JPWO2014157622A1 - Anti-non-membrane virus agent and use thereof - Google Patents

Abstract

Provided is an anti-non-membrane virus agent that can safely and effectively prevent non-membrane virus infection. A drug containing an epigallocatechin gallate derivative represented by the following chemical formula (1) is prepared. This can be used as an anti-non-membrane virus agent that inhibits non-membrane virus infection. In the following formula (1), in the chemical formula (1), R1 to R6 are each a hydrogen atom or a linear or branched saturated or unsaturated acyl group, which may be the same or different. May be further substituted with one or more substituents, and all of R1 to R6 are not simultaneously hydrogen atoms.

Description

  The present invention relates to inhibitors that inhibit membrane fusion of non-membrane viruses.

  Compared to other viral infections, norovirus infection is characterized not only by diarrhea but also by vomiting, and is known to cause acute gastroenteritis in a wide range of ages from infants to the elderly. Infection and reinfection are also regarded as problems. For this reason, attempts have been made to apply antiviral agents, but the reality is that norovirus exhibits strong drug resistance and there is no effective drug. This is a non-membrane virus that has no envelope and is resistant to alcohol and high temperatures. It also has a property of being resistant to drying and acid, and can survive in water for a long time. This is considered to be the cause.

  For this reason, provision of the new chemical | medical agent which shows the anti-non-membrane viral effect excellent in the norovirus which is a non-membrane virus is strongly calculated | required (patent documents 1 and 2). Such a problem is not limited to Norovirus but also applies to other non-membrane viruses.

WO2010 / 067873 JP 2009-29273

  Therefore, an object of the present invention is to provide an anti-non-membrane virus agent that is excellent in safety and anti-non-membrane virus effect.

In order to achieve the above object, the anti-non-membrane virus agent of the present invention comprises a derivative of epigallocatechin gallate (EGCG) represented by the following chemical formula (1), an isomer thereof, or a salt thereof. And
In the chemical formula (1),
R ^{1 to} R ⁶ are each a hydrogen atom, halogen, sodium, potassium, or a linear or branched saturated or unsaturated acyl group, which may be the same or different, and the acyl group further includes one or more Wherein at least one of R ^{1 to} R ⁶ is the acyl group, and R ^{7 to} R ¹⁶ are a hydrogen atom, a halogen, sodium, or potassium, and may be the same or different. Also good.

  The non-membrane virus infection prevention method of the present invention is a method for preventing non-membrane virus infection, and is characterized by administering the anti-non-membrane virus agent of the present invention to a subject.

  According to the present invention, non-membrane virus infection can be prevented safely and effectively. For this reason, it can be said that the present invention is useful in the medical field, the food hygiene field, and the like.

FIG. 1 is a graph showing the anti-non-membrane virus effect of an EGCG derivative against feline calicivirus in Example 1 of the present invention. FIG. 2 is a graph showing the anti-non-membrane virus effect of the EGCG derivative against feline calicivirus in Example 1 of the present invention. FIG. 3 is a graph showing the anti-non-membrane virus effect of an EGCG derivative against rotavirus in Example 2 of the present invention.

<Anti-non-membrane virus agent>
As described above, the anti-non-membrane virus agent of the present invention is characterized by containing a derivative of epigallocatechin gallate represented by the following chemical formula (1), an isomer thereof, or a salt thereof.
In the chemical formula (1),
R ^{1 to} R ⁶ are each a hydrogen atom, halogen, sodium, potassium, or a linear or branched saturated or unsaturated acyl group, which may be the same or different, and the acyl group further includes one or more Wherein at least one of R ^{1 to} R ⁶ is the acyl group, and R ^{7 to} R ¹⁶ are a hydrogen atom, a halogen, sodium, or potassium, and may be the same or different. Also good.

  In the chemical formula (1), “A to D” are notation of each ring in epigallocatechin gallate. In the present invention, epigallocatechin gallate is hereinafter referred to as “EGCG”, and a derivative of EGCG is referred to as “EGCG derivative”.

  In the present invention, EGCG derivatives include, for example, isomers such as salts, tautomers, stereoisomers, optical isomers, geometric isomers, and isomer mixtures of the compound represented by the chemical formula (1). It is. The salt is not particularly limited, and examples thereof include inorganic acid salts, organic acid salts, inorganic base salts, organic base salts, acidic or basic amino acid salts, and the like. The isomer can also be purified by a conventionally known separation method such as various types of chromatography. In the present invention, the EGCG derivative includes, for example, a compound that is produced by subjecting the compound represented by the chemical formula (1) to metabolism such as oxidation, reduction, hydrolysis, and conjugation.

In R ^{1 to} R ⁶ , the main chain length of the acyl group is not particularly limited, and includes, for example, carbonyl carbon and 2 to 20 atoms, preferably 4 to 20 and more preferably 8 to 18 Yes, more preferably 12-18, 14-18, and particularly preferably 16-18. The main chain length of the acyl group means the number of atoms of the longest chain in the acyl group, and includes, for example, a nitrogen atom, a sulfur atom, a phosphorus atom, an oxygen atom, a boron atom in addition to the carbon atom. But you can.

In R ^{1 to} R ⁶ , the number of atoms of the acyl group is not particularly limited. The number of atoms (for example, the number of carbon atoms) of the acyl group is, for example, 2 to 20, including carbonyl carbon, preferably 4 to 20, more preferably 8 to 18, still more preferably 12 to 18, 14 to 18, particularly preferably 16-18. The number of atoms (for example, the number of carbon atoms) is, for example, 4, 8, 12, 16, 18 or 20, preferably 12, 16 or 18, and more preferably 16 or 18. In addition, when the said acyl group is further substituted by the said substituent, it is preferable that the said carbon atom number is a number which does not contain the carbon atom number of the said substituent, for example. The unsaturated acyl group may be cis or trans, for example.

  The acyl group is not particularly limited, and for example, formyl group (C1), acetyl group (C2), propionyl group (C3), butyryl group (C4), isobutyryl group (C4), valeryl group (C5), isovaleryl group (C5), pivaloyl group (C5), hexanoyl group (C6), octanoyl group (C8), geranoyl group (3,7-dimethylocta-2,6-dienoyl group) (C10), trans-8-methyl-6 -Nonenoyl group (C10), undecanoyl group (C11), lauroyl group (dodecanoyl group) (C12), tridecanoyl group (C13), 12- (dimethylamino) lauroyl group (12- (dimethylamino) dodecanoyl group) (C14) Farnesoyl group (3,7,11-trimethyldodeca-2,6,10-trienoyl group) (C15), Mitoyl group (hexadecanoyl group) (C16), heptadecanoyl group (C17), stearoyl group (octadecanoyl group) (C18), linoleyl group (C18), linolenyl group (C18), nonadecanoyl group (C19), eicosanoyl group (Icosanoyl group) (C20) and the like. “C” in the parentheses of the listed acyl groups indicates the number of carbon atoms including the carbonyl carbon.

  Among the acyl groups, for example, an acyl group represented by the following chemical formula is particularly preferable. The position of the unsaturated bond in the unsaturated acyl group among the following acyl groups is not limited thereto. As a specific example, the unsaturated bond (double bond) of the trans-8-methyl-nonenoyl group (C10) is not limited to the 6-position shown below, and may be, for example, either the 2-5-position or the 7-position. May be.

  The kind of the acyl group is not particularly limited, and may be any of an unsaturated acyl group and a saturated acyl group as described above. The number of unsaturated bonds in the acyl group is not particularly limited and is, for example, 1, 2, or 3.

In the chemical formula (1), the number of the acyl groups is not particularly limited. For example, only one of R ^{1 to} R ⁶ may be the acyl group, or any two or more of the acyl groups may be the acyl group. But you can. When two or more locations are the acyl groups, the acyl groups at each site may be the same or different, for example. In the chemical formula (1), R other than the acyl group is not particularly limited, and for example, a hydrogen atom is preferable.

In the chemical formula (1), the site of the acyl group is not particularly limited among R ^{1 to} R ⁶ . As a specific example, for example, at least one of R ¹ and R ² of the B ring and R ⁵ and R ⁶ of the D ring preferably has the acyl group, and in particular, R ¹ , R ² , R ⁵ and R It is preferable that any one of ⁶ has the acyl group. In this case, R other than the acyl group is not particularly limited, and is preferably a hydrogen atom, for example.

Further, in Formula (1), it is preferable that at least one position of ^R 1, ^{R 2} and ^{R 3} of B ring is the acyl group, more preferably, ^R 1, ^{R 2} and ^{R 3} in the B ring It is preferable that only one of them is an acyl group.

In the chemical formula (1), the B ring rotates around a single bond between the B ring and the C ring, for example. Therefore, a derivative having an acyl group at R ¹ is substantially the same as a derivative having an acyl group at R ³ , for example. In the chemical formula (1), the D ring rotates around a single bond between the D ring and the ester, for example. For this reason, the derivative having an acyl group at R ⁶ is substantially the same as the derivative having an acyl group at R ⁴ , for example.

In the chemical formula (1), R ^{7 to} R ¹⁶ are a hydrogen atom, halogen, sodium or potassium as described above, and may be the same or different. For example, as shown in the chemical formula (2) below, A hydrogen atom is preferred. In the following formula (2), for example, any of R ^{1 to} R ⁶ may be the acyl group. As a specific example, for example, it is preferable that only one or two or more of R ^{1 to} R ⁶ are the above-described acyl groups, and more preferably R ¹ , R ² , R ⁵ and R ^6. Of these acyl groups, any one or two or more of them are preferably the aforementioned acyl groups. Among the acyl groups, for example, a butyryl group, an octanoyl group, a trans-8-methyl-6-nonenoyl group, and a geranoyl group. Group, lauroyl group, 12- (dimethylamino) lauroyl group, farnesoyl group, palmitoyl group, stearoyl group, linoleyl group, linolenyl group or eicosanoyl group is preferred.

In R ^{1 to} R ⁶ of the chemical formula (1), the acyl group may be substituted with one or more substituents as described above. Specifically, for example, a hydrogen atom of the acyl group is , May be substituted with the substituent. The substituent is not particularly limited, and examples thereof include an alkyl group, an amino group, an alkylamino group, and a dialkylamino group.

  Examples of the alkyl group include linear or branched alkyl groups having 1 to 6 carbon atoms, and a methyl group is preferable. Examples of the alkyl group in the alkylamino group include linear or branched alkyl groups having 1 to 6 carbon atoms, preferably a methylamino group. Examples of the alkyl group in the dialkylamino group include linear or branched alkyl groups having 1 to 6 carbon atoms, and preferably a dimethylamino group. These may be the same or different.

  In the present invention, “halogen” refers to any halogen element. Examples of the halogen include fluorine, chlorine, bromine and iodine. In the present invention, the “alkyl group” is not particularly limited. Examples of the alkyl group include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, a pentyl group, a hexyl group, a heptyl group, and an octyl group. Nonyl group, decyl group, undecyl group, dodecyl group, tridecyl group, tetradecyl group, pentadecyl group, hexadecyl group, heptadecyl group, octadecyl group, nonadecyl group, icosyl group and the like. For groups containing an alkyl group in the structure or derived from an alkyl group (alkylamino group, dialkylamino group, alkoxy group, carboxyalkyl group, alkoxycarbonylalkyl group, alkoxyalkyl group, alkenoxyalkyl group, etc.) It is the same.

  When the substituent or the like is a group having a chain structure, specifically, for example, an alkyl group, an alkylamino group, a dialkylamino group, an alkoxy group, a carboxyalkyl group, an alkoxycarbonylalkyl group, an alkoxyalkyl group, an alkenoxyalkyl In the case of a group or the like, it may be linear or branched unless otherwise specified. The same applies to the case where a part of the substituent or the like includes a chain structure, for example, when a substituent in a substituted alkyl group or a substituted aryl group includes a chain structure. When an isomer exists in a substituent or the like, any isomer may be used unless otherwise limited. For example, when it is simply referred to as “propyl group”, it may be either n-propyl group or isopropyl group, and when it is simply referred to as “butyl group”, any of n-butyl group, isobutyl group, sec-butyl group and tert-butyl group may be used. Well, when simply referred to as a “naphthyl group”, either a 1-naphthyl group or a 2-naphthyl group may be used.

In the present invention, the EGCG derivative may be, for example, one kind of EGCG derivative or a mixture of two or more kinds of EGCG derivatives. For example, a mixture of two or more types of EGCG derivatives having acyl groups at different sites among R ^{1 to} R ⁶ may be used, or a mixture of two or more types of EGCG derivatives having different acyl groups may be used. As specific examples, any one or more of EGCG derivatives in which R ¹ of the B ring is the acyl group, EGCG derivatives in which R ² is the acyl group, and EGCG derivatives in which R ³ is the acyl group, or It may be a mixture including all three types, among EGCG derivatives in which R ^{4 of} the D ring is the acyl group, EGCG derivatives in which R ⁵ is the acyl group, and EGCG derivatives in which R ⁶ is the acyl group, Any two or more kinds or a mixture containing all three kinds may be used. And a mixture of an EGCG derivative in which at least one of R ^{1 to} R ³ of the B ring is the acyl group and an EGCG derivative in which at least one of the R ^{4 to} R ⁶ of the D ring is the acyl group. Also good.

  The anti-non-membrane virus agent of the present invention can be used against non-membrane viruses as described above. The non-membrane virus is a virus having no so-called envelope (membrane). The non-membrane virus is not particularly limited, and for example, Norovirus genus, Feline calicivirus genus, Rotavirus genus, Beta Nodavirus genus, Aquavirnavirus genus, Ranavirus genus, Enterovirus genus, Mast adenovirus genus and Besivirus Genus and the like. The strain of the genus Feline calicivirus is not particularly limited. For example, FCV-F4, FCV-F9, FCV-255, FCV-2280, FCV-Diva, FCV-Kaos, FCV-Bellingham, FCV-M8, FCV-DD1 FCV-255 and the like. The type of rotavirus is not particularly limited, and when classified based on the antigenicity of the inner shell protein (VP6), for example, group A rotavirus, group B rotavirus, group C rotavirus, group D rotavirus, group E Examples include rotavirus, group F rotavirus, group G rotavirus and the like.

  The anti-non-membrane virus agent of this invention should just contain the said EGCG derivative, The form in particular is not restrict | limited. Examples of the form include liquids such as solutions and dispersions, solids, powders, and the like. The dosage form is not particularly limited, and can be appropriately set according to the administration method, and includes liquids, capsules, tablets, granules (fine granules), powders and the like. The administration method is not particularly limited, and examples thereof include oral administration and parenteral administration. The parenteral administration includes, for example, transdermal administration, intraperitoneal administration, intravenous administration such as intravenous injection, intramuscular administration, subcutaneous administration such as subcutaneous injection, rectal administration, etc., preferably transdermal administration. The anti-non-membrane virus agent of the present invention is not particularly limited, and can be administered, for example, as an internal medicine, a sublingual agent, a nasal drop, a mouthwash, a lacquer or the like containing the EGCG derivative, depending on the administration form. In addition, the EGCG derivative or a solution or dispersion containing the EGCG derivative can be administered using, for example, injection, nebulizer, aspirator or the like. Moreover, it can administer using a nebulizer, a suction device etc. as said EGCG derivative | guide_body and powder containing it, for example. In addition, since the anti-non-membrane virus agent of the present invention can reduce the infectivity of non-membrane viruses, examples of the detergent include hand-washing agents and wiping agents containing the EGCG derivative. With such an anti-non-membrane virus agent of the present invention, for example, by treating a place where a non-membrane virus is thought to exist, such as a hand or a desk, the infectivity of the existing non-membrane virus is reduced, and the non-membrane virus It is also possible to prevent infection. Further, the anti-non-membrane virus agent of the present invention may be carried on a mask, for example.

  The anti-non-membrane virus agent of the present invention can be used, for example, for prevention of non-membrane virus infection and treatment of non-membrane virus infection. The subject to which the anti-non-membrane virus agent of the present invention is administered includes, for example, humans or non-human animals, and examples of the non-human animals include non-human mammals such as pigs, ferrets, rats, mice, and cows, ducks. And birds such as chickens.

  In the anti-non-membrane virus agent of the present invention, the content of the EGCG derivative is not particularly limited, and can be appropriately determined according to, for example, the purpose of administration and the administration method. When the anti-non-membrane virus agent of the present invention is a mouthwash, for example, it is preferable to contain 20 to 2000 nmol / L of the EGCG derivative per time. Moreover, when the anti-non-membrane viral agent of this invention is a nasal drop, it is preferable that the said EGCG derivative is included 20-2000 nmol / L per time, for example.

  The anti-non-membrane virus agent of the present invention may further contain additives, bases and the like as appropriate depending on, for example, the dosage form and administration method. Examples of the additive include excipients, binders, lubricants, disintegrating agents, coloring agents, flavoring agents, flavoring agents, emulsifiers, surfactants, solubilizing agents, suspending agents, isotonic agents, Examples include buffers, preservatives, antioxidants, stabilizers, absorption promoters, and the like. These addition ratios are not particularly limited, and can be added within a range not impairing the effect of the EGCG derivative.

  The anti-non-membrane virus agent of the present invention may further contain other substances having non-membrane virus activity. Specific examples include natural catechins such as alcohol and broanthocyanin.

  In the present invention, the method for producing the EGCG derivative is not particularly limited. As the method, a conventionally known method such as an organic synthesis method, a chemical synthesis method using an enzyme or the like can be adopted. The chemical synthesis method using the enzyme is not particularly limited, and examples thereof include a method using lipase disclosed in WO2007 / 105280. That is, this is a method of acylating EGCG by carrying out an enzymatic reaction with lipase using EGCG and an acyl group donor as substrates in an organic solvent. According to this method, for example, EGCG can be selectively acylated. In addition, although the method of using a lipase is illustrated as an example below, in the present invention, the manufacturing method of the EGCG derivative is not particularly limited.

The lipase is, for example, IUB No. 3.1.1.3. Lipase can be used. Specific examples, Aspergillus sp lipase derived from such Aspergillus niger; Candida rugosa, Candida cylindracea , Candida sp lipase derived from such Candida antarctica; Pseudomonas fluorescens, Pseudomonas cepacia , Pseudomonas sp lipase derived from such Pseudomonas stutzeri; Alcaligenes sp lipase; Burkholderia cepacia Lipases derived from the genus Burkholderia, etc .; lipases derived from porcine pancreas and the like. These can be prepared by a conventionally known method. For example, Lipase AS “AMANO”, Lipase AYS “AMANO”, Lipase PS “AMANO”, Lipase AK “AMANO” 20, Lipase AH “AMANO” (all products Name: Amano Enzyme), Lipase MY, Lipase OF, Lipase PL, Lipase PLC, Lipase PLG, Lipase QLM, Lipase QLC, Lipase QLG, Lipase SL, Lipase TL Commercial products such as PPL, L4777 Lipase acrylic resin from Candida Antarctica, and L3126 Lipase from porcine pancreas (all trade names: Sigma Aldrich) can also be used. In addition, the physicochemical properties of each commercially available product are as described in each product description, and enzymes exhibiting similar physicochemical properties can be used in the same manner.

Moreover, the lipase which has the physicochemical characteristic and enzymological characteristic in any one of (1)-(8) as shown below may be sufficient.
(1) Molecular weight 35,000, isoelectric point 4.10 (for example, derived from Aspergillus niger )
(2) Inactivation by treatment with molecular weight 64,000, isoelectric point 4.30, 80 ° C. for 10 minutes (eg, derived from Candida rugosa )
(3) Optimal pH 8, optimal temperature 60 ° C., particularly stable in the range of pH 4-10, particularly stable below 70 ° C. (eg derived from Pseudomonas fluorescens )
(4) Particularly stable in solution at a molecular weight of 60,000, optimum pH 6-7, pH stability 3-8, optimum temperature 40-50 ° C., 37 ° C. or less (eg, derived from Candida cylindracea, derived from Candida rugosa )
(5) Particularly stable at a molecular weight of 30,000, an isoelectric point of 4.5, an optimum pH of 8 to 9.5, a pH stability of 7 to 10, an optimum temperature of 50 ° C. and 40 ° C. or less (eg, derived from the genus Alcaligenes )
(6) Molecular weight 31,000, isoelectric point 4.9, optimum pH 7-9, pH stability 6-10, optimum temperature 65-70 ° C., particularly stable at 50 ° C. or less (eg derived from Alcaligenes genus)
(7) Particularly stable at a molecular weight of 31,000, an isoelectric point of 5.2, an optimum pH of 7 to 9, a pH stability of 6 to 10, an optimum temperature of 65 to 70 ° C. and 60 ° C. or less (for example, derived from Pseudomonas cepacia , Burkholderia cepacia origin)
(8) Particularly stable at a molecular weight of 27,000, an isoelectric point of 6.6, an optimum pH of 7 to 8, a pH stability of 6 to 9, an optimum temperature of 50 ° C. and 40 ° C. or less (for example, derived from Pseudomonas stutzeri )

  The organic solvent is not particularly limited, and for example, acetonitrile, acetone, dimethylformamide (DMF), dimethyl sulfoxide (DMSO) and the like can be used. Further, for example, an organic solvent having a hydrophobicity parameter (log P value) in the range of −0.35 to 0.28 may be used. Such an organic solvent may be, for example, the above-mentioned acetonitrile (log P value: −0.45). To 0.19), acetone (log P value: -0.16 to 0.19), DMF (log P value: -1.01 to 0.28), DMSO (log P value: -1.35 to 0.28). Can be given. In addition to these, conventionally known solvents satisfying the above parameters can be used. Since logP is a value specific to the solvent, those skilled in the art can select a solvent that satisfies the parameters. In addition, logP is the one in which the target substance is added to a mixed solution of octanol and water, and the concentration ratio of the target substance in the octanol layer and the aqueous layer when equilibrium is reached is displayed in the common logarithm. As described above, it is a general parameter indicating the hydrophobicity of a substance.

In the present invention, examples of the acyl group (R—CO—) donor include carboxylic acid vinyl ester (R—CO—O—CH═CH ₂ ). Examples of the acyl group include linear or branched saturated or unsaturated acyl groups as described above.

  When DMF is used for the enzyme reaction solution, the addition ratio of EGCG is not particularly limited, and is, for example, 0.2 to 100 mmol / L, preferably 0.5 to 50 mmol / L, more preferably 0.5. ~ 20 mmol / L. The addition ratio of the acyl group donor is not particularly limited, and can be appropriately determined according to, for example, the addition ratio of EGCG in the reaction solution. As a specific example, the addition ratio (molar ratio) of EGCG to an acyl group donor is, for example, 1: 1 to 1:10, preferably 1: 1 to 1: 5, more preferably 1: 1 to 1. : 3. Further, the addition ratio of lipase in the reaction solution can be appropriately determined according to, for example, the addition ratio of EGCG or acyl group donor, the specific activity of lipase, etc., and is not particularly limited. For example, for EGCG 1 mmol / L, for example, 500 to 50,000 U / L, preferably 500 to 5,000 U / L, more preferably 1,000 to 2,500 U / L.

  The conditions for the enzyme reaction are not particularly limited, and the reaction temperature is, for example, in the range of 45 to 75 ° C. The reaction time is not particularly limited and can be appropriately determined depending on the amount of the substrate and the enzyme, and is, for example, 30 minutes to 24 hours (1440 minutes), preferably 1 hour (60 minutes) to 3 hours (180 minutes), More preferably, it is 1.5 hours (90 minutes) to 3 hours (180 minutes).

  A basic catalyst may be further added to the reaction solution. Examples of the basic catalyst include tertiary amines such as triethylamine, pyridine and the like. The addition rate of the basic catalyst in the reaction solution is not particularly limited, and is, for example, 5 to 720 mmol / L, preferably 12 to 240 mmol / L, more preferably 12 to 48 mmol / L.

  The position where the acyl group is introduced in EGCG can be selected depending on, for example, the type of lipase used. Further, the number of acyl groups introduced into EGCG can be determined, for example, depending on the type of organic solvent used and the reaction time. For example, the higher the hydrophobicity of the organic solvent (the lower the hydrophilicity), the lower the number of acyl groups introduced, and the higher the hydrophilicity of the organic solvent ( The lower the hydrophobicity), the greater the number of acyl groups introduced. Also, the number of acyl groups to be introduced can be adjusted by using a mixture of two or more organic solvents. As a specific example, it is preferable to use acetonitrile or the like when introducing one acyl group, for example, acetone or acetonitrile or the like is used when introducing one or two acyl groups. For example, when introducing 3 to 5 acyl groups, it is preferable to use DMSO, DMF or the like.

  Furthermore, even when the same organic solvent is used, the number of acyl groups to be introduced can be adjusted by combining with the control of temperature time and reaction temperature. Examples thereof are shown below, but are not limited thereto. When using DMF, for example, by setting the reaction temperature in the range of about 57 ° C. to about 70 ° C. and increasing the reaction temperature (eg, about 3 to 5 hours), two acyl groups are selected for EGCG. Can be obtained preferentially, while reducing the reaction temperature (eg, 57 ° C. to about 5 ° C. lower) and shortening the reaction time (eg, about 1-3 hours) Thus, one acyl group can be selectively introduced. Also, one acyl group can be selectively introduced into EGCG by using a mixed solvent in which acetone and DMF are mixed in the same amount (mass).

  The number of acyl groups to be introduced can be increased by adding the aforementioned basic catalyst to the reaction solution. In this case, to which site in EGCG the acyl group is further introduced depends on, for example, the regioselectivity of the lipase.

  The yield of the EGCG derivative by the enzyme reaction can be relatively improved, for example, by setting the reaction temperature relatively high. The reaction temperature is usually 45 to 75 ° C. as described above, but preferably 57 to 75 ° C., more preferably 57 to 70 ° C. from the viewpoint of improving the yield. In particular, when the reaction temperature is 57 to 70 ° C., the yield of the EGCG acylated derivative can be about 35 to 45%. In addition, the said yield means the ratio (conversion efficiency) of EGCG acylated derivatives (for example, all monoacylated derivatives) when EGCG used for reaction is 100%.

  In the present invention, the EGCG derivative may be, for example, any one type of EGCG derivative or a mixture of two or more types of EGCG derivatives as described above. When one kind of EGCG derivative is isolated from the mixture, it can be prepared by a conventionally known method using, for example, chromatography.

<Infection prevention method>
The infection prevention method of the present invention is a method for preventing non-membrane virus infection, and is characterized by administering the aforementioned EGCG derivative to a subject. In the present invention, the EGCG derivative is used, and other configurations and conditions are not particularly limited. The EGCG derivative and its usage are the same as described above, for example. In the present invention, for example, the anti-non-membrane virus agent of the present invention may be administered as the EGCG derivative.

  In the present invention, the subject is not particularly limited and is the same as described above, and includes a human or a non-human animal. The subject may be, for example, the living body itself, cells or tissues collected from the living body, or cultures thereof.

  When the subject is a living body, the administration method is not particularly limited, and examples thereof include parenteral administration and oral administration. Examples of the parenteral administration include transdermal administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, rectal administration, and the like, and preferably transdermal administration. In the case of parenteral administration, for example, the EGCG derivative can be administered by internal use, nasal drop, gargle, injection, nebulizer or aspirator. The transdermal administration method includes, for example, hand washing with a detergent containing the EGCG derivative, wiping with a wiping agent containing the EGCG derivative, and the like.

  When the subject is a cell or tissue collected from a living body, the administration method is not particularly limited, and examples thereof include addition to a medium and the like.

  The administration time of the EGCG derivative to the subject is not particularly limited, and may be, for example, before the non-membrane virus infection or after the non-membrane virus infection.

<Use of epigallocatechin gallate derivatives, or isomers or salts thereof>
The present invention is an epigallocatechin gallate derivative represented by the above chemical formula (1), an isomer thereof, or a salt thereof for preventing non-membrane virus infection. Moreover, this invention is use of the epigallocatechin gallate derivative represented by said Chemical formula (1), its isomer, or those salts for manufacture of the medicine for non-membrane viruses.

  Next, examples of the present invention will be described. However, the present invention is not limited by the following examples.

[Example 1]
(1) Preparation of EGCG Derivative As an anti-non-membrane virus agent, an EGCG derivative was prepared by the following method.

  EGCG (1 g), acyl group donor (927 mg) shown below and lipase (trade name Lipase PL, manufactured by Meika Sangyo Co., Ltd.) 50000U were mixed with DMF (100 mL), and incubated at 57 ° C. for 2 hours to carry out an enzyme reaction.

The reaction solution after the incubation is filtered and concentrated, and then subjected to column chromatography (spherical, neutral, 40-50 μm, trade name Silica gel N60, manufactured by Kanto Chemical Co., Inc.), and the unreacted acyl group donor as an impurity is obtained. Removed. As a result of performing electrospray ionization mass spectrometry (ESI-MS) on the obtained reaction product, the R ¹ or R ² of the B ring of EGCG or the R ⁵ or R ⁶ of the D ring was bonded to the above by an ester bond. It was confirmed that it was a monoester in which one acyl group shown in Table 1 (No. 1 to 4) was introduced and a diester in which two acyl groups (No. 5 to 7) were introduced.

Further, with respect to the monoester, the reaction product was analyzed by proton nuclear magnetic resonance (H ¹ NMR) in order to confirm which position of EGCG was esterified. The results are shown in the table below.

No. 1-No. The EGCG derivatives into which 7 acyl groups are introduced are hereinafter referred to as C16, C18, C18DE, C18TE, C12 × 2, C16 × 2, and C18DE × 2, respectively. These EGCG derivatives are EGCG derivatives represented by the chemical formula (2), and each site (R ¹ , R ² , R ⁵ or R ⁶ ) is an acyl group having the structural formula shown in Table 2. The following experiments were conducted using these EGCG derivatives.

(2) Comparison with known antiviral agents 1 (C16) was dissolved in DMSO and diluted with sterilized water to prepare a sample solution of the EGCG derivative 40 μmol / L, 2% DMSO. 200 μL of 1 × 10 ⁶ pfu / mL feline calicivirus (F-9 type feline calicivirus) suspended in 0.2% FBS-containing D-MEM was added to 800 μL of the sample solution, mixed, and then mixed at 20 ° C. For 30 seconds. Next, the reaction solution was diluted 1000 times with 0.2% FBS-containing DMEM.

On the other hand, cat kidney cultured cells (CRFK) were cultured until they became confluent in a 6-well plate containing an EMEM medium containing 10% FCS. The medium was removed from the plate, the obtained cell sheet was washed with D-PBS, and 1 mL of the diluted sample solution was applied. Then, after incubation at 37 ° C. for 1 hour, 0.8% agarose gel containing 6.0 × 10 ⁻⁴ % Trypsin and 0.2% BSA was overlaid on the cell sheet. Furthermore, after incubating at 37 ° C. for 52-60 hours in the presence of CO ₂ , the number of plaques appearing on the cell sheet was counted. As a control, the number of plaques was counted in the same manner without adding the EGCG derivative. The plaque formation ratio (%) was calculated with the number of plaques of the control (EGCG derivative not added: 0 μmol / L) as 100%. Plaque formation ratio (%) means virus infectivity titer (%).

  In the comparative example, the same treatment was performed except that, instead of the EGCG derivative, EGCG, benzalkonium chloride, or chlorhexidine, which were not introduced with an acyl group, were used as known antiviral agents. Instead of the sample solution of the EGCG derivative, the EGCG used was a 40 μmol / L solution, benzalkonium chloride used a 0.2% solution, and chlorhexidine used a 0.5% solution.

  These results are shown in FIG. FIG. 1 is a graph showing the virus infectivity titer (%), and the lower the value, the better the anti-non-membrane virus effect. As shown in FIG. 1, the EGCG derivative C16 showed an excellent anti-non-membrane virus effect at each stage as compared with the control (2% DMSO) and a known antiviral agent. From these results, it was found that it is difficult to prevent non-membrane virus infection with known anti-viral agents, but the anti-non-membrane virus agent of the present invention can effectively prevent non-membrane virus infections.

(3) Anti-non-membrane virus effect In the same manner as in (2) above, a sample solution of each EGCG derivative was prepared, and the virus infectivity titer (%) was measured.

  These results are shown in FIG. FIG. 2 is a graph showing the virus infectivity titer (%), and the lower the value, the better the anti-non-membrane virus effect. As shown in FIG. 2, each of the EGCG derivatives showed an anti-non-membrane virus effect superior to that of the control (2% DMSO) and a known antiviral agent. In particular, monoesters are unsaturated acyl groups having 16 or more carbon atoms and monoesters of saturated acyl groups (C16, C18, C18DE, C18TE), and diesters have 16 or more carbon atoms. The acyl ester diester showed a better effect.

[Example 2]
(1) Preparation of EGCG derivative 8 and no. An EGCG derivative having 9 acyl groups introduced therein was prepared in the same manner as in Example 1 (1).

  And as a result of performing electrospray ionization mass spectrometry (ESI-MS) about the obtained reaction product, the mono (1. 8) acyl group shown in the said Table 3 was introduce | transduced into EGCG by the ester bond. It was confirmed that the ester was a diester in which two acyl groups (No. 9) were introduced.

Further, with respect to the monoester, the reaction product was analyzed by proton nuclear magnetic resonance (H ¹ NMR) in order to confirm which position of EGCG was esterified. The results are shown in the table below.

  And the said No. of the said Example 1. 1-No. EGCG derivatives having an acyl group of 7 introduced therein, 8 and no. The following experiment was conducted using an EGCG derivative having 9 acyl groups introduced.

(2) Anti-rotavirus activity The EGCG and the EGCG derivatives (C12, C16, C18, C18DE, C18TE, C8x2, C12x2, C16x2) are each dissolved in DMSO, diluted with sterilized water, and the EGCG derivative 40 μmol / L A sample solution of 2% DMSO was prepared. On the other hand, rotavirus (Wa strain) was activated by incubating at 37 ° C. for 1 hour in EMEM containing 10 μg / mL trypsin. And the said process liquid was diluted with EMEM so that a trypsin density | concentration might be set to 2 microgram / mL, and the rotavirus liquid of 1 * 10 < ⁴ > pfu / mL was prepared. 15 μL of the rotavirus solution was added to 135 uL of the sample solution, mixed, and reacted at 20 ° C. for 10 minutes. Next, the reaction solution was diluted 10-fold with EMEM containing 1 μg / mL trypsin.

On the other hand, rhesus monkey fetal kidney cells (MA104) were cultured until they became confluent in a 6-well plate containing EMEM medium containing 10% FCS. The medium was removed from the plate, the obtained cell sheet was washed with D-PBS, and 1 mL of the diluted sample solution was applied. After incubating at 37 ° C. for 1 hour, 0.6% purified agarose gel containing 1.0 μg / mL trypsin and 0.01% DEAE dextran was overlaid on the cell sheet. Furthermore, after incubating at 37 ° C. for 52-60 hours in the presence of CO ₂ , the number of plaques appearing on the cell sheet was counted. As a control, the number of plaques was counted in the same manner without adding the EGCG derivative (2% DMSO). The plaque formation ratio (%) was calculated with the number of plaques of the control (EGCG derivative not added: 0 μmol / L) as 100%. Plaque formation ratio (%) means virus infectivity titer (%).

  These results are shown in FIG. FIG. 3 is a graph showing the virus infectivity titer (%), and the lower the value, the better the anti-non-membrane virus effect. As shown in FIG. 3, each EGCG derivative showed an anti-non-membrane virus effect superior to each stage as compared with control (2% DMSO) and EGCG. In particular, monoesters of unsaturated acyl groups having 12 or more carbon atoms and monoesters of saturated acyl groups (C12, C16, C18, C18DE, C18TE) showed more excellent effects. Moreover, as for the diester, the diester of an acyl group having 12 or more carbon atoms showed a more excellent effect, and the anti-non-membrane virus effect improved as the carbon number increased.

  As mentioned above, although this invention was demonstrated with reference to embodiment and an Example, this invention is not limited to the said embodiment and Example. Various changes that can be understood by those skilled in the art can be made to the configuration and details of the present invention within the scope of the present invention.

  This application claims the priority on the basis of Japanese application Japanese Patent Application No. 2013-070031 for which it applied on March 28, 2013, and takes in those the indications of all here.

  According to the present invention, non-membrane virus infection can be prevented safely and effectively. For this reason, it can be said that the present invention is useful in the medical field, the food hygiene field, and the like.

Claims (19)

An anti-non-membrane virus agent comprising an epigallocatechin gallate derivative represented by the following chemical formula (1), an isomer thereof, or a salt thereof.
In the chemical formula (1),
R ^{1 to} R ⁶ are
Each represents a hydrogen atom, halogen, sodium, potassium, or a linear or branched saturated or unsaturated acyl group, which may be the same or different;
The acyl group may be further substituted with one or more substituents,
At least one of the R ^{1 to} R ⁶ is the acyl group,
R ^{7 to} R ¹⁶ are a hydrogen atom, halogen, sodium or potassium, and may be the same or different. The anti-non-membrane virus agent according to claim 1, wherein the main chain length of the acyl group in R ^{1 to} R ⁶ is ¹ to 20 atoms. The anti-non-membrane virus agent according to claim 1 or 2, wherein in R ^{1 to} R ⁶ , the acyl group has a main chain length of 16 to 18 atoms. The anti-non-membrane virus agent according to any one of claims 1 to 3, wherein, in R ^{1 to} R ⁶ , the acyl group has 1 to 20 carbon atoms. The anti-non-membrane viral agent according to any one of claims 1 to 4, wherein at least one of R ¹ , R ² , R ⁵ and R ⁶ is the acyl group. The anti-non-membrane viral agent according to claim 5, wherein at least one of R ¹ , R ² , R ⁵ and R ⁶ is the acyl group, and the other is a hydrogen atom. The anti-non-membrane viral agent according to any one of claims 1 to 5, wherein at least two of R ¹ , R ² , R ⁵ and R ⁶ are the acyl group. The anti-non-membrane viral agent according to claim 7, wherein at least two of R ¹ , R ² , R ⁵ and R ⁶ are the acyl group, and the other is a hydrogen atom. The anti-non-membrane virus agent according to any one of claims 1 to 8, wherein R ^{7 to} R ¹⁶ are hydrogen atoms. The anti-non-membrane virus agent according to any one of claims 1 to 9, wherein the acyl group has a main chain length of 12 to 18 atoms. The anti-non-membrane virus agent according to claim 10, wherein the acyl group has a main chain length of 16 to 18 atoms. The anti-non-membrane virus agent according to any one of claims 1 to 11, wherein the acyl group has 12 to 18 atoms. The anti-non-membrane virus agent according to any one of claims 1 to 12, wherein the acyl group is a linear saturated acyl group. The acyl group is a butyryl group, octanoyl group, trans-8-methyl-6-octenoyl group, geranoyl group, lauroyl group, 12- (dimethylamino) lauroyl group, farnesoyl group, palmitoyl group, stearoyl group (C18), linoleyl. The anti-non-membrane virus agent according to any one of claims 1 to 9, which is at least one acyl group selected from the group consisting of a group, an eicosanoyl group and isomers thereof. In R < ¹ > -R < ⁶ >, the said substituent is at least 1 selected from the group which consists of an alkyl group, an amino group, an alkylamino group, and a dialkylamino group, It is any one of Claims 1-14 Anti-non-membrane virus agent. In R ^{1 to} R ⁶ , the alkyl group is a linear or branched alkyl group having 1 to 6 carbon atoms, and the alkyl group in the alkylamino group is a linear or branched group having 1 to 6 carbon atoms. The anti-non-membrane virus agent according to claim 15, which is an alkyl group, and the alkyl group in the dialkylamino group is a linear or branched alkyl group having 1 to 6 carbon atoms and may be the same or different. The anti-non-membrane virus agent according to claim 15 or 16, wherein in R ^{1 to} R ⁶ , the alkyl group is a methyl group, the alkylamino group is a methylamino group, and the dialkylamino group is a dimethylamino group. . The anti-non-membrane virus agent is a group consisting of Norovirus genus, Feline calicivirus genus, Rotavirus genus, Beta Nodavirus genus, Aquavirnavirus genus, Ranavirus genus, Enterovirus genus, Mast adenovirus genus and Vesivirus genus The anti-non-membrane virus agent according to any one of claims 1 to 17, which is an anti-non-membrane virus agent against at least one virus selected from. A method for preventing non-membrane viral infections,
A method for preventing non-membrane virus infection, comprising administering the anti-non-membrane virus agent according to any one of claims 1 to 18 to a subject.