JP2020196704A - Prophylactic and/or therapeutic agent for influenza virus infection or corona virus infection - Google Patents

Abstract

To provide a novel medicine for preventing and/or treating influenza virus infection or corona virus infection.SOLUTION: According to the present invention, provided are an anti-influenza virus agent, an anti-corona virus agent and a prophylactic and/or therapeutic agent for influenza virus infection or corona virus infection, each comprising, as an active ingredient, one or more kinds of 15-membered ring macrolide compounds selected from the group consisting of compound 1 to compound 4, a salt thereof or a hydrate of the same.SELECTED DRAWING: Figure 1

Description

The present invention relates to novel prophylactic and / or therapeutic agents for influenza virus infections or coronavirus infections.

Influenza infectious diseases caused by the influenza virus are infectious diseases that repeat a large epidemic due to their strong transmission power even in modern times, and cause enormous damage to society. While various infectious diseases have been overcome, there are few drugs that are effective and highly safe against influenza viruses, and the emergence of resistant viruses against them is also regarded as a problem, and the development of new anti-influenza drugs is expected. ..

The influenza virus causes acute respiratory infections, the clinical symptoms of which include systemic symptoms such as sudden fever, headache, joint pain and general malaise, as well as various respiratory symptoms of colds such as rhinorrhea and cough. It is characterized by high heat of ℃ or higher. In healthy people, it usually heals in about 1 to 2 weeks, but in infants, the elderly, patients with chronic diseases of the respiratory, circulatory, and kidneys, patients with metabolic diseases such as diabetes, and patients with weakened immune function, etc. It is not uncommon for people to die from secondary infections caused by bacteria and pneumonia. In addition to local respiratory infections, extremely serious cases such as severe nervous system complications such as influenza encephalitis and encephalopathy have also been reported. In addition, gastrointestinal symptoms such as abdominal pain, nausea / vomiting, and diarrhea may be observed, and caution is required especially in children. A major feature to be noted in diagnosis is that fever and other systemic symptoms are more prominent than respiratory symptoms.

Currently, vaccination is considered to be the basic measure against influenza from a preventive point of view. Anti-influenza drugs other than vaccines include amantadine (pharmaceutical name: Symmetrel), which has M2 protein ion channel function inhibitory activity, oseltamivir (pharmaceutical name: Tamiflu) and zanamivir (pharmaceutical name: Relenza), which are neuraminidase inhibitors. It is only licensed. For example, oseltamivir has excellent performance as a pharmaceutical product with EC ₅₀ = 0.03 and CC ₅₀ > 100.

Infection of cells with a virus begins with the following process. First, the hemagglutinin protein of the virus (hereinafter, also referred to as "hemagglutinin") binds to the sialic acid receptor on the cell side, and the virus is taken up into the cell by endocytosis. Subsequently, the viral gene enters the cytoplasm by fusion of the viral membrane and the endosome membrane caused by acidification in the endosome. This fusion requires cleavage of the peptide bond at a specific sequence site of hemagglutinin by an endoprotease derived from a host cell, and this cleavage may expose the membrane fusion domain of hemagglutinin and cause fusion with the endosome membrane. It has been reported (Latest Medicine, 2004, Vol. 59, 215-222). After the infection is established, the virus proliferates inside the cell and buds from the infected cell in order to spread the infection to new cells / tissues. Cleavage of hemagglutinin and sialic acid receptors by virus-derived neuraminidase is essential for this budding. Existing anti-influenza drugs, oseltamivir and zanamivir, act during virus budding after this infection and cannot prevent the establishment of the initial infection. Furthermore, in recent years, the emergence of influenza viruses resistant to the above-mentioned neuraminidase inhibitors and M2 protein ion channel inhibitors has also been reported (Matsuzaki Y, et al., Virol J. 2010; 7:53). , Dong G, et al., PloS One. 2015; 10 (3): e0119115).

By the way, the present inventors have previously reported that the 16-membered ring macrolide antibiotic spiramycin or josamycin (leucomycin A3) is effective in treating fulminant pneumonia caused by influenza virus infection. (Japanese Patent Laid-Open No. 2012-020953). There is also a report that clarithromycin, a 14-membered ring macrolide antibiotic, suppresses the growth of influenza virus (Yamaya M, et al., J Pharmacol Exp Ther. 2010 Apr; 333 (1): 81- 90). However, there have been no reports so far that 15-membered ring macrolide antibiotics such as azithromycin have anti-influenza virus activity. Rather, even if azithromycin is further intraperitoneally administered to mice orally administered with oseltamivir, improvement in therapeutic effect in terms of survival rate, viral titer, cytokine level, etc. is observed as compared with administration of oseltamivir alone. There is a report that it was not (Fage C, et al., J med Virol. 2017; 89 (12): 2239-43).

Moreover, unlike influenza viruses, some coronaviruses are known to cause a variety of disease symptoms in humans. Such pathogenic coronaviruses include human coronavirus 229E, NL63, OC43, and HKU1, which cause cold syndrome, SARS-CoV, which causes severe acute respiratory syndrome (SARS), and Middle East respiratory syndrome (MERS). Known causes include MERS-CoV and 2019 new coronavirus (SARS-CoV-2) that causes new coronavirus infection (COVID-19). However, it is not known that 15-membered ring macrolide antibiotics act effectively against these coronaviruses.

It is generally known that viruses easily mutate amino acids in their constituent proteins and, in some cases, mutate into drug-resistant viruses that are ineffective. And the emergence of such drug-resistant viruses can be a serious clinical problem. In fact, as reported in Matsuzaki Y, et al., Virol J. 2010; 7:53, Dong G, et al., PloS One. 2015; 10 (3): e0119115, as described above, drug-resistant viruses The emergence exists as a real threat.

Therefore, an object of the present invention is to provide a novel drug that can be effectively used for preventing and / or treating an influenza infection or a coronavirus infection.

The present inventors have conducted diligent research in view of the above-mentioned prior art. In the process, surprisingly, some compounds were found to have anti-influenza virus activity. Then, based on this finding, the present invention has been completed.

That is, according to the first aspect of the present invention, one or more 15-membered ring macrolide compounds selected from the group consisting of the following compounds 1 to 4 or salts or hydrates thereof are used as active ingredients. The anti-influenza virus agent contained as is provided.

Further, according to the second aspect of the present invention, one or more 15-membered ring macrolide compounds selected from the group consisting of the above compounds 1 to 4 or salts or hydrates thereof are used as active ingredients. Provided are prophylactic and / or therapeutic agents for influenza virus infections, which are contained as.

Furthermore, according to the third aspect of the present invention, one or more 15-membered ring macrolide compounds selected from the group consisting of the above compounds 1 to 4 or salts or hydrates thereof, and pharmaceuticals. Provided are pharmaceutical compositions for the prevention and / or treatment of influenza virus infections that contain a generally acceptable carrier.

Further, according to the fourth aspect of the present invention, an effective amount of one or more 15-membered ring macrolide compounds selected from the group consisting of the above compounds 1 to 4 or salts or hydrates thereof. Are provided for the prevention and / or treatment of influenza virus infections, including administration to patients in need.

Furthermore, according to the fifth aspect of the present invention, one or more selected from the group consisting of the above compounds 1 to 4 for the prevention and / or production of a therapeutic agent for influenza virus infection. The use of a 15-membered ring macrolide compound or a salt or hydrate thereof is provided.

Further, according to the sixth aspect of the present invention, one or more 15-membered ring macrolide compounds selected from the group consisting of the above compounds 1 to 4 or salts or hydrates thereof are used as active ingredients. Provided are a prophylactic and / or therapeutic agent for coronavirus infections such as 2019 Coronavirus (SARS-CoV-2) infections.

Further, according to the seventh aspect of the present invention, an effective amount of one or more 15-membered ring macrolide compounds selected from the group consisting of the above compounds 1 to 4 or salts or hydrates thereof. Is provided for the prevention and / or treatment of coronavirus infections such as the 2019 Coronavirus (SARS-CoV-2) infection, which comprises administering to a patient in need.

It was also found that among Compounds 1 to 4, Compounds 2 to 4 are novel compounds. Therefore, according to another embodiment of the present invention, a compound represented by the chemical formula (1) described later or a salt or hydrate thereof is also provided, and an anti-influenza virus agent containing these compounds and the like as an active ingredient. Alternatively, pharmaceuticals such as anti-coronavirus agents are also provided.

According to the present invention, novel medicines that can be effectively used for the prevention and / or treatment of influenza infection or coronavirus infection can be provided.

FIG. 1 shows the viral titer (FIG. 1a) and virus in the culture medium after administration of azithromycin (AZM) under four different conditions to A549 cells infected with human influenza A (H1N1) pdm09 virus. It is a graph which shows the result of having measured the expression level (FIG. 1b) of the matrix protein 1 (M1) gene in the cell by the virus plaque assay and the quantitative PCR method, respectively. Figure 2a, the infection and simultaneous treatment is a graph showing a result of calculating IC ₅₀ values AZM on the growth of the child virus infecting a A549 cell. FIG. 2b is a graph showing the results of an MTT assay under non-infectious or infectious conditions using a wide range of concentrations to determine the concentration at which AZM is toxic to host A549 cells. Is. Figure 3 is a graph showing the results of IC ₅₀ values were calculated based on the expression status of viral M1 genes of A549 cells. FIG. 4 is a photograph and graph showing the results of examining whether AZM inhibits the binding of viral hemagglutinin (HA) to sialic acid (SA) on red blood cells (RBC) by hemagglutinin inhibition assay. FIG. 5 shows the adhesion or internal migration stage of a virus infecting A549 cells for the purpose of measuring the effect of azithromycin (AZM) on the adhesion stage (FIG. 5a) or internal migration stage (FIG. 5b) in virus infection. It is a graph which shows the result of having extracted the total RNA from A549 cells after incubating AZM to synthesize cDNA, and measured the expression level of virus M1 and nucleoprotein (NP) by the quantitative PCR method. FIG. 6 is a graph showing the results of experiments conducted with the aim of demonstrating the hypothesis that AZM can inhibit the repetitive cycle of viral infection and proliferative growth of offspring. 6a and 6b show A549 cells infected with influenza A (H1N1) pdm09 virus, the cells cultured for 10 hours in the presence or absence of AZM, the titer of the offspring virus in culture and in the host cells. It is a graph which confirmed that the expression level of M1 of the virus was the same level in the presence and absence of AZM, respectively. In addition, FIG. 6c shows that the culture supernatant containing the germinated virus was collected, and the newly prepared A549 cells were infected in the presence or absence of AZM, and the virus-infected A549 cells were not added with AZM. It is a graph which shows the result of having compared the M1 expression level in the A549 cell cultured in the culture medium containing a child virus in the presence and absence of AZM after culturing in the culture medium of AZM for 7 hours. FIG. 7a is a protocol showing a method of nasally infecting mice with influenza A (H1N1) pdm09 virus. FIG. 7b is a graph showing the results of measuring the expression levels of viral M1 and NP genes from total RNA extracted from lung tissue after nasal administration of azithromycin (AZM) to mice by a quantitative PCR method. In addition, FIG. 7c is a graph comparing the rectal body temperature and body weight of the mice in this experiment. FIG. 8 uses azithromycin (AZM) and Compounds 2 to 4 and Compounds 5 to 7 as negative controls (although Compounds 5 and 6 have weak inhibitory activity), as shown in FIG. 1a, “simultaneously with infection”. It is a graph which shows the result of having performed the same experiment as shown in "processing". 9a-9c show the virus titers in the culture medium after administration of Compounds 2 to 4 under four different conditions to A549 cells infected with human influenza A (H1N1) pdm09 virus. It is a graph which shows the result measured by the virus plaque assay. Figure 10 uses the compound 2 Compound 4 showed anti-influenza virus activity 8, the same experiment as that shown in Figure 2a, is a graph showing the results of measurement IC ₅₀ values. FIG. 11 is a graph showing the results of conducting an experiment (MTT assay) similar to that shown in FIG. 2c using Compounds 2 to 4 showing anti-influenza virus activity in FIG. FIG. 12 shows the results of examining whether Compounds 2 and 4 inhibit the binding of viral hemagglutinin (HA) to sialic acid (SA) on erythrocytes (RBC) by a hemagglutinin inhibition assay. It is a photograph and a graph shown together with the result of AZM shown. FIG. 13 shows the adhesion or internal migration of a virus infecting A549 cells for the purpose of measuring the effect of Compounds 2 and 4 on the adhesion step (FIG. 13a) or internal migration step (FIG. 13b) in virus infection. After incubating each compound in the step, total RNA was extracted from A549 cells to synthesize cDNA, and the result of measuring the expression level of M1 of the virus by the quantitative PCR method was combined with the result of AZM shown in FIG. It is a graph shown. Figure 14, for azithromycin (AZM), except that the concentration of the dosing agent was changed to 68μM a _{IC 50} of AZM from 200 [mu] M, by the same method as the experiment shown in FIG. 1, and clarithromycin (CAM) It is a graph which shows the result of having performed the comparison experiment of.

The first embodiment of the present invention contains one or more 15-membered ring macrolide compounds selected from the group consisting of the above compounds 1 to 4 or salts or hydrates thereof as an active ingredient. It is an anti-influenza virus agent. In addition, the second embodiment of the present invention contains one or more 15-membered ring macrolide compounds selected from the group consisting of the above compounds 1 to 4 or salts or hydrates thereof as an active ingredient. It is a prophylactic and / or therapeutic agent for influenza virus infections. As described above, there has been no finding that the above active ingredient is effective in the prevention and / or treatment of influenza virus infection, and it is a novel finding first found by the inventors of the present application.

The active ingredient of the agent according to the first and second embodiments of the present invention is a 15-membered ring macrolide compound selected from the group consisting of the above compounds 1 to 4 or a salt or hydrate thereof. One or more of them are used. Of these, compound 1 is a 15-membered ring macrolide compound known as "azithromycin". In Japan, azithromycin hydrate is marketed under the trade name of "Zithromax (registered trademark)", and its indications are deep skin infection, lymphatic / lymphadenitis, pharyngeal / laryngitis, tonsillitis, etc. Acute bronchitis, pneumonia, etc. However, azithromycin is not known to have anti-influenza virus activity. Examples of the salts of the above compounds 1 to 4 include those in which an arbitrary cationic group such as an amino group is salt-formed with an arbitrary anion. The number of hydrated waters in the hydrates of the above compounds 1 to 4 is not particularly limited, and any hydrate may be in a form that can exist stably.

Of Compounds 1 to 4, from the viewpoint of anti-influenza virus activity, Compound 1 (AZM), Compound 3 or Compound 4 is preferably used, Compound 1 (AZM) or Compound 3 is more preferably used, and Compound 1 (AZM) is used. AZM) is particularly preferably used.

The type of influenza virus targeted by the anti-influenza virus agent according to the present invention is not particularly limited, and conventionally known influenza viruses and new types of influenza viruses that may occur in the future may be targeted. Among them, it is preferable to target influenza A virus, it is preferable to target H1N1 virus, and human influenza A (H1N1) pdm09 virus, which is a new type of influenza virus that caused a pandemic in 2009, is targeted. Is most preferable. Furthermore, it is preferable to target new influenza viruses that may spread in the future.

Further, based on the above-mentioned novel findings, according to the present invention, an anti-coronavirus agent is also provided in the same manner as the above-mentioned anti-influenza virus agent. Specifically, first, the 2019 new model containing one or more 15-membered ring macrolide compounds selected from the group consisting of the above compounds 1 to 4 or salts or hydrates thereof as an active ingredient. A prophylactic and / or therapeutic agent (sixth form) for coronavirus infections such as coronavirus (SARS-CoV-2) infections is provided. Similarly, according to the present invention, an effective amount of one or more 15-membered ring macrolide compounds selected from the group consisting of the above compounds 1 to 4 or salts or hydrates thereof is required. Also provided are methods of preventing and / or treating coronavirus infections, such as 2019 Coronavirus (SARS-CoV-2) infections, which include administration to a patient (seventh form).

The type of coronavirus targeted by the anti-coronavirus agent according to the present invention is not particularly limited, and a conventionally known coronavirus or a new type of coronavirus that may occur in the future may be targeted. Such pathogenic coronaviruses include human coronaviruses 229E, NL63, OC43, and HKU1, which cause cold syndrome, SARS-CoV, which causes severe acute respiratory syndrome (SARS), and Middle East respiratory syndrome (MERS). Examples include MERS-CoV that causes it, and 2019 new coronavirus (SARS-CoV-2) that causes the new coronavirus infection (COVID-19). Among them, it is preferable to target SARS-CoV, MERS-CoV or SARS-CoV-2, and it is most preferable to target SARS-CoV-2, which is a new type of coronavirus that caused a pandemic in 2019-2020. preferable. Furthermore, it is preferable to target the new coronavirus that may become epidemic in the future.

Anti-influenza virus agent according to the first form of the present invention, preventive and / or therapeutic agent for influenza virus infection according to the second form, prevention and / or prevention of coronavirus infection according to the sixth form of the present invention. Alternatively, the therapeutic agent is intended to be administered to a patient infected with or at risk of being infected with influenza virus or corona virus. In general, infected patients show symptoms such as sneezing, runny nose, stuffy nose, cough, sputum, chills, fever, headache, and sore throat. Therefore, in addition to the agent of the present invention, a symptomatic agent is administered (combined use). ) May. In addition to being used in combination, it can also be blended with the agent of the present invention to form a mixture (composition).

As an example of a symptomatic drug that can be administered together with the agent of the present invention, antitussive analgesics such as aspirin, aspirin aluminum, sazapyrin, etenzamid, salicylamide, ibprofen, acetaminophen, isopropylantipyrin; caffeine, anhydrous caffeine, benzo Central nervous system stimulants such as sodium caffeine acid; analgesics such as bromvalerylurea and allylisopropylacetylurea; chlorhexidine maleate, diphenhydramine, diphenhydramine salicylate, cremastine fumarate, carbinoxamine maleate, mekitadine, alimemazine tartrate, diphenylpyridinium hydrochloride , Antihistamines such as triprolysine hydrochloride; anti-inflammatory agents such as lysozyme chloride, bromeline, therapeptase, semi-alkali proteinase, glycyrrhizinic acid, dipotassium glycyridinium, ammonium glycyridinium, glycyrrhetinic acid, sodium azulene sulfonate; dihydrocodein phosphate, phosphorus Codein acid, noscapine hydrochloride, noscapine, dextrometholphan hydrobromide, dextrometholphan phenolphthaline salt, dimemorphan phosphate, tipepidin hibenzate, tipepidin citrate, epradinone hydrochloride, methylephedrine, methylephedrine hydrochloride, methoxyphenamine hydrochloride , Antitussives such as trimetokinol hydrochloride, phenylpropanolamine hydrochloride; expectorants such as L-ethylcysteine hydrochloride, potassium guayacol sulfonate, potassium cresolate, guayphenesin, bromhexidine hydrochloride, carbocysteine; bronchi such as theophylline, aminophyrin, diprophylline Analgesics; anti-acetylcholine agents such as veradonna (total) alkaloid, veradonna extract, isopropamide iodide, scopolamine hydrobromide, funnel extract, butylscopolamine bromide, methylbenactidium bromide, thymepidium bromide, pyrenzepine; cetylpyridinium , Cetylpyridinium chloride, popidone iodine, chlorhexidine gluconate, benzalkonium chloride, decalinium chloride, timol, iodine / potassium iodide, phenol, chlorhexidine hydrochloride, cleosort, benzethonium chloride and other bactericidal disinfectants; dibucaine hydrochloride, ethyl aminobenzoate , Lidokine, lidocaine hydrochloride, local anesthetics such as oxidine; vitamin A, liver oil, vitamin B ₁ , vitamin B ₂ , vitamin B ₆ , vitamin B ₁₂ , vitamin Vitamin preparations such as C, calcium ascorbate, vitamin D, vitamin E, tocopherol calcium succinate; pantothenic acid, pantenol, sodium pantothenate, calcium pantothenate, pantetin, nicotinic acid, nicotinic acid amide, glucuronic acid, glucuronolactone , Aminoethyl sulfonic acid, biotin, γ-orizanol and other metabolic components; Examples include, but are not limited to, extracts of biotins (extracts, tinctures, etc.). The symptomatic drug may contain a single component, or may be used in combination or in combination of two or more kinds.

As the route of administration of the agent according to the present invention, either systemic administration or topical administration can be selected. At this time, an appropriate administration route is selected according to the disease / symptom and the like. The agent according to the present invention can be administered by any of the nasal route, the oral route, and the parenteral route as transmucosal administration. Examples of the non-nasal route and the parenteral route include normal intravenous administration, intraarterial administration, and subcutaneous, intradermal, and intramuscular administration. Furthermore, it is also possible to carry out transdermal administration. Intranasal administration may preferably be employed.

The dosage form of the agent according to the present invention is not particularly limited, and various dosage forms such as an aerosol agent for nasal administration and tablets, capsules, powders, granules and pills for oral administration. , Liquids, emulsions, suspensions, solutions, liquor, syrups, extracts, or elixirs. Parenteral preparations include, for example, injections such as subcutaneous injections, intravenous injections, intramuscular injections, or intraperitoneal injections; transdermal or patches, ointments or lotions; tongue for oral administration. It can be, but is not limited to, a lotion, an oral patch; and a suppository. These formulations can be produced by known methods commonly used in the formulation process. Further, the agent according to the present invention may be in a long-acting or sustained-release dosage form.

When preparing an aerosol agent for nasal administration, a lubricant, a binder, a preservative, a preservative, a odorant, an excipient and the like can be added. For example, talc, stearic acid and sodium salt, calcium salt and other salts, carplex, etc. as lubricants, starch, dextrin, etc. as binders, citric acid, glycine, etc. as pH adjusters, ascorbic acid, etc. as preservatives, preservatives Examples of the agent include paraoxybenzoic acid esters, benzalkonium chloride, phenol, chlorobutanol and the like, menthol and citrus fragrance as a preservative, and cellulose acetate as an excipient.

When preparing an oral solid preparation, it is usual to add excipients and, if necessary, binders, disintegrants, lubricants, colorants, flavoring agents, and odorants to the active ingredient. According to the method, tablets, coated tablets, granules, powders, capsules and the like can be produced. Such additives may be those commonly used in the art, for example, excipients include lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, and. As the binder, water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl starch, methyl cellulose, ethyl cellulose, shelac, calcium phosphate, polyvinylpyrrolidone, etc. , Dry starch, sodium alginate, canten powder, sodium hydrogen carbonate, calcium carbonate, sodium lauryl sulfate, monoglyceride stearate, and lactose as disintegrants, and purified talc, stearate, borosand, etc. as lubricants. And polyethylene glycol and the like, and examples of the flavoring agent include sucrose, orange peel, citric acid, and tartaric acid.

When preparing an oral liquid preparation, a flavoring agent, a buffering agent, a stabilizer, a odorant and the like can be added to the active ingredient to produce an oral liquid preparation, a syrup agent, an elixir agent and the like by a conventional method. In this case, the above-mentioned flavoring agent can be used. In addition, examples of the buffer include sodium citrate, and examples of the stabilizer include tragant, gum arabic, and gelatin.

When preparing an injection, add a pH regulator, buffer, stabilizer, isotonic agent, local anesthetic, etc. to the active ingredient, and use a conventional method to make subcutaneous, intramuscular, and intravenous injections. Can be manufactured. Examples of the pH adjuster and buffer in this case include sodium citrate, sodium acetate, sodium phosphate and the like. Stabilizers include sodium metabisulfite, ethylenediaminetetraacetic acid (EDTA), thioglycolic acid, thiolactic acid and the like. Examples of the local anesthetic include procaine hydrochloride and lidocaine hydrochloride. Examples of the tonicity agent include sodium chloride and glucose.

When preparing suppositories, for the active ingredient, a carrier known in the art such as polyethylene glycol, lanolin, cocoa butter, and fatty acid triglyceride, and optionally Tween® After adding such a surfactant or the like, it can be produced by a conventional method.

When preparing an ointment, a base, a stabilizer, a wetting agent, a preservative, etc., which are usually used as an active ingredient, are blended as necessary, and are formulated by mixing or the like by a conventional method. Examples of the base include liquid paraffin, white petrolatum, beeswax, octyldodecyl alcohol, paraffin and the like. Preservatives include methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate and the like.

When preparing a patch, the ointment, cream, gel, paste, or the like may be applied to a normal support by a conventional method. Suitable supports include woven fabrics made of cotton, rayon, and chemical fibers, non-woven fabrics and films or foam sheets such as soft vinyl chloride, polyethylene, and polyurethane.

The amount of the active ingredient contained in the agent according to the present invention can be appropriately determined depending on the dose range of the active ingredient, the number of doses, and the like.

The dose range is not particularly limited, and the efficacy of the contained ingredients, the form of administration, the route of administration, the type of disease, the nature of the subject (weight, age, medical condition and the use of other drugs, etc.), and the doctor in charge It can be set as appropriate according to the judgment. Generally, an appropriate dose is preferably in the range of, for example, about 0.01 μg to 100 mg, preferably about 0.1 μg to 1 mg per 1 kg of the body weight of the subject. However, these dose changes can be made using common routine experiments for optimization well known in the art. The above dose can be administered once to several times a day.

From another point of view, one or more 15-membered ring macrolide compounds or salts or hydrates thereof selected from the group consisting of the above-mentioned compounds 1 to 4 as the active ingredient of the agent according to the present invention. It can be used in methods of preventing and / or treating influenza virus infections or coronavirus infections. Such a method can be carried out, for example, by administering to a subject (patient) a pharmaceutical composition containing the above-mentioned active ingredient together with a pharmaceutically acceptable carrier by an appropriate route of administration. The administration route and dose (effective amount) at this time can be appropriately set by those skilled in the art by taking into consideration the above-mentioned description of the drug and the common general knowledge as of the filing of the present application. From yet another point of view, one or more 15-membered ring macrolide compounds or salts or hydrates thereof selected from the group consisting of the above-mentioned compounds 1 to 4 are infected with influenza virus or coronavirus. It can also be used in the manufacture of prophylactic and / or therapeutic agents for disease.

The acquisition route of the 15-membered ring macrolide compound represented by the above-mentioned compounds 1 to 4 or a salt or hydrate thereof is not particularly limited, and if a commercially available product is available, the commercially available product is purchased. Alternatively, it may be synthesized by itself with reference to conventionally known findings. Here, as described above, among Compounds 1 to 4, Compounds 2 to 4 are novel compounds provided for the first time in the present application. According to the present invention, as a novel compound containing these compounds 2 to 4, a compound represented by the following chemical formula (1) or a salt or hydrate thereof is provided.

In the above chemical formula (1), X represents a hydrogen atom or an acyl group having 1 to 4 carbon atoms. Here, examples of the acyl group having 1 to 4 carbon atoms include a formyl group, an acetyl group, a propionyl group, and a butyryl group. Among them, as X, a hydrogen atom, an acetyl group or a propionyl group is preferable, and a hydrogen atom or an acetyl group is particularly preferable.

In the above chemical formula (1), Y ¹ is represented by a hydrogen atom or -C (= O) -NH- (CH ₂ ) _n- C≡CH (where n is an integer of 1 to 4). Represents a group. Here, when Y ¹ is a group represented by −C (= O) -NH- (CH ₂ ) _n −C≡CH, n is preferably an integer of 1 to 3, and n is 1 or 2 is more preferable, and n is particularly preferably 1. Further, from the viewpoint of anti-influenza virus activity, it is most preferable that Y ¹ is a hydrogen atom.

In the above chemical formula (1), Y ² and Y ³ are hydrogen atoms or -C (= O) -NH- (CH ₂ ) _n- C≡CH (where n is an integer of 1 to 4). It represents a group represented, or together, forms a group represented by the following chemical formula (2). The chemical formula (1) is represented by a group represented by -C (= O) -NH- (CH ₂ ) _n- C≡CH (where n is an integer of 1 to 4) or a chemical formula (2). Includes one of the groups to be. Here, when Y ² or Y ³ is a group represented by −C (= O) -NH- (CH ₂ ) _n −C≡CH, n is preferably an integer of 1 to 3. Is more preferably 2 or 3, and n is particularly preferably 3.

Here, in the above chemical formula (2), * represents a binding site. Among them, from the viewpoint of anti-influenza virus activity, it is preferable that Y ² and Y ³ together form a group represented by the above chemical formula (2), and one of them is −C (= O) −. It is more preferable that the group represented by NH- (CH ₂ ) _n- C ≡ CH and the other is a hydrogen atom, and Y ² is -C (= O) -NH- (CH ₂ ) _n -C ≡ CH. Y ³ group represented by is more preferably a hydrogen atom.

Hereinafter, an example of a method for producing the compound represented by the chemical formula (1) will be briefly described (see the production example described later).

Among the compounds represented by the chemical formula (1), Y ² is a group represented by -C (= O) -NH- (CH ₂ ) _n- C ≡ CH, for example, compound 4, and Y ³ is a hydrogen atom. compounds are the compounds 1 (AZM) and compound _{O = C = N- (CH 2} ) at heated under reflux conditions to a ^{compound Y 1} is acylated in one isocyanate compound represented by n -C≡CH It can be synthesized by reacting. The isocyanate compound represented by O = C = N- (CH ₂ ) _n- C≡CH bases the corresponding carboxylic acid HO-C (= O)-(CH ₂ ) _n- C≡CH. It can be obtained via acid azide by reacting with diphenylphosphoryl azide (DPPA) while heating and refluxing under sexual conditions.

Further, a compound such as compound 3 in which Y ² and Y ³ are combined to form a group represented by the chemical formula (2) is a compound in which X is acylated in compound 1 (AZM) or compound 1. It can be synthesized by reacting trimethylborozine under heating and refluxing. Then, using the compound in which Y ² and Y ³ thus obtained together to form a group represented by the chemical formula (2) is further used as a raw material, carbonyldiimidazole (CDI), propargylamine and the like are used. The group represented by -C (= O) -NH- (CH ₂ ) _n- C ≡ CH can be introduced into Y ¹ by reacting with the alkynyl amine of. The Y ² and Y ³ can then be converted to hydroxy groups by heating in the presence of silica.

Hereinafter, the present invention will be described in more detail with reference to Examples and the like, but the technical scope of the present invention is not limited thereto.

≪Manufacturing example≫
[Production Example 1: Synthesis of 5- (1-pentanyl) isocyanate (1)]

Diphenylphosphoryl azide (DPPA) (1.19 mL, 5.51 mmol) and TEA (0.77 mL, 5.51 mmol) in a solution of 5-caproic acid (600 mg, 5.35 mmol) in benzene (26.8 mL). Was added. After stirring at 85 ° C. for 2 hours, the reaction mixture was cooled to room temperature to obtain the crude product (1) (about 0.18 M) in benzene. This solution was used in the next step without purification.

[Production Example 2: Synthesis of Compound 4]

Azithromycin (AZM) (3.0 g, 4.01 mmol) is added to a benzene solution (about 0.18 M) of crude 5- (1-pentanyl) isocyanate (1) (~ 4.40 mmol) at room temperature to react. The mixture was heated to 80 ° C. After stirring for 105 min, the reaction mixture was cooled to room temperature, then quenched by the addition of saturated aqueous _NH 4 Cl (50 mL), and neutralized to pH7.0 with saturated aqueous NaHCO ₃ (28 mL). The mixture was extracted with ethyl acetate (80 mL x 2), the organic layers were combined, dried over Na ₂ SO ₄ , filtered and concentrated.

The crude product was purified by flash column chromatography (CHCl ₃ / MeOH / NH ₃ = 20/1 / 0.1) using silica gel to obtain compound 4 (1.42 g, 41%) as a colorless solid. ..

[Production Example 3: Synthesis of Compound 3]

2'-O-Ac-AZM was synthesized according to the previous report (Eur. J. Med. Chem. 2009, 44, 4010-4020).

To a solution of 2'-O-Ac-AZM (2.20 g, 2.78 mmol) was added trimethylborozine (0.055 mL, 0.391 mmol) at room temperature and the reaction mixture was heated to 120 ° C. .. After stirring for 2 hours, the mixture was cooled to room temperature and then concentrated under reduced pressure to give a nearly pure product (Compound 3) (2.27 g, equivalent) as a colorless powder.

[Production Example 4: Synthesis of Compound 2]

Carbonyldiimidazole (CDI) (3.0 g, 8.34 mmol) was added to a solution of compound 3 (2.27 g, 2.78 mmol) in THF (28.0 mL) at room temperature and the reaction mixture was added to 60 ° C. It was heated. After stirring for 2 hours and 45 minutes, propargylamine (1.8 mL, 27.8 mmol) was added to the reaction mixture, and the obtained mixture was further stirred at 60 ° C. for 24 hours. The mixture was then cooled to room temperature and concentrated under reduced pressure to give the crude product, which was used in the next step without purification.

SiO ₂ (2.8 g) was added to a solution of the crude product in MeOH (28.0 mL). The mixture was warmed to 50 ° C., stirred for 21 hours and then cooled to room temperature. This MeOH solution is concentrated and the residue is purified by flash column chromatography using silica gel (CHCl ₃ / MeOH / NH ₃ = 60/1 / 0.1 to 10/1 / 0.1) to be a colorless solid. Compound 2 (1.2 g, 52% in 3 steps) was obtained.

[Statistical analysis]
All experimental data were analyzed using GraphPrism7.02 by Mann-Whitney U-test (MWU), one-way (one-way) or two-way (two-way) analysis of variance (ANOVA).

≪Experimental method using azithromycin (AZM)≫
[Obtaining route for macrolide compounds]
For all experiments, azithromycin anhydride (AZM) purchased from Tokyo Chemical Industry Co., Ltd. was used.

[Cell culture]
Human lung cancer (A549) cells and MDCK (Madin-Darby canine kidney) cells at 37 ° C. in Dalveco supplemented with 10% fetal bovine serum (FBS), 20 μM / mL L-glutamine and 100 μg / mL penicillin and streptomycin. Cultured to 100% confluent in Modified Eagle's Medium (DMEM) or Minimal Essential Medium (MEM) (Sigma Life Sciences).

[Influenza virus]
As the human influenza A (H1N1) pdm09 (A / California / 7/2009 (H1N1)) virus, a virus managed by the National University Corporation Chiba University and A-CLIP Research Institute Co., Ltd. was used. MDCK cells were infected with this virus and cultured for 24 hours. Then, the virus titer in the culture solution was measured by the viral plaque assay described later, and the cells were divided into small portions and stored at −80 ° C. or −150 ° C. until use.

(A) Preparation of influenza A (H1N1) pdm09 virus when infecting host cells The required titer is obtained by thawing the influenza A (H1N1) pdm09 virus stored at -80 ° C or -150 ° C. The virus infected human lung cancer (A549) cells.

(B) Preparation of influenza A (H1N1) pdm09 virus compatible with mice Influenza A (H1N1) pdm09 virus was anesthetized with isoflurane in 8-week-old female mice with 1 × 10 ⁴ plaque forming units (pfu) human influenza A. (H1N1) pdm09 virus was nasally infected. After 4 days, lung tissue was homogenized in 1.5 mL phosphate buffered saline (PBS). After spinning down at 8000 rpm for 5 minutes, the supernatant was collected and diluted 3 times in RPMI 1640 medium supplemented with 2% FBS as the first passage virus for the next passage. A 30 μL aliquot was used for the second inoculation and the above step of substituting for mice was repeated 10 times. The last passed virus was used as a mouse-compatible virus in the following animal experiments.

[Different azithromycin treatments for viral infections in host cells]
AZM was dissolved in ethanol to adjust the final concentration of ethanol in DMEM to 0.2%. Confluent monolayer A549 cells in a 6-well plate were infected with human influenza A (H1N1) pdm09 virus at multiplicity of infection (MOI) 1 under four different treatment conditions: The concentration of AZM was set to 200 μM.
(I) Post-infection treatment: A549 cells were infected with the virus at 35 ° C. for 1 hour. After infection, cells were washed with PBS and cultured in 2 mL of supplemented DMEM medium supplemented with or without AZM at 37 ° C. for 48 hours.
(Ii) Cell pretreatment: Host cells were pretreated at 37 ° C. for 1 hour using 300 μL of AZM-added or AZM-free non-supplemented DMEM medium. After removing the medium, the cells were washed with PBS and infected with the virus at 35 ° C. for 1 hour. The cells were then washed with PBS and cultured in 2 mL of supplemented DMEM medium without AZM at 37 ° C. for 48 hours.
(Iii) Virus pretreatment: Virus was pretreated at 37 ° C. for 1 hour using 300 μL of AZM-added or AZM-free non-supplemented DMEM medium. After this pretreatment, A549 cells were infected with the virus at 35 ° C. for 1 hour. The cells were then washed with PBS and cultured in 2 mL of supplemented DMEM medium without AZM at 37 ° C. for 48 hours.
(Iv) Simultaneous treatment with infection: 300 μL of AZM-added or AZM-free non-supplemented DMEM medium was mixed with the virus, and A549 cells were rapidly infected with the virus at 35 ° C. for 1 hour. After infection, cells were washed with PBS and cultured in 2 mL of supplemented DMEM medium supplemented with or without AZM at 37 ° C. for 48 hours.

Viral titers in culture and intracellular expression levels of the viral matrix protein 1 (M1) gene were measured by viral plaque assay and quantitative PCR, respectively.

[Plaque assay as a virus titer evaluation method]
Confluent monolayer MDCK cells were serially diluted with the culture medium collected from each experiment and infected at 35 ° C. for 1 hour. After removing this culture, cells are washed with PBS and eagle containing 0.8% agarose, 40 mM HEPES, 0.15% sodium bicarbonate, 2 mM L-glutamine, 2 μg / mL trypsin and 50 μg / mL gentamicin. Overlaid with minimal essential medium (EMEM). After incubating at 37 ° C. for 48 hours, cells were fixed with 10% formaldehyde and then stained with 0.1% crystal violet solution to count viral plaques.

[Calculation of 50% inhibition concentration (IC ₅₀ )]
The 50% inhibitory concentration of AZM against viral growth was evaluated by the method described in the column of different azithromycin treatments for viral infections in host cells described above (iv; co-treatment with infection). The virus was premixed in 300 μL of non-supplemented DMEM medium containing various concentrations of AZM up to 600 μM, and the host cells, A549 cells, were rapidly infected at 35 ° C. for 1 hour. The cells were then washed with PBS and cultured in supplemented DMEM medium without AZM at 37 ° C. for 48 hours. The titer of the child virus in culture was measured by viral plaque assay, IC ₅₀ values were calculated.

[Evaluation of cytotoxicity]
Cytotoxicity of AZM to A549 cells using Cell Growth Kit I (Roche) according to the manufacturer's instructions MTT [3- (4,5-dimethylthiazole-2-yl) -2,5-diphenyltetrazolium bromide ] Evaluated by assay. Cells were incubated at 35 ° C. for 1 hour in 300 μL of non-supplemented DMEM medium containing viruses (1 MOI) in the presence or absence of various concentrations of AZM up to 600 μM. After treatment, cells were washed with PBS and cultured in supplemented DMEM medium without AZM at 37 ° C. for 48 hours. The medium was removed, 1 mL of DMEM containing MTT labeling reagent (0.5 mg / mL) was added, and the mixture was incubated for 3 hours. Then 1 mL of the solubilized solution was added and incubated at 37 ° C. for 14 hours. The solubilized formazan product was measured by spectrophotometry using an iMark microplate reader (Bio-Rad).

[Evaluation of AZM inhibitory effect on germinated virus]
Influenza A (H1N1) pdm09 virus (1MOI) was infected with A549 cells at 35 ° C. for 1 hour and cultured in the presence or absence of AZM (200 μM). After culturing for 10 hours, the gene expression of the virus in the cells and the titer of the germinated virus in the culture medium were measured by quantitative PCR and plaque assay, respectively. The collected culture broth containing the offspring virus was used to infect newly prepared A549 cells at 35 ° C. for 1 hour in the presence or absence of AZM. After infection, cells were cultured at 37 ° C. for 7 hours in a supplementary medium without AZM, which was subjected to M1 expression analysis.

[Hemagglutinin inhibition assay]
A PBS solution (1%) of fresh red blood cells (RBC) was prepared from whole avian blood (Biotest). 25 μL of a serially diluted solution of influenza A (H1N1) pdm09 virus (640 hemagglutinin units (HAU) / mL) in equal volume PBS or AZM / ethanol PBS solution at room temperature (20-22 ° C) for 30 minutes. Incubated. 5 μL of 1% RBC solution was added and then incubated at room temperature for 20 minutes. By observing red blood cell aggregation, it was investigated whether AZM inhibits the binding of the virus hemagglutinin (HA) to sialic acid (SA) on red blood cells (RBC).

[Inhibition assay to measure the effect of AZM on the adhesion or internal translocation step in viral infection]
Adhesion Step Assay: Confluent monolayer A549 cells were incubated with a mixture of 200 μM AZM and virus (1MOI) at 4 ° C. for 1 hour. After removing the mixture, the host cells were washed with cold PBS and total RNA was extracted.

Assay for internal transition stage: A549 cells were incubated with virus (1MOI) at 4 ° C. for 1 hour. Cells were washed with warm PBS and cultured at 37 ° C. for 1 hour in medium containing 200 μM AZM. After incubation, the cells were washed with PBS and treated with Proteinase K (Fujifilm Wako Pure Chemical Industries, Ltd.) PBS solution (final concentration 100 μg / mL) at 37 ° C. for 5 minutes to remove the virus remaining on the cell surface. Removed. CDNA was synthesized from the extracted total RNA, and the expression levels of viral M1 and nucleoprotein (NP) were measured by quantitative PCR.

[Quantitative real-time PCR]
Using a ReverTra Ace qPCR RT Master Mix (Toyobo) containing a genomic DNA remover, 1 μg of total RNA was reverse transcribed into cDNA in a 20 μL reaction mixture. The obtained cDNA was used for gene expression analysis of the virus by a quantitative PCR method using PowerUp SYBR green PCR master Mix (Thermofisher Scientific). At this time, PCR was carried out using the primer set shown in Table 1 below according to the following conditions: 50 ° C. for 2 minutes, 95 ° C. for 2 minutes, "95 ° C. for 15 seconds and 60 ° C. for 1 minute". 40 cycles.

[mouse]
The animal protocol for influenza virus infection was approved by the Teikyo University Animal Experiment Ethics Committee in accordance with the guidelines of Teikyo University (AUP number: 16-021). Wild-type 8-week-old BALB / c female mice were purchased from SLC and bred in a pathogen-free environment.

[Animal infection experiments and administration of AZM]
AZM was dissolved in ethanol and mixed with PBS (pH 7.0) to prepare a mixed solution containing 200 μg of AZM in a total volume of 50 μL. The final concentration of ethanol in the mixture was adjusted to within 3%. Anesthetized mice were nasally infected with 300 pfu of mouse-compatible influenza A (H1N1) pdm09 virus. Six hours after infection, all lung tissue was resected from one group of mice for control (untreated). Mice in the other group were nasally administered the above mixed solution with or without AZM twice daily under isoflurane anesthesia 3 days after infection. Rectal temperature and body weight of mice were monitored. At different time points, all lung tissue was excised from treated mice and homogenized in RNAiso Plus solution using a bead cell disruptor (MicroSmash ^TM MS-100, Tommy). A cDNA pool was synthesized from the extracted total RNA, and the expression levels of viral M1 and NP genes were measured by a quantitative PCR method.

≪Experimental results≫
(AZM inhibits the activity of influenza A (H1N1) pdm09 virus by direct interaction with the virus)
Experiments were conducted under four different treatment conditions to investigate the treatment conditions for AZM, which exhibits the most effective antiviral activity. As a result, as shown in FIG. 1, when AZM was administered after infection, the offspring virus titer (FIG. 1a) in the culture medium at the same level and the M1 gene in the host cell at the same level as compared with the control. The expression level (Fig. 1b) is shown. When the virus was pretreated with AZM 1 hour prior to infection, the titer and M1 expression of the offspring virus was significantly reduced. When AZM was administered at the same time as infection, the offspring virus titers were significantly reduced to levels comparable to those observed with virus pretreatment. In contrast, when host A549 cells were pretreated with AZM for 1 hour, significant differences were observed in both offspring virus titers and M1 expression levels compared to controls. These results suggest that AZM interacts with influenza A (H1N1) pdm09 virus in the early stages of infection to inhibit viral activity. Both 1-hour pretreatment and AZM treatment at the same time as infection showed comparable inhibitory effects on the growth of offspring virus.

(AZM show no cytotoxicity to the host cells in _{IC 50} range)
Then, _{IC 50} values were calculated for AZM on the growth of the child virus (Figure 2). Administration of AZM, child viruses released into the culture medium is decreased in a dose-dependent manner, the average _{IC 50} of from about 68MyuM (Figure 2a). The expression status of the viral M1 gene in A549 cells was correlated with the tendency of viral titers (Fig. 3).

To determine the concentration at which AZM is toxic to host A549 cells, an MTT assay was performed with a wide range of concentrations of AZM (FIG. 2b). Under non-infected conditions, no significant cytotoxicity was observed when co-cultured with AZM at concentrations below 200 μM (Fig. 2b, left panel). Similarly, under infectious conditions, no toxic effects were observed on A549 cells up to an AZM concentration of 600 μM (Fig. 2b, right panel). These results, in both uninfected state and infectious conditions, within the scope of IC ₅₀ AZM it was suggested not to adversely affect the viability of the host cell.

(AZM does not affect the adhesion state, but affects the internal migration of the virus)
To investigate the mechanism of AZM's antiviral activity, we investigated whether AZM inhibits the binding interaction between the viral hemagglutinin (HA) and sialic acid on the surface of erythrocytes. As can be seen from the results shown in the upper part of FIG. 4, hemagglutination was also observed in the virus diluted 1/16, but hemagglutination in this dilution range was not significantly inhibited even in the presence of AZM. This suggests that AZM does not affect the binding activity of the viral hemagglutinin (HA) to the sialic acid (SA) receptor on cells. The graph in the lower part of FIG. 4 is a graph of the hemagglutination unit as a numerical value from the photograph in the upper part of FIG.

In addition, the mechanism of inhibition of AZM against the process of viral adhesion and internal translocation was investigated based on the expression profile of the viral gene in the host cell (Fig. 5). As a result, as shown in FIG. 5a, in the group in which the virus was treated with AZM at the same time as the infection, the expression of M1 and NP was not changed in the virus adhered to the cell surface. In contrast, in the group in which AZM was administered after virus adhesion and the orphan virus was removed using proteinase K, the expression of M1 and NP in the host cells was significantly reduced (Fig. 5b). These results suggest that AZM does not affect the binding activity of the virus, but inhibits the internal translocation process in the early stages of virus invasion. Moreover, in view of the fact that AZM exhibits a growth inhibitory effect on influenza virus through such a mechanism, it is suggested that the growth of coronavirus is also suppressed by the same mechanism.

(AZM also attacks newly produced offspring virus)
From the inhibitory effect of AZM on the internal translocation of the parent virus during infection, we hypothesized that AZM could inhibit the repeated cycle of viral infection and proliferative growth of the offspring. Then, in order to test this hypothesis, the viral load at each time point of the primary infection of the parent virus and the secondary infection of the child virus was compared in the presence of AZM (Fig. 6). First, host A549 cells were infected with influenza A (H1N1) pdm09 virus, and the cells were cultured in the presence or absence of AZM for 10 hours. At this point, the titers of the offspring virus in the culture medium or the expression level of the virus M1 in the host cells were comparable in the presence and absence of AZM (FIGS. 6a and 6b). This result is consistent with the result shown in FIG. The culture supernatant containing the germinated virus was then collected and infected with freshly prepared A549 cells in the presence or absence of AZM. The virus-infected A549 cells were then cultured in AZM-free medium for 7 hours. At this point, M1 expression levels in A549 cells cultured in medium containing the offspring virus and AZM were significantly reduced (FIG. 6c). From these results, the above hypothesis that AZM can inhibit the internal translocation of extracellular virus when it invades the host cell has been substantiated.

(Viral load in infected mice was reduced by a single dose of AZM)
Based on the results of A549 cells in vitro described above, an experiment was conducted in which AZM was administered to virus-infected mice (in vivo and nasally) (Fig. 7a). As shown in FIG. 7b, it was observed that AZM administration tended to reduce the expression of viral M1 gene and NP gene in lung tissue 3 days after infection. Maximum inhibition of virus expression was observed 2 days after infection, when the virus proliferated dramatically (Fig. 7b). The decrease in body temperature caused by the administration of the virus was noticeable after 3 days and was suppressed by AZM (Fig. 7c). On the other hand, no effect was observed on the body weight of infected mice 3 days after infection (Fig. 7d). From the above, it was shown that nasal administration of AZM reduced the viral load in the lungs, thereby alleviating hypothermia during infection with the influenza A (H1N1) pdm09 virus.

≪Experimental method using azithromycin derivative≫
The following experiments were carried out using the compounds 2 to 4 synthesized above. The following compounds 5 to 7 were used as negative controls.

Specifically, first, the same experiment as shown in "Treatment at the same time as infection" in FIG. 1a was performed. As a result, as shown in FIG. 8, when treated with Compounds 2 and 4, a marked decrease in the titer of the offspring virus was confirmed in the same manner as in AZM. On the other hand, when treated with Compounds 5 and 7, the titers of the offspring virus did not decrease as much as Compounds 2 and 4, although the compounds 5 and 6 showed weak inhibitory activity. In addition, the same experiment was carried out using Compounds 2 to 4 for other treatment forms (post-infection treatment, cell pretreatment and virus pretreatment) shown in FIG. 1a. As a result, as shown in FIGS. 9A to 9C, the virus was pretreated with the compound 1 hour before infection in the same manner as in AZM regardless of which of the compounds 2 to 4 was used. In some cases (pretreatment of the virus) or when the compound was administered at the same time as the infection (treatment at the same time as the infection), the titer of the offspring virus was significantly reduced. These results suggest that Compounds 2 and 4, like AZM, also interact with influenza A (H1N1) pdm09 virus in the early stages of infection to inhibit viral activity.

In addition, the same experiment as shown in FIG. 2a was performed using Compounds 2 to 4. As a result, as shown in the upper panel of Figure 10, respectively the _{IC 50} values for the viral titer of the compound 2 to compound 4, it was calculated 130 uM, and 84MyuM and 84MyuM. Furthermore, a similar experiment was conducted using the expression level of the M1 gene in the host cell as an index. As a result, as shown in the lower panel of Figure 10, respectively the _{IC 50} values for M1 gene expression levels of the compound 2 to compound 4, it was calculated 140MyuM, and 140MyuM and 116MyuM.

In addition, the same experiment (MTT assay) as shown in FIG. 2c was performed using Compounds 2 to 4 showing anti-influenza virus activity above. As a result, as shown in FIG. 11, no toxic effect on A549 cells was observed up to an AZM concentration of 600 μM under both infectious conditions (upper panel) and non-infectious conditions (lower panel).

Furthermore, the same experiment (hemagglutinin inhibition assay) as shown in FIG. 4 was performed using Compounds 2 to 4. The results are shown in FIG. 12 together with the results of AZM in FIG. As shown in FIG. 12, for Compounds 2 and 4, hemagglutination was also observed in the virus diluted 1/16 in the same manner as for AZM, but the hemagglutination in this dilution range was observed even in the presence of AZM. It was not significantly inhibited. This suggests that Compounds 2 and 4 also do not affect the binding activity of viral hemagglutinin (HA) to cellular sialic acid (SA) receptors. The graph in the lower part of FIG. 12 is a graph of the hemagglutination unit as a numerical value from the photograph in the upper part of FIG.

In addition, the same experiments (adhesion assay and internal migration assay) as shown in FIG. 5 were performed using Compounds 2 to 4. Here, as shown in FIG. 5, AZM suppressed the stage of endocytosis without affecting the stage of adhesion by the virus. On the other hand, as shown in FIG. 13, all of Compounds 2 and 4 show an inhibitory effect on the adhesion stage in addition to suppressing the stage of virus internal translocation (endocytosis). You can see that there is. This suggests that AZM derivatives represented by Compounds 2 to 4 may have a mechanism that inhibits virus-host cell binding not mediated by hemagglutinin and sialic acid.

≪Comparison experiment between azithromycin (AZM) and clarithromycin (CAM)≫
Azithromycin anhydride for (AZM), except that the concentration of the dosing agent was changed to 68μM a _{IC 50} of AZM from 200 [mu] M, by the same method as the experiment shown in FIG. 1, compared with clarithromycin (CAM) An experiment was conducted. As a result, as shown in FIG. 14a, when the virus was pretreated with AZM 1 hour before infection and when AZM was administered at the same time as infection, the titer of the offspring virus was significantly reduced. On the other hand, when clarithromycin (CAM) was administered, such a decrease in the titer of the offspring virus was not observed (Fig. 14b). It has also been reported that the combined use of clarithromycin (CAM) with zanamivir did not inhibit the in vitro replication of human influenza A (H1N1) pdm09 virus (Lee ACY, et al. Arch Virus. 2018; 163). : 2349-2358). From these facts, it can be seen that clarithromycin (CAM) does not affect the activity of human influenza A (H1N1) pdm09 virus and does not show an inhibitory effect on the internal translocation of the virus.

This application is based on Japanese Patent Application No. 2019-100691 filed on May 29, 2019, the disclosure of which is incorporated by reference in its entirety.

[SEQ ID NO: 1]
It is a DNA sequence of a forward primer for amplifying a gene encoding the matrix protein 1 (M1) of influenza A (H1N1) pdm09 virus.

[SEQ ID NO: 2]
It is a DNA sequence of a reverse primer for amplifying a gene encoding the matrix protein 1 (M1) of influenza A (H1N1) pdm09 virus.

[SEQ ID NO: 3]
It is a DNA sequence of a forward primer for amplifying a gene encoding a nuclear protein (NP) of influenza A (H1N1) pdm09 virus.

[SEQ ID NO: 4]
It is a DNA sequence of a reverse primer for amplifying a gene encoding a nuclear protein (NP) of influenza A (H1N1) pdm09 virus.

[SEQ ID NO: 5]
It is a DNA sequence of a forward primer for amplifying the GAPDH gene which is a housekeeping gene in a mouse.

[SEQ ID NO: 6]
It is a DNA sequence of a reverse primer for amplifying the GAPDH gene which is a housekeeping gene in a mouse.

[SEQ ID NO: 7]
It is a DNA sequence of a forward primer for amplifying the GAPDH gene which is a housekeeping gene in humans.

[SEQ ID NO: 8]
It is a DNA sequence of a reverse primer for amplifying the GAPDH gene which is a housekeeping gene in humans.

Claims (17)

An anti-influenza virus agent containing one or more 15-membered ring macrolide compounds selected from the group consisting of the following compounds 1 to 4 or salts or hydrates thereof as an active ingredient.
Prevention of influenza virus infection and / or containing one or more 15-membered ring macrolide compounds selected from the group consisting of the following compounds 1 to 4 or salts or hydrates thereof as an active ingredient. Therapeutic agent.
Influenza virus infection containing one or more 15-membered ring macrolide compounds selected from the group consisting of the following compounds 1 to 4 or salts or hydrates thereof, and a pharmaceutically acceptable carrier. Pharmaceutical compositions for the prevention and / or treatment of disease.
Influenza virus infection, which comprises administering to a patient in need one or more 15-membered ring macrolide compounds or salts or hydrates thereof selected from the group consisting of Compounds 1 to 4 below. How to prevent and / or treat the disease.
One or more 15-membered ring macrolide compounds selected from the group consisting of the following compounds 1 to 4 or salts or hydrates thereof for the production of preventive and / or therapeutic agents for influenza virus infection. Use of things.
Prevention of coronavirus infection and / or containing one or more 15-membered ring macrolide compounds selected from the group consisting of the following compounds 1 to 4 or salts or hydrates thereof as an active ingredient. Therapeutic agent.
The prophylactic and / or therapeutic agent according to claim 6, wherein the coronavirus infection is a 2019 new coronavirus (SARS-CoV-2) infection. Coronavirus infection, comprising administering to a patient in need one or more 15-membered ring macrolide compounds or salts or hydrates thereof selected from the group consisting of Compounds 1 to 4 below. How to prevent and / or treat the disease.
The method according to claim 8, wherein the coronavirus infection is a 2019 new coronavirus (SARS-CoV-2) infection. Compound represented by the following chemical formula (1) or a salt or hydrate thereof:

In chemical formula (1),
X represents a hydrogen atom or an acyl group having 1 to 4 carbon atoms.
Y ¹ represents a hydrogen atom or a group represented by −C (= O) -NH- (CH ₂ ) _n −C≡CH (where n is an integer of 1 to 4).
Do Y ² and Y ³ represent a hydrogen atom or a group represented by -C (= O) -NH- (CH ₂ ) _n- C≡CH (where n is an integer of 1 to 4)? Or together to form a group represented by the following chemical formula (2),

In the chemical formula (2), * represents the binding site.
However, the chemical formula (1) is a group represented by −C (= O) -NH- (CH ₂ ) _n −C≡CH (where n is an integer of 1 to 4) or the chemical formula (2). Includes one of the groups represented by. A medicament containing the compound according to claim 10 or a salt or hydrate thereof. The medicament according to claim 10, which is an anti-influenza virus agent. The medicament according to claim 10, which is an anti-coronavirus agent. A prophylactic and / or therapeutic agent for coronavirus infection, which comprises the compound according to claim 10 or a salt or hydrate thereof as an active ingredient. The prophylactic and / or therapeutic agent according to claim 14, wherein the coronavirus infection is a 2019 coronavirus (SARS-CoV-2) infection. A method for preventing and / or treating a coronavirus infection, which comprises administering the compound according to claim 10 or a salt or hydrate thereof to a patient in need. The method according to claim 16, wherein the coronavirus infection is a 2019 new coronavirus (SARS-CoV-2) infection.