KR100792626B1 - Agent for preventing and treating infectious disease by influenza virus comprising cathechin derivative compounds - Google Patents

Abstract

The present invention relates to an agent for preventing and treating influenza virus infectious diseases using an catechin derivative compound as an active ingredient, and specifically, an catechin derivative compound that inhibits the growth of the flu virus and inhibits the binding between the flu virus and cells as an active ingredient. It is about the preventive and therapeutic agent of the flu virus infectious disease. The prophylactic and therapeutic agents of the present invention can be effectively used for the prevention and treatment of human influenza virus and avian influenza virus infectious diseases by inhibiting the binding action of cells with various kinds of influenza virus subtypes as well as exhibiting the above effects.

Catechin derivatives, flu virus.

Description

Agent for preventing and treating infectious disease by influenza virus comprising cathechin derivative compounds}

1 is a photograph showing the effect of inhibiting the activity of the flu virus by the catechin derivative compound:

A: Flu virus plaque photograph without catechin derivative;

B: Photograph of 10 uM of the catechin derivative compound No. 7;

C: photograph which 20 uM of the catechin derivative compound No. 7 was added;

D: photograph which 40 uM of the catechin derivative compound No. 7 was added;

E: photograph which added 80 uM of the catechin derivative compound No. 7; And

F: Photograph which added 160 uM of the catechin derivative compound No. 7.

2 is a graph showing the effect of inhibiting the binding ability between human subtypes of human influenza virus and cellular receptors by catechin derivative compounds.

The present invention relates to an agent for preventing and treating influenza virus infectious diseases using a catechin derivative compound as an active ingredient, and more particularly, to a catechin derivative compound that inhibits the growth of the flu virus and inhibits the binding between the flu virus and cells. The present invention relates to an agent for preventing and treating flu virus infectious diseases as an active ingredient.

Flu is a disease caused by influenza virus, which occurs regularly in winter every year, and is a representative respiratory infectious disease that causes high mortality in high-risk groups, including the elderly, and causes socio-economic losses for health workers with economic activities. It is classified as an infectious disease. In particular, the flu pandemic that occurs every 10 to 40 years has a high incidence and mortality rate. For example, the worldwide flu epidemic of 1918 caused about 20 million casualties. The flu pandemic is caused by the emergence of a new flu virus, and bird flu, which is continuously occurring in East Asia recently, has been identified as a cause of the pandemic. Therefore, development of a drug to control the flu is required.

Influenza viruses belong to the group of orosomyxoviruses that contain eight RNA fragments as genes (p605, Fundamental Virology 3rd edition, BN Fields et al Eds, Lippincott-Raven press). The genes are present in complex complexes with the replication enzymes PA, PB1, PB2, and NP proteins, which are required to replicate RNA genes. These RNP complexes are called Hemagglutinin (HA). ) And neuraminidase (hereinafter referred to as "NA"). Influenza viruses are classified into various subtypes according to the type of HA and NA. There are 15 types of HA and 9 types of NA.

Bird flu is an acute viral disease that infects chickens, ducks, turkeys, and wild birds, ranging from non-pathogenic to 100% highly pathogenic, and highly pathogenic avian influenza (poultry flu) is the first type of statutory hepatitis in Korea. It is classified as List A epidemic by OIE. The causative agent of avian influenza virus is a common virus and is divided into high pathogenic, drug pathogenic and non-pathogenic pathogens. Avian influenza virus is transmitted by droplets, air and water, but the main cause of transmission is contaminated feces. That is, feces are deposited on human hands, feet, feed cars, appliances, and equipment, and are indirectly transmitted. In addition, avian influenza virus may be infected by humans through intermediate hosts such as pigs and chickens. Avian influenza virus causes genetic mutations in the body of the intermediate host, and the mutated virus is transmitted to people through respiratory infections, thereby causing birds. It causes the flu. Such avian influenza virus has various serotypes and mutants so far it has not been developed in effective preventive medicines. Therefore, most countries have a policy of killing the highly pathogenic avian influenza virus.

Existing influenza drugs have been approved by the U.S. Food and Drug Administration as anti-influenza virus drugs such as Amantadine, Rimantadine, Oseltamivir and Zanamivir. received. Of these, amantadine and rimantadine have been pointed out as a problem due to the development of a virus that is easily tolerated during the process of being used for 40 years. Oseltamivir and zanamivir have been relatively recently developed and relatively effective, but both target the same mechanism of inhibiting the enzyme activity of NA, an external protein of the flu virus. It is highly likely that resistant strains will occur even within a few mutations, and studies on resistance strains already existing in nature have been reported (Mai Le, Q., et. al ., Nature 437: 1108, 2005) The development of new anti-flu virus therapies is urgent.

Green tea contains four major polyphenols: epigallocatechin gallate (hereinafter referred to as "EGCG"), epicatechin gallate (hereinafter referred to as "ECG") and epigallocatechin. (epigallocathechin: hereinafter referred to as "EGC") and epicatechin (hereinafter referred to as "EC"). In Korean Patent No. 554372, cell permeability was increased by introducing a functional group into catechin, which is known to have a lethal effect on existing cancer cells. Through this, the anti-cancer activity of the catechin derivative is increased, and the stability is improved, which indicates that the anti-cancer activity against prostate cancer, ovarian cancer, and brain cancer is administered to the mammal. However, there are no reported or generally known pharmacological and therapeutic properties for the anti-influenza virus activity of these compounds. In U.S. Patent No. 5,137,922, a polyphenol (catechin) compound extracted from green tea has been reported to aggregate the influenza virus on the surface of a host cell to inhibit the infection of the virus. However, it is only EGCG that the prior art has presented the invention through specific experimental examples, and no evidence for the effects of other ECGs, EGCs, and ECs is presented. In addition, there is a limit that the activity of only one type of type A (H3) and type B among the various subtypes of influenza viruses existing according to the type of HA and NA is confirmed. Related studies have shown that anti-viral virus treatments targeting HA proteins in a mechanism similar to polyphenols work effectively on H1 and H2 types, but not H3 types, depending on the type of HA (Guangxiang, L., et al ., Virology 226: 66-76, 1996). Therefore, there is a need for the development of antiviral therapeutics that have been proven effective against the recent influenza subtypes and avian influenza subtypes that are socially problematic.

Thus, the present inventors investigated the effect on the anti-influenza virus activity using the catechin derivative compound, and completed the present invention by confirming that the compounds inhibit the activity of various human influenza virus and avian influenza virus.

It is an object of the present invention to provide new uses for the use of catechin derivative compounds in the prevention and treatment of flu virus infectious diseases.

In order to achieve the above object, the present invention provides a flu virus infectious disease prevention and treatment comprising a catechin derivative compound or a pharmaceutically acceptable salt thereof as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention provides an agent for preventing and treating influenza virus infectious diseases comprising a catechin derivative compound or a pharmaceutically acceptable salt thereof as an active ingredient.

The flu virus is characterized by human flu virus and / or avian influenza virus.

The catechin derivative of the present invention preferably includes an epigallocatechin (hereinafter, referred to as "EGC") derivative of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

Wherein R is an unsubstituted or, independently or optionally alkyl to cycloalkyl of C _{1 ~ 15} linear or branched alkyl, C _{3 ~ 15} cycloalkyl, C _{1 ~ 15} haloalkyl, C _{3 ~ 15} of the, aryl of _{C 6 ~ 15, C 6 ~} 15 aryl-alkyl, C _{6 ~ 15} alkylaryl, at least one selected from the group consisting of aryloxy, halogen, and amino C _{1 ~ 15} alkoxy, C _{6 ~ 15} of the and of C _{1 ~ 18} substituted by a substituent straight or branched alkyl group or cyclic aliphatic or aromatic hydrocarbon,

Preferably unsubstituted or, independently or selectively C _{1 ~ 10} linear or branched alkyl, C _{3 ~ 10} cycloalkyl, C _{1 ~ 10} of haloalkyl, C _{3 ~ 10} alkyl, cycloalkyl to C of the substituent at least one selected from the group consisting of aryloxy, halogen and amino _6-10 aryl, C _6-10 arylalkyl, C _6-10 alkylaryl, C _{1 ~ 10} alkoxy, C _{6 to 10} of the a substituted C _{1 ~ 15} alkyl group or by a straight or branched, and cyclic aliphatic or aromatic tanhwasusoyi,

More preferably any one selected from the group consisting of a benzyl group substituted with an alkoxy-substituted C _{1 ~ 12} linear or branched alkyl group, a halogen and a C _{1 ~ 15} of the ring,

Most preferably, it is any one selected from the group consisting of a butyl group, a heptyl group, a decyl group, a dodecyl group, a difluorobenzyl group, a trifluorobenzyl group, and a dimethoxybenzyl group.

Preferably, the EGC derivative compound is selected from one or more of the group consisting of: 3-O-3,4,5-trifluorobenzyl epigallocatechin (3-O- (3,4,5-trifluoro) benzyl epigllao-catechin) (1); 3-O-3,5-dimethoxy benzyl epigallocatechin (2) (O) (3-O- (3,5-dimethoxy) benzyl epigllaocatechin) (2); 3-O-3,5-difluoro benzyl epigallocatechin (3-O- (3,5-difluoro) benzyl epigllaocatechin) (3); 3-O-butyl epigllaocatechin (4); 3-O-heptyl epigllaocatechin (5); 3-O-decyl epigllaocatechin (6); And 3-O-dodecyl epigllao-catechin (7). In addition, the epigallocatechin derivative compound is more preferably at least one selected from the group consisting of: 3-O-decyl epigallocatechin (6); And 3-O-dodecyl epigllao-catechin (7).

In addition, the catechin derivative of the present invention preferably includes a (+)-catechin (catechin: hereinafter “(+)-C”) derivative of Formula 2 or a pharmaceutically acceptable salt thereof as an active ingredient. .

Wherein R is an unsubstituted or, independently or optionally alkyl to cycloalkyl of C _{1 ~ 15} linear or branched alkyl, C _{3 ~ 15} cycloalkyl, C _{1 ~ 15} haloalkyl, C _{3 ~ 15} of the, aryl of _{C 6 ~ 15, C 6 ~} 15 aryl-alkyl, C _{6 ~ 15} alkylaryl, at least one selected from the group consisting of aryloxy, halogen, and amino C _{1 ~ 15} alkoxy, C _{6 ~ 15} of the and of C _{1 ~ 18} substituted by a substituent straight or branched alkyl group or cyclic aliphatic or aromatic hydrocarbon,

Preferably unsubstituted or, independently or selectively C _{1 ~ 10} linear or branched alkyl, C _{3 ~ 10} cycloalkyl, C _{1 ~ 10} of haloalkyl, C _{3 ~ 10} alkyl, cycloalkyl to C of the substituent at least one selected from the group consisting of aryloxy, halogen and amino _6-10 aryl, C _6-10 arylalkyl, C _6-10 alkylaryl, C _{1 ~ 10} alkoxy, C _{6 to 10} of the a substituted C _{1 ~ 15} alkyl group or by a straight or branched, and cyclic aliphatic or aromatic hydrocarbon,

More preferably any one selected from the group consisting of a benzyl group substituted with an alkoxy-substituted C _{1 ~ 12} linear or branched alkyl group, a halogen and a C _{1 ~ 15} of the ring,

Most preferably, it is any one selected from the group consisting of a butyl group, a heptyl group, a decyl group, a dodecyl group, a difluorobenzyl group, a trifluorobenzyl group, and a dimethoxybenzyl group.

Preferably, the (+)-C derivative compound is selected from one or more of the following groups: 3-O-3,4,5-trifluorobenzyl catechin (3-O- (3,4,5- trifluoro) benzyl catechin) (8); 3-O-3,5-difluoro benzyl catechin (9); 3-O-3,5-dimethoxy benzyl catechin (10); 3-O- (4-trifluoromethoxy) benzyl catechin (11); 3-O-benzyl catechin (12): 3-O-butyl catechin (13); 3-O-heptyl catechin (14); 3-O-decyl catechin (15); And 3-O-dodecyl catechin (16). Further, the (+)-catechin derivative compound is more preferably selected from one or more of the group consisting of: 3-O-decyl catechin (15); And 3-O-dodecyl catechin (16).

The EGC and / or (+)-C derivative compounds of the present invention represented by Chemical Formulas 1 and / or 2 may be used in the form of pharmaceutically acceptable salts, and as salts, pharmaceutically acceptable free acid Acid addition salts formed by) are useful. Inorganic acids and organic acids can be used as the free acid. Hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid, etc. may be used as the inorganic acid, and citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, gluconic acid, methanesulfonic acid, glyconic acid, succinic acid, 4-toluenesulfonic acid , Gluturonic acid, embonic acid, glutamic acid, aspartic acid, or the like can be used. In addition, the EGC and / or (+)-C derivative compounds of the formulas (1) and (2) may be pharmaceutically acceptable metal salts, in particular alkali metal salts, formed due to bases. Examples thereof include sodium salts and potassium salts.

The present inventors investigated the effects of the EGC of Formula 1 and the (+)-C derivative compound of Formula 2 on the flu virus. To this end, the catechin derivatives described in Table 1 were prepared by the method described in Korean Patent No. 554372 (see Table 1). Plaque formation inhibition experiments were performed in MDCK cell lines infected with the flu virus [A / Chile / 1/83 (H1N1), A / Sydeny / 5/97 (H3N2)] using the catechin derivatives prepared above. Plaques are a population of specifically killed cells that are produced because active viruses infect cells and then replicated viruses flow out of the cells and subsequently infect the immediate adjacent cells again. Plaques can be observed with the naked eye and under a microscope, and are easier to observe if a suitable staining reagent is used. Plaque formation experiments are experiments to determine whether the catechin derivative compound reduces the ability of the virus to form plaque by inhibiting the activity of the virus by adding the catechin derivative compound as a representative method of showing the activity of the virus. As a result, it was confirmed that the catechin derivative compounds of the present invention have high anti-influenza virus activity that their precursors (EGC, (+)-C) do not have (see Table 2 and Fig. 1).

The present inventors investigated the growth inhibitory effect of the bird flu virus of the catechin derivative compound of the present invention. The present inventors mixed the catechin derivative compound in the same amount with avian influenza virus (A / Chicken / Korea / MS96 / 96 (H9N2)) and cultured by injecting it into fertilized eggs. Thereafter, a hemagglutination assay (hereinafter referred to as “HA assay”) was performed to measure the cultured virus. The HA assay is a method of quantifying the amount of virus using the binding force between the flu virus and chicken red blood cells. As a result, it was confirmed that the catechin derivative compounds of the present invention effectively inhibited the growth of avian influenza virus even with 5.1 to 10.1 μM (see Table 3). In comparison, the MIC (Minimum inhibitory concentration) of green tea (EGCG, ECG, EGC, (+)-C) used as a control group, and oseltamivir and amantadine, which is a conventional influenza virus treatment, is higher than 500 μM. It was confirmed.

The present inventors investigated the effect of the catechin derivative compound of the present invention on the binding between influenza virus and cells. Hemagglutinin (hereinafter referred to as "HA"), one of the surface proteins of the flu virus, binds the receptor and the virus to cells in the early stages of infection, introducing the virus into the cell. However, when the virus is bound to the virus, the aggregation does not occur, and therefore, when the binding of the virus and the red blood cells is inhibited by the catechin derivative compound, the red blood cells are aggregated, which can be confirmed with the naked eye. After mixing with avian influenza virus (A / Chicken / Korea / MS96 / 96 (H9N2)) solution and reacting with chicken erythrocyte solution for 1 hour, we examined whether the virus-catechin derivative mixture solution inhibited the interaction between flu virus and erythrocytes. In addition, each of the catechin derivative compounds may The minimum concentration that inhibits the interaction between blood cells was determined, and as a result, it was confirmed that the catechin derivative compounds of the present invention inhibited cell receptors of the virus at concentrations lower than their precursors, EGC or (+)-C. Among them, compounds EGC derivatives 6 and 7 and (+)-C derivative compounds 15 and 16 exhibited the lowest inhibitory concentrations and ranged from 15.6 to 62.5 μM. These compounds showed lower inhibitory concentrations than oseltamivir and amantadine, which are known to be known as flu treatments, due to the different mechanisms of action and target positions for each treatment. Oseltamivir inhibits the enzymatic activity of neuraminidase (NA). In this regard, the catechin derivative compounds of the present invention have a novel effect on the activity of the influenza virus, which is completely different from existing inhibitors through the reaction of the virus with the HA protein that binds to cellular receptors. It can be seen that it inhibits.

The present inventors investigated the effects of the catechin derivative compounds of the present invention on inhibition of binding between flu virus and cells to various types of flu virus subtypes. Five human flu viruses were mixed with catechin derivative compounds and added to chicken erythrocyte solution. Thereafter, the concentration of each catechin derivative compound was checked for binding inhibition, and the minimum concentration of the inhibitory effect was determined for each of the influenza virus subtypes. As a result, although there are some differences between the subtypes of influenza virus, it was confirmed that four kinds of catechin derivative compounds effectively inhibit the binding between the cells and the human influenza virus and the cell receptor (see FIG. 2). In particular, 3-O-decyl epigllaocatechin (6), a derivative compound based on EGC, was used to bind viruses at concentrations of 50 μM or less for all five flu virus subtypes tested. It showed a high activity that inhibits. In addition, as a result of an acute toxicity test by parenteral administration to rats using the catechin derivative compound of the present invention, it was confirmed that the catechin derivative compound of the present invention is safe. As described above, the catechin derivative compound of the present invention may be usefully used as a prophylactic and therapeutic agent for influenza virus infectious diseases.

The present invention also provides an agent for preventing and treating influenza virus infectious diseases comprising the catechin derivative compound and a pharmaceutically acceptable carrier.

Flu virus infectious disease prevention and treatment of the present invention may further include conventionally used excipients, disintegrants, sweeteners, lubricants, flavoring agents, etc., tablets, capsules, powders, granules, forms by conventional methods It may be formulated as a suspension, emulsion, syrup, or other liquid.

Specifically, the prophylactic and therapeutic agents of the present invention may be formulated for oral administration, for example tablets, troches, lozenges, aqueous or oily suspensions, preparations or granules, emulsion hard or soft capsules, syrups or elixirs Formulated as elixirs. Lactose, saccharose, sorbitol, mannitol, starch, amylopectin, binders such as cellulose or gelatin, excipients such as dicalcium phosphate, disintegrants such as corn starch or sweet potato starch, magnesium stearate, for formulation into formulations such as tablets and capsules Lubricants such as calcium stearate, sodium stearyl fumarate or polyethylene glycol wax. Capsule formulations contain liquid carriers, such as fatty oils, in addition to the substances mentioned above.

Flu virus infectious disease prevention and treatment of the present invention can be administered parenterally or orally, parenteral administration is by subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection. To formulate into a parenteral formulation, the catechin derivative compound is mixed in water with a stabilizer or buffer to prepare a solution or suspension, which is formulated in unit doses of ampoules or vials.

The dosage of the active ingredient according to the catechin derivative compound of the present invention is appropriately selected depending on the absorption rate, inactivation rate and excretion rate of the active ingredient in the body, the age, sex and condition of the patient, and the severity of the disease to be treated, but generally adult Derivative compound of Formula 1 or Formula 2 per kg of body weight per day may be administered once to several times in an amount of 0.1 to 500 mg, preferably 0.1 to 100 mg.

In addition, the present invention provides an agent for preventing and treating flu virus infectious diseases in the form of a health food comprising a catechin derivative compound.

The health food containing the catechin derivative compound of the present invention includes folk remedies for the purpose of preventing and treating flu virus infectious diseases such as tea, jelly, juice, extract, and beverages, which contain the catechin derivative compound as an active ingredient. have. As described above, the healthy food for preventing and treating influenza virus infectious diseases processed in various forms is excellent in detoxification without side effects on the human body, and is easy to take and can be stored for a long time, thus preventing and treating influenza virus infectious diseases. It can be usefully used.

The catechin derivative compounds of the present invention may be added to health foods for the purpose of treating human flu and / or bird flu. When the catechin derivative compound of the present invention is used as a food additive, the derivative compound may be used as it is or may be used together with other food or food ingredients, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment). In general, in the manufacture of food or beverage, the catechin derivative compound of the present invention is added in an amount of 80 parts by weight or less, preferably 50 parts by weight or less based on 100 parts by weight of the raw material. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

There is no particular limitation on the kind of food. Examples of the food to which the catechin derivative compound of the present invention can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionary, pizza, ramen, other noodles, gum, ice cream, various soups, and beverages. , Teas, drinks, alcoholic beverages and vitamin complexes and the like, and includes all healthy foods in the conventional sense.

The health beverage composition of the present invention may contain various flavors, natural carbohydrates, and the like as additional components, as in a conventional beverage. The natural carbohydrates described above are glucose, monosaccharides such as fructose, maltose, disaccharides such as sucrose, and polysaccharides such as dextrin, cyclodextrin, sugar alcohols such as xylitol, sorbitol, and erythritol. As the sweetening agent, natural sweetening agents such as tautin and stevia extract, synthetic sweetening agents such as saccharin, aspartame, and the like can be used. The ratio of the natural carbohydrate is generally 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the catechin derivative compound of the present invention.

The above catechin derivative compounds of the present invention are various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin , Alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the catechin derivative compounds of the present invention may contain pulp for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components can be used independently or in combination. The proportion of such additives is not critical, but is generally selected from 0.01 to 0.1 parts by weight per 100 parts by weight of the formula catechin derivative compound of the present invention.

Hereinafter, the present invention will be described in detail by examples and formulation examples.

However, the following Examples and Preparation Examples specifically illustrate the present invention, and the contents of the present invention are not limited by Examples and Formulation Examples.

< Example  1> Preparation of catechin derivative compound

The present inventors produced the compounds shown in Table 1 by the method of Korean Patent No. 554,372.

Compounds 1 to 7 of the catechin derivative compounds are derivatives derived from epigallocatechin of Formula 1 (hereinafter referred to as “EGC”), and compounds 8 to 16 represent (+)-catechins of Formula 2 ( catechin: A derivative having a precursor of “(+)-C” hereinafter).

Catechin Derivative Compound Compound number Catechin Derivative Compound One 3-O-3,4,5-trifluoro benzyl epigallocatechin (3-O- (3,4,5-trifluoro) benzyl epigllaocatechin) 2 3-O-3,5-dimethoxy benzyl epigallocatechin (3-O- (3,5-dimethoxy) benzyl epigllaocatechin) 3 3-O-3,5-difluorobenzyl epigallocatechin (3-O- (3,5-difluoro) benzyl epigllaocatechin) 4 3-O-butyl epigllaocatechin 5 3-O-heptyl epigllaocatechin 6 3-O-decyl epigllaocatechin 7 3-O-dodecyl epigllaocatechin 8 3-O-3,4,5-trifluoro benzyl catechin (3-O- (3,4,5-trifluoro) benzyl catechin) 9 3-O-3,5-difluoro benzyl catechin (3-O- (3,5-difluoro) benzyl catechin) 10 3-O-3,5-dimethoxy benzyl catechin (3-O- (3,5-dimethoxy) benzyl catechin) 11 3-O- (4-trifluoromethoxy) benzyl catechin (3-O- (4-trifluoromethoxy) benzyl catechin) 12  3-O-benzyl catechin 13  3-O-butyl catechin 14 3-O-heptyl catechin 15 3-O-decyl catechin 16 3-O-dodecyl catechin

< Example  2> Inhibitory Effect of Catechin Derivative Compounds on Flu Virus Plaque Formation

Plaque formation inhibition experiment was conducted to determine the effect of the catechin derivative compound prepared in Example 1 on the activity of the flu virus.

One day before the experiment, 1.5 × 10 ⁵ was used for MDCK (Mardine Darbine Canine Kidney) cell line (ATCC, USA) in MEM (Minimum Essencial Medium: GIBCO Invitrogen Coporation, USA) medium containing FBS (Fetal Bovine Serum: GIBCO Invitrogen Coporation, USA). After inoculation at a concentration of cells / ml, the cells were incubated in a 6-well plate at 37 ° C. and 5% CO ₂ concentration. Two subtypes of the flu virus, A / Chile / 1/83 (H1N1) and A / Sydney / 5/97 (H3N2), were grown in an MDCK cell line that had grown to the bottom of the petri dish after 24 hours (National Institute for Medical Research, London). , UK) was infected at a concentration of 500 PFU (Plaque Forming Unit) / ml. After one hour, the treated virus solution was removed from the cells, and the cell lines were washed with 1 ml / well of PBS (Photasium Phosphate Buffered Saline) buffer. The catechin derivative compound of Table 1 was serially diluted 2-fold so that the final concentration was from 800 μM to 12.5 μM, followed by overlay media: 1% agarose, DMEM containing trypsin enzyme at a concentration of 10 μg / ml. Dulbecco's Modified Eagle's Medium: GIBCO Invitrogen Coporation, USA) medium added to the cells plated for plaque formation] and catechins (EGC and (+)-C), which are skeletons of catechin derivatives, instead of catechin derivative compounds as controls. EGCG, ECG and nothing added overlay medium from the group and green tea were used. The overlay medium was plated by 3 ml / well of influenza virus infected cells, and after 15 minutes, the overlay medium was solidified and incubated for 72 hours in an incubator maintained at 37 ° C. 5% CO ₂ concentration.

As a result, during the 72 hours, the flu virus formed plaques and plaque formation was inhibited in the experimental group to which the catechin derivative compound of the present invention (Table 1) was added. Typically, in the plaque formation process of A / sydney / 5/97 flu virus, the experimental results of the control group treated with or without the catechin derivative compound No. 7 at 160 μM to 10 μM concentration are shown in FIG. 1. After each plaque number was counted, the plaque formation inhibitory ability of the flu virus by the catechin derivative compound (Table 1) of the present invention was quantified by the ratio of the plaque number of the control group to the plaque number of the experimental group. The inhibitory ability at various concentrations of the catechin derivative compound of the present invention (Table 1) was repeated three times, and then treated statistically to obtain a concentration (EC ₅₀ : effective concentration) having a 50% inhibition ability of flu virus activity. (Table 2).

Compound Nos. 1 to 7 are EGC derivative compounds having EGC as precursors, and Nos. 8 to 16 are (+)-C derivative compounds having (+)-C as precursors.

From the following Table 2 it was confirmed that the catechin derivative compounds of the present invention have high anti-influenza virus activity that their precursors (EGC, (+)-C) do not have. Among derivative compounds having EGC of formula 1 as precursors, 5, 6, and 7 were all active against influenza viruses of different subtypes, and among derivative compounds having (+)-C of formula 2 as precursors, 14, 15 , 16 times had good activity. They differ only in the precursor, but the added functional groups are the same, so it can be seen that the anti-flu virus activity of the present invention is determined by the specific functional group possessed by the derivative.

Compound number EC ₅₀ (μM) (mean ± standard deviation, 3 repetitions) A / Sydney / 5/97 A / Chile / 1/83 One .9 ± 1.2 .4 ± 3.6 2 51.1 ± 1.1 87.2 ± 14.1 3 61.1 ± 1.2 79.4 ± 7.3 4 552 ± 1.5 304 ± 1.3 5 32.1 ± 1.1 45.0 ± 1.5 6 33.3 ± 1.4 25.1 ± 1.2 7 28.1 ± 1.1 60.2 ± 1.8 8 38.6 ± 1.2 57.3 ± 1.8 9 30.2 ± 1.1 40.0 ± 1.7 10 91.4 ± 5.0 71.2 ± 2.6 11 26.9 ± 1.1 56.6 ± 2.4 12 53.2 ± 1.2 79.7 ± 3.0 13 72.3 ± 1.2 95.9 ± 1.3 14 25.6 ± 1.1 57.9 ± 1.3 15 18.7 ± 1.1 18.6 ± 1.1 16 31.2 ± 1.2 19.5 ± 1.0 EGCG * 22.8 ± 1.1 28.4 ± 1.4 ECG * 22.2 ± 1.1 26.4 ± 2.0 EGC * 309 ± 1.2 318 ± 3.0 (+)-C * > 600 > 600

EGCG: epigallocatechin gallate (green tea leaf extract);

  ECG: epigallocatechin gallate (green tea leaf extract);

  EGC: epigallocatechin (Sigma, USA); And

  (+)-C (Sigma, USA): (+)-Catechin.

< Example  3> Investigation of the growth inhibitory effect of avian influenza virus by catechin derivative compounds

In order to investigate the effect of the catechin derivative compound of the present invention on the growth of avian influenza virus, a bird flu virus growth inhibition experiment using chicken eggs was performed.

For the four catechin derivative compounds of the present invention (6, 7, 15, and 16) that were effective in plaque inhibition experiments, 250 catechin derivative compounds of various concentrations, which were serially diluted twice from 500 μM concentration to 1 μM concentration, were 250 The mixture was mixed with avian influenza virus (A / Chicken / Korea / MS96 / 96 (H9N2)) (National Veterinary Research and Quarantine Service, Korea) at a concentration of HAU (Hemagglutination unit) and reacted at room temperature for 1 hour.

The reaction solution was inoculated into the capillary solution of fertilized eggs, and fertilized eggs were grown for 4 days in the incubator at 37 ° C to incubate the virus. After obtaining the systolic solution, the resultant was separated once using a centrifuge at low speed (1,200 rpm, 10 minutes), and the supernatant was filtered once using a filter (Minisart-plus, Satorius, Germany) and then hemagglutination. The grown virus was quantified using a Hemegglutination assay. As a control, it was quantified by culturing avian influenza virus that was not treated with the catechin derivative compound of the present invention. Also, using EGC, (+)-C and green tea extracted from oseltamivir (Roche, Swiss) and amantadine (Sigma, USA), which are used as anti-flu virus treatments, and precursors of derivative compounds, Tested.

As a result, it can be seen that the anti-influenza virus activity, which was not seen in precursors EGC and (+)-C, is shown in the catechin derivative compound of the present invention incorporating specific functional groups.

From Table 3 below, compounds No. 6 and No. 7 having EGC of Formula 1 as precursors and compounds No. 15 and 16 having (+)-C of Formula 2 as precursors among the catechin derivative compounds of the present invention are conventional as well as their precursors. It was confirmed that the growth inhibitory effect was at least 25 times better than the compound widely used as an anti-influenza virus treatment. In addition, it was confirmed that the difference in the effects of at least 400-fold with EGCG or ECG, which is a catechin compound extracted from green tea.

Compound number MIC (μM) 6 9.6 7 10.1 15 5.1 16 5.1 EGCG 4360 ECG 4520 EGC 〉 6000 (+)-Catechin 〉 6000 Oseltamivir 500 Amantadine 〉 1000

< Example  4> Flu virus and catechin derivative Intercellular  Binding inhibitory investigation

Hemagglutinin (hereinafter referred to as "HA"), one of the surface proteins of the flu virus, functions to bind the receptor and the virus of a cell in the early stage of infection. This process introduces the virus into the cell. The HA catechin derivative compound of the flu virus surface protein was introduced into the cell by binding the virus to the cell receptor of the early stage of infection and introducing the virus into the cell. Aggregation occurs, but when combined with the virus does not occur, therefore, when the binding of the virus and erythrocytes is inhibited by the catechin derivative compound, the erythrocytes aggregate, which can be visually confirmed.

A solution of avian influenza virus (A / Chicken / Korea / MS96 / 96 H9N2) was prepared at 500 HAU (hemagglutination unit / 25 μl), and the same catechin derivative compound diluted 2 times continuously from 1 mM to 7.8 uM was added. Mix by volume. After 1 hour of reaction, chicken red blood cell solution (1%) (Cambrex, USA) was added to the reaction solution of the virus and the catechin derivative compound of the present invention in the same volume. Chicken erythrocytes were reacted with a virus-catechin derivative mixture solution for 1 hour to see if the compound inhibited the interaction between the flu virus and erythrocytes. In addition, the minimum concentration at which each of the catechin derivative compounds of the present invention inhibited the interaction between the virus and the red blood cells was determined.

As a result, as shown in Table 4, compounds 6 and 7, EGC derivative compounds and compounds 15 and 16, which are (+)-C derivative compounds, inhibited cell receptors of the virus at lower concentrations than other compounds. It was confirmed that the concentration was 15.6 to 62.5 uM level. This activity is about 2 to 5 times higher than catechins EGCG and ECG extracted from green tea. In addition, since the precursor (EGC, (+)-C) is not shown to have an inhibitory effect even at the highest concentration, it can be seen that this activity is exhibited by a specific substituent according to the present invention. Oseltamivir and Amantadine, which are well known as anti-flu virus treatments, also had no effect. This is because each agent has a different mechanism of action and target position. That is, amantadine inhibits the role of the M2 protein, a protein of the influenza virus, and oseltamivir, which inhibits the enzyme activity of NA, has no effect on binding to cell receptors. Therefore, it can be seen from this experiment that the catechin derivative compound inhibits the activity of the influenza virus by a novel action that is completely different from existing inhibitors, in which the virus inhibits the reaction with the HA protein binding to the cell receptor.

Compound number MIC (μM) One 500 2 500 3 500 4 250 5 250 6 31.2 7 62.5 8 〉 1000 9 500 10 〉 1000 11 500 12 125 13 125 14 500 15 31.2 16 15.6 EGCG 68.5 ECG 1013 EGC 〉 2000 (+)-catechin 〉 2000 Oseltamivir 〉 2000 Amantadine 〉 2000

< Example  5> Investigation of the binding inhibitory activity between influenza virus cells of catechin derivative compounds against various influenza virus subtypes

Five kinds of human influenza virus solutions [A / Sydney / 5/97 (H3N2) (National Institute for medical Research, London, UK), A / X31 (H3N2) (Evans Biological Ltd., Liverpool, UK), A / PR / 8 (H1N1), A / Japan / 305/57 (H2N2), B / Lee / 40 (Oxford Univ. UK)] were prepared at the same concentration (500 HAU / 25 μl) and from 1 mM to 7.8 μM Two-fold serial dilutions of the catechin derivative compounds of the present invention were mixed in equal volumes. After 1 hour of reaction, chicken red blood cell solution (1%) is added to the reaction solution of virus and catechin derivative compound in equal volume. After confirming the inhibition of binding at each concentration of the catechin derivative compound of the present invention showed a minimum concentration showing the inhibitory effect for each influenza virus subtype (Fig. 2).

As a result, it was confirmed that four kinds of catechin derivative compounds (Nos. 6, 7, 15, and 16) effectively inhibit the binding between cells and human influenza virus and cellular receptors, although there are some differences between influenza subtypes. In particular, compound 6 showed high activity against virus binding at concentrations of 50 μM or less for all five subtypes tested. On the other hand, their precursors EGC and (+)-C did not show any inhibitory effect. Treatment with flu virus, oseltamivir and amantadine, did not inhibit the treatment with 800 μM.

< Example  6> On the rat  Acute toxicity test for oral administration

In order to determine the acute toxicity of the catechin derivative compound of the present invention was carried out the following experiment.

Acute toxicity experiments were performed using 6 week-old SPF SD rats. The catechin derivative compounds (Nos. 6, 7, 15, and 16) were each suspended orally in 0.5% methylcellulose solution and administered once orally at a dose of 500 mg / kg / 15 ml. After administration of the test substance, the mortality, clinical symptoms, and changes in body weight were examined, and hematological and hematological examinations were performed.

As a result, no significant clinical symptoms or closed animals were noted in all animals treated with the test substance, and no toxic changes were observed in weight changes, blood tests, blood biochemical tests, and autopsy findings. As a result, the catechin derivative compounds (Nos. 6, 7, 15, and 16) of the present invention did not show toxic changes up to 5 g / kg in rats and the minimum lethal dose (LD ₅₀ ) of parenteral administration was 5 g / kg or more as a safe substance. Judging.

Examples of preparations for the prevention and treatment of influenza virus infectious disease and health foods comprising the EGC of Formula 1 and / or the (+)-C derivative compound of Formula 2 of the present invention are illustrated.

< Formulation example  1> Preparation of Injection Solution

Injection solution containing 10 mg of the active ingredient was prepared by the following method.

1 g of the catechin derivative compound of the present invention, 0.6 g of sodium chloride, and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml. The solution was bottled and sterilized by heating at 20 ° C. for 30 minutes.

< Formulation example  2> Preparation of Syrup

A syrup containing the catechin derivative compound of the present invention as an active ingredient 2% (weight / volume) was prepared by the following method.

Acid addition salts, saccharin and sugar of the catechin derivative compound were dissolved in 80 g of warm water. After cooling the solution, it was prepared by mixing with a solution consisting of glycerin, saccharin, spices, ethanol, sorbic acid and distilled water. Water was added to this mixture to make 100 ml.

< Formulation example  3> Preparation of Tablet

A tablet containing 15 mg of active ingredient was prepared by the following method.

250 g of the catechin derivative compound of the present invention, 175.9 g of lactose, 180 g of potato starch, and 32 g of colloidal silicic acid were mixed. 10% gelatin solution was added to the mixture, which was then ground to pass 14 mesh bodies. It was dried and the mixture obtained by adding 160 g of potato starch, 50 g of talc, and 5 g of magnesium stearate was made into a tablet.

< Formulation example  4> Manufacture of food

Food containing the catechin derivative compound of the present invention was prepared as follows.

1. Preparation of Cooking Seasonings

Cooking seasoning was prepared by mixing 8 to 12 parts by weight of the catechin derivative compound in 100 parts by weight of the cooking seasoning for preventing and treating flu virus infectious diseases.

2. Preparation of soups and gravy

8 to 12 parts by weight of the catechin derivative compound of the present invention was added to 100 parts by weight of soup and juice to prepare meat products for preventing and treating flu virus infectious diseases, soups and noodles for noodles.

3. Manufacture of dairy products

8 to 12 parts by weight of the catechin derivative compound of the present invention was added to 100 parts by weight of milk, and various dairy products such as butter and ice cream were prepared using the milk.

4. Manufacture of wire

Brown rice, barley, glutinous rice, and yulmu were alphad by a known method, and then dried and roasted to prepare a powder having a particle size of 60 mesh.

Black beans, black sesame seeds, and perilla were also steamed and dried in a known manner, and then roasted to prepare a powder having a particle size of 60 mesh.

It was prepared by combining 8 to 12 parts by weight of the catechin derivative compound of the present invention in the following ratio.

Cereals (30 parts by weight brown rice, 15 parts by weight brittle, 20 parts by weight of barley),

Seeds (7 parts by weight perilla, 8 parts by weight black beans, 7 parts by weight black sesame seeds),

Catechin derivative compound (10 parts by weight),

Ganoderma lucidum (0.5 parts by weight), and

Sulfur (0.5 part by weight).

< Formulation example  5> Manufacture of beverage

1. Preparation of carbonated drinks

5 to 10 parts by weight of sugar, 0.05 to 0.3 parts by weight of citric acid, 0.005 to 0.02 parts by weight of caramel, 0.1 to 1 parts by weight of vitamin C, and 1 part by weight of the catechin derivative compound of the present invention are mixed with 79 to 94 parts by weight of purified water Mixing to make a syrup, the syrup was sterilized for 20 to 180 seconds at 95 ~ 98 ℃ mixed with cooling water in a ratio of 1: 4 and then injected into a carbon dioxide 0.5 to 0.82 parts by weight to prepare a carbonated beverage.

2. Manufacture of health drinks

0.5 parts by weight of liquid fructose, 2 parts by weight of oligosaccharide, 2 parts by weight of sugar, 0.5 parts by weight of salt, 75 parts by weight of water and 10 parts by weight of the catechin derivative compound of the present invention is homogeneously blended and then sterilized immediately by vial, Healthy drinks were prepared by packaging in small packaging containers such as plastic bottles.

3. Preparation of Vegetable Juice

10 g of the catechin derivative compound of the present invention was added to 1 L of tomato or carrot juice to prepare vegetable juice for health promotion.

4. Preparation of Fruit Juice

10 g of the catechin derivative compound of the present invention was added to 1 L of apple or grape juice to prepare a fruit juice for health promotion.

Flu virus infectious disease prevention and treatment agent comprising the catechin derivative compound of the present invention inhibits the growth of avian influenza virus, inhibits the binding between influenza virus and cells, and by inhibiting the binding between various flu virus subtypes and cells It can be usefully used as an agent for preventing and treating viral infectious diseases.

Claims (15)

An epigallocatechin derivative compound having the structure of Formula 1, or a pharmaceutically acceptable salt thereof, comprising: a flu virus infectious disease prevention and treatment agent. <Formula 1>

R of formula (1) is unsubstituted or, independently or optionally, C _1-15 linear or branched alkyl, C _3-15 cycloalkyl, C _1-15 haloalkyl, C _3-15 cyclohaloalkyl , any one is selected from aryloxy, the group consisting of halogen or amino of the C _{6 ~ 15} aryl group, C _{6 ~ 15} aryl, C _{6 ~ 15} alkylaryl, C _{1 ~ 15} alkoxy, C _{6 ~ 15} of the Flu virus infectious disease prevention and treatment agent selected from the group consisting of C _1-18 linear or branched alkyl groups substituted with the above substituents or cyclic aliphatic or aromatic hydrocarbons delete The compound of claim 1, wherein R is unsubstituted or, independently or optionally, C _1-10 straight or branched chain alkyl, C _3-10 cycloalkyl, C _1-10 haloalkyl, C _3-10 cyclo selected from haloalkyl, aryl, aryloxy, the group consisting of halogen or amino of the C _{6 ~ 10} alkylaryl, C _{1 ~ 10} alkoxy, C _{6 ~ 10} of the C _{6 ~ 10} aryl group, C _{6 ~ 10} of the Influenza virus infectious disease prevention and treatment agent selected from the group consisting of C _1-15 linear or branched alkyl group substituted by any one or more substituents or cyclic aliphatic or aromatic hydrocarbon. The agent for preventing and treating influenza virus infectious diseases according to claim 1, wherein R is selected from the group consisting of an unsubstituted C _1-12 straight or branched alkyl group, benzyl group substituted with halogen or C _1-15 alkoxy. . The agent for preventing and treating flu virus infectious diseases according to claim 1, wherein R is selected from the group consisting of a butyl group, heptyl group, decyl group, dodecyl group, difluorobenzyl group, trifluorobenzyl group, and dimethoxybenzyl group. . The method of claim 1, wherein the epigallocatechin derivative compound is at least one selected from the group consisting of: Flu virus infectious disease prevention and treatment agent: 3-O-3,4,5-trifluoro benzyl epigallocatechin (3-O- (3,4,5-trifluoro) benzyl epigllao-catechin) (1); 3-O-3,5-dimethoxy benzyl epigallocatechin (2) (O) (3-O- (3,5-dimethoxy) benzyl epigllaocatechin) (2); 3-O-3,5-difluoro benzyl epigallocatechin (3-O- (3,5-difluoro) benzyl epigllaocatechin) (3); 3-O-butyl epigllaocatechin (4); 3-O-heptyl epigllaocatechin (5); 3-O-decyl epigllaocatechin (6); And 3-O-dodecyl epigllao-catechin (7). A (+)-catechin derivative compound having the structure of Formula 2 or a pharmaceutically acceptable salt thereof, wherein the flu virus infectious disease prevention and treatment agent. <Formula 2>

R of formula (2) is unsubstituted or, independently or optionally, C _1-15 linear or branched alkyl, C _3-15 cycloalkyl, C _1-15 haloalkyl, C _3-15 cyclohaloalkyl , any one is selected from aryloxy, the group consisting of halogen or amino of the C _{6 ~ 15} aryl group, C _{6 ~ 15} aryl, C _{6 ~ 15} alkylaryl, C _{1 ~ 15} alkoxy, C _{6 ~ 15} of the Influenza virus infectious disease prevention and treatment agent selected from the group consisting of C _1-18 linear or branched alkyl groups substituted with the above substituents or cyclic aliphatic or aromatic hydrocarbons. The method of claim 7, wherein, R is unsubstituted or, independently or selectively C _{1 ~ 10} linear or branched alkyl, C _{3 to 10} cycloalkyl, cycloalkyl of C _{1 ~ 10} haloalkyl, C _3-10 of selected from haloalkyl, aryl, aryloxy, the group consisting of halogen or amino of the C _{6 ~ 10} alkylaryl, C _{1 ~ 10} alkoxy, C _{6 ~ 10} of the C _{6 ~ 10} aryl group, C _{6 ~ 10} of the prevention and treatment of influenza virus infections, wherein is selected from the group consisting of any one or more by a substituent of the substituted C _{1 ~ 15} linear or branched alkyl group or cyclic aliphatic or aromatic hydrocarbon. The method of claim 7, wherein, R is Beach straight chain of the unsubstituted C _{1 ~ 12,} or side-chain alkyl group, a halogen or influenza virus infectious diseases, preventive and therapeutic agent being selected from an alkoxy benzyl group the group consisting of substituted with a C _{1 ~ 15} . 8. The agent for preventing and treating influenza virus infectious diseases according to claim 7, wherein R is selected from the group consisting of a butik group, heptyl group, decyl group, dodecyl group, difluorobenzyl group, trifluorobenzyl group and dimethoxybenzyl group. . 8. The agent for preventing and treating influenza virus infectious diseases according to claim 7, wherein the (+)-catechin derivative compound is selected from the group consisting of: 3-O-3,4,5-trifluoro benzyl catechin (3-O- (3,4,5-trifluoro) benzyl catechin) (8); 3-O-3,5-difluoro benzyl catechin (9); 3-O-3,5-dimethoxy benzyl catechin (10); 3-O- (4-trifluoromethoxy) benzyl catechin (11); 3-O-benzyl catechin (12): 3-O-butyl catechin (13); 3-O-heptyl catechin (14); 3-O-decyl catechin (15); And 3-O-dodecyl catechin (16). The method of claim 1, wherein the flu virus is human flu virus or avian influenza virus, characterized in that the flu virus infectious disease prevention and treatment. The human influenza virus according to claim 12, wherein A / Sydney / 5/97 (H3N2), A / X31 (H3N2), A / PR / 8 (H1N1), A / Japan / 305/57 (H2N2), B / Lee / 40 flu virus infectious disease prevention and treatment. An agent for preventing and treating influenza virus infectious diseases comprising at least one of the derivative compounds of claims 1 or 7 and a pharmaceutically acceptable carrier. delete

Images