KR101419212B1 - Composition for skin whitening comprising 5-cyano-6-[(3-methoxybenzyl)sulfanyl]-2-methyl-N-phenyl-4-(2-thienyl)-1,4-dihydro-3-pyridinecarboxamide as effective component and uses thereof - Google Patents

Abstract

The present invention relates to a skin whitening composition and uses thereof. A 5-cyano-6-[(3- Methoxybenzyl)Sulfanyl]-Methyl-N-phenyl-4-(2- Thienyl)-1,4-Dihydro-3-Pyridine carboxamide compound which represents an excellent melanogenesis inhibition effect can be used helpfully as an effective component of a cosmetics composition, a food composition, and a medicinal composition for whitening skin since the compound has almost no toxicity.

Description

Methyl-N-phenyl-4- (2-thienyl) -1,4-dihydro-3-pyridinecarboxamide Compound The present invention relates to a composition for skin whitening comprising 5-cyano-6 - [(3-methoxybenzyl) sulfanyl] -2-methyl- 1,4-dihydro-3-pyridinecarboxamide as effective component and uses thereof.

More particularly, the present invention relates to a composition for skin whitening, and more particularly to a composition for skin whitening, Thienyl) -1,4-dihydro-3-pyridinecarboxamide compound as an active ingredient, to a cosmetic composition, a food composition and a pharmaceutical composition for skin whitening.

The skin color of a person is determined by various factors such as the activity of the melanocyte which forms the melanin pigment, the distribution of blood vessels, the thickness of the skin, and the presence or absence of pigment in and out of the body such as carotenoid and bilirubin. Among them, melanin, which is formed in melanocyte, is considered to be the most important factor for skin color formation. Melanin is converted to tyrosine by tyrosinase through 3,4-dihydroxyphenylalanine, dopaquinone, leucodopachrome, dopachrome, 5,6-dihydroxyindole and indole-5,6-quinone and finally to melanin It is the end product of the oxidation process being converted. That is, tyrosine and tyrosinase act as proteins which are very important in the production of melanin.

In addition, another protein important for melanin formation is the P protein. These P proteins are required for the production of eumelanin, which causes brown / black pigmentation rather than pheomelanin, which causes yellow / red pigmentation (Lamoreux et al. , Pigment Cell Res . 8: 263-70, 1995), and is known to play a role in regulating the pH of tyrosine transport and melanosome. However, it is known that P proteins play a crucial role in producing normal amounts of melanin in skin, hair and eyes, but the exact function of P proteins remains unclear in this process.

Thus, melanin biosynthesis is a very complex process and the precise mechanisms for inducing melanin synthesis have yet to be elucidated. Melanin provides a natural defense against harmful ultraviolet rays, but when melanin is overproduced, it induces hyperpigmentation such as spots and freckles. In addition, excessive production of melanin due to excessive UV exposure may cause skin cancer. Therefore, the necessity of development of a melanin biosynthesis inhibitor is increasing, and studies related to whitening are actively performed.

Active substances such as arbutin, deoxy arbutin, octadecendioic acid, conjugated linoleic acid, niacinamide, hydroquinone, kojic acid, glutathione, and azelaic acid are known as substances exhibiting a whitening effect by inhibiting pigmentation of skin. Such materials are known to have tyrosinase inhibitory activity and have in fact been used in whitening products. However, the above-mentioned active ingredients are limited in their use due to insufficient whitening effect, safety to skin, difficulty in formulation and stability, and the like. Accordingly, the present inventors intend to provide a compound having excellent whitening effect and high stability and skin safety as a cosmetic composition.

Korean Patent No. 1050484 discloses a composition for skin whitening comprising a lactone compound as an active ingredient and Korean Patent No. 0755742 discloses a composition for skin whitening comprising an acrylate compound Lt; / RTI > However, as in the present invention, when 5-cyano-6 - [(3-methoxybenzyl) sulfanyl] -2-methyl- - pyridinecarboxamide as an active ingredient has not been disclosed.

Disclosure of the Invention The present invention has been made in view of the above-mentioned needs, and the present inventors have conducted intensive studies to provide 5-cyano-6- [(3-methoxybenzyl) sulfanyl] (2-thienyl) -1,4-dihydro-3-pyridinecarboxamide compound exhibited excellent melanin production inhibitory effect and showed no cytotoxicity even at a concentration of 20 μM or higher By confirming, the present invention has been completed.

In order to solve the above problems, the present invention provides a process for producing 5-cyano-6 - [(3-methoxybenzyl) sulfanyl] A cosmetic composition for skin whitening, a food composition, and a pharmaceutical composition containing the dihydro-3-pyridinecarboxamide compound as an active ingredient.

According to the present invention there is provided a process for the preparation of 5-cyano-6 - [(3-methoxybenzyl) sulfanyl] The pyridinecarboxamide compound is excellent in the effect of inhibiting melanin formation and has almost no cytotoxicity, and thus can be usefully used as an effective ingredient of a skin whitening composition.

FIG. 1 shows the results of analyzing the effects of melanin production on the melanin production and cell viability after treating the melanin cells with 5, 10 and 20 μM concentrations of the compounds selected to analyze the melanin production inhibitory effect.
FIG. 2 shows the results of western blotting on the effect of treatment of a compound having an excellent melanin production inhibitory effect on tyrosinase expression of melanocytes.
FIG. 3 shows the results of HPLC analysis of the content of pheomelanin and eumelanin produced when a compound having excellent melanin production inhibitory effect and low cytotoxicity was treated in melanocytes.

The present invention provides a cosmetic composition for skin whitening comprising a compound represented by the following formula (1) as an active ingredient.

The compound represented by the above formula (1) can be produced by reacting 5-cyano-6 - [(3-methoxybenzyl) sulfanyl] 3-methoxybenzyl) sulfanyl] -2-methyl-N-phenyl-4- (2-thienyl) -1,4-dihydro-3- pyridinecarboxamide .

In order to exert an excellent whitening effect when a whitening component is applied to actual skin, it exhibits a highly effective skin whitening effect at a low concentration, has excellent ability to be permeated and absorbed through the skin, Is low, the active ingredient remains stable on the composition or on the skin, is easy to be formulated into medicines or cosmetics, and is safe for the skin. However, among known whitening ingredients, whitening ingredients that satisfy all of the above characteristics are not common. For example, some whitening agents have excellent whitening activity even at low concentrations in vitro, but they are difficult to apply to real skin because of their ability to permeate through the skin. Other whitening ingredients have low hydrophilicity and are difficult to formulate into cosmetics or pharmaceuticals. In addition, some of the whitening ingredients may be decomposed or transformed into other compounds when exposed to heat, light, or oxygen, leading to the disappearance of the whitening effect prior to application to the skin. However, the compound of formula (I) of the present invention has a melanin inhibitory effect comparable to that of arbutin, which is a known whitening ingredient, and is cytotoxic to arbutin, which is very safe for skin.

In the present invention, 'whitening' refers to inhibiting, inhibiting or alleviating hyperpigmentation of the skin. Hyperpigmentation of the skin includes freckles, stains, hyperpigmentation after exposure to ultraviolet light, hyperpigmentation after inflammation, aging black spots, brown spots or black spots.

In the cosmetic composition according to an embodiment of the present invention, the skin whitening cosmetic composition may contain the compound of Formula 1 in an amount of 0.0001 to 90 parts by weight based on the total weight of the composition, Including, but not limited to, an effective amount.

In the present invention, "effective amount" means the amount of a compound capable of inhibiting, inhibiting or alleviating hyperpigmentation of the skin. The effective amount of the compound contained in the composition for skin whitening of the present invention will vary depending on the form in which the composition for skin whitening is commercialized, the method in which the compound is applied to the skin, and the time on the skin. For example, when the composition for skin whitening is commercialized as a medicament for dermatological treatment due to hyperpigmentation of the skin, the composition of the present invention may contain a compound of the above formula 1 at a higher concentration than a cosmetic product that is routinely applied to skin You can do it. In the case of a wash-off type cosmetic such as a make-up remover, a detergent and the like, in which the active ingredient remains on the skin in a short period of time even when it is commercialized as a cosmetic product, it may contain a relatively high concentration of the compound. On the other hand, in the case of leave-on type cosmetics such as lotion, cream, essence and the like in which the active ingredient remains on the skin for a long period of time, the compound may be contained at a lower concentration than the wash-off type cosmetics will be.

In the cosmetic composition according to one embodiment of the present invention, the cosmetic composition for skin whitening may be at least one selected from the group consisting of ointment for external use, cream, softening agent, nutritional lotion, pack, essence, hair tonic, shampoo, rinse, hair conditioner, , Skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisturizing lotion, nutrition lotion, massage cream, nutritional cream, moisturizing cream, hand cream, foundation, nutrition essence, sunscreen, soap, cleansing foam, cleansing lotion , A cleansing cream, a body lotion, and a body cleanser. The composition of each of these formulations may contain various kinds of bases and additives necessary for formulation of the formulation, and the kinds and amounts of these ingredients can be easily selected by those skilled in the art.

The composition of the present invention may further contain at least one skin whitening active ingredient which exhibits the same or similar function to the compound of the present invention. Skin whitening active ingredients include kojic acid and its derivatives, arbutin, ascorbic acid and its derivatives, hydroquinone and its derivatives, resorcinol, cycloalkanone, methylenedioxyphenylalkanol, 2,7-dinitroindazole or Plant extracts such as mandarin orange extract, rice extract, licorice extract, and the like, but are not limited thereto.

When the formulation of the present invention is a paste, cream or gel, animal fiber, plant fiber, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier component .

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In the case of a spray, in particular, / Propane or dimethyl ether.

In the case of the solution or emulsion of the present invention, a solvent, a solvent or an emulsifier is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid esters.

When the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

When the formulation of the present invention is an interfacial active agent-containing cleansing, the carrier component is selected from aliphatic alcohol sulfates, aliphatic alcohol ether sulfates, sulfosuccinic acid monoesters, isethionates, imidazolinium derivatives, methyltaurate, sarcosinates, fatty acid amides Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, linolenic derivatives or ethoxylated glycerol fatty acid esters.

The formulation of the present invention may further contain an excipient including a fluorescent substance, a fungicide, a hygroscopic substance, a humidifier, a fragrance, a fragrance carrier, a protein, a solubilizing agent, a sugar derivative, a sunscreen, a vitamin, .

When the cosmetic composition for skin whitening as described above is formulated into pharmaceuticals or cosmetics, it may contain known excipients applicable to the skin acting as a carrier for the active ingredient. When formulating into pharmaceuticals, reference is made to the disclosure of [Remington ' s Pharmaceutical Science, Mack Publishing Company, Easton PA], and in formulation into cosmetics, International cosmetic ingredient dictionary, 6th ed., The cosmetic, Toiletry and Fragrance Association, Inc., Washington, 1995). Which are incorporated herein by reference.

The present invention also provides a skin whitening food composition comprising, as an active ingredient, a compound represented by the following formula (1).

[Chemical Formula 1]

When a composition containing the compound of Formula 1 of the present invention as an active ingredient is used as a food additive, the composition may be added as it is or may be used together with other food or food ingredients, and may be suitably used according to a conventional method . The amount of the active ingredient to be mixed can be suitably determined according to its intended use (prevention, health or therapeutic treatment). Generally, the composition of the present invention is added in an amount of not more than 15 parts by weight, preferably not more than 10 parts by weight, based on the raw material, when the food or beverage is produced. However, in the case of long-term consumption intended for health and hygiene purposes or for health control purposes, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range .

There is no particular limitation on the kind of the food. Examples of foods to which the above substances can be added include dairy products including meats, sausages, breads, chocolates, candies, snacks, confectionery, pizza, ramen noodles, gums, ice cream, soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include healthy foods in a conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Such natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like.

In addition to the above, the composition of the present invention may further comprise various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and its salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, A carbonating agent used in beverages, and the like. In addition, the composition of the present invention may contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. Although the ratio of these additives is not critical, it is generally selected from the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention, but the present invention is not limited thereto.

The present invention also provides a pharmaceutical composition for skin whitening comprising a compound represented by the following general formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Chemical Formula 1]

In the pharmaceutical composition for skin whitening according to the present invention, the compound of Formula 1, which is an effective ingredient, may be used as it is or in the form of a pharmaceutically acceptable salt. The salt is not particularly limited as long as it is pharmaceutically acceptable so long as it is pharmaceutically acceptable and includes, for example, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrofluoric acid, hydrobromic acid, formic acid acetic acid, tartaric acid, lactic acid, citric acid, fumaric acid, , Benzenesulfonic acid, toluenesulfonic acid, and naphthalenesulfonic acid. In addition to acid addition salts, additional base salts such as sodium hydroxide, potassium hydroxide, triethylamine, tertiary-butylamine may also be used.

In the formulation of the pharmaceutical composition according to the present invention, the content of the active ingredient may vary widely depending on the formulations, and is generally according to a conventional method.

The pharmaceutical composition according to the present invention may be formulated by adding various adjuvants such as carriers and other additives such as stabilizers, emollients, emulsifiers and the like, which are used in medicines, if necessary, so long as they do not adversely affect the active ingredients.

In addition, the composition according to the present invention can be administered parenterally or orally. As the route of parenteral administration, transdermal administration is preferable, and local application is most preferable. For example, it may be manufactured in the form of a bandage, but is not limited thereto. The formulations may be any formulations suitable for pharmaceutical preparations including ointments, creams, injections, powders, granules, tablets, and the like.

The preferred dosage of the pharmaceutical composition according to the present invention may be 0.001 to 1000 mg / Kg · day, but is not limited thereto. In addition, the composition according to the present invention may be administered alone, or may be administered together with other medicines in order to aid in the same or another medicament. In addition, in the composition of each formulation, components other than the composition, which is the above-mentioned essential ingredient, can be mixed and selected by a person skilled in the art according to the formulation or purpose of use of the other external preparation. In this case, Can happen.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Materials and methods

1. Cell culture

Melanocytes were cultured in RPMI medium containing 10% FBS, 1% penicillin / streptomycin and 200 nM TPA in an incubator at 37 ° C and 10% CO ₂ . Subculture was performed when melanocytes proliferated to 80-90% of the culture plate.

2. Melanin analysis

Experiments were conducted to select compounds that inhibit P protein activity and exhibit a whitening effect. In silico modeling was used to explore P protein inhibitors. Since there is no known crystal structure of P protein, we performed homology analysis using amino acid sequence and fragmental modeling of P protein using tyrosine transporter and other channel proteins. The generated model was verified using PROCHECK, WHAT IF, SUPERPOSE, and docking by Molegro virtual docker using ZINC database generated ligand library. ADME / TOX analysis was performed and a pharmacophore model was constructed to select the final 20 compounds. Each compound was purchased from www.specs.net.

The amount of melanin produced in the melanocytes was measured to determine the whitening effect of each of the 20 compounds, and the amount of melanin was measured using PBS, DMSO (dimethyl sulfoxide) and arbutin (200 μg / ml) as control. Cells were inoculated into 12-well plates and treated with 5, 10 and 20 μM concentrations of the compound and control material, respectively, after 24 hours, and incubated for 72 hours, then cells were recovered using trypsin / EDTA. To the recovered cells, 1 N NaOH containing 10% DMSO was added and reacted at 90 ° C for 1 hour to allow the melanin to sufficiently dissolve. Then, the absorbance was measured at 405 nm using an ELISA reader.

Melanin production (%) = absorbance of treatment / non-treatment absorbance × 100

3. Toxicity test ( MTT assay )

To determine whether or not each compound having melanin production inhibitory effect can be used as a whitening cosmetic composition, a cytotoxicity test was conducted. Cells were inoculated into 96-well plates and treated with 5, 10 and 20 μM concentrations of each compound and cultured in an incubator at 37 ° C and 10% CO ₂ for 2 days. After the culture, the medium was removed and 100 μl of a solution of 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide was added to the melanocytes and reacted at 30 ° C for 4 hours. After the reaction was completed, the solution was removed, 100 μl of DMSO was added to extract the formazan produced in the stained cells, and the absorbance at 540 nm was measured using an ELISA reader.

Cell survival rate (%) = (1-treated absorbance / untreated absorbance) x 100

4. Western Blot

To confirm whether or not each compound having an inhibitory effect on melanin production inhibits the expression of tyrosinase, melanin cells were treated with melanin cells at a concentration of 20 μM for each compound having an inhibitory effect on melanin production, and after 24 hours, . The extracted proteins were quantitated at the same concentration, the proteins were separated by SDS-PAGE, and Western blotting was performed using an anti-tyrosinase antibody.

5. HPLC  ( high performance liquid chromatography )

HPLC was performed to determine the amount of pheomelanin and eumelanin produced in the melanocytes. 1 × 10 ⁶ melanocytes were inoculated into a 100-pt Petri dish and cultured for 24 hours, treated with each compound, and cultured again for 72 hours to collect the cells. The collected cells were suspended in 100 μl of distilled water, transferred to a 10-ml tube, and 375 μl of 1M K ₂ CO ₃ and 25 μl of 30% H ₂ O ₂ were added and stirred at 25 ° C for 20 hours. A supernatant obtained by the addition of 10% Na ₂ SO ₃ and 6M HCl for 50 ㎕ and centrifuged for 1 minute to 4000 g was carried out an HPLC analysis.

HPLC (Waters Breeze system; Shisedio C18 column) was performed using thiazole-2,4,5-tricarboxylic acid (TTCA) as a standard compound and pyrrole-2,3,5-tricarboxylic acid ; Mobile phase: 0.1 M potassium phosphate buffer (pH 2.1) / methanol = 99: 1; flow rate: 0.7 ml / min).

Example  1. Melanogenesis inhibitor compound selection

The results of experiments conducted to inhibit the activity of P protein (P protein) involved in melanin production in melanocytes to reduce melanin production and to select compounds that are not toxic to cells are shown in FIG. The melanin content of the cells treated with each compound (5, 10 and 20 μM concentration) was converted to a relative value with the melanin content of the control cells not treated with the compound as 100%.

Melanin analysis showed that the compound of the formula (1) (1-methyl-2-thienyl) - Dihydro-3-pyridinecarboxamide), # 4 (methyl 5-cyano-6- {[2- (4- 4- (2-thienyl) -1,4-dihydro-3-pyridinecarboxylate), # 8 (3- (1-adamantyl) -6- ) -2,4-dihydropyrano [2,3-c] pyrazole-5-carbonitrile), # 13 (Methyl 6 - {[2- (1, 3-benzodi [iota], 4-dihydro- Oxoethyl] sulfanyl} -5-cyano-2-methyl-4- (2-thienyl) -1,4-dihydro-3-pyridinecarboxylate) Melanin inhibitory effect was high.

Analysis of cytotoxicity showed that the # 1 and # 4 compounds were not toxic at all three concentrations, and the # 8, # 13, and # 17 compounds were toxic at concentrations of 10 μM or more Melanin synthesis inhibitory effect was found to be high even at a concentration that is devoid of toxicity or shattering. On the basis of the above results, compounds # 1, # 4, # 8 and # 13 having high melanogenesis inhibitory effect and low cytotoxicity were selected as melanogenesis inhibitory compounds.

Example  2. The melanin inhibitory compound is tyrosinase ( tyrosinase ) Effect on protein expression

Analysis of tyrosinase protein expression of the cells treated with 20 μM of the compounds # 1, # 4, # 8, # 13 and # 17 selected in Example 1 revealed that the compounds # 1, # 4 and # 17 The amount of tyrosinase protein was decreased by treatment (Fig. 2). It is considered that the compounds # 1, # 4 and # 17 affect biosynthesis of tyrosinase and inhibit melanin production. On the other hand, in the case of the # 8 and # 13 compounds, there was no difference in expression of tyrosinase (FIG. 2). However, since the melanin production is inhibited by the treatment with the compounds # 8 and # 13, the compounds are expected to inhibit melanogenesis by affecting the maturation of the tyrosinase protein in a functional form.

Example  3. HPLC  Analysis of melanin content

The peomelanin and eumelanin contents of the cells treated with 20 μM of the compounds # 1, # 4, # 8 and # 13 selected in Example 1 were compared with those of the untreated control, It was confirmed that the content of melanin was remarkably decreased (Fig. 3).

Claims (4)

A cosmetic composition for skin whitening comprising a compound represented by the following formula (1) as an active ingredient.
[Chemical Formula 1]

The cosmetic composition according to claim 1, wherein the cosmetic composition is at least one selected from the group consisting of an ointment for external use, a cream, a softening agent, a nutritional lotion, a pack, an essence, a hair tonic, a shampoo, a rinse, a hair conditioner, a hair treatment, a gel, a skin lotion, Lotion, milk lotion, moisturizing lotion, nutrition lotion, massage cream, nutritional cream, moisturizing cream, hand cream, foundation, nutrition essence, sunscreen, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion and body cleanser Wherein the cosmetic composition has a formulation selected from the group consisting of: A food composition for skin whitening comprising a compound represented by the following formula (1) as an active ingredient.
[Chemical Formula 1]

A pharmaceutical composition for skin whitening comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.
[Chemical Formula 1]

Images