Phlebiarubrone derivative, and composition for preventing, alleviating or treating inflammatory diseases, comprising same as active ingredient

Abstract

The present invention relates to a phlebiarubrone derivative compound represented by structural formula 1. Therefore, the present invention prevents, alleviates or treats inflammatory diseases including atopic dermatitis through inhibition of IgE and IL-4 expression, inhibition of STAT6 phosphorylation, inhibition of degranulation of mast cells, and the like.

Description

Pleviarubron derivatives and compositions containing the same as active ingredients for preventing, improving or treating inflammatory diseases

[0001] The present invention relates to a novel pleviarubron derivative and a composition for preventing, improving or treating inflammatory diseases comprising the same as an active ingredient.

[0002] Allergic reactions can occur when a person develops IgE antibodies in response to a harmless allergen, and is subsequently exposed to the same allergen. These allergens activate IgE-binding mast cells in previously exposed tissues, triggering a cascade of allergic reactions. These allergic diseases can be divided into seasonal allergic diseases, which primarily occur in the spring, and chronic allergic diseases, which present symptoms year-round. If seasonal allergic diseases persist year after year, they can transition into chronic allergic diseases. Atopic dermatitis is a representative chronic allergic disease.

[0003] Atopic dermatitis is a chronic, relapsing inflammatory skin disease that typically begins in infancy or childhood, characterized by pruritus, dry skin, and characteristic eczema. When atopic dermatitis develops, various immunomodulatory mediators, such as IL-4, IL-5, IL-10, and IL-13, are excessively secreted by mast cells. While the exact mechanism of atopic dermatitis is not yet fully understood, it is believed to be caused by multiple factors, including impaired skin barrier function, immune system dysfunction, immunoglobulin E (IgE)-mediated hypersensitivity, and psychological, genetic, and environmental factors. Studies have shown that T cells, Langerhans cells, keratinocytes, eosinophils, and mast cells are key factors in atopic dermatitis, suggesting that an imbalance of TH1 and TH2 cytokines promotes IgE-mediated hypersensitivity, leading to atopic dermatitis.

[0004] Currently, topical medications such as steroids, calcineurin inhibitors, and T-cell suppressors are used to treat atopic dermatitis. However, long-term treatment can cause side effects such as numbness or burning, redness, fever, and swelling at the applied site, as well as suppression of pituitary and adrenal cortex function and glaucoma. For this reason, active research is being conducted to treat atopic dermatitis without side effects, utilizing the antioxidant and immune-modulating properties of natural products.

[0005] Meanwhile, p-terphenyl compounds have been reported to have various physiological activities, and have become the subject of much interest as synthetic targets for many organic synthesizers. Among them, phlebiarubrone was first reported in 1967 as a symmetrical terphenyl natural product with an o-benzoquinone skeleton and para-substituted phenyl groups on both sides, isolated from Punctularia strigozonata. It was later found to exist in the microorganism Punctularia atropurpurascens. However, little is known about the physiological activities of phlebiarubrone and its derivatives.

[0006] The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating inflammatory diseases including atopic dermatitis by suppressing IgE and IL-4 expression, suppressing STAT6 phosphorylation, and suppressing mast cell degranulation.

[0007] Another object of the present invention is to provide a food composition for preventing or improving inflammatory diseases including atopic dermatitis by suppressing IgE and IL-4 expression, suppressing STAT6 phosphorylation, and suppressing mast cell degranulation.

[0008] Another object of the present invention is to provide a cosmetic composition for preventing or improving inflammatory diseases including atopic dermatitis by suppressing IgE and IL-4 expression, suppressing STAT6 phosphorylation, and suppressing mast cell degranulation.

[0009] According to one aspect of the present invention,

[0010] A compound represented by the following structural formula 1 is provided.

[0011] [Structural formula 1]

[0012]

[0013] In structural formula 1,

[0014] R 1 Inland R 5 are each independently a hydrogen atom, , a substituted or unsubstituted C1 to C30 alkoxy group, a substituted or unsubstituted C1 to C30 alkyl group, a substituted or unsubstituted C3 to C30 cycloalkyl group, a substituted or unsubstituted C1 to C30 heterocycloalkyl group, a substituted or unsubstituted C6 to C30 aryl group, or a substituted or unsubstituted C1 to C30 heteroaryl group,

[0015] R 11 Inland R 13 are each independently a hydrogen atom, a halogen group, or a substituted or unsubstituted C1 to C30 alkyl group,

[0016] R 6 Inland R 10 are each independently a hydrogen atom, , a substituted or unsubstituted C1 to C30 alkoxy group, a substituted or unsubstituted C1 to C30 alkyl group, a substituted or unsubstituted C3 to C30 cycloalkyl group, a substituted or unsubstituted C1 to C30 heterocycloalkyl group, a substituted or unsubstituted C6 to C30 aryl group, or a substituted or unsubstituted C1 to C30 heteroaryl group,

[0017] R 14Inland R 16 are each independently a hydrogen atom, a halogen group, or a substituted or unsubstituted C1 to C30 alkyl group.

[0018] Preferably, in the above structural formula 1,

[0019] R 1 Inland R 5 are each independently a hydrogen atom, , a C1 to C10 alkoxy group, or a C1 to C10 alkyl group,

[0020] R 6 Inland R 10 are each independently a hydrogen atom, , may be a C1 to C10 alkoxy group, or a C1 to C10 alkyl group.

[0021] More preferably,

[0022] The compound represented by the above structural formula 1 can be represented by any one selected from the following structural formulas 2 to 4.

[0023] [Structural formula 2]

[0024]

[0025]

[0026] In structural formula 2,

[0027] R 1 and R 10 are each independently a C1 to C4 alkyl group.

[0028] [Structural formula 3]

[0029]

[0030]

[0031] In structural formula 3,

[0032] R 2 , R 4 , R 7 and R 9 are each independently a C1 to C4 alkyl group,

[0033] R 3 and R8 are each independently a C1 to C4 alkoxy group.

[0034] [Structural formula 4]

[0035]

[0036]

[0037] In structural formula 4,

[0038] R 3 and R 8 are each independently , or am.

[0039] Even more preferably,

[0040] The compound represented by the above structural formula 1 may be a compound represented by any one of the following chemical formulas 1 to 4.

[0041] [Chemical Formula 1]

[0042]

[0043]

[0044] [Chemical Formula 2]

[0045]

[0046]

[0047] [Chemical Formula 3]

[0048]

[0049]

[0050] [Chemical Formula 4]

[0051]

[0052]

[0053] According to another aspect of the present invention,

[0054] (a) a step of preparing compound H by reacting compound D and 1,2-dibromotetrachloroethane;

[0055] [Compound D]

[0056]

[0057]

[0058] [Compound H]

[0059]

[0060]

[0061] (b) a step of reacting the compound H with 4-trifluoromethylphenylboronic acid or 4-t-butylphenylboronic acid to produce a compound represented by the following structural formula 4-1; and

[0062] [Structural formula 4-1]

[0063]

[0064]

[0065] (c) A method for producing a compound represented by the following structural formula 4 is provided, including a step of reacting a compound represented by the above structural formula 4-1 with p-toluenesulfonic acid hydrate (p-TsOH·H20) and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ).

[0066] [Structural formula 4]

[0067]

[0068]

[0069] In structural formula 4,

[0070] R 3 and R 8 are each independently , or am.

[0071] According to another aspect of the present invention,

[0072] A pharmaceutical composition for preventing or treating an inflammatory disease is provided, which comprises a compound represented by any one of the following chemical formulas 1 to 4, or a pharmaceutically acceptable salt thereof, as an active ingredient.

[0073] [Chemical Formula 1]

[0074]

[0075]

[0076] [Chemical Formula 2]

[0077]

[0078]

[0079] [Chemical Formula 3]

[0080]

[0081]

[0082] [Chemical Formula 4]

[0083]

[0084]

[0085] The above inflammatory disease may be any one selected from atopic dermatitis, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, iritis, pharyngitis, tonsillitis, pneumonia, gastric ulcer, pancreatitis, gastritis, Crohn's disease, inflammatory bowel disease, colitis, hemorrhoids, gout, ankylosing spondylitis, lupus, fibromyalgia, psoriasis, and rheumatoid arthritis.

[0086] The above pharmaceutical composition may be used for suppressing the expression of at least one selected from IgE and IL-4.

[0087] The above pharmaceutical composition may be used for inhibiting STAT6 phosphorylation.

[0088] The above pharmaceutical composition may be used to inhibit degranulation of mast cells.

[0089] According to another aspect of the present invention,

[0090] A food composition for preventing or improving an inflammatory disease is provided, which comprises as an active ingredient a compound represented by any one of the following chemical formulas 1 to 4, or a food-wise acceptable salt thereof.

[0091] [Chemical Formula 1]

[0092]

[0093]

[0094] [Chemical Formula 2]

[0095]

[0096]

[0097] [Chemical Formula 3]

[0098]

[0099]

[0100] [Chemical Formula 4]

[0101]

[0102]

[0103] According to another aspect of the present invention,

[0104] A cosmetic composition for preventing or improving an inflammatory disease is provided, which comprises as an active ingredient a compound represented by any one of the following chemical formulas 1 to 4, or a cosmetically acceptable salt thereof.

[0105] [Chemical Formula 1]

[0106]

[0107]

[0108] [Chemical Formula 2]

[0109]

[0110]

[0111] [Chemical Formula 3]

[0112]

[0113]

[0114] [Chemical Formula 4]

[0115]

[0116]

[0117] The composition containing the phlebiarubrone derivative of the present invention as an active ingredient has an effect of preventing, improving or treating inflammatory diseases including atopic dermatitis by suppressing IgE and IL-4 expression, suppressing STAT6 phosphorylation, and suppressing mast cell degranulation.

[0118] Figure 1 shows the results of measuring the effect of inhibiting IL-4 mRNA expression.

[0119] Figure 2 shows the results of cytotoxicity evaluation.

[0120] Figure 3 shows the results of the analysis of the degranulation inhibition effect.

[0121] Figure 4 shows the results of the analysis of the STAT6 phosphorylation inhibition effect.

[0122] Figure 5 is a schedule for animal testing.

[0123] Figure 6 shows the results of H&E staining of the mouse dorsal tissue.

[0124] Figure 7 shows the results of measuring the number of mast cells in H&E staining.

[0125] Figure 8 shows the results of measuring the protein amounts of IgE and IL-4.

[0126] The present invention is susceptible to various modifications and embodiments. Specific embodiments are illustrated in the drawings and described in detail in the detailed description. However, this is not intended to limit the present invention to specific embodiments, but rather to encompass all modifications, equivalents, and alternatives falling within the spirit and technical scope of the present invention. In describing the present invention, detailed descriptions of related known technologies will be omitted if they are deemed to obscure the gist of the present invention.

[0127] The terminology used in this application is only used to describe specific embodiments and is not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly indicates otherwise. In this application, the terms "comprise" or "have" are intended to indicate the presence of a feature, number, step, component, or combination thereof described in the specification, but should be understood to not preclude the possibility of the presence or addition of one or more other features, numbers, steps, components, or combinations thereof.

[0128] In the present invention, "substituted" means at least one hydrogen atom is substituted with deuterium, a C1 to C30 alkyl group, a C3 to C30 cycloalkyl group, a C2 to C30 heterocycloalkyl group, a C1 to C30 halogenated alkyl group, a C6 to C30 aryl group, a C1 to C30 heteroaryl group, a C1 to C30 alkoxy group, a C3 to C30 cycloalkoxy group, a C1 to C30 heterocycloalkoxy group, a C2 to C30 alkenyl group, a C2 to C30 alkynyl group, a C6 to C30 aryloxy group, a C1 to C30 heteroaryloxy group, a silyloxy group (-OSiH3), -OSiR 1 H2 (R1 is a C1 to C30 alkyl group or a C6 to C30 aryl group), -OSiR 1 R 2 H(R 1 and R 2 are each independently a C1 to C30 alkyl group or a C6 to C30 aryl group), -OSiR 1 R 2 R 3 , (R 1 , R 2 , and R 3are each independently a C1 to C30 alkyl group or a C6 to C30 aryl group), a C1 to C30 acyl group, a C2 to C30 acyloxy group, a C2 to C30 heteroaryloxy group, a C1 to C30 sulfonyl group, a C1 to C30 alkylthiol group, a C3 to C30 cycloalkylthiol group, a C1 to C30 heterocycloalkylthiol group, a C6 to C30 arylthiol group, a C1 to C30 heteroarylthiol group, a C1 to C30 phosphoric acid amide group, a silyl group (SiR 1 R 2 R 3 )( R 1 , R 2 , and R 3 is independently a hydrogen atom, a C1 to C30 alkyl group, or a C6 to C30 aryl group), an amine group (-NRR') (wherein, R and R' are each independently a substituent selected from the group consisting of a hydrogen atom, a C1 to C30 alkyl group, and a C6 to C30 aryl group), a carboxyl group, a halogen group, a cyano group, a nitro group, an azo group, and a hydroxy group.

[0129] Additionally, two adjacent substituents among the above substituents may be fused to form a saturated or unsaturated ring.

[0130] In addition, the carbon number range of the alkyl group or aryl group in the above "substituted or unsubstituted C1 to C30 alkyl group" or "substituted or unsubstituted C6 to C30 aryl group" means the total number of carbon atoms constituting the alkyl portion or aryl portion when the substituent is viewed as unsubstituted without considering the substituted portion. For example, a phenyl group substituted with a butyl group at the para position means an aryl group having 6 carbon atoms substituted with a butyl group having 4 carbon atoms.

[0131] In this specification, unless otherwise defined, "hetero" means containing 1 to 4 heteroatoms selected from the group consisting of N, O, S and P in one functional group, and the remainder being carbon.

[0132] In this specification, “hydrogen” means singletium, deuterium, or tritium, unless otherwise defined.

[0133] In this specification, “alkyl group” means an aliphatic hydrocarbon group unless otherwise defined.

[0134] The alkyl group may be a "saturated alkyl group" that does not contain any double or triple bonds.

[0135] The alkyl group may be an "unsaturated alkyl group" containing at least one double bond or triple bond.

[0136] Alkyl groups, whether saturated or unsaturated, may be branched, straight-chain or cyclic.

[0137] The alkyl group may be a C1 to C30 alkyl group. More specifically, it may be a C1 to C20 alkyl group, a C1 to C10 alkyl group, or a C1 to C6 alkyl group.

[0138] For example, a C1 to C4 alkyl group indicates that the alkyl chain has 1 to 4 carbon atoms, i.e., the alkyl chain is selected from the group consisting of methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl and t-butyl.

[0139] For example, the alkyl group refers to a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a t-butyl group, a pentyl group, a hexyl group, an ethenyl group, a propenyl group, a butenyl group, a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, and the like.

[0140] A "cycloalkyl group" includes a monocyclic or fused ring polycyclic (i.e., a ring that shares adjacent pairs of carbon atoms) functional groups.

[0141] A "heterocycloalkyl group" means a cycloalkyl group containing 1 to 4 heteroatoms selected from the group consisting of N, O, S, and P, and the remainder being carbon. When the heterocycloalkyl group is a fused ring, at least one ring among the fused rings may contain 1 to 4 of the heteroatoms.

[0142] "Aryl group" includes monocyclic or fused ring polycyclic (i.e., rings that share adjacent pairs of carbon atoms) functional groups.

[0143] "Heteroaryl group" means an aryl group containing 1 to 4 heteroatoms selected from the group consisting of N, O, S, and P, and the remainder being carbon. When the heteroaryl group is a fused ring, at least one ring among the fused rings may contain 1 to 4 of the heteroatoms.

[0144] In aryl and heteroaryl groups, the number of ring atoms is the sum of the number of carbon atoms and the number of non-carbon atoms.

[0145]

[0146] Hereinafter, the novel compound phlebiarubrone derivative of the present invention will be described.

[0147] The Phlebiarubrone derivative of the present invention can be represented by the following structural formula 1.

[0148] [Structural formula 1]

[0149]

[0150]

[0151] In structural formula 1,

[0152] R 1 Inland R 5 are each independently a hydrogen atom, , a substituted or unsubstituted C1 to C30 alkoxy group, a substituted or unsubstituted C1 to C30 alkyl group, a substituted or unsubstituted C3 to C30 cycloalkyl group, a substituted or unsubstituted C1 to C30 heterocycloalkyl group, a substituted or unsubstituted C6 to C30 aryl group, or a substituted or unsubstituted C1 to C30 heteroaryl group,

[0153] R 11 Inland R 13 are each independently a hydrogen atom, a halogen group, or a substituted or unsubstituted C1 to C30 alkyl group,

[0154] R 6 Inland R 10 are each independently a hydrogen atom, , a substituted or unsubstituted C1 to C30 alkoxy group, a substituted or unsubstituted C1 to C30 alkyl group, a substituted or unsubstituted C3 to C30 cycloalkyl group, a substituted or unsubstituted C1 to C30 heterocycloalkyl group, a substituted or unsubstituted C6 to C30 aryl group, or a substituted or unsubstituted C1 to C30 heteroaryl group,

[0155] R 14 Inland R 16 are each independently a hydrogen atom, a halogen group, or a substituted or unsubstituted C1 to C30 alkyl group.

[0156] Preferably, in the above structural formula 1,

[0157] R 1 Inland R 5 are each independently a hydrogen atom, , a C1 to C10 alkoxy group, or a C1 to C10 alkyl group,

[0158] R 6 Inland R 10 are each independently a hydrogen atom, , may be a C1 to C10 alkoxy group, or a C1 to C10 alkyl group.

[0159] More preferably, the compound represented by the structural formula 1 may be represented by any one selected from structural formulas 2 to 4.

[0160] [Structural formula 2]

[0161]

[0162]

[0163] In structural formula 2,

[0164] R 1 and R 10 are each independently a C1 to C4 alkyl group.

[0165] [Structural formula 3]

[0166]

[0167]

[0168] In structural formula 3,

[0169] R 2 , R 4 , R 7 and R 9 are each independently a C1 to C4 alkyl group,

[0170] R 3 and R 8 are each independently a C1 to C4 alkoxy group.

[0171] [Structural formula 4]

[0172]

[0173]

[0174] In structural formula 4,

[0175] R 3 and R 8 are each independently , or am.

[0176] Even more preferably, the compound represented by the structural formula 1 may be a compound represented by any one of the following chemical formulas 1 to 4.

[0177] [Chemical Formula 1]

[0178]

[0179]

[0180] [Chemical Formula 2]

[0181]

[0182]

[0183] [Chemical Formula 3]

[0184]

[0185]

[0186] [Chemical Formula 4]

[0187]

[0188]

[0189] In addition, the present invention provides a method for producing a compound represented by structural formula 4 according to the following sequence. This method is highly suitable for mass production.

[0190] First, compound H is prepared by reacting compound D and 1,2-dibromotetrachloroethane (step a).

[0191] [Compound D]

[0192]

[0193]

[0194] [Compound H]

[0195]

[0196]

[0197] Next, the compound H is reacted with 4-trifluoromethylphenylboronic acid or 4-t-butylphenylboronic acid to produce a compound represented by the following structural formula 4-1 (step b).

[0198] [Structural formula 4-1]

[0199]

[0200]

[0201] Thereafter, the compound represented by the above structural formula 4-1 was reacted with p-toluenesulfonic acid hydrate (p-TsOH·H20) and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) to prepare a compound represented by the following structural formula 4 (step c).

[0202] Preferably, in this step, after the completion of the reaction, extraction, drying, concentration, and filtering are sequentially performed. Thereafter, it is preferable to obtain a compound represented by the following structural formula 4 through low-temperature crystallization without concentration.

[0203] [Structural formula 4]

[0204]

[0205]

[0206] In structural formula 4,

[0207] R 3 and R 8 are each independently , or am.

[0208]

[0209] When the above compound has isomers such as optical isomers, stereoisomers, positional isomers, rotational isomers, etc., any of the isomers and mixtures thereof are included in the compound. These isomers can be obtained as individual products according to synthetic and separation methods known in themselves (concentration, solvent extraction, column chromatography, recrystallization, etc.).

[0210]

[0211] Hereinafter, the pharmaceutical composition for preventing or treating inflammatory diseases of the present invention will be described.

[0212] The pharmaceutical composition for preventing or treating an inflammatory disease of the present invention comprises a compound represented by any one of the following chemical formulas 1 to 4, or a pharmaceutically acceptable salt thereof, as an active ingredient.

[0213] [Chemical Formula 1]

[0214]

[0215]

[0216] [Chemical Formula 2]

[0217]

[0218]

[0219] [Chemical Formula 3]

[0220]

[0221]

[0222] [Chemical Formula 4]

[0223]

[0224] The inflammatory disease may be any one selected from atopic dermatitis, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, iritis, pharyngitis, tonsillitis, pneumonia, gastric ulcer, pancreatitis, gastritis, Crohn's disease, inflammatory bowel disease, colitis, hemorrhoids, gout, ankylosing spondylitis, lupus, fibromyalgia, psoriasis, and rheumatoid arthritis, but the scope of the present invention is not limited thereto. Preferably, the inflammatory disease may be atopic dermatitis.

[0225]

[0226] The pharmaceutical composition of the present invention can be prepared using, in addition to the above-mentioned effective ingredient, a pharmaceutically suitable and physiologically acceptable auxiliary agent, and the auxiliary agent may be an excipient, a disintegrant, a sweetener, a binder, a coating agent, a swelling agent, a lubricant, a glidant, or a flavoring agent.

[0227] The above pharmaceutical composition may be preferably formulated as a pharmaceutical composition by additionally including one or more pharmaceutically acceptable carriers in addition to the above-described effective ingredients for administration.

[0228] The pharmaceutical composition may be in the form of a solution, syrup, juice, suspension, emulsion, drop, or the like. For example, if necessary, suitable binders, lubricants, disintegrants, and coloring agents may also be included in the mixture. Suitable binders include, but are not limited to, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tracheacanth, or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like. The pharmaceutical composition of the present invention is preferably administered transdermally.

[0229]

[0230] Meanwhile, the term "comprising as an active ingredient" in this specification means including a sufficient amount to achieve the efficacy or activity of a phlebiarubrone derivative. For example, the phlebiarubrone derivative may be used at a concentration of 10 to 1500 μg / ml, preferably 100 to 1000 μg / ml. The quantitative upper limit of the phlebiarubrone derivative can be selected and implemented by a person skilled in the art within an appropriate range.

[0231]

[0232] The present invention provides a cosmetic composition for preventing or improving atopic dermatitis, comprising the above-mentioned Phlebiarubrone derivative or its cosmetically acceptable salt as an active ingredient.

[0233] The components included in the cosmetic composition of the present invention include, in addition to the effective ingredients, components commonly used in cosmetic compositions, such as antioxidants, stabilizers, solubilizers, vitamins, fragrances, and conventional adjuvants, and carriers. The cosmetic composition of the present invention may be manufactured into any formulation commonly manufactured in the art, and may be formulated into, for example, a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, a surfactant-containing cleansing, an oil, a powder foundation, an emulsion foundation, a wax foundation, and the like, but is not limited thereto. More specifically, the cosmetic composition may be manufactured into formulations such as a facial cleansing, lotion, cream, massage cream, eye cream, essence, gel, pack, spray, powder, sunscreen, and the like.

[0234] When the formulation of the present invention is a paste, cream, or gel, vegetable oil, wax, starch, cellulose, silicone, bentonite, silica, zinc oxide, or the like can be used as a carrier component, and when the formulation is a powder, powders such as lactose, silica, aluminum hydroxide, and calcium silicate can be used as a carrier component. When the formulation of the present invention is a solution or emulsion, a solvent, a solubilizer, or an emulsifier is used as a carrier component, and examples thereof include water, ethanol, glycerin, ethyl carbonate, ethyl acetate, propylene glycol, butylene glycol, 1,2-hexanediol, glycerol aliphatic ester, and sorbitan fatty acid ester.

[0235] When the formulation of the present invention is a suspension, liquid diluents such as water, ethanol, and propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, and the like can be used as carrier components. In addition, when the formulation is a surfactant-containing cleansing agent, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, methyl taurate, sarcosinate, alkylamidobetaine, aliphatic alcohol, fatty acid glyceride, vegetable oil, or ethoxylated glycerol fatty acid ester, and the like can be used as carrier components.

[0236]

[0237] The present invention provides a food composition for preventing or improving inflammatory diseases, comprising the above phlebiarubrone derivative and a food-wise acceptable salt thereof as an active ingredient.

[0238] The food composition according to the present invention can be formulated in the same manner as the pharmaceutical composition described below and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, alcoholic beverages, confectionery, diet bars, dairy products, meat, chocolate, pizza, ramen, other noodles, gum, ice cream, vitamin complexes, health supplements, and the like.

[0239] The food composition of the present invention may include not only the effective ingredient of the present invention but also components commonly added during food manufacturing, such as proteins, carbohydrates, fats, nutrients, seasonings, and flavoring agents. Examples of the carbohydrates described above include monosaccharides such as glucose, fructose, etc.; disaccharides such as maltose, sucrose, oligosaccharides, etc.; and polysaccharides such as dextrin, cyclodextrin, etc., and conventional sugars and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents, natural flavoring agents [thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.]) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is manufactured into a drink or beverage, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, and various plant extracts may be additionally included in addition to the effective ingredient of the present invention.

[0240]

[0241] The present invention provides a health functional food comprising a food composition for preventing or improving inflammatory diseases, which comprises a phlebiarubrone derivative and a food-scientifically acceptable salt thereof as an active ingredient. A health functional food refers to a food prepared by adding a phlebiarubrone derivative to food materials such as beverages, teas, spices, gums, and confectionery, or by manufacturing the same in the form of capsules, powders, suspensions, etc., and which, when consumed, brings about a specific health effect. However, unlike general drugs, it has the advantage of not having side effects that may occur with long-term use of drugs made from food. The health functional food of the present invention obtained in this way is very useful because it can be consumed on a daily basis. The amount of phlebiarubrone derivatives to be added to such health functional foods varies depending on the type of the target health functional food and cannot be uniformly regulated, but may be added within a range that does not damage the original taste of the food, and is usually in the range of 0.01 to 50 wt%, preferably 0.1 to 20 wt%, with respect to the target food. In addition, in the case of health functional foods in the form of pills, granules, tablets or capsules, it may be added in the range of usually 0.1 to 100 wt%, preferably 0.5 to 80 wt%. In one specific example, the health functional food of the present invention may be in the form of pills, tablets, capsules or beverages.

[0242]

[0243] As used herein, the terms "pharmaceutically acceptable salt", "foodstuff acceptable salt", and "cosmetic acceptable salt" mean, unless otherwise defined, a formulation of a compound that does not cause serious irritation to an organism to which the compound is administered and does not impair the biological activity and physical properties of the compound. The pharmaceutically acceptable salt includes acid addition salts formed by acids that form non-toxic acid addition salts containing pharmaceutically acceptable anions, for example, inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, and hydroiodic acid; organic carboxylic acids such as tartaric acid, formic acid, citric acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, and salicylic acid; and sulfonic acids such as methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid. For example, pharmaceutically acceptable carboxylic acid salts include metal salts or alkaline earth metal salts formed by lithium, sodium, potassium, calcium, magnesium, etc.; amino acid salts such as lysine, arginine, guanidine, etc.; organic salts such as dicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine, diethanolamine, choline, and triethylamine, etc. The compound of structural formula 1, 2 or 3 according to the present invention may also be converted into its salt by a conventional method.

[0244]

[0245] The present invention also provides the use of a Phlebiarubrone derivative for the manufacture of a medicine or food for preventing, improving or treating an inflammatory disease.

[0246] Additionally, the present invention provides a method for improving, preventing, or treating an inflammatory disease, comprising administering an effective amount of a Phlebiarubrone derivative to a subject.

[0247] The term "subject" as used herein refers to a mammal, preferably a human, that is the subject of treatment, observation or experiment.

[0248] The term "effective amount" as used herein means the amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue, animal, or human, as conceived by a researcher, veterinarian, physician, or other clinician, including an amount that induces alleviation of the symptoms of the disease or disorder. The effective amount and frequency of administration of the active ingredient of the present invention may vary depending on the desired effect. Therefore, the optimal dosage to be administered can be readily determined by one skilled in the art, and may be adjusted according to various factors, including the type of disease, the severity of the disease, the content of the active ingredient and other ingredients contained in the composition, the type of formulation, and the patient's age, weight, general health, sex, and diet, the time of administration, the route of administration, and the excretion rate of the composition, the treatment period, and concurrently used drugs. In the method for preventing, treating, or improving a disease of the present invention, in the case of adults, it is preferable to administer the composition of the present invention once or several times a day at a dosage of 0.001 g / kg to 10 g / kg.

[0249] In the treatment method of the present invention, a composition containing a Phlebiarubrone derivative as an active ingredient can be administered in a conventional manner via oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, or intradermal routes.

[0250] Hereinafter, preferred examples are presented to help understand the present invention, but the following examples are only illustrative of the present invention, and it is obvious to those skilled in the art that various changes and modifications are possible within the scope and technical idea of ​​the present invention, and it is also natural that such changes and modifications fall within the scope of the appended patent claims.

[0251]

[0252] [Example: Preparation of Phlebiarubrone and its derivatives]

[0253] Manufacturing Example 1: Compound B

[0254] After dissolving 4.6 g (33.8 mmol) of KH2PO4 in 109 ml (0.2 M) of water, 12.8 g (47.7 mmol) of K2NO7S2 was added at 0 °C. Then, 3 g (21.7 mmol) of sesamol dissolved in 6 ml (3.8 M) of methanol was slowly added to the solution within 20 minutes. After 90 minutes, the precipitate formed at room temperature was filtered, mixed with 60 ml of water, and extracted three times with CH2Cl2. The obtained organic layer was dried over anhydrous Na2SO4, filtered, and concentrated to obtain 3.3 g of compound B. The mixture was used as is in the next reaction without purification by silica gel column chromatography.

[0255] [Compound B]

[0256]

[0257]

[0258] Manufacturing Example 2: Compound C

[0259] Compound B (3.3 g, 21.7 mmol) and Na2S2O4 (10.2 g, 4.89 mmol) were dissolved in MeCN (217 mL, 0.1 M) and water (72 mL, 0.3 M) and stirred at room temperature for 30 minutes. After 30 minutes, 100 mL of 1 NHCl aqueous solution was added to terminate the reaction, and the mixture was extracted three times with ethyl acetate (EtOAc). The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The mixture was dissolved in CH2Cl2, filtered, and concentrated again to obtain 2.17 g of Compound C. The mixture was used as is in the next reaction without purification by silica gel column chromatography.

[0260] [Compound C]

[0261]

[0262]

[0263] Manufacturing Example 3: Compound D

[0264] At 0℃, 2.17 g (14.1 mmol) of compound C was dissolved in 70 mL (0.2 M) of THF. 1.9 g (47.5 mmol) of 60% NaH was slowly added, and the mixture was stirred rapidly for 30 minutes. 3.2 mL (19.8 mmol) of methoxymethyl bromide dissolved in 10 mL of THF was slowly added over 10 minutes, and the mixture was stirred at room temperature overnight. The reaction was quenched with brine, extracted with CH2Cl2, and the organic layer was washed with 5% NaOH aqueous solution, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The mixture was purified by silica gel column chromatography (Hexane:EtOAc = 20:1) to obtain 2.1 g (8.72 mmol, 36%) of compound D.

[0265] 1 H NMR (500 MHz, CDCl3) δ 6.75 (s, 2H), 5.87 (s, 2H), 5.08 (s, 4H), 3.45 (s, 6H).

[0266] 13 C NMR (125 MHz, CDCl3) δ 142.38, 101.22, 100.74, 96.67, 56.09.

[0267] [Compound D]

[0268]

[0269]

[0270] Manufacturing Example 4: Compound E

[0271] Compound D (1.01 g, 4.17 mmol) was dissolved in THF (17 mL, 0.25 M), and 2.5 Mn-BuLi (5 mL, 12.5 mmol) was added at 0 °C, followed by stirring at the same temperature for 1 hour and 50 minutes. After 1 hour and 50 minutes, B(i-PrO)3 ​​(2.9 mL, 12.6 mmol) was slowly added, and the mixture was stirred for 50 minutes. Afterwards, 1.4 mL, 25.0 mmol) of acetic acid (AcOH) and 2.5 mL, 25.0 mmol) of H2O2 were added, and the mixture was stirred at room temperature for 16 hours. The reaction was quenched by adding (NH4)2SO4 aqueous solution and Na2SO3 aqueous solution, and extracted three times with ethyl acetate (EtOAc). The organic layer was washed with water and brine solution, dried, and concentrated. The mixture was purified by silica gel column chromatography (Hexane:EtOAc = 50:1) to obtain 578 mg (2.11 mmol, 53%) of compound E.

[0272] 1 H NMR (500 MHz, acetone-d6) δ 7.59 (s, 2H), 5.86 (s, 2H), 5.05 (s, 4H), 3.54 (s, 6H).

[0273] 13 C NMR (125 MHz, acetone-d6) δ 135.5, 133.1, 128.8, 102.1, 99.9, 57.4.

[0274] [Compound E]

[0275]

[0276]

[0277] Manufacturing Example 5: Compound F

[0278] Compound E (417 mg, 1.52 mmol) was dissolved in CH2Cl2 (30 mL, 0.05 M), and pyridine (0.37 mL, 4.1 M) was added at 0 °C. Trifluoromethanesulfonic anhydride (0.64 mL, 3.80 mmol) was slowly added little by little, and the mixture was stirred at room temperature for 2 hours. 1 N HCl aqueous solution was added to stop the reaction, and the mixture was extracted three times with ethyl acetate (EtOAc). The extracted organic layer was washed with NaHCO3 aqueous solution, then distilled water and brine solution in that order, dried, and concentrated. The mixture was purified by silica gel column chromatography (Hexane:EtOAc = 20:1) to obtain 0.79 g (1.46 mmol, 96%) of compound F.

[0279] 1 H NMR (500 MHz, CDCl3) δ 6.14 (s, 2H), 5.14 (s, 2H), 3.60 (s, 6H).

[0280] 13 C NMR (125 MHz, CDCl3) δ 138.3, 137.6, 126.4, 118.5.

[0281] [Compound F]

[0282]

[0283]

[0284] Manufacturing Example 6: Compound G-1

[0285] A mixture of 40 mg (0.074 mmol) of compound F and 36 mg (0.29 mmol) of phenylboronic acid was added 1.9 mL (0.04 M) of n-propanol and stirred for 30 minutes until dissolved. After that, 21 mg (3.7 x 10) of Pd(OAc) -3mmol), PPh 32.5 mg (0.011 mmol), 2M Na2CO 30.11 mL (0.22 mmol), and distilled water 0.37 mL (0.2 M) were added and stirred at 100 ℃ for 4 hours. After adding water and stirring for 1 hour, the mixture was extracted three times with ethyl acetate (EtOAc), and the organic layer was washed with water and brine solution, dried, and concentrated. The mixture was purified by silica gel column chromatography (Hexane:EtOAc = 20:1) to obtain 24.1 mg (0.061 mmol, 83%) of compound G-1.

[0286] 1 H NMR (500 MHz, CDCl3) δ 7.23 (d, 4H,J= 1.5 Hz), 7.45 (d, 4H,J= 1.0 Hz), 7.35 (d, 2H,J= 1.5 Hz), 5.95 (s, 2H), 4.88 (s, 4H), 2.99 (s, 6H).

[0287] 13 C NMR (125 MHz, CDCl3) δ 142.2, 141.4, 132.6, 130.5, 128.3, 127.8, 118.6, 101.3, 99.6, 57.2.

[0288] [Compound G-1]

[0289]

[0290]

[0291] Manufacturing Example 7: Compound G-2

[0292] A mixture of 40 mg (0.074 mmol) of compound F and 36 mg (0.29 mmol) of 4-methoxyphenylboronic acid was added 1.9 mL (0.04 M) of n-propanol and stirred for 30 minutes until dissolved. After that, 21 mg (3.7 x 10) of Pd(OAc) -3mmol), PPh32.5 mg (0.011 mmol), 2M Na2CO30.11 ml (0.22 mmol), and distilled water 0.37 ml (0.2 M) were added and stirred at 100 ℃ for 4 hours. After adding distilled water and stirring for 1 hour, the mixture was extracted three times with ethyl acetate (EtOAc), and the organic layer was washed with brine solution, dried, and concentrated. The mixture was purified by silica gel column chromatography (Hexane:EtOAc = 10:1) to obtain 28.1 mg (0.062 mmol, 82%) of compound G-2.

[0293] 1 H NMR (500 MHz, CDCl3) δ 7.56 (d, 4H,J= 1.0 Hz), 6.99 (d, 4H,J= 1.5 Hz), 5.93 (s, 2H), 4.88 (s, 4H), 3.86 (s, 6H), 3.07 (s, 6H).

[0294] 13 C NMR (125 MHz, CDCl3) δ 159.2, 142.2, 141.3, 131.7, 124.9, 116.2, 113.8, 101.3, 99.4, 55.4.

[0295] [Compound G-2]

[0296]

[0297]

[0298] Manufacturing Example 8: Compound G-3

[0299] A mixture of 40 mg (0.074 mmol) of compound F and 30.4 mg (0.22 mmol) of 2-methoxyphenylboronic acid was added 1.9 mL (0.04 M) of n-propanol and stirred for 30 minutes until dissolved. Afterwards, 21 mg (3.7 x 10) of Pd(OAc) -3mmol), PPh32.9 mg (0.011 mmol), 2M Na2CO30.11 ml (0.22 mmol), and distilled water 0.37 ml (0.2 M) were added and stirred at 100 ℃ for 4 hours. After adding distilled water and stirring for 1 hour, the mixture was extracted three times with ethyl acetate (EtOAc), and the organic layer was washed with brine solution, dried, and concentrated. The mixture was purified by silica gel column chromatography (Hexane:EtOAc = 10:1) to obtain 20.2 mg (0.048 mmol, 64%) of compound G-3.

[0300] 1 H NMR (500 MHz, CDCl3) δ 7.75 (d, 4H,J= 5.0 Hz, 7.71 (d, 4H,J= 10.0 Hz), 5.98 (s, 2H), 4.89 (s, 4H), 2.99 (s, 6H).

[0301] [Compound G-3]

[0302]

[0303]

[0304] Manufacturing Example 9: Compound G-4

[0305] A mixture of 42.5 mg (0.079 mmol) of compound F and 43 mg (0.29 mmol) of 3-tolueneboronic acid was added 2.0 mL (0.04 M) of n-propanol and stirred for 30 minutes until dissolved. After that, 21.1 mg (3.7 x 10) of Pd(OAc) -3mmol), PPh 32.7 mg (0.011 mmol), 2M Na2CO 3 0.119 mL (0.22 mmol), and distilled water 0.39 mL (0.2 M) were added and stirred at 100 ℃ for 4 hours. After adding distilled water and stirring for 1 hour, the mixture was extracted three times with ethyl acetate (EtOAc), and the organic layer was washed with brine solution, dried, and concentrated. The mixture was purified by silica gel column chromatography (Hexane:EA = 20:1) to obtain 20.7 mg (0.049 mmol, 63%) of compound G-4.

[0306] 1 H NMR (500 MHz, CDCl3) δ 7.41 (d, 4H,J= 1.5 Hz), 7.34 (t, 2H,J= 1.5 Hz), 7.17 (d, 2H,J= 1.0 Hz), 5.93 (s, 2H), 4.87 (s, 4H), 3.02 (s, 6H), 2.42 (s, 6H).

[0307] 13 C NMR (125 MHz, CDCl3) δ 144.7, 143.9, 140.3, 134.9, 133.6, 131.1, 130.0, 121.1, 103.8, 102.0, 59.7.

[0308] [Compound G-4]

[0309]

[0310]

[0311] Manufacturing Example 10: Compound G-5

[0312] A mixture of 40 mg (0.074 mmol) of compound F and 30.4 mg (0.22 mmol) of 4-methylphenylboronic acid was added 1.9 mL (0.04 M) of n-propanol and stirred for 30 minutes until dissolved. Afterwards, 21 mg (3.7 x 10) of Pd(OAc) -3mmol), PPh32.9 mg (0.011 mmol), 2M Na2CO30.11 ml (0.22 mmol), and distilled water 0.37 ml (0.2 M) were added and stirred at 100 ℃ for 4 hours. After adding distilled water and stirring for 1 hour, the mixture was extracted three times with ethyl acetate (EtOAc), and the organic layer was washed with brine solution, dried, and concentrated. The mixture was purified by silica gel column chromatography (Hexane:EtOAc = 10:1) to obtain 26 mg of compound G-5. Compound G-5 was unstable, so it was used as is in the next reaction without purification by silica gel column chromatography.

[0313] [Compound G-5]

[0314]

[0315]

[0316] Manufacturing Example 11: Compound G-6

[0317] A mixture of 40.4 mg (0.079 mmol) of compound F and 54 mg (0.29 mmol) of 4-t-butylphenylboronic acid was added 1.9 mL (0.04 M) of n-propanol and stirred for 30 minutes until dissolved. After that, 21.1 mg (3.7 x 10) of Pd(OAc) -3 mmol), PPh32.5 mg (0.011 mmol), 2M Na2CO30.11 ml (0.22 mmol), and distilled water 0.37 ml (0.2 M) were added and stirred at 100 ℃ for 4 hours. After adding distilled water and stirring for 1 hour, the mixture was extracted three times with ethyl acetate (EtOAc), and the organic layer was washed with brine solution, dried, and concentrated. The mixture was purified by silica gel column chromatography (Hexane:EtOAc = 10:1) to obtain 24.8 mg (0.049 mmol, 60%) of compound G-6.

[0318] 1H NMR (500 MHz, CDCl3) δ 7.54 (d, 4H,J= 1.0 Hz), 7.47 (d, 4H,J= 1.0 Hz), 5.94 (s, 2H), 4.87 (s, 4H), 4.62 (s, 2H), 2.99 (s, 6H), 1.36 (s, 18H).

[0319] 13 C NMR (125 MHz, CDCl3) δ 150.8, 142.3, 141.4, 130.0, 129.6, 125.2, 119.3, 101.3, 99.6, 57.1, 34.8, 31.5.

[0320] [Compound G-6]

[0321]

[0322]

[0323] Manufacturing Example 12: Compound G-7

[0324] To a mixture of 40.4 mg (0.079 mmol) of compound F and 54 mg (0.29 mmol) of 3,5-dimethyl-4-methoxyphenylboronic acid, 1.9 mL (0.04 M) of n-propanol was added and stirred for 30 minutes until dissolved. Afterwards, 21.1 mg (3.7 x 10) of Pd(OAc) -3 mmol), PPh32.5 mg (0.011 mmol), 2M Na2CO30.11 ml (0.22 mmol), and distilled water 0.37 ml (0.2 M) were added and stirred at 100 ℃ for 4 hours. After adding distilled water and stirring for 1 hour, the mixture was extracted three times with ethyl acetate (EtOAc), and the organic layer was washed with brine solution, dried, and concentrated to obtain 31 mg of compound G-7. Compound G-7 was unstable, so the mixture was used as is in the next reaction without purification by silica gel column chromatography.

[0325] [Compound G-7]

[0326]

[0327]

[0328] Manufacturing Example 13: Compound G-8

[0329] To a mixture of 1.03 g (1.91 mmol) of compound F and 1.93 g (7.65 mmol) of (4-t-butyldimethylsilyloxyphenyl) boronic acid, 0.49 g (4.11 mmol) of potassium bromide and 1.68 g (7.91 mmol) of potassium bromide were dissolved in 12 mL of dioxane, and 110 mg (0.10 mmol) of tetrakis(triphenylphosphine)palladium was added and stirred at 100 °C for 26 hours. Distilled water was then added, stirred for 1 hour, and extracted three times with diethyl ether. The organic layer was washed with brine solution, dried, and concentrated. The mixture was purified by silica gel column chromatography (Hexane:EtOAc = 200:1) to obtain 1.20 g (96%) of compound G-8.

[0330] 1 H NMR (500 MHz, CDCl3) δ 7.47 (d, 4H,J= 8.6 Hz), 6.90 (d, 4H,J= 8.6 Hz), 5.93 (s, 2H), 4.86 (s, 4H), 3.02 (s, 6H), 1.00 (s, 18H), 0.21 (s, 12H).

[0331] 13 C NMR (125 MHz, CDCl3) δ 155.1, 141.9, 141.1, 131.5, 125.3, 119.7, 117.8, 101.0, 99.2, 57.1, 25.7, 18.2.

[0332] [Compound G-8]

[0333]

[0334]

[0335] Manufacturing Example 14: Compound G-9

[0336] A mixture of 40.4 mg (0.079 mmol) of compound F and 42.5 mg (0.22 mmol) of 3-trifluoromethylphenylboronic acid was added 1.9 mL (0.04 M) of n-propanol and stirred for 30 minutes until dissolved. After that, 20.8 mg (3.7 x 10) of Pd(OAc) -3 mmol), PPh 32.9 mg (0.011 mmol), 2M Na2CO 30.11 mL (0.22 mmol), and distilled water 0.37 mL (0.2 M) were added and stirred at 100 ℃ for 4 hours. After adding distilled water and stirring for 1 hour, the mixture was extracted three times with ethyl acetate (EtOAc), and the organic layer was washed with brine solution, dried, and concentrated. The mixture was purified by silica gel column chromatography (Hexane:EtOAc = 10:1) to obtain 37.9 mg (0.071 mmol, 96%) of compound G-9.

[0337] 1 H NMR (500 MHz, CDCl3) δ 7.47 (d, 4H,J= 8.6 Hz), 6.90 (d, 4H,J= 8.6 Hz), 5.93 (s, 2H), 4.86 (s, 4H), 3.02 (s, 6H), 1.00 (s, 18H), 0.21 (s, 12H).

[0338] [Compound G-9]

[0339]

[0340]

[0341] Manufacturing Example 15: Compound G-10

[0342] A mixture of 40.4 mg (0.079 mmol) of compound F and 42.5 mg (0.22 mmol) of 4-trifluoromethylphenylboronic acid was added 1.9 mL (0.04 M) of n-propanol and stirred for 30 minutes until dissolved. After that, 20.8 mg (3.7 x 10) of Pd(OAc) -3 mmol), PPh 32.9 mg (0.011 mmol), 2M Na2CO 30.11 mL (0.22 mmol), and distilled water 0.37 mL (0.2 M) were added and stirred at 100 ℃ for 4 hours. After adding distilled water and stirring for 1 hour, the mixture was extracted three times with ethyl acetate (EtOAc), and the organic layer was washed with brine solution, dried, and concentrated. The mixture was purified by silica gel column chromatography (Hexane:EtOAc = 10:1) to obtain 36.4 mg of compound G-10. Compound G-10 was unstable and was used as is in the next reaction without purification by silica gel column chromatography.

[0343] [Compound G-10]

[0344]

[0345]

[0346] Manufacturing Example 16: Compound G-11

[0347] A mixture of 40.4 mg (0.079 mmol) of compound F and 54 mg (0.29 mmol) of 4-ethoxycarbonylphenylboronic acid was added 1.9 mL (0.04 M) of n-propanol and stirred for 30 minutes until dissolved. After that, 21.1 mg (3.7 x 10) of Pd(OAc) -3mmol), PPh32.5 mg (0.011 mmol), 2M Na2CO30.11 ml (0.22 mmol), and distilled water 0.37 ml (0.2 M) were added and stirred at 100 ℃ for 4 hours. After adding distilled water and stirring for 1 hour, the mixture was extracted three times with ethyl acetate (EtOAc), and the organic layer was washed with brine solution, dried, and concentrated to obtain 25 mg of compound G-11. Compound G-11 was unstable, so the mixture was used as is in the next reaction without purification by silica gel column chromatography.

[0348] [Compound G-11]

[0349]

[0350]

[0351] Example 1: Compound P-1

[0352] 41.2 mg (0.10 mmol) of compound G-1 was dissolved in 1.3 ml (0.08 M) of benzene, and 29.8 mg (0.16 mmol) of p-TsOH·H2O and 71.2 mg (0.31 mmol) of DDQ were added. The mixture was stirred at 50°C for 2 hours and 30 minutes. The reaction was quenched with aqueous NaHCO3 solution and extracted three times with ethyl acetate (EtOAc). The organic layer was washed with brine solution, dried, and concentrated. The mixture was purified by silica gel column chromatography (Hexane:EtOAc = 20:1) to obtain 30.3 mg (0.10 mmol, 95%) of compound P-1.

[0353] 1 H NMR (500 MHz, CDCl3) δ 7.62-7.64 (m, 4H), 7.37-7.44 (m, 6H), 6.14 (s, 2H).

[0354] 13 C NMR (125 MHz, CDCl3) δ 176.3, 156.4, 129.7, 129.0, 128.9, 128.5, 114.0, 103.0.

[0355] [Compound P-1]

[0356]

[0357]

[0358] Example 2: Compound P-2

[0359] 15.1 mg (0.033 mmol) of compound G-2 was dissolved in 0.42 ml (0.08 M) of benzene, and 9.5 mg (0.050 mmol) of p-TsOH·H2O and 22.6 mg (0.10 mmol) of DDQ were added. The mixture was stirred at 50°C for 2 hours and 30 minutes. The reaction was quenched with aqueous NaHCO3 solution and extracted three times with ethyl acetate (EtOAc). The organic layer was washed with brine solution, dried, and concentrated. The mixture was purified by silica gel column chromatography (Hexane:EtOAc = 5:1) to obtain 7.4 mg (0.020 mmol, 61%) of compound P-2.

[0360] 1 H NMR (500 MHz, CDCl3) δ 7.61 (d, 4H,J= 1.0 Hz), 6.97 (d, 4H,J= 1.0 Hz), 6.11 (s, 2H), 3.85 (s, 6H).

[0361] 13 C NMR (125 MHz, CDCl3) δ 176.8, 159.8, 155.8, 131.1, 121.5, 113.9, 113.3, 102.7, 55.5.

[0362] [Compound P-2]

[0363]

[0364]

[0365] Example 3: Compound P-3

[0366] 17.2 mg (0.041 mmol) of compound G-3 was dissolved in 0.51 ml (0.08 M) of benzene, and 11.6 mg (0.061 mmol) of p-TsOH·H2O and 27.7 mg (0.12 mmol) of DDQ were added. The mixture was stirred at 50°C for 2 hours and 30 minutes. The reaction was quenched with aqueous NaHCO3 solution and extracted three times with ethyl acetate (EtOAc). The organic layer was washed with brine solution, dried, and concentrated. The mixture was purified by silica gel column chromatography (Hexane:EtOAc = 5:1) to obtain 8.8 mg (0.027 mmol, 65%) of compound P-3.

[0367] 1 H NMR (500 MHz, CDCl3) δ 7.35-7.23 (m, 8H), 6.04 (s, 2H), 2.26 (s, 6H).

[0368] [Compound P-3]

[0369]

[0370]

[0371] Example 4: Compound P-4

[0372] 34.1 mg (0.081 mmol) of compound G-4 was dissolved in 1.01 ml (0.08 M) of benzene, and 23.0 mg (0.12 mmol) of p-TsOH·H2O and 55.0 mg (0.24 mmol) of DDQ were added. The mixture was stirred at 50°C for 2 hours and 30 minutes, quenched with aqueous NaHCO3 solution, and extracted three times with ethyl acetate (EtOAc). The organic layer was washed with brine solution, dried, and concentrated. The mixture was purified by silica gel column chromatography (Hexane:EtOAc = 10:1) to obtain 24.2 mg (0.073 mmol, 90%) of compound P-4.

[0373] 1H NMR (500 MHz, CDCl3) δ 7.43-7.33 (m, 6H), 7.20 (d, 2H,J= 0.5 Hz), 6.13 (d, 2H), 2.40 (d, 2H).

[0374] 13 C NMR (125 MHz, CDCl3) δ 176.5, 156.4, 138.1, 130.3, 129.7, 128.9, 128.3, 126.8, 114.0, 102.9, 21.7.

[0375] [Compound P-4]

[0376]

[0377]

[0378] Example 5: Compound P-5

[0379] 26.6 mg (0.063 mmol) of compound G-5 was dissolved in 0.79 ml (0.08 M) of benzene, and 18.0 mg (0.095 mmol) of p-TsOH·H2O and 42.9 mg (0.19 mmol) of DDQ were added. The mixture was stirred at 50°C for 2 hours and 30 minutes, quenched with aqueous NaHCO3 solution, and extracted three times with ethyl acetate (EtOAc). The organic layer was washed with brine solution, dried, and concentrated. The mixture was purified by silica gel column chromatography (Hexane:EtOAc = 10:1) to obtain 18.5 mg (0.056 mmol, 89%) of compound P-5.

[0380] 1 H NMR (500 MHz, CDCl3) δ 7.52 (d, 4H,J= 8.2 Hz), 7.25 (d, 4H,J= 8.2 Hz), 6.10 (s, 2H), 2.38 (s, 6H).

[0381] [Compound P-5]

[0382]

[0383]

[0384] Example 6: Compound P-6

[0385] 23.1 mg (0.046 mmol) of compound G-6 was dissolved in 0.57 ml (0.08 M) of benzene, and 13.0 mg (0.068 mmol) of p-TsOH·H20 and 31.1 mg (0.14 mmol) of DDQ were added. The mixture was stirred at 50°C for 2 hours and 30 minutes, quenched with aqueous NaHCO3 solution, and extracted three times with ethyl acetate (EtOAc). The organic layer was washed with brine solution, dried, and concentrated. The mixture was purified by silica gel column chromatography (Hexane:EA = 20:1) to obtain 16.4 mg (0.039 mmol, 86%) of compound P-6.

[0386] 1 H NMR (500 MHz, CDCl3) δ 77.59 (d, 4H,J= 1.0 Hz), .48 (d, 4H,J= 1.0 Hz), 6.12 (s, 2H), 1.36 (s, 18H).

[0387] 13 C NMR (125 MHz, CDCl3) δ 176.6, 156.2, 151.9, 129.4, 126.1, 125.5, 113.8, 102.8, 34.9, 31.4.

[0388] [Compound P-6]

[0389]

[0390]

[0391] Example 7: Compound P-7

[0392] 42.1 mg (0.083 mmol) of compound G-7 was dissolved in 1.0 ml (0.08 M) of benzene, and 23.5 mg (0.12 mmol) of p-TsOH·H2O and 56.2 mg (0.25 mmol) of DDQ were added. The mixture was stirred at 50°C for 2 hours and 30 minutes, quenched with aqueous NaHCO3 solution, and extracted three times with ethyl acetate (EtOAc). The organic layer was washed with brine solution, dried, and concentrated. The mixture was purified by silica gel column chromatography (Hexane:EtOAc = 5:1) to obtain 22.8 mg (0.054 mmol, 66%) of compound P-7.

[0393] 1 H NMR (500 MHz, CDCl3) δ 7.30 (s, 4H), 6.12 (s, 2H), 3.76 (s, 6H), 2.32 (s, 12H).

[0394] 13 C NMR (125 MHz, CDCl3) δ 176.7, 157.4, 155.8, 131.0, 130.2, 130.1, 124.3, 102.7, 59.7, 16.3.

[0395] [Compound P-7]

[0396]

[0397]

[0398] Example 8: Compound P-8

[0399] 269 ​​mg (0.41 mmol) of compound G-8 was dissolved in 5.1 mL (0.08 M) of benzene, and 117 mg (0.62 mmol) of p-TsOH·H20 and 280 mg (1.23 mmol) of DDQ were added. After stirring at 50°C for 2 hours and 30 minutes, 5.0 mL of 10% HCl was added, and the mixture was reacted for an additional 12 hours at room temperature. After concentrating the mixture, it was purified by silica gel column chromatography (Hexane:EtOAc = 10:1 -> pyridine only) to obtain 117.7 mg (0.35 mmol, 85%) of compound P-8.

[0400] 1 H NMR (500 MHz, acetone-d6) δ 8.59 (s, 2H), 7.53 (d, 4H,J= 8.6 Hz), 6.90 (d, 4H,J= 8.6 Hz), 6.35 (s, 2H).

[0401] 13 C NMR (125 MHz, acetone-d6) δ 177.4, 159.2, 156.5, 131.9, 121.2, 116.1, 113.0, 103.7.

[0402] [Compound P-8]

[0403]

[0404]

[0405] Example 9: Compound P-9

[0406] 284 mg (0.54 mmol) of compound G-9 was dissolved in 6.7 ml (0.08 M) of benzene, and 153 mg (0.80 mmol) of p-TsOH·H20 and 365 mg (1.61 mmol) of DDQ were added. The mixture was stirred at 50°C for 2 hours and 30 minutes, quenched with aqueous NaHCO3 solution, and extracted three times with ethyl acetate (EtOAc). The organic layer was washed with brine solution, dried, and concentrated. The mixture was purified by silica gel column chromatography (Hexane:EtOAc = 5:1) to obtain 211 mg (0.48 mmol, 89%) of compound P-9.

[0407] 1 H NMR (500 MHz, CDCl3) δ 7.90 (s, 2H), 7.81 (d, 2H,J= 7.8 Hz), 7.63 (d, 2H,J= 7.8 Hz), 7.56 (t, 2H,J= 7.8 Hz), 6.24 (s, 2H).

[0408] [Compound P-9]

[0409]

[0410]

[0411] Example 10: Compound P-10

[0412] 24.6 mg (0.046 mmol) of compound G-10 was dissolved in 0.58 ml (0.08 M) of benzene, and 13.2 mg (0.070 mmol) of p-TsOH·H20 and 31.6 mg (0.14 mmol) of DDQ were added. The mixture was stirred at 50°C for 2 hours and 30 minutes, quenched with aqueous NaHCO3 solution, and extracted three times with ethyl acetate (EtOAc). The organic layer was washed with brine solution, dried, and concentrated. The mixture was purified by silica gel column chromatography (Hexane:EtOAc = 10:1) to obtain 16.8 mg (0.038 mmol, 82%) of compound P-10.

[0413] 1 H NMR (500 MHz, CDCl3) δ 7.78 (d, 4H,J= 8.1 Hz), 7.71 (d, 4H,J= 8.1 Hz), 6.23 (s, 2H).

[0414] [Compound P-10]

[0415]

[0416]

[0417] Example 11: Compound P-10

[0418] This embodiment is a method suitable for mass production.

[0419] The method for preparing compound P-10 in Example 10 involves synthesis in the order of compound D -> compound E -> compound F -> compound G-10 -> compound P-10, whereas in this Example, synthesis is carried out in the order of compound D -> compound H -> compound G-10 -> compound P-10.

[0420] The specific method is as follows.

[0421] (1) Preparation of compound H

[0422] Compound D (1.51 g, 6.2 mmol) was dissolved in THF (31.0 mL, 0.2 M), and 2.0 Mn-BuLi (7.5 mL, 15.5 mmol) was added at 0°C, followed by stirring at the same temperature for 1 hour and 50 minutes. After 1 hour, 1,2-dibromotetrachloroethane (1,2-dibromotetrachloroethane) (5.3 g, 16.1 mmol) was slowly added, and the mixture was stirred for 2 hours. After the reaction, NH4Cl aqueous solution was added to quench the reaction, and the mixture was extracted three times with ethyl acetate (EtOAc). The organic layer was washed with water and brine solution, dried, and concentrated. The mixture was purified by silica gel column chromatography (Hexane:EtOAc = 50:1) to obtain compound H (1.55 g, 3.91 mmol, 63%).

[0423] 1H NMR (500 MHz, acetone-d6) δ 5.89 (s, 2H), 5.03 (s, 4H), 3.54 (s, 6H).

[0424] [Compound H]

[0425]

[0426]

[0427] (2) Preparation of compound G-10

[0428] To a mixture of 1.55 g (3.89 mmol) of compound H and 2.96 g (15.56 mmol) of 4-trifluoromethylphenylboronic acid, 62.2 ml (0.0625 M) of toluene was added and stirred for 30 minutes until dissolved. Then, 250 mg (0.19 mmol) of PdCl2(MeCN), 159.7 mg (0.39 mmol) of SPhos, and 3.3 g (15.6 mmol) of K3PO4 were added and stirred at 110 ℃ for 4 hours. After adding distilled water and stirring for 1 hour, the mixture was extracted three times with ethyl acetate (EtOAc), and the organic layer was washed with brine solution, dried, and concentrated. The mixture was filtered through a silica gel pad (Hexane:EtOAc = 10:1) to obtain 1.44 g. Compound G-10 was unstable and was used as a mixture in the next reaction without purification by silica gel column chromatography.

[0429] [Compound G-10]

[0430]

[0431]

[0432] (3) Manufacture of compound P-10

[0433] 1.44 g (2.72 mmol) of compound G-10 prepared in the above (2) was dissolved in 34 ml (0.08 M) of benzene, and 775 mg (4.1 mmol) of p-toluenesulfonic acid hydrate (p-TsOH·H20) and 1.85 g (8.15 mmol) of DDQ were added. The mixture was stirred at 50°C for 2 hours and 30 minutes, quenched with an aqueous NaHCO3 solution, and extracted three times with ethyl acetate (EtOAc). The organic layer was washed with brine solution, dried, and concentrated. The mixture was first filtered through a silica gel pad (Hexane:EtOAc = 10:1), and then, without concentration, 0.902 mg (2.04 mmol, 75%) of compound P-10 was obtained through low-temperature crystallization.

[0434] 1 H NMR (500 MHz, CDCl3) δ 7.78 (d, 4H,J= 8.1 Hz), 7.71 (d, 4H,J= 8.1 Hz), 6.23 (s, 2H).

[0435] [Compound P-10]

[0436]

[0437]

[0438] Example 12: Compound P-11

[0439] 25.0 mg (0.046 mmol) of compound G-11 was dissolved in 0.58 ml (0.08 M) of benzene, and 13.3 mg (0.070 mmol) of p-TsOH·H2O and 31.6 mg (0.14 mmol) of DDQ were added. The mixture was stirred at 50°C for 2 hours and 30 minutes, quenched with aqueous NaHCO3 solution, and extracted three times with EtOAc. The organic layer was washed with brine solution, dried, and concentrated. The mixture was purified by silica gel column chromatography (Hexane:EtOAc = 10:1) to obtain 15.2 mg (0.034 mmol, 73%) of compound P-11.

[0440] 1 H NMR (500 MHz, CDCl3) δ 8.10 (d, 4H,J= 1.0 Hz), 7.73 (d, 4H,J= 1.0 Hz), 6.12 (s, 2H), 4.43-4.38 (m, 4H), 1.42 (s, 6H).

[0441] 13 C NMR (125 MHz, CDCl3) δ 175.7, 166.2, 156.9, 133.2, 130.6, 129.7, 129.6, 113.5, 103.5, 61.3, 14.5.

[0442] [Compound P-11]

[0443]

[0444]

[0445] Among the abbreviations for the substituents used in the above chemical formula, OTBS is tert-butyldimethylsilyloxy group, MOM is methoxymethyl group, TfO is trifluoromethanesulfonyl group, Me is methyl group, and Et is ethyl group.

[0446]

[0447] [In vitro experiments]

[0448] Experimental method

[0449] (1) Cell culture

[0450] i. RBL-2H3 cell line culture

[0451] Rat basophilic leukemia cells, RBL-2H3, were cultured in DMEM (GenDEPOT, Dallas, TX, USA) containing 10% FBS (fetal bovine serum, Invitrogen), 200 IU / mL penicillin, 200 mg / mL streptomycin, 25 mM glucose, 4 mM L-glutamine, 0.042 mM phenol red, and 1 mM sodium pyruvate at 37°C in a 5% CO2 incubator. The culture medium was changed every two days.

[0452] ⅱ. HaCaT cell line culture

[0453] Human keratinocyte cell line HaCaT cells were cultured in DMEM (GenDEPOT, Dallas, TX, USA) containing 10% FBS (fetal bovine serum, Invitrogen), 200 IU / mL penicillin, 200 mg / mL streptomycin, 25 mM glucose, 4 mM L-glutamine, 0.042 mM phenol red, and 1 mM sodium pyruvate at 37°C in an incubator with 5% CO2. The culture medium was changed every two days.

[0454]

[0455] (2) RNA isolation and real-time PCR analysis

[0456] RBL-2H3 cells were seeded in 6-well at 3×10 5The cells were seeded at a concentration of 10 cells / well and cultured at 37°C and 5% CO2. After 20 hours, the cells were pretreated with 1 μM cyclosporine A (Sigma-aldrich, Louis, Mo, USA) and phlebiarubrone derivative at a concentration of 10 μg / ml for 1 hour, followed by treatment with PMA (50 ng / ml) and ionomycin (1 mM: Sigma-aldrich, Louis, Mo, USA) and cultured for 8 hours. Afterwards, RNA was extracted using TRIzol Reagent (ThermoFisher, USA). The extracted RNA was quantified at a concentration of 1 μg / ml, and cDNA was synthesized using Bioneer AccuPower RT PreMix & Master Mix. PCR of IL-4 was performed using TaqMan probe IL-4 (Rn01456866_m1) and 18 S (Hs03003631_g1), TaqMan™ Fast Advanced Master Mix (Applied Biosystems™) on a QuantStudio™ 3 Real-Time PCR Instruments device under the following conditions: 50°C for 2 min, 95°C for 2 min, 95°C for 2 s, and 60°C for 20 s.

[0457]

[0458] (3) Measurement of β-hexosaminidase secretion

[0459] RBL-2H3 cells were seeded in 24-well plates at 5 × 10 in DMEM medium containing 10% FBS, 200 IU / mL penicillin, and 200 mg / mL streptomycin. 5After dispensing at 10 cells / ㎖, the cells were cultured in a 37 ℃, 5% CO2 incubator, and then treated with 0.5 ㎍ / ㎖ dinitrophenyl-ImmunoglobulinE (DNP-IgE) (Sigma, USA) for 4 hours. After removing the supernatant, the cells were washed twice with Siraganian buffer (119 mM NaCl, 25 mM PIPES, 5.6 mM glucose, 5 mM KCl, 1 mM CaCl2, 0.4 mM MgCl2, 0.1% BSA, pH 7.2), and then treated with various concentrations of Phlebiarubrone derivatives in DMEM medium excluding FBS, and reacted for 20 minutes in a 37 ℃, 5% CO2 incubator. Afterwards, 2 μg / ml DNP-HSA (Sigma, USA) was treated, and the reaction was performed in an incubator at 37°C, 5% CO, for 30 min, and the reaction was terminated by incubation in an ice bath for 10 min. 50 μl of the supernatant was transferred to a 96-well plate, and 50 μl of substrate buffer (4-p-nitrophenyl-N-acetyl-β-D-glucosaminide 1 mM, sodium citrate, 0.05 M, pH 4.5) was added, and the reaction was incubated at 37°C for 1 h. 100 μl of stop solution was added to each well to terminate the reaction, and the absorbance was measured at 405 nm using an ELISA reader.

[0460]

[0461] (4) Western blotting analysis

[0462] HaCaT cells were seeded in 6-well plates at 5X10 5Cells / well were dispensed at a concentration of 10 cells / well and cultured at 37°C, 5% CO2 conditions. After 24 hours, phlebiarubrone derivatives were treated at various concentrations and cultured for 1 hour. After that, recombinant IL-4 (R&D systems, USA) was treated at a concentration of 10 ng / ㎖ and cultured for 30 minutes. After culture, proteins were extracted using 1 X RIPA buffer (Tech&innovation, Chuncheon, Korea). The extracted proteins were quantified as 20 μg of protein using the BCA (Bicinchoninic acid; Sigma co.) quantitative method and separated using SDS gel (sodium dodecyl sulfate polyacrylamide gel). The separated proteins were transferred to a PVDF membrane (Immobilon-P membrane, Merck Millipore, Damstadt, Germany) and antibody reaction was performed. The primary antibodies used in the experiment were STAT6 (1:1000, cell signaling, #5397S), phospho-STAT6 (1:1000, cell signaling, #56554S), β-Actin (1:2000, cell signaling, #3700). The secondary antibodies used were anti-rabbit antibody (1:10000, GeneTex, #GTX26721) and anti-mouse antibody (1:10000, GeneTex, #GTX213111-01) conjugated with horseradish peroxidase. The bands were measured using Amersham ImageQuant 800 (Cytiva, Marlborough, MA, USA), and the expression levels of the bands were confirmed by dividing them into β-Actin using the Image J program.

[0463]

[0464] Experimental results

[0465] (1) Inhibitory effect on IL-4 mRNA expression

[0466] RBL-2H3 cells, rat basophil cells, are known as an excellent cell line for studying the immune response of type-2 helper T cells (Th2) because they exhibit characteristics similar to mast cells, such as the expression of IgE receptors on the cell surface, cytokines that induce immune responses, and the secretion of various granules. Therefore, qPCR (quantitative polymerase chain reaction) was performed to investigate the effect of compounds P-1 to P-11 on the expression of IL-4 mRNA in RBL-2H3 cells induced by an immune response induced by PMA (phorbol 12-myristate 13-acetate) / Ionomycin (PI). The results were expressed as 100% with the PI treatment group as the negative control, and the relative value was measured. The positive control group was treated with 1 μM of the immunosuppressant CsA (cyclosporine) to compare the activity with the groups treated with Phlebiarubrone and its derivatives, and the results are shown in Figure 1. According to this, the IL-4 mRNA expression level of the PI treatment group increased by approximately 72% compared to the untreated group, and no decrease in IL-4 mRNA expression was observed in the group treated with compound p-1 at a concentration of 30 μM, but it was confirmed that the expression levels of IL-4 in the groups treated with compound p-3, compound p-6, compound p-7, and compound p-10 at a concentration of 30 μM were inhibited by 30%, 51%, 27%, and 59%, respectively, compared to the PI treatment group (# p < 0.05 vs. vehicle control; * p < 0.05 vs. PI).

[0467]

[0468] (2) Cytotoxicity evaluation

[0469] In order to confirm the cytotoxicity of the phlebiarubrone derivative of the present invention, HaCaT and RBL-2H3 cells were treated with various concentrations (1, 5, 10, 30, 50 μM), and the cytotoxicity was analyzed using the WST assay, and the results are shown in Fig. 2. According to this, in RBL-2H3 cells, compound p-6 had no cytotoxicity up to a concentration of 50 μM, while compounds p-3 and p-7 showed cytotoxicity of about 30% or more from a concentration of 30 μM, and compound p-10 showed cytotoxicity of about 72% or more at a concentration of 50 μM. In addition, in HaCaT cells, compound p-6 had no cytotoxicity up to a concentration of 50 μM, while compounds p-3, p-7, and p-10 showed cytotoxicity of about 25%, 12%, and 53%, respectively, from a concentration of 50 μM.

[0470]

[0471] (3) Degranulation inhibition effect

[0472] The degranulation inhibitory effect of the compound of the present invention was confirmed through the secretion of β-Hexosaminidase, a degranulation indicator of RBL-2H3 cells, which are mast cells. β-Hexosaminidase was observed to have an inhibitory effect on β-Hexosaminidase secretion by treating RBL2H3 cells induced with immunoglobulin E (IgE) with DNP-BSA, and the results are shown in Fig. 3. According to this, when compounds p-3, p-6, p-7, and p-10, which showed an inhibitory effect on IL-4 expression in RBL-2H3 cells, were treated at a concentration of 30 μM, inhibition of β-Hexosaminidase secretion was observed in compounds p-3 and p-10, with a decrease of 59.3% and 69.9%, respectively. In addition, as the intracellular treatment concentration of compound p-3 and compound p-10 increased, the inhibitory effect on β-Hexosaminidase secretion became more evident (# p < 0.05 vs. vehicle control; * p < 0.05 vs. DNP-BSA).

[0473]

[0474] (4) Inhibitory effect of STAT6 phosphorylation in keratinocytes

[0475] In keratinocytes, the expression level of STAT6 is increased by IL-4, and overactivated STAT6 inhibits the production of filaggrin, loricrin, and involucrin proteins that constitute the skin barrier, thereby weakening the function of the skin barrier. The results of measuring the degree of STAT6 phosphorylation in HaCaT cells are shown in Fig. 4. According to this, when treated with hIL-4 (recombinant human IL-4) for 15 minutes, STAT6 protein phosphorylation increased approximately 6.7-fold compared to the IL-4 non-treated group, and when treated with compound p-3 and compound p-10 at concentrations of 5, 10, and 30 μM and treated with hIL-4, it was observed that STAT6 phosphorylation decreased in a concentration-dependent manner compared to the IL-4 treated group. Additionally, we observed that compound p-10 showed approximately 2-fold more inhibition of STAT6 phosphorylation compared to the IL-4 treatment group than compound p-3 (# p < 0.05 vs. vehicle control; * p < 0.05 vs. IL-4).

[0476]

[0477] [Experimental Example: In Vivo Experiment]

[0478] Experimental method

[0479] (1) Method for measuring the improvement effect of atopic dermatitis in a DNCB-induced mouse model

[0480] Six-week-old female hairless mice (SKH1-hr, Orientbio) were divided into six groups, with five mice per group. The mice were housed in an air-conditioned animal room with a temperature of 22±2℃, a humidity of 50±10%, and a 12 / 12-h light / dark cycle. They were also provided with experimental food and water ad libitum. DNCB (2,4-dinitrochlorobenzene; Sigma-Aldrich Co.) was used as a solvent in a 3:1 mixture of 99% acetone and olive oil. The vehicle was a 7:3 mixture of propylene glycol and ethanol. After group separation and acclimation for one week, 200 μl of 1% DNCB solution was applied to the backs of the mice daily for 7 days. After 7 days, 0.1% DNCB solution was applied seven times at 2-day intervals in the same manner. Mice were treated with 0.1% DNCB solution for 14 days, and 0.05%, 0.1%, and 0.5% solutions of compound P-10 using vehicle as a solvent were applied to the backs of the mice twice a day, in the morning and afternoon (100 μl each). On the 21st day, the mice were dissected, and dorsal tissue, spleen, and blood were collected. The dorsal tissue used for tissue staining was fixed with 4% paraformaldehyde (Paraformaldehyde; Sigma Co.). This experiment was conducted in accordance with the Animal Ethics Guidelines, and the schedule is shown in Fig. 5.

[0481]

[0482] (2) Dyeing

[0483] Mouse skin tissue fixed in a 4% paraformaldehyde solution was made into a paraffin block. Hematoxylin & eosin staining was performed to observe changes in the epidermis of the tissue, and toluidine blue staining was performed to observe immune cells. Slides were photographed using a slide scanner, and staining analysis was performed using the Caseviewer program.

[0484]

[0485] (3) Blood analysis

[0486] On the final day of the animal experiment, blood was collected from the untreated group, vehicle-treated group, and phlebiarubrone derivative-treated group, and the collected blood was centrifuged at 8,000 rpm for 15 minutes to separate the serum. The protein levels of immunoglobulin E (IgE), one of the indicators of atopic dermatitis, and interleukin-4 (IL-4), a Th2 cytokine of immune cells, were measured in the serum using the EnzymeLinked Immuno Assay (ELISA) test method. The IgE ELISA kit (Mouse IgE ELISA kit, abcam, USA) and the IL-4 ELISA kit (mIL-4 Quantikine ELISA kit, R&D systems) were used.

[0487]

[0488] Experimental results

[0489] (1) Improvement effect in DNCB atopic dermatitis model

[0490] Atopic dermatitis-like lesions were induced with DNCB in 6-week-old female Hairless mice. The condition of the back was observed while treating them with compound p-10 for 14 days. As a result, the back skin began to peel and keratinization 5 days after lesion induction with 1% DNCB, and hyperkeratosis, in which the skin peeled and the keratin became hard, was observed 7 days after induction. In addition, it was confirmed that the lesions gradually decreased after sample treatment. When visually observed from 14 days after lesion induction, it was observed that the compound p-10-treated group had less keratin and the skin condition improved compared to the vehicle-treated group. After 21 days of lesion induction, partial keratinization was observed in the vehicle group, but a phenotype similar to that of the untreated group was observed in the compound p-10-treated group.

[0491] For morphological analysis of the epidermis, the results of H&E (Hematoxylin and eosin) staining of the dorsal tissue of the mouse are shown in Fig. 6. According to this, the thickness of the epidermis increased 3.5 times in the vehicle group treated with DNCB to induce atopic dermatitis compared to the untreated group, and an increase in the number of inflammation-related cells in the dermal layer of the skin was observed. This appears to be due to an increased immune response in the atopic dermatitis lesion area, resulting in infiltration of immune cells into the dermis. However, the compound p-10 treatment group showed a thinner epidermis and a decrease in the number of inflammation-related cells compared to the vehicle group (# p < 0.05 vs. control; *p < 0.05 vs. vehicle).

[0492] The H&E staining results confirmed an increase in the number of immune-related cells in the dermal layer of the DNCB-treated group, and to observe whether the increased cells were cells involved in the immune response, the number of mast cells was measured by toluidine blue staining, and the results are shown in Fig. 7. According to this, the number of mast cells increased 2.6-fold in the vehicle-treated group compared to the untreated group, and decreased in the compound p-10-treated group compared to the vehicle group (# p < 0.05 vs. control; *p < 0.05 vs. vehicle).

[0493]

[0494] (2) Analysis of blood and atopic dermatitis-related factors in the DNCB model

[0495] In order to measure the protein amounts of IgE and interleukin-4 (IL-4), known as diagnostic target markers for atopic dermatitis, the serum of mice was separated and observed through an ELISA experiment. The results are shown in Fig. 8. According to this, the IgE protein amount in the vehicle group increased 5.61-fold compared to the untreated group. However, the compound p-10 treatment group decreased 1.6-fold compared to the vehicle treatment group. In addition, the protein amount of IL-4 increased 2.2-fold in the vehicle group compared to the untreated group, and decreased to the level of the untreated group in the compound p-10 treatment group.

[0496]

[0497] Above, embodiments of the present invention have been described, but those of ordinary skill in the art will be able to modify and change the present invention in various ways by adding, changing, deleting or adding components, etc., within the scope that does not depart from the spirit of the present invention described in the claims, and this will also be considered to be included within the scope of the rights of the present invention.

[0498] The composition containing the phlebiarubrone derivative of the present invention as an active ingredient can be used to prevent, improve or treat inflammatory diseases including atopic dermatitis by suppressing IgE and IL-4 expression, suppressing STAT6 phosphorylation, and suppressing mast cell degranulation.

Claims

1. A compound represented by the following structural formula 1; [Structural formula 1] In structural formula 1, R 1 Inland R 5 are each independently a hydrogen atom, , a substituted or unsubstituted C1 to C30 alkoxy group, a substituted or unsubstituted C1 to C30 alkyl group, a substituted or unsubstituted C3 to C30 cycloalkyl group, a substituted or unsubstituted C1 to C30 heterocycloalkyl group, a substituted or unsubstituted C6 to C30 aryl group, or a substituted or unsubstituted C1 to C30 heteroaryl group, R 11 Inland R 13 are each independently a hydrogen atom, a halogen group, or a substituted or unsubstituted C1 to C30 alkyl group, R 6 Inland R 10 are each independently a hydrogen atom, , a substituted or unsubstituted C1 to C30 alkoxy group, a substituted or unsubstituted C1 to C30 alkyl group, a substituted or unsubstituted C3 to C30 cycloalkyl group, a substituted or unsubstituted C1 to C30 heterocycloalkyl group, a substituted or unsubstituted C6 to C30 aryl group, or a substituted or unsubstituted C1 to C30 heteroaryl group, R 14 Inland R 16 are each independently a hydrogen atom, a halogen group, or a substituted or unsubstituted C1 to C30 alkyl group.

2. In paragraph 1, In the above structural formula 1, R 1 Inland R 5 are each independently a hydrogen atom, , a C1 to C10 alkoxy group, or a C1 to C10 alkyl group, R 6 Inland R 10 are each independently a hydrogen atom, , a compound characterized by a C1 to C10 alkoxy group, or a C1 to C10 alkyl group.

3. In paragraph 2, The compound represented by the above structural formula 1 is a compound characterized by being represented by any one selected from the following structural formulas 2 to 4; [Structural formula 2] In structural formula 2, R 1 and R 10 are each independently a C1 to C4 alkyl group. [Structural formula 3] In structural formula 3, R 2 , R 4 , R 7 and R 9 are each independently a C1 to C4 alkyl group, R 3 and R 8 are each independently a C1 to C4 alkoxy group. [Structural formula 4] In structural formula 4, R 3 and R 8 are each independently , or am.

4. In paragraph 3, A compound represented by the above structural formula 1 is a compound characterized in that it is a compound represented by any one of the following chemical formulas 1 to 4; [Chemical Formula 1] [Chemical Formula 2] [Chemical Formula 3] [Chemical Formula 4] 5. A step of preparing the following compound H by reacting the following compound D with 1,2-dibromotetrachloroethane; [Compound D] [Compound H] A step of preparing a compound represented by the following structural formula 4-1 by reacting the above compound H with 4-trifluoromethylphenylboronic acid or 4-t-butylphenylboronic acid; and [Structural formula 4-1] A method for producing a compound represented by the following structural formula 4, comprising the step of reacting a compound represented by the above structural formula 4-1 with p-toluenesulfonic acid hydrate (p-TsOH·H20) and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ); [Structural formula 4] In structural formula 4, R 3 and R 8 are each independently , or am.

6. A pharmaceutical composition for preventing or treating an inflammatory disease, comprising a compound represented by any one of the following chemical formulas 1 to 4, or a pharmaceutically acceptable salt thereof, as an active ingredient; [Chemical Formula 1] [Chemical Formula 2] [Chemical Formula 3] [Chemical Formula 4] 7. In paragraph 1, A pharmaceutical composition for preventing or treating an inflammatory disease, characterized in that the inflammatory disease is any one selected from among atopic dermatitis, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, iritis, pharyngitis, tonsillitis, pneumonia, gastric ulcer, pancreatitis, gastritis, Crohn's disease, inflammatory bowel disease, colitis, hemorrhoids, gout, ankylosing spondylitis, lupus, fibromyalgia, psoriasis, and rheumatoid arthritis.

8. In paragraph 1, A pharmaceutical composition for preventing or treating inflammatory diseases, characterized in that the pharmaceutical composition is for suppressing the expression of at least one selected from IgE and IL-4.

9. In paragraph 1, A pharmaceutical composition for preventing or treating inflammatory diseases, characterized in that the above pharmaceutical composition is for inhibiting STAT6 phosphorylation.

10. In paragraph 1, The above pharmaceutical composition is a pharmaceutical composition for preventing or treating inflammatory diseases, characterized in that it inhibits degranulation of mast cells.

11. A food composition for preventing or improving inflammatory diseases, comprising a compound represented by any one of the following chemical formulas 1 to 4, or a food-wise acceptable salt thereof, as an active ingredient; [Chemical Formula 1] [Chemical Formula 2] [Chemical Formula 3] [Chemical Formula 4] 12. A cosmetic composition for preventing or improving inflammatory diseases, comprising a compound represented by any one of the following chemical formulas 1 to 4, or a cosmetically acceptable salt thereof, as an active ingredient; [Chemical Formula 1] [Chemical Formula 2] [Chemical Formula 3] [Chemical Formula 4]

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