Pharmaceutical composition for preventing, ameliorating or treating atopic disease comprising flavonoid derivative as active ingredient and method for preparing flavonoid derivative

Abstract

Provided are a pharmaceutical composition for preventing, ameliorating or treating atopic diseases comprising a flavonoid derivative as an active ingredient, and a method for preparing the flavonoid derivative, and more specifically, provided are a pharmaceutical composition for preventing, ameliorating or treating atopic diseases comprising a flavonoid derivative 3, 3 ', 3', 3 ', 3', 3 ', 3', 3 ', 3', 3 ', 3', 3 ', 3', 3 ', 3', 3 ' The invention relates to a pharmaceutical composition containing 3, 4-dihydroxyflavonol as an active ingredient and a method for preparing the 3, 4-dihydroxyflavonol.

Description

Technical Field

[0001] The present disclosure relates to a pharmaceutical composition comprising a flavonoid derivative as an active ingredient for preventing, improving or treating atopy and a method for preparing the flavonoid derivative, and more particularly, to a pharmaceutical composition comprising a flavonoid derivative 3,4-dihydroxyflavonol as an active ingredient for preventing, improving or treating atopy and a method for preparing the 3,4-dihydroxyflavonol. Background Art

[0002] Atopic dermatitis is a chronic, relapsing inflammatory skin condition that typically begins in infancy or childhood and presents with symptoms including itching, dry skin, and distinctive eczematous features. As of 2020, the number of atopic dermatitis patients in South Korea is estimated to have reached 1 million. This number is expected to increase in the future, as it is ranked as the seventh most common disease.

[0003] In the past, steroids or immunosuppressants were primarily used to treat atopic dermatitis. Steroids relieve itching by reducing inflammation, but the amount of steroids that can be used and the potential for steroid resistance are limited. Additionally, conventional immunosuppressants can alleviate atopic symptoms by suppressing excessive immune responses, but their efficacy varies among patients, and long-term use can lead to side effects including abnormalities in blood pressure, blood sugar levels, and kidney function.

[0004] Therefore, there is a growing need for new atopic dermatitis treatments, and recently, the use of small molecule drugs has attracted attention. Small molecule drugs, which include small compounds composed of dozens of bonded atoms and are derived from plants or microorganisms or chemically synthesized, enable essential treatment by targeting only the pathogen. Therefore, the use of small molecule drugs can treat atopy by intercepting the erroneous inflammatory signals that cause atopy, and can treat atopy without the side effects associated with existing steroid and immunosuppressant therapies.

[0005] Protease-activated receptor 2 (PAR2) is a receptor activated by specific proteases that causes itching and is particularly abundant in atopic patients. Trypsin activates PAR2, leading to allergic and inflammatory reactions. Therefore, the use of small molecule drugs that can act as trypsin antagonists can have a therapeutic effect on atopic dermatitis by inhibiting the activation of PAR2. Therefore, the present disclosure discloses flavonoid derivative small molecule drugs that effectively inhibit PAR2 as trypsin antagonists. Summary of the Invention

[0006] [Technical Issues]

[0007] The present disclosure aims to provide a pharmaceutical composition for preventing, improving or treating atopy, which comprises a flavonoid derivative 3,4-dihydroxyflavonol as an active ingredient.

[0008] Another object of the present disclosure is to provide a cosmetic composition for preventing or improving atopy, comprising a flavonoid derivative 3,4-dihydroxyflavonol as an active ingredient.

[0009] Yet another object of the present disclosure is to provide a method for preparing 3,4-dihydroxyflavonol, which is a flavonoid derivative.

[0010] The technical problems to be solved by the present invention are not limited to the above-mentioned technical problems, and other technical problems not described can be clearly understood by those skilled in the art from the description of the present disclosure.

[0011] [Technical solution]

[0012] In one general aspect, a pharmaceutical composition or cosmetic composition for preventing, improving or treating atopy is provided, comprising a compound represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof:

[0013] [Chemical Formula 1]

[0014]

[0015] The compound represented by Chemical Formula 1 can function as a trypsin antagonist to inhibit protease-activated receptor 2 (PAR2).

[0016] The pharmaceutical composition or cosmetic composition can reduce the expression of at least one gene selected from the group consisting of COX-2, MCP-1, IL-6, TNF-α, ICAM-1 and MMP1.

[0017] In another general aspect, a method for preparing a compound represented by the following Chemical Formula 1 is provided, comprising the following steps:

[0018] (S1) preparing a compound represented by the following Chemical Formula 3 from a compound represented by the following Chemical Formula 2;

[0019] (S2) preparing a compound represented by the following Chemical Formula 4 from the compound represented by Chemical Formula 3;

[0020] (S3) preparing a compound represented by the following Chemical Formula 5 from the compound represented by Chemical Formula 4; and

[0021] (S4) preparing the compound represented by Chemical Formula 1 from the compound represented by Chemical Formula 5.

[0022] The step S4 may include reacting the compound represented by Chemical Formula 4 with dichloromethane, trifluoroacetic acid, an aqueous sodium bicarbonate solution, and a mixed solution of methanol and dichloromethane in sequence to prepare a compound represented by Chemical Formula 5:

[0023] [Chemical Formula 1]

[0024]

[0025] [Chemical Formula 2]

[0026]

[0027] [Chemical Formula 3]

[0028]

[0029] [Chemical Formula 4]

[0030]

[0031] [Chemical Formula 5]

[0032]

[0033] [Beneficial Effects]

[0034] The pharmaceutical composition for preventing, improving or treating atopy according to the present disclosure, comprising the flavonoid derivative 3,4-dihydroxyflavonol as an active ingredient, can be used as a novel therapy for atopic dermatitis without the side effects caused by conventional atopy treatments.

[0035] Effects obtained from the present disclosure are not limited to the above-described effects, and other effects that are not described will be clearly understood by those skilled in the art from the description of the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 These are images showing the results of the cytotoxicity evaluation of 3,4-dihydroxyflavonol.

[0037] Figure 2 The graph shows that 3,4-dihydroxyflavonol causes a decrease in COX-2 gene expression.

[0038] Figure 3 These are images showing the reduction in MCP-1 gene expression caused by 3,4-dihydroxyflavonol.

[0039] Figure 4 The graph shows that 3,4-dihydroxyflavonol causes a decrease in IL-6 gene expression.

[0040] Figure 5These are images showing the reduction in TNF-α gene expression caused by 3,4-dihydroxyflavonol.

[0041] Figure 6 These are images showing the reduction in ICAM-1 gene expression caused by 3,4-dihydroxyflavonol.

[0042] Figure 7 These are images showing the reduction in MMP-1 gene expression caused by 3,4-dihydroxyflavonol.

[0043] Figure 8 These are images showing the reduction in COX-2 protein expression caused by 3,4-dihydroxyflavonol.

[0044] Figure 9 These are images showing the reduction in MCP-1 protein expression caused by 3,4-dihydroxyflavonol.

[0045] Figure 10 These are images showing the reduction in IL-6 protein expression caused by 3,4-dihydroxyflavonol.

[0046] Figure 11 These are images showing the reduction in TNF-α protein expression caused by 3,4-dihydroxyflavonol.

[0047] Figure 12 These are images showing the reduction in ICAM-1 protein expression caused by 3,4-dihydroxyflavonol. DETAILED DESCRIPTION

[0048] The terms used in this specification are general and are intended to be consistent with current widespread usage, while also taking into account their function in this disclosure. However, it must be recognized that these terms may change based on the intentions of those skilled in the art or precedents, the emergence of new technologies, etc. In addition, there are specific cases where the applicant chooses terms on his own, and in such cases, their meanings will be explained in the relevant descriptions of this disclosure. Therefore, the terms used in this disclosure should not be defined merely by the simple name of the term, but should be defined based on its meaning and the entire content of the invention.

[0049] In addition, unless otherwise defined, all terms used herein (including technical or scientific terms) have the same meaning as commonly understood by those skilled in the art to which the present disclosure belongs. Terms (such as those defined in commonly used dictionaries) should be interpreted as having a meaning consistent with that in the relevant art and should not be interpreted in an idealized or overly formal sense unless explicitly defined in this application.

[0050] Numerical ranges include the values defined within the above ranges. Each maximum numerical limit provided in this specification essentially includes all smaller numerical limits, as if each smaller limit were expressly stated. Each minimum numerical limit provided in this specification essentially includes all higher numerical limits, as if each higher limit were expressly stated. All numerical limits provided in this specification will include all better numerical ranges within the wider numerical ranges, as if each narrower limit were expressly stated, including all better ranges.

[0051] The present disclosure will be described in more detail below.

[0052] Method for preparing 3,4-dihydroxyflavonol

[0053] The present disclosure provides a method for preparing a 3,4-dihydroxyflavonol compound represented by the following Chemical Formula 1, using compounds represented by the following Chemical Formulas 2 to 5:

[0054] [Chemical Formula 1]

[0055]

[0056] [Chemical Formula 2]

[0057]

[0058] [Chemical Formula 3]

[0059]

[0060] [Chemical Formula 4]

[0061]

[0062] [Chemical Formula 5]

[0063]

[0064] In the present disclosure, the compound represented by Chemical Formula 3 may be prepared using the compound represented by Chemical Formula 2, and a specific preparation method is as follows, but is not limited thereto.

[0065] After dissolving 1g to 3g of the compound represented by Chemical Formula 2 in 50mL to 70mL of N,N-dimethylformamide, 12g to 16g of potassium carbonate may be added at 15°C to 25°C. When using less than 1g or more than 3g of the compound represented by Chemical Formula 2, crystals may precipitate during the compound formation process, and crystallization may terminate the compound synthesis. 4g to 7g of 4-methoxybenzyl chloride may be slowly added to the potassium carbonate reaction product, and then stirred at 60°C to 80°C for 15 to 25 hours. The 4-methoxybenzyl chloride reaction product may be cooled to 15°C to 25°C and made into a solid by adding ice. The resulting solid may be filtered under reduced pressure and washed with 80mL to 120mL of water to obtain a solid, which is then recrystallized from n-hexane to obtain the compound represented by Chemical Formula 3 as an apricot crystalline solid.

[0066] In the present disclosure, the compound represented by Chemical Formula 4 may be prepared using the compound represented by Chemical Formula 3, and a specific preparation method is as follows, but is not limited thereto.

[0067] 600 mg to 700 mg of sodium hydride can be added to 30 mL to 50 mL of N,N-dimethylformamide under nitrogen, and then 1 g to 2 g of 2-hydroxyacetophenone dissolved in 10 mL to 20 mL of N,N-dimethylformamide can be slowly added to the sodium hydride reaction product at -5°C to 5°C and stirred at -5°C to 5°C for 5 to 20 minutes. Next, 4 g to 5 g of the compound represented by Chemical Formula 3 above can be dissolved in 10 mL to 20 mL of N,N-dimethylformamide at -5°C to 5°C. A solution of the compound represented by Compound 3 dissolved in N,N-dimethylformamide can be slowly added to the stirred product, stirred at -5°C to 5°C for 20 to 40 minutes, and then stirred at 15°C to 25°C for 15 to 25 hours. Water can be slowly added to the stirred product at -5 ° C to 5 ° C until no bubbles are generated, and the resulting mixture can be diluted with 80 mL to 120 mL of ethyl acetate, washed with 40 mL to 60 mL of water, and then washed three times with 40 mL to 60 mL of water. The organic layer of the washed product can be dried over anhydrous sodium sulfate, filtered under reduced pressure and concentrated under reduced pressure to obtain a yellow solid. The yellow solid can be recrystallized from a mixed solution of 20 mL to 40 mL of dichloromethane and n-hexane mixed in a ratio of 1: 8 to 1: 10 to obtain a compound represented by Chemical Formula 4 as a yellow crystalline solid.

[0068] In the present disclosure, the compound represented by Chemical Formula 5 may be prepared using the compound represented by Chemical Formula 4, and a specific preparation method is as follows, but is not limited thereto.

[0069] 4g to 5g of the compound represented by Chemical Formula 4 can be added to 60mL to 80mL of anhydrous ethanol, and 60mL to 80mL of 4N-sodium hydroxide aqueous solution can be added at -5°C to 5°C to dissolve the compound. After the compound is dissolved, 8mL to 16mL of hydrogen peroxide can be slowly added at -5°C to 5°C, and the hydrogen peroxide reaction product can be stirred at -5°C to 5°C for 20 minutes to 40 minutes, or can be stirred at 15°C to 25°C for 15 hours to 25 hours. 2N-hydrochloric acid aqueous solution can be slowly added to the stirred product at -5°C to 5°C, and when the pH reaches 0.5 to 1.5 by adding the hydrochloric acid aqueous solution, the resulting solid can be filtered under reduced pressure and washed with 20mL to 40mL of water to obtain a yellow solid. The solid can be recrystallized from 10mL to 30mL of anhydrous ethanol to obtain a compound represented by Chemical Formula 5 as a yellow crystalline solid.

[0070] In the present disclosure, the 3,4-dihydroxyflavonol compound represented by Chemical Formula 1 may be prepared using the compound represented by Chemical Formula 5, and the specific preparation method is as follows, but is not limited thereto.

[0071] 4g to 5g of the compound represented by Chemical Formula 5 can be added to 50mL to 70mL of dichloromethane, and 10mL to 20mL of trifluoroacetic acid can be slowly added to the dichloromethane reaction product, followed by stirring at -5°C to 5°C for 20 minutes to 40 minutes, and at 15°C to 25°C for 8 hours to 16 hours. The stirred product can be concentrated under reduced pressure, and the pH can be adjusted to 9 to 11 by slowly adding an aqueous sodium bicarbonate solution at -5°C to 5°C. After pH adjustment, the resulting solid can be filtered under reduced pressure and washed with 40mL to 60mL of water to obtain a yellow solid. The solid can be recrystallized from a mixed solution of methanol and dichloromethane in a ratio of 1:15 to 1:25. Through the above recrystallization, the 3,4-dihydroxyflavonol compound represented by Chemical Formula 1 can be obtained as a yellow crystalline solid.

[0072] Pharmaceutical composition

[0073] The present disclosure provides a pharmaceutical composition for preventing, improving or treating atopy, comprising a 3,4-dihydroxyflavonol compound represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof:

[0074] [Chemical Formula 1]

[0075]

[0076] In the present disclosure, the 3,4-dihydroxyflavonol compound represented by the above Chemical Formula 1 or a pharmaceutically acceptable salt thereof can function as a trypsin antagonist to inhibit protease-activated receptor 2 (PAR2), but is not limited thereto.

[0077] In the present disclosure, the pharmaceutical composition can reduce the expression of at least one gene selected from the group consisting of COX-2, MCP-1, IL-6, TNF-α, ICAM-1 and MMP1, but is not limited thereto.

[0078] PAR2 receptor is a receptor activated by a specific protease, which causes itching and is particularly enriched in atopic patients. Trypsin can cause allergic and inflammatory reactions by activating PAR2, so trypsin antagonists can be used for the treatment of atopy. In addition, since COX-2, MCP-1, IL-6, TNF-α, ICAM-1 and MMP1 genes are associated with inflammation, their expression can be increased by the activation of PAR2 receptor, and their expression can be reduced by according to the pharmaceutical composition of the present disclosure.

[0079] More specifically, activation of pro-inflammatory cytokines (e.g., MCP-1, TNF-α, and IL-6) can increase the expression of inflammatory enzymes (e.g., COX-2), the cell adhesion molecule ICAM-1, and the skin tissue-degrading enzyme MMP-1. With increased expression of these genes, the inflammatory response can be exacerbated, inducing tissue damage.

[0080] Therefore, reducing the expression level of the corresponding gene may be beneficial in the treatment of atopic dermatitis, which causes itching and scratching, leading to skin inflammation and damage.

[0081] As used herein, the term "pharmaceutically acceptable salt" refers to any organic or inorganic addition salt that has an effective effect at a concentration that is relatively non-toxic and harmless to the patient, wherein the side effects caused by these salts do not impair the effect of 3,4-dihydroxyflavonol in preventing, improving or treating atopy. For example, organic acids, inorganic acids, non-toxic salts, etc. can be used as free acids. Examples of inorganic acids may include hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid, etc., and examples of organic acids may include methanesulfonic acid, p-toluenesulfonic acid, acetic acid, trifluoroacetic acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carboxylic acid, vanillic acid, hydroiodic acid, etc.

[0082] The pharmaceutical composition of the present disclosure may further comprise a pharmaceutically acceptable carrier, excipient or diluent in addition to the active ingredient. Examples of the carrier, excipient and diluent may include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate / ester, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, methylparaben, propylparaben, talc, magnesium stearate and mineral oil.

[0083] According to the pharmaceutical composition of the present disclosure, powders, granules, sustained-release granules, enteric-coated granules, solutions, eye drops, elixirs, emulsions, suspensions, spirits, lozenges, spices, lemonade, tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric-coated capsules, pills, tinctures, soft extracts, dry extracts, liquid extracts, injections, capsules, perfusions, external preparations (such as plasters, lotions, pastes, sprays, inhalants, patches, sterile injection solutions or aerosols) can be used. External preparations can have the dosage form of, for example, creams, gels, patches, sprays, ointments, plasters, lotions, liniments, pastes or cataplasms.

[0084] The pharmaceutical composition of the present disclosure can be administered orally or parenterally according to the desired method. The parenteral administration may include intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc., and specifically, can be administered orally or transdermally.

[0085] The pharmaceutical composition of the present disclosure can be used alone or in combination with methods using surgery, hormone therapy, chemotherapy, and biological response modifiers to prevent or treat atopic dermatitis.

[0086] Cosmetic composition

[0087] The present disclosure provides a cosmetic composition for preventing or improving atopy, comprising: a 3,4-dihydroxyflavonol compound or a pharmaceutically acceptable salt thereof.

[0088] In addition to the above-mentioned active ingredients, the components contained in the cosmetic composition of the present disclosure may include any components commonly used in cosmetic compositions. For example, the components may include conventional adjuvants and carriers, such as stabilizers, solubilizers, vitamins, colorants and fragrances.

[0089] The cosmetic composition of the present disclosure can be used in various atopic dermatitis cosmetics, which have the effect of alleviating or inhibiting skin inflammation and / or itching.

[0090] The cosmetic composition of the present invention can be prepared in any formulation commonly prepared in the art. For example, the cosmetic composition of the present invention can be prepared into an emulsion, a cream, a toner, a facial mask, a foundation, a lotion, a beauty essence, a hair cosmetic, etc. Specifically, the cosmetic composition of the present invention can be in the form of a lotion, a softener, a toner, a astringent, an emulsion, a lotion, a milk lotion, a moisturizing lotion, a nutrient lotion, a massage cream, a nutrient cream, a moisturizing cream, a hand cream, a foundation, an essence, a nutrient essence, a facial mask, a soap, a cleansing foam, a cleansing milk, a cleansing cream, a body lotion, a body cleanser, an eye cream, etc.

[0091] When the cosmetic composition of the present disclosure is formulated as a paste, cream, or gel, animal fiber, plant fiber, wax, paraffin, starch, tracant, cellulose derivatives, polyethylene glycol, silicone, bentonite, silicon dioxide, talc, zinc oxide, etc. may be used as carrier components.

[0092] When the cosmetic composition of the present disclosure is formulated as a powder or a spray, lactose, talc, silicon dioxide, aluminum hydroxide, calcium silicate, polyamide powder, etc. can be used as a carrier component. In particular, when the cosmetic composition of the present disclosure is formulated as a spray, the composition may further contain a propellant such as chlorofluorocarbons, propane / butane, or dimethyl ether.

[0093] When the cosmetic composition of the present disclosure is formulated as a solution or emulsion, a solvent, solubilizer, or emulsifier may be used as a carrier component. For example, water, ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol oil, glycerol fatty acid ester, polyethylene glycol, sorbitan fatty acid ester, etc. may be used.

[0094] When the formulation of the cosmetic composition of the present disclosure is a suspension, a liquid diluent (e.g., water, ethanol, or propylene glycol), a suspending agent (e.g., ethoxylated isostearyl alcohol, polyoxyethylene sorbitan esters, and polyoxyethylene sorbitan esters), microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, tracant, etc. can be used as a carrier component.

[0095] When the cosmetic composition of the present disclosure is formulated as a surfactant-containing cleanser, fatty alcohol sulfates / esters, fatty alcohol ether sulfates / esters, sulfosuccinic acid monoesters, isethionates / esters, imidazoline derivatives, methyl taurates / esters, sarcosinates / esters, fatty acid amide ether sulfates / esters, alkylamide betaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives, ethoxylated glyceryl fatty acid esters, and the like can be used as carrier components.

[0096] Unless there is any conflict between the contents described in the pharmaceutical composition or cosmetic composition of the present disclosure, they are all equally applicable.

[0097] Treatment

[0098] The present disclosure provides a method for preventing or treating atopy, comprising: administering 3,4-dihydroxyflavonol or a pharmaceutically acceptable salt thereof to a subject having atopy or at risk of developing atopy.

[0099] For preventive uses, the compositions of the present disclosure are administered to a subject believed to be suffering from or at risk of developing a disease, disorder, or condition described herein. For therapeutic uses, the compositions of the present disclosure are administered to a subject (e.g., a patient already suffering from a disorder described herein) in an amount sufficient to treat or at least partially arrest the symptoms of a disease, disorder, or condition described herein. The effective amount for such uses will depend on the severity and course of the disease, disorder, or condition, previous treatment, the individual's medical condition and response to drugs, and the judgment of the physician or veterinarian.

[0100] In the treatment methods of the present disclosure, the composition may be administered to a subject in a pharmaceutically effective amount.

[0101] As used herein, the term "subject" refers to all animals that suffer from or may develop atopy, including humans, dogs, cats, etc., and can generally be human or non-human animals that show beneficial effects by treatment with the 3,4-dihydroxyflavonol or a pharmaceutically acceptable salt thereof disclosed herein, and can include, but is not limited to, any subject that has atopic symptoms or is prone to such symptoms. As described above, by administering the pharmaceutical composition of the present disclosure to a subject, atopy can be effectively prevented, improved, or treated.

[0102] As used herein, the term "administer" means introducing a predetermined substance into a human or non-human animal (e.g., dog and cat) by any appropriate method, and the administration route of the composition of the present disclosure can be oral or parenteral administration by any common route, as long as the composition can reach the target tissue. In addition, the composition of the present disclosure can be administered by any device capable of delivering the active ingredient to the target cell.

[0103] Specifically, "pharmaceutically effective amount" means an amount sufficient to treat the disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dosage level can be determined according to factors including the type and severity of the subject, age, sex, type of disease, drug activity, sensitivity to the drug, time of administration, route of administration, rate of excretion, duration of treatment, concurrent medications, and other factors well known in the medical field.

[0104] The composition can be given as a separate treatment or in combination with other treatments, and can be given sequentially or simultaneously with conventional treatment. In addition, the composition of the present disclosure can be given in a single dose or multiple doses. Taking all of the above factors into account, it is important to give the composition in an amount that can achieve the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

[0105] Unless otherwise stated, the contents described in the pharmaceutical compositions and methods of treatment disclosed herein are equally applicable.

[0106] use

[0107] The present disclosure provides use of 3,4-dihydroxyflavonol or a pharmaceutically acceptable salt thereof in preventing, improving or treating atopy.

[0108] In addition, the present disclosure provides use of 3,4-dihydroxyflavonol or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for preventing, improving or treating atopy.

[0109] Unless otherwise specified, the contents described in the pharmaceutical compositions and uses of the present disclosure are equally applicable.

[0110] Example

[0111] The following will describe the embodiments of the present disclosure in detail, but it is obvious that the present disclosure is not limited to the following embodiments.

[0112] Preparation Example. Preparation of 3,4-dihydroxyflavonol compound

[0113] Preparation of the compound represented by Chemical Formula 3

[0114] To prepare the compound represented by the following Chemical Formula 3, the compound represented by the following Chemical Formula 2 was used:

[0115] [Chemical Formula 2]

[0116]

[0117] [Chemical Formula 3]

[0118]

[0119] Specifically, the compound (2g, 14.480mmol) represented by chemical formula 2 is dissolved in 60mL N, N-dimethylformamide, and potassium carbonate (14g, 101.360mmol) is subsequently added at room temperature. Then, 4-methoxybenzyl chloride (5.7g, 36.200mmol) is slowly added to the potassium carbonate reaction product and stirred at 70°C for 20 hours. The 4-methoxybenzyl chloride reaction product is cooled to room temperature and made into a solid on the ice. The gained solid is filtered under reduced pressure, and washed with 100mL water to obtain a solid, which is then recrystallized from n-hexane to obtain the compound (5.66g, quantitative) represented by chemical formula 3 as an apricot crystalline solid.

[0120] The NMR results of the compound represented by Chemical Formula 3 are described below.

[0121] 1H NMR (400MHz, CDCl3) δ9.83 (s, 1H), 7.50 (d, J = 1.6Hz, 1H), 7.43-7.36 (m, 5H), 7 .04(d,J=8.0Hz,1H),6.93-6.90(m,4H),5.19(s,2H),5.15(s,2H),3.83(s,6H)

[0122] Preparation of the compound represented by Chemical Formula 4

[0123] To prepare the compound represented by the following Chemical Formula 4, the compound represented by Chemical Formula 3 prepared above was used:

[0124] [Chemical Formula 4]

[0125]

[0126] Specifically, sodium hydride (661mg, 16.525mmol) is added to 40mL N, N- dimethylformamide under nitrogen. Then, 2-hydroxyacetophenone (1.5g, 11.017mmol) dissolved in 15mL N, N- dimethylformamide is slowly added to the sodium hydride reaction product at 0°C, and the resulting mixture is stirred at 0°C for 10 minutes. Next, the compound (4.17g, 11.017mmol) represented by chemical formula 3 prepared above is dissolved in 15mL N, N- dimethylformamide at 0°C. The solution of the compound represented by compound 3 dissolved in 15mL N, N- dimethylformamide is slowly added to the stirred product, stirred at 0°C for 30 minutes, and then stirred at room temperature for 20 hours. Water is slowly added to the stirred product at 0°C until no bubble is generated, diluted with 100mL ethyl acetate, washed with 50mL water, and then washed three times with 50mL water. The organic layer of the washed product was dried over 10 g of anhydrous sodium sulfate, filtered under reduced pressure, and concentrated under reduced pressure to obtain a yellow solid. The solid was recrystallized from 30 mL of a mixed solution of dichloromethane and n-hexane in a ratio of 1:9 to obtain the compound represented by Chemical Formula 4 (4.27 g, 78%) as a yellow crystalline solid.

[0127] The NMR results of the compound represented by Chemical Formula 4 are described below.

[0128] 1H NMR (400MHz, CDCl3) δ7.91 (d, J = 8.4Hz, 1H), 7.84 (d, J = 15.2Hz, 1H), 7.51-7.37 (m, 6H), 7.28-7.23 (m,2H),7.05(d,J=8.4Hz,1H),6.98(d,J=8.4Hz,2H),6.95-6.92(m,4H),5.16(s,4H),3.84(s,6H)

[0129] Preparation of the compound represented by Chemical Formula 5

[0130] To prepare the compound represented by the following Chemical Formula 5, the compound represented by Chemical Formula 4 prepared above was used:

[0131] [Chemical Formula 5]

[0132]

[0133] Specifically, the compound (4.27g, 8.599mmol) represented by chemical formula 4 prepared above is added to 70mL of anhydrous ethanol, and 70mL of 4N-sodium hydroxide aqueous solution is added at 0°C to confirm that the compound dissolves. After the above-mentioned compound dissolves, 12mL of hydrogen peroxide is slowly added at 0°C. The hydrogen peroxide reaction product is stirred at 0°C for 30 minutes, and then stirred at room temperature for 20 hours. 2N-hydrochloric acid aqueous solution is slowly added to the stirred product at 0°C, and when pH reaches 1, the gained solid is filtered under reduced pressure, and washed with 30mL of water, to obtain a yellow solid. The solid is recrystallized from 20mL of anhydrous ethanol to obtain a compound (4.24g, 96%) represented by chemical formula 5 as a yellow crystalline solid.

[0134] The NMR results of the compound represented by Chemical Formula 5 are described below.

[0135] 1 H NMR(400MHz, DMSO-d6)δ9.49(s,1H),8.11(d,J=7.6Hz,1H),7.93(d,J=2.0Hz,1H),7.88-7.76(m,3H),7.49-7.4 0(m,5H),7.26(d,J=8.8Hz,1H),6.95(dd,J=8.8,2.4Hz,4H),5.14(s,2H),5.12(s,2H),3.76(s,3H),3.75(s,3H)

[0136] Preparation of 3,4-dihydroxyflavonol compound represented by Chemical Formula 1

[0137] To prepare the compound represented by the following Chemical Formula 1, the compound represented by Chemical Formula 5 prepared above was used:

[0138] [Chemical Formula 1]

[0139]

[0140] Specifically, the compound (4.24g, 8.305mmol) represented by Chemical Formula 5 prepared above is added to 60mL of dichloromethane. 15mL of trifluoroacetic acid is slowly added to the dichloromethane reaction product at 0°C, and the resulting mixture is stirred at 0°C for 30 minutes, and then stirred at room temperature for 12 hours. The stirred product is concentrated under reduced pressure, and sodium bicarbonate aqueous solution is slowly added at 0°C to adjust the pH to 10. After pH adjustment, the resulting solid is filtered under reduced pressure and washed with 50mL of water to obtain a yellow solid. The solid is recrystallized from a mixed solution in which methanol and dichloromethane are mixed in a ratio of 0.5: 9.5 to obtain a 3,4-dihydroxyflavonol compound (1.16g, 50.3%) represented by Chemical Formula 1 as a yellow crystalline solid.

[0141] The NMR results of the 3,4-dihydroxyflavonol compound represented by Chemical Formula 1 are described below.

[0142] 1 H NMR (400MHz, DMSO-d6) δ8.10(dd,J=7.6,1.2Hz,1H),7.80-7.70(m,3H),7.61(dd,J=8.4,2.0Hz,1H),7.45(t,J=7.2Hz,1H),6.91(d,J=8.4Hz,1H)

[0143] Experimental Example 1. Cytotoxicity Evaluation of 3,4-Dihydroxyflavonol Compounds

[0144] HaCaT cells (human keratinocytes) were purchased from the Korean Cell Line Bank (KCLB) and cultured in DMEM (Gibco, USA) supplemented with 10% FBS (Gibco, USA) in a cell culture incubator maintained at 37°C and 5% CO2. The cells were subcultured every 2-3 days.

[0145] HaCaT cells were cultured at 2×10 4Cells / well were distributed in a 48-well plate and cultured for 24 hours. 3,4-dihydroxyflavonol prepared in the above preparation example was treated at a concentration of 1 μM, 5 μM, 10 μM, 20 μM, 25 μM or 50 μM and cultured for another 24 hours. After all the cultures were completed, 10 μl of EZ-Cytox solution was added to every 100 μl of culture medium and reacted in a cell culture incubator for 30 minutes. After the reaction, the absorbance was measured at 450 nm, and the cell viability was expressed as a percentage relative to the control group. The results of the above experimental example 1 are shown in Tables 1 and Figure 1 shown.

[0146] [Table 1]

[0147]

[0148] As shown in the results of Experiment 1, no cytotoxicity was observed for 3,4-dihydroxyflavonol at concentrations below 20 μM, but cytotoxicity was observed at concentrations above 25 μM. Therefore, subsequent experiments were performed using 3,4-dihydroxyflavonol at concentrations below 20 μM.

[0149] Experimental Example 2. Evaluation of Calcium Channel Inhibitory Ability of 3,4-Dihydroxyflavonol Compounds

[0150] Increased intracellular calcium concentration causes itching, which is caused by the activation of PAR2. PAR2 is one of the G protein-coupled receptors and is expressed in a variety of cells (such as muscle cells, bone cells, nerve cells, immune cells, epithelial cells, vascular endothelial cells and fibroblasts). When inflammation occurs, the PAR2 receptor is activated by trypsin, causing an increase in intracellular calcium concentration, leading to the generation of itching. Therefore, in this experimental example, a trypsin-induced calcium flux cell assay was performed to evaluate the degree of inhibition of calcium flux using 3,4-dihydroxyflavonol, which acts as a PAR2 antagonist.

[0151] The HaCaT cells used in this experiment were prepared in the same manner as in Experimental Example 1, and the HaCaT cells were cultured at a density of 2 × 10 4 50 μL of the solution was dispensed into a 96-well black culture plate at 100 cells / well and incubated in a 37°C, 5% CO2 incubator for 24 hours. The cells were then stained for 2 hours by adding 50 μL of FLIRP Calcium 6 assay reagent. The cells were treated twice with 100 μM 3,4-dihydroxyflavonol prepared in the above Preparation Example and treated with trypsin at a concentration of 20 μM using a syringe in a FlexStation 3. Fluorescence changes before and after sample treatment were measured on the FlexStation 3 in Flex mode at 485 / 535 nm.

[0152] As shown in the results of Experimental Example 2, 3,4-dihydroxyflavonol exhibited excellent inhibitory activity on calcium ion channels by inhibiting the calcium flux caused by trypsin by 40% or more (46.74% on average).

[0153] Experimental Example 3. Reduction of COX-2, MCP-1, IL-6, TNF-α, ICAM-1, and MMP1 gene expression induced by 3,4-dihydroxyflavonol compounds

[0154] The HaCaT cells used in the above experimental example were prepared in the same manner as in Experimental Example 1, with a density of 2×10 5 Cells / well were distributed into 6-well plates and cultured for 24 hours, followed by treatment with 3,4-dihydroxyflavonol (LYB) prepared in the above-mentioned preparation example at a concentration of 5 μM, 10 μM or 20 μM. Then, lipopolysaccharide (LPS, Sigma, USA) was added at a concentration of 1 μg / ml, and the cells were cultured for another 24 hours. After all cultures were completed, RNA was extracted from the cells obtained by centrifugation using a total RNA prep kit (Total RNA Prep Kit, Intronbio, South Korea). The extracted RNA was mixed with a reverse transcription premix (Bioneer, South Korea) and cDNA synthesis was performed at 45 ° C for 60 minutes using a PCR cycler, followed by reaction at 95 ° C for 5 minutes. Real-time PCR was performed to amplify and verify specific genes from the synthesized cDNA, wherein the process involved mixing cDNA, gene-specific primers, and SYBR green premix (PCR Biosystems, USA) and initializing at 95°C for 2 minutes, followed by 40 cycles of 5 seconds at 95°C and 30 seconds at 62.5°C to amplify specific genes. Based on the gene expression levels in the control group, relative quantification of gene expression levels was performed. The information of the primers used is shown in Table 2 below.

[0155] [Table 2]

[0156]

[0157] The results of this experiment are expressed as mean ± standard error of the mean and analyzed using SPSS Statistics version 21.0 (IBM, USA). Statistical comparisons between two groups were first performed using the independent sample t-test, followed by analysis of variance (ANOVA) for multiple group comparisons. Next, the Tukey honestly significant difference (HSD) test was performed to test for significance at a significance level of 0.05, with significance divided into three levels: p < 0.05, p < 0.01, and p < 0.001.

[0158] Changes in gene expression caused by 3,4-dihydroxyflavonol were confirmed at the RNA levels of COX-2, MCP-1, IL-6, TNF-α, ICAM-1, and MMP1, and the results are described below.

[0159] Cyclooxygenase-2 (COX-2)

[0160] The COX-2 gene expression level measured by 3,4-dihydroxyflavonol showed a significant decrease compared to the control group. The detailed results are shown in Tables 3 and Figure 2 Shown in.

[0161] [Table 3]

[0162]

[0163] Monocyte chemoattractant protein 1 (MCP-1)

[0164] The expression level of MCP-1 gene was significantly decreased in the presence of 3,4-dihydroxyflavonol compared to the control group. Figure 3 Shown in.

[0165] [Table 4]

[0166]

[0167] Interleukin-6 (IL-6)

[0168] The IL-6 gene expression level measured by 3,4-dihydroxyflavonol showed a significant decrease compared to the control group. The detailed results are shown in Tables 5 and Figure 4 Shown in.

[0169] [Table 5]

[0170]

[0171] Tumor necrosis factor-α (TNF-α)

[0172] The TNF-α gene expression level measured by 3,4-dihydroxyflavonol showed a significant decrease compared to the control group. The detailed results are shown in Tables 6 and Figure 5 Shown in.

[0173] [Table 6]

[0174]

[0175] Intercellular adhesion molecule-1 (ICAM-1)

[0176] The ICAM-1 gene expression level measured by 3,4-dihydroxyflavonol showed a significant decrease compared to the control group. The detailed results are shown in Tables 7 and Figure 6 Shown in.

[0177] [Table 7]

[0178]

[0179] Matrix metalloproteinase-1 (MMP-1)

[0180] The measurement of MMP-1 gene expression level based on 3,4-dihydroxyflavonol showed a significant decrease compared to the control group. The detailed results are shown in Table 8 and Figure 7 Shown in.

[0181] [Table 8]

[0182]

[0183] Experimental Example 4. Reduction of COX-2, MCP-1, IL-6, TNF-α, and ICAM-1 protein expression induced by 3,4-dihydroxyflavonol compounds

[0184] The HaCaT cells used in the above experimental example were prepared in the same manner as in Experimental Example 1, with a density of 2×10 5 Cells were plated at 10 μM / well in a 6-well plate and cultured for 24 hours. They were then treated with 3,4-dihydroxyflavonol (LYB) prepared in the above-mentioned Preparation Example at concentrations of 10 μM or 20 μM. Lipopolysaccharide (LPS) was then added at a concentration of 1 μg / ml, and the cells were cultured for an additional 24 hours. After all cultures were completed, the cells, obtained by centrifugation, were reacted with RIPA buffer (Thermo Fisher, USA) containing protease inhibitor cocktail I (Sigma, USA) and phosphatase inhibitors II and III (Sigma, USA) to extract proteins. The extracted proteins were quantified using a BCA protein assay kit (Thermo Fisher, USA) and prepared by mixing with loading buffer (ELPISbiotech, South Korea) and reacting at 95°C for 5 minutes. The prepared proteins were size-separated by SDS-PAGE using a 10% acrylamide gel and transferred to a PVDF membrane. The membrane with the transferred proteins was soaked in 3% BSA, reacted at room temperature for 2 hours, and washed with TBS-T buffer. Then, the primary antibody (Cell Signaling, USA) was added and reacted at 4°C for 16 hours. The membrane after the reaction with the primary antibody was washed three times, and then the secondary antibody (Jackson ImmunoResearch, USA) was added and reacted at room temperature for 1 hour, followed by washing again. Then, the protein was developed using enhanced chemiluminescence (ECL) solution. Then, the protein expression level was analyzed by Chemidoc Fusion FX.

[0185] The results of Experimental Example 4 were statistically analyzed in the same manner as in Experimental Example 3.

[0186] Changes in protein expression caused by 3,4-dihydroxyflavonol were confirmed in COX-2, MCP-1, IL-6, TNF-α, and ICAM-1, and the results are described below.

[0187] Cyclooxygenase 2 (COX-2)

[0188] The expression level of COX-2 protein was significantly decreased in the 3,4-dihydroxyflavonol group compared to the control group. Figure 8 Shown in.

[0189] [Table 9]

[0190]

[0191] Monocyte chemoattractant protein 1 (MCP-1)

[0192] The expression level of MCP-1 protein was significantly decreased in the 3,4-dihydroxyflavonol group compared to the control group. Figure 9 Shown in.

[0193] [Table 10]

[0194]

[0195] Interleukin-6 (IL-6)

[0196] The IL-6 protein expression level measured by 3,4-dihydroxyflavonol showed a significant decrease compared to the control group. The detailed results are shown in Tables 11 and Figure 10 Shown in.

[0197] [Table 11]

[0198]

[0199] Tumor necrosis factor-α (TNF-α)

[0200] Compared with the control group, the expression level of TNF-α protein was significantly decreased according to the measurement of 3,4-dihydroxyflavonol. The detailed results are shown in Table 12 and Figure 11 Shown in.

[0201] [Table 12]

[0202]

[0203] Intercellular adhesion molecule-1 (ICAM-1)

[0204] The expression level of ICAM-1 protein was significantly decreased in the 3,4-dihydroxyflavonol group compared to the control group. Figure 12 Shown in.

[0205] [Table 13]

[0206]

[0207] Through the above description, those skilled in the art will understand that the present disclosure can be embodied in other specific forms without changing its technical spirit or essential features. In this regard, it should be understood that the above embodiments are illustrative in all aspects and not restrictive.

Claims

1. A pharmaceutical composition for preventing, improving or treating atopy, comprising 3,4-dihydroxyflavonol represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof: [Chemical Formula 1] 2. The pharmaceutical composition according to claim 1, wherein The 3,4-dihydroxyflavonol inhibits protease activated receptor 2 (PAR2).

3. The pharmaceutical composition according to claim 1, wherein The pharmaceutical composition reduces the expression of at least one gene selected from the group consisting of COX-2, MCP-1, IL-6, TNF-α, ICAM-1 and MMP1.

4. A cosmetic composition for preventing or improving atopy, comprising 3,4-dihydroxyflavonol represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof: [Chemical Formula 1] 5. A method for preparing the 3,4-dihydroxyflavonol according to claims 1-4, comprising: (S1) preparing a compound represented by the following Chemical Formula 3 from a compound represented by the following Chemical Formula 2; (S2) preparing a compound represented by the following Chemical Formula 4 from the compound represented by Chemical Formula 3; (S3) preparing a compound represented by the following Chemical Formula 5 from the compound represented by Chemical Formula 4; as well as (S4) preparing a 3,4-dihydroxyflavonol compound represented by the following Chemical Formula 1 from the compound represented by Chemical Formula 5: [Chemical Formula 1] [Chemical Formula 2] [Chemical Formula 3] [Chemical Formula 4] [Chemical Formula 5] 6. The method according to claim 5, wherein: The step S4 includes preparing the compound represented by Chemical Formula 5 by reacting the compound represented by Chemical Formula 4 with dichloromethane, trifluoroacetic acid, an aqueous sodium bicarbonate solution, and a mixed solution of methanol and dichloromethane in sequence.

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