TW202110461A - 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. 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.

Description

Preventive and/or therapeutic agent for influenza virus infection or coronavirus infection

The present invention relates to a novel preventive and/or therapeutic agent for influenza virus infection or coronavirus infection.

Influenza infection with influenza virus as the pathogen, even in modern times, is still an infectious disease that has repeated pandemics due to its powerful transmission power, and has caused great harm to society. In the gradual overcoming of various infectious diseases, there are few drugs that are effective and safe against influenza viruses. In addition, the emergence of viruses resistant to these drugs will also be regarded as a problem, and new developments are expected. Of anti-influenza drugs.

Influenza virus can cause acute respiratory infections, and its clinical symptoms are characterized by sudden fever, headache, joint pain, general fatigue and other systemic symptoms. It is also accompanied by various respiratory symptoms such as nasal mucus and cough that can be seen in colds, and 38 High fever above ℃. In healthy people, the cure usually takes about 1 to 2 weeks. However, in infants, the elderly, patients with chronic diseases in the respiratory tract, circulatory system, and kidneys, patients with metabolic diseases such as diabetes, and immunocompromised patients, etc. There are many deaths caused by secondary infections of bacteria and pneumonia. In addition, not only local infections of the respiratory tract, but also reports of extremely serious cases of so-called severe neurological complications represented by influenza encephalitis/encephalopathy. In addition, symptoms of digestive organs such as abdominal pain, nausea/vomiting, and diarrhea may also be observed, especially in children. At the time of diagnosis, the major feature that should be noted is that fever and other systemic symptoms are more significant than respiratory symptoms.

At present, the countermeasures against influenza, from the point of view of prevention, vaccination can be regarded as basic. Anti-influenza drugs other than vaccines include amantadine (drug name: symmetrel), which has M2 protein ion channel inhibitory activity, and only neuraminidase inhibitor oseltamivir (drug name: tamiflu), Zanamivir (pharmaceutical name: relenza) etc. are licensed. For example, oseltamivir has excellent properties as a medicine _{with EC 50} =0.03 and CC _{50 >100.}

Virus infection into cells starts with the following process. First, the hemagglutinin protein (hemagglutinin, hereinafter also referred to as "hemagglutinin") possessed by the virus binds to the sialic acid receptor on the cell side, and the virus is taken up into the cell through endocytosis. Then, the fusion of the viral membrane and the endosomal membrane caused by the acidification of the endosome causes the viral genes to enter the cytoplasm. It has been reported that in this fusion, the cleavage of the peptide bond on the specific sequence of hemagglutinin caused by the host cell endoprotease is necessary. Through this cleavage, the hemagglutinin membrane is fused The domain will be exposed and fusion with the endosomal membrane will occur (Latest Medicine, 2004, Vol. 59, 215-222). After the infection is established, the virus multiplies in the cell, and in order to expand the infection to new cells/tissues, it will bud from the infected cells. During this budding, neuraminidase derived from the virus requires the cleavage of hemagglutinin and sialic acid receptors. The existing anti-influenza drugs, oseltamivir and zanamivir, act when the virus germinates after the infection, and therefore cannot prevent the establishment of the initial infection. Furthermore, there have been reports in recent years that there have been influenza viruses that are resistant to the above-mentioned neuraminidase inhibitors and M2 protein ion channel inhibitors (Matsuzaki Y, et al., Virol J. 2010; 7:53, Dong G, et al., PloS One. 2015; 10(3):e0119115).

However, the inventors of this case have previously reported that spiramycin or josamycin (leucomycin A3), which is a 16-membered cyclomacrolactone, is effective in the treatment of severe pneumonia caused by influenza virus infection. (Japanese Patent Publication No. 2012-020953). In addition, there are also reports that the 14-membered macrolide anti-biomass clarithromycin can inhibit the proliferation of influenza virus (Yamaya M, et al., J Pharmacol Exp Ther. 2010 Apr; 333(1): 81-90). However, there is no report that azithromycin and other 15-membered cyclomacrolide antibiotics have anti-influenza virus effects. On the contrary, there are reports that for mice that have been orally administered oseltamivir, even if azithromycin is further administered intraperitoneally, compared with oseltamivir alone, the survival rate, viral power, and cytokine level From the viewpoint of others, no improvement in the treatment effect has been observed (Fage C, et al., J med Virol. 2017; 89(12): 2239–43).

It is further known that, unlike influenza viruses, some coronaviruses cause various disease symptoms to humans. Such pathogenic coronaviruses are known to cause cold syndrome human coronaviruses 229E, NL63, OC43, and HKU1, as well as SARS-CoV, which can cause severe acute respiratory syndrome (SARS), and MERS, which can cause Middle East respiratory syndrome (MERS). -CoV, the 2019 novel coronavirus (SARS-CoV-2) that can cause novel coronavirus infection (COVID-19), etc. However, it is not known that the 15-membered cyclomacrolide anti-biomass is still effective against these coronaviruses.

[The problem to be solved by the invention] Generally speaking, known viruses are prone to mutations in amino acids that constitute proteins, and depending on the circumstances, can mutate into drug-resistant viruses that render drugs ineffective. Then, the emergence of such drug-resistant viruses may become a serious clinical problem. In fact, as reported by Matsuzaki Y, et al., Virol J. 2010; 7:53, Dong G, et al., PloS One. 2015; 10(3):e0119115, the emergence of drug-resistant viruses has Existence becomes a real threat.

In this regard, the purpose of the present invention is to provide a novel medicine that can be effectively used to prevent and/or treat influenza infection or coronavirus infection.

[Means to solve the problem] The inventors of this case have conducted intensive research in view of the above-mentioned prior art. During this process, it was surprisingly discovered that some compounds showed anti-influenza virus effects. Therefore, based on this finding, the present invention has been completed.

阿奇黴素(AZM) [化合物3]                                                       [化合物4]

That is, according to the first aspect of the present invention, there is provided an anti-influenza virus agent containing one or more 15-membered cyclomacromoids selected from the group consisting of the following compounds 1 to 4 A cyclic lactone compound or its salt or hydrate is used as an active ingredient. [Chemistry 1] [Compound 1] [Compound 2]

Azithromycin (AZM) [Compound 3] [Compound 4]

Furthermore, according to the second aspect of the present invention, there is provided a preventive and/or therapeutic agent for influenza virus infection, containing one or more selected from the group consisting of the above-mentioned compound 1 to compound 4 The 15-membered ring macrolide compound or its salt or hydrate as the active ingredient.

Furthermore, according to the third aspect of the present invention, there is provided a pharmaceutical composition for the prevention and/or treatment of influenza virus infection, which contains one selected from the group consisting of the above-mentioned compound 1 to compound 4 or Two or more 15-membered ring macrolide compounds or their salts or hydrates, and a pharmaceutically acceptable carrier.

Furthermore, according to the fourth aspect of the present invention, there is provided a method for the prevention and/or treatment of influenza virus infection, comprising administering to a patient in need an effective amount selected from the group consisting of the above-mentioned compounds 1 to 4 One or two or more 15-membered ring macrolide compounds in the group or their salts or hydrates.

Furthermore, according to the fifth aspect of the present invention, there is provided a 15-membered ring macrolide compound or salt thereof, or a hydrated compound selected from one or two or more of the above-mentioned compound 1 to compound 4 It is used to produce preventive and/or therapeutic agents for influenza virus infection.

In addition, according to the sixth aspect of the present invention, there is provided a preventive and/or therapeutic agent for coronavirus infections such as the 2019 novel coronavirus (SARS-CoV-2) infection, which contains selected from the above-mentioned compounds 1 to 4 One or two or more 15-membered ring macrolide compounds or their salts or hydrates in the constituted group are used as active ingredients.

Further, according to the seventh aspect of the present invention, there is provided a method for preventing and/or treating coronavirus infections such as the 2019 novel coronavirus (SARS-CoV-2) infection, which includes the option of administering an effective amount to patients in need One or two or more 15-membered ring macrolide compounds or their salts or hydrates from the group consisting of the above-mentioned compound 1 to compound 4.

Then, it was also discerned that among compound 1 to compound 4, compound 2 to compound 4 were novel compounds. Therefore, according to other aspects of the present invention, there is also provided a compound or its salt or hydrate represented by the following chemical formula (1); and an anti-influenza virus agent or an anti-influenza agent containing these compounds as effective ingredients. Coronavirus agents and other medicines.

Invention effect According to the present invention, a novel medicine that can be effectively used to prevent and/or treat influenza infection or coronavirus infection can be provided.

The first aspect of the present invention is an anti-influenza virus agent containing one or more 15-membered ring macrolide compounds selected from the group consisting of the above-mentioned compound 1 to compound 4 or Salts or hydrates are used as active ingredients. In addition, the second aspect of the present invention is a preventive and/or therapeutic agent for influenza virus infection, containing one or more of 15 selected from the group consisting of the above-mentioned compound 1 to compound 4 A membered ring macrolide compound or its salt or hydrate is used as an active ingredient. As mentioned above, there is no knowledge that the above-mentioned active ingredients are effective for preventing and/or treating influenza virus infections, but this is the novel knowledge discovered by the inventors of the case for the first time.

The active ingredient of the agent of the first and second aspect of the present invention is a 15-membered ring macrolide compound or its salt or hydrate selected from the group consisting of the above-mentioned compound 1 to compound 4, and can be used One or more of these. Among them, compound 1 is a 15-membered ring macrolide compound known as "azithromycin". In Japan, azithromycin hydrate is marketed under the trade name "Zithromac (registered trademark)", and its indications are deep skin infections, lymphatics/lymphadenitis, pharyngeal/laryngopharyngitis, tonsillitis, acute bronchitis, pneumonia, etc. . However, it is unknown that azithromycin has an anti-influenza virus effect. In addition, examples of the salts of the above-mentioned compound 1 to compound 4 include those in which any cationic group such as an amino group forms a salt with any anion. In addition, the number of hydration water in the hydrate of the above-mentioned compound 1 to compound 4 is not particularly limited, as long as it is a hydrate that can exist in a stable form.

Among compounds 1 to 4, from the viewpoint of anti-influenza virus activity, compound 1 (AZM), compound 3, or compound 4 is preferably used, and compound 1 (AZM) or compound 3 is preferably used. 1(AZM) is particularly preferred.

There are no particular restrictions on the types of influenza viruses targeted by the anti-influenza virus agent of the present invention, and influenza viruses that are known in the past can be targeted, and new types of epidemics that may occur in the future can also be targeted. Influenza virus as a target. Among them, the type A influenza virus should be the target, and the H1N1 type virus should be the target, and the human influenza A(H1N1)pdm09 virus, a new type of influenza virus that brought a pandemic in 2009, should be used as the target. The object is the best. Furthermore, it is also appropriate to target a new type of influenza virus that may become epidemic in the future.

In addition, based on the above-mentioned novel knowledge, according to the present invention, an anti-coronavirus agent similar to the above-mentioned anti-influenza virus agent is also provided. Specifically, first, a preventive and/or therapeutic agent (sixth form) for the prevention and/or treatment of coronavirus infections such as the 2019 novel coronavirus (SARS-CoV-2) infection is provided, which contains selected from the above-mentioned compounds 1 to 4 One or two or more 15-membered ring macrolide compounds or their salts or hydrates in the constituted group are used as active ingredients. Similarly, according to the present invention, there is also provided a method for preventing and/or treating coronavirus infections such as the 2019 novel coronavirus (SARS-CoV-2) infection (the seventh form), which is effective for administering to patients in need The amount is selected from one or two or more 15-membered ring macrolide compounds or their salts or hydrates from the group consisting of the above-mentioned compounds 1 to 4.

There are no particular restrictions on the types of coronaviruses targeted by the anti-coronavirus agent of the present invention, and known coronaviruses can be targeted, and new coronaviruses that may occur in the future can also be targeted. Such pathogenic coronaviruses include human coronaviruses 229E, NL63, OC43, and HKU1 that cause cold syndrome, SARS-CoV that causes severe acute respiratory syndrome (SARS), and MERS- which causes Middle East respiratory syndrome (MERS). CoV, the 2019 new coronavirus (SARS-CoV-2) that can cause new coronavirus infection (COVID-19), etc. Among them, SARS-CoV, MERS-CoV or SARS-CoV-2 should be targeted, and SARS-CoV-2, a new type of coronavirus that will bring a pandemic from 2019 to 2020, is the best target. Furthermore, it is also appropriate to target a new type of coronavirus that may have an epidemic in the future.

Related to the anti-influenza virus agent of the first aspect of the present invention, related to the prevention and/or treatment of influenza virus infection of the second aspect of the present invention, and related to the prevention and/or treatment of coronavirus infection of the sixth aspect of the present invention The drug is used to administer to patients who have been infected with influenza virus or coronavirus, or to administer to those who are (already) at risk of infection. Generally speaking, infected patients will show symptoms such as sneezing, nasal water, nasal congestion, cough, sputum, chills, fever, headache, sore throat, etc. Therefore, in addition to the agent of the present invention, it can also be administered (combined) symptomatic therapy medicine. Moreover, not only can it be used in combination, but it can also be blended with the agent of the present invention to form a mixture (composition).

An example of a symptomatic therapy agent that can be administered together with the agent of the present invention includes aspirin, aspirin aluminum, sasapyrine, ethenzamide, water Antipyretic and analgesic drugs such as salicamide, ibuprofen, acetaminophen, and propantipyrine; central nervous system stimulants such as caffeine, anhydrous caffeine, sodium benzoate and caffeine, such as sodium caffeine; bromovalerylurea bromide (bromovalerylurea) ), allylisopropylacetylurea and other sedatives; chlorpheniramine maleate, diphenhydramine, diphenhydramine salicylate, clemastine fumarate, malic acid Antihistamines such as carbinoxamine maleate, mequitazine, alimazine tartrate, diphenylpyraline hydrochloride, triprolidine hydrochloride, etc.; chlorinated Anti-inflammatory drugs such as lysozyme, bromelain, serrapeptase, semi alkaline proteinase, glycyrrhizic acid, dipotassium glycyrrhizinate, ammonium glycyrrhizinate, glycyrrhetinic acid, sodium azulene sulfonate; Codeine Dihydrogen Phosphate, Codeine Phosphate, Noscapine Hydrochloride, Noscapine, Dexmethorphan Hydrobromide, Dexmethorphene Phenolphthaleate, Dememophen Phosphate (dimemorfan phosphate), tipepidine hibenzate, tipepidine citrate, eprazinone hydrochloride, methylephedrine, methylephedrine hydrochloride, methylephedrine hydrochloride Cough suppressants such as methoxyphenamine hydrochloride, trimetoquinol hydrochloride, phenylpropanolamine hydrochloride; L-ethylcysteine hydrochloride, potassium guaiacol sulfonate, potassium cresol , Guaifenesin, bromhexine hydrochloride (bromhexine hydrochloride), carboxymethylcysteine and other expectorants; theophylline (theophylline), aminophylline (aminophylline), diprophylline (diprophylline) and other bronchial Expansion drugs; belladonna (total) alkaloids, belladonna extract, isoiodamine, scopolamine hydrobromide, scopolamine extract, bromobutyl scopolamine, diethylaminoethyl Diphenylglyceride methoxy bromide (methylbenactyzium bromide), timepidium bromide, pirenzepine (pirenzepine) and other anti-acetylcholine agents; cetylpyridinium (cetylpyridinium), ten chloride Hexaalkylpyridinium, povidone iodine, chlorhexidine gluconate, benzalkonium chloride, dequalinium chloride, thymol, iodine/potassium iodide, phenol, chloride hydrochloride Disinfectants such as chlorhexidine hydrochloride, creosote, and benzethonium chloride; dobucaine hydrochloride, ethyl aminobenzoate, lidocaine, lidocaine hydrochloride, Océ Local anesthetics such as oxethazaine; vitamin A, liver oil, vitamin B ₁ , vitamin B ₂ , vitamin B ₆ , vitamin B ₁₂ , vitamin C, calcium ascorbate, vitamin D, vitamin E, tocopherol calcium succinate and other vitamin agents ; Pantothenic acid, panthenol, sodium pantothenate, calcium pantothenate, pantothenate, nicotinic acid, nicotine amide, glucuronic acid, glucuronic acid lactone, ethanesulfonic acid, biotin, gamma-glutitol (γ -oryzanol) and other metabolic components; Rehmannia glutinosa, cinnamon, bezoar, ginger, platycodon, ephedra, licorice, almond, pinellia, plantain, Polygala, Bupleurum, Poria, Xinyi and other crude drugs and extracts of these crude drugs (Essence, tincture, etc.), but not limited to these. The symptomatic medicine may be blended with a single component, or two or more of them may be used in combination or blended.

Regarding the route of administration of the agent of the present invention, either systemic administration or local administration can be selected. At this time, an appropriate route of administration can be selected according to the disease, symptoms, etc. Regarding the transmucosal administration of the agent of the present invention, it can also be administered by any of the nasal route, the oral route, and the non-oral route. The non-nasal route and the non-oral route can include general intravenous administration and intraarterial administration, and in addition, subcutaneous, intradermal, intramuscular, etc. administration can also be cited. Furthermore, transdermal administration can also be implemented. Preferably, intranasal administration can be used.

The dosage form of the agent of the present invention is not particularly limited. It can be made into various dosage forms, for example, an aerosol for nasal administration, and when it is administered orally, it can be made into tablets, capsules, Powders, granules, pills, liquids, emulsions, suspensions, solutions, alcohols, syrups, extracts or elixirs. Parenteral preparations can be made into, for example, injections such as subcutaneous injections, intravenous injections, intramuscular injections, or intraperitoneal injections; transdermal administration or patch, ointment or lotion; for intraoral administration Sublingual agents, oral patches; and suppositories, but not limited to these. These preparations can be manufactured by well-known methods generally used in preparation steps. In addition, the medicament related to the present invention may also be in the form of sustained or aspiration.

When preparing an aerosol for nasal administration, lubricants, binders, preservatives, preservatives, odorants, excipients, etc. can be added. For example, talc, stearic acid, sodium salt, calcium salt and other salts, Carplex, etc. as lubricants; starch, dextrin, etc. as binding agents; citric acid, glycine etc. as pH regulators; as preservation Ascorbic acid, etc. as preservatives; parabens, benzalkonium chloride, phenol, chlorobutanol, etc. as preservatives; menthol, citrus flavors, etc. as corrigents; cellulose acetate as excipients Vegetarian etc.

When preparing a solid preparation for oral use, excipients and optional binders, disintegrants, lubricants, coloring agents, flavors, and odorants can be added to the active ingredients, and then the tablets can be manufactured by conventional methods. Tablets, coated tablets, granules, powders, and capsules. Such additives can be used by general users in the field, such as lactose, white sugar, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose and silicic acid as excipients; as binding agents Of water, ethanol, propanol, monosyrup, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl starch, methyl cellulose, ethyl cellulose, shellac, Calcium phosphate, polyvinylpyrrolidone, etc.; as a disintegrant, dry starch, sodium alginate, cold sky powder, sodium bicarbonate, calcium carbonate, sodium lauryl sulfate, stearic acid monoglyceride, and lactose, etc.; as a lubricant The purified talc, stearate, borax, and polyethylene glycol, etc.; as the flavoring agent, white sugar, orange peel, citric acid, and tartaric acid.

When preparing oral liquid preparations, flavoring agents, buffering agents, stabilizers, and flavoring agents can be added to the active ingredients, and oral liquid preparations, syrups, elixirs, etc. can be manufactured by conventional methods. In this case, the above-mentioned flavoring agent can be used. In addition, examples of buffering agents include sodium citrate, and examples of stabilizing agents include tragacanth, gum arabic, and gelatin.

When preparing injections, pH regulators, buffers, stabilizers, isotonic agents, local anesthetics, etc. can be added to the active ingredients, and subcutaneous, intramuscular, and intravenous injections can be manufactured by conventional methods. In this case, the pH adjuster and buffer may include sodium citrate, sodium acetate, and sodium phosphate. Examples of stabilizers include sodium metabisulfite, ethylenediaminetetraacetic acid (EDTA), thioglycolic acid, and thiolactic acid. Examples of local anesthetics include procaine hydrochloride and lidocaine hydrochloride. Examples of isotonic agents include sodium chloride and glucose.

When preparing suppositories, the active ingredients can be added with well-known preparation carriers in the industry, such as polyethylene glycol, lanolin, cocoa butter, and fatty acid triglycerides, and further, if necessary, such as Tween (Registered trademark) surfactants, etc., afterwards, are manufactured using common methods.

When preparing an ointment, if necessary, the active ingredients may be blended with commonly used bases, stabilizers, wetting agents, and preservatives, and mixed by conventional methods to prepare them. The base may include fluidized paraffin, white petrolatum, white beeswax, octyldodecanol, and paraffin. Examples of the preservative include methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, and propyl p-hydroxybenzoate.

When preparing a patch, it is sufficient to apply the aforementioned ointment, cream, gel, paste, etc., to a normal support by a conventional method. The support is suitably a woven fabric or non-woven fabric composed of cotton, staple fiber, and chemical fiber, and a film or foam sheet such as soft vinyl chloride, polyethylene, and polyurethane.

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

The dosage range is not particularly limited, and it can be based on the effectiveness of the ingredients contained, the form of administration, the route of administration, the type of disease, the nature of the subject (weight, age, symptoms and other medical use, etc.), and the judgment of the undertaking physician Wait for suitable settings. Generally, the appropriate dosage is preferably, for example, about 0.01 μg to 100 mg per 1 kg of the subject's body weight, and preferably in the range of about 0.1 μg to 1 mg. However, general routine experiments for optimization, which are widely known in the field, can be used to change these amounts. The above dosage can be administered once a day to several times.

From another point of view, as the effective ingredient of the agent of the present invention, one or two or more 15-membered ring macrolide compounds or their salts selected from the group consisting of the above-mentioned compound 1 to compound 4, Or hydrates can be used in methods for preventing and/or treating influenza virus infection or coronavirus infection. Such a method can be implemented by, for example, the following method: a pharmaceutical composition containing the above-mentioned active ingredients and a pharmaceutically acceptable carrier is administered to a subject (patient) through an appropriate administration route. At this time, the injection route and dosage (effective amount) can be appropriately set by those who are acquainted with this art by referring to the above description of the relevant medicine and the technical common sense at the time of application of this case. Further from another point of view, one or two or more 15-membered ring macrolide compounds or their salts or hydrates selected from the group consisting of the above-mentioned compounds 1 to 4 can also be used for manufacturing Preventive and/or therapeutic agent for influenza virus infection or coronavirus infection.

There are no special restrictions on the way to obtain the 15-membered ring macrolide compound represented by compound 1 to compound 4, or its salt, or hydrate. The commercial product can be purchased when the commercial product is available, or you can refer to it. In the past, the well-known knowledge was synthesized by itself. Here, as mentioned above, among compounds 1 to 4, compounds 2 to 4 are novel compounds provided for the first time in this case. According to the present invention, a novel compound containing these compounds 2 to 4 can be provided, and is a compound represented by the following chemical formula (1) or a salt or hydrate thereof. [化2]

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 formyl, acetyl, propionyl, and butyryl. Among them, X is preferably a hydrogen atom, an acetyl group, or a propyl group, and a hydrogen atom or an acetyl group is particularly preferable.

In the above chemical formula (1), Y ¹ represents a hydrogen atom or a group represented by -C(=O)-NH-(CH ₂ ) _n -C≡CH (here, n is an integer of 1 to 4). 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 preferably 1 or 2, and n is 1 is particularly good. Also, from the viewpoint of anti-influenza virus activity, Y ^{1 is} preferably a hydrogen atom.

In the above chemical formula (1), Y ² and Y ³ represent a hydrogen atom or a group represented by -C(=O)-NH-(CH ₂ ) _n -C≡CH (here, n is an integer of 1 to 4), Or together they form a group represented by the following chemical formula (2). The chemical formula (1) includes either the group represented by -C(=O)-NH-(CH ₂ ) _n -C≡CH (here, n is an integer of 1 to 4) or the group represented by the chemical formula (2) . 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, and n is preferably 2 or 3. , N is 3 particularly preferably. [Chemical formula 3] [Chemical formula 2]

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

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

Among the compounds represented by the chemical formula (1), for example, compounds where Y ² of compound 4 is -C(=O)-NH-(CH ₂ ) _n -C≡CH and Y ³ is a hydrogen atom can be The isocyanate compound represented by O=C=N-(CH ₂ ) _n -C≡CH is synthesized by reacting compound 1 (AZM) and the compound whose Y ^{1 in} compound 1 is acylated under heating and refluxing conditions. In addition, the isocyanate compound represented by O=C=N-(CH ₂ ) _n -C≡CH can make the corresponding carboxylic acid HO-C(=O)-(CH ₂ ) _n -C≡CH under alkaline conditions Under heating and refluxing, it reacts with diphenyl azide phosphate (DPPA) and obtains it through acid azide.

In addition, as in compound 3, Y ² and Y ³ are compounds that together form the group represented by the chemical formula (2). Trimethylcycloboroxane can be heated to reflux compound 1 (AZM) and X in compound 1 The acylated compound reacts to synthesize. Then, the compound in which Y ² and Y ³ are obtained together to form the group represented by the chemical formula (2) is further used as a raw material, and is sequentially reacted with alkynyl amines such as carbonyl diimidazole (CDI) and propargyl amine , By this, the group represented by -C(=O)-NH-(CH ₂ ) _n ^{-C≡CH can be introduced into Y 1.} Afterwards, it can also be heated in the presence of silicon dioxide to convert Y ² and Y ³ to hydroxyl groups.

[Examples] Hereinafter, the present invention will be explained in more detail through examples and the like, but the technical scope of the present invention is not limited to them. ≪Production example≫ [Production example 1: Synthesis of 5-(1-pentynyl)isocyanate (1)] [Chemical 4]

To 5-hexanoic acid (600mg, 5.35 millimoles) in benzene (26.8mL) solution add diphenyl azide phosphate (DPPA) (1.19mL, 5.51 millimoles) and TEA (0.77mL, 5.51 millimoles) . After stirring at 85°C for 2 hours, the reaction mixture was cooled to room temperature to obtain a crude product (1) (about 0.18 M) in benzene. The solution was not purified and used in the next step. [Production Example 2: Synthesis of Compound 4] [化5]

對粗5-(1-戊炔基)異氰酸酯(1)(~4.40毫莫耳)之苯溶液(約0.18M)在室溫下添加阿奇黴素(AZM)(3.0g、4.01毫莫耳)，並將反應混合物加溫到80℃。攪拌105分鐘後，將反應混合物冷卻至室溫，接著，添加飽和NH₄ Cl水溶液(50mL)並終止(quench)，使用飽和NaHCO₃ 水溶液(28mL)中和到pH7.0。將該混合物使用乙酸乙酯(80mL×2)萃取，合併有機層並使用Na₂ SO₄ 乾燥，進行過濾、濃縮。

Add azithromycin (AZM) (3.0g, 4.01 millimoles) to the crude 5-(1-pentynyl) isocyanate (1) (~4.40 millimoles) in benzene (about 0.18M) at room temperature, and The reaction mixture was warmed to 80°C. After stirring for 105 minutes, the reaction mixture was cooled to room temperature, then, a saturated _{aqueous NH 4} Cl solution (50 mL) was added and quenched, and a saturated aqueous NaHCO ₃ solution (28 mL) was used to neutralize to pH 7.0. The mixture was extracted with ethyl acetate (80 mL×2), and the organic layers were combined _{, dried with 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) with silica gel to obtain compound 4 (1.42g, 41%) as a colorless solid ). [Chemistry 6] mp: 95.5-96.3 ℃; [α] ²⁷ _D ( c = 1.0, CHCl ₃ ); -42.5 °; IR (KBr) ν (cm ^-1 ): 3370, 2939, 2360, 1712, 1520, 1458, 1373, 1319, 1242, 1049 ¹ H NMR (500 MHz, DMSO- d ₆ , 60 ºC) δ (ppm): 5.97 (s, 1H), 4.93 (m, 1H), 4.82 (app s, 1H) , 4.45 (d, J = 6.9 Hz, 1H), 4.33 (app s, 1H), 4.03-4.00 (complex m, 3H), 3.90-3.84 (complex m, 3H), 3.65 (m, 1H), 3.22 ( s, 3H), 3.05-3.04 (complex m, 3H), 2.92 (ddd, J = 8.6, 8.0, 7.5 Hz, 1H), 2.83 (dd, J = 7.5, 6.9 Hz, 1H), 2.69-2.62 (complex m, 2H), 2.45 (m, 2H, overlapped the peak of DMSO- d ₆ ), 2.27-2.20 (complex m, 7H), 2.15 (s, 3H), 2.06-1.97 (complex m, 2H), 1.65- 1.50 (complex m, 8H), 1.23-1.15 (complex m, 12 H), 1.11-1.02 (complex m, 10 H), 0.94 (d, J = 5.2 Hz, 3H), 0.88 (d, J = 5.7 Hz , 2H), 0.85 (m, 3H) [Production Example 3: Synthesis of Compound 3] [Chemical Formula 7]

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

To a solution of 2'-O-Ac-AZM (2.20g, 2.78 millimoles) at room temperature, add trimethylboroxine (0.055mL, 0.391 millimoles), and warm the reaction mixture to 120°C. After stirring for 2 hours, the mixture was cooled to room temperature, and then concentrated under reduced pressure to obtain an almost pure product (compound 3) (2.27 g, equivalent) as a colorless powder. [Chem. 8] ¹ H NMR (500 MHz, CDCl ₃ ) δ (ppm): 7.52 (s, 1H), 5.08 (d, J = 4.6 Hz, 1H), 4.78 (m, 1H), 4.60 (dd, J = 9.2, 3.4 Hz, 1H), 4.53 (d, J = 7.5 Hz, 1H), 4.40 (m, 1H), 4.13 (s, 1H), 4.02 (m, 1H), 3.55 (d, J = 6.9 Hz , 1H), 3.48 (m, 1H), 3.36 (s, 3H), 3.04 (m, 1H), 2.72 (m, 1H), 2.71-2.62 (complex m, 3H), 2.37-2.34 (complex m, 3H) ), 2.31-2.24 (complex m, 6H), 2.24 (s, 3H), 2.18 (d, J = 10.3 Hz, 1H), 2.08-1.99 (complex m, 4H), 1.98-1.92 (complex m, 2H) , 1.82 (m, 1H), 1.72 (bs, 1H), 1.58 (dd, J = 15.5, 5.2 Hz, 1H), 1.48 (m, 1H), 1.34-1.19 (complex m, 4H), 1.32 (d, J = 5.8 Hz, 3H), 1.27-1.23 (complex m, 12H), 1.20 (d, J = 7.5 Hz, 3H), 0.89-0.84 (complex m, 12H). ESI-MS; 815.4 [M+H] ⁺ [Manufacturing Example 4: Synthesis of Compound 2] [Chemical 9]

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

_{SiO 2} (2.8 g) was added to the MeOH (28.0 mL) solution of the crude product. The mixture was heated to 50°C, stirred for 21 hours, and then cooled to room temperature. The MeOH solution was concentrated, and the residue was purified by rapid column chromatography with silica gel (CHCl ₃ /MeOH/NH ₃ =60/1/0.1 to 10/1/0.1) to obtain compound 2 as a colorless solid ( 1.2g, 52% in 3 steps). [Chem. 10] ¹ H NMR (500 MHz, CDCl ₃ ) δ (ppm): 5.12 (d, J = 4.6 Hz, 1H), 4.86 (m, 1H), 4.70 (dd, J = 9.8, 2.3 Hz, 1H ), 4.55 (d, J = 9.8 Hz, 1H), 4.51 (d, J = 7.5 Hz, 1H), 4.39-4.36 (complex m, 2H), 4.28 (d, J = 2.9 Hz, 1H), 4.02- 4.00 (complex m, 2H), 3.72-3.68 (complex m, 2H), 3.65 (d, J = 6.9 Hz, 1H), 3.37 (bs, 1H), 3.32 (s, 3H), 3.24 (dd, J = 9.8, 7.5 Hz, 1H), 2.81-2.78 (complex m, 2H), 2.70 (m, 1H), 2.54-2.50 (complex m, 2H), 2.39 (d, J = 14.9 Hz, 1H), 2.32-2.31 (complex m, 8H), 2.25 (m, 1H), 2.08-1.98 (complex m, 3H), 1.89 (m, 1H), 1.78 (d, J = 14.4 Hz, 1H), 1.68 (m, 1H), 1.64 (dd, J = 14.8, 5.2 Hz, 1H), 1.48 (m, 1H), 1.32-1.24 (complex m, 6H), 1.21-1.18 (complex m, 10 H), 1.16 (s, 3H), 1.10 -1.05 (complex m, 9H), 0.91-0.88 (complex m, 6H). ESI-MS: m/z calcd for C ₄₂ H ₇₆ N ₃ O ₁₃ [M+H] ⁺ 830.5378. Found: m/z 830.5379 . [Statistical Analysis]

All experimental data are analyzed using GraphPrism7.02 and analyzed by Mann-Whitney U test (MWU), one-way or two-way analysis of variance (ANOVA). ≪Experimental technique using azithromycin (AZM)≫ [Access to macrolide compounds]

For all experiments, Azithromycin Anhydrate (AZM) was purchased from Tokyo Chemical Industry Co., Ltd. [Cell Culture]

Human lung cancer (A549) cells and MDCK (Madin-Darby canine kidney) cells were added with 10% fetal bovine serum (FBS), 20μM/mL L-glutamic acid and 100μg/mL penicillin and chain at 37°C. In Dulbecco Modified Eagle Medium (DMEM) or Minimal Essential Medium (MEM) (Sigma Life Science), cultured to 100% confluent. [Influenza virus]

The human influenza A(H1N1)pdm09 (A/California/7/2009(H1N1)) virus is a virus managed by the National University Corporation Chiba University and the A-CLIP Research Institute Co., Ltd. The virus was infected into MDCK cells and cultured for 24 hours. Next, the virus plaque analysis described later is used to determine the virus power value in the culture medium, and until before use, it is divided into small portions and stored at -80°C or -150°C. (A) Influenza A (H1N1) pdm09 virus when it is infected into host cells

Thaw the influenza A (H1N1) pdm09 virus pre-stored at -80°C or -150°C, and infect human lung cancer (A549) cells with the required amount of virus. (B) Preparation of influenza A (H1N1) pdm09 virus suitable for mice

Influenza A (H1N1) pdm09 virus was nasally infected with 1×10 ⁴ plaque forming units (pfu) of human influenza A (H1N1) pdm09 virus in 8-week-old female mice anesthetized with isoflurane. After 4 days, the lung tissue was homogenized in 1.5 mL of phosphate buffered saline (PBS). After spinning down at 8000 rpm for 5 minutes, the supernatant was recovered and diluted with RPMI1640 medium supplemented with 2% FBS three times as the primary virus for the next subculture. An aliquot of 30 μL was used for the second inoculation, and the subculture procedure was repeated 10 times for the above-mentioned mice. The virus that was finally succeeded was used as a mouse suitable virus and used in the following animal experiments. [For virus infection in host cells, different azithromycin treatments]

Dissolve AZM in ethanol and adjust the final concentration of ethanol in DMEM to 0.2%. For the confluent monolayer of A549 cells in the 6-well plate, the human influenza A (H1N1) pdm09 virus was infected with the multiplicity of infection (MOI) 1 and the following 4 different treatment conditions. The concentration of AZM is set to 200 μM.

(i) Treatment after infection: A549 cells were infected with virus at 35°C for 1 hour. After infection, the cells were washed with PBS, and cultured at 37°C for 48 hours in 2 mL of supplemented DMEM medium with or without AZM addition.

(ii) Pre-treatment of cells: Use 300 μL of AZM supplemented or non-supplemented DMEM medium without AZM supplementation to pre-treat the host cells at 37°C for 1 hour. After removing the medium, the cells were washed with PBS and allowed to infect the virus at 35°C for 1 hour. Next, the cells were washed with PBS and cultured in 2 mL of supplemented DMEM medium without AZM at 37°C for 48 hours.

(iii) Virus pretreatment: Use 300 μL of AZM supplemented or non-supplemented DMEM medium without AZM supplement to pre-treat the virus at 37°C for 1 hour. After this pretreatment, A549 cells were infected with virus at 35°C for 1 hour. Next, the cells were washed with PBS and cultured in 2 mL of supplemented DMEM medium without AZM at 37°C for 48 hours.

(iv) Treatment simultaneously with infection: Mix 300 μL of AZM-added or AZM-free non-supplemented DMEM culture with virus, and quickly use it to infect A549 cells with virus at 35°C for 1 hour. After infection, the cells were washed with PBS, and cultured at 37°C for 48 hours in 2 mL of supplemented DMEM medium with or without AZM addition.

The virus valency in the culture medium and the expression degree of the virus matrix protein 1 (M1) gene in the cells were determined by virus plaque analysis and quantitative PCR methods, respectively. [As a spot analysis of the virus power price evaluation method]

The confluent monolayer of MDCK cells were infected with the following culture medium at 35°C for 1 hour, which was the culture medium recovered from each experiment and serially diluted. After removing the culture medium, wash the cells with PBS, and wash the cells with 0.8% agarose, 40mM HEPES, 0.15% sodium bicarbonate, 2mM L-glutamic acid, 2μg/mL trypsin and 50μg/mL gentamicin (gentamicin) Eagle's minimum essential medium (EMEM) overlay (overlay). After culturing at 37°C for 48 hours, the cells were fixed with 10% formaldehyde, followed by staining with 0.1% crystal violet solution and counting the virus plaques. [Calculate 50% inhibition concentration (IC ₅₀ )]

The 50% inhibitory concentration of AZM on virus proliferation was evaluated by the method described in the column of azithromycin treatments for different azithromycin treatments against virus infection in host cells (iv; treatment at the same time as infection). The virus was pre-mixed in 300 μL of non-supplemented DMEM medium containing various concentrations of AZM up to 600 μM, and quickly allowed to infect the host cell A549 cell virus at 35°C for 1 hour. Next, the cells were washed with PBS and cultured in AZM supplemented DMEM medium without addition at 37°C for 48 hours. The power value of the progeny virus in the culture medium was measured by virus plaque analysis, and the IC ₅₀ value was calculated. [Evaluation of Cytotoxicity]

The cytotoxicity of AZM to A549 cells is evaluated as follows: Use cell proliferation kit I (Roche), and follow the manufacturer’s instructions through MTT[3-(4,5-dimethylthiazol-2-yl) -2,5-Diphenyltetrazolium bromide] analysis and evaluation. Cells were cultured in 300 μL of non-supplemented DMEM medium containing various concentrations of AZM up to 600 μM in the presence or absence of virus (1 MOI) at 35° C. for 1 hour. After the treatment, the cells were washed with PBS and cultured in AZM supplemented DMEM medium 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 culture was cultured for 3 hours. Next, 1 mL of the solubilizing solution was added, and cultured at 37°C for 14 hours. The solubilized formazan product was measured by spectrophotometry using iMark microplate reader (Bio-Rad). [Evaluation of the inhibitory effect of AZM on the germination 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 (200μM). After culturing for 10 hours, the gene expression of the virus in the cells and the power value of the germination progeny virus in the culture medium were determined by quantitative PCR and spot analysis, respectively. The culture medium containing the progeny virus was recovered, and the recovered A549 cells were infected with the reconstituted A549 cells at 35°C in the presence or absence of AZM for 1 hour. After infection, the cells were cultured in supplemented medium without AZM at 37°C for 7 hours, and then used for M1 performance analysis. [Analysis of Hemagglutinin Inhibition]

Prepare a fresh red blood cell (RBC) PBS solution (1%) from chicken whole blood (Biotest). Put 25μL of the serially diluted influenza A (H1N1) pdm09 virus solution (640 hemagglutination unit (HAU)/mL) in an equal volume of PBS or AZM/ethanol PBS solution at room temperature (20~22℃) ) Incubate for 30 minutes. Add 5 μL of 1% RBC solution, and then incubate at room temperature for 20 minutes. By observing hemagglutination, investigate whether AZM can inhibit the combination of hemagglutinin (HA) of the virus and sialic acid (SA) on red blood cells (RBC). [To determine the inhibition analysis of the effect of AZM on the attachment phase or internal movement phase during virus infection]

The next stage of analysis: the confluent monolayer of 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.

Analysis of internal migration stage: A549 cells were incubated with virus (1MOI) at 4°C for 1 hour. The cells were washed with warm PBS and cultured in a medium containing 200 μM AZM at 37°C for 1 hour. After culturing, the cells were washed with PBS, and treated with proteinase K (Fuji Film Wako Pure Chemical Industries, Ltd.) in PBS solution (final concentration 100 μg/mL) at 37°C for 5 minutes to remove viruses remaining on the cell surface. Synthesize cDNA from the extracted total RNA, and use quantitative PCR to determine the expression level of virus M1 and nuclear protein (NP). [Quantitative real-time PCR]

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

[Mouse]

The animal protocol for influenza virus infection is in accordance with the guidelines of Teikyo University and is recognized by the ethics committee of Teikyo University's animal experiments (AUP number: 16-021). Wild-type 8-week-old BALB/c female mice were purchased from SLC Company and bred in a pathogen-free environment. [Animal infection experiment and AZM administration]

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 is adjusted to within 3%. The anesthetized mice were nasally infected with 300 pfu mice suitable for influenza A (H1N1) pdm09 virus. After 6 hours of infection, all lung tissues were excised from the 1 group of mice and set as a control group (untreated). To mice in other groups, 3 days after infection, the above-mentioned mixed solution with AZM addition (10 mg/kg) or no addition was administered nasally under isoflurane anesthesia at 12-hour intervals a day and twice. Monitor the rectal body temperature and body weight of the mice. At different time points, all lung tissues were excised from the treated mice, and ^{homogenized in RNAiso Plus solution using a bead cell crusher (MicroSmash TM} MS-100, Tomy Company). Synthesize a cDNA pool from the extracted total RNA, and use a quantitative PCR method to determine the degree of expression of the virus's M1 and NP genes. ≪Experimental results≫ (AZM inhibits the activity of influenza A(H1N1)pdm09 virus through direct interaction with the virus)

In order to investigate the AZM treatment conditions that showed the most effective antiviral activity, experiments were conducted under 4 different treatment conditions. The results are shown in Figure 1. Compared with the control group, the AZM administration after infection showed the same degree of viral power in the offspring in the culture medium (Figure 1a) and the same degree of expression of the M1 gene in the host cell (Figure 1a). Figure 1b). When the virus is pre-treated with AZM 1 hour before infection, the power value and M1 performance of the offspring virus are significantly reduced. In the case of AZM administered at the same time as the infection, the viral power of the offspring was also significantly reduced to the same level as that observed during the previous treatment of the virus. In contrast, when the A549 cells of the host were treated with AZM for 1 hour before treatment, compared with the control group, significant differences were observed in both the power of the offspring virus and the degree of M1 expression. These results suggest that AZM interacts with influenza A (H1N1) pdm09 virus in the initial stage of infection to inhibit viral activity. Both the treatment 1 hour before and the AZM treatment at the same time as the infection showed the same inhibitory effect on the proliferation of the offspring virus. (AZM does not show cytotoxicity to host cells within the _{IC 50 range)}

_{Next, the IC 50} value of AZM on the proliferation of the offspring virus was calculated (Figure 2). Through administration of AZM, to reduce the discharged dose-dependent generation of virus after culture solution, and the average IC ₅₀ of about of 68 m (FIG. 2a). The expression status of the virus M1 gene in A549 cells is correlated with the tendency of virus power (Figure 3).

In order to determine the concentration at which AZM exhibits toxicity to host A549 cells, a wide range of AZM concentrations were used and MTT analysis was performed (Figure 2b). Under non-infectious conditions, even if it is co-cultured with AZM at a concentration of less than 200 μM, significant cytotoxicity will not be observed (Figure 2b, left panel). Similarly, under infection conditions, no toxic effect on A549 cells was observed until the AZM concentration of 600 μM (Figure 2b, right panel). From these results suggest that in both non-infected state and the state of infection, in the range of IC _50, of AZM have a bad effect on the survival of host cells does not. (AZM will not affect the connection status, but it will affect the internal migration of the virus)

In order to investigate the mechanism of AZM's antiviral activity, it was investigated whether AZM could inhibit the binding interaction between viral hemagglutinin (HA) and sialic acid on the surface of red blood cells. As can be seen from the results shown in the upper part of Figure 4, even in the virus diluted to 1/16, hematocrit can still be observed, but in this dilution range, even in the presence of AZM, hematocrit cannot be significantly inhibited. This suggests that AZM does not affect the binding activity of viral hemagglutinin (HA) to sialic acid (SA) receptors on cells. In addition, the graph in the lower part of FIG. 4 is based on the upper part of the photograph in FIG.

In addition, based on the profile of viral genes in the host cell, the mechanism of AZM's inhibition of virus adhering and internal migration process (Figure 5) was explored. 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, no change was seen in the expression of M1 and NP in the virus that followed to the cell surface. In contrast, in the group where AZM was administered after the virus followed and the orphan virus was removed with proteinase K, the expression of M1 and NP in the host cell was significantly reduced (Figure 5b). These results suggest that although AZM does not affect the binding activity of the virus, it does inhibit the internal migration process in the initial stage of virus invasion. In addition, in view of the fact that AZM exhibits a proliferation inhibitory effect on influenza viruses through this mechanism, it is implied that through the same mechanism, the proliferation of coronavirus can also be inhibited by the same mechanism. (AZM will also attack new generation viruses)

The AZM inhibitory effect from the internal migration of the parent virus at the time of infection sets a hypothesis that AZM can inhibit the repeated cycle of virus infection and proliferation of the offspring virus. Then, in order to verify this hypothesis, the amount of virus in the presence of AZM at each time point of the primary infection of the parent virus and the secondary infection of the offspring virus was compared (Figure 6). First, A549 cells of the host are infected with influenza A (H1N1) pdm09 virus, and the cells are cultured for 10 hours in the presence or absence of AZM. At this point in time, the strength of the offspring virus in the culture medium or the degree of M1 expression of the virus in the host cell is the same in the presence or absence of AZM (Figure 6a and Figure 6b). This result is an integrator of the result shown in Figure 1. Next, the culture supernatant containing the germinative progeny virus is recovered and infected with the reconstituted A549 cells in the presence or absence of AZM. Next, the virus-infected A549 cells were cultured in AZM non-supplemented medium for 7 hours. At this time point, in A549 cells cultured in a medium containing progeny virus and AZM, the degree of M1 expression was significantly reduced (Figure 6c). These results actually confirmed the above hypothesis, that is, AZM inhibits the internal migration of extracellular viruses when they invade host cells. (The amount of virus in infected mice will be reduced by a single administration of AZM)

Considering the above in vitro results in A549 cells, experiments were performed to administer AZM to virus-infected mice (in vivo, nasally) (Figure 7a). As shown in Figure 7b, by administering AZM, the expression of the virus's M1 gene and NP gene can be observed to decrease in the lung tissue after 3 days of infection. The maximum inhibition of viral performance can be observed after 2 days of infection in which the virus proliferates vigorously (Figure 7b). The hypothermia caused by the virus administration appeared significantly after 3 days and was suppressed by AZM (Figure 7c). On the other hand, at the time point 3 days after the infection, no effect was seen on the body weight of the infected mice (Figure 7d). The above shows that AZM administered through the nose can reduce the amount of virus in the lungs, thereby alleviating the hypothermia during influenza A(H1N1)pdm09 virus infection. ≪Experimental method using azithromycin derivatives≫

Using the above synthesized compound 2 to compound 4, the following experiments were carried out. In addition, the following compound 5 to compound 7 were used as negative controls. [化11]

Specifically, first, the same experiment as that shown in "treatment simultaneous with infection" in Fig. 1a was performed. As a result, as shown in Fig. 8, when treated with compound 2 to compound 4, similar to AZM, it was confirmed that the progeny virus valence decreased significantly. On the other hand, when compound 5 to compound 7 were treated with compound 5 and compound 6, although compound 5 and compound 6 showed weak inhibitory activity, the potency of the offspring virus did not decrease as much as compound 2 to compound 4. In addition, for the other treatment forms described in Fig. 1a (treatment after infection, treatment before cells, and treatment before virus), the same experiment was also performed using Compound 2 to Compound 4. The results are shown in Fig. 9a~c. Regardless of which compound 2 to compound 4 are used, they are the same as AZM. In the case of pre-treatment of the virus with the compound 1 hour before infection (pre-treatment of the virus), and infection In the case of simultaneous administration of the compound (treatment at the same time as the infection), the power value of the offspring virus is significantly reduced. These results suggest that, like AZM, compound 2 to compound 4 will interact with influenza A (H1N1) pdm09 virus in the initial stage of infection to inhibit viral activity.

Also, using compound 2 to compound 4, the same experiment as that shown in Fig. 2a was performed. As a result, as shown in the upper section of Fig. 10, the calculated IC ₅₀ values of compound 2 to compound 4 against the virus valency were 130 μM, 84 μM, and 84 μM, respectively. Furthermore, using the degree of expression of the M1 gene in the host cell as an index, the same experiment was performed. As a result, as shown in the lower section of FIG. 10, the IC ₅₀ values of compound 2 to compound 4 for the expression level of the M1 gene were calculated to be 140 μM, 140 μM, and 116 μM, respectively.

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

Furthermore, using compound 2 to compound 4, the same experiment as that shown in FIG. 4 (hemagglutinin inhibition analysis) was performed. The result is shown in Fig. 12 together with the result of AZM in Fig. 4. As shown in Figure 12, for compound 2 to compound 4, similar to AZM, even when the virus is diluted to 1/16, red blood cell agglutination can still be observed. However, in this dilution range, even in the presence of AZM, red blood cell agglutination can be observed. Agglutination cannot be significantly suppressed. This suggests that compound 2 to compound 4 will not affect the binding activity of viral hemagglutinin (HA) to sialic acid (SA) receptors on cells. In addition, the graph in the lower part of FIG. 12 is a graph based on the upper part of the photograph in FIG. 12 for the value of hemagglutination unit.

In addition, using compound 2 to compound 4, the same experiment as that shown in FIG. 5 was performed (analysis and internal migration analysis followed). Here, as shown in Fig. 5, AZM suppresses the stage of internal migration (endocytosis) while it does not affect the subsequent stage of the virus. On the other hand, as shown in FIG. 13, it can be seen that compound 2 to compound 4 all inhibit the internal migration (endocytosis) stage of the virus, and also show an inhibitory effect on the subsequent stage. This suggests that AZM derivatives represented by compound 2 to compound 4 may have a mechanism that does not inhibit virus-host cell binding by hemagglutinin and sialic acid. ≪Comparison experiment of azithromycin (AZM) and clarithromycin (CAM)≫

[For Azithromycin Anhydrate (AZM), except that the concentration of the administered agent was changed from _{200μM to 68μM of the IC 50} of AZM, the rest was performed by the same method as the experiment shown in Figure 1 with clarithromycin (CAM) The comparative experiment. As a result, as shown in Fig. 14a, when the virus was pretreated with AZM one hour before infection and when AZM was administered at the same time as the infection, the power value of the offspring virus was significantly reduced. On the other hand, in the case of administration of clarithromycin (CAM), no reduction in the power value of such progeny viruses was observed (Figure 14b). In addition, there are reports that even if clarithromycin (CAM) is used in combination with zanamivir, it still cannot inhibit the replication of human influenza A (H1N1) pdm09 virus in vitro (Lee ACY, et al. Arch Virol. 2018; 163: 2349–2358). It can be seen that clarithromycin (CAM) does not affect the activity of human influenza A (H1N1) pdm09 virus, nor does it show an inhibitory effect on the internal migration of the virus.

This application is based on the Japanese Patent Application No. 2019-100691 filed on May 29, 2019, and the disclosure content is incorporated as a whole by reference. Sequence table non-keyword text (free text) [Serial Number: 1]

The DNA sequence of a pre-primer used to amplify the gene encoding the matrix protein 1 (M1) of the influenza A (H1N1) pdm09 virus. [Serial Number: 2]

The DNA sequence of a reverse primer used to amplify the gene encoding the matrix protein 1 (M1) of the influenza A (H1N1) pdm09 virus. [Serial Number: 3]

The DNA sequence of a pre-primer used to amplify the gene encoding the nuclear protein (NP) of influenza A (H1N1) pdm09 virus. [Serial Number: 4]

The DNA sequence of a reverse primer used to amplify the gene encoding the nuclear protein (NP) of influenza A (H1N1) pdm09 virus. [Serial Number: 5]

The DNA sequence of a pre-primer used to amplify the GAPDH gene of the housekeeping gene in mice. [Serial Number: 6]

The DNA sequence of a reverse primer used to amplify the GAPDH gene of the housekeeping gene in mice. [Serial Number: 7]

The DNA sequence of a pre-primer used to amplify the GAPDH gene of the housekeeping gene in humans. [Serial Number: 8]

The DNA sequence of a reverse primer used to amplify the GAPDH gene of the housekeeping gene in humans.

Figure 1 is a graph and shows that after the human influenza A (H1N1) pdm09 virus was infected into A549 cells, the culture solution after administration of azithromycin (AZM) under 4 different conditions was analyzed by virus plaque assay (virus plaque assay). ) The results of the determination of the viral power (Figure 1a), and the results of the quantitative PCR method of determining the expression level of the virus's matrix protein 1 (M1) gene in the cells (Figure 1b).

_{Fig. 2a of Fig. 2 is a graph showing the result of calculating the IC 50} of AZM on the proliferation of the virus after infection in A549 cells by the treatment at the same time as the infection. Figure 2b is a graph and shows that in order to determine the concentration at which AZM exhibits toxicity to host A549 cells, a wide concentration range of AZM was used and the results of MTT analysis performed under non-infectious or infected conditions.

FIG 3 is a chart and displays, based on the M1 gene expression in A549 cells by the antiviral state ₅₀ the calculation result value IC.

Fig. 4 is a photograph and a graph and both show the results of investigating whether AZM can inhibit the binding of hemagglutinin (HA) of the virus and sialic acid (SA) on red blood cells (RBC) through hemagglutinin inhibition analysis.

Figure 5 is a diagram and shows that for the purpose of measuring the effect of azithromycin (AZM) on the attachment phase (Figure 5a) or internal migration phase (Figure 5b) of virus infection, the attachment phase or internal migration of the virus infected A549 cells After stage incubation of AZM, total RNA was extracted from A549 cells to synthesize cDNA, and quantitative PCR was used to determine the expression level of virus M1 and nuclear protein (NP).

Figure 6 is a graph and shows the results of experiments conducted under the purpose of confirming the hypothesis that AZM can inhibit viral infection and proliferation of progeny viruses. Figures 6a and 6b are graphs and respectively confirm that when the influenza A (H1N1) pdm09 virus is infected into A549 cells, and the cells are cultured for 10 hours in the presence or absence of AZM, the progeny virus in the culture medium The strength and the degree of M1 expression of the virus in the host cell are the same in the presence of AZM and the absence of AZM. In addition, Fig. 6c is a graph and shows that the culture supernatant containing the germination progeny virus is recovered, and the newly prepared A549 cells are infected in the presence or absence of AZM, and the virus-infected A549 cells are added to the AZM medium without the addition of AZM. After culturing for 7 hours in medium, compare the results of M1 expression level in A549 cells cultured in a medium containing progeny virus in the presence or absence of AZM.

Fig. 7a of Fig. 7 is an experimental procedure and shows the method of nasally infecting mice with influenza A (H1N1) pdm09 virus. Figure 7b is a graph and shows the results of total RNA extracted from lung tissue after nasal administration of azithromycin (AZM) to mice, and the results of quantitative PCR to determine the expression level of virus M1 and NP genes from the total RNA . In addition, Figure 7c is a graph and compares the rectal body temperature and body weight of the mice in the experiment.

Figure 8 is a graph and shows that azithromycin (AZM) and compound 2~compound 4 are used, as well as compound 5~compound 7 as negative controls (although compound 5 and compound 6 have weak inhibitory activity), and are compared with Figure 1a. Simultaneous infection treatment" shows the results of the same experiment.

Figures 9a-9c of Figure 9 are graphs and show that after the human influenza A (H1N1) pdm09 virus is infected into A549 cells, the medium after compound 2 to compound 4 is administered under 4 different conditions, and the virus Spot analysis is the result of determining the power value of the virus.

Figure 10 is a graph and shows the results of using Compound 2 to Compound 4 that showed anti-influenza virus activity in Figure 8 to perform the same experiment as that shown in Figure 2a, and to determine the IC ₅₀ value.

Fig. 11 is a graph and shows the results of the same experiment (MTT analysis) as shown in Fig. 2c using Compound 2 to Compound 4 showing anti-influenza virus activity in Fig. 8.

Figure 12 is a photo and graph, and is the result of investigating whether compound 2~compound 4 can inhibit the binding of viral hemagglutinin (HA) and sialic acid (SA) on red blood cells (RBC) by hemagglutinin inhibition analysis , Which is displayed together with the AZM result shown in Figure 4.

Figure 13 is a graph and shows the results of the AZM shown in Figure 5 together with the following results: This result is the attachment stage (Figure 13a) or the internal movement stage (Figure 13a) when the virus is infected by compounds 2 to 4 For the purpose of the effect of 13b), after incubating each compound during the attachment stage or the internal migration stage of the virus infected A549 cells, total RNA was extracted from the A549 cells to synthesize cDNA, and the performance of the virus M1 was measured by quantitative PCR degree.

Figure 14 is a graph and shows that, for azithromycin (AZM), except that the concentration of the administered drug was changed from 200 μM to _{68 μM of the IC 50} of AZM, the rest was performed by the same method as the experiment shown in Figure 1 The results of comparative experiments on clarithromycin (CAM).

Claims (17)

An anti-influenza virus agent containing one or more 15-membered ring macrolide compounds or their salts or hydrates selected from the group consisting of the following compounds 1 to 4 as effective Ingredients: [Chemical 1] [Compound 1] [Compound 2]

Azithromycin (AZM) [Compound 3] [Compound 4]

. A preventive and/or therapeutic agent for influenza virus infection, containing one or two or more 15-membered cyclic macrolide compounds selected from the group consisting of the following compounds 1 to 4 Salt or hydrate as the effective ingredient: [Chemical 2] [Compound 1] [Compound 2]

Azithromycin (AZM) [Compound 3] [Compound 4]

. A medicinal composition for the prevention and/or treatment of influenza virus infection, containing one or more 15-membered cyclic macrolides selected from the group consisting of the following compounds 1 to 4 The compound or its salt, or hydrate, and a pharmaceutically acceptable carrier: [form 3] [compound 1] [compound 2]

Azithromycin (AZM) [Compound 3] [Compound 4]

. A method for the prevention and/or treatment of influenza virus infection, comprising administering to a patient in need one or more 15-membered ring macrocycles selected from the group consisting of the following compounds 1 to 4 Lactone compound or its salt, or hydrate: [Chem 4] [Compound 1] [Compound 2]

Azithromycin (AZM) [Compound 3] [Compound 4]

. A use of one or two or more 15-membered ring macrolide compounds or their salts or hydrates selected from the group consisting of the following compounds 1 to 4, which is used to make popular Preventive and/or therapeutic agent for influenza virus infection: [化5] [Compound 1] [Compound 2]

Azithromycin (AZM) [Compound 3] [Compound 4]

. A preventive and/or therapeutic agent for coronavirus infection, which contains one or more 15-membered ring macrolide compounds or their salts selected from the group consisting of the following compounds 1 to 4, Or hydrate as the effective ingredient: [Chemical 6] [Compound 1] [Compound 2]

Azithromycin (AZM) [Compound 3] [Compound 4]

. The preventive and/or therapeutic agent according to claim 6, wherein the aforementioned coronavirus infection is a 2019 novel coronavirus (SARS-CoV-2) infection. A method for the prevention and/or treatment of coronavirus infection, comprising administering to a patient in need one or more 15-membered cyclomacrolides selected from the group consisting of the following compounds 1 to 4 Compound or its salt, or hydrate: [Chemical Formula 7] [Compound 1] [Compound 2]

Azithromycin (AZM) [Compound 3] [Compound 4]

. Such as the method of claim 8, wherein the aforementioned coronavirus infection is a 2019 novel coronavirus (SARS-CoV-2) infection. A compound represented by the following chemical formula (1) or its salt or hydrate: [Chemical Formula 8]

In the chemical formula (1), X represents a hydrogen atom or an acyl group having 1 to 4 carbon atoms, and Y ¹ represents a hydrogen atom or -C(=O)-NH-(CH ₂ ) _n -C≡CH (here, n is an integer from 1 to 4), Y ² and Y ³ represent a hydrogen atom or -C(=O)-NH-(CH ₂ ) _n -C≡CH (here, n is an integer from 1 to 4 ) Represents the group, or together form the group represented by the following chemical formula (2), [Chemical formula 9] [Chemical formula 2]

In the chemical formula (2), * represents the bonding site, but the chemical formula (1) contains -C(=O)-NH-(CH ₂ ) _n -C≡CH (here, n is an integer from 1 to 4) Either the group represented by the formula (2) or the group represented by the chemical formula (2). A medicine containing the compound of claim 10 or its salt or hydrate. Such as the medicine of claim 10, which is an anti-influenza virus agent. Such as the medicine of claim 10, which is an anti-coronavirus agent. A preventive and/or therapeutic agent for coronavirus infection, which contains the compound of claim 10 or its salt or hydrate as an active ingredient. Such as the preventive and/or therapeutic agent of claim 14, wherein the aforementioned coronavirus infection is a 2019 novel coronavirus (SARS-CoV-2) infection. A method for the prevention and/or treatment of coronavirus infection, which comprises administering the compound of claim 10 or its salt or hydrate to a patient in need. Such as the method of claim 16, wherein the aforementioned coronavirus infection is a 2019 novel coronavirus (SARS-CoV-2) infection.

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