JP6630887B2 - Viral replication inhibitor - Google Patents

Description

  The present invention relates to a replication inhibitor for a plus-strand RNA virus including tomato mosaic virus, rubella virus, porcine epidemic diarrhea virus and human hepatitis E virus. In particular, a compound having an inhibitory activity on NTP (nucleotide triphosphate) degrading enzyme activity of SF1 helicase of tomato mosaic virus or the like, a salt thereof, a solvate thereof, or a hydrate thereof as an active ingredient. The present invention relates to a strand RNA virus replication inhibitor.

  To date, drugs that inhibit the replication of human herpes simplex virus, human acquired immunodeficiency syndrome virus (HIV) and influenza virus have been developed, and have been put to practical use as drugs for controlling infection by the viruses. However, development of drugs for controlling infection by viruses other than the above has not been sufficiently advanced. For example, for plant virus infection, there is no effective drug that does not affect the host and suppresses only virus replication, so there is no means to fundamentally suppress virus infection. Countermeasures such as extermination of organisms, disinfection of soil, and introduction of virus resistance genes into plants have been taken. However, extermination of the vector and disinfection of the soil require a large amount of money, have a large economic loss, and have a great impact on the environment. In addition, there is a concern about the safety of genetically modified crops in the technique of genetically modifying crops, and it cannot be said that this is a practical method. Few drugs are effective against animal viruses such as swine epidemic diarrhea virus, and at present, measures such as early diagnosis of infection, isolation and culling such as culling, thorough disinfection, and vaccine prevention are taken. Have been. However, it is hard to say that detection and selection requires a great deal of labor and is preferable from the viewpoint of animal protection. In addition, excessive disinfection may cause a burden on the environment as in the case of plant viruses. In prophylaxis by vaccination, conventional vaccines are ineffective due to mutations in the virus, and there are many viruses for which no effective vaccine is available in the first place.

  Tomato mosaic virus (ToMV), which is a kind of tobamovirus, is a pathogenic plus-strand RNA virus that causes tomato mosaic disease and belongs to the category of plus-strand RNA virus. In general, a plus-strand RNA virus enters host cells such as eukaryotes, and after genomic RNA is translated through uncoating and a replication protein that controls replication is synthesized, the replication protein becomes its own replication template. It is known that genomic RNA is recruited on a biological membrane, forms a replication complex with host factors, and replicates plus-strand RNA via minus-strand RNA.

  In addition, a helicase domain is encoded in the ToMV genome. Helicase is a generic term for enzymes that unfold nucleic acids that move and entangle while moving along the phosphate backbone of nucleic acids. The helicase domain has NTP hydrolase activity, and the NTPase activity is a helicase activity (hydrogen between bases). The helicase domain is essential for virus replication by dissolving the binding and unwinding the double helix of the DNA and the RNA having a secondary structure, and the host factor (low molecular weight GTPase protein ARL8 ( It has already been clarified that by playing an important role in the interaction with the non-patent document 1) and the membrane protein TOM1 (non-patent document 2), it is essential for the replication of the virus. Therefore, if a compound that targets the helicase domain and inhibits the helicase activity or the NTPase activity can be found, the compound is expected to be a plus-strand RNA virus replication inhibitor.

In general, helicases are classified into six types from superfamily (SF) 1 to SF6 based on the similarity of the three-dimensional structure and the homology of the conserved amino acid motif (Non-Patent Document 3). There are three types of helicase domains, SF1 to SF3 (for example, Non-Patent Document 4).
Dengue virus, hepatitis C virus, and the like are known as viruses encoding SF2, and drug development based on the structure is being promoted (Non-Patent Document 5). Examples of the virus encoding SF3 include papilloma virus. These three-dimensional structures have already been elucidated (Non-Patent Document 6). On the other hand, SF1 is expected to be encoded by many plant and animal viruses including ToMV, such as coronavirus and rubella virus, but the three-dimensional structure of SF1 has not been elucidated. Patent Document 7).

  The inventors have succeeded in determining the three-dimensional structure of SF1 helicase (ToMV-Hel) encoded by tomato mosaic virus for the first time in the world in 2012 (Non-Patent Document 7). From the three-dimensional structure of ToMV-Hel revealed thereby and the comparison of the amino acid sequence of ToMV-Hel with the amino acid sequences of helicases of various positive-strand RNA viruses such as coronavirus, It was presumed that the three-dimensional structure of the NTP binding site of the SF1 helicase was similar to that of ToMV-Hel.

  However, development of a replication inhibitor for various positive-strand RNA viruses such as ToMV and coronavirus has not been reported so far.

Nishikiori et al. (2011) PLoS Pathog. 7: e1002409 Yamanaka et al. (2000) PNAS 97: 10107. Shingleton et al. (2007) Annu Rev Biocheme 76:23. Koonin and Dolja (1993) Biochem Mole Biol 28: 375. Kwang et al. (2001) Curr Top Microbiol Immunol 242: 171. Norder et al. (2011) Antiviral Res 89: 204. Nishikiori et al. (2012) J.C. Virol. 86: 7565

In view of the above circumstances, an object of the present invention is to provide an inhibitor that inhibits the NTPase activity of the SF1 helicase domain of tomato mosaic virus and the like.
Furthermore, an object of the present invention is to provide a medicament or an agricultural chemical useful for treating and / or preventing infectious diseases caused by a virus having the SF1 helicase, which contains an inhibitor of “SF1 helicase” as an active ingredient.

If the NTPase activity of ToMV-Hel can be specifically inhibited, the helicase activity of the protein can be inhibited, and thus the replication of the virus encoding the helicase can be inhibited. As a result of the above-mentioned three-dimensional structure analysis, the three-dimensional structure of the NTP binding site in ToMV-Hel has been clarified.
The present inventors have selected, by computer simulation, about 300 types of low-molecular-weight organic compounds that may have an activity of specifically binding to the NTP-binding site based on the three-dimensional structure information of SF1 helicase possessed by ToMV. As a result of examining the activity of the selected compound to inhibit the replication of ToMV and the activity of inhibiting the NTP hydrolase activity of ToMV-Hel, it was found that five kinds of organic compounds had the activity of inhibiting the replication of ToMV. Was found. Furthermore, the inventors have found that these compounds have a replication inhibitory activity against a positive-strand RNA virus such as rubella virus encoding SF1 helicase. Based on these results, the inventors have completed the invention of a novel replication inhibitor against various plus-strand RNA viruses.

That is, the present invention is a plus-strand RNA virus replication inhibitor comprising, as an active ingredient, a compound that inhibits the NTPase activity of SF1 helicase, a salt thereof, or a solvate or hydrate thereof.
More specifically, the present invention relates to the following general formula (1)
[In the formula (1), X represents a methoxy group or a direct bond, and R ₁ , R ₂ , R ₄ and R ₅ each independently represent a hydrogen atom, a methoxy group, an ethoxy group, a fluorine atom, a chlorine atom or R ₃ represents a methyl group, an ethyl group, an ethenyl group or a substituent represented by the formula (2);
(In the formula (2), A ₁ represents a hydrogen atom, a methyl group, a fluorine atom, a chlorine atom or a bromine atom, and A ₂ represents a hydrogen atom, a methyl group, a 3-oxoethyl group, a 3-oxopropyl group, a fluorine atom, A represents a chlorine atom or a bromine atom, A ₃ represents an arbitrary substituent, and preferably A ₂ and A ₃ each independently represent a chlorine atom or a substituent represented by the formula (3) to (5).
More preferably, when A ₂ is a chlorine atom, A ₃ is a substituent represented by the formula (3), and when A ₂ is a substituent represented by the formula (4) In the formula, A ₃ is a substituent represented by the formula (5), and A ₂ and A ₃ may together form a condensed ring, and the condensed ring is preferably a group represented by the formula (6) Or a substituent represented by (7)
It is. Incidentally, doublet moieties described repeatedly wavy line in the formula (6) and (7) in indicates a double bond A ₂ and A ₃ are attached in formula (2). ),
Or a substituent represented by the following general formula (8)
(In the formula (8), Y represents a carbon atom or a nitrogen atom, B ₁ and B ₂ each represent an arbitrary substituent, and B ₁ and B ₂ may form a condensed ring together. , Preferably a substituent represented by the formula (9)
),
And a solvate or hydrate thereof as an active ingredient.

  In addition, the present invention relates to a method for preventing infectious diseases in animals caused by a virus having an SF1 helicase containing a compound represented by the general formula (1) or a salt thereof, or a solvate or hydrate thereof as an active ingredient. It is a medicament or a pharmaceutical composition useful for treatment and / or prevention, and the animal preferably includes human, pig, horse and the like.

  Furthermore, the present invention provides a method for treating infectious diseases caused by a virus having SF1 helicase, comprising a compound represented by the above general formula (1) or a salt thereof, or a solvate or hydrate thereof as an active ingredient. And / or pesticides or pesticidal compositions useful for prevention.

  The compounds of the present invention can inhibit the replication of viruses having the SF1 helicase. As a result, the use of the compound of the present invention makes it possible to prevent and / or treat an infectious disease of a virus having SF1 helicase.

  In addition, the compound of the present invention and a medicament or pesticide for an animal containing the compound as a pharmaceutically active ingredient can be used for prevention and / or treatment of an animal or plant infectious disease of a virus having SF1 helicase. it can.

Flow of in silico screening. Evaluation of ToMV replication inhibitory activity using an in vitro ToMV RNA replication system. Evaluation of ATP hydrolase inhibitory activity using isothermal titration calorimetry. Evaluation of pig pig epidemic diarrhea swine coronavirus (PEDV) replication inhibitory activity. Evaluation of rubella virus replication inhibitory activity. Evaluation of human hepatitis E virus (HEV) replication inhibitory activity.

In the general formula (1), X represents a methoxy group or a direct bond, and R ₁ , R ₂ , R ₄ and R ₅ each independently represent a hydrogen atom, a methoxy group, an ethoxy group, a fluorine atom, a chlorine atom or a bromine atom. R ₃ represents a methyl group, an ethyl group, an ethenyl group, a substituent represented by the formula (2) (in the formula (2), A ₁ represents a hydrogen atom, a methyl group, a fluorine atom, a chlorine atom or a bromine atom. A ₂ represents a hydrogen atom, a methyl group, a 3-oxoethyl group, a 3-oxopropyl group, a fluorine atom, a chlorine atom or a bromine atom, A ₃ represents an arbitrary substituent, preferably A ₂ and A ₃ Is independently a chlorine atom or any of the substituents represented by formulas (3) to (5). More preferably, when A ₂ is a chlorine atom, A ₃ is represented by the formula (3) is a substituent, the substituents a ₂ are represented by the formula (4) If, A ₃ is a substituent represented by the formula (5), further it may form a condensed ring in cooperation A ₂ and A _3, condensed rings, preferably, formula (6) or (7) is a substituent represented by. Incidentally, doublet moieties described repeatedly wavy line in the formula (6) and (7) in the bonded is a ₂ and a ₃ in formula (2) Or a substituent represented by the following general formula (8) (in the formula (8), Y represents a carbon atom or a nitrogen atom, and B ₁ and B ₂ are each independently arbitrary) indicates the substituent, further B ₁ and B ₂ may form a condensed ring in cooperation, preferably exhibit a substituent represented by the formula (9)).
Unless otherwise specified, the tautomers and geometric isomers of the compound represented by the general formula (1) contained as an active ingredient of a virus replication inhibitor having an SF1 helicase according to the present invention and a medicament are included. Stereoisomers such as (for example, E-form and Z-form) and enantiomers are also included.

Examples of the compound represented by the general formula (1) and a salt thereof include, but are not limited to, the following.
2- {4-[(1Z) -2-chloro-2- (5-chlorobenzimidazol-2-yl) vinyl] -2-methoxyphenoxy} acetic acid (Compound A) and an acceptable salt thereof,
2- {4-[(5-imino-7-oxo-3-phenyl (4-hydro-1,3-thiazolino [3,2-a] pyrimidine-6-ylidene)) methyl] -2-methoxyphenoxy} Acetic acid (compound B) and its acceptable salts,
2- {4-[(1Z) -2- (3-cyclohexyl (7H-1,2,4-triazolo [3,4-b] 1,3,4-thiadiazin-6-yl))-2- ( Ethoxycarbonyl) vinyl] phenoxydiacetic acid (compound C) and its acceptable salts,
2- [4-({1-[(4-chlorophenyl) methyl] -2,5-dioxo (1,3-diazolidine-4-ylidene)} methyl) -2-methoxyphenoxy] acetic acid (compound D) Acceptable salts,
4-{[6-methyl-5- (4-methylphenyl) -4-oxo-3-hydrothiopheno [2,3-d] pyrimidin-3-yl] methyl} benzoic acid (Compound E) and its tolerance Salt.

  As the compound represented by the general formula (1) of the present invention, commercially available compounds can be purchased. For example, commercially available products from Enamine, MolPort, Namiki Shoji and the like can be used.

  According to a preferred embodiment of the present invention, there is provided a plus-strand RNA virus replication inhibitor comprising a compound represented by the general formula (1), a salt thereof, a solvate thereof, or a hydrate thereof as an active ingredient. Is done. Further, according to another preferred embodiment of the present invention, a method for preventing and / or treating infectious diseases caused by a virus having SF1 helicase, comprising a compound represented by the general formula (1) as a pharmacologically active ingredient. Or a pesticide or a pesticide composition for an animal.

  The SF1 helicase according to the present invention is a virus having the characteristics of GDxxQ as motif III and T / SxxxxQ / KG (x can be any amino acid) as motif V. As a virus having SF1 helicase as an essential factor for its replication, For example, as a plant virus, tobacco rattle virus, tobacco mosaic virus, tobacco mild green virus, turnip yellow mosaic virus, alfalfa mosaic virus, cucumber mosaic virus, beet yellowing virus, potato X virus, wheat dwarf virus, pig as animal virus Epidemic diarrhea virus, SARS coronavirus, MERS coronavirus, feline peritonitis virus, coronavirus including mouse hepatitis virus, togavirus including rubella virus, hepatitis E virus , Can be exemplified. See Non-Patent Document 4 for details.

The subject to be treated by the medicament of the present invention may be any animal as long as it is infected with a virus having SF1 helicase as an essential factor for its replication. For example, non-human animals infected with swine epidemic diarrhea virus, feline peritonitis virus, mouse hepatitis virus, etc., humans infected with rubella virus, etc., and hepatitis E, a zoonotic virus, equine encephalitis virus Human and non-human animals infected with E. coli or the like are treated with the medicament of the present invention, and are preferably swine in swine epidemic diarrhea virus or the like, or human in rubella virus or the like.
The target of the disease control by the pesticide of the present invention may be any plant, for example, tomatoes infected with tomato mosaic virus, tobacco rattle virus, tobacco mosaic virus, tobacco infected with tobacco mild green virus, etc., Wheat that is infected with the wheat dwarf virus can be mentioned.

As the active ingredient of the medicament of the present invention, a physiologically acceptable salt thereof may be used in addition to the compound represented by the general formula (1). Examples of the salt include, when an acidic group is present, salts of alkali metals and alkaline earth metals such as lithium, sodium, potassium, magnesium, and calcium; ammonia, methylamine, dimethylamine, trimethylamine, dicyclohexylamine, and tris ( A salt of an amine such as (hydroxymethyl) aminomethane, N, N-bis (hydroxyethyl) piperazine, 2-amino-2-methyl-1-propanol, ethanolamine, N-methylglucamine, L-glucamine; or lysine; Salts with basic amino acids such as δ-hydroxylysine and arginine can be formed. When a basic group is present, salts of mineral acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid; methanesulfonic acid, benzenesulfonic acid, paratoluenesulfonic acid, acetic acid, propionate, and tartaric acid With organic acids such as fumaric acid, maleic acid, malic acid, oxalic acid, succinic acid, citric acid, benzoic acid, mandelic acid, cinnamic acid, lactic acid, glycolic acid, glucuronic acid, ascorbic acid, nicotinic acid, salicylic acid, etc. Salts; and salts with acidic amino acids such as aspartic acid and glutamic acid.
Furthermore, as the active ingredient of the medicament of the present invention, a solvate or hydrate of the compound represented by the general formula (1) or a salt thereof can be used.

  The medicament of the present invention may be administered with a compound represented by the general formula (1) as an active ingredient and a pharmacologically acceptable salt thereof, or a solvate or hydrate thereof itself. However, in general, it is desirable to administer in the form of a pharmaceutical composition containing one or more pharmaceutical additives in addition to the compound represented by the general formula (1), which is an active ingredient. As the active ingredient of the medicament of the present invention for treating an animal, two or more of the above substances can be used in combination, and the above-mentioned pharmaceutical composition contains an active ingredient of another therapeutic agent for a viral infectious disease. It is also possible to mix.

  The type of the pharmaceutical composition is not particularly limited, and the dosage forms include tablets, capsules, granules, powders, syrups, suspensions, suppositories, ointments, creams, gels, patches, inhalants, Injections and the like can be mentioned. These preparations are prepared according to a conventional method. In the case of a liquid preparation, it may be in the form of being dissolved or suspended in water or another appropriate solvent at the time of use. Tablets and granules may be coated by a known method. In the case of an injection, the compound of the present invention is prepared by dissolving it in water, but may be dissolved in a physiological saline solution or a glucose solution as necessary, or may be added with a buffer or a preservative. Good. It is provided in any formulation for oral or parenteral administration. For example, pharmaceutical compositions for oral administration in the form of granules, fine granules, powders, hard capsules, soft capsules, syrups, emulsions, suspensions or solutions, intravenous administration, intramuscular administration, Alternatively, it can be prepared as a pharmaceutical composition for parenteral administration in the form of injections for subcutaneous administration, drops, transdermal absorbents, transmucosal absorbents, nasal drops, inhalants, suppositories and the like. Injections, drops, and the like can be prepared as a powdery dosage form such as a lyophilized form, and dissolved in an appropriate aqueous medium such as physiological saline before use. In addition, a sustained-release preparation coated with a polymer or the like can be directly administered into the brain.

  A person skilled in the art can appropriately select the type of pharmaceutical additive used in the production of the pharmaceutical composition, the ratio of the pharmaceutical additive to the active ingredient, or the method of producing the pharmaceutical composition, depending on the form of the composition. It is. Inorganic or organic substances, or solid or liquid substances, can be used as pharmaceutical additives, and can be generally blended at 1 to 90% by weight based on the weight of the active ingredient. Specifically, examples of such substances include lactose, glucose, mannitol, dextrin, cyclodextrin, starch, sucrose, magnesium aluminate metasilicate, synthetic aluminum silicate, sodium carboxymethyl cellulose, hydroxypropyl starch, carboxymethyl cellulose calcium , Ion exchange resins, methylcellulose, gelatin, gum arabic, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, light silicic anhydride, magnesium stearate, talc, tragacanth, bentonite, veegum, titanium oxide, sorbitan fatty acid esters, Sodium lauryl sulfate, glycerin, fatty acid glycerin ester, purified lanolin, glycerogelatin, polysodium Bate, macrogol, vegetable oils, waxes, liquid paraffin, white petrolatum, fluorocarbons, nonionic surfactants, propylene glycol, water and the like.

  To prepare a solid preparation for oral administration, the active ingredient and excipient components such as lactose, starch, microcrystalline cellulose, calcium lactate, and silicic acid are mixed to form a powder, or if necessary, sucrose, A binder such as hydroxypropylcellulose and polyvinylpyrrolidone, and a disintegrant such as carboxymethylcellulose and carboxymethylcellulose calcium are added, and the mixture is granulated by wet or dry granulation. In order to produce tablets, these powders and granules may be compressed as they are or by adding a lubricant such as magnesium stearate or talc. These granules or tablets should be coated with an enteric base such as hydroxypropylmethylcellulose phthalate, methacrylic acid-methyl methacrylate polymer and enteric coated preparations, or coated with ethylcellulose, carnauba wax, hardened oil, etc. to form a sustained release preparation. You can also. To produce capsules, powders or granules are filled in hard capsules, or the active ingredient is dissolved in glycerin, polyethylene glycol, sesame oil, olive oil, or the like, and then coated with a gelatin film to form soft capsules. Can be.

  To manufacture an injection, the active ingredient may be used as required, such as hydrochloric acid, sodium hydroxide, lactose, lactic acid, sodium, sodium monohydrogen phosphate, sodium dihydrogen phosphate, etc., a pH adjuster, sodium chloride, glucose, etc. It may be dissolved in distilled water for injection together with an isotonic agent and sterile-filtered and filled into ampoules, or mannitol, dextrin, cyclodextrin, gelatin or the like may be added and freeze-dried in vacuum to obtain a working-solution-type injection. . In addition, reticin, polysorbate 80, polyoxyethylene hydrogenated castor oil and the like can be added to the active ingredient and emulsified in water to prepare an emulsion for injection.

  In order to manufacture a rectal preparation, the active ingredient is moisturized and dissolved with a suppository base such as cocoa butter, tri-, di- and monoglycerides of fatty acids, and polyethylene glycol and poured into a mold and cooled, or the active ingredient is polyethylene. After dissolving in glycol, soybean oil or the like, it may be coated with a gelatin film.

  To produce an external preparation for the skin, the active ingredient is added to white petrolatum, beeswax, liquid paraffin, polyethylene glycol, etc. and, if necessary, humidified and kneaded to form an ointment, or rosin, an alkyl acrylate polymer After kneading with a pressure-sensitive adhesive such as, for example, it is spread on a nonwoven fabric such as polyalkyl to form a tape.

  The dose and the number of times of administration of the medicament of the present invention are not particularly limited. It can be appropriately selected by judgment. In general, the daily dose for an adult in oral administration is about 0.01 to 1000 mg (weight of the active ingredient), and it may be administered once or several times a day or every few days. it can. When used as an injection, it is desirable to continuously or intermittently administer a daily dose of 0.001 to 100 mg (weight of the active ingredient) to an adult.

  The medicament of the present invention can be prepared as sustained-release preparations such as implantable tablets and delivery systems encapsulated in microcapsules, using a carrier capable of preventing immediate removal from the body. For example, biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Such materials can be readily prepared by those skilled in the art. Also, suspensions of liposomes can be used as pharmaceutically acceptable carriers. Useful liposomes are prepared through a filter of appropriate pore size to a size suitable for use as a lipid composition comprising, but not limited to, phosphatidylcholine, cholesterol and PEG-derived phosphatidylethanol (PEG-PE). Purified by evaporation.

Further, the present invention provides a method for preventing or treating a viral infection using the medicament of the present invention.
The method for preventing a viral infection of the present invention comprises administering the medicament or the pharmaceutical composition of the present invention to an animal which may be infected with a virus having SF1 helicase, and suppressing the onset of symptoms caused by the virus infection. Is included. In addition, the method for treating a viral infection of the present invention comprises administering the medicament or the pharmaceutical composition of the present invention to an animal infected with a virus having SF1 helicase, thereby preventing the progress and worsening of symptoms caused by the virus infection or This includes mitigating.

  The `` animal '' to be prevented or treated refers to any animal classified as a mammal, and is not particularly limited, but includes, for example, humans, pet animals such as dogs, cats, rabbits, cows, pigs, Livestock animals such as sheep and horses.

In the present application, the pesticide is not used in a special meaning, but refers to a chemical used for controlling viruses and the like infecting agricultural crops (including trees and agricultural and forestry products).
The pesticide of the present invention may be administered with a compound represented by the general formula (1) as an active ingredient and a pharmacologically acceptable salt thereof, or a solvate or hydrate thereof itself. However, in general, in addition to the compound represented by the general formula (1) as an active ingredient, it may be sprayed in the form of an agricultural chemical composition containing one or more additives for agricultural chemical formulations, or And fertilizer components. When used as an agricultural chemical or a fertilizer, it may be mixed with other components such as a surfactant, an emulsifier, a bulking agent, a humectant, and the like. When used as a pesticide or fertilizer mixture, the dosage form is not particularly limited, but may be a liquid, a water solvent, a powder, a granule, a wettable powder, an emulsion, a flowable, a microcapsule and the like. Examples of the method of use include a method of spraying these as they are or diluting them with water.

  The pesticide or the pesticide composition of the present invention can be treated with a compound fertilizer containing three elements of nitrogen, phosphoric acid, and potassium to form a formulation, and sprayed on a plant to be treated to thereby treat a virus infection. it can. In addition, process the mixture into a mixed preparation using foliar spray fertilizer (liquid or powdered compound fertilizer), etc., dissolve them in water and spray directly on the foliage of the crop to simultaneously control the disease and apply fertilizer. Can be. Furthermore, by mixing and formulating with a microelement fertilizer containing the required microelements such as manganese, boron, iron, copper, zinc, molybdenum, chlorine, nickel, etc., and spraying it to control virus infection be able to.

  The medicament or pesticide of the present invention can be included in the form of a kit as a pharmaceutical composition or a pesticide composition in a container or a pack together with instructions for administration. When the pharmaceutical composition according to the present invention is supplied as a kit, different components of the pharmaceutical composition are packaged in separate containers and mixed just before use. The separate packaging of the components in this way is to allow long term storage without losing the function of the active components.

  The reagents contained in the kit are supplied in any type of container in which the components maintain their activity effectively for a long period of time, are not adsorbed by the material of the container, and do not undergo deterioration. For example, sealed glass ampules include a buffer packaged under a neutral, non-reactive gas such as nitrogen gas. The ampoule may be comprised of glass, organic polymers such as polycarbonate, polystyrene, ceramic, metal, or any other suitable material commonly used to hold reagents. Examples of other suitable containers include simple bottles made from similar materials, such as ampules, and foil-lined wraps, such as aluminum or alloys. Other containers include test tubes, vials, flasks, bottles, syringes, or the like. The container may have a sterile access port, such as a bottle with a stopper pierceable by a hypodermic injection needle, in the case of a pharmaceutical kit.

  Instructions for use are also attached to the kit. Instructions for use of the kit comprising the pharmaceutical or pesticidal composition may be printed on paper or other material and supplied as an electrically or electromagnetically readable medium such as a CD-ROM or DVD-ROM. Good. The detailed instructions may be actually attached to the kit, or may be posted on a website specified by the manufacturer or distributor of the kit or notified by e-mail or the like.

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

1. Selection of Specific Binding Compound to Tomato Mosaic Virus (ToMV) SF1 Helicase NTP-Binding Site by Computer Simulation Based on the three-dimensional structural information of SF1 helicase possessed by ToMV, specific to ATP-binding site, one of NTP, by computer simulation Approximately 300 kinds of low-molecular-weight organic compounds that may have the activity of binding to are selected. Specifically, based on the three-dimensional structure information of SF1 helicase possessed by ToMV, the site where NTP binds was determined as the binding pocket of the compound. The pocket is subjected to a virtual screening using a known simulation method (Consensus DOCK; Okamoto, et al. (2010) Chem. Pharm. Bull. 58; 1655) to obtain a commercially available compound (about 5 million compounds, for example, Enamine, A large-scale virtual screening was carried out on a database consisting of about 500,000 compounds, which were selected from a group of compounds available and available from MolPort, Namiki Shoji, etc.). Docking was performed for each compound, and the 5000 compounds ranked in the top ranking of the score were set as the first selected compound group. After that, whether (i) there are many interactions with proteins centering on hydrogen bonds, (ii) whether there is no intramolecular distortion in the bonding pose, and (iii) whether there is any interaction other than pharmacophore , Etc. were visually evaluated, and 300 compounds were narrowed down as a second selection compound group. Thereafter, 177 low molecular weight organic compounds available from the second selection compound group were used as final candidate compounds and subjected to the following evaluation.

2. Evaluation of ToMV replication inhibitory activity using in vitro ToMV RNA replication system The activity of the compound selected in the above for inhibiting ToMV replication was evaluated based on the following procedure.
175 ng of ToMV RNA was translated using 14.5 μL of an in vitro translation solution derived from detoxified protoplast extract (mdBYL) of tobacco BY-2 cultured cells from which the biological membrane had been removed. 0.5 μL of the candidate compound (50 μl DMSO solution), 5 μL of the biomembrane fraction, and 5 μL of 5 × R buffer were mixed with this reaction solution to replicate ToMV RNA. At this time, the concentrations of ATP, GTP, UTP, and CTP in the 5 × R buffer were each 0.5 mM. The complementary strand RNA of the replication product ToMV was detected by Northern blot hybridization (FIG. 2).
As a result of this experiment, five compounds (compounds AE) were shown to have replication inhibitory activity on ToMV.

3. 1. Evaluation of ATP hydrolase inhibitory activity Of the five compounds (compounds AE) selected in 1 above, which are active in inhibiting the NTPase activity of the ToMV helicase domain, were examined by the following method using the inhibition of hydrolase on ATP, one of the NTPs, as an index. .
Xiang et al. (2012) The region encoding the helicase domain (S666-Q1116) of the tomato mosaic virus replication protein was expressed and purified by the method described in Protein Expression and Purification 67: 1649-1652 and used in all experiments. The measurement was performed using a high-sensitivity isothermal titration calorimetry device (VP-ITC: GE Healthcare). The concentration of the purified helicase determined by _{UV 280 (ε = 41340L · μM-} 1 · cm- 1), was adjusted to a concentration of 1.5μM by diluting. After centrifugation at 20400 g at 4 ° C. for 15 minutes, the mixture was filled in a syringe. A sample cell (cell volume: 1.44 mL) was filled with a 0.2 mM ATP solution prepared using an external dialysate after centrifugation at 20400 g at 4 ° C. for 30 minutes. Concentration determination exact ATP solution _UV 259: was determined by ^{(ATP ε = 15.4L · μM- 1} · cm- 1). All measurements were performed at a temperature of 15 ° C. to prevent protein denaturation, and were performed by a single titration method in which 100 μL of a helicase solution was dropped at a time in 250 sec. In the cell under measurement, the rotation speed of the stirring blade was set to 310 rpm, and the DP was set to 34.5 μcal / sec. In the ATP hydrolase inhibition experiment with the compound, an equal amount of the compound was added to each of the helicase and the ATP solution, and the measurement was similarly performed. FIG. 3 shows the measurement results of compound A and compound B. After the measurement, the enzyme activity (k _cat / K _m ) was determined using an analysis program ITCENZWC (Karim et al. (2004) Thermochimica Acta 412: 91). Table 1 shows the values of k _cat , K _m and k _cat / K _m of the compounds A to E, and 2. 5 shows the replication inhibitory activity measured in the above.
In addition, the value of ATP hydrolase inhibitory activity is shown as a comparison value with a control experiment, and the replication inhibitory activity by the in vitro translation system is a compound in which a strong inhibitory activity was observed when the compound was administered to the reaction system at a final concentration of 10 μM. ◎, and those with weak inhibitory activity are indicated by ○.
As a result of this experiment, it was shown that Compounds A to E have an activity of inhibiting ATPase activity.

4. Evaluation of porcine epidemic diarrhea virus (PEDV) replication inhibitory activity Vero cells (ATCC CCL-81) and Vero / TMPRSS2 (Shirogane et al. (2008) Journal of Virology 82: 8942) were used in all experiments. These Vero cells express the membrane-type serine protease TMRPSS2 (GenBank accession no. AF12345). Cells were incubated at 37 ° C., 5% CO ₂ , 5% fetal calf serum (FCS, Gibco), 1% penicillin streptomycin (P4333, Sigma), and 1 μg / mL G418 sulfate (380−) in DMEM (D5796, Sigma). 013, Enzo). PEDV was replicated and titered using Vero cells. Specifically, Vero cells prepared in a 10 cm Petri dish were infected with PEDV virus, and after adsorbing for 60 minutes, the cells were washed with phosphate buffer (PBS), and a 10% tryptose phosphate bouillon solution in DMEM (260300, Difco ). On the third day after the infection, the culture supernatant was discarded, 2 mL of DMEM was added, the cells were collected, and sonication was performed for 5 minutes. Thereafter, centrifugation was performed at 8000 rpm at 4 ° C., and the supernatant was used as a virus stock. A 10-fold serially diluted virus solution was inoculated into each well of Vero cells prepared in a collagen I-coated 6-well plate (Iwaki), and the cells were washed twice with PBS after adsorption for 60 minutes. Thereafter, the cells were cultured for 18 hours in a solution containing 10% tryptose phosphate bouillon solution and 1.25 μg / mL trypsin (T8802, sigma) in DMEM. After the culture, the cells were stained and fixed with a solution containing 20% formalin solution (064-00406, Wako) and 0.1% crystal violet in PBS for 2 hours, and giant cells formed by the virus were counted under a microscope. Virus titers were expressed as plaque forming units (PFU).
The drug inhibition experiment was performed as follows: After dissolving each of the five compounds selected in the above (Compounds AE) in DMSO, a solution containing 5% FCS, 1% penicillin streptomycin, and 100 μM of each drug in DMEM was prepared and prepared in a 96-well plate. The culture supernatant of Vero cells was once discarded, and 50 μL of each well was inoculated. As a negative control, a culture solution containing only DMSO at the same concentration was used. Thirty minutes later, the medium was discarded, and 50 μL of a solution containing 5% FCS, 1% penicillin streptomycin, and 150 PFU of PEDV in DMEM was inoculated. Cells were cultured at 37 ° C., 5% CO ₂ , and on day 3 after virus infection, infected cells were fixed with a 4% formaldehyde solution in PBS for 1 hour, 0.1% boric acid, 10% methanol, 1% After staining with a solution containing 5% crystal violet for 5 minutes, giant cell formation by the virus was observed under a microscope and compared with a negative control.
As a result, it was shown that the proliferation of PEDV in the cells treated with Compound E was significantly inhibited, and it was revealed that Compound E had the activity of inhibiting the replication of PEDV (FIG. 4).

5. Evaluation of rubella virus replication inhibitory activity Introduction of rubella virus replicon RNA, in which the structural protein gene of rubella virus was recombined into a puromycin N-acetyltransferase, a foot-and-mouth disease virus 2A protein self-cleaving active region and a fusion protein gene of Renilla luciferase, were introduced into BHK cells. And Wen-Pin et al. (2012) Rubella virus replicon cells were established according to the method of Virology 429; 29. The cells were cultured in a cell culture [DMEM (D5796, Sigma), 5% fetal calf serum (FCS, Gibco), 1% penicillin streptomycin (PS, 15140-122, Life technologies) at 35 ° C., 5% CO _2. To the solution], and a solution in which Puromycin Dihydrochloride (1113802, Life technologies) was added at 0.5 μg / mL. 2. The effect of each of the five compounds (compounds AE) selected in Example 1 on anti-rubella virus replication was evaluated by changes in Renilla luciferase activity in rubella virus replicon cells. That is, rubella virus replicon cells adjusted to 20,000 cells / 100 μL in a cell culture solution were seeded at 100 μL per well, and cultured overnight at 35 ° C. and 5% CO ₂ . Each compound dissolved in DMSO was adjusted to 100, 20, and 4 μM in a cell culture solution, and added to the rubella virus replicon cells in which the culture supernatant was discarded, 100 μL per well. As a negative control, a culture solution containing only DMSO at the same concentration was used. After culturing at 35 ° C. and 5% CO ₂ for 3 days, the Renilla virus replicon intracellular Renilla luciferase activity and cell viability were determined by ViviRen Live cell substrate (E6491, Promega) and cell viability by CellTiter-Glo Luminescent Cell Viable Cell Viability, respectively. (G7570, Promega), and the effect of the compound on anti-rubella virus replication was evaluated by intracellular Renilla luciferase activity / cell viability, and compared with a negative control.
As a result, it was shown that the treatment of compound A significantly inhibited rubella virus replicon replication (FIG. 5).

6. Evaluation of human hepatitis E virus (HEV) replication inhibitory activity HEV was obtained from EMBL Accession No. : AB740232: HEV replicon has a secretory Gaussia Luciferase (pNL1.3 (Accession No .: JQ433372, Promega), which has a structural protein region of the HEV sequence (nucleic acid numbers 5173 to 7155). , Madison, WI) and replaced by nucleic acid numbers from 100 to 699). The HEV replicon sequence was introduced into the modified plasmid pUC19 (Promega, Madison, WI) using standard molecular biology techniques. Since this replicon sequence has a T7 promoter sequence (AATACGACTCACTATA) added upstream thereof, replicon RNA can be synthesized by using Ambion mMESSAGE mMMACHINER Kit (Life Technologies).
The HEV replicon RNA synthesized by the above method was introduced into human hepatoma-derived cells PLC / PRF / 5 by electroporation (voltage 270 V, capacity 950 μF) using GenePulser Xcell manufactured by BIO-RAD. 1. A 96-well plate was prepared such that the number of cells per well was 2.5 × 10 ⁴ cells / well and the amount of replicon RNA was 30 ng, and electroporation was performed. Each of the five compounds (compounds AE) selected in was added after 3 hours. Thereafter, after culturing at 37 ° C., 5% CO ₂ , and 100% relative humidity for 5 days, the culture supernatant was collected, and the secreted luciferase activity was measured using a Promega Nano-Glo Luciferase Assay System.
As a result of this experiment, it was shown that out of the five compounds evaluated, compounds B and C had an activity of inhibiting the replication of HEV (FIG. 6).

  The compound according to the present invention can be used as an agent for protecting and treating infection by various plus-strand RNA viruses having SF1 helicase, such as ToMV and rubella virus. In particular, under the current situation where measures against virus infection to plants are not progressing slowly, the compounds of the present invention are greatly expected as a new means for effectively suppressing damage to agricultural crops. In addition, livestock viruses for which new viruses are becoming a problem one after another, but for which effective measures have not been taken, are also expected as a new anti-domestic virus strategy.

Claims (7)

A compound that inhibits NTPase activity of SF1 helicase or a salt thereof, or a solvate or hydrate thereof, as a positive-strand RNA virus replication inhibitor comprising as an active ingredient ,
Wherein the compound is
2- {4-[(1Z) -2-chloro-2- (5-chlorobenzimidazol-2-yl) vinyl] -2-methoxyphenoxy} acetic acid,
2- {4-[(5-imino-7-oxo-3-phenyl (4-hydro-1,3-thiazolino [3,2-a] pyrimidine-6-ylidene)) methyl] -2-methoxyphenoxy} Acetic acid,
2- {4-[(1Z) -2- (3-cyclohexyl (7H-1,2,4-triazolo [3,4-b] 1,3,4-thiadiazin-6-yl))-2- ( Ethoxycarbonyl) vinyl] phenoxy diacetic acid,
2- [4-({1-[(4-chlorophenyl) methyl] -2,5-dioxo (1,3-diazolidine-4-ylidene)} methyl) -2-methoxyphenoxy] acetic acid, or
4-{[6-methyl-5- (4-methylphenyl) -4-oxo-3-hydrothiopheno [2,3-d] pyrimidin-3-yl] methyl} benzoic acid,
Is one of
The positive-strand RNA virus is a tomato mosaic virus.
Positive-strand RNA virus replication inhibitor. A compound that inhibits NTPase activity of SF1 helicase or a salt thereof, or a solvate or hydrate thereof, as a positive-strand RNA virus replication inhibitor comprising as an active ingredient,
Wherein the compound is
4-{[6-methyl-5- (4-methylphenyl) -4-oxo-3-hydrothiopheno [2,3-d] pyrimidin-3-yl] methyl} benzoic acid
And
The plus-strand RNA virus replication inhibitor, wherein the plus-strand RNA virus is a swine coronavirus. A compound that inhibits NTPase activity of SF1 helicase or a salt thereof, or a solvate or hydrate thereof, as a positive-strand RNA virus replication inhibitor comprising as an active ingredient,
Wherein the compound is
2- {4-[(1Z) -2-chloro-2- (5-chlorobenzimidazol-2-yl) vinyl] -2-methoxyphenoxy} acetic acid
And
A positive-strand RNA virus replication inhibitor, wherein the positive-strand RNA virus is rubella virus. A compound that inhibits NTPase activity of SF1 helicase or a salt thereof, or a solvate or hydrate thereof, as a positive-strand RNA virus replication inhibitor comprising as an active ingredient,
Wherein the compound is
2- {4-[(5-imino-7-oxo-3-phenyl (4-hydro-1,3-thiazolino [3,2-a] pyrimidine-6-ylidene)) methyl] -2-methoxyphenoxy} Acetic acid, or
2- {4-[(1Z) -2- (3-cyclohexyl (7H-1,2,4-triazolo [3,4-b] 1,3,4-thiadiazin-6-yl))-2- ( Ethoxycarbonyl) vinyl] phenoxy diacetic acid
Is one of
The positive-strand RNA virus replication inhibitor, wherein the positive-strand RNA virus is human hepatitis E virus. A pesticide or a pesticidal composition useful for treating and / or preventing infectious diseases caused by a plus-strand RNA virus having SF1 helicase, comprising the plus-strand RNA virus replication inhibitor according to claim 1 as an active ingredient. A medicament useful for the treatment and / or prevention of infectious diseases in animals by an SF1 helicase-containing plus-strand RNA virus, comprising the plus-strand RNA virus replication inhibitor according to any one of claims 2 to 4 as an active ingredient. Or a pharmaceutical composition. Comprising as an active ingredient a positive-strand RNA viral replication inhibitor according to claim 3 or claim 4, wherein the infection by the positive-strand RNA virus is an infectious disease to humans, according to claim 6 Pharmaceutical or pharmaceutical composition.