used herein, the term “mammal”, “mammalian subject” or “mammalian cell” includes mammals and cells in which treatment against virus infection is desirable. The term “subject” includes domestic animals (e.g., cats, dogs, horses, pigs, cows, goats, sheep), rodents (e.g., mice or rats), rabbits, squirrels, bears, primates (e.g., chimpanzees, monkeys, gorillas, and humans), wild animals such as those living in zoos (e.g., lion, tiger, elephant, and the like), and transgenic species thereof. Preferably, the mammalian subject is a human, more preferably a human patient in need of treatment. Even more preferably the mammalian subject is a human patient diagnosed or susceptible to suffer from a RNA virus, e.g., a coronavirus disease such as COVID-19, SARS and/or MERS.

[00052] As used herein, the terms “treatment” or “treating” of a subject include one or more of administration of an antiviral compound or composition to a subject with the purpose of stabilizing, curing, healing, alleviating, relieving, altering, remedying, less worsening, ameliorating, improving, or affecting the disease or condition, the symptom of the disease or condition, or the risk of (or susceptibility to) the disease or condition. The term “treating” refers to any indication of success in the treatment or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement; remission; lessening of the rate of worsening; lessening severity of the disease; stabilization, diminishing of symptoms or making the injury, pathology or condition more tolerable to the subject; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; or improving a subject’s physical or mental well-being. In one embodiment, the treatment method comprises administering to the subject a therapeutically effective amount of an antiviral siRNA as defined herein.

[00053] In embodiments, the term treatment encompasses “prophylaxis”, i.e. the compound or composition is administered for prevention of the onset of the disease (the whole pathology, or one or more pathologies), and delay of the onset of the disease. A “prophylactically effective amount” refers to a dose of the compound of the present invention which is sufficient to achieve such object.

[00054] When one or more of the antiviral compound of the invention is to be administered to humans, a physician may for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. [00055] In embodiments, the effective amount for a human subject may be a single administration of a high concentration of the antiviral compound. Alternatively, the antiviral compound may be administered more frequently (e.g., daily, weekly, monthly) and/or until the patient is tested negative for the infection and even afterwards.

[00056] The methods of treatments, antiviral compounds and compositions of the present invention may also be used in combination with other medicament(s), preferably to provide superior prophylactic and/or treatment effect associated to the combined use with other medicament(s). In addition, such combination therapy may also reduce the dose of other medicaments and reduce the side effects thereof.

[00057] In embodiments, the methods of treatments, antiviral compounds and compositions of the present invention are used in combination with antiviral therapies (e.g., antivirals, anti-inflammatory agents, etc.) and/or vaccines, especially already approved therapies, steroids, proteolytic enzyme inhibitors, drugs for the treatment of severe cytokine storm syndrome and acute respiratory distress syndrome, interferon(s), currently approved antiviral agents, antiviral compounds being tested in human subjects for treatment of viral infection(s), etc., including those particular compounds mentioned hereinbefore.

[00058] In embodiments, methods of treatments in accordance with the present invention comprises concomitant administration of one or more existing therapies (e.g., antiviral therapies, anti-inflammatory agents, vaccines, etc.) as mentioned hereinbefore. As used herein, the term “concomitant” or “concomitantly” as in the phrases “administering concomitantly” or “concomitantly with” or “concomitant administration” includes administering a first compound in the presence of a second compound. A concomitant administration includes methods in which the first or second compounds are co-administered. A concomitant administration treatment method may be executed step- wise by different actors. For example, one actor may administer to a subject a first compound and as a second actor may administer to the subject a second compound. The administering steps may be executed at the same time, or nearly the same time (e.g., within 1 , 2, 5, 10, 20 or 30 minutes), or at distant times (e.g., more than 1 hour, more than 2 hours, more than 3 hours, more than 6 hours, more than 12 hours, more than 24 hours, etc.). The actor and the subject may be the same entity (e.g., a human).

[00059] The dose of the concomitant drug can be appropriately selected based on the dose used clinically. The mixing ratio of the compound of the present invention and the concomitant drug can be appropriately selected according to factors such as the disease and symptom of the subject, the administration route, the kind of the concomitant drug to be used, and the like.

[00060] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents are considered to be within the scope of this invention, and covered by the claims appended hereto. The invention is further illustrated by the following examples, which should not be construed as further or specifically limiting.

EXAMPLES

[00061] Example 1 : In vitro study of RK derivatives against SARS-CoV-2

[00062] The present study was carried out in accordance with Matsuyama, S. et al. (PNAS, 117, 7001 (2020) “Enhanced isolation of SARS-CoV-2 by TMPRSS2”). The inhibitory effect (in vitro) by RdRP inhibitors against SARS-CoV-2 infection was carried out by measuring SARS-CoV-2 RNA in the culture supernatant after SARS-CoV-2 infection to VeroE6/TMPRSS2 cells. VeroE6/TMPRSS2 cells are available at the JCRB Cell Bank under number JCRB1819.

[00063] TMPRSS2 overexpressing VeroE6 cell line was cultured in each well of a 96- well cell culture plate manufactured by IWAKI. The following were tested: (i) RdRP inhibitors RK-X, RK-Y or RK-Z (as defined hereinbefore), each at a concentration of 16 pM, 24 pM, 36 pM; (ii) remdesivir 10 pM (manufactured by MedChemExpress) as a positive control drug; and (iii) dimethyl sulfoxide 0.5% (manufactured by Sigma) as negative control compound. The compounds were added, and the cells were cultured under 5% CO₂ condition at 37°C for 1 hr. The cells were culture in DMEM supplemented with Fetal bovine serum (Life Technologies).

[00064] The cultured cells were contacted with SARS-CoV-2 (obtained from National Institute of Infectious Diseases, Department of Virology III). After 1 hr, the cells were washed to remove excess SARS-CoV-2, and cultured at 37°C for 24 hr in the presence of the above-mentioned drugs and the like. An outline of the testing method is depicted in Figure 1A.

[00065] RNA of SARS-CoV-2 in the culture medium supernatant in each well was quantified using a real-time RT-PCR system (Applied Biosystems), and the relative viral RNA were determined.

[00066] Results of virus RNA quantifications are shown in Figure 1 B. As shown, tested compounds RK-X, RK-Y and RK-Z all decreased cells viral RNA, without obvious cytotoxicity. Particularly, the following percentage were calculated, these percentages representing a percentage (%) with respect to the viral load in the DMSO-administered control group: 0.27% for RK-X 24 pM, 0.0087% for RK-Y 36 pM, and 0.26% for RK-Z 24 pM. These results confirm that the three tested compounds all had a significant inhibitory activity on cell infection by SARS-CoV-2 and/or replication of SARS-CoV-2.

[00067] Example 2: Inhibition of RNA-dependent RNA polymerase (RdRp) activity

[00068] RNA-dependent RNA polymerase (RdRp) activity inhibitory effect of compound RK-X on the SARS-CoV-2-derived nsp7-nsp8-nsp12 complex was evaluated by means of a primer extension assay based on the method described by Shanoon et al. (Rapid incorporation of Favipiravir by the fast and permissive viral RNA polymerase complex results in SARS-CoV-2 lethal mutagenesis, Nature Communications 11 , 4682 (2020)).

[00069] nsp12 was expressed in baculovirus-infected insect cells and purified by various chromatographies. The purified nsp12 was dissolved in buffer A (20 mM HEPES pH 7.5, 200 mM NaCI, 1 mM TCEP). nsp7 and nsp8 were each expressed in Escherichia coli and purified by various chromatographies. nsp7 and nsp8 were dissolved in buffer B (20 mM HEPES pH 8.0, 150 mM NaCI, 1 mM DTT). RNA oligonucleotides used as a template (40 mer) and a 5’-Cy5-labeled primer (10 mer) in the primer extension assay were purchased from Japan Bio Services and dissolved in water at a concentration of 100 pM.

[00070] Equal amounts of the template and primer solutions were mixed in buffer C (20 mM HEPES pH 7.5, 50 mM NaCI). The RNA solution was incubated at 75°C for 4 min and then slowly cooled to 25°C. Subsequently, the RNA solution was placed at 4°C over 10 min. Next, 9 pl of 2.5 pM RNA solution, 1.5 pl of 15 pM nsp12, 1.5 pl of 45 pM nsp7, 1.5 pl of 45 pM nsp8, 15 pl of buffer D (20 mM HEPES pH 7.5, 50 mM NaCI, 10 mM MgCI₂, 2 mM DTT, 20% (v/v) glycerol) and 0.5 pl of DMSO or RK-X were mixed and then placed at room temperature for 20 min. To start the primer extension, 1 ul of an NTP-containing solution (1 .5 mM ATP, 1 .5 mM GTP, 1 .5 mM CTP, 1 .5 mM UTP) was added to the reaction mixture and then placed at room temperature for 40 min. The primer extension was stopped by adding buffer E (89 mM Tris, 12 mM EDTA, 89 mM borate, 10 M urea) to the reaction mixture. The reaction mixture was heated at 75°C for 3 min and subjected to denaturing urea polyacrylamide gel electrophoresis analysis. The fluorescence of Cy5 in the gel was detected using Amersham Typhoon™ scanner. The fluorescence intensity of Cy5-labeled extended primers in the image data was quantified using ImageQuant™ TL. The experiments in this section were performed three times.

[00071] Figure 2 shows results of the primer extension assay. The fluorescence intensity of Cy5-labeled extended primers decreased with increasing concentration of RK- X in the reaction mixture, strongly suggesting that RK-X directly interferes with the RNA extension mediated by the nsp7-nsp8-nsp12 complex.

[00072] Example 3: Infection and treatment of K18 mice

[00073] Compound RK-X was assessed for efficacy on clinical score/survival data of mice infected with SARS2-HK.

[00074] Six-week-old male or female B6.Cg-Tg(K18-ACE2)2Primn/J mice were purchased from Jackson Labs. The mice were quarantined for 72 hours. Following quarantine, mice were weighed and randomly grouped into groups of 5 mice/cage. For this study there are 3 groups of mice (Table 1). Mice were infected (intranasal route) with SARS2-HK at LD o dose. Mice were treated by Per os (oral gavage) BID at least 12 hours apart for 4 days beginning at either -2 hours or +8 hours following infection with either control or compound RK-X. Mice were monitored daily for body weight and clinical symptoms: ruffled fur, hunched, lethargic and respiratory distress. All IACUC endpoints were followed and mice exhibiting a score of 3 or have a loss of more than 20% of their body weight will be euthanized. All SARS2-HK work was done in an approved ABSL-3 animal suite.

[00075] Table 1 : Groups of mice and treatment overview

Control vehicle: 0.5% methyl cellulose (MC); RK-X (50 mg/kg) suspended in 0.5% MC [00076] As shown in Figure 3A, the clinical score data show protection of infected mice with the 50 mg/kg dose of RK-X (p=0.047).

[00077] As shown in Figure 3B, the 50 mg/kg dose of RK-X confers survival advantage: median survival of infected mice increased from 6 to 7 days and the time maximum survival have doubled to about 14 days.

[00078] Headings are included herein for reference and to aid in locating certain sections. These headings are not intended to limit the scope of the concepts described therein, and these concepts may have applicability in other sections throughout the entire specification. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

[00079] The singular forms “a”, “an” and “the” include corresponding plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a compound" includes one or more of such compounds and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.

[00080] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, concentrations, properties, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the present specification and attached claims are approximations that may vary depending upon the properties sought to be obtained. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the embodiments are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors resulting from variations in experiments, testing measurements, statistical analyses and such.

[00081] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the present invention and scope of the appended claims.

Claims

CLAIMS:

1. A pharmaceutical composition for the treatment of a virus infection, said composition comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof:

wherein

2. The pharmaceutical composition of claim 1 , wherein the compound of Formula (I) is a compound represented by Formula (la):

wherein

Ria, R , and R_2a are each independently a halogen atom, and R_2b is a hydrogen atom or a halogen atom.

3. The pharmaceutical composition of claim 1 , wherein the compound represented by the Formula (I) is represented by one of the following formulas (RK-X), (RK-Y) or (RK-Z):

4. The pharmaceutical composition of any one of claims 1 to 3, wherein said compound of Formula (I) comprises at least one of the following antiviral activity: (i) it impairs cellular replication of RNA viruses; (ii) it inhibits virus infection; and (iii) reducing cellular viral load.

5. The pharmaceutical composition of any one of claims 1 to 3, wherein said compound of Formula (I) inhibits virus RNA dependent RNA polymerase activity.

6. The pharmaceutical composition of any one of claims 1 to 5, wherein the virus is a RNA virus.

7. The pharmaceutical composition of any one of claims 1 to 5, wherein the virus is a single-stranded RNA virus.

8. The pharmaceutical composition of any one of claims 1 to 5, wherein the virus is selected from the group consisting of corona virus, hepatitis C virus (HCV), influenza virus, Ebola virus, polio virus, dengue virus, enterovirus, coxsackievirus, measles virus, and West Nile fever virus.

9. The pharmaceutical composition of any one of claims 1 to 5, wherein the virus infection is selected from the group consisting of severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), COVID-19, hepatitis C, influenza, Ebola hemorrhagic fever, polio, dengue fever, hand foot and mouth disease, measles, and West Nile fever.

10. The pharmaceutical composition of any one of claims 1 to 5, wherein the virus is a corona virus.

11 . The pharmaceutical composition of any one of claims 1 to 5, wherein the virus is SARS-CoV-2.

12. The pharmaceutical composition of any one of claims 1 to 11 , wherein said composition is for treating a human subject suffering, or susceptible to suffer, from a viral infection.

13. The pharmaceutical composition of any one of claims 1 to 12, wherein said pharmaceutical composition further comprises at least of an antiviral drug, a steroid, an anti-inflammatory agent, a proteolytic enzyme inhibitor, a drug for the treatment of severe cytokine storm syndrome and acute respiratory distress syndrome, and interferon.

14. The pharmaceutical composition of any one of claims 1 to 13, wherein said pharmaceutical compositions is formulated for oral administration.

15. The pharmaceutical composition of claim 14, wherein said pharmaceutical compositions is formulated as a tablet or capsule.

16. The pharmaceutical composition of any one of claims 1 to 15, wherein said pharmaceutical composition is for the treatment of a disease or condition other than depression, diabetes, obesity, CNS disorders, non-alcoholic fatty liver diseases (NAFLD) and cancer.

17. Use of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for the treatment of a virus infection:

wherein

Ri and R₂ are each independently a halogen atom, and m and n are each independently an integer between 0 - 3.

18. The use according to claim 17, wherein the compound of Formula (I) is a compound represented by Formula (la):

wherein

Ria, Rib, and R_2a are each independently a halogen atom, and R₂b is a hydrogen atom or a halogen atom.

19. The use according to claim 17, wherein the compound represented by the Formula

(I) is represented by one of the following formulas (RK-X), (RK-Y) or (RK-Z):

20. The use according to any one of claims 17 to 19, wherein said compound of Formula (I) comprises at least one of the following antiviral activity: (i) it impairs cellular replication of RNA viruses; (ii) it inhibits virus infection; and (iii) reducing cellular viral load.

21. The use according to any one of claims 17 to 19, wherein said compound of Formula (I) inhibits virus RNA dependent RNA polymerase activity.

22. The use according to any one of claims 17 to 21 , wherein the virus is a RNA virus.

23. The use according to any one of claims 17 to 22, wherein the virus is a singlestranded RNA virus.

24. The use according to any one of claims 17 to 21 , wherein the virus is selected from the group consisting of corona virus, hepatitis C virus (HCV), influenza virus, Ebola virus, polio virus, dengue virus, enterovirus, coxsackievirus, measles virus, and West Nile fever virus.

25. The use according to any one of claims 17 to 21 , wherein the virus infection is selected from the group consisting of severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), COVID-19, hepatitis C, influenza, Ebola hemorrhagic fever, polio, dengue fever, hand foot and mouth disease, measles, and West Nile fever.

26. The use according to any one of claims 17 to 21 , wherein the virus is a corona virus.

27. The use according to any one of claims 17 to 21 , wherein the virus is SARS-CoV-2.

28. The use according to any one of claims 17 to 27, for treating a human subject suffering, or susceptible to suffer, from a viral infection.

29. The use according to any one of claims 17 to 28, wherein said compound of Formula (I) is used concomitantly with at least one of an antiviral drug, a steroid, an antiinflammatory agent, a proteolytic enzyme inhibitor, a drug for the treatment of severe cytokine storm syndrome and acute respiratory distress syndrome, and interferon.

30. The use according to any one of claims 17 to 29, wherein said compound of Formula (I) is formulated for and oral administration.

31 . The use according to any one of claims 17 to 30, for the treatment of a disease or condition other than depression, diabetes, obesity, CNS disorders, non-alcoholic fatty liver diseases (NAFLD) and cancer.

32. A method for interfering with virus replication, comprising contacting said virus with a compound of Formula (I), or a pharmaceutically acceptable salt thereof:

wherein

33. A method for the treatment of a virus infection, comprising administering to a subject in need thereof a compound of Formula (I), or a pharmaceutically acceptable salt thereof:

wherein

34. The method of claim 32 or 33, wherein the compound of Formula (I) is a compound represented by Formula (la):

wherein

Ria, R , and R_2a are each independently a halogen atom, and R₂b is a hydrogen atom or a halogen atom.

35. The method of claim 32 or 33, wherein the compound represented by the Formula

(I) is represented by one of the following formulas (RK-X), (RK-Y) or (RK-Z):

36. The method as defined in any one of claims 32 to 35, wherein said compound of Formula (I) comprises at least one of the following antiviral activity: (i) it impairs cellular replication of RNA viruses; (ii) it inhibits virus infection; and (iii) reducing cellular viral load.

37. The method as defined in any one of claims 32 to 35, wherein said compound of Formula (I) inhibits virus RNA dependent RNA polymerase activity.

38. The method as defined in any one of claims 32 to 37, wherein the virus is an RNA virus.

39. The method as defined in any one of claims 32 to 35, wherein the virus is a singlestranded RNA virus.

40. The method as defined in any one of claims 32 to 35, wherein the virus is selected from the group consisting of corona virus, hepatitis C virus (HCV), influenza virus, Ebola virus, polio virus, dengue virus, enterovirus, coxsackievirus, measles virus, and West Nile fever virus.

41 . The method as defined in any one of claims 32 to 35, wherein the virus infection is selected from the group consisting of severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), COVID-19, hepatitis C, influenza, Ebola hemorrhagic fever, polio, dengue fever, hand foot and mouth disease, measles, and West Nile fever.

42. The method as defined in any one of claims 32 to 35, wherein the virus is a corona virus.

43. The method as defined in any one of claims 32 to 35, wherein the virus is SARS- CoV-2.

44. The method of claim 33, wherein said subject is a human subject suffering, or susceptible to suffer, from a viral infection.

45. The method as defined in any one of claims 32 to 45, wherein the compound of Formula (I) is formulated for oral administration.

46. The method as defined in any one of claims 32 to 45, wherein said method is for the treatment of a disease or condition other than depression, diabetes, obesity, CNS disorders, non-alcoholic fatty liver diseases (NAFLD) and cancer.

47. The method as defined in any one of claims 32 to 46, wherein said method comprises concomitant administration of at least additional compound selected from the group consisting of: antiviral drugs, steroids, anti-inflammatory agents, proteolytic enzyme inhibitors, drugs for the treatment of severe cytokine storm syndrome and acute respiratory distress syndrome, and interferons.