A therapeutic agent for coronavirus infections comprising a combination of a pyrazine derivative and another therapeutic agent for coronavirus infections

Abstract

A coronavirus infection therapeutic agent comprising a combination of a pyrazine derivative or a salt thereof and another coronavirus infection therapeutic drug.

Description

[Technical Field]

[0001] The present invention relates to a therapeutic agent for coronavirus infection, which comprises a pyrazine derivative or a salt thereof in combination with one or more other therapeutic agents for coronavirus infection. [Background technology]

[0002] The novel coronavirus (SARS-CoV-2), first reported in China at the end of 2019 (Non-Patent Document 1), is an RNA virus belonging to the Coronaviridae family of the Nidovirales order (Non-Patent Document 2). Infection with this virus causes acute respiratory symptoms such as fever, cough, and difficulty breathing (Non-Patent Document 1), and if the condition worsens, pneumonia develops. As of March 22, 2020, more than 290,000 cases of SARS-CoV-2 infection (COVID-19) have been reported worldwide, with more than 12,000 deaths. 1,046 cases of COVID-19 have been reported in Japan, with 36 confirmed deaths (Non-Patent Document 3).

[0003] Baricitinib, lopinavir, ritonavir, darunavir, favipiravir (a pyrazine derivative), remdesivir, and ribavirin have been reported as potentially effective treatments for COVID-19 (Non-Patent Document 4). However, there have been no studies that have systematically evaluated the combined effects of two or more of these agents against coronaviruses. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Wang C, Horby PW, Hayden FG, Gao GF. A novel coronavirus outbreak of global health concern. Lancet. 2020;395:470-3. [Non-patent document 2] Coronaviridae Study Group of the International Committee on Taxonomy of Viruses. The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2. Nat Microbiol. 2020 Mar 2. PubMed PMID: 32123347. [Non-patent document 3] Coronavirus disease 2019 (COVID-19) situation reports - 62 (22 March 2020). [Internet]. Geneva: World Health Organization. Available from: https: / / www.who.int / docs / default-source / coronaviruse / situation-reports / 20200322-sitrep-62-covid-19.pdf?sfvrsn=f7764c46_2 [Non-patent document 4] ACS Central Science (2020), 6(3), 315-331 Summary of the Invention [Problem to be solved by the invention]

[0005] The object of the present invention is to provide a new combination of substances that is effective against coronaviruses. [Means for solving the problem]

[0006] Under these circumstances, the present inventors have conducted extensive research and have found that the general formula [1] [ka] (In the formula, R 1 and R 2are the same or different and represent a hydrogen atom or a halogen atom; R 3 represents a hydrogen atom or an amino-protecting group.) or a salt thereof, when used in combination with other therapeutic drugs for coronavirus infection, the antiviral activity against coronavirus is enhanced, and the present invention has been completed based on this finding.

[0007] That is, the present invention provides the following. [1] The general formula: [ka] (In the formula, R 1 and R 2 are the same or different and represent a hydrogen atom or a halogen atom; R 3 represents a hydrogen atom or an amino-protecting group) or a salt thereof, and one or more therapeutic agents for coronavirus infection selected from the following (1) to (19): (1) Interferon (2) Nucleic acid analogs (3) Protease inhibitors (4) Furin convertase cleavage inhibitors (5) Reverse transcriptase inhibitors and / or other RNA polymerase inhibitors (6) Neuraminidase inhibitor (7) Angiotensin II receptor blockers (8) Endosome fusion inhibitors and / or endosome alkalinizers (9) AAK1, GAK, and clathrin A, B, C (endocytosis) inhibitors (10) Hemagglutinin esterase inhibitors (11) Cytokine or inflammation inhibitors and regulators (12) Cathepsin B inhibitor (13) Cathepsin L inhibitor (14) Helicase nsp13 inhibitor (15) MBL2 gene agonist (16) TP53 inhibitors (17) Selective estrogen receptor modulators (18) Steroids (19) amantadine, foscarnet, triazavirine, umifenovir, rapamycin, everolimus, nitazoxanide, tizoxanide, caraphenol A, ivermectin, VIR-2703, tocilizumab, bamlanivimab, etesevimab, casirivimab / imdevimab, AZD7422, VIR-7831, VIR-7832, or BI 767551 [2] The pharmaceutical composition according to [1], wherein the interferon is interferon α-2A, interferon α-2B, interferon α-n1, interferon α-n3, interferon β-1a or interferon β-1b. [3] The pharmaceutical composition according to [1], wherein the nucleic acid analog is acyclovir, ganciclovir, ribavirin, or taribavirin. [4] The pharmaceutical composition according to [1], wherein the protease inhibitor is indinavir, nelfinavir, saquinavir, camostat, lopinavir-ritonavir combination drug (product name: Kaletra), epigallocatechin gallate, kaempferol-7-glucoside, mycophenolic acid, darunavir, mercaptopurine, disulfiram, nafamostat, or PF-07321332. [5] The pharmaceutical composition according to [1], wherein the furin convertase cleavage inhibitor is tenofovir disoproxil, dolutegravir, boceprevir, andrographolide, luteolin, or baicalein. [6] The pharmaceutical composition according to [1], wherein the reverse transcriptase inhibitor and / or other RNA polymerase inhibitor is remdesivir, sofosbuvir, dactinomycin, galidesivir, baloxavir marboxil, molnupiravir, sangivamycin, or AT-527. [7] The pharmaceutical composition according to [1], wherein the neuraminidase inhibitor is oseltamivir or zanamivir. [8] The pharmaceutical composition according to [1], wherein the angiotensin II receptor blocker is valsartan, telmisartan, losartan, irbesartan, azilsartan, olmesartan or emodin. [9] The pharmaceutical composition according to [1], wherein the endosome fusion inhibitor and / or endosome alkalinizer is baicalin, chloroquine, hydroxychloroquine, Griffithsin, quinine or lactoferrin.

[10] The pharmaceutical composition according to [1], wherein the AAK1, GAK and clathrin A, B, C (endocytosis) inhibitor is baricitinib, sunitinib, erlotinib, fedratinib, gefitinib or silibinin.

[11] The pharmaceutical composition according to [1], wherein the hemagglutinin esterase inhibitor is 3,4-dichloroisocoumarin.

[12] The pharmaceutical composition according to [1], wherein the cytokine or inflammation inhibitor and regulator is ligustrazine, a statin, melatonin, eplerenone or methylprednisolone.

[13] The pharmaceutical composition according to [1], wherein the cathepsin B inhibitor is salvianolic acid B.

[14] The pharmaceutical composition according to [1], wherein the cathepsin L inhibitor is MOL736, chelidocystatin, astaxanthin, curcumin, or vitamin D.

[15] The pharmaceutical composition according to [1], wherein the helicase nsp13 inhibitor is valsartan, bananin, iodobananin, vanillinbananin, eubananin, or silvestrol.

[16] The pharmaceutical composition according to [1], wherein the MBL2 gene agonist is β-glucan or vitamin A.

[17] The pharmaceutical composition according to [1], wherein the TP53 inhibitor is vitexin or gossypol.

[18] The pharmaceutical composition according to [1], wherein the selective estrogen receptor modulator is toremifene or equilin.

[19] The pharmaceutical composition according to [1], wherein the steroid drug is ciclesonide or dexamethasone.

[20] The pharmaceutical composition according to [1], wherein the one or more therapeutic agents for coronavirus infection are reverse transcriptase inhibitors and / or other RNA polymerase inhibitors.

[0008] The present invention further provides the following. [A] General formula [1] [ka] (In the formula, R 1 and R 2 are the same or different and represent a hydrogen atom or a halogen atom; R 3 A method for treating coronavirus infection, comprising administering to a subject a pyrazine derivative represented by the formula (I) or a salt thereof, and another therapeutic agent for coronavirus infection. [B] For producing a coronavirus infection treatment agent, a compound represented by the general formula [1] [ka] (In the formula, R 1 and R 2 are the same or different and represent a hydrogen atom or a halogen atom; R 3 represents a hydrogen atom or an amino-protecting group) or a salt thereof, together with other therapeutic agents for coronavirus infection. [C] A compound of the general formula [1] for use in the treatment of coronavirus infections [ka] (In the formula, R 1 and R 2 are the same or different and represent a hydrogen atom or a halogen atom; R3 represents a hydrogen atom or an amino-protecting group) or a salt thereof with another therapeutic agent for coronavirus infection. [Effects of the Invention]

[0009] A therapeutic agent for coronavirus infections that combines a pyrazine derivative or a salt thereof with another therapeutic agent for coronavirus infections is useful for treatments such as the treatment or prevention of coronavirus infections. DETAILED DESCRIPTION OF THE INVENTION

[0010] The present invention will be described in detail below.

[0011] In this specification, a numerical range indicated using "to" or "-" means a range that includes the numerical values ​​before and after "to" or "-" as the minimum and maximum values, respectively.

[0012] The halogen atom means a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom. 1-6 Alkyl groups include straight or branched C alkyl groups such as methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isopentyl, 2-methylbutyl, 2-pentyl, 3-pentyl, and hexyl groups. 1-6 It means an alkyl group.

[0013] The amino-protecting group includes all groups that can be used as ordinary amino-protecting groups, and examples thereof include groups described in W. Greene et al., Protective Groups in Organic Synthesis, 5th Edition, pp. 895-1193, 2014, John Wiley & Sons, INC.

[0014] Specific examples include an acyl group, an alkyloxycarbonyl group, an arylalkyloxycarbonyl group, an aryloxycarbonyl group, an arylalkyl group, an alkoxyalkyl group, an arylalkyloxyalkyl group, an arylthio group, an alkylsulfonyl group, an arylsulfonyl group, a dialkylaminoalkylidene group, an arylalkylidene group, a nitrogen-containing heterocyclic alkylidene group, a cycloalkylidene group, a diarylphosphoryl group, a diarylalkylphosphoryl group, an oxygen-containing heterocyclic alkyl group, and a substituted silyl group.

[0015] Salts of the compound of general formula [1] include commonly known salts of the hydroxyl group. For example, salts with alkali metals such as sodium and potassium; salts with alkaline earth metals such as calcium and magnesium; ammonium salts; and salts with nitrogen-containing organic bases such as trimethylamine, triethylamine, tributylamine, N-methylpiperidine, N-methylmorpholine, diethylamine, dicyclohexylamine, procaine, dibenzylamine, N-benzyl-β-phenethylamine, 1-ephenamine, and N,N'-dibenzylethylenediamine. Preferred salts include pharmacologically acceptable salts, and salts with sodium are more preferred.

[0016] In the compound of the general formula [1], preferably, R 1 is a hydrogen atom; R 2 is a fluorine atom; R 3 is a hydrogen atom. A preferred compound is T-705 (favipiravir).

[0017] Alternatively, in the compound of the general formula [1], preferably, R 1 is a hydrogen atom; R 2 is a hydrogen atom; R 3 is a hydrogen atom. A preferred compound is T-1105.

[0018] The compound of the general formula [1] can be produced by combining methods known per se, for example, by the production method described in WO 00 / 10569.

[0019] A drug for treating coronavirus infection means a substance that is effective in treating or preventing coronavirus infection through some mechanism of action, such as inhibiting the proliferation of coronavirus within host cells, inhibiting the invasion of coronavirus into host cells, inhibiting the release of coronavirus outside host cells, activating the body's immune function, suppressing inflammation, or suppressing cytokine storm. Examples of such a drug include the substances listed below in (1) to (19). (1) Interferon (2) Nucleic acid analogs (3) Protease inhibitors (4) Furin convertase cleavage inhibitors (5) Reverse transcriptase inhibitors and / or RNA polymerase inhibitors (6) Neuraminidase inhibitor (7) Angiotensin II receptor blockers (8) Endosome fusion inhibitors and / or endosome alkalinizers (9) AAK1, GAK, and clathrin A, B, C (endocytosis) inhibitors (10) Hemagglutinin esterase inhibitors (11) Cytokine or inflammation inhibitors and regulators (12) Cathepsin B inhibitor (13) Cathepsin L inhibitor (14) Helicase nsp13 inhibitor (15) MBL2 gene agonist (16) TP53 inhibitors (17) Selective estrogen receptor modulators (18) Steroids (19) Other drugs

[0020] Examples of interferons include interferon α-2A, interferon α-2B, interferon α-n1, interferon α-n3, interferon β-1a, and interferon β-1b. Interferons can be produced by combining known methods, or commercially available ones can be used.

[0021] Examples of nucleic acid analogs include acyclovir, ganciclovir, ribavirin, and taribavirin. They may be produced by combining known methods, or commercially available products may be used. Nucleic acid analogs may be produced by combining known methods, or commercially available products may be used.

[0022] Examples of protease inhibitors include indinavir, nelfinavir, saquinavir, camostat, lopinavir-ritonavir combination drug (product name: Kaletra), epigallocatechin gallate, kaempferol-7-glucoside, mycophenolic acid, darunavir, mercaptopurine, disulfiram, nafamostat, and PF-07321332. Protease inhibitors can be produced by combining known methods, or commercially available products can be used.

[0023] Examples of furin convertase cleavage inhibitors include tenofovir disoproxil, dolutegravir, boceprevir, andrographolide, luteolin, and baicalein. Furin convertase cleavage inhibitors can be produced by combining known methods, or commercially available products can be used.

[0024] Examples of reverse transcriptase inhibitors and / or RNA polymerase inhibitors include remdesivir, sofosbuvir, dactinomycin, galidesivir, baloxavir marboxil, molnupiravir, sangibamycin, and AT-527. Reverse transcriptase inhibitors and / or RNA polymerase inhibitors may be produced by combining known methods, or commercially available products may be used.

[0025] Examples of neuraminidase inhibitors include oseltamivir and zanamivir. Neuraminidase inhibitors can be produced by combining known methods, or commercially available ones can be used.

[0026] Examples of angiotensin II receptor blockers include valsartan, telmisartan, losartan, irbesartan, azilsartan, olmesartan, and emodin. Angiotensin II receptor blockers can be produced by combining known methods, or commercially available products can be used.

[0027] Examples of endosome fusion inhibitors and / or endosome alkalizing agents include baicalin, chloroquine, hydroxychloroquine, Griffithsin, quinine, and lactoferrin. Endosome fusion inhibitors and / or endosome alkalizing agents can be produced by combining known methods, or commercially available products can be used.

[0028] Examples of AAK1, GAK, and clathrin A, B, and C (endocytosis) inhibitors include baricitinib, sunitinib, erlotinib, fedratinib, gefitinib, and silibinin. AAK1, GAK, and clathrin A, B, and C (endocytosis) inhibitors can be produced by combining known methods, or commercially available products can be used.

[0029] An example of a hemagglutinin esterase inhibitor is 3,4-dichloroisocoumarin. Hemagglutinin esterase inhibitors can be produced by combining known methods, or commercially available products can be used.

[0030] Examples of cytokine or inflammation inhibitors and regulators include ligustrazine, statins, melatonin, eplerenone, and methylprednisolone. Cytokine or inflammation inhibitors and regulators can be produced by combining known methods, or commercially available products can be used.

[0031] An example of a cathepsin B inhibitor is salvianolic acid B. Cathepsin B inhibitors can be produced by combining known methods, or commercially available products can be used.

[0032] Examples of cathepsin L inhibitors include MOL736, Chelidocystatin, astaxanthin, curcumin, and vitamin D. Cathepsin L inhibitors can be produced by combining known methods, or commercially available products can be used.

[0033] Examples of helicase nsp13 inhibitors include valsartan, bananin, iodobananin, vanillinbananin, eubananin, and silvestrol. Helicase nsp13 inhibitors can be produced by combining known methods, or commercially available inhibitors can be used.

[0034] Examples of MBL2 gene agonists include β-glucan and vitamin A. MBL2 gene agonists can be produced by combining known methods, or commercially available products can be used.

[0035] Examples of TP53 inhibitors include vitexin and gossypol. TP53 inhibitors can be produced by combining known methods, or commercially available products can be used.

[0036] Examples of selective estrogen receptor modulators include toremifene and equilin. The selective estrogen receptor modulators may be produced by combining known methods, or commercially available products may be used.

[0037] Examples of steroid drugs include ciclesonide and dexamethasone. Steroid drugs can be produced by combining known methods, or commercially available drugs can be used.

[0038] Other drugs include, for example, amantadine, foscarnet, triazavirine, umifenovir, rapamycin, everolimus, nitazoxanide, tizoxanide, caraphenol A, ivermectin, VIR-2703, tocilizumab, bamlanivimab, etesevimab, casirivimab / imdevimab, AZD7422, VIR-7831, VIR-7832, and BI 767551. Amantadine, foscarnet, triazavirine, umifenovir, rapamycin, everolimus, nitazoxanide, tizoxanide, caraphenol A, ivermectin, VIR-2703, tocilizumab, bamlanivimab, etesevimab, casirivimab / imdevimab, AZD7422, VIR-7831, VIR-7832, and BI 767551 may be produced by combining methods known per se, or commercially available products may be used.

[0039] In the present invention, a pyrazine derivative is used in combination with another therapeutic agent for coronavirus infection. The term "combination" includes administration of the pyrazine derivative and another therapeutic agent for coronavirus infection simultaneously, separately, or in a specific order (concomitant administration), and administration as a mixture (combined drug).

[0040] That is, "combined use" does not necessarily mean that the administration times of a pyrazine derivative or a salt thereof and another therapeutic drug for coronavirus infection are the same, but also includes administering a pyrazine derivative or a salt thereof and another therapeutic drug for coronavirus infection within a single administration schedule. The administration routes of a pyrazine derivative or a salt thereof and another therapeutic drug for coronavirus infection may be the same or different.

[0041] The quantitative ratio of the pyrazine derivative or its salt to the other therapeutic drug for coronavirus infection may be any ratio that allows the effects of both to be exhibited additively or synergistically, and preferably a quantitative ratio that allows the effects to be exhibited synergistically. Preferably, the quantitative ratio (molar ratio) of the pyrazine derivative or its salt to the other therapeutic drug for coronavirus infection is 1:500 to 500:1, more preferably 1:200 to 200:1, even more preferably 1:50 to 50:1, and still more preferably 1:10 to 10:1.

[0042] When using the pyrazine derivative or a salt thereof of the present invention and other therapeutic drugs for coronavirus infection, formulation adjuvants such as excipients, carriers, and diluents normally used in formulations may be appropriately mixed, and these can be made into the form of tablets, capsules, powders, syrups, granules, pills, suspensions, emulsions, liquids, powder preparations, suppositories, eye drops, nasal drops, ear drops, patches, ointments, injections, and the like according to conventional methods.

[0043] When used as a combination preparation, the pyrazine derivative or a salt thereof and another therapeutic drug for coronavirus infection may be mixed and homogenized during the above-mentioned formulation process, and then the mixture may be made into an appropriate formulation.

[0044] The route of administration of the therapeutic agent for coronavirus infection of the present invention is not particularly limited, and it can be administered intravenously, orally, intramuscularly, subcutaneously, by inhalation, nebulization, or other routes. In addition, the pyrazine derivative or a salt thereof may be administered simultaneously with other therapeutic agents for coronavirus infection or in a specific order.

[0045] The administration method, dosage, and frequency of administration of the pyrazine derivative or salt thereof of the present invention can be appropriately selected depending on the age, weight, and symptoms of the patient. Typically, for adults, 10 to 50,000 mg, or preferably 200 to 24,000 mg of the active ingredient pyrazine derivative or salt thereof is administered orally or parenterally (e.g., by injection, infusion, or rectal administration) once or several times a day. Administration is typically monitored, and repeated administration as needed is preferred. Specifically, for adults, 100 to 6,000 mg is administered once, twice, three, or four times a day (on the first day), and 100 to 6,000 mg is administered once, twice, three, or four times a day (on the second day and thereafter). It is preferable that the dose on day 1 is higher than the dose on days 2 and thereafter (loading dose), and it is preferable that the dose on day 1 is at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, or at least 5 times the dose on days 2 and thereafter. More specifically, 1000 to 5000 mg may be administered once, twice, three or four times a day (Day 1), or 400 to 2400 mg may be administered twice, twice, three or four times a day (Day 2 and thereafter). For adults, 1600 mg may be administered twice a day (Day 1) and 600 mg may be administered twice a day (Day 2 and thereafter), or 1800 mg may be administered twice a day (Day 1) and 800 mg may be administered twice a day (Day 2 and thereafter), or 1800 mg may be administered twice a day (Day 1) and 1000 mg may be administered twice a day (Day 2 and thereafter), and more preferably 1800 mg may be administered twice a day (Day 1) and 800 mg may be administered twice a day (Day 2 and thereafter). The interval between twice-daily administrations is preferably at least 4 hours after the first administration, and more preferably at least 6 hours after the second administration. The administration period is determined appropriately depending on the progression of symptoms, and can be selected from, for example, up to 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 1 month, 2 months, and 3 months. A maximum of 10 days, 13 days, 14 days, or 22 days is preferred, with a maximum of 14 days being more preferred. When administered for preventive purposes, it is preferable to administer continuously during the period when the risk of infection is high. Furthermore, it has been reported that the 50% effective concentration (EC50) of T-705, a pyrazine derivative of the present invention, against SARS-CoV-2 using Vero E6 cells is 61.88 μM (Wang M, Cao R, Zhang L, Yang X, Liu J, Xu M, et al. Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro. Cell Res. 2020;30:269-71.). This corresponds to 9.72 μg / mL, so it is preferable to administer the pyrazine derivative or a salt thereof so that the blood concentration is 9.72 μg / mL or higher. However, this does not apply to forms in which a synergistic effect can be expected in the present invention.

[0046] The administration method, dosage, and frequency of administration of the other coronavirus infection therapeutic agents of the present invention can be appropriately selected depending on the age, weight, and symptoms of the patient. It is preferable to select them based on the known clinical trial design and results. Specific examples include the following: For hydroxychloroquine, for example, 800 mg twice a day (day 1), 400 mg twice a day (from day 2 up to day 10), 1200 mg divided into four doses a day (for 5 to 10 days), 800 mg (as the sulfate) divided into two doses a day (day 1), 400 mg divided into two doses a day (days 2 to 5), 600 mg twice a day (day 1), 400 mg twice a day (days 2 to 7) can be administered. For lopinavir-ritonavir combination drugs, for example, lopinavir 200 mg / ritonavir 100 mg twice daily (7 to 10 days), lopinavir 400 mg / ritonavir 100 mg once daily (up to 14 days), lopinavir 400 mg / ritonavir 100 mg twice daily (up to 14 days), or 400 mg / ritonavir 200 mg twice daily (5 to 14 days) may be administered. For ciclesonide, for example, 320 μg of ciclesonide for inhalation may be administered every 12 hours (for a maximum of 14 days), or 200 μg of ciclesonide for inhalation may be administered every 12 hours (for a maximum of 14 days). For remdesivir, for example, 200 mg once daily (day 1), 100 mg once daily (days 2 to 10, can be extended up to day 13), 200 mg once daily (day 1), 100 mg once daily (days 2 to 5), or 400 mg twice daily (day 1), 400 mg once daily (2 to 11 days). For children weighing less than 40 kg, 5 mg / kg once daily (day 1), 2.5 mg / kg once daily (days 2 to 10, can be extended up to day 13) can be administered. Molnupiravir may be administered at a dose of, for example, 200 mg, 400 mg, 600 mg, 800 mg, 1000 mg, 1200 mg, 1400 mg, 1600 mg, 1800 mg, or 2000 mg twice daily for 5 days. Preferably, 800 mg is administered twice daily for 5 days.

[0047] The therapeutic agent for coronavirus infection of the present invention is useful for treatment such as the treatment or prevention of coronavirus infection.

[0048] As used herein, the term "coronavirus" refers to an RNA virus belonging to the Coronaviridae family of the Nidovirales order. Coronavirus infection refers to an infectious disease caused by a coronavirus. A "novel coronavirus" refers to a novel coronavirus that has been newly identified and reported since 2019, such as SARS-CoV-2, which was reported at the end of 2019. Infections caused by SARS-CoV-2 are also referred to as COVID-19. "Novel coronavirus" naturally includes mutated species and strains of known coronaviruses. "Coronavirus infection" naturally includes infections caused by novel coronaviruses, including COVID-19. [Example]

[0049] Next, the present invention will be described with reference to examples, but the present invention is not limited to these examples.

[0050] Test Example 1 The combined efficacy of pyrazine derivatives and remdesivir was tested in a viral cell infection model. Specifically, the antiviral effect was evaluated by detecting the cytopathic effect (CPE). In addition to the CPE method, the antiviral effect can also be evaluated by the viral antigen-antibody method or real-time polymerase chain reaction (PCR) of viral RNA extracted from viral culture supernatant.

[0051] T-705 was selected as the pyrazine derivative, remdesivir was selected as the drug for treating other coronavirus infections, and the novel coronavirus (SARS-CoV-2) was selected as the RNA virus.

[0052] (1) Cultivation of Vero E6 cells African green monkey kidney Vero E6 cells, which had been subcultured in 10% fetal bovine serum-supplemented Eagle's MEM / kanamycin 60 μg / mL (Eagle's MEM / kanamycin) medium under 5% carbon dioxide at 37°C, were detached by the ethylenediaminetetraacetic acid trypsin method and cultured at 2 × 10 cells / mL in 100 μL of the same medium. 4 The suspension containing 100 cells was seeded onto a 96-well plate and cultured overnight at 37°C under 5% carbon dioxide to obtain a monolayer of Vero E6 cells.

[0053] (2) SARS-CoV-2 infection and drug administration The test medium was Eagle's MEM / kanamycin medium supplemented with 2% fetal bovine serum. The culture supernatant of the Vero E6 cells obtained in (1) was removed, and the following (A) and (B) were added to each well and cultured at 37°C under 5% carbon dioxide for 2 hours. (A) 100 μL of SARS-CoV-2 solution prepared in test medium so that the final multiplicity of infection is 0.02 (B) 100 μL of test medium containing 0.5% DMSO containing combinations of T-705 and remdesivir at twice the set concentration. T-705 set concentration (μM): 0, 0.1, 0.3, 1, 3, 10, 30, 100, 300, 1000 Remdesivir target concentration (μM): 0, 0.1, 0.3, 1, 3, 10, 30, 100

[0054] Two hours after infection, the culture supernatant was removed from each well, and 100 μL of test medium containing 0.5% DMSO containing a combination of T-705 and remdesivir at a concentration 1x the set concentration was added. After drug addition, the cells were cultured at 37°C under 5% carbon dioxide for 2 days.

[0055] (3) Determination of cytopathic effect (CPE) CPE, which occurs with the proliferation of SARS-CoV-2, was determined using the following method.

[0056] After incubation, 25 μL of 100% formalin solution was added to each well to inactivate the virus and fix the cells. After allowing the plate to stand at room temperature for at least two hours, the aqueous solution was removed and the plate was gently washed with water. 50 μL of 0.02% methylene blue solution was added per well and the plate was then left to stand at room temperature for one hour. The aqueous solution was removed, the plate was gently washed with water, and the plate was air-dried. The absorbance (660 nm) was then measured using a microplate reader (Tecan). For the non-infected control, 100 μL of test medium was added instead of the SARS-CoV-2 solution, and the same procedures as for the test group were repeated to measure the absorbance.

[0057] The test was performed on two plates (8 cases per plate for the infected and non-infected controls) with 1 case per plate. The value obtained by subtracting the absorbance of the infected control from the absorbance of the non-infected control was used as the value for complete inhibition of viral growth, and the CPE inhibition rate for each test was calculated using the formula below.

[0058] CPE inhibition rate = 100 × [(absorbance when used alone or in combination) - (absorbance of infected control)] / [(absorbance of non-infected control) - (absorbance of infected control)]

[0059] The 50% CPE inhibitory concentration was calculated using the FORECAST function (linear regression method) of Microsoft Office Excel 2016.

[0060] By combining these drugs, a 50% inhibitory concentration was confirmed at a combination of a T-705 concentration lower than the 50% inhibitory concentration when T-705 was used alone and a remdesivir concentration lower than the 50% inhibitory concentration when remdesivir was used alone.

[0061] Similar tests will be conducted on the coronavirus infection treatment drugs listed in Table 1 other than remdesivir. By combining these drugs, the 50% inhibitory concentration will be confirmed at a combination of a T-705 concentration lower than the 50% inhibitory concentration when T-705 is used alone and a remdesivir concentration lower than the 50% inhibitory concentration when remdesivir is used alone. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0062] Test Example 2

[0063] T-705 was selected as the pyrazine derivative, remdesivir was selected as the treatment for other coronavirus infections, and the common cold coronavirus (HCoV-OC43), which belongs to the same betacoronavirus genus as the new coronavirus, was selected as the RNA virus.

[0064] (1) Culture of BHK-21 cells Hamster kidney BHK-21 cells were subcultured in 10% fetal bovine serum-supplemented EMEM / kanamycin 60 μg / mL (EMEM / kanamycin) medium at 37°C under 5% carbon dioxide conditions. They were detached by the ethylenediaminetetraacetic acid trypsin method and then resuspended in 100 μL of the same medium at 4 × 10 4 The suspension containing 100 cells was seeded onto a 96-well plate and cultured overnight at 37°C under 5% carbon dioxide to obtain a monolayer of BHK-21 cells.

[0065] (2) HCoV-OC43 infection and drug administration The test medium was EMEM / kanamycin medium supplemented with 2% fetal bovine serum. The culture supernatant of the BHK-21 cells obtained in (1) was removed, and the following (A) to (C) were added to each well. (A) 100 μL of test medium (B) The final infectious titer in the test medium was 4.0 × 10 3 TCID 50 50 μL of HCoV-OC43 solution prepared to (C) 50 μL of test medium containing 1% DMSO containing combinations of T-705 and remdesivir at 4-fold the set concentration. T-705 setting concentration (μM): 0, 1, 10,100,1000 Remdesivir concentration (μM): 0, 0.1, 0.3

[0066] After adding the drug, the cells were cultured under 5% carbon dioxide conditions at 33°C for 3 to 4 days.

[0067] (3) Determination of cytopathic effect (CPE) CPE observed during HCoV-OC43 proliferation was determined using the following method.

[0068] After incubation, 50 μL of 100% formalin solution was added to each well to inactivate the virus and fix the cells. After allowing the plate to stand at room temperature for at least two hours, the aqueous solution was removed and the plate was gently washed with water. 50 μL of 0.02% methylene blue solution was then added to each well and allowed to stand at room temperature for one hour. The aqueous solution was removed, the plate was gently washed with water, and then air-dried. The absorbance (660 nm) was then measured using a microplate reader (Tecan). For the uninfected control, 50 μL of test medium was added instead of the HCoV-OC43 solution, and the same procedures as for the test group were repeated to measure the absorbance.

[0069] The test was performed on two plates (six infected and non-infected controls) with one sample per plate. The value obtained by subtracting the absorbance of the infected control from the absorbance of the non-infected control was used as the value for complete inhibition of viral growth, and the CPE inhibition rate for each test was calculated using the formula below.

[0070] CPE inhibition rate = 100 × [(absorbance when used alone or in combination) - (absorbance of infected control)] / [(absorbance of non-infected control) - (absorbance of infected control)]

[0071] The 50% CPE inhibitory concentration was calculated using the FORECAST function (linear regression method) of Microsoft Office Excel 2016.

[0072] The CPE inhibition rates were compared between single-agent and combined use of these drugs. T-705 at 1000 μM inhibited CPE by 79%, while remdesivir at 0.1 or 0.3 μM inhibited CPE by 51% and 80%, respectively. Meanwhile, the combined use of T-705 at 1000 μM and remdesivir at 0.1 μM or 0.3 μM inhibited CPE by 92% and 102%, respectively.

[0073] The combined use of T-705 and remdesivir demonstrated a higher CPE inhibition rate than either T-705 or remdesivir alone, demonstrating a nearly 100% CPE suppression effect. These results also suggest that the combined use of T-705 with drugs that have a similar mechanism of action to remdesivir, i.e., reverse transcriptase inhibitors and / or other RNA polymerase inhibitors (e.g., molnupiravir), is also effective. [Industrial Applicability]

[0074] A therapeutic agent for coronavirus infection comprising a combination of a pyrazine derivative or a salt thereof and another therapeutic agent for coronavirus infection is useful in the field of the pharmaceutical industry.

Claims

[Claim 1] of the following general formula: 【Chemistry 1】 (In the formula, R １ and R ２ are the same or different and represent a hydrogen atom or a halogen atom; R ３ represents a hydrogen atom or an amino-protecting group) or a salt thereof and remdesivir, the amino protecting group is selected from an acyl group, an alkyloxycarbonyl group, an arylalkyloxycarbonyl group, an aryloxycarbonyl group, an arylalkyl group, an alkoxyalkyl group, an arylalkyloxyalkyl group, an arylthio group, an alkylsulfonyl group, an arylsulfonyl group, a dialkylaminoalkylidene group, an arylalkylidene group, a nitrogen-containing heterocyclic alkylidene group, a cycloalkylidene group, a diarylphosphoryl group, a diarylalkylphosphoryl group, an oxygen-containing heterocyclic alkyl group, and a substituted silyl group; Pharmaceutical compositions.

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